Q*p£oi in ttxe $it$ of Stew WiaxU College of pijvstctans an& burgeons Reference itibrarp i.»'i '. jv. I x-,:, r :'%u. I ■ ' cm ■ ■k/ ■ i Digitized by the Internet Archive in 2010 with funding from Columbia University Libraries http://www.archive.org/details/enzymestheirapplOOeffr '-«*&* V ENZYMES AND THEIR APPLICATIONS. BY DR. JEAN EFFRONT, PROFESSOR IN THE NEW UNIVERSITY IN BRUSSELS AND DIRECTOR OF THE FERMENTATION INSTITUTE. ENGLISH TRANSLATION BY SAMUEL C. PRESCOTT, S.B., INSTRUCTOR IN INDUSTRIAL BIOLOGY, MASSACHUSETTS INSTITUTE OF TECHNOLOGY, BOSTON. Volume I. THE ENZYMES OF THE CARBOHYDRATES. THE OXIDASES. FIRST EDITION. FIRST THOUSAND. NEW YORK: JOHN WILEY & SONS. London: CHAPMAN & HALL, Limited. 1902. Copyright, 1902, BY JOHN WILEY & SONS. ROBERT DRUMMOND PRINTER NEW VORK. AUTHOR'S PREFACE. The study of chemical ferments affords the double advan- tage of presenting a broad scientific interest and having at the same time' numerous industrial applications. The phe- nomena of assimilation and respiration which take place in the interior of the living cell are in direct relation to the diastatic secretions, the study of which is consequently of as great importance to physiologists as to botanists and bac- teriologists. A knowledge of the reactions caused by the diastases is also of first importance to chemists, for whom these physiological agents may become reagents of an ex- ceptional sensitiveness. The science of chemical ferments comprises also the knowledge of certain microbial poisons, which, by their properties, are singularly allied to ordinary diastases. To study these poisons from the point of view of their diffusion, conservation, and destruction in the organ- ism, one must also possess an accurate knowledge of en- zymes. Finally, a whole class of soluble ferments have found, at the present time, industrial application, and undoubtedly the future will add many others ; here, then, is a further in- terest which attaches to the study of enzymes. The present work, which is a summary of the course given at the Institute of Fermentations of the new Univer- sity of Brussels, is designed both for persons who give them- selves up to purely scientific studies and for those who are occupied particularly in fermentation industries. So, while reserving the largest place for theoretical questions, we have 347505 iv AUTHOR'S PREFACE. not neglected the practical results. Our work is divided into two parts. In the first, which constitutes the present volume, we deal with the enzymes of carbohydrates and with the oxidases, as well as with their industrial applications. In the second part, now in preparation, we shall study the pro- teolytic enzymes and the toxins. We have personally verified the greater part of the ex- perimental data which this first volume contains, in which the reader will find a certain number of hitherto unpublished ex- periments, methods of preparation, methods of analysis, and technical processes. Brussels, 1898. TRANSLATOR'S NOTE. Up to the present time very few works upon Enzymes have appeared in our language. In the translation of Profes- sor Effront's book I have been actuated by the desire to make available in English the valuable material contained in the original French edition. I have endeavored to reproduce the author's ideas with clearness and simplicity, without at the same time deviating too much from his own form of ex- pression. I have made no additions or changes, and the book is therefore presented in a form as nearly as possible like that in which it came from the pen of the author. I wish to acknowledge my indebtedness to my friend, Mr. Percy G. Stiles of Johns Hopkins University, for much as- sistance in proof revision. Samuel C. Prescott. Massachusetts Institute of Technology, December, 1901. TABLE OF CONTENTS. PAGE Preface "*• CHAPTER I. GENERAL REMARKS. Synthetical and analytical work of the living cell.— Synchronism of the two phenomena. — Difference between chemical and physio- logical activity. — Chemical agents and physiological agents. — In- tervention of vital energy.— Necessity of studying the physical and chemical conditions of the medium. — Definition of enzymes. ■ — Their part in assimilation and disassimilation. — Enzymes as producers of heat I CHAPTER II. GENERAL PROPERTIES. History of the knowledge of enzymes. — Works of Reaumur and Spallanzani,Kirchoff, Dubrunfaut and Payen. — General properties of diastases. — Means of distinguishing a diastatic action from a purely chemical action. — Test with tincture of guaiacum. — Law of proportionality in diastatic action. — Means of distinguishing the work of organized ferments from diastatic action. — Means of isolating the diastase from the medium which contains it. — Chem- ical composition of enzymes. — Zymogenesis. — Method of action of diastases ifc CHAPTER III. MANNER OF ACTION OF DIASTASES. Manner of action of diastases. — Different opinions on this subject. — The diastatic property and the diastase itself. — Works of vii Tin TABLE OF CONTENTS. Bunzen, Hiifner, Naegeli, Wittich and Fick, de Jager, Arthus. — Analogy between organized ferments and soluble ferments. — Hypothesis of Armand Gautier on the nature of enzymes. 27 CHAPTER IV. INDIVIDUALITY OF ENZYMES. Difficulties encountered in proving the individuality of enzymes. — In- fluence of the manner of nutrition of the cells on the nature of the enzymes they secrete. — Direct proofs of the individuality of en- zymes. — Relation between the diastases; the chemical constitution arid the structure of the bodies on which they act. — Nomenclature of enzymes. — Classification 4 action of each of these enzymes is limited to a certain number of bodies. These facts are sufficiently numerous for us to draw a general conclusion in favor of the individuality of enzymes. And as a matter of fact, it is difficult to believe that the same active substance can in one special case act on two or three chemical substances, while in another case its action is limited to a single one of these substances. As we have just seen, a diastase having a hydrating or oxidizing function does not act on all the substances capable of being hydrated or oxidized ; the diastatic agent differs completely from a chemical agent having a definite function and exercising it independently of the constitution of the bodies on which it acts. For example, by the action of a mineral acid is obtained the splitting of saccharose, the saponification of fatty matters, the decomposition of gluco- sides, the peptonization of albuminoid substances, in a word all the phenomena which we meet in diastatic hydrations. Among diastases, on the contrary, decompositions and hy- drations are caused by numerous agents, each capable of a special diastatic work and acting upon only a very limited number of substances. The action of acids is, then, up to a certain point, independent of the constitution of the bodies on which they act, while diastases exercise their hvdrating or oxidizing action only on bodies of a strictly definite struc- ture. A hydrating enzyme may sometimes exert its action on different bodies, but only when the chemical constitution of these bodies is very much like that of the diastase, and when they can furnish the same products of decomposition. It is thus that amylase acts on starch, glycogen, and dextrin, giv- ing always the same end-product, maltose. Pepsin acts on a great number of bodies, for example, on all albuminoid substances. Xow all these bodies resemble each other and have a very similar structure, since their products of decomposition by the diastase are always the same: proteoses and peptones. The enzymes of glucosides appear capable, at first sight, 44 THE ENZYMES AND THEIR APPLICATIONS. of a more energetic action, and one extending to chemically different bodies, but this anomaly is only apparent : emulsin, -which acts on very complex bodies, affects only that part common to all the molecules of glucosides. The action of emulsin is due to the affinity it has for glucose, and, as Emil Fischer has demonstrated, this affinity is explained by the stereochemical structure of the carbohydrate molecules. Emulsin acts, not only on natural glucosides, but also on artificial ethers, which are obtained with glucose. In studying the action of enzymes on artificial ethers, Emil Fischer has found this very interesting fact : that the ac- tion or inaction of an enzyme depends, not only on the com- position of the substance on which it is made to act, but also on its configuration. By treating glucose with methyl alcohol in the presence of hydrochloric acid one obtains two isomeric ethers, differing in their geometrical structure on account of the asymmetrical carbon atoms of the glucoside chain. The formation of two isomeric ethers is easy to explain : the aldehydic glucose group disappears by the action of the alcohol in presence of hydrochloric acid, and dehydration is produced in the glucose chain, giving rise to an intra- molecular ethereal group. The carbon of the aldehydic group thus becomes asymmetrical, and in consequence the appearance of two stereo-isomers becomes comprehensible. The two isomeric glucosides H-C-O-CH, HCOH HCOH CH HCOH CH.-C-O-H HCOH \ HCOH CH HOCH H 2 COH HOCH a. Methyl dextroglucoside. /5. Methyl dextroglucoside. behave differently under the action of enzymes. INDIVIDUALITY OF ENZYMES. 45 Emulsin, which acts on certain derivatives of dextrose and galactose, acts also on the /3-methyl glucoside, but has no action on the isomer a. In the beer-yeasts is found another soluble ferment which acts on natural glucosides, but this ferment has absolutely no effect on the /?-methyl glucoside, while it acts on the isomer a. This example is another proof of the individuality of en- zymes and shows in a striking way the influence which the chemical structure of bodies has on the diastatic action. Emil Fischer has evolved the hypothesis that a diastatic action cannot be produced except on condition that there shall be a stereochemical relation between the acting sub- stance and the body acted upon. According to him, it is necessary that the ferments and the substances they act upon shall have a like geometrical structure, or at least a certain structural resemblance. We believe that this hypothesis will explain very happily the development of different diastases from cells which have been fed with different substances. A cell nourished by starch will secrete an enzyme having the stereochemical structure of starch, while if the cell is nourished by cane-sugar, the diastase which it will form will have the geometrical constitution of cane-sugar. Our knowledge of oxidizing enzymes is much less exten- sive than that which we possess of hydrating enzymes. But the facts hitherto observed demonstrate unquestionably that in this case, as in the preceding, there are found to be present different specific ferments, all acting like oxidizing agents, but, individually, on different materials. For this class of bodies also, it is evident that the arrangement of the various chemical groups in the molecules of the oxidizable substances considerably influences the activity of the enzymes. Ex- amples are known of oxidizing enzymes acting on an entire series of homologous bodies, whose action is still possible, if one group is substituted for another, while the action of these 46 THE ENZYMES AND THEIR APPLICATIONS. same enzymes ceases when the arrangement of the groups is changed. Thus it is that laccase, which oxidizes diphenol, its homologues and the products of substitution of these sub- stances, exercises its action on all their derivatives in which the two hydroxyl groups are found in the ortho position, while the same diastase does not act on the isomeric products in which these groups occupy the meta position. Classification of Enzymes. — Now that we have acquired some general knowledge of enzymes and their mode of ac- tion, we can turn our attention to the individual properties of each known enzyme. But before attempting this description, it is necessary to agree upon a nomenclature and a classifica- tion of diastases. The chemists who discovered the first dia- stases designated them by very different names, according to their different points of view. As long as the study of enzymes was confined to a small number of substances, the drawbacks of this nomenclature were not very great. But at the pres- ent time a considerable number of diastases are known, and this number is certain to increase. Under these conditions it would be desirable to have a logical nomenclature seeking to designate a ferment by a name giving a clear idea of its own characteristics. Recognizing this need, Duclaux has suggested a rational nomenclature, by designating an enzyme by the name of the body on which its action was first observed ; and in order to distinguish the substance on which the diastase performs its action from the enzyme itself, he has proposed to add to the root of the word the termination asc. Thus the diastase acting on casein would be called casease, and the diastase which transforms. starch (amylum) becomes amylase. Unfortunately, the nomenclature of Duclaux has not been adopted by all workers, and a certain number of new diastases have received from their discoverers a name hav- ing, to be sure, the termination asc, but whose root is not derived from the name of the substance on which the diastase acts, but from that of its product. Thus the glucase of Cuse- INDIVIDUALITY OF ENZYMES. 47 nier is not a diastase acting on glucose, but an active sub- stance transforming starch and maltose into glucose. This new nomenclature has the great disadvantage of bringing about confusion, and it would have been better to have ad- hered to the nomenclature of Duclaux, although that was not all that could be desired. It is not well to take as the root of the name that of the product formed, because different dia- stases may, at the end of the reaction, produce identical re- sults, while acting on very different bodies. Thus we know, in addition to glucase, many ferments which transform cer- tain carbohydrates into glucose. It is true that the nomen- clature of Duclaux also gives rise to confusion. Thus, the action of glucase was first observed on starch ; one should then designate this enzyme by the name of amylase, a name applied to the diastase from malt. It is then necessary to take account, not only of the substance on which the dia- stase acts, but also of the substance produced by the diastase. From this point of view one would name the glucase of Cuisenier amylo-glucase, that is to say, indicating that it is a diastase acting on starch and producing glucose. The diastase of malt, on the contrary, ought to be called amylo- maltase, because the product finally resulting from the action of this diastase on starch is maltose. However, in the present work we shall retain the old nomenclature and give to the diastases the names which are generally met in the literature. The reason for this method is that we know very well that every change of nomenclature, although aiming to simplify matters, only adds new compli- cations, and on the whole produces a result contrary to that which was intended. The most rational classification of en- zymes consists in distinguishing them according to the chem- ical work which they produce. We already know that dia- stases can produce hydration, oxidation, or molecular trans- formation. We shall then describe the diastases, grouping them according to the chemical character of their action. The study of the diastases of proteid matters will be the sub- 4 8 THE ENZYMES AND THEIR APPLICATIONS. ject of the second volume of the present work. In the first we shall only occupy ourselves with the diastases producing either hydration, oxidation, or molecular change. Hydrating diastases act on carbohydrates, fatty sub- stances, glucosides, proteins, and urea. Oxidases act on bodies of very diverse nature: alcohols, phenols, amides, fatty substances, etc. Enzymes causing molecular transformations are so few that not many bodies susceptible to their action can be named. CLASSIFICATION OF SOLUBLE FERMENTS. A. Soluble Hydrating Ferments. ist. Soluble Ferments of Carbohydrates. Names of the Enzymes. Invertin or sucrase. Amylase or diastase. Glucase or maltase. Lactase. Trehalase. Inulase. Cytase. Pectase. Caroubinase. Substances on which the Enzyme Acts. Cane-sugar. Starch and dextrin. Dextrin and maltose. Lactose. Trehalose. Inulin. Cellulose. Pectin. Caroubin. Products of the Reaction. Invert-sugar. Maltose. Dextrose. Dextrose and galactose. Glucose. Fructose, levulose. Sugars. Pectates and sugars. Caroubinose. 2d. Soluble Ferments of Glucosides. Emulsin. Myrosin. Betulase. Rhamnase. Steapsin. Lipase. Rennet. Plasmase. Casease. Pepsin. Trypsin. Papain. Amygdalin and other glucosides. Potassium myronate. Gaultherin. Xanthorhamnin. jd. Soluble Ferments of Fatty Substances > Fatty substances. 4th. Soluble Ferments of Proteins. Glucose, oil of bitter almonds, and hydro- cyanic acid. Glucose and allyl iso- sulphocyanate. Oil of wintergreen. Glucose. Rhamnetine, isodulcite. Glycerin and fatty acids. Caseinogen. (Casein, Hammarsten.) Fibrinogen. Casein. Albuminoid substances. Casein. (Para casein.) Fibrin. Proteoses, peptones. ( Proteoses, peptones* \ amides. INDIVIDUALITY OF ENZYMES. 49 Urease. Laccase. Oxidin. Malase. Olease. Tyrosinase. Oenoxidase. jth. Ferments of Urea. Urea. | Ammonium carbonate. B. Soluble Oxidizing Ferments. Uruschic acid. Tannin, anilin, etc. Coloring matters of cereals. Coloring matters of fruits. Olive oil. Tyrosin. Coloring matter of wine. Oxyuruschic acid. Products of oxidation. Products of oxidation. C. Ferment Causing Molecular Decomposition. Zymase or alcoholic diastase. Various sugars. Alcohol and carbonic acid. BIBLIOGRAPHY. Em. Bourquelot. — Sur l'identite de la diastase chez les etres vivants. Comptes Rendus des seances de la Soc. de Biologie, 1885, p. 73. Duclaux. — Individuality des diverses diastases. Microbiologic, 1883, p. 141- Em. Fischer. — Einfluss der Configuration auf die Wirkung des Enzymes. Berichte der deutschen chemischen Gesellschaft, 1894, p. 2071, 2985, 1429, 3479. Ueber die Verbindungen des Zuckers mit den Alkohol und Ketonen. Berichte der deut. chemischen Gesellschaft, 1895, p. 1145, 1429. CHAPTER V. SUCRASE. Extraction of sucrase from yeast. — Secretion by Aspergillus niger. — Prep- aration of sucrase in the dry state. — Influence of the quantity and of time. — Influence of temperature. — Difference between the properties of sucrase of different origin. — Effect of the acidity or alkalinity of the medium. — Action of oxygen and of light. — Action of chemical sub- stances. — Mode cf secretion of sucrase in the cells. — Measurement of sucrase. — Method of Fernbach. — Method of Effrcnt. Sucrase is a diastase capable of transforming cane-sugar into invert-sugar. Saccharose, under the action of sucrase, is decomposed with addition of a molecule of water, giving two monosaccharids : dextrose and levulose, CjsHsaOn + H 2 = C G H 12 6 + C 6 H 12 6 . Saccharose. Dextrose. Levulose. Sucrase is very widely distributed in nature. For ex- ample, we find its existence in the saliva, in the gastric juice, and in the small intestine. Cane-sugar, retained for some time in the mouth, is trans- formed by the action of the saliva into invert-sugar. How- ever, this transformation is not due to the action of a secretion of the salivary glands, but rather to the sucrase evolved by the numerous bacteria which are found in the saliva, for the active substance which occurs in the mouth transforms only very limited quantities of cane-sugar. The diastases of the gastric juice are endowed with much stronger inverting power. However, in spite of this energy, the inversion of saccharose does not proceed actively in the 50 SUCRASE. 5 1 stomach. A considerable part of the cane-sugar absorbed reaches the circulation without having undergone the action of the diastases, and it is only in the small intestine that the transioi iiiation becomes complete. In the blood the pres- ence of active substances capable of transforming saccharose has not been observed. Sugar injected in the veins or in the cellular tissue of an animal is eliminated in the urine; but this elimination does not occur when sugar is injected in the portal vein. It then traverses the liver and there undergoes a strong diastatic ac- tion which completely transforms it. Sucrase is also of very common occurrence in the vege- table kingdom: it is found in buds and flowers, as well as the leaves of a very great number of plants. Furthermore, numerous moulds, such as Aspergillus niger, Mucor raccmosus, Penicillium glaucum, Penkillium Duclauxi, Aspergillus oryzae, yeasts, and many other ferments, also effect the inversion of saccharose. As a general rule, a cell nourished by sugar must neces- sarily contain a sucrase. This rule has, however, been op- posed by Hansen, who has called attention to the fact that the mould called Monilia Candida, while nourished by sac- charose, does not secrete sucrase. This assertion has been successfully refuted by E. Fischer, who in a more thorough study of this mould discovered that it really contains a su- crase, but that the enzyme is retained in the cells, and is diffi- cult to isolate. In the literature sucrase is designated under different names: it is called glucose ferment, cytozymase, zymase, and invertin. This enzyme was discovered by Dobereiner and Mitscherlich. These investigators first found that beer- yeast inverted saccharose. They also remarked that this ac- tive substance can be extracted from the yeast by washing with water. Berthelot first succeeded in isolating the dia- 9tase in the solid form by precipitating from yeast extract by alcohol. 5 2 THE ENZYMES AND THEIR APPLICATIONS. J Mode of Preparation. — Several different methods of preparation of sucrase may be employed. The enzyme can easily be obtained by putting some beer-yeast in water with the addition of a few drops of chloroform ; after a time the active substance is dissolved in the water. Then the liquid is filtered to remove the suspended yeast-cells. The solution thus obtained is necessarily far from being com- posed solely of sucrase, the yeast containing, besides the su- crase, other extractive matters which enter into solution at the same time. Notwithstanding this, the infusion is very active, and may very well serve for the study of sucrase. A better method of preparation of this enzyme consists in the extraction of a culture of Aspergillus niger in Raulin's solution. Yet, the extraction of the diastase from Aspergillus niger demands, in order to give sufficient quantities of en- zyme, the maintenance of certain conditions without which the results would not be satisfactory. The best method of procedure has been suggested by Duclaux. He advises al- lowing a culture of Aspergillus niger to develop on a large sur- face of Raulin's solution for about four days, and when the moulds formed have taken on a green or light brown color, drawing off the liquid and replacing it by pure water or water containing sugar. On this new liquid one allows the mould to grow further for two or three days up to the complete ex- haustion of the nutritive medium. Then the enzymes secreted by the plants enter into solution and it only remains to filter the liquid to free it from the fragments of mould which may be found in suspension there. The solution of sucrase prepared in this way is very active and contains rela- tively few impurities. To prevent the liquid changing dur- ing the growth of the plant, one may add a few drops of mus- tard oil which, acting as an antiseptic, preserves the medium from the invasion of bacteria without destroying the diastase. However, it is better to cultivate the mould in sterilized liquid, and to inoculate this liquid with a pure culture of Aspergillus niger. When the plant has sufficiently developed SUCRASE. 53 on the Raulin's solution, this is replaced by sterilized distilled water. To obtain sucrase in a dry state, E. Donath recommends the following method : extract the beer-yeast for some time in absolute alcohol ; then decant the alcohol, filter, and dry by exposing to the air. In this way a brittle mass is obtained which is pulverized and treated with distilled water. This infusion is filtered to remove the yeast-cells which are present. However, as the cells easily pass through the filter, one must make sure by a microscopic examination that they have all disappeared from the liquid. If cells are still present, the solution must be filtered several times through a double filter. When the liquid is free from cells, ether is added and it is shaken. A viscid substance appears which remains in suspension in the upper part of the liquid and which must be separated from the rest of the infusion. This substance is then treated with distilled water and dropped slowly into absolute alcohol, where a pulverulent precipitate is produced. This precipitate, separated from the liquid, is washed -with alcohol and dried in vacuo. This procedure furnishes a white powder, swelling in water and dissolving in it with great diffi- culty. It can be kept for a long time and possesses great diastatic power. It appears beyond doubt, however, that a considerable part of the active substance must be coagulated by the treatments with alcohol and ether, and consequently rendered inactive. The rapidity of inversion of saccharose by sucrase de- pends upon the quantity of active substance employed, as well as upon the physical and chemical conditions of the medium in which the transformation takes place. The study of special conditions which favor or retard diastatic action is still more interesting in that it furnishes valuable data from the theoretical as well as from the practical point of view. We shall, therefore, accord to this question the develop- ment which it merits. We shall first study the influence exerted on the rate of inversion by the quantity of sucrase, 54 THE ENZYMES AND THEIR APPLICATIONS. and the temperature at which it works. We shall next de- termine the time-factor in the inversion, as well as the in- fluence of the acidity or alkalinity of the medium. Finally, we shall see how light, oxygen, and certain other chemical substances influence the rate of the transformation. Influence of Quantity and of Time. — When sucrase acts on a solution of saccharose, the results obtained are very dif- ferent according to the quantity of active substance em- ployed. If one holds to definite conditions one may observe an almost constant relation between the quantity of sucrase em- ployed and the quantity of invert-sugar. This proportion is, up to a certain point, independent of the concentration of the sugar solution in which the diastase works. If, for ex- ample, i and 2 cubic centimetres of sucrase are made "to act during the same time and at the same temperature upon equal quantities of saccharose, it is found that with 2 cubic centimetres of sucrase twice as much invert-sugar is obtained as with 1 cubic centimetre. However, this proportionality between the quantity of acting substance and the quantity of product formed is not always constant. Duclaux has observed that the law of pro- portionality is true only when the sucrase is used in very small amounts, and if the inversion is arrested at an early stage. The ratio holds good until 10 to 20 per cent of the sugar is inverted, after which it fails. When one studies the influence of time on the action of sucrase, the same rule holds good. Sucrase is a very energetic enzyme. According to Duclaux, 1 gram of active substance transforms 4000 times its weight of sugar. However, while very energetic, the ac- tion of this diastase is relatively slow. One hundred cubic centimetres of 10 per cent solution of saccharose to which 1 cubic centimetre of sucrase was added, when kept at 50 , yielded the following results : SUCRASE. 55 After i hour 20 gr. Invert-sugar. " 2 hours 41 " 3 " 60 " " 4 " 80 " This experiment illustrates the remarkable slowness with which inversion is produced. It is noteworthy in this con- nection that the amount of invert-sugar increases propor- tionally to the duration of the action. After two hours, one finds about twice as much invert-sugar as after one hour, and after five hours almost five times as much. But from this time on the proportion ceases to exist. If we continue in fact to follow the action of sucrase in the preceding experi- ment, we obtain: After 10 hours 1.72 gr. Invert-sugar. " 20 " 3.12 " If the transformation had continued with the same speed as at the beginning of the action, we should have had : After 10 hours 2.00 gr. Invert-sugar. " 20 " 4.00 " The retardation which we observe commences when about 20 per cent of sugar has been transformed, and in proportion, as the inversion proceeds, the abatement continues to be marked. The irregular course which we observe in the action of sucrase has been the subject of different researches and has occupied many investigators. It has given rise to various hypotheses which we will examine later. It is sufficient now to note the fact before we pass to the action of temperature. Influence of Temperature. — Temperature plays a very important part in the inversion of saccharose, and exerts a considerable effect on the degree of activity of sucrase. At o° sucrase exercises only a very feeble action, but it in- creases considerably with increase in temperature. This in- 56 THE ENZYMES AND THEIR APPLICATIONS. crease is gradual between 5 and 30 . Above this tempera- ture, from 30 to 50 , diastatic activity increases rapidly. By allowing sucrase from yeast to act for an hour upon a 20 per cent solution of sugar, we have obtained with the same quantity of sucrase at different temperatures the following figures : Temperature, Gr. Invert-sugar Degrees Centigrade. formed per ioo c*c. solution. o O O 5° 0.05 IO° O.I I 15° 0.18 20° O.35 3°° • 0.40 40° 1.65 50 2.20 6o° 2.10 The temperature at which inversion proceeds with the greatest rapidity is, according to Kjeldahl, 52. 5 ; beyond that the diastase becomes more and more weakened. In trying to determine the temperature at which sucrase is destroyed, it is important to have conditions absolutely constant, because the concentration of the liquid and the re- action, as well as the other special qualities of the medium, have a considerable influence on the activity of the diastase. Sucrase from yeast, much diluted, can be maintained for an hour at 52 without losing its inverting power; on the contrary, the more concentrated solutions of sucrase weaken very perceptibly when they are kept at that temperature even for a short time. When yeast is placed for an hour in water at 65 , its diastase is completely destroyed ; while at the same temperature a part of the active substance remains un- changed, when a very dilute solution of sucrase is used. The reason for this difference of resistance is that other bodies unfavorable to the action of sucrase are found with it in the SUCRASE. 5 7 extract, and the retarding action of these substances evidently diminishes with the degree of dilution of the solution. The presence of sugar in the liquid containing sucrase perceptibly increases the power of the enzyme to resist heat. On the whole, the variations observed between the opti- mum temperature and the destructive temperature are quite considerable. The optimum temperature is found, accord- ing to different authors, between 50 and 56 , and the destructive temperature between 65 and 70 . But the ac- tivity of the sucrase is weakened as the destructive tempera- ture is approached. Sucrases of Different Origin. — Kjeldahl has observed that sucrase extracted from bottom yeasts possesses an optimum temperature different from that of the active substances of top yeasts. For the latter he has found that the optimum temperature is 3.5 higher than that of bottom yeasts. It is not only the optimum temperature which varies with the origin of the sucrase : most of the properties of the enzyme depend upon its origin as well as upon the mode of prepara- tion. Thus, sucrase extracted from yeasts can be filtered by the Chamberland filter, while the active substance of Asper- gillus niger is completely held back by the filter. In beer-yeast sucrase is found in an uneombined state and can easily be extracted by water; in Monilia Candida, on the contrary, the enzyme is retained in the cells, where it is found combined with other substances which render it insoluble. Sucrases obtained from different yeasts may also differ by their greater or less sensitiveness to chemical reagents. Fernbach has found, for example, that the enzyme of the yeast of Tantonville is fifty times more sensitive than sucrase extracted from other kinds which had been given him to study. These differences of properties, found in sucrase, are not confined to it. We shall meet with similar facts when we study pepsin as well as many other soluble ferments. These differences may be explained by the presence of various foreign substances having the property of lowering the opti- .58 THE ENZYMES AND THEIR APPLICATIONS. mum temperature and the destructive temperature of chang- ing the solubility of enzymes, and of influencing their sensi- tiveness towards physical and chemical agents. This explanation leads to the conclusion that the enzyme lias in itself constant properties, and that if two sucrases, for example, show different characteristics and act in different ways, one must simply seek the cause in the conditions of the medium, — in the presence of substances endowed with an ac- celerating or retarding power. But all authors do not concur in this opinion. The differ- ence which exists between the properties of two enzymes of the same nature, but of different origin, has sometimes been interpreted quite otherwise. One may suppose, for example, that the medium in which the enzyme is secreted influences :not only the mode of action, but also the very composition of the diastase. By this hypothesis, the difference existing between the modes of action of various enzymes must be regarded as re- sulting from a series of changes in the composition or in the structure of the diastase, whereby one simply is dealing with "various modifications of the same enzyme. We have just said that sucrase of top yeasts produces its maximum effect at a higher temperature than the optimum temperature of low yeasts. This difference may be attributed to a phenomenon of adaptation of the yeast to the medium in which it works, adaptation having as its consequence the formation of different diastases at the different temperatures. This adaptation to the medium is manifested still more clearly when one studies the action of gastric juice. The pepsin of warm-blooded animals does not act at o°, and its maximum effect is produced at 50 , while the gastric juice of cold-blooded animals produces a manifest action at o°, and lias an optimum temperature of 40 . Many facts analogous to the preceding are known which support the hypothesis of the adaptation of diastases to their ■environment. SUCRASE. 59 But the existence of different varieties of the same en- zyme is very difficult to demonstrate exactly, because we always have to deal with mixtures of enzymes and more or less clearly recognized foreign substances. However, we are the more led to deny the existence of different varieties of the same enzyme, because the variations in the properties of the same enzyme are generally not very pronounced, and are susceptible of being artifically reproduced by starting with a definite diastase, and simply changing the conditions of the medium. We think it more logical to assume, until the con- trary is proved, that the variations observed in enzymes of different origin are due to the presence of foreign substances. We shall have occasion to return to this question re- peatedly in studying the enzymes individually. Part Played by the Acidity or Alkalinity of the Medium. — The acidity or alkalinity of the medium has con- siderable influence on the sucrase. Kjeldahl has demon- strated that a slight acidity is favorable to its action, while large amounts of acid or alkali diminish its diastatic power. In a very comprehensive work, Fernbach has studied a sucrase derived from Aspergillus niger and has examined with great care the influence of the medium. His study has given valuable information on the question which we have to con- sider. Fernbach found that the solution of sucrase extracted from Aspergillus niger always possesses an acid reaction due to the oxalic acid elaborated, in greater or less quantity, by the mould. While this acid reaction is in reality very weak, the diastatic solution may have dilute soda added in con- siderable amount before it will turn litmus paper blue. The sucrase still shows itself very sensitive to the action of quanti- ties of alkali too weak to be revealed by litmus paper and other indicators of alkalinity. Fernbach made the follow- ing experiment to show this sensitiveness to the acids and alkalies of the medium : Into each of eight test-tubes he poured two cubic cen- timetres of an infusion of sucrase, added to each of them a 60 THE ENZYMES AND THEIR APPLICATIONS. different quantity of a solution of soda (1:15000), then brought the volume of liquid in each tube up to 10 cubic cen- timetres by the addition of sugar solution. At the end of an hour of action at 56°, he determined the quantity of invert- sugar formed in each of the tubes and obtained the following results : Numbers of Quantity of soda Invert-sugar the tubes. added. formed. 1 occ 35 mgr. 2 0.5 31 3 1 • 25 4 1-5 l 7 5 2 12 6 ,: 2.5 7 7. •■••' 3 5 8 3-4 3 The liquid in the tubes 1, 2, 3, 4, showed an acid reaction ; after tube 4, to tube 7, the solution was neutral; it gave a slightly alkaline reaction in tubes 7 and 8. We notice in this table that the invert-sugar formed diminishes in proportion as the quantity of soda in the solu- tion increases. When soda is not added 35 milligrams of invert-sugar are obtained, while the addition of only 1.5 cubic centimetres of soda (1 115000), an amount hardly sufficient to neutralize the solution, reduces the quantity of invert-sugar to 17 milligrams. This diminution of about 50 per cent is. here effected by the action of a quantity of soda equivalent to about 1 gram per hundred litres. This extreme sensitiveness of the diastase to the alka- linity or acidity of the medium suggests one of the causes of the non-proportionality between the quantity of active sub- stance employed in an inversion and the quantity of invert- sugar which results. In fact, when the sucrase is neutral and used in small amount, the quantity of matter transformed is, as we have seen, proportional to the quantity of active sub- SUCRASE. 61 stance allowed to act, but this proportion ceases to exist when the experiments are made with an acid or slightly alka- line solution of sucrase. It is evident that by using these increasing quantities of sucrase, there are at the same time introduced increasing quantities of acid or alkali, which in- fluence more and more strongly the degree of the inversion and change the ratio. Fernbach has determined, in his work, the optimum amount of different acids for the greatest activity of the dia- stase. To this end he first neutralized, as exactly as possible, a sucrase solution, and then acidified it, using increasing quantities of different acids. He obtained the results shown in the following table : Acids. Optimum quantity (No. of grams per litre). Inhibiting quantity (No. of grams per litre). O.025 I O.066 2 5 10 0.2 2 O.I 4 10 50 Lactic " It is seen that the optimum amount depends upon the na- ture of the acid employed. The activity of the enzyme increases in the presence of small amounts of acid up to the moment when the optimum amount is reached; but this once passed, the presence of the acid becomes destructive to the diastatic action, which de- creases perceptibly. The amount of oxalic acid producing the greatest effect on the inversion does not, by itself, possess inverting power at 56 ; but the other acids, by their own action, invert a cer- tain amount of sugar. The invert-sugar formed in the pres- ence of acids results then from the combined actions of the acid and of the diastase. From this it results that by em- ploying different acids, each in its own optimum amount, there will of necessity be obtained different quantities of in- 6 2 THE ENZYMES AND THEIR APPLICATIONS. vert-sugar with the same amount of sucrase. This differ- ence is due to the action of the acid alone and not to that of the sucrase, for the latter is always influenced in the same de- gree by the different acids. Fernbach made a series of comparative experiments for studying the combined actions of acid and sucrase. He performed, on different sugar solutions, two experiments, A and B, for each acid. In experiment A he used the optimum amount of acid with the addition of a certain amount of sucrase; in experiment B, he allowed the acid to act alone. By afterwards determining the amount of in- vert-sugar formed in each experiment, he has been able by subtraction to determine the amount of invert-sugar which may be attributed to the special actio ( n of the diastase. These experiments, carried out with different acids, have given him the following results : Quantities of Acid per litre. Sugar Inverted by Acid and Diastase. Sugar Inverted by Acid. Difference or Sugar Inverted by Diastase. Sulphuric acid.o.osgr. Oxalic " 0.066 Tartaric " 1 Succinic " 2 Lactic " 5 Acetic " 10 31-3 30 40 34-2 4i-5 37-9 0.7 O 8.6 3-7 12.2 7.2 30.5 30 31.4 30.5 29-3 30.7 It will be seen that the figures of the last column, which designate the results of the diastatic action, properly speak- ing, are the same for all the acids, for the slight differences observed may be attributed to errors in measurement. This experiment demonstrates then the fact that we have stated above, namely, that the diastase is always influenced in the same degree by the different acids. However, the data fur- nished by Fernbach upon the influence of the medium apply exclusively to sucrase secreted by Aspergillus nigcr cultivated on Raulin's solution. It is probable that the same mould cultivated in other media would furnish sucrase solutions not having the same sensitiveness to reagents. Furthermore, SUCRASE. 63 the determination of the amounts of acid checking or favor- ing diastatic action, has always been made by him at a tem- perature of 56 ; it is, therefore, presumable that the figures which he has calculated are only correct for that temperature. In reality, from 30 to 40 , the quantities of acid which correspond to the maximum of result are entirely different from the amounts necessary at a temperature of 56 . At these temperatures the amounts of acid must be multiplied by 5 to produce the same result as at 65 . According to O'Sullivan and Tompson, the maximum acid amount de- pends also upon the quantity of sucrase used, for they have found that with increased amounts of sucrase, increasing amounts of acid must also be Used. On the whole, the influence of the reaction of the medium on the rate of inversion is not a simple one. Sucrase derived from yeasts differs from that obtained from Aspergillus niger in the resistance to the action of acids. The solution of sucrase which is obtained by extracting yeast with cold water is generally more sensitive to varying reac- tions of the medium than the diastatic solution extracted from Aspergillus niger. The sensitiveness of the sucrase of yeasts varies, furthermore, with the nature of the yeast used, and, for the same yeast, with the nutrition to which it has been subjected. Fernbach has determined the amount of acid which is most favorable to the action of the sucrase in three kinds of yeasts (see the table on page 64). It is seen by an inspection of this table that the maximum amount of acid is about 0.2 cubic centimetres for the champagne yeast, about 0.5 cubic centimetres for Saccharomyccs Pastorianus and the yeast of pale ale, while sucrase extracted from Aspergillus niger gives a maximum result only when much larger quantities of acid are present. The considerable influence which the content of alkali in the medium exercises on the course of transformation has caused it to be supposed that the accelerating action of the 64 THE ENZYMES AND THEIR APPLICATIONS. acid arises from a modification in the nature of the enzyme brought about by the action of this chemical agent. Quantity of Acetic Acid per litre. Champagne Yeast. Saccharomyces Pastorianus. Yeast of Pale Ale. O 33.3 29.7 18.8 0.02 38-7 3i-9 19.8 O.05 63-9 32-4 22.3 O.I 74-3 32.4 25-5 0.2 0.5 79-4 78.4 32-9 33 28.3 29.4 I 7-5 31-3 28.9 2 71.9 29.6 27.6 5 10 50.4 But this transformation in the nature of the diastase is very difficult to prove, and in any case appears to be slight. The quantity of alkali clearly checking the inversion does not really cause an appreciable change in the active sub- stance. The diminution of activity is due to the abnormal conditions of the medium, rendered refractory to the action of the enzymes by the addition of alkali. But the active sub- stance evidently remains unchanged because it is only neces- sary to neutralize the liquid again to have the diastatic work resumed with all its initial intensity. It is only by increasing the amount of alkali to very large proportions that the dia- stase is destroyed, just as albuminoid substances are destroyed with the same amounts of the same agents. Action of Oxygen and of Light. — Duclaux was the first to find that air exercised a very appreciable action on sucrase. He observed that a solution of sucrase in ordinary water changed color in contact with air and became inactive as a result of oxidation. This oxidation of sucrase is influenced to a very great de- gree by the presence or absence of light, as well as by the acidity or alkalinity of the medium. Sheltered from light and in a slightly alkaline medium, de- SUCRASE. &5 composition by the oxygen of air is produced very rapidly ; it is less pronounced in a neutral medium, and is manifested slowly in the presence of an acid. By exposing a solution of sucrase to the action of the air at 35 , 50 per cent of the active substance is destroyed in about 48 hours ; at a temperature of 50 oxidation is more rapid and the same degree of change is reached after 4 or 5 hours exposure to oxygen. Light alone, in the absence of oxygen, is without action on sucrase. Fernbach has shown this by exposing to sunlight exhausted tubes containing sucrase. The sucrase remained unchanged for several months. We have just seen that in darkness acids give to the dia- stase a considerable resistance to the action of air. When sucrase is not protected from light, this ceases to be the case and there are alkalies which become capable of protecting the enzyme against oxidation. By leaving in contact with air and light two solutions of sucrase, one slightly acid and the other slightly alkaline, it is found that the acid liquid under- goes a very rapid alteration, while the alkaline solution is pre- served for a long time. This fact has been observed by Fernbach, who, by exposing three sugar solutions of different reactions to the action of the air and sunlight for 48 hours, found that they possessed at the end of that time the follow- ing diastatic powers : Slightly acid solution 3.7 Neutral " 6.6 Slightly alkaline " 7.4 The favorable or unfavorable influence of the acidity or alkalinity of the medium on the oxidation of the sucrase has been very well shown by the following experiment: Five solutions of sucrase showing a diastatic power of 18, some acid, others alkaline, in different degrees, were sub- jected, in darkness, to the action of the air at a temperature of 35 for 48 hours. Determination of diastatic powers at the end of the experiment gave the following results : 66 THE ENZYMES AND THEIR APPLICATIONS. Numbers of the Quantity of acid Diastatic experiments. in millionths. power. I 420 18 2 270 18 3 Neutral 17 4 75 soda I4.6 5 150 soda IO.6 We clearly see from this table the preservative action which acids exercise and also the destructive influence of alkalies. The study of the effects of light and oxygen on inversion of saccharose leads us to a practical conclusion relative to the preservation of the diastase. To preserve a solution of sucrase it is of foremost import- ance to avoid oxidation and consequently contact with air. For this purpose a vacuum is made in the partially filled flask, or else the diastatic solution is covered with a layer of oil. I have found in my experiments that a solution of sucrase pre- pared in this way still possesses all its energy after three months of preservation. Action of Chemical Substances. — Sucrase is very sensi- tive to different chemical reagents. Duclaux has completely elucidated this question and has given figures which, without being absolute, are sufficiently accurate. Calcium chloride markedly suppresses the action of su- crase and its retarding influence increases with the amount. Chlorides of sodium and potassium, after having pro- duced a favorable effect in slight quantities, such as 0.4 per cent, lessen the diastatic action when the quantity is in- creased. Ammonium chloride, according to Nasse, acts very favor- ably in 10 per cent solution, and in small amounts is indif- ferent. The action of alkaline salts and of bases is, according to Duclaux, retarding and destructive in the following amounts : SUCROSE. 67 Salts. 0.1% 0.2% 0.4* 0.5* o.8£ 4 i-4 7.2 9-3 25 1 5-6 1-3 Sodium salicylate up to 0.2 per cent seems to be without effect ; with 0.4 per cent, the diastatic action is reduced, for we have to employ 1.3 of the diastase instead of 1.0 to obtain the same effect. Sodium borate and sodium arseniate retard hydration in a marked degree. The presence of o. 1 per cent reduces the efficiency of diastase to one-fourth the normal. Antiseptics affect very differently the diastatic power of sucrase. Chloroform, ether, and oil of wintergreen, when in excess, reduce the activity of sucrase by about ten per cent. Toxic substances have also an inhibiting action, as appears from the following table (Duclaux): Salts. Per cent. o.oiif 0.02# 0.04^ O.I# 0.256 I.03 I.30 I6.3O I.O4 1.25 44 1.25 O.70 62 1.40 Potassium cyanide.. 1.26 Mercuric chloride has then a very slight retarding influ- ence; in the presence of 0.1 per cent, the diastase is but lit- tle weakened. Potassium cyanide is a very strong retarding agent ; in the presence of 0.02 per cent, the power of the ferment is re- duced to one-sixteenth the original. Silver nitrate first checks, then accelerates the transformation, due, according to Duclaux, to the acidity which it produces in the sugar solution. Finally, 10 per cent alcohol produces a retardation ex- pressed by the figure 1.3. Oil of garlic and other essential oils produce only inappreciable effects on the degree of inversion. 68 THE ENZYMES AND THEIR APPLICATIONS. Formation of Sucrase in Living Cells. — We have seen above that all cells which are nourished with cane-sugar secrete sucrase. We will now discuss the most favorable conditions for the secretion of this enzyme by living cells. In the cells of beer-yeast cultivated in a nutrient solution containing saccharose, sucrase appears. To explain this phenomenon, it must be stated that the cells found in the presence of non-assimilable substances produce a secretion capable of transforming these substances into assimilable materials. But on studying the phenomenon more closely, it is found that the secretion of sucrase does not strictly de- pend on the manner of nutrition of the cell ; that it seems rather to be intimately allied to the nature of the organism, and that it is produced independently of the real needs of the cell. If, for example, in the nutrient solution cane-sugar is replaced by directly assimilable carbohydrate the secretion of sucrase continues. In this case, however, the nutrition does not at all necessitate the presence of this enzyme. Though the nature of the sugar does not possess any influ- ence on the secretion of sucrase, one must not conclude that, as a general rule, the secretion of diastase is independent of the mode of nutrition of the cell. Experiment has shown, on the contrary, that the diastatic secretion is directly allied to the nature of the food, while being independent of the carbohydrate employed. Yeasts cultivated in beer-wort secrete much more sucrase than yeasts cultivated simply in sugar solutions; the secretion of sucrase is favored in this case by the nitrogenous materials of the malt. Experiment has shown, for example, that the addition of peptones in- creases the quantity of sucrase in the culture medium. The substances most favorable for the growth of yeast are not always those which most favor the formation of sucrase Phosphates, for example, which influence yeast very favor- ably are, on the contrary, unfavorable to the formation of su- crase. Nitrogenous materials are not then the only ones having an influence on the secretion of sucrase. Unfortu- SUCRASE. 69 nately, the conditions which favor the formation of diastase are imperfectly known. They merit thorough study, for they are of a nature to afford very interesting information from a theoretical point of view. If the conditions favorable to the formation of sucrase are little known, we are much better informed as to the manner of diffusion of sucrase through the cells. To study the mode of formation of sucrase in Aspergillus niger, Fernbach proceeded in the following manner: He sowed a certain number of dishes containing equal parts of Raulin's solution with a definite number of spores, all coming from the same culture of Aspergillus. He then subjected the liquid thus sown to a constant temperature of 35 . He determined daily in one of his experiments the weight of the plants, the sugar remaining, and the acidity, as well as the amount of sucrase produced. Each dish contained 400 cubic centimetres of Raulin's solution, 17.6 gr. of saccharose, and 0.72 gr. of free tartaric acid. The results which he obtained are tabulated below : Weight of the plant. Ash. 3.105 O. 116 6.200 O.171 7-835 O.191 6.870 0.200 5-58o O.198 By following in this table the figures placed under the heading " Sucrase," it is seen that at the beginning of the development of the young plant, when it is using great quan- tities of sugar, the sucrase does not appear in the culture liquid, and that one cannot detect its appearance until the sugar is exhausted and inversion no longer takes place. 7o THE ENZYMES AND THEIR APPLICATIONS. This fact is of great interest ; it shows us that inversion is not produced in the liquid which surrounds the mould. The presence in the liquid of 8.3 grams of invert-sugar after two days tends to confirm the opinion that the transformation takes place inside the cell. If we accept this hypothesis, we must at the same time assume that sucrase exists in the cell from the beginning, and the diffusion noted is produced as the result of a modification of the cell contents. In fact, Fernbach, in looking for sucrase in the plant,, found that the greatest quantity of diastase secreted by the cells appeared at the beginning of its development, and that the moment of its appearance outside coincided with the in- stant when the plant had already caused the greatest quantity of sugar to disappear. After 1 day. . . . " 2 days. . . " 3 " •■• " 4 " ... " 5 " ... Saccha- rose re- maining-. Invert- sugar. Sugar con- sumed. Acidity. Sucrase of the iiquid. Sucrase of the cells. Weight of the plant. I.36 2.36 O.92 O.293 2 53 O.65 0.22 I.65 2-57 O.368 3 47 I.265 O 0.7 3-74 O.267 5 45 I.78 O O 4.44 O.143 IO 44 I.65 O O O.I35 13 35 I. 6l The diffusion of the sucrase at the moment of the disap- pearance of the sugar may then be considered as a conse- quence of the disintegration of the plant. When we reflect a little, we realize that, in cells well nourished and fortified with reserve food, the diffusion must be accomplished with great difficulty. With the disappearance of the invert-sugar in the liquid, the cells begin to consume their reserve food ; vacuoles are formed and filled with water, which certainly facilitates diffusion. Moreover, there are some very conclusive experiments which prove that the diffusion of diastases characterizes a pathological state of the cells: SUCRASE. 7 1 Place two young and identical cultures of Aspergillus niger, one in water, the other in a rich nutritive medium. After 48 hours examine the two liquids and it will be found that the first medium contains a great quantity of active enzymes, while the second has no trace of any. Denutrition then favors the secretion of sucrase. One may, moreover, take a culture of Aspergillus niger and exclude it from the ac- tion of the air. Thus fructification is prevented and this ab- normal condition causes, like inanition, an abundant diffu- sion of the diastase in the culture medium. Finally, beer- yeast suspended in water may be heated for several seconds at ioo°. Thus the active substance is completely destroyed as well as a majority of the yeast cells. On allowing the liquid to cool one notes, after a while, the appearance of su- crase. The secretion of the enzyme may be attributed to the cells which have escaped the destructive action of the heat, although greatly injured by the high temperature to which they have been subjected. They are then in a certain path- ological state and diffuse with ease the active substance they contain. As we see by these experiments, the lack of sugar or oxygen, the elevation of the temperature, etc., may equally favor the diffusion of the sucrase secreted by the cells. Measurement of Sucrase. — We may easily ascertain the transformation of saccharose into invert-sugar by the aid of Fehling's solution. Cane-sugar does not reduce this solu- tion, while 0.4941 gr. of invert-sugar reduce 100 cubic cen- timetres of Fehling's solution. The transformation of saccharose into invert-sugar may likewise be ascertained by the change of optical activity that accompanies the transformation. Cane-sugar rotates to the right and the mixture produced by hydrolysis on the con- trary, rotates to the left. Saccharose gives a rotation to the right of aj-\- 73.8, and invert-sugar a rotation to the left of — 44. The measurement of sucrase requires, first, the de- termination of the quantity of invert-sugar. Now, the same 72 THE ENZYMES AND THEIR APPLICATIONS. quantity of sucrase may furnish greater or less quantities of invert-sugar. These variations are due to the different fac- tors which we have indicated ; the reaction of the medium, temperature, duration of the action, etc. It must not be for- gotten that the ratio between the quantities of ferment employed and of invert-sugar obtained only exists in early stages of the action, and before 20 per cent of the total amount of sugar submitted to the action has been trans- formed. Hence, it is absolutely indispensable, in order to be able to compare two diastatic products, to place them in identical conditions. To avoid errors arising from the acidity, care must be taken to neutralize the liquid as exactly as possible, then to acidulate with 1 per cent of acetic acid. The choice of acetic acid is not arbitrary ; it is due to the fol- lowing reasons : acetic acid can be used in considerable, and, consequently, easily measureable quantities. It does not dis- place the other organic acids of the solution, and, finally, it has little influence on the sucrase. In the measurement, the greatest care should be taken to prevent oxidation of the sucrase, and to this end the analysis should be made as rapidly as possible. Generally one should let the sucrase act only for one hour. To avoid the errors which may come from the deviations from the law of proportionality between the quantity of enzyme used and the quantity of sugar inverted, one must seek the quantity of sucrase capable of transforming a certain quantity of cane-sugar, and not the amount of sugar which a given quantity of sucrase can invert. In the method of measurement suggested by Fernbach, one takes as the unit the quantity of sucrase capable of in- verting 20 centigrams of saccharose in one hour at a tem- perature of 56 in the presence of 1 per cent of acetic acid. To perform this measurement the solution of sucrase is pre- viously neutralized, then in a series of reaction tubes, each containing 4 cubic centimetres of a 50 per cent solution of saccharose, is added 1, 2, 3, 4, 5, cubic centimetres of the SUCRASE. 73 sucrase solution to be analyzed ; one cubic centimetre of acetic acid (i :io) is added ; the volume in each tube is brought up to 10 cubic centimetres. The tubes are left for one hour at a temperature of 56 , then quickly cooled; and several drops of a soda solution are added to arrest inversion, and the quantity of invert-sugar formed in each is estimated by the use of Fehling's solution. It can thus be seen in which of the tubes the 20 centigrams of sugar have been in- verted. Let us suppose that this result was obtained in the tube containing 5 cubic centimetres of sucrase : one then finds present a solution containing only traces of saccharose. Since the amount of acetic acid used in the experiment might itself have inverted some centigrams of sugar, it may be that the whole of the inversion is due to foreign substances and not to the diastase. In order to be certain that the transformation of sac- charose is due to the effect of a diastase, the experiment must be made once without heating, and again with a solution heated to ioo°, to see if the results are the same. In case 1, 2 or even 3 cubic centimetres of solution are sufficient to obtain the transformation of 20 centigrams of sugar, the inverting power of the solution is considerable, and by repeating the experiment with 1^, if, 2, 2.\, etc.. cubic centimetres of the solution experimented with, a very accurate measurement of the diastatic activity may be ob- tained. In case \\ cubic centimetres of solution must be used to obtain 20 centigrams of invert-sugar, we say the unit amount of sucrase is found in 1^ cubic centimetres and, consequently, that the solution possesses two-thirds of the diastatic power of the standard solution. The method of Fernbach gives fairly accurate results, but it demands many trials and a long series of measurements which take a great deal of time. When it is a question of a qualitative rather than quanti- 74 THE ENZYMES AND THEIR APPLICATIONS. tative estimate, the measurement of the sugar may be com- pletely omitted. In order to test for sucrase in liquids we make use of a very expeditious method requiring only half an hour, and in which the inversion is ascertained by the color which the in- serted solution gives with soda. For this kind of experiment we make use of a 10 per cent solution of sugar. The liquid in which the sucrase is meas- ured is neutralized as accurately as possible with soda (i : iooo). In two test tubes, A and B, are poured 10 cubic centimetres of sugar solution ; to A is added one cubic cen- timetre of diastatic solution and to B one cubic centimetre of the same solution previously heated for several minutes to ioo°. The two tubes are left for thirty minutes at 50 . One cubic centimetre of ordinary soda is added to each of the lubes and heated 5 minutes at 98 . If a solution of sucrase is ■present, tube A takes a much deeper color than tube B. It is possible to use this procedure as a colorimetric method. BIBLIOGRAPHY. A. Fernbach. — Recherches sur la sucrase, diastase inversive du sucre de canne. These, Paris, 1890. J. Kjeldahl. — Recherches sur les ferments producteurs de sucre. Mad- delelser fra Carlsberg Laboratoriet, Copenhague, 1879. Duclaux. — Microbiologic, 1883. CHAPTER VI. SUCRASE (Continued). Retarding factors and their explanation. — Deterioration and alteration of sucrase. — Experiments of Effront on the influence exerted by invert- sugar in the medium in which the inversion is produced. — Hypothesis of O'Sullivan and Tompson. — Arguments for and against this hypothesis. — Theory of Effront on the decomposition of cane- sugar, and experiments on the manner of action of acids in the inver- sion of saccharose. Factors Retarding Inversion and their Explanation. — When we examined the course of the transformation of saccharose by sucrase, we noted that the quantity of invert- sugar formed during a given time diminished constantly with the progress of the inversion. This diminution is produced in such a manner that the last portions of saccharose which remain in the solution are transformed very slowly, whereas at the beginning of the transformation the inversion is ac- complished much more rapidly. Various hypotheses have been put forth for explaining the irregularities which are observed in the hydration of cane-sugar. Certain authors attribute the observed retarda- tion to a deterioration or alteration of the sucrase, a change taking place in proportion as the work of hydration pro- gresses. According to other authorities the retardation in the inversion proceeds from the disappearance of cane-sugar, whose presence favors diastatic action. Finally, the hypoth- esis that the products of transformation accumulated in the liquid check diastatic action may give a probable explanation 75 "2 6 THE ENZYMES AND THEIR APPLICATIONS. of the irregularity with which the transformation proceeds. In this last hypothesis, the efficiency of the enzyme would be hindered by the invert-sugar formed during the action. Let us see on what facts these different hypotheses rest, and seek a rational interpretation of the irregular course of the inversion. Deterioration and Alteration of the Diastase.— The hy- pothesis explaining the retarding of the inversion by a dete- rioration of the active substance during its work does not ap- pear to us to merit a serious discussion. The ratio observed at the beginning between the duration of the action and the quantity of sugar transformed affords a conclusive proof of the permanence of the diastase. In fact, if after the second hour of action we can ascertain that the quantity of sugar transformed is double that which we have found after the first hour, it is very evident that the diastase has acted during the second period of operation with the same energy as dur- ing the first. The work done during the first hour has not then caused any destruction of the active substance, and it appears difficult to us to believe that weakening, which is not found at the beginning, can be produced during the course of the operation. Moreover, the mode of action of enzymes excludes all idea of deterioration of the active substance in the course of transformation. In studying the mode of action of amylase, we have had occasion to make evident by direct experiments the enduring character of the diastase during activity, and we believe that the explanation which we have given of this phenomenon can be generalized and extended to all analogous phenomena, for the slackening has identical characteristics in a great number of diastatic actions. The hypothesis of the alteration of the diastase during its activity appears to be very probable. In fact, numerous chemical agents, as well as various physical conditions, in- fluence the sucrase differently and to a very great degree. SUCRASE. 7 7 In the experiments -cited in the preceding- chapter, for ex- ample, the slackening in the transformation must unques- tionably be attributed to the combined action of oxygen and light. Still, we cannot attribute the irregularity in inversion en- tirely to physical or chemical causes, for, even when avoiding the action of light and oxygen, it is still found that irregu- larity occurs. Furthermore, the alteration of sucrase caused by oxygen does not become appreciable till after a prolonged contact with the air, while the ratio soon ceases to hold when the diastatic action takes place in a very dilute solution of sugar, or for that matter, when large quantities of sucrase are put in action. We have seen that in placing, under certain fixed con- ditions, a mass of sucrase in the presence of any quantity of sugar it is found that just as much invert-sugar is formed during the second hour as during the first. If, under the same conditions, ten times as much sucrase is employed, this equality of work during the first and second hours of the action no longer holds good, but the proportion- ality will be observed if quantities of invert-sugar are com- pared after 10 and 20 minutes of work. If the amount of sucrase is again augmented, the ratio is found to hold during the first part of the action, but to cease after ten minutes. As we see, the retarding force may appear in the liquid at different times according to the amount of sucrase used. If then we accept the hypothesis attributing the slackening to an alteration of the active substance, we must assume at the same time that the same sucrase may alter very rapidly or very slowly according as it is used in a large or small amount. Finally, as the proportionality ceases at different times for the same quantity of sucrase put in sugar solutions of different densities, it must be assumed that the quickness of the alteration depends, not only on the amount of enzyme 78 THE ENZYMES AND THEIR APPLICATIONS. used, but also on the concentration of the sugar solution. The improbability of this theory can be seen. It results from the facts which we have just presented that neither wearing out by working nor alteration by physical or chemical agents can be the true cause of the slackening of the diastatic action. Experiments on the Influence of the Invert-sugar. — Most authors have attributed the failure of the proportion- ality during the course of the transformation to the invert- sugar formed, which, according to them, checks the diastatic action. We have sought to verify by a direct experiment this re- tarding action of the invert-sugar. To this end two solutions, A and B, were made, each con- taining ioo cubic centimetres of water, 5 grams of sac- charose, 1 cubic centimetre of acetic acid, and 10 cubic cen- timetres of yeast sucrase. In solution B was added 2 grams of invert-sugar. These solutions were left in a water-bath, and from time to time samples were taken in which the quan- tity of reducing sugar formed was determined. Solution A. Solution B. Minutes. Reducing sugar Reducing sugar formed. formed. 15 O.26 O.25 30 O.51 O.52 45 0.79 0.74 60 0.9 I.I I 90 1.2 1.2 I20 I.4 I.32 l8o -I-75 L89 It is seen from* this table that the proportionality ceased after 45 minutes of action in solution A, which contained only cane-sugar, and that the weakening of the diastatic power began when about one-twentieth of the total sugar SUCRASE. 79 contained in the solution had been transformed. In solution B, which contained 40 per cent of invert-sugar at the begin- ning" of the action, inversion was not at all retarded during the first 45 minutes. On the contrary, the transformation appeared to conform very nearly to the law of ratio, and the slackening in the transformation was not manifested until after an hour of action. By comparing the quantities of sugar inverted during the first hour in experiments A and B, we find that only 18 per cent of saccharose had been transformed in the experi- ment made with pure sugar, and 22 per cent in the experi- ment made with a mixture of saccharose and invert-sugar. The slackening in the action of the diastases must not then be attributed to the presence of the products of transforma- tion in the medium where the diastatic work is performed. Hypothesis of O'Sullivan and Tompson. — O'Sullivan and Tompson have put forth the hypothesis that the effect produced by sucrase is constantly proportional to the weight of the cane-sugar present in the liquid at the time of action. Starting from that, they attribute the retardation produced in the transformation to the diminution of the quantity of saccharose as the inversion proceeds. According to this view, the sucrase would act in the same way and with the same energy from the beginning to the end of the action, and the slackening would be exclusively due to the reduction of the supply of saccharose. Then, if we invert a solution containing 10 grams of sugar by the aid of a quantity of sucrase able to produce a gram of invert-sugar in the first 10 minutes, we may expect to have produced during each of the following ten minutes a hydra- tion corresponding to one-tenth of the total quantity of cane- sugar contained in the solution at that time. According to this theory, the mode of action of sucrase would not change during the work; the course of the inver- sion would be on the whole regular and the slackening ob- served would be the direct and inevitable consequence of the So THE ENZYMES AND THEIR APPLICATIONS. regularity of the phenomenon itself. For, if after the first 10 minutes of the action we have found the production of one gram of invert-sugar, after the following 10 minutes, we shall have obtained only 0.9 grams, inasmuch as the action is produced in this case no longer upon 10 grams, but only upon 9 grams of cane-sugar. After 20 minutes, there will remain in the solution 8.1 grams of saccharose and, by acting always under the same conditions, the sucrase will invert dur- ing the following 10 minutes 10 per cent of the sugar remain- ing, or 0.81 grams. This hypothesis is, these authors say, fully confirmed by the measurement of the quantities of sugar inverted at the end of periods varying in arithmetical progression. Arguments for and Against this Hypothesis. — The theory of O'Sullivan and Tompson is very attractive; it has. not, however, found many adherents, and various objections have been raised against it. First, it has been objected that the experimental proofs which they bring forward in favor of their theory do not at all prove that the slackening comes from the diminution of the quantity of cane-sugar. In fact, the results of their experi- ments may equally well be explained by the gradual increase, during the action of the sucrase, of the quantity oi invert- sugar. Our experiments, cited above, on the influence of the in- vert-sugar, show the invalidity of this argument. But, still another objection may be raised to the theory of O'Sullivan and Tompson. If the gradual disappearance of cane-sugar is really the retarding cause, the quantity of sugar inverted by any amount of sucrase will be in direct relation with the weight of the cane-sugar present in the liquid. In- crease in the amount of cane-sugar will then cause a corre- sponding increase in the amount of sugar inverted. We al- ready know that these expectations are not always realized, and that the same quantity of sucrase produces the same amount of invert-sugar, independently of the concentration SUCRASE. St of the sugar solution. There, then, is a serious argument against the hypothesis that we are considering, but it is none the less true that the quantity of sugar contained in the medium is not without influence on the slackening. By studying the phenomenon more closely, we find that the de- gree of hydration depends upon two factors. At the beginning of the action it is the quantity of su- crase used which plays a predominant part, and the quantity of invert-sugar formed is proportional to the quantity of ac- tive substance used. When the inversion is more advanced, the influence of the quantity of sucrase becomes less. The transformation becomes directly proportional to the sugar content. The successive influence of the two factors may be shown by the following experiment: To ioo cubic centimetres of three liquids, A, B, and C, containing respectively 5, 10, and 20 grams of saccharose, add the same quantity of sucrase. Then leave these solu- tions in a water-bath at a temperature of 50 . From time to time, take samples, and determine the saccharose remaining in them and, when in solution A 15 per cent of saccharose has been transformed, begin to measure the invert-sugar in the two other specimens. There is then obtained the follow- ing figures : Invert-sugar at the end of A B C 2 hours 0.75 0.74 0.78 4 " 1.1 1.4 1.6 One finds then at the beginning of the action almost the same quantities of sugar transformed in the three liquids, A, B, and C, but at the end of four hours the conditions change and there is found in the solution containing 20 per cent of sugar 1.6 gr. of transformed sugar, while the 5 per cent liquid affords only 1.1 gr. of invert-sugar. Th« concentration of the sugar solution, then, influences &2 THE ENZYMES AND THEIR APPLICATIONS. the action of the sucrase up to a certain point. The course of the hydration of the sugar in solutions of different concen- trations is shown to be rather favorable to the theory of O'Sullivan and Tompson, especially if the beginning of the transformation is excluded. Still this theory does not appear to us to be based on very well established data. By noting the transformation of the sugar at different periods we have, indeed, found that the slackening in the in- version increases in proportion as the action advances, but we have never been able to see the regularity which the authors of the hypothesis claim, and which is the very basis of their theory. Even admitting that it can be demonstrated experiment- ally that the decrease in the inversion varies in geometric progression, this demonstration would show the way in which the retardation takes place, but it would not at all re- veal the real cause. By making the same quantity of sucrase act on sugar solutions of different concentration, it is seen that the retarding force is manifested very differently. In a dilute solution, the proportionality between the duration of the action and the. quantity of sugar formed is lost at the end of a relatively short time. In a concentrated solution, on the contrary, it persists for a longer time. These great differences in the action of sucrase are easily explained if one determines in liquids of different concentra- tion the quantitative relation which exists between the sugar inverted and that which is not yet inverted. By studying the variations of this relation for a dilute solution and a concentrated solution, it is found that the slackening of hydration does not become really appreciable until the sugar solutions contain about 15 parts of invert- rugar to 85 parts of unchanged saccharose. It being granted that sucrase produces, at the beginning f the transformation a hydrating effect proportional to its iiuantity, it is quite evident that in the dilute solution the ratio SUCRASE. 83 -|4 will be reached much more quickly than in the concen- trated solution. In other words, when the slackening of hydration is first observed, the concentrated solution contains more invert- sug-ar than the dilute solution, although the quantity of su- crase put in action would be the same in the two solutions. The retardation in the inversion depends directly upon the composition of the liquid in which the diastase acts. It is not caused by the diminution of the quantity of cane-sugar contained in the solution, and neither does it arise from the increase of the quantity of invert-sugar. It is rather caused by the combination of the two circumstances. Theory of Inversion of Cane-sugar. — We must, we be- lieve, seek in the structure of the molecules of saccharose the real origin of the retarding force. It is generally supposed that the action of the sucrase is manifested by the successive hydration of molecules of sugar with which it comes in con- tact. It is, however, probable that the mechanism of inver- sion does not possess this simple character. It is more probable that the sucrase acts from the begin- ning on the whole mass of sugar with which it comes in con- tact, and that, in the transformation of cane-sugar into in- vert-sugar, it produces a series of modifications. It is easily conceived that, by successive hydrations, there may be formed, besides invert-sugar, a series of substances very similar to saccharose, but which may have a different degree of sensitiveness to the sucrase. Such are the intermediate substances produced by hydration which afterwards appear more or less suitable for the transformation, and it is from this greater or less susceptibility to hydration that the check- ing of the inversion comes. It may be, too, that the changes undergone by the sac- charose consist in changes in the configuration of the mole- cules, and that stereochemical isomers are formed in the solution. Still it is impossible for us to bring forward convincing 84 THE ENZYMES AND THEIR APPLICATIONS. facts in favor of our hypothesis. Having predicted the formation, in the course of inversion, of products inter- mediate between the saccharose and the invert-sugar, we have sought to isolate these products, or at least to charac- terize them. But the various experiments attempted with this in view have remained without results. Still the hy- pothesis, as we shall see, finds support in the process of hy- dration by acids. Experiments on Transformation by Acids. — By studying the inversion of sugar in the presence of increasing amounts of acid, we have been able to ascertain that there is produced at certain periods a noticeable retardation in the progress of hydration. The slackening takes place at times which always coincide with certain stages in the hydration of saccharose. Thus there is a striking analogy between the action of acids and that of sucrase. The retarding force is found in both cases, in the action of the acids as well as in the diastatic ac- tion, and the moment when the slackening begins corre- sponds to the instant when the relation between the quanti- ties of invert-sugar and of non-transformed sugar reaches a certain value. This resemblance between the mode of action of chemical and of physical agents proves that the retarding force does not come from the sucrase, and that the origin of the retarda- tion is not necessarily to be attributed to the manner of de- composition of the cane-sugar and to the formation of transi- tory products which resist differently the agents of trans- formation. Some details of the experiments which we have made are here given : Dissolve a gram of cane-sugar in distilled water, add 2 n cubic centimetres of — sulphuric acid, and bring the volume 10 F & up to 100 cubic centimetres. Leave in water-bath at 6o°, for one hour, then neutralize accurately with normal soda, and determine the quantity of invert-sugar formed by the action SUCRASE. 85 of the acid. This experiment is then repeated with 4, 6, 8, n 10, etc., cubic centimetres of — sulphuric acid, and the fol- io L lowing;' results are obtained : Cubic centimetres of acid. Percentage of invert-sugar. . .. 5.71... .. .II.36.. . 6 15.29. 8 22.12. 10 26.34. 12 32.00. 14 37-!4- 16 46.76. 18 51.36. 20 53.33. 22 52.00 44 65.20 Increase. ••571 ••5.65 ••3-93 ..6.83 . .4.22 ..5.16 ..5.14 . .9.62 . .4.60 ..1.97 ••i-33 . . 1.20 The heading- " Percentage of invert-sugar " indicates the quantity of sugar transformed during the experiment. Under the heading " Increase " we have written the in- crease in the quantity of sugar transformed by each addition of two cubic centimetres of acid. By following, in the table, the progress of the hydration in the presence of increasing amounts of acid, it is found that the ratio between the quantities of acid and invert-sugar formed is not at all constant. This ratio exists in the first experiments and disappears completely in those where 50 per cent of cane-sugar is transformed. Thus 2 cubic cen- timetres of acid have formed 5.71 centigrams of invert-sugar; with a double amount, 11.36 eg. of invert-sugar are formed, or practically double the preceding quantity. Tf we increase the amount of acid and use 20 cubic centimetres we cause a hydration of 53 per cent of sugar; but beyond this amount of acid the hydration slackens and 44 cubic cen- S6 THE ENZYMES AND THEIR APPLICATIONS. timetres of acid hydrate only 65 per cent of the cane-sugar present in the liquid. If the ratio really existed, with this amount of acid a complete inversion of all the saccharose contained in the liquid would be obtained. The action of increasing amounts of acid is still better shown when the. heading "Increase" is followed. In the first experiments the increase gradually falls from 5.71 to 3.93, but in the following ones, when nearly a quarter of the whole quantity of cane-sugar has been transformed, a com- plete change is found in the progress of the inversion. The increase rises to 6.83 to fall again to 5.14. The increase is again augmented when half the cane-sugar has been transformed, then it undergoes another decline, and is represented by the number 1.2 in the presence of 65 per cent of invert-sugar. The course of hydration by acids is then far from being regular. A great number of analogous experiments, made under the same conditions, have always confirmed in our work the non-existence of a ratio between the quantities of acid used and invert-sugar formed. We have always found a slackening in hydration, which coincides with the appearance of a definite ratio between the quantities of sugar inverted and of non-transformed sugar in the liquid. The action of acids is then, in the main, identical with that of the diastase, and the retardation observed in hydration by sucrase must rather be attributed to a structural transforma- tion of the saccharose molecule. BIBLIOGRAPHY. Mitscherlich. — Rapport annuel de Berzelius. Paris, 1843. Berthelot. — Sur la fermentation glucosique du sucre de canne. Chim. org. fondee sur la synthese. Paris. Comptes Rendus, L. i860, p. 980. Dumas.— Sur les ferments appartenant au groupe de la diastase. Comptes Rendus. 1872. Nasse.— Bemerkungen zur Physiologie der Kohlenhydrate. Pfliig. Arch., 1877. SUCRASE. 8 j J. O'Sullivan. — L'invertase de la levure de biere, 1893. Monit. scient. J. Kjeldahl. — Carlsberger Laboratorium, 1879 et 1881. E. Donath. — Ueber den invertirenden Bestandtheile der Hefe. Berichte der deutsch. chemisch. Gesellschaft, 1875, VIII, p. 975. Kossman. — Etudes sur les ferments solubles contenus dans les plantes. Comptes Rendus, 2 e ser., 1875, p. 406. Em. Bourquelot. — Sur la physiologie du gentianose et son dedoublement par las ferments solubles. Comptes Rendus, 1898, p. 1045. O'Sullivan et Tompson. — Sur un ferment non-organise, l'invertase. Comptes Rendus, 1872, 2 e ser., p. 295. Fernbach. — Recherches sur la sucrase, diastase inversive du sucre de canne. These, Paris, 1890. Duclaux. — Sur Taction de la diastase. Annales de l'lnstitut Pasteur, 1897. J. O'Sullivan. — L'invertase. Journal of the Chem. Soc, 1890, I, p. 834-931. Beitriige zur Geschichte eines Enzymes. Berichte der deutsch. chemisch. Gesellschaft, 1890, p. 743. Ad. Mayer. — Die Lehre von den chemischen Fermenten oder Enzymol- ogie. Heidelberg, 1882. Wasserzug. — Ann. de l'lnstitut Pasteur, 1887. E. Fischer et Lintner. — Verhalten der Enzyme gegen Melibiose, Rohr- zucker und Maltose. Berichte der deutsch. chemisch. Gesellschaft, 1895, 3. 3055- Miura. — Inversion des Rohrzuckers. Berichte der deutsch. chemisch. Gesellschaft, 1895, p. 623. V. Tieghem. — Inversion du sucre de canne par le pollen. Societe bota- nique de France, 1866. CHAPTER VII. FERMENTATION OF MOLASSES. Industrial application of sucrase. — Fermentation of molasses. The enzyme producing the inversion of saccharose does not constitute a commercial article. It has a very limited manufacture and serves exclusively for study and for labora- tory experiments. But. if specially prepared sucrase is not used in industrial operations, this diastase nevertheless plays an important part in fermentations, and especially in the manufacture of alcohol from molasses. The fermentation of molasses, a substance which contains nearly 50 per cent of saccharose, is a relatively simple opera- tion. It is effected in the following manner : The molasses is first diluted with water acidified with sul- phuric acid, and brought to from 9 to 12° Beaume. Thus a mash is formed ready to undergo the action of the beer-yeast which inverts the saccharose and causes the in- vert-sugar produced to ferment. The transformation of molasses into alcohol appears then to be a simple industrial operation. The apparatus required is, in fact, less complex than that of a grain distillery. Moreover, the working of the molasses requires relatively little supervision, and much less practical knowledge on the part of the operators than does the distillation of grains. Yet there are few manu- factories which utilize rationally the former materials and secure results even approximating the theoretical yield. Manufacturers attribute the difficulties they encounter ss FERMENTATION OF MOLASSES. 89 either to the quality of molasses used, or to the inefficiency of yeasts, or to infection by foreign ferments, and they seek to remedy these imperfect conditions by a strong acidifica- tion of the mash. They sometimes try also to regulate the work by a preliminary heating of the molasses in order to eliminate the volatile organic acids. These acids are freed by the addition of sulphuric acid to the mash at the time of acidification. The causes which occasion these troubles in the working of molasses are very numerous. We cannot here enter upon a thorough study of this question, but, nevertheless, we think we ought to call the attention of manufacturers who are oc- cupied with fermentations to some of the very frequent causes of the smallness of the output, especially the insuffi- ciency of inversion. In the practice of distilling molasses this point is wholly neglected. Although it is known that non-inverted sugar is not fermentable, little importance is attached to the inversion of the saccharose of molasses, because of the current opinion that inversion is very easily accomplished owing to various conditions of the medium. If the question is studied more closely, it is seen that, on the contrary, inversion is very slow, and that in most cases it is not completed at the end of the fermentation. The reason that in practice little attention is paid to hy- dration is that during fermentation two factors are generally counted on : first, the sulphuric acid which has been intro- duced into the molasses and which is considered sufficient in itself to produce inversion ; second, the yeast which is sup- posed to be an inexhaustible source of sucrase. Let us see to what extent each of these two factors con- tributes to the inversion, and let us first study the part played by the acid. By the addition of acid to mashes of molasses there is secured in practice an acidity corresponding to from 1 to 2.5 grams per litre of sulphuric acid. go THE ENZYMES AND THEIR APPLICATIONS. The acidification of the musts is done at a low or high temperature according to the distillery. To give an idea of the inverting power which these amounts of acid possess, let us add to a certain number of ioo-gram specimens of a 10 per cent solution of cane-sugar different amounts of sulphuric acid and let us submit these experiments for 24 hours to a temperature of 30 . Numbers of Number of grams of Grams of the specimens. sulphuric acid per litre. invert-sugar. I 2.5 I 2 5 • 1.8 3 10 3-3 4 25 6.7 Thus when we submit our experiment to the action of 2.5 grams of sulphuric acid, the greatest amount used in manu- facture, we obtain at the end of 24 hours only 10 per cent of invert-sugar, and in order to obtain 67 per cent, we must use 25 grams of acid, or a quantity 10 times greater. The action of the cold acid is not then an important factor in inversion. The results obtained by boiling the sugar solu- tions appear quite different, it is true. If, to ascertain the influence of high temperatures, we re- peat the preceding experiments at 90 , we shall observe that with the smallest quantity of acid (0.5 gr. per litre) we pro- duce a complete inversion of the saccharose. We may conclude from this, that heating the molasses with slight amounts of acid present is very important from the point of view of inversion. But molasses does not act towards the different factors in the same manner as the solu- tions of pure sugar. In fact, the acidity found in mashes of molasses comes, not from mineral acids which have been added to them, but rather from organic acids which have been freed by the sulphuric acid, and which act on the sac- charose with much less energy than inorganic acids. More- FERMENTATION OF MOLASSES. 9 1 over, the presence of salts in the molasses weakens the action of the acids. The effect produced practically by heating acid molas- ses may be shown by the following experiments: ioo grams of molasses are diluted in 400 grams of water. Different specimens of this must are taken ; they are acidified with varying amounts of sulphuric acid; they are kept boil- ing for some time, then cooled and brought up to their original volume. By examining the rotatory power of these specimens, one may study the course of the transformation in the presence of different amounts of acid. The solution be- fore inversion gives a rotation of 38 to the right, and after complete inversion a rotation of SV to the left. Moreover here are the intermediate results: Numbers of Grams of sulphuric Rotation to the specimens. acid per litre. the right. 1 1-25 37° 2 2.5 36 3 5 35 4 10 24 5 I2 -5 3-6 With the amount of acid used in manufacture, or two grams and a half, inversion is then trifling; rotation de- creases only from 38 to 36 . We find, indeed, that by using five times as much acid, we are still far from obtaining com- plete inversion; sulphuric acid used in an amount of 12.5 gr. still gives us a rotation to the right of 3. 6°, while complete in- version would have given a rotation to the left of 8.5 . In many manufactories it is the custom to boil acidified molasses after diluting it with water. It is seen that in this case practically no inversion takes place. We have been able, however, to ascertain that the inversion is hastened when the acid molasses is heated before dilution. In the practice of distilling molasses it is the sucrase of 92 THE ENZYMES AND THEIR APPLICATIONS. the yeasts and not the acids used which produces hydration of the saccharose, and the course of the fermentation de- pends, in very large measure, on the manner in which the secretion of diastases by the cells is accomplished. Now, the action of the sucrase is considerably influenced by the saline substances contained in the molasses. The following experi- ment is well calculated to demonstrate this fact : ' Acidify a sugar solution at 12 Balling with sulphuric acid in the proportion of 0.5 grams per litre, and take from this must two specimens, A and B. To specimen A, which is the control specimen, add 10 cubic centimetres of yeast su- crase. The second specimen receives the same quantity of sucrase, then the accurately neutralized ash of 100 cubic centimetres of a must of molasses of 12 , Ball. The two specimens are then left in a water-bath at 30 . Here is ; the comparative progress of inversion in the two specimens : Minutes. A - B - Invert-sugar. Invert-sugar, 40 min 4-7% 2.4% 2 h 5-79 2.9 3 h 7.0 3.2 4 h 9.2 4.6 These figures prove in a conclusive manner that the min- eral substances of the molasses retard inversion considerably. After four hours of action, there is found in the control solu- tion 9.2% of invert-sugar, while in the solution to which ash. of molasses has been added there is found only 4.6%. These data show the nature of the difficulties encountered in the fermentation of molasses. Still it may be objected that in the fermentation indus- tries, a solution of diastase is not used : but that inversion is accomplished by living cells. It must then be admitted that the conditions of transformation are completely different; that since inversion by yeasts can be accomplished inside the FERMENTATION OF MOLASSES. 93 cells, the composition of the exterior liquid naturally has a much smaller influence on that account. To answer this objection we have made the following ex- periments: To a 10 per cent solution of cane-sugar is added yeast ash. This solution serves as the basis of two specimens, A and B, each of 500 cubic centimetres. In specimen A there are introduced the neutralized ash of 50 grams of molasses and 5 grams of yeast. Specimen B is submitted to the influ- ence of the same quantity of yeast, but without the addition of salts. Here is the comparative course of the fermentation of the two specimens : A B .... ( Invert-sugar 0.5% 1.8% After 6 hours \ . , , , fe ° ' ( Alcohol 0.4 0.65 Ar , ( Invert-sugar 0.2 3 After 12 hours < ■ , , & ° , { Alcohol 1.5 2.6 A . , ( Invert-sugar 0.5 0.2 After 24 hours ■{.,,, [ Alcohol 3 5.9 By comparing the quantity of invert-sugar after six hours in the two solutions, we see that inversion proceeds much more slowly in solution A, treated with the salts contained in the molasses. It is true that after 24 hours we find a greater quantity of invert-sugar in solution A than in solu- tion B, but if we take into account the quantity of alcohol present at this time in the two solutions, it becomes evident that hydration has followed a much more regular course in B than in A. Phenomena of this nature — slowness in fermentation, ir- regularities in the progress of the transformation — are often found in molasses distilleries and are generally attributed to the degeneracy of the yeasts. This opinion is absolutely erroneous; yeast does not gen- erally degenerate in molasses mashes ; on the contrary, it re- 94 THE ENZYMES AND THEIR APPLICATIONS. produces abundantly and the cells formed under these con- ditions generally have great activity. These yeasts cause a very rapid fermentation of grain mashes, but the quantity of sucrase which they secrete diminishes. It is to this diastatic weakening that we must attribute the difficulties encountered in fermenting unchanged sac- charose with yeasts cultivated in molasses. Not all the beer-yeasts contain the same quantity of su- crase; the quantity of the enzyme secreted varies with the special variety. In the choice of a yeast for molasses, one should first take account of the inverting power, as well as of the degree of resistance of the active substance which it con- tains. Generally the distiller seeks to substitute quantity for quality in the yeasts. This practice is far from rational. The expense in yeast is thus rendered quite large and the alcoholic yield is diminished, for the yeast consumes a part of the carbohydrate for the construction of its tissues and for their maintenance. To test a yeast with a view to its action on molasses it is not enough to determine its inverting power in a solution of pure saccharose. It is better to make the experiment with saline substances present. The advantage of this method is that it gives more certain results, because it approximates actual industrial conditions. We have had occasion to make experiments with yeasts from different sources, and these ex- periments have shown us that the degree of resistance of the sucrase contained in the cells differs much according to the variety. These experiments have proved to us, moreover, that the resistance of the enzyme plays a very important part as influencing the yield. Compressed yeasts as well as beer-yeasts have been re- placed in the fermentation of molasses by leaven. The dis- tiller cultivates his yeasts himself and for this purpose uses mashes of grain prepared either by sulphuric acid or by malt. There is generally used for the preparation of the mash FERMENTATION OF MOLASSES. 95 leaven 3 to 5 kilograms of grain for 100 kilograms of molasses. In many distilleries this amount of grain is in- creased. Sometimes it is thought best to add to the molasses or to the yeast mash a certain amount of nutritive nitroge- nous materials, such as rootlets of malt, amides, and pep- tones. Undoubtedly the use of grains and nitrogenous materials furnishes appreciable results with certain varieties of yeasts which demand a special medium to acquire their inverting power. It must, however, be recognized that the very prin- ciple of this practice is wholly false and that the results ob- tained are far from being satisfactory from an economic point of view. Molasses contains all the nutritive substances necessary to feed the yeast-cells abundantly. If a variety of yeast cannot become accustomed to acting upon molasses, if a special manner of nutrition must be adopted so that it can live in this medium, this yeast should be given up and another less delicate kind should be used. While visiting the molasses distilleries at Breslau, Leip- zig, Darmstadt, etc., in 1895. we found out that the leaven used for the fermentation of molasses in these manufactories cost from 8 to 10 francs per hundred litres of alcohol manu- factured. This expense, a total loss, arose from the fact that the distilleries used, for the making of their leaven, malt and grains without which their yeasts did not work well. We advised them to take a yeast suitable for action upon molas- ses, and we have since had the satisfaction of learning of the almost complete suppression of the use of grain for leaven in those places. The leaven is at present made with pure molasses and the yield in alcohol is unquestionably greater. Most kinds of beer-yeast furnish a sucrase of little resist- ance, but their greater or less alterability depends especially on the culture medium in which they are developed. Sucrase secreted by yeasts cultivated in molasses possesses a resist- 9 6 THE ENZYMES AND THEIR APPLICATIONS. ance inferior to that of the same yeasts cultiyatecl in a must of grains or malt. This weakening of the resistance is due neither to the nature of the enzyme secreted nor to the prolonged contact with saline substances ; its true cause is rather the sudden pas- sage of the cells from one medium to another. We have found that yeasts capable of producing the fer- mentation of dilute molasses can be brought to effect the complete fermentation of very concentrated mashes by ac- climating these yeasts to the new medium by gradually fur- nishing them with solutions of increasing concentration. The changes which are produced in the resistance of a sucrase by acclimatization to the medium may be demon- strated by the following experiments: . Sucrase is extracted from yeasts in different stages of habituation and tried simultaneously on a solution of pure sugar, and on a solution of sugar to which ash of molasses has been added. It is thus found that the yeast has acquired new properties and furnishes an enzyme which is little changed when saline substances are present. These new properties acquired by the yeasts are, however, transitory properties. * * These experiments throw a special light on the mechanism of ac- climatization as well as on the individuality of diastases. By studying the sensitiveness of beer-yeasts to the action of different antiseptics, we have established the fact that one can acclimatize the yeasts to relatively large amounts of these agents. Thus, a beer-yeast which shows itself very sensitive to the action of 10 milligrams of hydrofluoric acid and which no longer gives any fermentation in the nutritive mash can be made to reproduce in the presence of 30 times as much of this acid, and to give rise to very active fermentation. This acclimatization requires that one accustom the yeast to increasing amounts of this anti- septic. The cells thus obtained acquire characteristic properties: The fermenting power is considerably augmented, while the power of multiplication is reduced to its lowest limits. Yeast acclimated to antiseptics preserves the characteristic property of resisting them for several months, even when it is daily cultivated in solutions free from the agent to which they have become accustomed. FERMENTATION OF MOLASSES. 97 The difficulty of inversion in the fermentation of molasses may also arise from some other cause than the insufficiency of the sucrase of the yeast. Thus, strong acidity of the musts or a great concentration of the sugar may produce a slackening" in the fermentation. The excess of sugar acts unfavorably on account of the accumulation of alcohol in the musts. Even with 5 per cent of alcohol present, the diastatic force is influenced, and with 10 per cent the inversion proceeds very slowly indeed. The acidity.of the musts, such as is found in practice, does not act directly on the sucrase and the retardation observed must rather be attributed to foreign ferments, which, favored by the acidity, develop in the mash. From molasses which is fermentable with difficulty we have isolated bacteria which produced a slight acidity in Acclimatization has then produced a profound change in the cells, and one which is transmitted from one generation to another. Quite a different thing is observed in the action of saline substances on yeast. The resistance which sucrase acquires by acclimatization commences to be weakened as soon as the yeast is again found in a medium free from salts. The properties of sucrase are here then closely related to the composi- tion of the medium. This fact contradicts the hypothesis of the existence of different enzymes acting on the same body and producing the same chemical reactions. According to this hypothesis, indeed, there exist different sucrases. •The sucrase of Aspergillus nigcr, for example, would be a different sub- stance from the sucrase of yeast, etc. One would also have to distinguish between the sucrases of different yeasts, because these are not equally sensitive to the temperature and to the reaction of the medium. The variable resistance of sucrase to chemical agents would evidently lead to new distinctions, but these latter distinctions would be absolutely illusory. The difference in the manner of action in the presence of chemical sub- stances arises, not from a change in the nature of the sucrase, but rather from difference in the external media. The same thing must occur with sucrases of different origin, showing- different properties. In fact, the diastase is accompanied in the cells which secrete it by different substances which modify its properties. 98 THE ENZYMES AND THEIR APPLICATIONS. sugar solutions. Alcoholic fermentation is manifestly ar- rested by such ferments. These micro-organisms act also on yeast in grain mashes, but they are shown to be specially dan- gerous in the fermentation of saccharose. In the presence of these organisms the fermentation of the sugar solutions is arrested when the must has still only a slight acidity and a small quantity of sugar has as yet been transformed. The difficulties in fermentation of molasses caused by lack of sucrase are accompanied by the following symptoms : The fermentation begins regularly, but when about 50 per cent of the sugar is transformed, that is, before the end of the principal fermentation, there is suddenly found a noticeable slackening, then a stop which is prolonged for several hours. The yeast is deposited slowly ; there is pro- duced little by little new amounts of sucrase and the fermen- tation recommences, often with great energy. Then a second stop occurs which is generally final. The mash in fermenta- tion often contains at this period considerable quantities of non-inverted sugar. Such is the course of events when the work is carried on in the presence of antiseptics. In the opposite case, the fermentation has an entirely dif- ferent aspect. When the slackening occurs, the mash, invaded by bacteria, becomes decidedly acid. The yeast degenerates and the fermentation, once stopped, does not recommence, or at least, does so only very feebly. From the practical point of view, it is well for the distiller of molasses to pay more attention than he usually does to the manner of inversion of cane-sugar during fermentation. It is especially necessary to exercise great care in the choice of yeasts. It is then indispensable to protect the mashes by antiseptics against foreign ferments. It is also well to filter or decant the molasses mashes after their acidification. Not all the ferments are destroyed by simply boiling. The bacteria found in difficultly fermentable molasses are only FERMENTATION OF MOLASSES. 99 destroyed at a temperature of no°, though they may be easily removed either by filtration or by decantation. BIBLIOGRAPHY. Effront.— Etude sur la fermentation des melasses. Moniteur scientifique, 1894 Bui 1894 189.4, p. 461. . . Bulletin de la Societe d'encouragement pour 1 Industrie national CHAPTER VIII. AMYLASE. Presence of amylase in vegetable and animal cells. — Preparation. — Cohn- heim's method. — Lintner's method. — Effront's method. — Wroblew- sky's method. — Properties. — Influence of quantity, time, and tempera- ture. — Influence of chemical agents; acids, alkalies, salts. — Substances which accelerate diastatic action. The enzyme called amylase, or simply diastase, is a solu- ble ferment hydrating starch and transforming it into mal- tose and dextrins. The existence of this enzyme was first observed by Kirchoff, in 1814, in gluten. Dubrunfaut, Pay en, and Persoz have since studied this substance thoroughly. Amylase is widely distributed in nature. It is found in barley, oats, rice, maize, and in general, in all cereals. The raw grains have little amylase, the enzyme being formed especially in the course of germination. The presence of amylase has been observed in the tubers of potatoes, as well as in the leaves and shoots of different plants. The transformation of starch into carbohydrates assimi- lable by living cells being generally accompanied by the ac- tion of amylase, it may be believed that this substance plays a very important part in the formation of vegetable tissues. Yet the transformation of starch is not always produced by the aid of amylase, and because a cell brings about the transformation of amylaceous materials does not at all prove that it secretes this diastase. We shall see later that other enzymes exist which act on AMYLASE. ioi starch and render it assimilable and suitable for the construc- tion of tissues. Wortmann asserted that the assimilation of starch is not always accompanied by the action of enzymes, and he be- lieved that he demonstrated that protoplasm alone can by itself produce a hydrating and dissolving action on starch. This view may be seriously questioned, however. It is true that in the leaves of plants where a very active transforma- tion of starch is brought about, there are generally found quantities of diastase small in comparison with the work ob- served. It is equally true that often the stalks and stems do not secrete active substances, while in these organs is found an energetic assimilation of starch. But these facts are not sufficient to prove the direct action of protoplasm in the hy- dration of starch. The fact that amylase has not been found in the cells may simply arise from difficulties like those which occur in the study of sucrase ; in other words, amylase may be more or less retained within the cells, or it may enter into combination with other substances and thus become more or less soluble. As we have found for tannin, amylase is sometimes present in an inactive form, because the environmental conditions under which it is found are unfavorable for its action. In this case, it again acquires its normal prop- erties as soon as it is placed under favorable conditions. We know, moreover, that the effect produced by an enzyme de- pends especially upon the conditions of the medium, and we have reason to believe that in living cells the action of dia- stases is more energetic than in our experiments. It is prob- able, moreover, that with a very small quantity of active sub- stance one could obtain marked results if the conditions of the medium are favorable. If, then, amylase has not been found in the various vegetable organs examined, these nega- tive results may be attributed to the different circumstances which we have just enumerated. Then it is not at all estab- lished that protoplasm would be capable, without the inter- IQ2 THE ENZYMES AND THEIR APPLICATIONS. vention of enzymes, of hydrating starch, and all the data which we possess on enzymes tends rather to prove that dia- static action takes place here also. Again, amylase is found, according to some authors, in moulds. Thus, Aspergillus niger, or Penicillium glaucum, if cultivated under certain conditions, would secrete a certain quantity of amylase ; the presence of this diastase in moulds is very rare, however. The diastase which hydrates starch is. met with, not only in the vegetable kingdom, but also in animal secretions. It is present in the saliva, the pancreatic juice, and the liver. The constant, presence of the active substance in the saliva may be explained by two different theories ; first, that amylase is secreted by the salivary glands ; or, second, that it is due to organized ferments which are found in the mouth, and feed upon amylaceous materials. Claude Bernard, who studied this question, was a supporter of the latter theory. By heating saliva to ioo°, he found the complete destruction of the active substance. This saliva, having lost the property of acting on starch, became active when left for some time at the ordinary temperature. He attributes this phenomenon to the development in the saliva of new ferments capable of furnishing to the surrounding liquid new quantities of diastase. The appearance of the dia- stase could, however, be explained by the action of the tem- perature on the ferments of the saliva. Certain cells which have not been destroyed by the heat might still retain amy- lase, and the enzyme, when the temperature is lowered, would diffuse into the liquid. However this may be, the ap- pearance of amylase in the saliva may be explained otherwise than by a secretion of the glands. To settle this question definitely, the experiment of Claude Bernard must be repeated under such conditions that the action of any organized ferments can be avoided. Preparation of Amylase. — Amylase can be precipitated from its solutions, either by mechanical precipitation or by the action of alcohol. According to Cohnheim, the diastase AMYLASE. 103 can be extracted from the saliva by the following method: quicken salivation by rinsing - the mouth with ether. The saliva is then collected and a slight amount of phosphoric acid is added. The acid liquid is then neutralized with great care by the aid of very dilute lime-water. Thus calcium phosphate is formed, which carries down the diastase as well as other nitrogenous materials. This precipitate is removed by filtration, then washed on the filter with a volume of water equal to that of the saliva used. By the washing, the dia- stase goes into solution. It is precipitated from this solution by a suitable addition of alcohol. The preparation of amy- lase is accomplished much more easily by the use of an in- fusion of malt. Payen was the first to find that the active substances of an infusion can be precipitated from the solu- tion by a suitable addition of alcohol. The product thus obtained is unfortunately far from pure; furthermore, it changes readily in the air, oxidizes, and becomes inactive very quickly, and assumes a very dark color. The alteration of amylase is facilitated by foreign sub- stances precipitated at the same time as the diastase by the alcohol. Different means have been proposed to avoid these difficulties. Payen and Persoz, for example, advise adding to the infusion of malt a quantity of alcohol insuffi- cient to precipitate the diastase, then bringing the alcoholic solution to a temperature of 70 , which, according to them, would cause the coagulation of foreign substances, especially albuminoid matter. This operation finished, the coagulated substances are separated from the liquid which contains them and an excess of alcohol is added to cause a precipitation of the amylase. By this method there is obtained a white prod- uct which is not very changeable but which possesses only a very slight activity. Much more satisfactory results are obtained by Lintner's method : One part of finely grouwl malt is mixed with four parts of 20 per cent alcohol ; it is allowed to stand for 24 104 THE ENZYMES AND THEIR APPLICATIONS. hours ; then the liquid is poured off from the malt, and fil- tered, and to each volume of filtered liquid 2 volumes of ab- solute alcohol is added. Thus a flocculent precipitate is formed; the clear solution is decanted and the precipitate collected on a filter. After a first washing with alcohol and ether, the precipitate is ground in a small mortar with a little alcohol ; it is then replaced on the filter and washed a second time with alcohol and ether, then dried in vacuo. By this method is obtained a product which Lintner calls raw dia- stase and which can be still further purified by solution in water and precipitation by alcohol. This purification leads to a product of constant composition but of slight activity. This method gives good results if the operations are per- formed rapidly, in order to prevent the precipitated diastase irom coming in contact with the air before it is completely dehydrated. Nevertheless, the product obtained is very rich in ash as well as foreign materials which have been precipi- tated from the infusion of malt by the alcohol. To obtain purer and more active products we advise using another method. To diminish the quantity of extractive substances in the infusion of malt which do not possess diastatic power, fer- ment this infusion with yeasts which have been previously limited to a scant supply of nitrogen. The alcoholic fermen- tation, caused by these yeasts in the infusion of malt, destroys a great part of the carbohydrates, eliminates a con- siderable quantity of albuminoid matter and salts, but leaves the diastase absolutely intact. This is accomplished as fol- lows : macerate 100 grams of malt reduced to powder with 300 grams of water at a temperature of 30° for 18 hours. Stir the mixture every half hour. The mass freed from the liquid T^y pressure is thoroughly washed with water, — which, to- gether with the original extract, is filtered. The filtrate is made up to 300 cubic centimetres, 10 grams of beer-yeast added and left at a temperature of 28 for 48 hours. It is then filtered, and to the clear liquid is added 700 cubic centimetres AMYLASE. 105 of alcohol. The yeast used for this preparation must first of all have remained for 24 hours in a 10 per cent solution of sugar. The fermentation causes the yeast to lose a part of its nitrogen and gives it an avidity for albuminoid materials. With 100 grams of malt we have obtained from 3 to 3.5 grams of a white substance having the same activity as 80 grams of the malt used. A new method of preparation of diastase has been re- cently proposed by Wroblewsky, who considers that diastase prepared by the ordinary methods is always found mixed with a pentose, arabinose. The method consists of a par- tial precipitation caused by the action of salts. The author adds, drop by drop, to a solution of amylase, ammonium sulphate until a turbidity is produced in the solu- tion. Then the liquid contains 50 per cent of ammonium sulphate; allowed to stand for some time there is produced a yellowish flaky precipitate, which is separated and washed with a 54 per cent solution of ammonium sulphate. This precipitate is very active: added to a solution of starch, it completely and almost instantaneously transforms it into sugar. According to the author this deposit would be com- posed of pure diastase. To the liquid from which the precipitate has been separ- ated is added again ammonium sulphate up to the amount of 60 per cent ; a new deposit is then produced which, separ- ated, washed, and examined, has been recognized as being a mixture of a pentose, arabinose, and diastase. Finally, in a third operation the second liquid separated from its precipi- tate is again taken, saturated with ammonium sulphate and a new product obtained composed wholly of the pentose. When one wishes to obtain a very active product then, one must take the first precipitate obtained in the solution con- taining 50 per cent of ammonium sulphate. Amylase prepared in this manner is very soluble in water. It is not coagulated by heating, either in a neutral solution,, or after acidification with acetic acid or slight amounts of 106 THE ENZYMES AND THEIR APPLICATIONS. hydrochloric acid. A great addition of hydrochloric acid, however, produces, by heating, a coagulation in the form of light flakes. The solution of amylase, with a certain amount of nitric acid added, gives a light precipitate which is redis- solved in an excess of the reagent. It gives Millon's reac- tion, also the biuret and xanthoproteic reactions. Its solu- tion gives a light precipitate with mercuric chloride. Tannic acid added to the solution of amylase produces a voluminous precipitate soluble in dilute soda. This alkaline solution be- comes somewhat discolored when left to the air at 50 , but does not entirely lose its hydrating power. The amylase obtained by Wroblewsky gave on analysis 16.53 P er cent °f nitrogen. Properties of Amylase. — Amylase is endowed with two distinct properties : it liquefies starch, and transforms starch, as well as dextrin, into maltose. The two properties of this diastase may easily be shown t>y the following experiments: To 100 cubic centimetres of water kept at the boiling point add 10 grams of potato-flour diluted in 20 cubic cen- timetres of tepid water. The mixture forms a thick paste which acquires still more resistance when it is kept for some time in the neighborhood of ioo°. To this starch is added several cubic centimetres of an infusion of malt and the whole is left in a water-bath at a temperature of 70°-75°. The pasty mass quickly becomes fluid, and in a longer or shorter time, according to the diastatic power of the infusion, the starch is transformed into a transparent liquid passing through filter paper. This liquid, of an insipid taste, con- tains dextrins and traces only of sugar. With tincture of iodine it takes a deep-blue color. This solution of dextrins is cooled, a little more of the infusion added, and is left to act at a temperature of 50°-6o°. If samples of this are taken from time to time and analyzed, it is found that the dextrin gradually disappears and there appears in the liquid a re- ducing sugar : maltose. The successive changes which oc- AMYLASE. 10 7 cur in the mixture of dextrins, under the action of the in- fusion of malt, may be easily followed by the aid of tincture of iodine. The deep blue color, obtained in the solution of starch by the iodine, is gradually weakened as the saccharin- cation proceeds. Several hues are obtained in the course of the saccharification. From the deep blue which the starch gives we pass to violet, then to red, then to yellow. Finally, when the saccharification is well advanced, the iodine no longer causes any coloration. The action of the amylase, at the same time saccharifying and liquefying, has raised a doubt as to the individuality of this enzyme. The hypothesis has been suggested that two different enzymes are present, because the two diastatic functions of amylase appear at very different temperatures and because the saccharification and liquefaction are very differently influenced by the chemical and physical conditions of the medium. Still we must discard, at least until it is es- tablished by sure proofs, this interpretation which brings a new complication to the study of amylase. This hypothesis would only be acceptable if one could isolate completely each of the functions of amylase, that is to say obtain two prod- ucts, the one having simply the liquefying power, the other simply the saccharifying power. But this separation has never been made. By keeping the malt infusion at yo° one specially favors the liquefaction, but the product obtained also contains a slight quantity of sugar. If, on the contrary, one maintains the temperature in such a way that saccharifi- cation is favored, that is at 50°-6o°, a slight liquefaction is produced at the same time. Influence of Quantities. — The study of the action of amy- lase leads to conclusions analogous to those furnished by the study of the mode of action of sucrase. It is found in fact if the course of saccharification is followed, that the quantity of sugar formed at the beginning of hydration is propor- tional to the quantity of the enzyme used. Afterwards, when the decomposition of the starch is more advanced, this pro r lo8 THE ENZYMES AND THEIR APPLICATIONS. portionality ceases to exist. The course of the saccharifica- tion when, for the same quantity of starch, increasing quan- tities of amylase are used, is shown by the following table: Quantity of infusion Quantity of maltose of malt used. produced. I CC O. I 3 0.31 5 °-49 10 0.82 15 I-I 20 I.I 30 1.2 By checking the action of the diastase after one hour, in a saccharification made at 50 with different amounts of amy- lase, it is found that with 3 cubic centimetres of an infusion of malt there is obtained fully 3 times as much maltose as with 1 cubic centimetre. If the amount of diastase is still further increased, there is observed an increase in the quan- tity of maltose formed, an increase which is, however, less and less regular. Beyond a certain limit the quantity of the infusion no longer influences the progress of saccharification, even though at that moment not all the dextrins contained in the solution have been transformed. In inversion by sucrase we have observed an analogous course. Still the analogy is not complete. With amylase the proportion is maintained until nearly 40 per cent of the starch is transformed, while with sucrase the proportionality is no longer shown when 15 per cent of the sugar is inverted. Moreover, the slackening at the end of the action is much more pronounced in the case of amylase than in that of su- crase. Influence of Time. — When one studies the effect of time on the progress of saccharification, one finds, as before, that at the beginning of the action a constant ratio exists, and AMYLASE. 109 then a slackening which is more and more marked as the transformation of starch advances. To show the influence of time we cause a slight quantity of diastase to act upon 1 per cent starch ; we take samples from time to time and de- termine the quantity of sugar formed. The following are the results obtained at a temperature of 50 : Numbers of 'he Duration of action Maltose samples. in minutes. produced. 1 15 0-05 gr. 2 30 0.097 3 60 0.21 4 120 0.39 5 240 0.63 6 480 0.82 In the four first samples the quantity of maltose formed is almost proportional to the duration of the action. In the others the proportionality ceases to exist and it is interesting to observe that when the slackening commences there is formed in the liquid nearly 40 per cent of maltose. It is the ratio between the quantities of transformed and non-trans- formed product which influences the progress of hydration. It is this ratio which determines the cessation in the propor- tionality. If, instead of using, as we have just done, a very slight quantity of diastase, we repeat the experiment with double amounts of infusion, while employing the same quantity of starch, we find that the constant ratio ceases after the first hour of action. If the quantity of diastase is still further in- creased, the course of the transformation becomes irregular after some minutes. Influence of Temperature. — By saccharifying starch with infusions of malt at different temperatures for 15 minutes,. Kjeldahl has obtained the following results: Ho THE ENZYMES AND THEIR APPLICATIONS. Temperature. Reducing powers. 18.5 i7-5 35 30.5 54 4i-5 63 42 66.5 34 68 29 70 18 The action of amylase is very slow at o°. Towards 30 saccharification begins to progress rapidly and the activity of the enzyme then increases very rapidly in intensity up to 6o°. Above 6o° the production of maltose diminishes, and at a temperature of 70 , which is the most favorable tem- perature for liquefaction, the quantity of sugar produced be- comes insignificant. Kjeldahl and Bourquelot have observed that amylase, kept for some time at temperatures higher than 6o°, acts differently from a diastase that has not been heated. An infusion of malt kept for 10 minutes at different temperatures and then introduced in starch at 50 produces very different reactions. Infusion of malt t at 63 furnishes 63^ maltose, 37$ dextrin, heated for 10 -j at 68° " 35$ " 65$ " minutes ' at 70° " 17.4$ " 82.6$ " The temperature to which the infusion has previously been brought has then produced a change in the mode of ac- tion of the amylase. The heated diastase causes a decom- position of starch according to equations which differ ac- cording to the temperatures to which the infusion has been, brought. To show the influence of temperature on the progress of saccharification, the following experiment may also be made: Heat an infusion of malt for 12 hours at 68°, then try its fermenting power by comparing it with that of the same infusion not heated. At 50 allow 10 cubic centime- AMYLASE. 1.1 1 tres of non-heated infusion and 10 cubic centimetres of the same infusion previously heated to 68° to act on I per cent starch. In the first case 0.6 of maltose is obtained and in the second 0.3. The diastatic power has then diminished by half. Now the power of the infusion heated to 68° may be tried in starch of different degrees of concentration : 10 c.c. of infusion have given in a 1% starch 0.3 maltose. 2% " 0.6 3% " 0.9 In 2 per cent starch the heated infusion gives a normal result, that is it furnishes the same quantity of sugar as if it had not been heated. In 3 per cent starch the infusion, kept at 68°, furnishes a still larger amount of sugar. The variation in the energy of the infusion, according to the concentration of the starch, becomes more striking if one observes that in the three experiments cited above there are formed quantities of maltose proportional to the amounts of starch contained in the paste. There is always obtained 30 per cent of maltose. The heated infusion then preserves all its properties when it is a question of producing limited decompositions, as long as hydration does not exceed 30 per cent, but this in- fusion cannot produce greater hydration. Workers have sought to explain the difference of action of the heated diastase and that which is not heated by the hypothesis that there exist different kinds of amylase. These different diastases would possess different temperatures of destruction and coagulation and would decompose starch differently. According to this theory, by heating the infusion to 68°, an unfavorable action would be exerted on the dia- es producing complete hydration, that is, giving little dextrin and much sugar, but the diastases performing the opposite work, that is, forming much dextrin and little sugar, would be left intact. The diastases which produce a slight saccharification would, according to this hypothesis, act very favorably when H2 THE ENZYMES AND THEIR APPLICATIONS put in a heated infusion of large quantities of starch. In this particular case it would be found that the height of the temperature produced no change in the diastases. We shall have occasion to return to this hypothesis, but let us say, at this point, that it does not at all accord with the facts which we shall set forth later. Influence of Chemical Agents. — The conditions of the medium influence to a very great degree the action of amy- lase, which shows itself very sensitive towards many chemi- cal substances. For a long time it has been observed that the least change in the reaction of the medium has a visible influence on the progress of saccharification by the diastase. It is generally admitted that diastatic action is favored by very slight amounts of acid and that, by a greater acidity, it is possible to slacken and then arrest completely the progress, of hydration. Kjeldahl was the first to study with exactness the influ- ence of the acidity of the medium. For this experiment he made use of dextrin solutions. In a series of samples of ioo cubic centimetres he added, to the same quantities of in- fusion of malt and of starch, different quantities of sulphuric acid, allowed the saccharification to continue for 20 minutes at a temperature of 59 , and then determined the sugar formed. The influence of the different amounts of acid upon the diastase is shown in the following table : Milligrams of H 2 S0 4 Increase in per 100 c.c. of solution. sugar. 0.44 1 O.47 2 O.49 2-5 , O.48 3 0-43 3-5 o- 2 7 4 0.13 6 0.02 10 0.01 AMYLASE. 113 Thus it is seen that an amount of 1 to 2.5 milligrams of sulphuric acid produces a favorable action, while an amount of 3.5 milligrams causes a slackening which larger amounts accentuate more and more. With 10 milligrams there is ob- tained an almost complete arrest. If one compares sucrase and amylase from the point of view of their sensitiveness towards the reaction of the medium, one finds a very noticeable difference between the two enzymes. We have seen that sucrase produces its maximum effect in an acid medium. On the other hand, the favorable influ- ence exerted by acids is extremely slight for amylase. For the diastase which inverts cane-sugar the natural medium is acid. On the contrary the enzyme producing maltose derives little benefit from a slight acidity, and shows an extraordinary sensitiveness towards larger quantities of" acid. The figures indicated by Kjeldahl must not, however, be considered as constant, for, under conditions other than his, we have found entirely different results. We have taken an infusion of filtered malt and have added to it different quanti- ties of sulphuric acid and hydrochloric acid, then we have de- termined the diastatic power of the infusion before and after acidification. We have thus obtained the following results: ... Milligrams per Diastatic 100 c.c. power. O IOO 2 I08 Sulphuric acid J 3 104 5 100 10 98 f 3 107 Hydrochloric acid .J 5 104 [ IO 97 Now, with 10 milligrams of sulphuric acid, Kjeldahl found an almost complete arrest in the saccharification, while in H4 THE ENZYMES AND THEIR APPLICATIONS. our experiments this amount of acid showed itself almost without effect. One may then conclude that the medium by itself possesses here also' an influence upon the sensitiveness of the enzyme. When a mineral acid is added to an infusion of malt, a part of this acid combines with the bases of the infusion, thus displacing organic acids which, varying in na- ture in different infusions, act more or less energetically on amylase. The action of lactic acid on amylase deserves special at- tention because the infusion of malt, as well as the grain mashes generally, contain this acid. It is proper then, in in- dustrial saccharification, to take account of these factors. We have studied the action of lactic acid under very varied con- ditions, and these experiments have led us to the following conclusions : The effect of a given amount of acid on dia- stase differs according to the duration of the action and also according to the temperature. Acids, furthermore, act dif- ferently upon the saccharifying power and the liquefying- power. The combined influences of time and acids may be shown by the following experiment: To an infusion of malt, fil- tered by. the Chamberland filter, add different quantities of lactic acid, and determine the diastatic power after i hour and after 12 hours. Saccharifying power of the infusion. Lactic acid per ioo c.c. J ° Centigrams. ^~ fter x faoun After M hours _ IO 48 . 42 100 53 24 400 57 21 By leaving the infusion of malt for an hour with 400 cen- tigrams of acid at a temperature of 30 , a perceptible increase of the saccharifying power is observed, while the same amount of acid produces a disastrous effect if the action is al- lowed to be prolonged for 12 hours. The saccharifying power falls off in this case from 57 to 21. AMYLASE. 115 Then the same experiment is repeated, only changing the temperature. The infusion is left at a temperature of 55 for 1 hour and the diastatic power determined. Acid in centigrams. Diastatic power. 10 44 100 41 400 20 The amounts of acid which produced an increase of cMastatic power at 30 act quite otherwise at a temperature of 55 °. At this temperature the 400 centigrams of acid have made the saccharifying power fall off from 40 to 20. The sensitiveness of amylase becomes still more evident if we examine the changes produced in the liquefying power, after 1 and 12 hours of action, with different quantities of acid. T . . , Liquefying power. Lactic acid * >. in centigrams. After I hour. After 12 hours. IO IOO IOO 100 100 50 400 51 20 The changes observed in the liquefying power show us that under the influence of acids the increase in the sacchari- fying power of amylase is made with a parallel reduction of the liquefying power. After 1 hour at 30 , 400 centigrams cause the saccharify- ing power to rise from 48 to 57, but at the same time the liquefying power is found to be reduced almost one-half. We shall see further on, in the chapter devoted to indus- trial applications, that the real value of the diastase exists in its liquefying power and that consequently the rise caused by acid is more apparent than real. Alkaline media are unfavorable to the action of amylase. Still, the diastase can endure a certain alkalinity without n6 THE ENZYMES AND THEIR APPLICATIONS. changing, for, when the alkali is neutralized, the diastase resumes its activity. Sodium carbonate acts upon amylase in extremely small amounts. By adding 5 milligrams of sodium carbonate to 100 cubic centimetres of neutral starch, we have found the diastatic power to diminish almost 20 per cent. With .25 gr. of soda we have obtained only a quarter of the quantity of sugar which the diastase would have given without the addition of alkali. According to the experiment of Duggan, caustic soda in the amount of 2 milligrams produces a disas- trous effect : under its influence amylase loses almost 75 per cent of its activity. Salts also influence diastatic action, by either increasing or decreasing the activity of the amylase. Mercuric chloride in the amount of one-millionth paralyzes its action. Calcium chloride in the amount of one-hundredth dimin- ishes the activity of amylase by half. According to Kjeldahl, the salts of lead, zinc, and iron, as well as alum, check the action of the diastase and their more or less destructive influence may be expressed by the following figures which indicate the proportion between the normal action, expressed by 100, and the action with salts: Potassium nitrate, 10 centigrams 20 Zinc sulphate 20 Ferrous sulphate 20 Alum 3 According to different authors, sodium chloride in the amount of one-half per cent would cause a noticeable slack- ening. Our experiments have not confirmed these data. With commercial salt we have often observed a paralyzing ac- tion, but the same thing has not occurred when we have used chemically pure salt. The checking action of commercial common salt must then be attributed rather to impurities. AMYLASE. 117 Alcohol and most antiseptics must also be placed in the class of inhibiting agents. Salicylic acid, phenol, and formic aldehyde, used in the smallest amounts, act upon the dia- stase. Still we cannot definitely place all antiseptics in the class of destructive substances. Picric acid, for example, as is shown by our experiments, does not act at all as a depressor ; its action is shown rather in the opposite direction. The study of the conditions which may influence the prog- ress of saccharification presents a genuine interest from a practical point of view. It furnishes valuable information to distillers and brewers as well as other manufacturers who utilize the properties of amylase. In seeking to determine conditions favoring diastatic ac- tion, we have attempted to effect the saccharification by the aid of a great number of chemical substances. These experi- ments have not fulfilled all our hopes. We have not suc- ceeded in enhancing the efficiency of a malt by chemical sub- stances, but the results of our experiments throw a special light on the mode of action of amylase and afford a firm basis for analysis of the diastase. We have found that many chemical substances may re- inforce to a very great degree the progress of saccharifica- tion by the diastase. This reinforcing action is, however, of a peculiar nature, and can only be shown under certain con- ditions. To the list of favorable substances belong the salts of vanadium and aluminium, the phosphates, asparagin, albu- minoid substances, and picric acid. To study the action of these different substances on diastatic fermentation we have used two different methods. The diastase has first been put in direct contact with the reagent and then introduced into the starch. After sac- charification the quantity of sugar formed has been meas- ured. In a parallel experiment, made with the same quan- tity of diastase not having undergone the influence of the reagent, the quantity of maltose produced has been deter- n« THE ENZYMES AND THEIR APPLICATIONS. mined and finally the results of the two experiments have been compared. In the second series of experiments the re- agents have been added directly to the starch, in which is then poured the infusion of malt. In our experiments we used an infusion of malt prepared cold with one part of malt and forty parts of water. The starch solution had a density of 1.015. For each experiment we used 1 cubic centimetre of filtered infusion and 100 cubic centimetres of starch. The saccharification took place at 50 for 1 hour. Here are some figures which sum up the influence of the different chemical substances : Maltose per ioo of starch. Without addition 8.63 With 0.7 gr. of ammonium phosphate.. 51.62 0.5 acid calcium phosphate 46.12 0.25 aluminium acetate 62.40 0.25 ammonium alum 56.30 0.25 potassium alum 54-3 2 0.05 asparagin 61.20 By the addition of 50 milligrams of asparagin the sac- charification was nearly 7 times more extensive than in the proof experiments. Aluminium acetate can produce the same effect, but it must be used in a larger quantity. The two methods which we have used have afforded dif- ferent results for calcium phosphate as well as for alum. For the other substances we could find no difference. The results are not changed if instead of an infusion of malt, diastase precipitated by alcohol is used. Neither do they change if the saccharification is effected at different temperatures. It is very evident that the quantities of sugar found vary according to the temperature of saccharification, Imt the difference between the proof experiments and the AMYLASE. 119 experiments made with various substances always remains practically the same. A series of experiments made under widely differing con- ditions has led us to the following conclusions: 1st. Substances which act favorably, act in proportion to their quantity up to a certain maximum amount. Thus, by taking 0.005 of asparagin, we have obtained 25.5 maltose. 0.02 " " 37 " 0.05 " " 61.2 " " 1 gr. " " 61.2 " 2nd. The maximum is not the same for all substances favoring diastatic action. Thus, asparagin and aluminium acetate in maximum quantities may influence diastatic fermentation much more strongly than phosphates. 3rd. The exciting action of chemical substances is mani- fested only in the first phase of hydration of starch ; when saccharification is very advanced it ceases to act. It results from these facts that the same substance may possess a very different activity according to the conditions of the experiment. If amylase is present in a very small proportion in rela- tion to the starch to be transformed, the effect of chemical substances is easy to ascertain. In the opposite case, that is with a greater quantity of amylase, the effect of chemical substances is reduced, and, for a quantity of diastase capable of itself transforming nearly 60 per cent of starch, the ac- celerating substances have no longer any influence on the enzyme. The following experiment shows the influence of aspar- agin with different quantities of infusion. In each of two portions of 100 cubic centimetres of starch paste is introduced 1 cubic centimetre of malt infusion and saccharification is allowed to proceed for an hour at 50 . One of the specimens is saccharified without addition of as- paragin; to the other is added, when the enzyme is intro- duced, 5 centigrams of that substance. 120 THE ENZYMES AND THEIR APPLICATIONS At the end of the time interval, one determines the pro- portion of maltose formed ia the solution of starch submitted to diastatic action. These same experiments are then re- peated under* the same conditions with 10 cubic centimetres of infusion in place of one, and the followiing results are ob- tained : Maltose per ioo. j i c.c. without asparagin 18 " with asparagin 62 10 c.c. without asparagin 79- 2 5 with asparagin 79- 2 5 It appears from this table that in the experiment contain- ing 10 cubic centimetres of infusion, the asparagin no longer acts, although the saccharification must still be far from finished. An analogous result may be obtained, even with a very small quantity of diastase. For this, instead of checking sac- charification after an hour, as was just done, the diastase is left in contact with the starch for twelve hours. Per cent of maltose. f Saccharification, 1 hour at 30 . A -{ 1) 1 c.c. of infusion without asparagin. . 6.4 t 2) " with asparagin 45,0 f Saccharification, 12 hours at 30 . B -^ 1) 1 c.c. without asparagin 74.8 I 2) " with asparagin 74.9 Another characteristic property of substances favoring diastatic fermentation is that they act exclusively on the sac- charifying power, while they never influence the liquefying power of amylase. As the liquefying power exerts its action exclusively on starch and not on dextrins, it must be assumed that the ac- tion of favoring substances is exerted only on the latter bodies. CHAPTER IX. CHEMICAL WORK OF AMYLASE. Chemical work of amylase. — Theories of Payen and Musculus. — Existence of different dextrins. — Theory of Duclaux on the nature of the dif- ferent dextrins. — Preservation of the diastases during saccharification. — Experiments of Effront. When grains of starch are submitted for a short time at a low temperature to the action of amylase, they are very slightly attacked by the diastase. On the contrary, if the action is prolonged, a very complete work is effected; the grains are corroded, pass into solution and are then trans- formed into sugars and related bodies. The action of amy- lase is nevertheless more energetic and much more rapid when it is produced on starch paste. By allowing amylase to act at a suitable temperature on starch paste the solution and saccharification proceed rapidly. The chemical reactions that the diastase cause in the paste, as well as on the grains of starch, may be expressed by the following equation : (C 12 H 20 Oi ) + H 2 = C 12 H 22 11 . Starch. Maltose. This formula shows us that starch, under the influence of amylase, is hydrated and transformed into maltose, but it does not indicate the mechanism of the transformation. In reality the phenomenon is much more complex. In the products of the reaction there are always found dextrins whose presence shows that the reaction was com- plicated by the formation of intermediate products. 122 THE ENZYMES AND THEIR APPLICATIONS. The saccharification of starch by malt has been the object, of much research. However, in spite of the number of re- searches in this field, the complete solution of the problem is far from being found at present. The simplest and also the oldest interpretation of the course of saccharification is that of Payen. According to him, diastase exercises on starch two successive actions : it first transforms it into dextrin, then into maltose. There is first produced an isomeric modification of starch, then a hy- dration of that isomer. According to the interpretation of Payen the transformation of starch into dextrin and into maltose would of necessity be produced not only gradually but regularly, from the beginning to the end of the action. Now, the course of saccharification presents quite a different aspect. We know, in fact, that as hydration proceeds the action of amylase becomes slower and slower. The theory of Payen is then in disagreement with the facts. Further- more, it cannot explain to us the formation during sacchari- fication of dextrins endowed with different properties. According to Musculus, saccharification takes place, not by the successive transformation of starch into dextrin and then into maltose, but by hydration followed by cleavage. This author maintains that the molecule of starch is first hydrated and then decomposed into a molecule of maltose and a molecule of dextrin. 2C 12 -ti 20 O 10 T" xi 2 " — ^12^22^11 "T ^12^20^'l0* Starch. Maltose. Dextrin. In support of this theory Musculus endeavors to establish that the dextrin and sugar formed during saccharification are in a constant ratio. He insists, moreover, that dextrin cannot be attacked by the diastase. These facts do not with- stand criticism. In fact, dextrins can be transformed into sugar by amylase, and the ratio between the quantities of maltose and of dextrins does not remain at all constant dur- CHEMICAL WORK OF AMYLASE. 1 23 ing the transformation. This ratio changes for the same temperature according to the duration of the action and the quantity of diastase used, and also depends upon the tem- perature of saccharification. The ratio between the prod- ucts formed, the maltose and the dextrins, is then neither simple nor constant. The theory of Musculus, based upon observations which are not very exact and reasonings which are not welt founded, has however had unmerited acceptance, and at the present time it still serves as a basis for almost all the theories of saccharification. To reconcile this theory with the data on saccharification actually possessed, a repetition of the two operations, hydration and cleavage, is believed to occur. The starch is supposed to possess a very great molec- ular weight. This complex molecule, by hydrating, is de- composed into maltose and a primary dextrin. This dextrin, of a complicated constitution, then furnishes in its turn a second molecule of maltose and a new dextrin of a molecular weight less than that of the first, and so on. The saccharification is then brought about by giving rise successively to dextrins of smaller and smaller molecular weight. The ideas of Musculus as to the course of saccharification. explaining it as a progressive degradation of the dextrins, were adopted by Brown and Morris as well as by Lihtner. They are, however, far from sharing the opinion of Musculus as to the formation of the intermediate products of the re- action, and as to the molecular weight of the starch and the dextrins. According to other chemists, among the products of sac- charification there are found not only dextrins and maltose, but also substances formed by the combination of these two bodies. Existence of Different Dextrins. — It does not enter into the scope of the present work to discuss all the theories evolved to explain the saccharification of starch. Let us 324 THE ENZYMES AND THEIR APPLICATIONS. confine ourselves to the essential facts underlying these theories, especially the formation, during sacchanfication, of dextrins which act differently towards reagents. To show the difference between the dextrins, one may proceed in the following manner : Treat with alcohol a paste of saccharified starch contain- ing from 10 to 20 parts of sugar for 100 parts of starch. Dis- solve the precipitate obtained, precipitate again with alcohol and repeat this operation a number of times. Thus a prod- uct is secured which contains only traces of sugar. On the other hand, precipitate in the same manner the dextrins contained in a paste of starch in an advanced stage of saccharification and containing nearly 80 parts of maltose ior 100 parts of starch. With the two kinds of dextrins thus obtained and which we will call dextrins A and B, prepare two solutions of the same concentration ; add to them the same quantity of infusion of malt and allow saccharification to proceed at 50 for 2 hours. A measurement of the maltose in the two solutions shows the difference existing between the two dextrins. The dextrin A, extracted from slightly saccharified starch, 5s hydrated very easily, while dextrin B furnishes very little maltose. The two dextrins then differ in their sensitiveness towards amylase. The difference in the nature of the two dextrins can he still further shown by the action of acids. Large amounts •of a mineral acid, acting while warm on the two dextrins, give with both equal quantities of dextrose, but different results are obtained if very slight amounts of acid are used. In this case, very marked differences are observed in the progress of hydration: the dextrins coming from slightly saccharified mashes are transformed under the influence of acids, much more easily than others. Another difference between the two dextrins is shown by the clearly different action exerted upon them by diastases with accelerating substances. Take two specimens of the CHEMICAL WORK OF AMYLASE. 125 solution of dextrin A, to which add equal quantities of malt infusion and to one add also a slight amount of asparagin. By letting the diastase act for some time and then measuring the sugar formed, it is found that the saccharifica- tion is produced in a very different manner in the two cases. The specimen containing the asparagin shows itself much richer in sugar than the specimen lacking the accelerating substance. If the same experiments are now repeated with the dex- trin B, it is ascertained that the progress of saccharification is entirely different. The two experiments, that without asparagin as well as that which contains it, have, after sac- charification, the same quantity of sugar. This proves that dextrin B is not susceptible to the combined actions of amy- lase and asparagin, while the transformation of dextrin A is influenced by the united action of these two substances. The difference in sensitiveness of the dextrins A and B explains to us the irregularity in the progress of saccharifica- tion. It gives us at the same time the reason of the lack of ratio between the quantities of diastase used and of maltose formed. It is the formation, at the end of the reaction, of dextrins of a special nature which produces the slackening in the progress of hydration and which destroys the proportionality between the quantities of active substance and product formed, which exists at the beginning of hydration before the final dextrins are formed. The existence of different dextrins is an argument in favor of the theory which regards saccharification as hydra- tion followed by cleavage. The authors of this hypothesis are, however, wrong in bringing forward, to demonstrate the existence of different dextrins, arguments of little value, and to attribute to these different dextrins properties which they do not have. Thus, according to many authors, dextrins would be dis- tinguished from each other by the difference in their rotatory 126 THE ENZYMES AND THEIR APPLICATIONS. and reducing powers. Now, in reality, these differences do not exist at all. The differences found between the reduc- ing powers and the rotatory powers of different dextrins come solely from the impurities which these dextrins con- tain. They arise specially from the admixture of sugar which is removed with difficulty, even by repeated precipitations with alcohol. To obtain dextrins free from sugar, we submit impure dextrins to an alcoholic fermentation as w T ell as to lactic fer- mentation. The different dextrins obtained by this method possess neither rotatory nor reducing power. Moreover, the characteristic properties of the dextrins A and B prove sufficiently the existence of different bodies of this class and is is not at all necessary to attribute to them still other char- acteristics which they do not possess. The theory of Musculus in its modern form assumes that the dextrins differ in their molecular weight. By employing Raoult's method of freezing it has not been possible to es- tablish with certainty that dextrins obtained after more or less extensive saccharification really possess different molec- ular weight. Lintner and Dull have found for erythro- dextrin a molecular weight of 6000, and for the other dex- trins a molecular weight of 2000. These figures must, how- ever, be taken with a certain reserve, for Lintner and Dull ascertained at the same time, for various dextrins, the exist- ence of rotatory and reducing powers. It is, therefore, to be presumed that their determinations were made with impure products, and that under those conditions they have only a relative value. Theory of Duclaux on the Origin of the Different Dex- trins. — The existence of different dextrins being demon- strated, one may ask from whence they arise and by what mechanism they are produced during the course of sacchari- fication. According to Duclaux it is in the structure of the mole- cules of starch that one must look for the origin of the dif- CHEMICAL WORK OF AMYLASE. 127 ferences found between the various products of its trans- formation. According to this author dextnns differ from each other, not by their chemical structure but by their physical consti- tution. These differences have for cause the structure of the grains of starch, which are composed of non-homoge- neous superposed layers, unevenly compact, and offering a different resistance to physical and chemical agents. This hypothesis, very attractive because of its simplicity, is sup- ported by very weighty arguments. It has been known for a long time that starch acts differently, according to its origin, in the presence of amylase in the cold. Potato-starch is very difficultly attacked, while the starches of barley and wheat saccharify with great ease. This difference, which evidently comes from the more or less compact state of the layers forming the grain of starch, is again met with in the action of this diastase at relatively high temperatures. Lintner, by saccharifying raw starches of different ori- gins, has found that the attack varies considerably in energy according to the origin of the starch. The proportions of starch dissolved at different tempera- tures are here tabulated : Potato Barley Fresh malt , Brewed " . Wheat Rice Maize Rye Temperature of Action. O 12 29 13 6 2 25 5 53 58 56 62 9 52 92 92 91 91 19 18 40 90 96 96 93 94 3i 54 94 Temperature of Gelatimzation 65 80 75-80 80 75 At a temperature of 50 , 12 per cent of barley-starch is dissolved, 2 per cent of corn-starch, and 25 per cent of rye- starch. At a temperature of 6o°, 92 per cent of barley- starch is dissolved and only 18 per cent of corn-starch. The 128 THE ENZYMES AND THEIR APPLICATIONS. quantity of starch which can be dissolved at a given tem- perature, "therefore, depends distinctly upon the origin of the starch. Noticeable differences are also found between the temperatures necessary for the gelatinization of starches of different origin. Potato-starch gelatinizes at 65 °, and the diastase at this temperature dissolves 90 per cent, while the barley-starch, which gelatinizes at much higher tempera- tures, yields at 65 , 96 per cent of dissolved substances. These figures prove that gelatinization does not change the properties of starch which result from the variable de- gree of compactness of the different layers of grains. Really, a granule of starch is irregularly attacked by dia-. stase : corrosion occurs in very different directions and places. This manner of corrosion arises from the inequality of resistance of the surface of the grains, so that the differ- ence existing in the compactness of the various parts of the grains is, on the whole, the initial cause of the variations in resistance to diastatic action. Potato-starch and barley-starch are both composed of non-homogeneous granules differing in the degree of com- pactness of the layers which compose them. In the granules of potato-starch more resisting layers are found than in the granules of barley-starch. Now, we have observed that with different kinds of starch, pastes are ob- tained which saccharify with more or less difficulty. We must then suppose that the difference of compactness be- tween parts of the same granule does not disappear when the starch gelatinizes and that consequently the starch cannot offer an equal resistance in all its parts. The most coherent parts of the granules will form a paste more difficult to liquefy and will then give, even when enter- ing into solution, a dextrin which will offer more resistance. From this point of view, the dextrins do not exist as chemically distinct bodies, but the different constituents of the starch-grains of varying coherence yield more or less re- fractory dextrins. CHEMICAL IVORK OF AMYLASE. 1 29 According to Duclaux, the phenomena occur in the fol- lowing order : By the action of amylase on the paste there is first pro- duced an almost instantaneous liquefaction. There is de- struction of a coagulum, analogous to that observed when a few drops of acid or of ammonium citrate are added to a gelatinous mass of calcium phosphate. Saccharification then commences in the least resistant portion of the paste. This portion is first transformed into dextrin, then into mal- tose, but at the same time other portions of starch are at- tacked and increase the quantities of dextrin and maltose in the solution. When iodine no longer gives color, the starch is com- pletely transformed, but there are still left some dextrins arising from the portions of starch which were least easily attacked. Some of the dextrins are so slow to disappear that they still remain at the end of the operation. Still, these dextrins disappear in their turn and are transformed into maltose if the diastatic action is sufficiently prolonged. When precautions are taken to avoid alteration of the diastase, the course of saccharification conforms to the theory of Payen. The starch is first transformed into dex- trin and then into maltose. The hypothesis of the varying compactness of the con- stituents of the starch-grains has then brought a new sup- port to Payen's theory, which, at first sight, appeared to be in direct contradiction to all the data on saccharification. Deterioration of Diastases by Work. — In the rapid glance which we have just given at the manner of action of amylase, we have been exclusively occupied with a single factor, the progressive transformation of starch, without troubling our- selves about the fate of the acting bodies. The question which arises first of all in considering the active substance is this : When the diastase has carried out a considerable chem- 130 THE ENZYMES AND THEIR APPLICATIONS. ical work, is it still in the same state as at the beginning of its action, and has it preserved its activity? This question has been discussed by different authors. According to some, the diastase undergoes a weakening during the work; others claim that it possesses at the last the same fermenting power as at first. Unfortunately, the two opinions are founded altogether on very questionable general considerations, while the ex- perimental method alone can furnish the true solution of this question, so interesting from a theoretical point of view and so rich in practical consequences. The following experiment gives the solution of the problem: In 200 cubic centimetres of starch paste put 3 cubic cen- timetres of a malt infusion and let saccharification proceed for 4 hours at a temperature of 30 . The volume of liquid thus saccharified is brought up to 300 cubic centimetres, so that 100 cubic centimetres of the liquid contain exactly 1 cubic centimetre of infusion of malt which has already produced a work of saccharification. To see if the work done by the infusion has really caused weakening of the active substances, we compare the fer- menting power of 100 cubic centimetres of this liquid with the fermenting power of 1 cubic centimetre of the original infusion. For this, 100 cubic centimetres of saccharified paste are mixed with 200 cubic centimetres of starch. The mixture is placed in a water-bath at 50 for an hour. This we will call specimen A. We also take a second specimen of 100 cubic centimetres of saccharified paste ; bring it very rapidly up to a tempera- ture of ioo° to destroy the diastase, after which w T e pour it into 200 cubic centimetres of starch with 1 cubic centimetre of fresh infusion added and place it in the water-bath. This will be specimen B. The two saccharifications are then made during the same time, with the same quantities of infusion, but with this dif- CHEMICAL WORK OF AMYLASE. 131 ference, that the infusion of specimen A has already been used once, while the infusion of specimen B has not. The quantities of maltose obtained in a series of parallel experiments are here shown: Specimens. 123 A 1.48 1.31 1.92 B 1.46 1.32 1.92 Thus the quantity of maltose obtained is the same in the specimens A and B. Weakening'does not, therefore, occur, and all the theoretical considerations from which other con- clusions are drawn must be rejected. It is true that in changing the conditions of the experi- ment, exactly opposite results may easily be obtained, but in such cases there is alteration and not weakening of the dia- stase. Thus, by repeating the same experiments, with the same infusion and the same starch and letting the action be pro- longed, not 4 hours at 30 , but only half an hour at 6o° or 65 °, we reach very different results. Temperature 60°. Temperature 68°. A 2.19 maltose 2.00 maltose B 3-^5 " 3-i5 " The differences found between specimens A and B arise here from the action of heat on the diastase and not from weakening. The aqueous solution of amylase, left to a tem- perature of 6o°, loses in fact, as is known, a great part of its diastatic power. CHAPTER X. AMYLASES OF DIFFERENT SOURCES. Different amylases. — Ptyalin. — Diastase of raw grains and diastase of sprouted grains. — Action of translocation diastase on starch. — Reich- ler's diastase. — Mode of action of diastase brought up to a tempera- ture of 70°. — Conditions of secretion of amylase. — Quantitative analy- sis of amylase. — Comparative value. — Absolute value. — Methods of Effront. When we study amylases of different sources from the point of view of their action on starch, we are struck with' certain characteristic peculiarities, which tend to confirm the existence of different kinds of amylase. The authors who have studied this question closely, first of all distinguish the salivary diastase called ptyalin and the diastase of grains. They then discriminate between the amylase of raw grains and that of sprouted grains. The characteristic of the salivary diastase is, according to certain authors, its resistance to the action of alkaline and acid media. This assertion is erroneous. Ptyalin really behaves to- ward the reactions of the medium in exactly the same man- ner as malt diastase. Saliva, in reality, often possesses a very pronounced al- kaline reaction which corresponds to 97 milligrams of bicar- bonate of sodium per 100 c.c. Now, Chittenden and Smith have shown that this alkalin- ity weakens the fermenting power of the diastase, and its power increases in a certain proportion when the saliva is neutralized. 132 AMYLASES OF DIFFERENT SOURCES. 133 The resistance to acids which has been claimed for ptyalin is especially based on the part played by the salivary diastase in digestion, but here also the observations made have not been exact. In fact ptyalin acts only in the first phase of digestion ■when the gastric contents are not yet acid. The reaction of this enzyme is checked when acidity is developed. The distinction between amylase of raw grains and amy- lase of malt appears at first sight to be based on more reliable data. Lintner and Eckhard have ascertained a per- ceptible difference between the action of amylase of sprouted barley and that of the diastase of barley which has not sprouted. At low temperature the diastase of raw grains accomplishes a more complete work than the diastase of malt. At the optimum temperature, on the contrary, which is practically the same for the two diastases, it is the amylase of the sprouted grain which forms the greatest amount of sugar. A still more appreciable difference results from the study of the liquefying power of amylases of different origin. Malt diastase liquefies starch very quickly, while the amy- lase of raw grains, though possessing an energetic sacchari- fying power, shows itself almost inactive as regards liquefac- tion. Lintner and Eckhard have compiled a comparative table of the action of temperatures on the two diastases and have brought forward many distinctive properties, which, they say, characterize the two enzymes. But here, as in the case of ptyalin, the conclusions which the experimenters have drawn from their observations are not well founded. The difference found between an infusion of raw grain and an infusion of malt comes really not from the existence of two distinct diastases, but from the presence of different foreign bodies in the two liquids. In the infusion of raw grain there is very little amylase, but the liquid is very rich in substances favoring diastatic action. 134 THE ENZYMES AND THEIR APPLICATIONS. When we studied the influence of substances which favor diastatic action, we showed that this action is manifested especially in the first phase of hydration and that it ceases in the presence of residuary dextrins. It is for this reason that the low temperature is shown to be favorable to the action of amylase of raw grain and unfavorable to that of malt diastase, which under these conditions produces an in- complete hydration. At the optimum, temperature the conditions are entirely different. The small quantity of diastase contained in raw grain can by itself cause a considerable saccharification which proceeds up to the point where the foreign substances have no more influence on the course of hydration. By using diastase of raw grain one rarely observes such a degree of saccharification that the iodine test for starch is no longer obtained. The same phenomenon is observed when the diastase is used with accelerating substances. To obtain a decomposition of starch corresponding to 40 or 50 per cent of maltose, a very small amount of infusion is sufficient if there is present a body accelerating its action. To reach 70 per cent of maltose it needs 10 to 20 times as much malt infusion, even if the work is done in the presence of asparagin. We shall find later that there are really in raw grains sub- stances which accelerate the diastase. These are the cause of all the differences observed by Lintner between the dia- stase of malt and that of grains which have not sprouted. Brown and Morris also make a distinction between malt amylase and the enzyme of raw grains. They call the first " secretion diastase " and the second " translocation dia- stase." According to these authors, the two diastases act in an entirely different manner on raw starch. Secretion dia- stase corrodes the granules of starch, channels them irreg- ularly and disintegrates them. Translocation diastase, on the contrary, produces neither corrosion nor disintegration. The solution of starch takes place layer by layer; the AMYLASES OF DIt-FERENT SOURCES. I3£ grains retain their original shape, but diminish gradually in size as long as they are visible. This singular difference in the mode of digestion appears at first sight completely to confirm the hypothesis of the existence of different amylases. But this new argument ap- pears much less conclusive if we study with more care the different modes of attack of starch by diastase. According to Krabbe, the attack on the starch is produced in a very dif- ferent manner in different plants. In potato-starch and in that of grains the digestion is accomplished in successive layers; the corrosion is centripetal and uniform. In the case of leguminous plants the amylase produces at the sur- face of the grain fissures which extend towards the center of the grain where they unite, forming a cavity which con- stantly increases. Corrosion then takes place here in two directions; it is first centripetal, then it becomes centrifugal. With grains, on the contrary, the starch is unequally at- tacked ; channels and grooves are formed which extend to- wards the center. These facts show us that the manner of digestion of starch varies in different plants. And, in reality, the manner of action of amylase is very complex, even when it is a ques- tion of the digestion of grains of starch of the same origin. Here also is found a very variable manner of working and it is observed that the grains are not all attacked in the same manner. When cold starch is treated with an infusion of malt, digestion occurs without any regularity. In certain cells the corrosion is accompanied by clefts and holes, in others the attack is made in a regular manner. These dif- ferences evidently come from the compactness and the non- homogeneity of the granules of starch. Moreover, the man- ner of digestion may also be influenced by the reaction of the medium as well as by the presence of foreign bodies. Starch, difficultly dissolved in the cold, easily digests in slightly acid media. As the acid reaction favors but slightly the saccharification of starch, the action observed on whole 136 THE ENZYMES AND THEIR APPLICATIONS. grains can only be explained by the change which the acid produces in the physical state of the grains ; it is probable that this acid reaction favors the contact of the diastase with the starch. The facts announced by Brown and Morris have not, moreover, received sufficient confirmation. It has not yet been demonstrated that two diastases of different origin always act differently on raw starch and, even if this had been proved, one ought not to conclude from it that different amylases exist. The difference in action of various diastases might in fact come from the foreign sub- stances which accompany them. The artificial diastase of Reichler is also cited as consti- tuting another variety. This worker, on digesting gluten in a certain quantity of slightly acidified water, found that the saccharifying power of the liquid gradually increased. The enzyme obtained in this way shows all the properties of diastases of raw grain, and it is admitted that it is formed by the action of the acid on the gluten. According to Lintner the formation of that enzyme is due to a hypothetical substance contained in the gluten, practi- cally a zymogen, which under the action of acids is trans- formed into amylase. Malt amylase is, in fact, present in a very small quantity, and the increase of diastatic power results simply from the change in the medium produced by the action of the acid. Changes Produced in the Activity of Diastases at a Temperature of 70 . — By saccharifying starch with an in- fusion of malt, very different quantities of maltose are ob- tained according to the temperature at which one works. Analogous results are obtained by merely heating the in- fusion to different temperatures. According to O'Sullivan, there is for each temperature a certain degree of hydration of staich which is easily at- tained but cannot be exceeded. AMYLASES OF DIFFERENT SOURCES. 137 The infusion heated to K /would cause a decomposition \* maltose and > dextrin. DO > , , , . . « - I " "2 " _ o 1 ut starch corresponding to J 70 ) ^ & (1 " 5 By keeping the diastase successively at temperatures of 64 , 68°, and 70°, a total change is each time produced in the manner of work, and it may be concluded that either real transformations of the active substance have taken place, or that there is an artificial formation of different types of amy- lase. Our knowledge of the effect of chemical conditions upon diastases leads us to quite another interpretation. The temperature has no other effect than to reduce the diastatic power. The nearer the temperature approaches to 70 , the greater is this reduction. Only, while the diastase is losing its real activity, there is still an apparent activity, owing to foreign substances contained in the infusion, which act with a still greater energy as the diastase becomes weaker. To sum up, we have here a phenomenon which we have already observed concerning the diastase of raw grains, only the action is more complicated in the present case. The diastase, maintained at a temperature of 68° to 70 , has not the same properties as the amylase of raw grains : the saccharifying power has largely disappeared but the liquefy- ing power has not been affected. From this it results that the heated infusion, though act- ing like the diastase of raw grains, differs from the latter in the ease with which it liquefies starch. Condition of the Secretion of Amylase. — After having studied the action of physical and chemical agents on amy- lase, we will briefly discuss the method of secretion of this enzyme, as well as the conditions which favor its produc- tion. In grain in germination, it is the embryo alone which plays an active part; the role of the endosperm is entire! v secondary. J3 8 THE ENZYMES AND THEIR APPLICATIONS. The embryo of grains of barley, detached with care, may jDe transformed, if it is put in a damp place and under suitable conditions of temperature, into a little plant. The vegeta- tion produced under these conditions is very delicate and short-lived, but the germ nevertheless consumes its reserve food and secretes amylase. If the germ is placed on its own endosperm reduced to pulp, the vegetation becomes normal and the course of diastatic secretion may be followed by the chemical transformation which is produced in the amy- laceous matter. By cultivating the germ in different nutri- tive media and under different conditions, very interesting data may be obtained upon the conditions which regulate the secretion of diastase. Brown and Morris, by adopting this method, have made some interesting discoveries upon the influence of different carbohydrates and of the acidity of the medium upon the production of the diastase. By cultivating the same number of embryos, in simple gelatine on the one hand, in gelatine with six thousandths of a part of formic acid added on the other hand, they have found a noteworthy difference in the quantities of diastase secreted. Fifty embryos cultivated in simple neutral gelatine have furnished a quantity of diastase corresponding to o. 118 gr. of oxide of copper. The diastase was found distributed in the following manner: In the germs 0.0708 gr., in the gelatine 0.0478 gr. The 50 germs cultivated in the acidu- lated gelatine produced a quantity of diastase corresponding to 0.145 gr. of oxide of copper. It was distributed as fol- lows: In the germs 0.0904 gr., in the gelatine 0.0546 gr. The acidity of the medium, therefore, clearly favors the secretion of diastase. By adding to gelatine different assim- ilable carbohydrates other than starch, they have found that these substances act very unfavorably on the secretion. The property of secreting diastase is, therefore, not a fundamental property of cells. AMYLASES OF DIFFERENT SOURCES. 139 The appearance of the diastase depends upon the method of nutrition, but let us note, however, that this appearance does not always correspond to the real needs of the cells, and that it must not be considered as an indication of in- telligence of the cells which, by the aid of a diastatic secre- tion, would adapt themselves to different media. A barley germ cultivated in gelatine in which it cannot obtain nutri- tive matters secretes the same quantity of amylase as if it were cultivated in starch. The secretion is always abundant when the germ is found in poor nutritive conditions and it is checked as soon as an assimilable substance appears. Here, as in the case of sucrase which we studied above, the secretion of diastase is a consequence of mal-nutrition, and the primary cause of all the variations observed in the secretion is nothing else than the reaction of the medium. The secretion of amylase, as we have just seen, is favored by acidity of the medium. The degree of acidity of the cel- lular substances, therefore, influences considerably the inten- sity of the secretions. Starting with this statement, w r e can explain why secre- tion is favored where nutriment is lacking. The cells, when they find non-assimilable substances present, consume their reserves, and this consumption produces in their interior a change of composition which favors osmosis. The saline substances of the surrounding medium then penetrate more easily into the cells and, as a result of dissociation, there is produced an accumulation of acids which favor secretion. Analysis of Amylase. — The method used to determine the diastatic power of a solution is based on the following observation of Kjeldahl : As long as the diastase is in the presence of a large amount of non-transformed starch, the quantity of malt pro- duced is proportional to the quantity of diastase contained in the solution: in other words, there is a constant ratio be- tween the quantities of maltose formed and diastase em- J40 THE ENZYMES AND THEIR APPLICATIONS. ployed, as long as the latter acts in the presence of a great quantity of non-transformed starch. This observation has been verified and confirmed by var- ious experimenters, and it is incontestable that by submitting various specimens of the same starch, at the same tempera- ture, to the action of increasing amounts of diastase, quanti- ties of maltose are obtained proportional to the quantities of diastase used. The condition essential to the success of this determination is that in all the specimens a minimum amount of diastase is used, an amount capable of transforming, at the most, 40 to 50 per cent of the starch into sugar. Starting with this principle, it is easy to determine the fermenting power of a liquid. It is sufficient to have a standard diastatic solution of known value and to make com- parative experiments with starch. A 2 per cent solution of soluble starch is generally used. To 100 cubic centimetres of solution containing 2 grams of starch add 2 cubic centimetres of a solution of standard amylase. In another dish also containing 100 cubic cen- timetres of a solution of soluble starch add 2 cubic centime- tres of the solution to be tested. Place the two specimens, in a water-bath at 50 , and, after an hour of saccharification, measure the maltose in the two solutions. The diastatic power of the solution is expressed by the ratio between the quantities of sugar formed with equal quantities of the experimental diastatic solution and the standard solution. If there is found, for example, 0.4 of maltose in the prod- uct saccharified with standard amylase and 0.2 of maltose in the second specimen, we should say that the activity of the solution is 50 per cent, meaning that the solution examined is half as active as the standard liquid. This method of analysis permits a comparison of the value of two products, but it does not permit the absolute ex- pression of the fermenting power of a diastase, because it is very difficult to maintain in a solution of amylase a constant AMYLASES OF DIFFERENT SOURCES. 14* diastatic energy. The results are, therefore, often uncer- tain. To determine absolute diastatic values, we use a method in which we take for unity the quantity of diastase which, acting for an hour at 6o° on i gram of soluble starch, gives 50 centigrams of maltose. The following is the regular procedure: Ten grams of anhydrous, neutral starch are dissolved in 700 cubic centimetres of boiling water. It is cooled and the volume of the solution brought up to 750 c.c. From this solution are taken a series of specimens of 75 cubic centime- tres each. To these specimens are added different quanti- ties of the active liquid to be examined and they are left for an hour in a water-bath at 6o°. The saccharification finished, all the specimens are rapidly brought up to the boiling point, cooled, brought up to 100 cubic centimetres and in each of them the quantity of sugar produced is determined. The specimen in which 50 centigrams of maltose is formed is re- garded as the standard unit. If these 50 centigrams are formed in the specimen to which was added 1 cubic centime- tre of the solution experimented with, we say that the dia- static power of that solution is 100. If these 50 centigrams are found in the tube to which 2 cubic centimetres of the solution were added, we say that the diastatic power is 50, and so on. It is often difficult with a single series of experiments to succeed in producing exactly % gram of maltose. So it is of advantage to make first an approximate experiment with 1, 2, 4, 6, 8, 10 cubic centimetres of active substance. If the unit of diastatic activity is approximated, for example, in the experiment made with 4 cubic centimetres of infusion, we repeat the experiments with 2.5, 2.75, 3, 3.25, 3.50, 3.75 c.c. of liquid. One must also take into account in these experiments the quantity of reducing substances which may be found in the active solution. One must of course subtract from the 142 THE ENZYMES AND THEIR APPLICATIONS. total quantity 'of maltose found after saccharification the quantity of sugar which was introduced with the infusion. This method may also be applied to an analysis of malt. To estimate. the diastatic power of malt we must first ex- tract the active substances. For that we reduce the malt to fine powder, add 20 parts of water and leave it for 6 hours at a temperature of 30 , shaking the solution every quarter of an hour. With the filtered infusion, saccharification is ac- complished as has just been indicated. A malt of excellent quality affords under these conditions an infusion producing 50 centigrams of maltose per cubic centimetre of infusion. Still, this method does not furnish precise data on the value of a malt from a practical point of view. In the chapter treating of the industrial applications of amylase, we shall treat particularly of such analyses. The determination of the saccharifying power of liquids containing slight amounts of amylase often presents great difficulties. To obtain an appreciable quantity of maltose, it is necessary to use a great quantity of solution which often contains reducing materials. In such cases it is better first to precipitate the diastase by alcohol, but this method is applicable only when one has a quite considerable volume of solution at his disposal, for when this precipitation is practiced on a small quantity of in- fusion, a very fine precipitate is obtained which passes thiough the filter and consequently gives rise to perceptible losses. To remedy this difficulty, we have sought to pro- duce in the active solution precipitates which are more voluminous and more easily separated. We have found that tannin leads to this result. In fact our experiments have shown us that this substance completely precipitates the dia- stase and that the inactive precipitate becomes active again when it is carefully treated with a dilute solution of sodium •carbonate. The method of procedure is here given : To 10 cubic centimetres of active liquid add 4 centigrams AMYLASES OF DIFFERENT SOURCES. 143 of tannin dissolved in a few cubic centimetres of water; stir it and leave it for a half-hour. The solution is then filtered and the precipitate, well washed with water and alcohol, is placed, without being separated from the filter, in a glass capsule containing 5 cubic centimetres of sodium carbonate (1 : 10,000). The filter is washed in the solution for one or two minutes; as soon as the precipitate is redissolved add a few drops of a solution of lactic acid (1 : 1000) to neutralize it, and filter. All these manipulations must be made as rapidly as pos- sible because the tannin precipitate changes by a prolonged exposure to the air and becomes insoluble in the alkaline -solution. The contact of the precipitate with the carbonate of sodium must also be of very short duration. When the precipitate does not redissolve except after 4 or 5 minutes of contact, the experiment must be repeated because the dia- stase is already changed. The solution can be greatly facili- tated by triturating the filter in a mortar with the alkaline solution. By working rapidly it is possible to redissolve all the active substances precipitated and avoid all loss. The precipitate obtained by the tannin, washed in water, alcohol, and ether, and then dried, gave on analysis the following figures, a deduction being made of 2,2.2% of tannin : Water 5.53% Nitrogen 8.83 Ash 1.32 This method is of special use when one wishes to deter- mine amylase in vegetable cells. In analyses of this kind the substances are reduced to powder. They are left to soak in 1 to 2 parts of water for 6 hours; the liquid is expressed from the substances not dis- solved. The residue is again soaked with a volume or two of water and expressed a second time. The combined liquids are filtered and the diastase is precipitated from the mixture by tannin, in the same way as with the malt infusion. 144 THE ENZYMES AND THEIR APPLICATIONS. The activity of the precipitate dissolved in the water gives an idea of the diastatic value of the substances sub- mitted to examination. Here, for 'example, is an analysis of bean-leaves: Ten grams of ■ bean-leaves are reduced to a paste in a mortar. Ten cubic centimetres of water is added to the mass and a few drops of chloroform, then it is allowed to stand for 6 hours. The leaves are then pressed and filtered in a cloth. To the residue is then added 10 cubic centimetres of water and a small drop of chloroform, after which the whole is left quiet for three hours. Then the liquid is separated and the residue washed again with water; the liquids of the two macerations and of the washing are combined, and the total volume made up to 50 cubic centimetres. It is again filtered and precipitated with 16 cubic centimetres of tannin. The precipitate is redissolved in alkaline water and the solution is made up to 10 cubic centimetres. It is found necessary to use 2 cubic centimetres of this solution to produce 50 milli- grams of maltose. Therefore, the solution has a diastatic power of 50. If we compare the diastatic power of bean- leaves with that of a good quality of malt we shall obtain the following- results : Ten grams of malt furnish 200 cubic cen- timetres of infusion, one centimetre of which produces 50 milligrams of maltose. Ten grams of bean-leaves furnish 10 cubic centimetres of liquid of which 2 cubic centimetres are needed to furnish 50 milligrams of maltose. Since the malt has a diastatic power of 100, the bean-leaves have one of 2.5. The malt contains consequently 40 times as much active sub- stance as the bean-leaves. BIBLIOGRAPHY. Sig. Kirchoff. — Ueber die Zuckerbildung beim Malzen des Getreides. Schweiggers Journal, 1815, p. 389. Dubrunfaut. — Memoire sur la sacchariflcation des fecules, 2 e edition, Gau- thier-Villars, Paris, 1882. Guerin Varry. — Memoire concernant Taction de la diastase sur l'amidon de pomme de terre. Ann. de chimie et de phys., 1835, p. 32. AMYLASES OF DIFFERENT SOURCES. M5 Leuchs. — Ueber die Verzuckerung des Starkemehles durch Speichel. Kastners Archiv. fur die Ges. Naturlehre, 1831. Biot. — Memoire sur l'amidon. Ann. des Sciences nat., 1838, p. 5. Clement Desormes. — Ann. de chimie et phys., IV, p. 473. Payen et Persoz. — Memoire sur la diastase et les principaux produits de sa reaction. Ann. de chimie et phys., 1833. Miahle. — De Taction de la salive sur l'amidon. Comptes Rendus, XX, 1845, p. 1485. De la digestion et de Tassimilation des matieres sucrees. Comptes Rendus, 1845, p. 954, t. XX. Musculus. — Sur la transformation de la matiere amylacee en glucose et en dextrine. Ann. de chimie et de phys., i860, LX, p. 203. Bouchardat and Sandras. — Des fonctions du pancreas et de son influence sur la digestion des fecules. Comptes Rendus, 1845, XX, p. 1085. O'Sullivan. — Sur le produit de transformation des amidons. Journ. of the Chemical Society, 1872-1874. Kossmann. — Recherches chimiques sur les ferments contenus dans les vegetaux. Bull, de la Soc. chim. de Paris, 1877. Baranetsky.— Die Starke umwandelnder Fermente, 1878. Ch. Richet. — Du sue gastrique chez l'homme et les animaux. These, Paris, 1878. Kjeldahl. — Recherches sur le ferment producteur du sucre. Comptes Rendus des trav. du laboratoire de Carlsberg, 1879. Brown and Heron. — Beitrage zur Geschichte der Starke und der Ver- wendung derselben. Liebig's Annalen, 1879. Brown and Morris. — Journal of the Chem. Soc, 1890. J. Lintner. — Studien iiber Diastase. Journ. f. prakt. Chemie, 1886, p. 378. Ueber das diastatische Ferment des ungekeimten Weizen. Zeit. fur das ges. Brauwesen, 1888. Em. Bourquelot. — Sur la separation et le dosage du glycogene dans les tissus. Journal des connaissances med., 1884. Chittenden and Smith. — The diastatic action of saliva as modified by various conditions, studied quantitatively. Chemical News, 1886. Brown and Morris. — Untersuchung iiber die Keimung einiger Graser. Zeit. fur das gesammte Brauwesen, 1890. Moritz and Glandening. — Sur Taction de la diastase. The Chemical Society, 1892. Lintner and Dull. — Ueber den Abbau der Starke unter dem Einfluss der Diastasewirkung. Berichte der deutschen chem. Gesellsch., 1893, p. 2533- Sohiffer. — Sur les produits incristallisables de Taction de la diastase sur l'amidon. Moniteur scientifique, 1893, p. 712. J. V. Egoroff. — Sur la diastase des grains crus. Journal de la Soc. de chimie et phys. Saint-Petersbourg, 1893, t. XXV. Effront. — Contribution a Tetude de la saccharification. Moniteur scien- tifique. 146 THE ENZYMES AND THEIR APPLICATIONS. Effront. — Actions des acides mineraux dans la saccharification par le malt. Moniteur scientifique, 1890. Sur les conditions chimiques de Taction des diastases. Comptes Ren- dus, 1892, p. 1324. ■ Sur l'amylase. Comptes Rendus, 1895. L'Influence des antiseptiques sur les ferments. Moniteur scien- tifique, 1894. Contribution a l'etude de l'amylase. Moniteur scientifique, 1895, VIII, p. 54i; X, 711. Henri Pottevin. — Sur la saccharification de l'amidon par l'amylase du malt. Comptes Rendus, 1898, p. 17. Duclaux. — Sur la saccharification. Ann. de l'lnst.. Pasteur, 1895, 56. • Les theories de la saccharification. Ann. de l'lnst. Pasteur, 1895, 170.' ■ Amidon, dextrine et maltose. Ann. de l'lnst. Pasteur, 1895, p. 215. Brown and Morris. — Einwirkung der diastase auf Starke. Berichte der deutschen chemischen Gesellschaft, 1895, p. 642. H. Seyffert. — Untersuchungen fiber Gerste und Malz Diastase. Zeit- schrift fur das gesammte Brauwesen, 1898. A. Wroblewsky. — Ueber die chemischen Eigenschaften der Diastase und fiber das Vorkommen eines Arabans in der Diastase Preparaten. Berichte der deutschen chem. Gesellsch., 1897, 2, p. 2289; 1897, 3, p. 3048. Osborne and Campbell. — Wirkung der Diastase bei fortschreitender Keimung. Berichte, 1896, p. 1159, Journal Amer. Chem. Soc, 18, p. 536-542. O. Nass and Framm. — Bemerkungen zur Glycolyse. Pflug. Archiv, 63, p. 203-208. A. Sing and Baker. — Journal Chem. Soc, 67, p. 702-708. CHAPTER XI. INDUSTRIAL APPLICATIONS OF AMYLASE. Malting. — Chemical transformations which accompany germination. — ^ Methods of malting, sorting, steeping, germination, brewing. Amylase is formed in considerable quantities in the grains of cereals during germination. It is for this reason that the industries which utilize diastase as an agent of hydration make use of sprouted grain' called malt, a product which at the present time is the "only agent of that kind capable of being manufactured economically. All cereals produce amy- lase during germination, but barley furnishes the greatest yields in active substance. When one heaps up barley pre- viously soaked, one observes a series of phenomena which, all together, characterize germination. First is found an in- crease in temperature, an absorption of oxygen, and a libera- tion of carbonic anhydride, which is augmented as the tem- perature of the mass increases. Along with this phenom- enon of respiration are observed considerable changes in the various constituents of the grain. The reserve materials, cellulose, starch, protein matters and fatty substances as well as sugars are partially transformed by hydration. These transformations are due to a secretion of enzymes acting on the albumen and transforming it into assimilable substances, which are in part absorbed by the embryos in the course of their development. After 24 or 48 hours of germination, there are seen to appear, on the outside of the grains, little roots which then grow quite rapidly. The development of the plumule is much 147 14§ THE ENZYMES AND THEIR APPLICATIONS. slower. After 8 or 10 days of germination, the length of the plumule reaches half or three quarters that of the grain. It is at this phase of development that germination is generally considered as ended. The development of the germ is largely at the expense of the starch. Under normal conditions the expenditure in amylaceous materials is 8 to 10 per cent of the starch con- tained in the grains, but this proportion is considerably ex- ceeded when germination is accomplished at a temperature higher than 20 . During germination the grain secretes, besides amylase, other active substances, among them peptase, which trans- forms albuminoid substances into amides, and cytase which acts on certain kinds of cellulose. The role of cytase is very important from the point of view of malting. The starch in the grains is in the form of granules enclosed within resistant cell-walls. These cell-walls pro- tect the starch against the action of the amylase, and the at- tack on the carbohydrate would not be very strong without the intervention of the cytase which disintegrates the envelope of the granules. The action on the starch, during germination, is accom- plished in two successive phases. In the first phase the cel- lulose envelopes of the starch cells are liquefied by cytase, and it is then only that the amylase begins to act on the starch. To the action of cytase must also be attributed the differences found between the starch of raw grains and the starch of malt. On account of the destruction of the membrane of the cell the malt-starch is liquefied at a lower temperature than the starch of raw grains. When germination is produced at a temperature of 15-17 , the secretion of amylase commences after 35 or 40 hours and the diastatic power then gradually increases for 8 or 10 days. INDUSTRIAL APPLICATIONS OF AMYLASE. 149 In the practice of malting the grains are submitted to a succession of operations. The first part of the work consists in sorting and cleans- ing the grains. Then they are soaked, then allowed to ger- minate. The germinated grains are utilized in a fresh state in distilleries as well as in the manufacture of maltose; for the purposes of the brewery the sprouted grains are malted. Without entering into all the details of these different manipulations, let us consider the principal points. The sorting of the grains is done in special apparatus which eliminates foreign substances as well as broken grains. Moreover, this apparatus separates the grains according to their dimensions. The grain intended for germination must not be too fresh. Grain taken immediately after the harvest has a small germinating power. It is only after some time that it be- comes good for germination. Grain coming from different harvests must not be mixed, nor grain having different den- sities. To obtain a good germination it is really indispen- sable that the grains should be as far as possible of a uniform weight. In sorting, the grains are separated according to their sizes. Grains of different sizes could not be put together in germination because they would soak unequally. Grains of a different weight are not suitable for the same use. Heavy grains are preferable for use in the brewery, while light grains, containing less starch and furnishing a much greater yield in diastase, are suitable rather for the work of the distillery. The sorted barley is then steeped. This operation is generally carried on in special vats which easily permit of changing the water. The aim of the soaking is to make the grains absorb the quantity of water necessary for a good ger- mination. The grains, in contact with water, swell, absorb a certain amount of oxygen, and undergo different modifica- tions. 150 THE ENZYMES AND THEIR APPLICATIONS. They also lose a part of their soluble substances, espe- cially salts and carbohydrates other than starch. The loss in extractive substances varies from 0.8 to 1 per cent. The elimination of sugar by the soaking of the grains is very favorable to the secretion of the enzymes during ger- mination. In ordinary water, there is not sufficient oxygen for normal germination, so it is advisable to pass a current of air into the mass during the soaking. The soaking water must be frequently renewed so that" the dissolved substances shall not enter into fermentation. Generally, the grains are washed in water before soaking them to free them from germs and ferments which may adhere to their surface. The grains are left in water for 3 to 5 days, and the water is carefully renewed every 12 or 24 hours. The duration of the soaking' depends upon many factors ; it depends on the temperature as well as the quality of the water, but it de- pends especially on the quality of the grains. Thick-glumed barley absorbs water more slowly than thin-glumed grain. The operation of soaking the grains may be considered as ended when they have absorbed nearly 50 per cent of water. By prolonging the soaking the grains would absorb a still greater quantity of water, but in that case germination would be less regular and there would be danger of getting mouldy malts. It is very difficult to stop the soaking at exactly the right point. This difficulty comes from the dif- ferences in the grains employed. It is, therefore, ad- visable to stop the process before the grains are sufficiently soaked. The danger of soaking too long is especially great when rye is used. The grains having had too long a soak- ing become sticky, acquire a pasty aspect, and the malt they furnish is of a doubtful quality. The grains, after having been soaked, are carried to the malt-house, where they are spread in layers of 30 to 80 cen- timetres in depth, according to the kind of malt-house and INDUSTRIAL APPLICATIONS OF AMYLASE. I5 1 the manner of aeration. Malt-houses must conform to the two following conditions : They must be (1) well ventilated, and (2) capable of being kept at a constant temperature. The heaped-up grains heat quite rapidly. The oxidation of starch and fatty materials frees a quantity of heat sufficient to bring the entire mass to a temperature of ioo°. It is then necessary to avoid raising the temperature. This is accom- plished, either by frequently changing the position of the grains, or by spreading them in thinner and thinner layers as the action becomes more energetic. In the system called " pneumatic " the layers are cooled by a current of moist air. Germination lasts from 8 to 10 days. It is desirable to work always at the lowest possible temperature. Generally the germination is begun at a temperature of 10-11 and is continued up to 17-18 , which limit is not passed. When the malt is spread on a cement floor, the layers are made at the beginning of 40 to 50 centimetres in thickness, and then are progressively made thinner. The fourth day a thickness of 10 to 12 centimetres is reached. In the pneu- matic system the depth of the layers remains constant, but the grain is often turned over to prevent the little roots from tangling. During germination the moisture of the grains constantly diminishes and at the end of the operation they have lost from 50 to 60 per cent of the water which they have ab- sorbed during the soaking. It often happens that the water absorbed during soaking is insufficient to ensure germina- tion. In this case the layers must be sprinkled from the third or fourth day. The sprinkling is done systematically in small quantities and at frequent intervals. Generally the germination is checked when the length of the plumules reaches half or three quarters that of the grains. It is generally assumed that at this time the grains con- tain the greatest quantity of active substances. In reality it is not so. The researches which we have made in this sub- J 52 THE ENZYMES AND THEIR APPLICATIONS. ject show that one cannot trust to the length of the plumules to determine the time when the quantity of diastase con- tained in the grains reaches its maximum, and that it is only analysis which can show when germination should be checked. The following table traces the course of germination, at i2°-iy°, of four different malts conducted under the same conditions. At the beginnin 1 day 2 days 3 " 4 " 5 " 6 " 7 " 8 " io " Diastatic Power. 41 50 60 60 70 81 85 95 100 96 60 70 95 95 97 95 98 100 100 100 52 70 80 81 85 87 88 86 89 85 35 40 57 62 80 85 97 100 94 These experiments, as well as a very great number of ob- servations made in different manufactories, have led us to the following conclusion : It is when the malt possesses plumules twice as long as the grains that the diastatic power reaches its maximum; however, in some cases the maximum is not reached at this time. The quantity of diastase contained in the grain increases gradually in the course of germination ; but often reaches its maximum before the plumules have reached the length in- dicated above. The quantity of diastase developed in the malt often re- mains stationary for a certain time. In other cases, on the contrary, a very rapid diminution of the quantity of diastase is observed. This diminution may, in fact, be observed in the table reproduced above. We have sought the cause of this decrease and have found that it INDUSTRIAL APPLICATIONS OF AMYLASE. 153 comes from the energetic oxidation which occurs when ger- mination is very far advanced. It is really in the pneumatic malt-houses that diminution of diastase is most frequently found, while in common malt-houses the alteration of the diastase is much rarer. It may be that in addition to the oxygen of the air other factors also come into play to produce the diminution of the diastatic power of the malt. When a very active malt is desired, it is indispensable that it shall be analyzed after the eighth or ninth day, and the variations of its diastatic power observed twice a day. It is only in this way that losses of diastase can be avoided. In the brewery, fresh malt cannot be employed. To make it suitable for the manufacture of beer it must pass through the malt-kiln where, under the influence of a high temperature, certain principles contained in the grains un- dergo transformations which give to the malt a characteris- tic flavor, as well as a more or less dark color. The drying is done by the aid of hot air and, according to the kind of malt which it is proposed to make, the drying is accomplished at higher or lower temperatures. The fundamental principle of malting consists in raising" the temperature gradually, especially at the beginning of drying. Although the grain contains from 10 to 12 per cent of water, it is extremely dangerous to go above a temperature of 50 . In fact the malt diastase changes under the action of heat and this change is the more rapid as the grain con- tains a greater quantity of water. The grains dehydrated below 50 may then be brought up to a temperature of ioo° without completely destroying; the diastase. The highest temperature reached during the malting is from 103 to 104 for malt of the Munich type and only from 62 ° to 63° for malt of the Pilsen type. Drying always destroys a part of the diastase, even when all possible precautions are taken. In drying the malt at the 154 THE ENZYMES AND THEIR APPLICATIONS. maximum temperature of 50 and avoiding raising the tem- perature at the beginning, we have found that nearly 20 per cent of active substances are destroyed during the process. The loss is hence seen to be considerable. Finally, there exists a great difference between the dis- tillery malt and brewery malt. As we have said above, it is well to choose for brewing- malt, grain which is very heavy and very rich in starch. For the distillery, on the other hand, light grain which furnishes more diastase is to be preferred. The germination of brew- ing-malt should be arrested when the plumules have acquired half or three quarters the length of the grains. When, how- ever, it is a question of distillery malt one should allow the plumule to grow as long as possible. Brewery malt may be aerated up to the last moment, while for distillery malt, aeration should cease during the last two or three days. Finally, there is a great difference in the drying of malt according as it is destined for the brewery or the distillery. For the distillery the temperature must be the lowest pos- sible, while for the brewery it must be quite high. BIBLIOGRAPHY. Moritz and Morris. — Handb. d. Brauwissenschaft. Prior. — Chemie und Physiologie des Maizes und des Bieres. • CHAPTER XII. ROLE OF AMYLASE IN THE BREWERY. The brewing industry was at first carried on by following empirical methods, and it is only within thirty years that the manufacture of beer has drawn the attention of investigators. The works of Pasteur, Dubrunfaut, and Hansen have brought to this domain valuable data which form at the present time the scientific basis of this industry. The re- searches of these workers have brought about noticeable im- provements in the methods of manufacturing beer. It must be recognized that at the present time empirical methods have not entirely disappeared from the business of the brewery, and that science cannot yet explain all the phenomena observed in the manufacture of beer. To carry out this manufacture successfully, it still requires more prac- tice than science. The brewery uses as raw materials malt, hops, water, and yeast. With these simple materials an almost infinite variety of fermented beverages is made. The variations in beers come, in the first place, from differences in the quality of the raw materials. Brewing-malt is far from being a substance of constant composition. It varies according to the origin and quality of the barley, and also according to the method of malting employed. The same is true of the other factors which enter into the manufacture of beer. In fact, different yeasts act very differently in the same mash and give very different results. 155 I5 6 THE ENZYMES AND THEIR APPLICATIONS. The difference in the character of the beers may also be influenced by the quality of the water or that of the hops. The taste and the appearance of the fermented product may also change on account of the intervention of bacteria and of foreign yeasts. All these causes undoubtedly influ- ence the manufacture, but variation in raw materials does not explain all the differences observed among fermented beverages. The character of a beer depends in reality on a number of factors : on the method of work, the manner in which the malting and brewing are conducted, the methods of extrac- tion and saccharification, as well as the mode of fermenta- tion. As may be seen, brewing is an exceedingly complicated industry. To understand the process, a very complete scien- tific knowledge is necessary, and even then one often finds problems which have not been scientifically solved. Fortu- nately, the brewer solves the difficulty by observation as well as by the routine he has acquired. Malt is generally very ricfL in enzymes and the amylase it contains can hydrate 10 to 20 times as much amylaceous matter as the malt contains. Liquefaction and saccharification take place without s diffi- culty when there is a great deal of diastase. If complete sac- charification were the sole object sought, the problem would be easily solved. But, in reality, the brewer has in view, not merely a complete transformation of starch into sugar; indeed, he often wishes to prevent complete saccharification. In fact, it is especially important to him to succeed in a par- ticular decomposition of starch and to obtain certain dex- trins which resist the action of yeasts. He often desires the production of difficultly fermentable sugars which remain in- tact during the principal fermentation and come into play only in the after fermentation. The method of decomposition of the starch influences to a great degree the character of the beer, and, according to the ROLE OF AMYLASE IN THE BRELVERY. 157 type of beer that the brewer proposes to produce, more or less dextrins and easily fermentable sugars must be formed. Under these conditions, the presence of a large quantity of diastase is undesirable rather than useful. For this reason the brewer, even before he could have known the scientific reasons, always sought conditions which hinder saccharifica- tion and the action of an excess of amylase. Thus in drying, the formation of dextrins is favored, and by saccharification at a high temperature the excess of enzymes is destroyed. The influence of the temperature of saccharification on the quantities of maltose and dextrins formed is shown in the following table from Petit, which shows the quantities of mal- tose and of dextrins formed at different temperatures as well as the relation between these quantities. Temperature of Saccharification. 6o-6l° 65-66. 68-69. 72-73- Maltose. 72 71-4 44-7 24.7 Ratio. 30 1 10.4 31.8 1 -.0.44 57 1 : 1.27 76-3 1 : 3 We have said above that the methods of saccharification and of drying influence, not only the quantity and nature of the dextrins, but also the nature of the sugar. In fact, by saccharifying starch under certain condi- tions, combinations of maltose and dextrins are procured which act differently from maltose and dextrins alone. Thus, when a beer-wort is left to the action of yeast, it is found that the liquid still contains, when fermentation is fin- ished, a certain quantity of maltose. The non-fermentation of the remaining sugar is not at all due to the exhaustion of the yeasts, as one might think at first. Hence it is that the addition of pure maltose to the fermented wort induces a new fermenation which exhausts the added sugar, while the sugar remaining in the wort is hardly attacked by the yeasts during the new fermentation. 158 THE ENZYMES AND THEIR APPLICATIONS. To explain this fact, it is supposed that maltose can form combinations with dextrins, which are called malto-dextrins. These bodies have not been isolated in a pure state and their chemical individuality is far from being demonstrated. Yet it is beyond doubt that a considerable difference exists in the fermentability of the various sugars obtained by saccharify- ing starch by malt under different conditions. This difference may be attributed either to the actual existence of different maltoses having different geometrical structures, or to the formation of more or less stable com- binations of maltose and dextrins. The authors who have studied especially the decomposi- tion of starch by malt generally assume the existence of differ- ent types of malto-dextrins which are characterized by the rel- ative quantities of maltose and dextrins which they contain. Malto-dextrins containing a great quantity of maltose are called malto-dextrins of a low type; while malto-dex- trins containing dextrins in large quantity and little maltose are of a high type. The dextrin contained in malto-dextrins is transformed by diastase at temperatures higher than 55 , while above 63 malto-dextrins remain unattacked. Beer yeast decomposes these combinations into fermentable materials and dextrins. This decomposition is always produced more or less slowly according as the yeast acts on a low or high type of malto- dextrin. The formation of combinations of maltose and dextrins depends on the temperature of saccharification. By the ac- tion of the diastase below 50 maltose and free dextrins are formed without malto-dextrins. By allowing the diastase to act between 55 and 62 , the appearance of maltose com- bined with dextrins is observed and the malto-dextrins in- crease considerably when this temperature is exceeded. The composition of wort, from the point of view of its amount of maltose combined with dextrins, may consequently be regu- lated by the choice of the temperature of saccharification. ROLE OF AMYLASE IN THE BREIVERY. 159 According to Petit, there is obtained, with the same malt successively saccharified at 6o°, 65 , and 69°, the following" respective quantities of malto-dextrins : Temperature 6o° 65° 69° Malto-dextrins 2.4% 6.6% 16.2% Temperature, while influencing the formation of malto- dextrins, does not greatly influence the kind of malto-dex- trins transformed. Thus the temperatures comprised between 6o° and 65 ° all produce the same type, and it is only at a temperature of 69 that one succeeds in appreciably increasing the amount of dextrin in the malto-dextrins formed. The temperature of malting also has a manifest influence on the course of the hydration of the starch. Brown and Morris, by analyzing worts obtained with four malts prepared at different temperatures in ascending series, have found the following figures. Experiments. I 2 3 4 47 45 34 17 4-25 7-9 14.9 22.4 1 :o.5 1 : 1.5 1 : 2 1 : 2 Diastatic power Per cent of malto-dextrins Type of malto-dextrins obtained ... As is seen, the temperature of drying acts both on the quantity and the nature of the malto-dextrins. The malt containing the least diastase furnishes both the maximum of combined maltose and the highest type of malto-dextrin. The qualities and the properties of the beer are influenced to a great degree by the quantity and the type of malto-dextrins formed during manufacture. These substances exert an in- fluence on the attenuation and the taste, as well as the pres- ervation of the beer. We cannot in the present volume describe the different methods of brewing and we prefer to refer the reader to special works. Let us only remark that by modifying the mariner of hydration of the starch, beers of different kinds 160 THE ENZYMES AND THEIR APPLICATIONS. are produced. In fact, the method of conducting the brew- ing influences to a great degree the composition of the wort which in its turn acts on the quality and type of the beer. Even before the decomposition of starch had been ex- plained theoretically brewers understood the conditions necessary to procure a wort having the qualities required in each case. When the brewer proposed to make beers of great attenuation and rich in alcohol, he found it necessary to effect the brewing in such a way as to avoid the formation of great quantities of malto-dextrins. When it was a ques- tion, on the contrary, of a beer of low fermentation followed by a prolonged secondary fermentation, he sought to obtain a great quantity of malto-dextrins of a very high type. For top-fermentation beers the manner of conducting saccharification also depends upon the degree of density of the wort. Wort intended for the manufacture of light beers is generally completely saccharified, while for strong beers,, on the contrary, it is sought to produce dextrins in much larger proportion. It is then by drying at a suitable temperature and by the duration of saccharification that one succeeds in producing worts of .very different compositions, while using the same primary materials. BIBLIOGRAPHY. Carl Lintner. — Lehrbuch der Bierbrauerei. Verlag von Friedrich Wieweg und Sohn, Braunschweig. P. Petit. — La biere et l'industrie de la brasserie. Paris, 1896. Wilhelm Windisch. — Das chemische Laboratorium des Brauers. Berlin. Paul Parey. Paul Lindner. — Mikroskopische Betriebskontroll in den Garungsgewer- ben. Paul Parey, Berlin. CHAPTER XIII. MANUFACTURE OF MALTOSE. By the action of malt on starch one can obtain accord- ing to the duration of the process and the temperature which is employed, a series of products differing in the degree of hydration. By saccharifying a paste containing from 5 to 7 per cent of starch with an infusion of malt at a temperature of 40°-45°, an almost complete transformation of starch into maltose is obtained after 12 to 15 hours. The saccharin liquid, evaporated to the consistency of 40°-42° Baume, forms a white crystalline mass containing only 1 to 2 parts of dextrin to 100 parts of sugar. A product of an entirely dif- ferent nature is obtained by saccharification at 6o°-62°. If the duration of saccharification is limited to 30 or 60 minutes and the work is performed with an excess of diastase, a strongly saccharified syrup is obtained containing from 20 to 25 parts of dextrin for 100 of sugar. By saccharification at 68° products are obtained which possess only 60 per cent of maltose. These different products owe their industrial applications to the work of Dubrunfaut and Cuisenier. These workers made a thorough study of saccharification by malt, and they projected an industrial process which ap- pears to have a very great future in store for it. Dubrunfaut, in promoting the manufacture of maltose, hoped that the different products of saccharification would find many applications in different industries. He believed that pure maltose could replace with advantage cane-sugar 161 1 62 THE ENZYMES AND THEIR APPLICATIONS. in wine-making and the manufacture of liqueurs. The sac- charified syrup was to have a place in all the industries which made use of glucose, for example in pastry, in preparation of preserves, bon-bons, etc. Products containing a large pro- portion of dextrin would be especially useful in the brewery where they would replace a great part of the malt. The expectations of Dubrunfaut have not been complete- ly realized. The maltose industry had a great development at a cer- tain period. Manufactories were started in France, Belgium, Holland, and England, and the production of this sugar reached very large proportions. Of late years, however, this industry, for various reasons, has undergone a considerable falling off". Nevertheless, the state of the maltose industry does not warrant the prediction that it is destined to disappear. The advantages which saccharification by malt afford over saccharification by acids are unquestionable, and we are absolutely certain that eventually this industry will replace glucose manufacture. As the industrial preparation of maltose is very little known, we will give here some information concerning its technique. Potato-flour, rice-flour or maize-flour is used as the source of the product. From an economic point of view maize is the raw material offering the most advantages. Un- fortunately the handling of this cereal presents great difficul- ties as to filtering and decoloration of the syrup. To pro- cure products of a good appearance and to obtain satisfac- tory yields it is necessary to have strictly constant conditions. The successive operations are as follows : ist. Grinding. 2nd. Drying. 3rd. Saccharification. 4th. Filtration. 5th. Clarification. MANUFACTURE OF MALTOSE. 163 6th. Second filtration. 7th. Evaporation. 8th. Second clarification. 9th. Evaporation at 40 . The maize, coarsely ground, is introduced into a horizon- tal receptacle furnished on the inside with a paddle. Each cooker receives 750 kilograms of meal and enough water to give after cooking 45 hectolitres of liquid. The pressure is quickly raised while the mass is agitated, and remains 40 minutes at 3 atmospheres. As it takes about 40 minutes to arrive at this pressure, the cooking is ended after about 80 minutes. The cooked maize is sent into a second horizontal recep- tacle furnished with a double wall, a Bohm crusher, and a paddle. A small amount of malt is added at a temperature of 7°°~7S°> an< J in 5 or 10 minutes liquefaction of the mass takes place. Then it is cooled by the water jacket, the rest of the malt is added at a temperature of 65°, it is left about 20 minutes at this temperature, heated again to 70 , and the mass filter-pressed. For the manufacture of syrup contain- ing dextrin saccharification is prolonged for 1 hour at 68°. Malt-sugar syrup requires for its manufacture 25 per cent of fresh malt. For dextrin syrup the quantity of malt is re- duced to 15 per cent. Great importance is attached to the filtering, and this operation influences to a great degree the quality of the product as well as the yield. The passage through the filter-press should be made very rapidly, and the filtrate should be perfectly clear. An incom- plete filtration causes a change in the juices and shows at the same time a poor extraction. The slight turbidity found in badly filtered solutions re- veals the presence of a certain quantity of starch capable of producing trouble during the concentration of the juices. To get a good filtration it is essential to use a malt whose 1 64 THE ENZYMES AND THEIR APPLICATIONS. plumule is very long, and to reheat the saccharified wort at a temperature of 70 ° '. In maltose manufactories filter-presses of 70 square cen- timetres are generally used, furnished with 12 frames cov- ered with linen. A battery of 7 filters furnishes in 15 minutes 45 hectolitres of juice of 2.5 ° to 3 Baume. The filtered juices are placed in copper reservoirs fur- nished with a double wall for the entrance of the steam. They are reheated rapidly to 75 ° and left for about half an hour at this temperature for clarification. An abundant precipitate is formed which is separated by a second passage through the filter. This second filtration presents no difficulties. It is carried on in a filter-press of small dimensions. The clear juices are evaporated in a triple-effect appa- ratus where they are concentrated to 22 Baume. The syrups are then submitted to purification and to a bone-black treatment. The syrups are placed in special reservoirs in which are added 10 kilograms of powdered bone-black and 500 grams of dried blood per 25 hectolitres of syrup. It is kept boiling for 10 minutes, filtered and concen- trated in a vacuum up to 40°-42° Baume. For the manu- facture of products which are highly clarified the syrups, after purification, are placed in the battery of bone-black fil- ters where they remain from 5 to 8 hours. The yields usually obtained in the manufactories are from 92 to 94 kilograms of syrup at 40 per 100 kilograms of maize, but to secure this result a very well-conducted opera- tion and much attention are needed. To give an idea of the influence of the method of work on the output we may state that in the first years of the. manufacture of maltose the yield was only from 60 to 65 kilograms of syrup per 100 kilograms of maize, and that it was only later, owing to successive improvements, that the results mentioned above were reached. MANUFACTURE OF MALTOSE. 165 Properly prepared syrup generally keeps well, but better in the open air than in closed reservoirs. In large reser- voirs exposed to the air, change is never found, while syrup placed in barrels often ferments. The analysis of the indus- trial products is given below : LUMP MALTOSE. Water ^.o. Maltose 80.6 Dextrin 0.2 WHITE SYRUP (FECULA). Dry substances 77. 1 Maltose 50.2 Dextrin *74 SACCHARIFIED MAIZE. Water 20.2 Maltose 4c Dextrin 33. Nitrogenous matter 2.2 Mineral substances 0.91 DEXTRINATED SYRUPS. Water 20. Maltose 30.2 Dextrin 48. Nitrogenous matter 2. 1 Mineral substances 0.91 SYRUPS OF RICE. Water !8.8 Maltose yi Dextrin 2.4 Foreign substances 8.2 1 66 THE ENZYMES AND THEIR APPLICATIONS. The maltose syrup affords very great advantages over that of glucose in its purity and economy. Maltose is a nutritive substance of great value. In the living organism it is transformed into assimilable sugar more rapidly than is saccharose. It is very easy to digest and, having not so sweet a taste as cane-sugar, it can be taken in much greater quantities than the latter. By the action of acids on starch industrial glucoses are ob- tained which contain, besides dextrins, foreign bodies formed under the influence of the acids at high temperature. These bodies give a disagreeable taste to glucoses and often possess poisonous properties. The dextrins formed under the influence of acids have a scant nutritive value. The pancreatic juice acts very slowly on these dextrins and its action is always incomplete. As shown by the experiments made by Soxhlet and Stut- zer, dextrins formed by malt act quite differently; they are much more easily transformed by diastases. Saccharification by malt affords another great advantage: that of being able to utilize amylaceous materials directly without going through the manufacture of starch. By treating maize with acid great modifications are caused in the nitrogenous matters as well as in the fatty mat- ters. The products obtained are black, of a disagreeable taste, and not suitable for the manufacture of beer. To obtain the purest products it is necessary first to extract the starch, which entails great losses. Out of 60 kilograms of starch, contained in 100 kilograms of maize, only 50 to 52 kilograms are recovered in practice. One loses, therefore, 8 to 10 kilograms of starch, as well as other nutri- tive substances, organic and mineral, which enter into the composition of the grain and which are utilized in the manu- facture of maltose. The maltose industry also furnishes a more wholesome and more nutritive malt than that furnished by the glucose industry. It is, therefore, indisputable from a hygienic as MANUFACTURE OF MALTOSE. 167 well as economic point of view that maltose is preferable to glucose. The crisis through which the maltose industry is at pres- ent passing is not likely to lead to its abandonment. This manufacture presents certain advantages and the efforts made by Dubrunfaut and Cuisenier will not have been in vain. The patents which protected this industry have fallen into disuse and this circumstance will certainly not fail to give it a new impetus. CHAPTER XIV. PANARY FERMENTATION. Dumas' theory of panary fermentation. — Cerealin of Mege-Mouries: — The part played by bacteria in panary fermentation. — The origin of the sugar in flour. The work of bread-making is done in three successive stages : kneading, fermentation (raising), and cooking. The first of these operations has for its aim to make with the flour an elastic and homogeneous dough. To this end a little yeast is diluted in warm water, flour is added little by little, then the mixture is stirred and the mass is kneaded. Thus a dough is formed into which a certain quantity of salty water is uniformly worked. The kneading finished, the mass is left for some time. The incorporated yeast then causes a fermentation which modifies the structure and the chemical composition of the dough. This fermentation constitutes the second period, which is called " raising." It takes place in the kneading-trough and generally lasts from 20 to 30 minutes. The dough is then divided into parts of a certain size to which is given the form of a loaf of bread. They are sprinkled with flour and again left quietly for 30 to 40 minutes, after which they are baked in ovens brought tip to 250 or 300 . The leaven used in the preparation of bread comes from a previous operation. After kneading the baker takes away a small quantity of the dough and uses it as leaven in the 168 PANARY FERMENTATION. 169 next operation. The same ferment is used in this way for an indefinite number of times. The principal agent in panary fermentation is a Sac- charomyces. But this is not the only factor; others come in play, and here, too, are found diastatic actions. Corn, rye, and all other cereals, contain considerable quantities of amylase and substances which accelerate dia- static action. By grinding, it is true, a great part of the dia- stase is eliminated with the bran, but the flour is not com- pletely deprived of active substances. These remaining en- zymes play successively an important part in the various stages in the making of bread. The action of the diastases of the grains begins during the milling. This action is con- tinued during panary fermentation, and may even be evident during baking. The part played by the yeast, as well as the physical and chemical phenomena which are manifested dur- ing bread-making, have given rise to different theories. Dumas regards panary fermentation as an alcoholic fer- mentation. According to him, the starch and gluten of the flour have been already partially hydrated as a result of mix- ing with water. This hydration would also be favored by the kneading which scatters the yeast uniformly throughout the mass and brings it into contact with the air, a condition which favors fermentation. During the raising the carbonic acid formed in the mass is imprisoned in the cavities of the dough, to which the gluten has given coherence. During baking the sudden ele- vation of temperature expands the gases enclosed in the dough, and produces a swelling of the mass as well as a closer adherence among the hydrated materials, the starch, gluten, and albumen. According to Dumas, the carbonic acid produced by panary fermentation remains almost entirely in the bread, of which it occupies about half the volume, at a temperature of ioo°. The yeast then, according to him, would act by the car- T7° THE ENZYMES AND THEIR APPLICATIONS. bonic acid it produces and the fermentation would occur at the expense of the sugar already existing in the flour. The theory of Dumas likening panary fermentation to an alcoholic fermentation has met with various objections; cer- tain authors have objected that, in panary fermentation, there is neither production of alcohol nor multiplication of yeasts. According to Mege-Mouries, the bran contains an active substance which he calls cerealin and which has the property of transforming starch successively into dextrin, glucose, and lactic acid. This substance is not met with in the flour, but, according to Mege-Mouries, the gluten itself can saccharify the starch and make it ferment. The presence of alcohol in the dough after raising escaped detection for a very long time. Moreover, different experimenters have arrived at the conclusion that the yeasts introduced with the leaven do not multiply during the rais- ing. Relying on these data, and on the almost invariable pres- ence of bacteria in the leaven, some bacteriologists have evolved the hypothesis that it is the bacteria and not the yeasts which produce the fermentation. In 1883, Chicandard described the Bacillus glutinis which lie considers is the agent of panary fermentation. Laurent, in his later works, has described the Bacillus paniUcans. Popoff has isolated from baker's dough an anaerobic bacillus which, in the presence of sugar, produces carbonic acid and lactic acid. The bacteriological analyses of leavens made by Peters and Boutroux have demonstrated the constant presence in the leaven of bacteria secreting diastase and acting on starch and albuminoid matters. Furthermore, the presence of bac- teria of the same nature in corn-meal has been ascertained. The constant intervention of ferments in bread-making may then be considered as demonstrated. PANARY FERMENTATION. ill According to some, the bacteria alone cause fermenta- tion; according to others, the bacteria act in symbiosis with the yeast : the former, by the aid of their diastase, would furnish sugar to the yeasts. Wolffin succeeded in producing normal bread by replac- ing the leaven by a culture of Bacillus levans. Analogous ex- periments have been made by Popoff with the same success. Boutroux, who has taken up these experiments again, and carefully studied bakery yeast, has reached the following conclusions : ist. Alcoholic yeast is always present in the leaven of bread. 2nd. This yeast is cultivated from dough to dough in such a way that by sowing a first dough with imponderable traces of yeast, there will be found, at the end of several operations, a uniform distribution of yeast in the dough. 3rd. The other micro-organism found in the dough, and to which may be hypothetically attributed the power of making it rise, acts quite differently : transferred from dough to dough, it ceases to produce fermentation after the second or third operation. The presence in the leaven of bacteria favoring bread- making is on the whole an exceptional phenomenon. It appears from the studies of Boutroux that generally the presence of bacteria is unfavorable: they attack the glu- ten and prevent the bread from rising. In practical baking the destructive action of these bacteria is checked by the presence of the yeast which, in a normally constituted dough, finds an excellent field for development, antagonizes the foreign organisms, and is alone of importance in panary fer- mentation. The opinion of Dumas is also confirmed by the experi- ments of Moussette and Aime Girard, who have succeeded in ascertaining the presence of alcohol in the products of panary fermentation. Moussette, by condensing the steam of bread-ovens dur- 172 THE ENZYMES AND THEIR APPLICATIONS. ing baking, has obtained an alcoholic solution containing 1.6 per cent of alcohol. According to Girard, the same weight of alcohol as of carbonic acid is formed during fermentation. He finds nearly 2.5 grams of each of these substances per kilogram of bread. According to some authors, the sugar consumed in pan- ary fermentation, and which equals nearly 1 per cent of the. weight of the flour, comes directly from the grain. To support this opinion we may mention barley, which, always contains appreciable quantities of fermentable sugars. But the amount of sugar in corn is really very variable, though it is observed that cereals which contain quantities of fermentable materials insufficient for panary fermentation nevertheless ferment as energetically as cereals which are rich in sugar. On the other hand, flour which is deprived of certain constituent parts of the grains is found, as a result, poorer in sugars. According to Aime Girard, Boutroux, and Morris, there is produced during the growth of gramineous plants an ac- cumulation of sugar in the stem; this sugar, at the time of the formation of starch, passes into the embryo of the grains and is there transformed into starch as the grain ripens. As a result, there will be found only traces of sugar in the ripe corn, and the flour will be free from natural sugars since during grinding the greater part of the germ is carried away. In view of this fact, it is pertinent to ask whence comes the sugar which serves for fermentation. According to Poehl, the fermentable sugar found in flour is produced dur- ing the grinding of the grains, as the result of a diastatic ac- tion on the starch. This diastatic action is manifested only with the grains containing a certain quantity of water, while the dry grains do not furnish any. Thus, when one treats a grist of corn containing 11 to 13 per cent of water with 90 alcohol, one finds in the liquid PA NARY FERMENTATION. 173 the presence of reducing sugar. The same grain, previously dried and then submitted to the same treatment by alcohol, furnishes no sugar. There is really then a transformation of starch into sugar and the action of amylase is consequently shown at the time of grinding. It is quite reasonable to suppose that hydra- tion once begun continues during kneading and raising, although the amount of sugar does not perceptibly increase during these stages of the work. The intervention of diastase is shown with more clearness during baking. The dough, once introduced into the oven, heats very unequally. At the surface the temperature rises abruptly and causes the formation of a crust which prevents the volatilization of the gases and the water vapor formed. Inside the temperature rises very slowly, a circumstance which favors alcoholic fermentation as well as diastatic ac- tion, then the diastases continue to act up to a temperature of 8o°. Under the action of the water vapor and the heat, the grains of starch are transformed into soluble starch and amylo-dextrins. The small quantity of diastase contained in the flour is in excellent condition to cause hydration of the starch paste, which cannot be formed except in very small quantity, owing to the lack of water. It is especially during baking that mal- tose and dextrins are formed in the bread and give to it a characteristic taste and consistency. Flour of superior qual- ity generally contains small quantities of diastase, while flours containing a certain quantity of bran are richer in ac- tive materials which influence to a great degree the character of the bread. Thus the soft crumb of brown bread is due ex- clusively to the diastase of the bran. White bread, soaked in warm water, furnishes a half-solid mass and only about 6 per cent of the materials dissolve. Brown bread, treated in the same way, gives to the water a milky aspect and 45 to 50 per cent of the dry matter is dis- solved. This difference in solubility comes from the differ- 174 THE ENZYMES AND THEIR APPLICATIONS. ence between the modes of action of the diastase in the two kinds of bread. In bran, in the germs of corn, and consequently in the flour too, there are still other enzymes which take part in the bread-making. The transformation which the gluten undergoes during the raising and baking appears to us to be due to a dia- static action, but this question is not yet very clearly demon- strated. The intervention of enzymes is much more evident in the coloring of flour. In flour, there are found oxidizing enzymes to which we shall have occasion to return in studying oxidases. BIBLIOGRAPHY. Dumas. — Traite de chimie applique aux arts. Paris, 1843. Birnbaum. — Das Brotbacken. Leon Boutroux. — Le pain et la panification; chimie et technologie de la boulangerie et de la meunerie. Aime Girard. — Sur la fermentation panaire. Comptes Rendus, t. CI, p. 605. Boutroux. — Contribution a. l'etude de la fermentation panaire. Comptes Rendus, 1883, p. 116. Moussette. — Observations sur la fermentation panaire. Comptes Rendus, 1865, XCV. Lehman. — Ueber die Sauerteiggahrung und die Beziehungen des Bacillus levans zum Bacillus coli communis. Centralbl. fur Bakteriologie, 1894. W. L. Peters. — Die Organismen des Sauerteigs und ihre Bedeutung fiir die Brotgahrung. Botanische Zeitung, 1889. CHAPTER XV. ROLE OF AMYLASE IN THE DISTILLERY. Treatment of grains by acid and by malt.— Influence of heating on sac- charification. — Choice of temperature of saccharification — Principal and secondary saccharification.— Experiments of Effront on change in diastases during saccharification.— The infusion process.— Change in diastases during the successive stages of the work.— Control of the work in the distillery. Amylaceous materials do not ferment directly by the ac- tion of yeast. To make them readily subject to the alcoholic fermentation, it is necessary to submit them to a previous saccharification. To produce this transformation, the distiller has for a long time used mineral acids, and it is only in recent years that these agents have almost wholly disappeared from manufactories, where they have been replaced by malt. The use of acids as saccharifying agents presents, as a matter of fact, some notable disadvantages. To obtain a complete saccharification without considerable loss of the sugar formed, it is necessary to have very dilute mashes, to keep them for a very long time at a temperature in the neigh- borhood of ioo°, and to use considerable quantities of acid which must necessarily be neutralized before the addition of the yeast. Saccharification by acids is, therefore, not very economical, moreover it is never complete, and the greatest yields which can be obtained are never above 50 or 53 litres of alcohol per 100 kilograms of starch used. Working with acid presents still another disadvantage: it gives a residue which cannot be utilized for food for cattle, a disadvantage sufficient to condemn the method. 175 I? 6 THE ENZYMES AND THEIR APPLICATIONS. By using malt all the disadvantages of the acid process disappear, and saccharification takes place comparatively rapidly. The malts obtained in this way are of good quality and the yield in alcohol exceeds 65 litres for each 100 kilo- grams of starch used. Nevertheless, the malt process has its difficulties, also. It is not always easy to prepare a malt corresponding to the needs of the distillery, and it is often very difficult to use it to the best advantage. Of all the industries which use diastase as a saccharifying agent, the distillery undoubtedly has the most difficulties to contend with in the use of amylase. It is, in fact, the amy- lase which plays the principal part in this industry, for it reg- ulates the course of fermentation, and influences all the stages of the work. A thorough knowledge of the method and conditions of the action of this diastase is, therefore, indispensable in order to direct the work suitably. For this reason, in studying the process of distillation solely from the point of view of the part played by the malt, we may review the successive operations in that industry. Cooking. — Starch removed from the cells is not easily attacked by amylase but, when it is not freed from the grains which enclose it, its transformation by diastase is still more difficult. The intercellular substances and the cellu- lose membrane of starch-containing cells prevents contact of the enzyme with the granules of starch. To render efficacious the action of the diastase on amy- laceous materials, it is necessary to submit them to a cook- ing which dissolves the intercellular substances and frees the grains of starch. By working with finely ground amylaceous materials, the combined actions of heat and water favor to a great degree the action on the starch, and cooking in contact with the air is sufficient to obtain a paste which is easily saccharified by amylase. However, in working with whole grains, it is necessary to work under pressure. ROLE OF AMYLASE IN THE DISTILLERY. 177 In practice, the steaming is done in closed vessels, where the grains are submitted for about 2 hours to a pressure of 3 to 4 atmospheres. Increase in temperature is very favorable to the solution of the starch, but it presents great disadvantages from other points of view. The principal part of the grains, the starch, resists high temperatures without decomposing, but this is not true of the other substances constituting the grain, of the sugars, for example, which are destroyed at high temperature. By cooking a mash containing sugar at different temperatures, it is found that the destruction of the sugar increases in a great degree in proportion as the pressure increases. Thus a mash containing 15 per cent of maltose kept for £ hour at 2 atmospheres loses 0.85 of sugar. « " 3 " 1.7 " " 4 " 3-4 Grains, and especially potatoes, contain quite consider- able quantities of fermentable sugars, and the destruction of these must necessarily bring about a perceptible loss in al- cohol. High pressure also has the effect of dissolving different substances which enter into the composition of the grains. The increase, in the mash, of the quantity of extractive substances under the influence of high pressures is consid- ered by different authors as a proof of the efficiency of steam- ing. It is on this basis that we are sometimes advised to ex- ceed the pressure of 3 atmospheres during cooking. It is unquestionable that high pressure increases the density of the mash, and that it favors increase in the quantity of reduc- ing substances, but this fact does not necessarily mean an increase in the alcoholic output. On the contrary, numer- ous experiments made to this end have shown that a mash of much cooked grains, while giving a good saccharification with amylase, furnishes a yield in alcohol inferior to that of 1 78 THE ENZYMES AND THEIR APPLICATIONS. a mash prepared at a moderate pressure. Thus three grain mashes, prepared at different pressures, but with other con- ditions constant, give the following results : Density Alrnhnl Diastatic Balling. Alconoi. Powen 2 atmospheres 17 10.5 40 3 " 18.1 10.3 28 4 " 18.6 9.8 13 It is seen that the mash cooked at 4 atmospheres pos- sesses a density of 18.6, while the mash cooked at two atmos- pheres shows a density of only 17. We may observe at the same time that the maximum amount of sugar does not cor- respond to the greatest yield of alcohol. Indeed the must prepared at 2 atmospheres furnishes 10.5% of alcohol, while the must prepared at 4 atmospheres gives only 9.8%. Un- der the heading " Diastatic Power " we find an explanation of this anomaly. The grains cooked at 2 atmospheres and then sacchari- fied under the same conditions show a fermenting power of 40; the diastatic power diminishes with the increase of pres- sure and at 4 atmospheres a fermenting power of only 13 is found. The cooking of the must gives rise to certain sub- stances which weaken the enzymes during saccharification. The cooking under high pressure therefore brings about, as immediate consequence, an incomplete fermentation. The nature of the harmful substances is not exactly known, neither can it be determined what are the bodies which give rise to them ; nevertheless the formation of substances impeding diastatic action cannot be doubted. It is well to take note of this fact, especially when it is proposed to work with a limited quantity of diastase. The most rational method of w r ork consists in making a very fine meal of the grains, and in cooking this meal for 1^ to 2 hours with water at i| or 2 atmospheres at the most. Under these conditions, mashes are obtained which do not weaken the diastase. This mode of work also offers the ROLE OF AMYLASE IN THE DISTILLERY. 179 great advantage of furnishing a much more wholesome malt than that obtained by cooking at high pressure. It is very difficult to show in a conclusive way the un- favorable influence which cooking at high pressure has upon the quality of the malts. Chemical analysis gives us data on the amount of nitrogen, phosphates, and organic materials, in the malts, but it does not give us any information as to their nutritive value, and the comparative value of the malts cannot be determined except by experiments on animals. Experiments of this kind would have to be made in an agricultural station having a distillery at its disposal. We do not think that experiments of this kind have been at- tempted, and at any rate we do not know the results which they have given. Still, our opinion on the comparative value of different malts, according to the temperatures at which the cooking has been done, results from an inquiry we have made. Information we have gained from different distillers and agriculturists proves that cattle eat more readily malts obtained by cooking the grains at slight pres- sure. These malts can be consumed by them in greater quantities than the malts cooked under high pressure. These same malts have not, like those prepared at high pres- sure, an unfavorable effect on the quantity and the quality of the milk. The influence of cooking on the nutritive value of the malts may be particularly observed in towns possessing several distilleries. The farmer who buys liquid malts always ends, after longer or shorter trials, by giving prefer- ence to one of the distilleries, and this preference is always in favor of the manufactory using low pressure. It appears to us probable that the same substances which influence saccharification unfavorably hinder digestion of the malts obtained by cooking at high pressure. Saccharification of Amylaceous Materials. — By cook- ing, the substances which in the grains are interposed be- tween the starch-bearing cells are partially dissolved, and 180 THE ENZYMES AND THEIR APPLICATIONS. the starch-cells are liberated from the tissues where they were enclosed. Inside the cells the grains of starch first swell and then liquefy. To remove this starch from the cells, it is necessary to have recourse to a mechanical action which bursts the cellulose membrane, which resists very strongly the action of heat. This bursting is necessary because, if the liquid starch remains enclosed in the cells, it will undergo an in- complete saccharification only. To this end the cooked mass is vigorously stirred, the mash is expelled from the macerater by strong pressure, and the action is completed by a crushing which breaks up the mass and bursts the most resistant cells. The mash thus prepared is suitably cooled, malt is added, and it is left to saccharify. The determination of the temperature at which saccharifi- cation should be carried on has been made both by manu- facturers and by scientists. Still, in spite of all the efforts of this kind, the question is yet unsettled on account of the divergence of opinion on the subject. To understand the difficulties which are met with in the choice of the tempera- ture for saccharification, one must first of all realize the many and varied results which it is desired to obtain by this operation, namely: the liquefaction of the starch of the raw grains and the proper utilization of the starch of the malt. Finally one must take account of the presence of germs and bacteria in the malt, as well as the acidity of the medium and the change in the diastase. Theoretically, by a very prolonged action of the malt on the starch, a complete saccharification is obtained, but in practice it is absolutely impossible to procure a complete transformation and the saccharification, under the best con- ditions, only furnishes 80 parts of maltose per 100 of starch. The hydration of the starch is accomplished in the work of the distillery in two different stages : saccharification ROLE CF AMYLASE IN THE DISTILLERY. i«t proper, then the secondary saccharification, which is pro- longed throughout the duration of fermentation. Of these two saccharifications, the last is the most dif- ficult to regulate, and it is generally believed that it is best to produce the greatest part of the sugar in the first stage of hydration, so as to leave the fewest possible dextrins for the secondary saccharification. To this end the most favor- able conditions must be employed during the principal sac- charification and a temperature must be adopted which furnishes the greatest effect in the least time. It is the determining of this temperature that offers the first difficul- ties. The optimum temperature of diastases is far from being constant. In fact, if we compare the quantities of maltose formed during the same period of time with a given quantity of malt, at different temperatures, we shall find that the maximum of sugar formed takes place at very different temperatures according to the duration of saccharification. Thus, when different specimens of the same starch are saccharified for I hour with the same quantity of malt, work- ing at temperatures increasing from 30 to 70 , an optimum temperature of 6o° to 63 is found. When the same experiment is repeated by prolonging the duration of saccharification for three hours, the optimum temperature is reduced to 50 , and it descends to 30 if saccharification is made to last for 12 hours. From this it results that the selection of the tempera- tures of saccharification depends on the duration of the latter, and that the longer the duration of the action, the lower should be the temperature of saccharification. In practice the duration of saccharification is very varied. This operation lasts, in different manufactories, from 20 minutes to 2 hours. Its duration is determined by the kind of plant, and by the general conditions of the work. If a complete hydration of the starch is desired, it is always well to choose, when saccharification lasts a half- hour, a temperature of 62 to 63 , while one must lower the 182 THE ENZYMES AND THEIR APPLICATIONS. temperature to 57°-58° for saccharifications lasting from i to 2 hours. We now know the conditions necessary, in the principal saccharification, for a complete hydration. But in reality the quantity of sugar formed during the first stage of saccharification has very little influence on the final result of the operation. A dextrinated mash affords as- much alcohol as one strongly saccharified.' Moreover, the quantity of diastase indispensable for the secondary saccha- rification is no greater for the dextrinated mash than for the mash which already contains a large amount of sugar. A very long series of experiments performed in this way- have shown us that the intensity of the first saccharification is of little importance, and that the final result depends especially on the more or less complete preservation of the diastase during fermentation. Still, the principal saccharification cannot be completely suppressed. It has a reason for existing, especially from the point of view of liquefaction. In fact, it is this first opera- tion which gives to the cooked mass the necessary fluidity. Moreover, it succeeds in attacking the cells which have escaped the action of steam, and it liquefies the particles of starch which adhere to the spent malts. By raising the temperature of saccharification above 60% excellent conditions are obtained for liquefaction. We may now consider the change in the diastase under the action of heat, for the active substance which is to serve for the secondary saccharification must, after the principal saccharification, be absolutely unchanged. The tempera- ture of saccharification must, therefore, necessarily be lower than that at which the diastase commences to weaken. All the authors who have investigated saccharification are com- pletely in accord on this point, but their opinions are very different when it is a question of saying at what temperature the change begins. According to some, the active sub- ROLE OF AMYLASE IN THE DISTILLERY. 183 stance of the malt can withstand temperatures of 62 with- out changing. According to others, the degree of resist- ance of the malt to high temperature depends on the dura- tion of the action as well as upon the concentration and the composition of the mash. According to some chemists, a temperature of 6o°-62° would bring about in diluted mashes a pronounced change of the diastase, while in concentrated mashes the amylase would resist much better. Others finally, make a notable difference, from the point of view of the preservation of the enzyme, between a dextrinated must and a saccharified must. It is assumed that the presence of great quantities of maltose in the solution protects the diastase against the disastrous effect of high temperatures and, on that basis, it would be advisable to conduct saccharification in two stages, during the first 30 minutes of the action of the malt at a temperature of 58°-6o°, then raising it to 6 4 °-67°. For the support of this theory numerous experiments are cited which, however, do not lead to very clear conclu- sions. The various determinations, made by several chem- ists with different raw materials, under conditions neces- sarily varied, and by diverse methods, cannot furnish data of sufficient accuracy to solve this question. The greater or less change in the diastase at different temperatures may be demonstrated by a very simple method. To a starch paste is added a quantity of malt just suffi- cient to produce, under favorable conditions, a complete saccharification. After this addition, two samples are taken; one is left for 12 hours at a temperature of 30°, the other is first kept for an hour at a high temperature, then for 1 1 hours at 30 . If under these conditions a difference is found in the amount of maltose, this proves the influence of high tem- peratures. 1 84 THE ENZYMES AND THEIR APPLICATIONS. Here are three experiments made at different tempera- tures: Maltose formed : After i hour. After 12 hours. . j 12 hours at 30 C 2.4 9.6 ( 1 hour at 50° C. and 11 hours at 30 C 8.3 10.2 R j 12 hours at 30° C 2.2 9.8 ( 1 hour at 55° C. and n hours at 30 C 9.1 11. 6 P \ 12 hours at 30 C 2.2 9.9 • 1 hour at 59° C. and n hours at 30 C 9.5 9.7 The maltose, in all these experiments, was measured after 1 hour and after 12 hours of saccharification. Mashes kept at 45 , 50 , and 59° furnished, after the first hour of saccharification, a quantity of sugar much greater than that produced in the sample specimen left at 30 . After saccharification at 59 for 1 hour, 9.5% of maltose is ob- tained, in place of 2.2% obtained in the same length of time at 30 . If the diastase had not changed during the first hour of saccharification at 59 , at the end of the 11 subsequent hours a much larger quantity of sugar would be obtained than in the sample specimens, since in the first hour of saccharifica- tion it was already much more advanced than in the sample specimens. But such was not the case. After 12 hours of saccharification, there was found in the sample specimen 9.9% of maltose, while in the experiment where the diastase was carried for an hour at 59 there was found only 9.7% of sugar. The temperature of 57 is, therefore, the limit to which amylase can be carried for 1 hour without producing a noticeable change. The influence of high temperatures of saccharification may be equally well shown by the following experiments : In different experiments, at different temperatures, digest a litre of paste containing 10 grams of starch and 5 cubic centimetres of malt infusion. ROLE OF AMYLASE IN THE DISTILLERY. 185 Experiment. Duration of Saccharification. Starch transformed. i 12 hours at 30 85% 2 i hour at 45 and 1 1 hours at 30 . 97 3 " 5o° 96 4 " 64 68 By repeating the same experiments with mashes of dif- ferent concentrations and containing different proportions of dextrins and maltose, we have been able to ascertain definitely the concentration and the amount of maltose exer- cising a protective action on the diastase, but that this ac- tion is slight and that it may be entirely neglected when 58 is exceeded. At temperatures higher than 58 , even when the mashes are very concentrated, a great destruction of diastase occurs. By working, as is the case in most distilleries, with a great quantity of malt, the lack of diastase in the secondary fer- mentation is not perceived, but the result is quite different when there are rational conditions of work and when one seeks to reduce the quantity of malt to what is strictly neces- sary. Those who recommend high temperatures of saccharifi- cation bring other arguments to the support of their opinion- According to them, one must employ a temperature of 60 ° or even a higher one, because otherwise the starch of the malt will not be utilized to the best advantage, and because only a high temperature can weaken the bacterial ferments which are always present in the malt. The utilization of the starch in malt involves great diffi- culties, because its complete saccharification is secured only at a temperature of 70 . At 65 one finds 4% of starch still undissolved. " 6o° " 8% " 50° " 73% Hence in the choice of a temperature of saccharification *86 THE ENZYMES AND THEIR APPLICATIONS. one must take into account the starch of the malt. By choosing a temperature of 55 ° one runs the risk of losing 42 per cent of the starch contained in the malt, while at a tem- perature of 6o° the loss is considerably less. At this temperature there remains only 8 per cent of amylace- ous material not attacked. When using from 12 to 16 per cent of malt one is obliged to choose a high temperature of saccharification, but when working with a very much re- duced quantity, one may choose a lower temperature, be- cause the loss of starch is reduced in this case to a minimum. Furthermore, the losses in amylaceous materials which may come as the result of a poor extraction of starch are never -as detrimental to the yield as the weakening in the diastase under the influence of temperature. It is always prefer- able, therefore, to give up the attempt to secure a com- plete extraction of the starch and to seek to control the diastase, especially as the undissolved starch is not com- pletely lost. The starch of malt, which escapes solution during sac- charification, is partially dissolved during fermentation. High temperatures must, therefore, be avoided, and sac- charification carried out between 55 and 6o°. In certain cases, and especially when one is dealing with raw materials of doubtful quality giving mashes which con- tain 0.25 to 0.35 per cent of lactic acid, it is necessary to keep the temperature of saccharification still lower (not above 55°), because in an acid medium the diastase becomes more sensitive to the action of heat. In practice, unfortunately, these principles are entirely set aside. With a mouldy malt of poor quality much higher temperatures are adopted than in ordinary work, because it is supposed that by raising the temperature the micro-organisms which prevent the fermen- tation are killed. The results obtained by thus raising the temperature are not very satisfactory, but the distiller is con- soled by the thought that if he had not employed high tem- perature the final result would have been still worse. In ROLE OF AMYLASE IN THE DISTILLERY. 1S7 reality, an increase of several degrees in temperature has not much influence on the purity of the fermentation and does not kill the germs at all, but destroys the diastase and hinders normal fermentation. When working with materials of poor quality, one must have recourse to antiseptics or employ only very active yeasts which can protect the mash from the in- vasion of foreign ferments without hindering the secondary saccharification. The Infusion Process. — As we have just seen, the choice of temperature of saccharification presents great difficulties. The starch paste formed during steaming must be lique- fied at a temperature higher than 65 . The starch of the malt, to be completely dissolved, re- quires a temperature of 70 , while the diastase cannot be brought to a temperature of 6o° without undergoing a per- ceptible weakening. Under these circumstances, it is always necessary to sac- rifice either the enzyme or the starch, and the temperature of saccharification must necessarily vary according to the con- ditions and the quality of the raw materials. An ideal process requires the separation of the active sub- stances from the starch of the malt and their separate treat- ment at different temperatures. By leaving the malt in contact with wat^r under suitable conditions, the diastase passes into solution, is separated from the starch and can serve afterwards for saccharification. As to the residue, it is still impregnated with sufficient quantities of the enzyme to produce liquefaction. The method, thus put in practice, leaves nothing to be desired. The mashes cooked under pressure are liquefied at a tem- perature of 70 with the soaked malt and then cooled to 45°— 50 . At that temperature the solution of the enzyme is added; the mixture is kept for some minutes at 45 — 50 ; then cooled to the temperature of fermentation; the yeast is added and it is left to ferment. This mode of operation cannot fail to give good results 1 88 THE ENZYMES AND THEIR APPLICATIONS. provided that the extraction of the diastase has been as com- plete as possible. Let us now see how one must proceed to extract the dia- stase from the malt. It is wrongly assumed that malt amylase dissolves easily in water. In reality the extraction is difficult; it depends on the temperature of the water and on the thoroughness with which the malt is ground. We may emphasize this fact by the two following experi- ments: make two mixtures of malt and water and submit them to a temperature of 30°. Specimen A is not disturbed, while specimen B is constantly stirred. From time to time a few cubic centimetres of each liquid is taken and the dia- static power determined, which enables us to follow the course of the solution of the diastase. Dlastatic Power. Experiments. After S hours, i- hours. 2c hours. 4- hours. 52 hours. Liquid A ^ 45 4§ 60 55 B 39 58 52 50 42 The quantity of diastase dissolved in the infusion at first increases with the duration, reaches a maximum, and then decreases. In the liquid A it is after 47 hours that the dia- static power attains its maximum. Shaking renders the ex- traction more rapid in the liquid B, where the diastatic power reaches its maximum in 17 hours. Numerous experiments made with different malts have shown that this maximum is reached earlier as the tempera- ture of the infusion is higher. Our observations are summed up in the following table : An infusion prepared at 45 reaches its maximum of diastase dissolved after 7 or 8 hours. li " " from 55 to 59° " " *' " 3 hours. " " " " 6o° to 6 5 ° " " " " J£ hour. The time necessary for a good extraction therefore depends on the temperature. There is. moreover, a critical time which must be borne in mind, since from this time onward, the diastase begins to disappear. ROLE OF AMYLASE IN THE DISTILLERY. 189 The maximum quantity of active substance which can be dissolved in the infusion at the critical time is not at all con- stant. It varies considerably for the same malt according to the temperature, as may be seen in the following table : Diastatic Power of the Infusion. Temperature of After U hour. 3 hours. 8 hours. 17 hours. 2s hours. Intusion. '* J 30 .. .. 31 60 49 45 ..44 5 6 5 1 55 46 55 65 36 20 It is at a temperature of 30 that the most active solutions are obtained; from 45 to 55 the quantity of diastase which can be extracted remains almost the same, while at 65 ° the destruction of the ferment occurs as it passes into solution, and even at the maximum, a very weak infusion is obtained. The preparation of a cold infusion during 17 hours offers cer- tain practical difficulties. To utilize the malt to advantage, it is well to make the solution at 55 ° for 3 hours. The infusion process is especially recommended in the case of malts of maize. These malts generally give from 8 to 20 per cent of ungerminated grains, and their diastatic power is only from one fifth to one third that of barley-malt. To employ this malt it is necessary to use large quantities, and the loss in starch is greater, because the starch of maize-malt is much more slowly attacked by the enzyme than the barley- starch. An infusion of this malt must be made in the follow- ing way : Reduce the malt to powder; dilute it in 4-5 volumes of water at a temperature of 55°. Then place it in a conical ves- sel and stir it during the first hour, then leave it for an hour or an hour and a half. Deposition takes place very readily and the liquid can be removed without carrying along the malt. A filter-press may be used for the same purpose. The infusion of malted maize yields a liquid which filters easily. J 9° THE ENZYMES AND THEIR APPLICATIONS. The infusion of barley-malt is made in most manufactories with crushed malt, and at a temperature of io°-i5°. It is prepared in a crushing apparatus. The malt is bruised for 15-30 minutes, then put in water for one or two hours, after which the liquid which is to be used for saccharification is decanted. The diastatic power of an infusion prepared in this way is very variable. It depends more upon the special nature of the malt than upon its richness in amylase. The quantity of diastase extracted is between 10 and 50 per cent of that contained in the malt. This method of preparing an infu- sion of malt is not one to be recommended. Much more satisfactory results are obtained by prepar- ing the infusion at a temperature of 45°-50°, and allowing the solution to proceed for 2 or 3 hours. By this method, from 70 to 80 per cent of the enzymes contained in the malt are dissolved. The infusion process is as yet scarcely in practice, but it is unquestionably the method of the future. The invention of a contrivance for separating barley-malt from its infusion is to be desired, because the principal dif- ficulty always lies in this operation. Concerning the Change which the Diastases Undergo during the Successive Stages of the Work. — From the study we have just made of the conditions of the action of amylase, it appears that a part of the enzymes of malt are destroyed during saccharification and that the varying re- sistance of the diastase to temperatures from 6o°-62° de- pends upon the degree of acidity of the medium. The acidity of the musts is not the only factor which produces a change in the diastase ; other conditions must be taken into account and these are not always easy to recognize. Two malts possessing the same saccharifying power, used in the same quantity and producing in identical musts the same quantity of sugar may nevertheless yield infusions containing different quantities of diastase. ROLE OF AMYLASE IN THE DISTILLERY. *9 l Besides richness in active materials, other factors must be taken into consideration in estimating the value of a malt. The origin of differences in resistance is perhaps to be found in the degree of natural acidity of the grains, perhaps also in the kind of acid or in the nature of other foreign sub- stances contained in the malt. We have made a series of experiments for the purpose of finding the cause of the dif- fering resistance of malts and we can furnish some informa- tion on this subject, though unfortunately it is very incom- plete. The resistance of malts depends on the temperature at which germination is conducted. Thus, by malting two portions of the same barley at different temperatures, one for 8 days at i(f-22 , the other for 9 days at I2°-I5°, we have obtained malts which differed in their resistance at a temperature of 6o°. Malted barley, worked in the cold and for 9 hours, gave better results than barley malted at higher temperatures. On the other hand we found that barley, giving from 7 to 10 per cent of non-germinated grains after malting, possessed not only a saccharifying power less than that of completely germinated barley, but also a widely dif- ferent resistance to the reaction of the medium. Incom- pletely germinated barley offers less resistance. The richness of the mashes in enzymes after the principal saccharification consequently depends upon the quantity of diastase which is found in the malt, the temperature of saccharification, and finally the degree of resistance of the diastase. The loss of enzymes wnich occurs during saccharification at high temperatures may, under favorable conditions, be limited to 20 per cent, but generally this limit is exceeded and the destruction reaches 30 per cent. The secondary saccharification is made with the diastase which escapes destruction during the first stage of the opera- tion. This saccharification is very slow and must be pro- longed at least three days. 19 2 THE ENZYMES AND THEIR APPLICATIONS. The diastase is generally preserved much better in mashes in fermentation than in sweet mashes. The dia- static power of the latter weakens considerably, even in the presence of antiseptics. The diastatic power of a mash which has fermented under favorable conditions remains almost constant for more than 70 hours. The favorable progress of fermentation depends principally on the preser- vation of the diastase. This preservation can be insured only in musts free from foreign ferments and for this reason the use of antiseptics is necessary in the distillery. In fact it is absolutely impossible to avoid infection by any other means. Control of the Process in the Distillery. — The normal progress of a fermentation depends upon various factors and, besides those which have to do with the cooking and the temperature of fermentation (which is always easy to control), we must take note of the quality of the malt used, and of the nature of the yeasts as well as of the degree of infection of the must by foreign ferments. Each of these three factors gives rise to a problem which is complicated by the interaction of the others. And it is often very hard, when there is some difficulty with the work,, to recognize its cause and point out its origin. A poor fermentation usually coincides with an infection by foreign ferments, but this is not always the first cause of the trouble observed; on the contrary, it is more often only the consequence either of lack of diastase or of weak- ness of the yeast. Nor should the lack of enzymes in a fer- menting must always be attributed to a poor quality of malt; the destruction of enzymes may have been caused by the invasion of foreign ferments. So, too, if in a bad fermenta- tion a degeneracy or a weakening of the yeast is found, this must not be considered as the direct cause of the trouble; the lack of diastase, among its pernicious effects, may have brought the yeast into this state. To go back to the beginning and discover the real cause ROLE OF AMYLASE IN THE DISTILLERY. 1 93 of the trouble, it is necessary to follow the course of the weakening of the enzyme in all the stages of the work. A quantitative determination of the diastase contained in the malt gives an idea of the quantity of germinated grain necessary for a normal operation. Then by determining the diastatic power of the saccharified mash produced with the malt under examination, one may find the extent of the change produced during saccharification and be in a posi- tion to judge if the quantity of amylase remaining is suffi- cient for the secondary saccharification. The determination of the diastatic power of the mash at different stages of fer- mentation furnishes data on the weakening of the diastase; it makes it possible to ascertain the point at which activity begins to diminish and to recognize clearly the cause of this diminution. A perceptible weakening of the enzyme in the first period of fermentation must lead to an irregular progress. The cause of the phenomenon is generally the initial acidity of the must and it is well, in such cases, to choose a saccharification temperature much below 6o°. The weakening of the diastase during fermentation may be due to other causes. It may come from the quality of the grain and in this case high pressure during cooking must be avoided, because it is generally during this process that substances which weaken the diastase are formed. During the secondary fermentation, the acidity must be determined at the same time with the weakening of the dia- stase, for a perceptible increase in acidity is always followed by partial destruction of the diastase. The weakening of the diastase may be averted in this case by an addition of antiseptics. An entirely opposite state of affairs is sometimes ob- served: the diastase first weakens and acidification is pro- duced only from 6 to 10 hours later. The appearance in the musts of foreign ferments here results from weakening of the diastase. In this case the 194 THE ENZYMES AND THEIR APPLICATIONS. addition of more of the infusion to the fermenting must may prevent acidity and keep up the yield of alcohol. Finally, if an incomplete fermentation is met with in the musts which are not abnormally acid and are rich in diastase, the cause may lie in the yeasts. This case often arises when antiseptics are used which leave the diastase intact, but act very unfavorably on certain kinds of yeasts. From the foregoing, it is evident that determinations of the fermenting power of malt and of musts may be of great service to distillers. In the following chapter will be found the methods to be followed in such analyses. BIBLIOGRAPHY. Effront. — Sur les conditions chimiques de Taction des diastases. Comptes- Rendus, 1892, t. 115, p. 1524. ■ Sur certaines conditions chimiques de Taction des Ievures de biere. Comptes Rendus, 1893, t. 117, p. 559. Sur la formation de Tacide succinique et de la glycerine dans la fer- mentation alcoholique. Comptes Rendus, 1894, t. 119, p. 92. Accoutumance des ferments aux antiseptiques et influence de cette accoutumance sur leur travail chimique. Comptes Rendus, 1894, t. 119, p. 169. De Tinfluence des composes du fluor sur les Ievures de biere. Comptes Rendus, 1894, t. 118, p. 1420. Etude sur les Ievures lactiques. Annales de l'Inst. Pasteur, 1896, p. S24. De Tinfluence des fluorures sur Taccroissement et le developpement des cellules de la levure alcoholique. Moniteur scientifique, 1891, P ; 254. Etude sur les Ievures. Monit. scientifique, XI, p. 1138, 1891. Des conditions auxquelles doivent satisfaire les solutions fermen- tescibles pour que les fluorures y produisent un maximum d'effet. Monit. scientifique, 1892, t. VI, p. 81. Maercker. — Spiritusfabrikation. Paul Parey, Berlin, 1894. Max Biicheler. Die Branntwein Industrie. Zweite vollstandig umgear- beitete Auflage des Lehrbuches der Branntweinbrennerei von Stam- mer. Braunschweig. Leitfaden fur den landwirthschaftlichen Brennereitreib. Braun- schweig, 1898. CHAPTER XVI. QUANTITATIVE STUDY OF MALT. Determination of the diastatic power of malt and mashes by the methods of Effront. — Determination of saccharifying and liquefying powers. — Determination of the diastatic power of sweet and fermented mashes. The methods generally used for the quantitative study of malt take account only of its saccharifying power and en- tirely neglect its liquefying power as well as the resistance of the enzymes. Our researches have shown that it is indis- pensable to take account of these two properties. The sac- charifying power of malt is subject to the influence of foreign substances contained in the grains. The intensity of the sac- charifying power of a malt does not, therefore, afford an exact measure of the quantity of amylase it contains. The effect obtained in a saccharirication by diastase is often the result of the combined action of the enzyme and of accelerat- ing substances which accompany it. The following experiment gives a clear idea of the influ- ence of these extractive materials of the grain on the saccha- rifying power: Prepare an infusion, using one part of malt and twelve parts of water, and at the same time an infusion of non- malted barley with one part of the grain and four parts of water. Filter these two infusions: from each take a certain number of cubic centimetres, which are introduced into a starch paste. Saccharify for one hour at 50 . The quantity of maltose obtained under these conditions furnishes a basis of comparison between the diastatic value of the two infusions. 195 196 THE ENZYMES AND THEIR. APPLICATIONS. In a second series of experiments, add to the starch, at the same time with the infusions of malt and of fresh barley, a certain quantity of infusion previously boiled. The saccharification is made at the same temperature in all the experiments. Number of the Experiment. Fresh Infusion. 1 C.C. 2 " 6 " " 1 " I " Boiled Infusion, Maltose formed. ' I 2 3 4 5 6 0.37 g- O.65 " O.85 " " O.6 " O.72 " I 6 c.c. r " 2 " r 1 *7 / 8 9 10 0.5 C.C. 0.5 " " 0.5 " 2 c.c. o-5 " 1 " 0.07 g. O " 1 1 O.O95" O. II " Six cubic centimetres of the barley infusion, not boiled, give in the starch 0.85 g. of maltose (Experiment No. 3), The same quantity of infusion, previously boiled, used with- out fresh infusion, remains absolutely without action on the starch (Experiment No. 4). But this liquid, inactive by it- self, influences saccharification to a great degree if it is with active diastase. Thus, a cubic centimetre of infusion of bar- ley gives 0.37 g. of maltose and this same quantity of in- fusion produces 0.72 g. of maltose when it has 2 cubic cen- timetres of boiled infusion added. The same thing is true with an infusion of malt heated to ioo°; 0.5 cubic centimetres of this infusion furnish 0.07 g. of maltose and the same quantity of infusion gives o. 11 g. of maltose when saccharification occurs with a cubic cen- timetre of boiled infusion. Thus it is seen that the extractive materials of raw grain have a considerable action on the amylase of malt and that with them saccharification can produce ten times as much sugar as by the action of the ferment alone. Analogous experiments with barley of different origin QUANTITATIVE STUDY OF MALT. 197 have shown us that no constant ratio exists between the real saccharifying power (due to amylase) and the accelerating power latent in raw grain. It follows that the active constit- uents of grains of different origin influence the saccharifying power to varying degrees. It must be admitted that in ascertaining the value of a malt it makes no difference whether the saccharifying power comes from the amylase or some other substance, but amy- lase must not be confounded with the substances which ac- celerate saccharification; the mode of action of the latter is wholly different from that of the diastase. The substances which excite or accelerate hydration do not always increase the quantity of sugar formed and they have absolutely no in- fluence on the distillery mash. In practice, — and that is the important point for us, — it is amylase alone which comes into play. This results from the fact that the exciting substances act only in mashes containing little maltose, and because the ef- fect they produce becomes weaker and weaker as sacchari- flcation advances. In distillery and brewery mashes a large proportion of maltose is always present and the effect of exciting substances is negligible. The determination of the amylase of malt, looking solely to its saccharifying power, will consequently always give un- reliable results. To reach more trustworthy results, we have sought a method which will allow of measuring quantitatively the liquefying power. The liquefying power of malt is not influenced by foreign substances. For this reason we ex- press the value of a malt by both its powers : its saccharify- ing power and its liquefying power. In the preceding chapter we have seen that malts differ much in their resistance to a temperature of 6o°. This cir- cumstance forces us, in determinations, to take account of the degree of resistance of the amylase. These factors be- come especially important in the case of malts destined for the distillery. I9 8 THE ENZYMES AND THEIR APPLICATIONS. A malt of high diastatic power, but of little resistance to high temperatures, gives a less satisfactory result in the dis- tillery than a malt less rich in amylase but which endures without change a high temperature of saccharification. To form an idea of the greater or less resistance of malt at a high temperature, we keep the malt for an hour at 6o° and determine the saccharifying power in mashes where the diastase has previously been destroyed. We have thus established a method of determination which, we believe, answers to the needs of the industry. The determination is made in three stages : 1st. Preparation of an infusion. 2nd. Determination o'f the saccharifying power. 3rd. Determination of the liquefying power. Preparation of the Infusion. — To prepare the infusion, weigh out 6 grams of crushed malt, put them in a flask con- taining 100 cubic centimetres of water at 6o° ; keep the flask in a water-bath for an hour at a temperature of 6o°. During saccharification, shake the flask from time to time ; the sac- charincation ended, cool to 30 and filter; to 50 cubic cen- timetres of filtered liquid then add 50 cubic centimetres of distilled water and determine the saccharifying power of this diluted infusion. The remainder of the non-diluted infusion is used to determine the liquefying power. Determination of the Saccharifying Power. — The sac- charifying power is determined by the aid of a standard solu- tion of starch. Dissolve 2 grams of starch in water and make the solu- tion up to 100 cubic centimetres. To 100 cubic centimetres of this 2 per cent solution, add 55 cubic centimetres of distilled water and 5 cubic centimetres of an infusion of malt diluted as described above. The whole is then placed in a water-bath at 6o° for an hour. After saccharification, it is rapidly cooled and the content of sugar immediately determined. To determine the maltose in the saccharine liquid, 2 cubic QUANTITATIVE STUDY OF MALT. ^99 centimetres of a solution of cupro-potassic tartrate is used, which corresponds to 0.01498 grams of maltose. The 2 cubic centimetres of cupro-potassic solution is put in a test- tube to which is added 3 cubic centimetres of water and a few fragments of pumice-stone. The number of cubic cen- timetres of saccharine solution necessary for the reduction of the copper salt varies, according to the malts, from 3 to 20; comparative experiments have shown that where 3 to 5 cubic centimetres of the saccharified solution reduce 2 cubic cen- timetres of cupro-potassic tartrate under the conditions in- dicated, the malt may be considered as having a maximum saccharifying power ; 6 to 8 cubic centimetres correspond to a good malt, 9 to 12 to a malt of medium value, and if the quantity of saccharified solution necessary for the reduction is from 14 to 20 cubic centimetres, the malt may be consid- ered poor. The small quantity of maltose introduced with the in- fusion does not have much influence on the results, which, moreover, do not serve as a basis for accurate calculations, but merely as supplementary data for the estimation. The standard starch solution which we use for the deter- mination of the saccharifying power is prepared in the follow- ing manner : Let potato-flour soak at a temperature of 40 in a solution of 7 per cent hydrochloric acid, shaking the liquid every 6 hours. After 3 days decant the liquid, wash the mass with water to a neutral reaction, and dry at the ordinary temperature. The product obtained contains from \y\ to 18 per cent of water and is completely dissolved in warm water. By submitting to this operation different potato-flours of the same origin, the same product is constantly obtained, but the result differs much when starches from different sources are used, as the diastase, in this case, shows itself more active in some samples than in others. It is necessary that the standard starch, before being used for a determination, should be tried with a malt of known 200 THE ENZYMES AND THEIR APPLICATIONS. saccharifying power. If the same power is found in the starch thus tested, this can be considered as a standard mix- ture. Otherwise, one must, by repeated trials, determine the composition of a new starch solution which must be taken to replace 2 grams of the standard. Given, for example, a malt which possesses a saccharify- ing power of 4.5, measured by the standard starch, we will suppose that the same malt with another starch mixture would have a saccharifying power of 4.1. The question is to determine what quantity of the new starch must be taken to give the same result as 2 grams of the standard. To this end solutions are prepared which contain, instead of 2 grams per 100 of starch, 1.9, 1.8, 1.7 grams per 100, and the sacchari- fying power of the malt is tried with these solutions. If it is found that in the solution containing 1.7 g. of starch the saccharifying power is 4.5, 1.7 g. of the new starch will be constantly used instead of 2 grams, and under these condi- tions alone can it be used in place of the standard. One must always use a fresh starch solution, for we have found that this solution, although it keeps fairly well, acts differently with the same malt, according as it is fresh, or has been prepared for some time. This peculiarity is the more unexpected since we find no difference in acidity in the two solutions of starch. Determination of the Liquefying Power. — Weigh out 40 grams of standard rice-starch; dilute with a little water in a capsule; introduce the mixture into a 100 cubic centimetre calibrated flask ; rinse the capsule with a fresh portion of water which is poured into the flask and make the volume up to 100 cubic centimetres. From the mixture of starch and water briskly stirred, take with a pipette 8 specimens of 5 cubic centimetres each, and introduce them into numbered test-tubes of 10 cubic centimetres capacity; add to the con- tents of each tube the same quantity of infusion of malt pre- pared in the manner indicated above. For each of the num- bered tubes, containing 2 grams of starch and the infusion, QUANTITATIVE STUDY OF MALT. 201 prepare a second larger tube having a diameter of 19 milli- metres, a height of 19 centimetres and similarly numbered. In each of the large tubes place 14 cubic centimetres of dis- tilled water, and put them in a water-bath at 8o° ; then bring them one after another rapidly to the boiling point, and pour into the boiling liquid the starch paste and infusion contained in the smaller tube having the same number. Stir rapidly with a glass rod, rinse the tube which contained the starch with a cubic centimetre of water and add it to the contents of the large tube. Stir again with the rod, mark the hour exactly and leave in the water-bath at 8o° for 10 minutes. Take them one by one, stir the contents once more with a glass rod and plunge them into a water-bath at ioo°, where they stay exactly 10 minutes. After this operation all the tubes are rapidly cooled. A thermometer placed in one of them indicates the moment when the temperature reaches 1 5°, and it is at this point that the degree of liquefaction is ascertained. The tubes, thus cooled to 15 , are inverted one after the other. If the contents of the tube runs out in- stantly and without difficulty, the specimen is considered liquefied; a tube which empties entirely, but whose contents presents the consistency of a thick syrup, shows a three-quar- ters liquefaction ; a tube which does not become entirely empty shows a partial liquefaction. If the tube whose contents are entirely liquid has, for ex- ample, received 2 cubic centimetres of the non-diluted in- fusion, the liquefying power is expressed by 2. Comparative experiments with different malts have shown that a liquefying power of 1.5 to 2 shows a malt of excellent quality. A liquefying power of 2.5 to 3 corresponds to a malt of good quality, while a liquefying power of 3.5 to 4 shows a malt of doubtful quality whose value will depend 011 its saccharifying power. A malt with a liquefying power of 4 and a saccharifying power of 4 to 5 does passable work in the distillery, while a malt having the same liquefying power as the preceding and a saccharifying power of 7 to 9 must be 202 THE ENZYMES AND THEIR APPLICATIONS. considered poor. The difference between the saccharifying and liquefying powers is especially marked in dry malt. By drying, the saccharifying power is considerably weakened, while the liquefying power is much less changed. In the dry malt a liquefying power of 2 to 3 does not necessarily prove an excellent product : all depends on the saccharifying power. With a moderate saccharifying power the malt may hiave a great value, but with a saccharifying power of 12 the malt, even when it has a great liquefying power, cannot be used in the distillery. To determine the liquefying power, rice-starch, chosen with great care, is used. The rice-starches of different origin act differently with diastase at 8o° ; so rice-starches xnay be grouped in two classes. In the first are placed the products which, at the moment of liquefaction, become com- pletely colorless and transparent ; in the second, those which preserve a whitish tint and give an opaque liquefaction. The ffrst liquefy with much more difficulty than the second and the liquefying power of an infusion may vary much according as one or the other type of starch is used. At the beginning of our investigation we worked with a starch of the second type; we afterwards abandoned it be- cause we found that the starches which give a transparent liquefaction are preferable, the exact time of liquefaction being more easy to observe. If one wishes to obtain constant results the same starch must always be used in testing amylase. As a standard we use Hoffman starch and whenever we make a new mixture we verify the starch with the standard specimen. The verification is made with infusion of malt. Two grams of standard starch and 2 grams of the starch to be tested are liquefied with the same quantity of infusion at a temperature of 8o° for ten minutes. If the number of cubic centimetres of infusion necessary to liquefy completely the standard starch and the starch to be tested is the same, the two starches may be considered identical. Otherwise, in- QUANTITATIVE STUDY OF MALT. 203 crease or diminish the quantity of starch tested in such a way as to obtain liquefaction with the same quantity of infusion. If it happens, for example, that for the liquefaction of 2 grams of standard starch it takes 2.5 c.c. of an infusion of malt and that for the liquefaction of the same quantity of starch under examination it takes 3 cubic centimetres of the same infusion, one must weigh out 1.9 gr., 1.8 gr., 1.7 gr., of the starch to be tested and see which of these amounts is liquefied by 2.5 c.c. of infusion. If complete liquefaction is produced by 1.9 gr., it must be concluded that instead of 2 grams of standard starch one must take only 1.9 gr. of the starch which is being tested. Another method for transforming a certain starch into standard starch consists in acidifying it or making it alkaline. This method, which is preferable to the other, has for its basis the following observation : The standard starch is slightly alkaline, and if rice-starch is brought to the same degree of alkalinity it acquires all the properties of the standard starch. The liquefying power is so sensitive to the alkalinity of the liquid that the quantity of soda to be added cannot be determined by a single alkalimetric measurement. If the difference in alkalinity of the two starches corresponds to 2 cubic centimetres of decinormal solution of soda, one must add only half of the alkaline solution for 50 grams of starch, and then keep adding tenths of cubic centimetres until the two starch pastes liquefy with the same quantity of infusion of malt. The standard starch for saccharification, as well as the standard for liquefaction * keeps without change in bottles with ground stoppers and can be used for determinations, for at least two years. We have also observed that dry malt, kept in the same * Standard starches which we use are for sale at Drosten's, rue du Marais, Brussels, and at H. Koenig's, manufacturer of chemicals, Leipzig. 204 THE ENZYMES AND THEIR APPLICATIONS. way, preserves for years its saccharifying and liquefying- powers. We give here two determinations of malt made accord- ing to the method indicated above. Malt A. — Russian barley, soaked 2\ days with aeration,, malted in revolving drums. Minimum temperature i8°, maximum 21 °. Duration of germination, 4 days. Water 48.04%. General aspect and odor, normal. 3 non-germinated grains. 34 grains whose plumule was shorter than- the grain, having a plumule of the length of the grain, having a plumule i^ times as long as the grain, having a plumule more than twice as long as the grain. The liquefying power and the saccharifying power of this malt were determined in three different specimens: 1st. In unsorted grains. 2nd. In grains in which the plumules are twice as long as the grains. 3rd. In grains in which the length of the plumules does- not exceed that of the grain. 100 grains include 30 21 12 Unsorted Malt. Malt with Plumule twice the Length of the Grains. Malt with Plumule^ not longer than Grains. 0.6 O.585. 0.53 17. C.C. 9.5 c.c. 20.7 C.C. f L 2.5 c.c. not liq. 3 " " " 3-5 " . i." 4 " liquid 2.5 c.c. not liq. 3 " " " 3.5 " liquid 3.5 c.c. not liq- 4 " i " QUANTITATIVE STUDY OF MALT. 205 The maltose indicated for each infusion shows the sugar content of the dilute infusion which was used for determining the saccharifying power. In the grains not classified, when coming out of the apparatus, there is found a saccharifying power of 17 and a liquefying power of 4. Therefore the malt is decidedly me- diocre; the trial of fermentation with different quantities of malt has shown us that it takes 18 parts of this malt per 100 of rice to insure a complete transformation. The comparative analysis of the three specimens confirms for us the facts established by the Institute of Berlin, namely, that the development of the plumules coincides with an in- crease in the quantity of the enzymes. Malt B. — Saladin pneumatic system malt-house. Malt- house of Buir, near Cologne. Small Russian barley, soaked 2\ days without aeration. Duration of malting, 9 days. Temperature, minimum, 18°; maximum, 23 . The sprouting is uniform and the plumules do not ex- ceed the grain. Non-germinated grains, 3. Water, 47%. Maltose of infusion, 0.74. Saccharifying power, 4.65. 25 c.c. = liquid. Liquefying power . (2 c.c. = /a liquid. The liquefying power as well as the saccharifying power shows a malt of excellent quality. The quantity of malt necessary for fermenting 100 kilograms of rice is 8 kilo- grams. The method we have just indicated is applicable to the investigation of malts of barley and rye. For the analysis of malt of maize other conditions are necessary because this malt always contains relatively small quantities of diastase. For the preparation of the infusion 12 grams of ground malt are taken instead of 6, and, to de- 206 THE ENZYMES AND THEIR APPLICATIONS. termine the saccharifying power, only i cubic centimetre in- stead of 2 of the cupro-potassic solution is used. A maize malt of fine quality always contains 4 to 8 per cent of non-germinated grains. It possesses, under the con- ditions indicated, a saccharifying power of 4 to 6 and a lique- fying power of 2.5 to 3. By comparing the malt of maize with barley malt of first quality, it is found that it is only one fourth as active and in practice it is necessary to use 4 to 5 times as much of this as of barley-malt to secure the same result. Method of Analysis of Sweet and Fermented Mashes. — The saccharifying power of the mash may be determined by aid of the coloration which the mash gives with iodine. This is done' in the following manner: take 6 specimens of 20 cubic centimetres each of a fresh 2% solution of soluble starch and place each sample in a numbered test-tube; add with a pipette divided into tenths of a cubic centimetre .25 c.c, .50 c.c, .75 c.c, 1 c.c, 1.25 c.c, and 1.5 c.c, of the sac- charine or fermented must to be tested; place the tubes in a water-bath at 6o° for an hour; cool and add to the contents of each tube half a cubic centimetre of a very dilute solution, of iodine and observe the coloration at the moment the iodine is added to the liquid. A saccharification made with .25 c.c. of sweet mash, not colored by iodine, corresponds to the maximum of sacchari- fying power, and if this result is reached, one may be sure that a greater quantity of malt than necessary has been used for the fermentation. The absence of coloration in a tube which has received .75 c.c. of solution shows that the sweet mash possesses a saccharifying power sufficient for fermen- tation, at least if the liquefying power of this must is normal. If a coloration appears- in the liquid containing 1.25 c.c. of the saccharified mash, it is certain that this mash does not contain the necessary quantity of malt and it is useless to pay any attention to the liquefying power. Failure to give color with iodine of solutions which have received 1 cubic QUANTITATIVE STUDY OF MALT. 207 -centimetre of mash shows a sufficient quantity of diastase if the liquefying power is very great. Otherwise, the saccha- rified mash is not rich enough in active materials. This method is of great service for the control of mashes during fermentation; the saccharifying power is determined by coloration with the solution of iodine at the beginning of fermentation, and this operation is repeated after 30 and 60 hours. The saccharifying power determined by this method at the beginning of fermentation should not change much up to the end of the operation. If it is found that, at a certain time, twice as much liquid is needed as at the be- inning to have no coloration with iodine, one may be cer- tain that there is a change in the diastase and it is important to add more infusion. A mash fermented for 86 hours should have a sacchari- fying power of between 0.75 and 1, that is, with 0.75 to 1 cubic centimetre of mash no coloration by iodine should be obtained. A saccharifying power of 1.5 at the end of fer- mentation shows a lack of diastase. These data are applicable to mashes of rice of a density of 17 to 19 Balling. Mashes of grains and potatoes act dif- ferently. In these, the weakening of the diastases during saccharification and the fermentation occur much more rap- idly. One must seek to have mashes of a saccharifying power of I to 1.25 at the beginning of the operation and of 2 at the end of fermentation. Our method of determination of malt and mash is now introduced in the central stations of the Association of Dis- tillers of Bavaria and Austria-Hungary. The directors of these stations, Professors Kruis and Biicheler 'have ex- pressed to us their entire satisfaction. With a little experience, one can succeed in having a complete control of the work by means of this method. BIBLIOGRAPHY. J. Effront. — Contributions a l'etude de l'amylase. Monit. scientifique, tome VIII, p. 541, et tome X, p. 711. CHAPTER XVII. MALTASE. Glucase of Cusenier. — Maltase of yeast. — Properties. — Differences be- tween the optimum temperatures of different glucases. — Maltase of moulds. — Manner of action upon starch. — Processes of secretion. — In- fluence of nitrogenous food. — Influence of carbohydrates. — The dif- ferent amylomaltases of Laborde. Maltase or glucase is an enzyme which acts upon starch, dextrins, and maltose. The existence of an enzyme acting - on maltose was doubted for a long time. It is, however, evident that mal- tose, to be assimilated by living cells, must be hydrated and transformed into glucose. In 1865, Bechamp found in the urine the presence of an enzyme acting on maltose which he called nefrozymase. Brown and Heron discovered an analogous active principle in the pancreatic juice and the small intestine of the pig. Later Emile Bourquelot confirmed the observation of Brown and Heron, demonstrating the presence of the same principle in the pancreas and small intestine of the rabbit. The diastatic liquids obtained by these workers presented very varied properties. They evidently contained diastases of very different natures and it was very difficult to establish definitely the presence, in the liquids studied, of a special ferment acting solely upon maltose. The discovery of the active principle which decomposes maltose into two molecules of glucose dates from 1886. It belongs to Leon Cusenier, who named this enzyme glucase. Cusenier, by soaking ground maize in water at a tern- 208 MALTASE. 209 perature of 50 , found that a great part of the amylaceous matter passed into solution, and that the rotatory power of the saccharine liquid decreased as the soaking was pro- longed. This observation led to researches in regard to the nature of the sugar formed, as well as that of the agent pro- ducing this transformation. A series of experiments undertaken with this aim resulted in finding that the maize contains a special ferment which acts on starch, giving glucose and dextrins, which in the end are themselves transformed into dextrose. The optimum temperature of this enzyme is 6o° ; its tem- perature of destruction, about 70 . This enzyme acts likewise on maltose and transforms it into glucose. Its presence has been observed in almost all the cereals, but in a much smaller quantity than in maize. There exists in the latter an amount of glucase which is more than enough to transform into glucose all the starch it contains. According to Gedulde, it is possible to isolate the glucase of maize by soaking a grist with water and then precipitating the filtered liquid with alcohol. The product obtained, and dried in vacuo, is a brownish mass which is friable and has the following properties: It contains about 8 to 12 per cent of nitrogen. It gives the reaction of guaiacum and hydrogen peroxide. Pre- cipitated by alcohol, it is redissolved in water with difficulty. It possesses a relatively weak activity: with one part of precipitated active substance only 100 parts of maltose are transformed into glucose. Its optimum temperature is from 56 to 6o°. Above 6o° there is found a perceptible slackening in the hydration it produces. Above 70 glucase is without action. This enzyme acts more energetically on the products of decomposition of starch than on the starch itself. According to Beijerinck, glucase can easily be prepared from maize which is hulled and deprived of its oil. The fol- 2io THE ENZYMES AND THEIR APPLICATIONS. lowing method is employed: Three kilograms and a half of maize thus prepared are treated with 5 litres of water, with the addition of 500 cubic centimetres of 96% alcohol and of 2 grams of tartaric acid. This mixture is kept for 30 hours at 15 or 20°, then filtered. Thus 4-J litres of a clear liquid are obtained in which is produced a partial precipitation by adding to it an equal volume of 96% alcohol. The deposit thus procured is treated with acidulated water in the pro- portion of 0.4 grams of tartaric acid per litre, then a little alcohol is added. The precipitate is partially redissolved in the liquid and the insoluble part is collected on a filter. This, insoluble product is very rich in glucase according to Beijerinck. It contains 1.11% of nitrogen. Other products may be obtained by adding alcohol to the filtered liquid. The precipitates which still contain a certain quantity of diastases in solution are collected. But the pre- cipitates obtained by these treatments, while showing a nitro- gen content of 4.78 and 2.20 per cent, are less active than the insoluble part spoken of before. The glucase obtained by Beijerinck is not, however, an absolutely pure product, as he himself found. Its impurities must be due to mucilages. According to Beijerinck, glucase acts on maltose, on starch, and on dextrins, but more actively on maltose than on dextrins; it causes the transformation of starch with much difficulty. According to Gonnerman, glucase or an analogous fer- ment exists in beets frozen or in germination. Dubourg and Rhomann have discovered its presence in the blood. It is also found in the urine and in yeasts, as well as in a great number of moulds. The secretion of maltase by yeast is of particular in- terest. Maltose has been considered for a long time as a directly fermentable sugar and, in reality, during fermentation it is impossible to tell when the maltose is transformed into glucose. This is why the fermentation of maltose has been MALTASE. 2H regarded as an intracellular transformation in which the solu- ble ferment has no part. Bourquelot, Lintner, and Emil Fischer have studied the question very closely and established the fact that yeast always contains a certain quantity of maltase which is re- tained in the cells and which is diffused with difficulty into the surrounding liquid. To extract the enzymes of yeasts, the cells must be crushed with pumice-stone or pounded glass and the mass then soaked in water. One may also have recourse to an- other means which seems more expeditious: fresh yeast is spread in a very thin layer ; it is slowly dried at 40 and then soaked in water. Under these conditions, the maltase of the yeast becomes soluble. The enzyme extracted from the yeasts by this method differs in many respects from the active substance of the maize which hydrates the maltose. According to Gedulde, the glucase of maize can be pre- cipitated from its solution in the active state by alcohol; the maltase of yeast, on the contrary, is almost completely destroyed by this reagent. Maltases of different origin also posses* a very different sensitiveness towards heat. Lintner, by exposing to dif- ferent temperatures 3 specimens of a solution of maltose, to which had been added the same quantity of glucase ex- tracted from yeasts, obtained quantities of glucose varying according to the temperature at which the action took place. Temperature. q{ ^AcXn. GluC ° SC formed ' 35°. 2 hours 2.90 gr. 40 " 3-°9 45 " 2 -° 8 The optimum temperature, according to these experi- ments, would be 40°, while Cusenier's glucase possesses art optimum temperature of 56° to 6o°. By trying yeast maltase at temperatures of'40 and 50 2 12 THE ENZYMES AND THEIR APPLICATIONS. Lintner found the formation after two hours of action of the following quantities of glucose: Temperature. Glucose formed. 40 1.8 5o° 0.3 Therefore the temperature of 50 almost entirely de- stroys the maltase of yeast, while if glucase of maize is used, the maximum result is not reached at that temperature. A great number of moulds possess the property of trans- forming starch into sugar. By studying the action of Aspergillus orizce, Atkinson was the first to find that the diastase of this mould brings about the transformation of amylaceous materials in a dif- ferent way from that of the diastase of malt. The final prod- uct of this reaction of Aspergillus orizcc is dextrose and not maltose. Since then the same fact has been ascertained by Bodin and Rolants for Amylomyces Rouxii, then by Bourquelot and Laborde for Aspergillus rtiger, Penicillium glaucum and Euro- tiopsis Gayoni. There is every reason for believing that it is a general fact and that many other moulds render starch assimilable by the aid of the glucases they secrete. Diastatic liquids obtained by soaking moulds act on starch, dextrins, and maltose, forming glucose. According to Atkinson, the transformation of starch by Aspergillus orizcc is done by successive hydrations of the molecules, with formation of maltose as an intermediate product, but the analyses cited for the support of this opinion are not at all conclusive. Laborde, on the contrary, found that there is a direct transformation without the for- mation of maltose. He has found the same facts in the case of Aspergillus niger, Penicillium glaucum and Eurotiopsis Gayoni. To demonstrate the presence of glucase in an active liquid, a 2 per cent solution of maltose is added to a certain MALTASE. 213 quantity of the liquid under examination. A trace of chlo- roform or thymol is added and the solution is left at a tem- perature of 45 for 24 hours. By examining the rotation of the liquid before and after the experiment, one can easily follow the course of the hydration. The rotatory power of the maltose is (a)d -f- 138.4 and that of glucose 52.4 The glucase of moulds acts more strongly on starch than on maltose. The formation as well as the diffusion of glucases of moulds take place under the same conditions as secretion of sucrase by Aspergillus n'iger. During the development of the plant, the quantity of glucase increases as the nutritive substances diminish and the maximum amount of enzyme appears in the plant at the moment when it begins to utilize the reserve substances. Thus the glucase produced in the moulds is retained inside the cells and diffuses with great difficulty until the nutritive-medium begins to be exhausted. According to Pfeffer and Katz, the addition of sugar to the culture medium generally diminishes the production of glucase, but it is observedthat the different kinds of moulds are more or less sensitive to this action. Thus Penicillium glaucum does not secrete glucase when there is 10 per cent of saccharose, while in an amount of 30 per cent this sugar does not entirely stop its secretion by Aspergillus niger. Glucose acts in the same way as saccharose. The presence of maltose in the nutritive medium influ- ences the secretion in a less degree. Penicillium glaucum still produces glucase in a medium containing 10 per cent of sugar. Nitrogenous food also has a great influence on the pro- duction of glucase. The well-nourished cells yield the enzyme in largest quantity. Moulds may take nitrogen from very different sources. Thus alkaline nitrates, peptone, casein, and urea are equally favorable to the cultivation of Eurotiopsis and furnish practi- 214 THE ENZYMES AND THEIR APPLICATIONS. cally the same quantity of glucase. Sulphate and chloride of ammonium, however, furnish decidedly less, and these sub- stances act very unfavorably from the point of view of the formation of diastase. According to PfefTer and Katz, the secretion of glucase by Penicillium glaucum and Aspergillus niger is limited by the quantity of enzyme already present in the nutritive medium. By removing the glucase from the medium with tannin, they have found a more abundant secretion of diastase. It is, however, not very probable that taking away the active substance can cause a further formation of diastase. It is more plausible to think that in precipitating the diastase by tannin, one favors the diffusion of the active substances al- ready formed but retained by the cells. Of all the moulds studied from the point of view of their action on amylaceous materials, Aspergillus orizce is the most active, and it is this which really secretes the greatest quan- tity of maltase. Mucor altcrnens and Amylomyccs Rou.vii both belong to the class of moulds which is rich in maltose. Aspergillus niger and Penicillium glaucum possess a diastatic power which is much weaker, and Eurotiopsis occupies the last place as re- gards the secretion of glucase According to Laborde, the saccharifying diastases of As- pergillus niger, Penicillium glaucum and Eurotiopsis Gayoni, and which he designates under the name of amylo-maltases, have different characteristics. Starting with this observa- tion, Laborde conceives the existence of 3 different diastases having common characteristics but distinguished by their sensitiveness towards physical and chemical agents, as well as by the intensity of their action. By allowing enzymes of the three moulds to act, under the same conditions on 2 per cent starch paste, Laborde found perceptible differences for the 3 diastases, which are given in the following table : MALTASE. "5 Origin of the Diastatic Liquids. Aspergillus niger. Penicillium elaucum. Eurotiopsis Gayoni. Duration in Hours of the Action. 96 12 48 96 12 48 96 Folariscopic Rotation of Glucose. the Liquids. % gr. 17-5 i-3i 14.0 1. 61 14.0 1.66 12.5 I.3I 12.0 1. 61 12.0 1.72 7.0 0.S0 9.0 1. 61 9-3 1.92 Dextrins. gr. O.56 O.3I O.30 0.31 0.2I O.18 O.16 O.06 O.OO The difference in the course of hydration is especially- shown by the rotatory powers of the liquids as well as by the relation of the quantity of maltose and that of dextrins. For 1.60 of glucose formed with the three active liquids, there are found noticeably different quantities of dextrins as well as different rotations. These differences probably come from the fact that the starch is not liquefied with the same facility by diastases of different origin. Maltases of different moulds are further differentiated by their optimum temperatures as well as by their temperatures of destruction: Temperature Temperature Optimum. of Destruction. Aspergillus niger 6o° 8o° Penicillium glaucum .45 70 Eurotiopsis Gayoni. 50 75 As regards the action of heat, the maltase of Aspergillus niger approaches the maltase of maize, while the active sub- stance of Eurotiopsis Gayoni resembles rather the diastase of Penicillium glaucum and the yeasts. There is also found another difference between the mal- tase of cereals and the ferments of moulds. While the first acts with more difficulty on starch than on maltose, the second acts more vigorously on starch than on the products of hydration. 216 THE ENZYMES AND THEIR APPLICATIONS. Moreover, these differences between the manner of ac- tion of diastases are apparent rather than real. An extract of maize acts energetically in the cold on starch and on mal- tose. But if, on the other hand, one precipitates the active substance of an infusion of maize with alcohol, one obtains a product which hardly acts at all on starch, but which, on the contrary, acts very strongly on maltose. This difference evidently comes from the change of medium. In the first case the action is due to the diastase accompanied by foreign substances which influence its ac- tion. In the second, it is the effect of the diastase alone, or of this diastase accompanied only by substances exercising a very slight influence on the transformation. It must then be admitted that all the differences which we have found in maltases of different origin comes exclu- sively from foreign substances which accompany them and influence their sensitiveness towards reagents. Most moulds secreting maltases develop very easily in the mashes of the brewery and the distillery, as well as in yeast-water to which carbohydrates have been added. Sanguinetti made a comparative study of Aspergillus crizce, Mucor alternans and Amylomyces Rouxii as regards sac- charifying and oxidizing powers. He cultivated them in mashes containing starch, dextrin or other carbohydrates, and observed the progress of diastatic secretions as well as the influence of nutrition on these secretions. Here are some of his experiments: In a flask of a capacity of 1,500 cubic centimetres he placed 500 cubic centimetres of yeast-water and added 15 grams of starch or dextrin. He sterilized the liquid, and after cooling it, sowed dif- ferent flasks at a temperature of 30 with the spores of dif- ferent moulds. He allowed the plants to develop for 10 days, and shook the flasks twice a day to prevent the formation of MAL TASE. 217 spores. He then determined the weight of the plants formed and determined the sugar and alcohol in the liquid. The results of experiments made with yeast-water and starch are here given : Weight of plant Dry extract at ioo" Total acidity, H 2 S0 4 Weight of alcohol Reducing sugar (as glucose) Total reducing sugar after saccha- rification by hydrochloric acid. . . Loss of alcohol per 100 of starch . . Control. 19.00 O.127 16.67 Aspergillus orizae. gr. 2.081 5-20 0.670 2.77 I.30 Mucor alternans. 2.25 O.667 6.27 O.980 1.58 Traces 2.99 41// Amylo- myces. gr. 2.080 4- 50 O.660 3-96 Traces 3-75 25. 7# When the quantity of carbohydrate which has disap- peared is compared with the quantity of alcohol formed, it is found that there is obtained: With Aspergillus oriza for 14.42 of sugar transformed, 2.77 alcohol. Mucor alternans " 13.68 " " " 1.58 Amylomyces Rouxii " 12.92 " " " 3.06 " Aspergillus orizee shows itself the most active and leaves in the 15 per cent solution of starch .85 gr. of untransformed dextrin, while the Mucor alternans and Amylomyees furnish twice as much unattacked dextrin. When in these experiments starch is replaced by dextrins a still less complete saccharification is observed. After 10 days of development there is still found 3.80 of dextrins. If the time of the action and the weight of the plant are taken into consideration, it is seen that the quantity of active substance secreted by the moulds is relatively very slight, even with Aspergillus orizee; 2.081 gr. of plant do not suf- fice, as we have just seen, to transform 15 grams of starch. while .5 gr. of malt produce, under similar conditions, a com- plete transformation. The maltase of fungi is very sensitive to the medium, fiodin and Rolants have studied the action of oxygen and of 2i; THE ENZYMES AND THEIR APPLICATIONS. the acidity of the medium. The following experiment af- fords some data on this subject: Bring a distillery residue to different degrees of acidity and, in the sterilized liquid, cultivate Amylomyccs under dif- ferent conditions. In one culture let the plant develop at the surface of the liquid (culture S) ; in another let the development take place deep down (culture P) ; in a third (culture A) let a current of air pass for 48 hours. The following results were obtained after a fermentation of 4 days at a temperature of 26 : Alcohol, per litre Acidity Reducing sugar in glucose Total sugar ■ Weight of amylomyces obtained in a pressed state Neutral Residue. 3-4C.C. O.36 4-83 10.23 10.23 5.5CC. 0.83 2-33 7-3 4.6gr. 3 c.c. 0.4 1.64 5-57 8.15 gr Residue with Acidity equivalent to 3.4 gr. of Sulphuric Acid. 3 C.C. 2.69 4-31 17.71 17.71 i.Sc.c 3-13 3-5 17.71 o.25gr 1.7C.C. 2.69 3-4 13-28 2.30gr It is clear that aeration is very favorable to the develop- ment of the plant, since there are 8.15 gr. of plants in the aerated liquid, while a culture from which the air was ex- cluded furnishes only 4.6 gr. The quantity of acid formed during the development is in direct proportion with the initial acidity and is smaller ac- cording as the acidity of the medium at the beginning is stronger. Maltase acts very well in a slightly acid medium, but its action is stopped by an amount of organic acid correspond- ing to 2 grams per litre of sulphuric acid. BIBLIOGRAPHY. Dubourg. — Recherches sur l'amylase de l'urine. These, Paris, 1889. Bourquelot. — Recherches sur les proprietes physiologique du maltose. Comptes Rendus, 1883. MALTASE. 219 Cusenier. — Sur une nouvelle matiere sucree diastasique et sa fabrication. Monit. scientif., 1886, p. 718. Brown and Heron. — Ueber die hydrolitischen Wirkungen des Pancreas und des Diinndarms. An. chim. et pharm., 1880, 228. Lintner und Kroeber. — Verschiedenheit der Hefeglucase von Maisglucase und Invertin. Ber. der deutsch, chem. Gesellsch., 1895, p. 1050. G. H. Morris. — Hydrolyse de la maltase par la levure. Proc. Chem. Soc, 1895. Laborde. — Recherches physiol. sur une moisissure d'Eurotiopsis Gayoni. Ann. de l'lnst. Pasteur, 1898. Sanguinetti. — Contrib. a l'etude de l'amylomyces Rouxii. Ann. de l'lnst. Pasteur, 1897. Bodin et Rolants. — Contrib. a l'etude de l'utilisation de l'amylomyces Rouxii. Biere et boissons fermentees. Mars. 1897. Pfeffer und Katz. — Schriften der konig. sach. Gesellschaft der Wissen- schaft. Leip., 1896. Fischer und Lindner. — Enzyme von Schizosaccharomyces octosporus und Saccharomyces Marxii. Berichte der deutschen chemischen Gesell- schaft, 1895, I, p. 984. Beijerinck. — Centralblatt fur Bakteriologie, n. Abth., 2. Jahrg., 1898. Wroblewsky. — Ueber die chemische Beschaffenheit der amylolytischen Fermente. Berichte der deutsch. chem. Gesellschaft, 1898. CHAPTER XVIII. INDUSTRIAL APPLICATIONS OF MALTASE. CEREALOSE. Industrial Manufacture of Glucose by Enzymes. — Cuse- nier, having found glucase in maize, sought to apply this dis- covery to the glucose industry. By replacing, in this manu- facture, acid by enzymes contained in the grains, he suc- ceeded in making a product of great value which is found in commerce under the name of cerealose. Cerealose is obtained in the form of a crystalline mass containing maltose and glucose. The manufacture of cerealose is as yet little developed,, for the process of Cusenier leaves much to be desired from the point of view of yield and cost of production, which is. greater than that of the glucose obtained with acids. The difficulties encountered in this industry are various. The maize contains hydrating ferments in a quantity more than sufficient to transform all the starch into sugar, but practically it is very difficult to have conditions favorable to the action of these ferments. It results that the transfor- mation is far from complete. When ground maize is soaked with 4 volumes of water at a temperature of 6o° for 24 hours, 60 to 65 per cent of amy- laceous materials is extracted in the form of sugar. A more prolonged soaking does not bring about a perceptibly bet- ter result. The enzyme acts only on a part of the starch and unattacked grains of starch always remain, although the saccharine solution obtained is very rich in active substances. It is this peculiarity in particular which makes the manufac- 220 INDUSTRIAL APPLICATIONS OF MALTASE. 221 ture of glucose by glucase difficult. The operation can really never be carried to perfection. One is obliged to have recourse to a continuous method of work. The method of procedure is here outlined : Five hundred kilograms of coarsely ground maize are poured into an apparatus furnished with a double wall and a stirrer. Twenty hectolitres of water at 65 are added. The temperature is kept at 58°-6o° for 6 to 8 hours, the stirrer being kept in motion. The transformation of amylaceous materials into glucose is followed by the observation of the density and rotatory power of the mash. During all the course of the operation, the density of the liquids increases, while the rotatory power diminishes. The operation is considered as finished when the mash containing 10 per cent of dry materials marks 40 to 45 on the Soleil polarimeter. Then the saccharine solu- tion is separated by filtration from the extracted grain, which still contains quite considerable quantities of unattacked starch. The juice is decolorized with bone-black and evap- orated in a vacuum to a concentration of 40 to 42 ° Baume, after which the syrup is placed in vessels, where it solidifies at once after it has been primed with crystallized glucose. The starch not attacked in the first operation is subjected to further treatment. The grist of maize remaining on the filter is submitted to cooking under slight pressure. The starch obtained by this operation is saccharified at a temperature of 63 , by the aid of a small quantity of malt (1 to 2 per cent), and the dextrinated mashes replace water in the following operation. With the malts coming from 500 kilograms of maize, nearly 20 hectolitres of dextrinated mash is obtained. Into this mash are introduced 400 kilo- grams of ground maize and saccharification is allowed to pro- ceed for 6 to 8 hours at a temperature of 58°-6o°. The maltase contained in the barley malt can also be utilized for the manufacture of glucose. 222 THE ENZYMES AND THEIR APPLICATIONS. The infusion of malt has very little action on the maltose, but the crushed malt acts vigorously on the maltose syrups, which are transformed into dextrose syrups. For this kind of work it is well to put the malt in contact with the syrups at 20°-25° Baume and not with diluted juices. In saccharified and very concentrated mashes, the extrac- tion of the diastases of the malt is made more easily than in syrups of low concentration. Cerealose has the following average composition : Maltose 2.5% Glucose 72 " Dextrin 2.5 " Water 20 " CHAPTER XIX INDUSTRIAL APPLICATIONS OF MALTASE.— (Continued.) JAPANESE AND CHINESE YEASTS. Manufacture of Japanese yeast. — Preparation of koji. — Changes produced in the rice. — Composition of koji. — Action of salts. — Manufacture of " moto " leaven. — Manufacture of " sake " beer. — Composition of moto. — Composition of sake. — Manufacture of Chinese yeast. — Prop- erties of Chinese yeast. — Influence of temperature and chemical agents. — Oriental methods of distillation. — Utilization of Oriental processes in the distilleries of Western countries. — Works of Taka- mine, Collette, and Boidin. Japarjese Yeast. — In certain countries of the far East al- coholic beverages are manufactured with amylaceous ma- terials. In Japan a kind of beer is made which is called sake. In China and Cochin-China a brandy is prepared from rice, " choum-choum," which contains from 34 to 42% of alcohol. The methods employed by the Orientals differ radically from European methods. The saccharification of the amy- laceous materials and the fermentation are brought about by special ferments which are cultivated as an industry. The active agent which the Japanese use is called koji. The ferment which serves for the manufacture of Cochin- China brandy is called niigcn or men. Chinese and Japanese yeasts owe their activity to moulds which secrete maltase and probably zymase. In the Chinese yeast, the predominating organism is Am\lomyccs Rouxii. Koji owes its activity to Eurotium orizce. Korschelt and Atkinson published the first data on the preparation and utilization of Japanese yeasts. 223 224 THE ENZYMES AND THEIR APPLICATIONS. Preparation of Koji. — The grains of rice destined for the manufacture of koji are first cleansed and beaten so as to free them of their covering, then they are submitted to a soaking of a dozen hours. The grains are then cooked in a current of steam until they have reached a certain consist- ency, then are spread on mats, which are vigorously shaken to prevent the grains from uniting in lumps. The rice is then sown with the spores of a mould, Eurotium orizcE. The spores of this mould, which form a commercial product* in Japan, are mixed with the rice in the proportion of I part of spores to 40,000 parts of rice. They are distributed throughout the mass by a vigorous shaking of the mat and the whole is taken to the malt-house. Atkinson, professor at the University of Tokio, to whom we owe the description of this industry, describes the special construction of these malt-houses. They are long subter- ranean passages, joining each other, 4 to 10 metres long, from 2.10 m. to 2.40 m. wide and 1.20 m. high. These malt- houses are never heated except at the beginning of the cold season. The rice, mixed with spores, is heaped up in the malt-house, covered with mats and left in this state over night. On the second day it is sprinkled with a certain quantity of water, if it is not to be used for the manufacture of the beer called sake. The koji is then spread in a very thin layer and left. The third day the rice is again heaped up for about 4 hours. At the end of this time the grains are covered with a light fleece coming from the mycelium of the mould. The rice is then cooled by shaking, and disposed in thin layers on mats, to which is given a lateral motion to prevent the formation of lumps. Under these conditions, the vegetation develops ; the mycelium winds about among the grains and the fourth day the koji forms a kind of cake which is all ready to be used. Koji is used in different branches of Japanese industry; in INDUSTRIAL APPLICATION OF MALTASE. 225 the making of bread, in the preparation of the " soy " sauce; but especially in the brewery for the preparation of sake. Temperature of Germination. — In Atkinson's account are found some data on the variations of temperature in the course of this manufacture. When after soaking, the grains of rice have been dried by shaking the mats, they have a tem- perature of 28°-30°. The second day, after being scattered, the temperature falls to 23°-26°, to rise again afterwards in the malt-house to 30 . The next day, the third, Atkinson observed it to reach 40°-4i°. The rice is then cooled, but it heats again to 37 . These figures are, however, only ap- proximate ; they change according to the time of year. In the month of May the temperature of the malt-house has been observed to be from 24°-26° ; the temperature of the koji was then from 25°-26°. In the month of December the thermometer marked 2j° in the malt-house and the rice showed a temperature of 39 . This increase in temperature is explained by very vigorous oxidation produced by the moulds : often a difference of 10 degrees is found between the temperature of the koji and the external temperature. At a temperature of 40 considerable losses of starch must occur, as well as a perceptible change in the diastase secreted by the moulds. According to more recent data on the preparation of koji, the Japanese manufacturers take pains not to exceed a tem- perature of 25 . The whole duration of the manufacture from the moment of sowing to that of the complete develop- ment of the plants is only three days. Changes Produced in the Rice. — The transformation of the rice into koji appears then like a true phenomenon of oxidation. In fact, the rice used in this process loses as much as 1 1 per cent of its starch ; this carbohydrate is oxi- dized with liberation of carbonic acid and formation of water. The koji presents the aspect of a cake formed of grains of rice bound together by fungus threads. The grains ex- tracted from this cake are covered with a sort of down, and 226 THE ENZYMES AND THEIR APPLICATIONS. an incision made on one of them shows that the exterior cells are penetrated by the threads of the mycelium, while the in- terior remains unattacked and even acquires a certain hard- ness. The mould in the course of development attacks the al- buminoid materials which are found in the rice ; these sub- stances become soluble and the fermenting power of the koji increases with their solution. The composition of koji dried at ioo°, according to At- kinson, is here given : f Dextrose 25.02 r, , ., . _,ri 1 Dextrin (by difference) 3.88 Part soluble in water: 37.76%... -{ c , , , v / J ■" ' j Soluble ash 0.52 [ Soluble albuminoids 8.34 f Insoluble albuminoids 1.50 j Insoluble ash 0.09 Part insoluble in water: 62.24$. i Fatty bodies 0.45 I Cellulose 4.20 (^ Starch (by difference) 56.00 The fresh koji contains 25.82 per cent of water. The growth of Enrotium orizce on the grain causes the proportion of soluble nitrogen in the latter to increase. In dried koji there is found a total of 9.84 per cent of albuminoid materials, the soluble albuminoid materials being represented by 8.34 per cent. In the rice not transformed into koji the quantity of soluble albuminoid materials is only 1.38 per cent. There is also a difference of solubility between rice and koji. When the koji has not been heated to ioo°, it for the most part dissolves. After a short contact with cold water 12 or 15 per cent of its total weight dissolves. If the contact with water is prolonged the diastase continues to act and at the end of a longer or shorter time 30 to 60 per cent of the koji enters into solution. Action of Salts. — The enzyme of koji is influenced by the acidity of the medium. Lactic acid, in an amount of 0.05 per cent, is favorable; the amount of 0.1 per cent possesses a re- tarding action. The diastase is equally sensitive to the action of sodium hloride. The influence of this salt was determined by INDUSTRIAL APPLICATION OF MALTASE. 227 Watanabe. To 5 grams of dry starch, gelatinized and cooled, he added different amounts of sodium chloride and then added to each of these specimens the same quantity of extract of koji. He then left it at the ordinary temperature for 1 hour, then brought the volume up to 250 cubic cen- timetres and filtered. The action of the salt is determined by the aid of the reducing power and the rotatory power of the solution. Common salt Reducing power Specific rotatory per 100 of starch. (on copper oxide). power. o 30.8 173-8° 10 28.6 179.3 30 25.1 182.6 50 23.8 187.6 75 2 °-9 190-3 100 20.1 189.1 150 19. 1 190.2 200 18.0 192.2 300 16.9 194.1 500 14-4 197-5 The increase in rotatory power is easily seen and the diminution in reducing power as the amount of sodium chloride increases. Manufacture of Moto. — Koji is used in Japanese industry as agent of saccharification, fermentation, and the manufac- ture of the beer called " sake." The operations necessary for this manufacture are divided into two classes : first the prep- aration of a strong ferment, called moto, then the manufac- ture of the mash and its fermentation. For the manufacture of moto, there are used as raw materials rice cooked by steam, koji, and water mixed in the following proportions: Rice 68 parts. Koji 21 " Water 72 " 2 28 THE ENZYMES AND THEIR APPLICATIONS. The manufacture of moto is carried on in two stages. In the first stage, which lasts from 5 to 6 days, the mixture of grains and koji is distributed in different vessels. Under the influence of the koji, the rice-starch becomes liquefied and saccharified. The temperature does not exceed a few de- grees, and the fermentation is extremely slow. In the second stage, the combined liquids are heated to about 25 ° ; fermen- tation commences, and the temperature rises, but never ex- ceeds 30 . The manufacture of moto lasts from 16 to 18 days, and the mature leavens show as much as 10 per cent of alcohol. The following table shows the composition of moto in the first stage of the manufacture : After 3 days. After 5 days. Per cent. Per cent. Dextrose 7.35 12.25 Dextrin 5.12 5.69 Glycerin ~\ Ash J- Trace 0.48 Albumen J Fixed acids 0.017 0.019 Volatile acids .... 0.008 Water by difference 87.513 81.553 Undissolved starch 20.43 1 5A& Its composition, during the second stage, is shown by the following table : 7 days. 10 days. 12 days. 14 days. Alcohol 5.2 8.61 9.41 0.62 Dextrose 5.4 0.99 0.49 0.50 Dextrin 7.0 2.81 2.72 2.57 • Glycerin 1.14 2.82 2.35 1.93 Fixed acids 0.31 0.24 0.31 0.30 Volatile acids 0.15 0.11 0.05 0.03 Water by difference 80.80 84.42 84.67 85.47 Undissolved starch. 10.68 12.46 11.55 I2 -05 INDUSTRIAL APPLICATION OF MALTASE. 229 Manufacture of Sake. — For the manufacture of sake the same primary materials are used as for the preparation of moto. The rice is saccharified by means of koji, and mo/o-yeast is added. Saccharification and fermentation are produced simultaneously. After a few days of slow fermentation, the mash becomes heated and begins to ferment very vigorously. The mashes of sake have a very strong concentration, reaching 35 ° Balling. The fermentation of these mashes lasts from 15 to 17 days. Generally the amount of alcohol produced is from 12 to 13 per cent, and in some manufac- tories from 14 to 15 per cent. The table shows the composition of the must after 28 days of fermentation : Alcohol !3- 2 3 Dextrose Dextrin 0.41 Glycerin 1.99 1 Fixed acids o. 107 Volatile acids 0.061 Water 84.202 Undissolved starch 4.18 The fermented mashes are filtered, and not further used ; in certain manufactories, however, the mash is kept, and made to undergo a secondary fermentation. The starch contained in the residues is used again after cooking. To preserve the sake, the fermented liquids are reheated to 50°-66°. The Japanese, therefore, adopted a method of sterilization before Europe had any knowledge of the pro- cess of Pasteurization. As, after sterilization, they put the liquid back into an unsterile receptacle, they lose a part of the good of this operation. Sake differs from beer by the small quantity of dextrin and dextrose which it contains. 230 THE ENZYMES AND THEIR APPLICATIONS. In the manufacture of moto, a very strong alcoholic fer- mentation is developed. The explanations as to the cause of this fermentation are very contradictory. According to certain authors, it is due to a Saccharomyces which is developed spontaneously in the mash ; others claim that it is due to a mucor which, under certain culture conditions, is transformed into an alcoholic ferment. It is incontestable that Aspergillus orizoz as well as numer- ous other moulds can acquire a fermenting power when cul- tivated under certain conditions ; but these moulds generally give little alcohol and ferment very slowly. Moreover, as. Saccharomyces have always been found in koji, there is every reason for believing that the fermentation comes prin- cipally from the yeast. Chinese Yeast. — Chinese yeast has been particularly studied by Calmette, who published detailed data on the work in the distilleries of the far East. It is from these works that we obtain the following information : Chinese yeast possesses the double property of sacchari- fying and causing to ferment the amylaceous materials with which it comes in contact. It is found in commerce in the form of little loaves of rice which give out a musty odor. These loaves are filled with bacteria, yeasts, and different kinds of moulds. The active agent contained in these loaves is a mould which penetrates all through the mass by its mycelial ramifi- cations, a mould called by Calmette Amylomyces Rouxii. The Amylomyces is very abundant in the loaves which con- stitute the Chinese yeast. When this mould is cultivated in glucose agar-agar, it develops very rapidly and forms at the end of 48 hours a sort of veil extending over the whole sur- face of the culture. Potato and sweet potato, sown with the spores of this mould, become covered with a light floury coating which at last becomes transparent and invisible. The Amylomyces develops normally in gelatine, peptone, and INDUSTRIAL APPLICATION OF MALTASE. 231 beef broth which is peptonized and alkaline, although a slight acidity is more favorable for it. The mould coagulates milk in 24 hours and reddens it when it has been previously colored blue by litmus. As a general rule saccharine mashes containing potas- sium phosphate are suitable for the development of the plant ; however, the media where it grows the best are beer-worts, liquid or gelatinized, and amylaceous substances which have been cooked by steam. When the mould is developed away from the air, it takes on a fleecy aspect and produces small quantities of alcohol. If, on the contrary, it lives at the surface of the mash or wort, it consumes the sugar and produces oxalic acid. Cultivated in the air in a medium containing dextrins or starch, it trans- forms cane-sugar into fermentable sugar. Amylomyccs is, according to Calmette, the ferment which transforms starch into sugar with most energy. Calmette, by following the growth of this plant, has found that in contact with the air the mycelium forms conidia; when the air is excluded they extend their hyphae in every direction and are reproduced by direct budding. This mould differs, from botanical and physiological points of view, from all other known species: it seems to ap- proach the trichophytes, while by its mode of reproduction as well as by its physiological properties it recalls the branched Saccharomyccs. Diastase of Chinese Yeast. — The diastase contained in the cells of Amylomyccs presents, according to Calmette, all the characteristics of the amylase of malt. This diastase is secreted by the hyphae. Calmette also attributes to the Amylomyccs the property of secreting sucrase. In reality the diastase secreted is glu- case, and this enzyme has nothing in common with either amylase or sucrase. To obtain a diastatic solution of this ferment, recourse is 232 THE ENZYMES AND THEIR APPLICATIONS. had to a method similar to that recommended by Fernbach for the preparation of the sucrase of yeasts. First the mould is cultivated in sterilized Raulin's medium, or better still in beer-wort, and when the plant has reached its normal growth, the liquid is replaced by sterilized water. After a stay of nearly 60 hours in a thermostat at 38 , the diastases contained in the cells are diffused into the surrounding liquid ; then the water is taken away, and shows active diastatic properties. The following experiment has been made by Calmette to determine the activity of the enzyme of Amylomyces. The diastatic solution is divided into several portions of 30 cubic centimetres each, which are added to a 1 per cent solution of starch, sterilized, and weighing 120 grams. Each portion re- ceives a drop of oil of garlic, which plays the part of anti- septic. The whole is taken to the thermostat, where sac- charification proceeds. The quantities of sugar obtained are here shown : After 1 hour 0.12 gr. " 6 " 0.28 "12 " 0.33 "24 " 0.35 It is seen that the proportion between the duration of the action and the quantity of product formed ceases after 12 hours ; it appears, therefore, that the diastase is altered after that length of time. To estimate the fermenting power of the plant cultivated on rice, Calmette- suggests the following method : One hundred grams of steamed rice are sown with hyphse from a pure culture of Amylomyces ; it is left 3 days at a tem- perature suitable for the development of the mould. Then the mass is ground with 500 grams of water and the whole poured on the membrane of a dialyser floating on distilled and sterilized water. The starch paste does not dialyse and the membrane al- INDUSTRIAL APPLICATION OF MALTASE. ^t, lows only the glucose and the diastases in solution to be transfused. There is then formed under the dialyser a new diastatic solution in which the sugar is measured ; then cer- tain quantities of these solutions are stirred in a i per cent solution of starch paste. After saccharification the sugar formed is measured and the sugar introduced with the infusion of Amylomyces is deducted. The diastase extracted from fresh cultures produces a more intense hydration than the diastase extracted from old cultures. The filtration of diastatic solutions in Chamberland bougies takes away all their fermenting power. The method adopted by Calmette for determining the diastase leaves much to be desired. It is evident that by this process only a small part of the diastase contained in the plants is obtained. To take account of the diastatic power of the amylaceous materials on which moulds have devel- oped, it is necessary to crush them, reduce them to powder or paste, and use the substance prepared in this way. For example, i gram of this substance may be taken, mixed with 10 grams of gelatinized starch and saccharification allowed to proceed for an hour at 40 . From the quantity of sugar found must then be deducted the sugar formed under the in- fluence of the active materials alone, for it is particularly a question of determining the quantity of sugar which the gram of active matter can give by itself under the conditions of the experiment. Influence of Temperature and Chemical Agents.— The temperature most favorable for the development of Amy- lomyces is from 35°-38°. At this temperature the plant produces the strongest hydration. Above 38 , or lower than 2 3°, growth weakens ; at 72° the diastase is destroyed. The plant itself is destroyed by a stay of a half-hour at' 75° or 15 minutes at 8o°. The presence of salts appears to be of little disadvantage to the diastase. Calmette has determined the amounts of 234 THE ENZYMES AND THEIR APPLICATIONS. different substances which do not influence the diastase; he lias found : 1.10% of phenol. 0.05 " of silver nitrate. 0.10 " of copper sulphate. o. 10 " of iron sulphate. 0.10" of zinc sulphate. Oil of mustard, used in small quantities, has no influence on the development of the plant. Five per cent of glycerin produces a favorable effect. Oil of garlic in very small quan- tity and mercuric chloride in 0.005 P er cent, on the contrary, check the growth of the mould. Manufacture of Chinese Yeast. — Chinese yeast, the prep- aration of which demands quite complicated operations, is in the far East the object of a very interesting industry. The apparatus needed for this preparation is quite simple ; being composed of mats, shelves, sieves, a granite mortar, and a cir- cular trough. The raw materials are hulled rice and various Icinds of aromatic plants which give a special perfume to the alcohol formed and which, furthermore, undoubtedly act as antiseptics. These plants are exceedingly numerous; the best known are the Sinapis alba, Caryophyllus aromaticus, cinnamon, Juper nigrum, cloves, etc. The aromatic plants and the rice are separately ground and after pulverization mixed and ground with water to form a soft paste. This paste is formed into little disks a centimetre thick, which are placed on a mat after having been sown with mould by the aid of balls of rice "which are introduced into the paste. The mats are then put on shelves, covered with straw matting, and the mould al- lowed to develop at a temperature of 28 or 30 . After two days the moulds have covered the disks with a fine down ; the yeast is then dried in the sun and prepared for sale. The rice used for the manufacture of the yeast is not of the very first quality ; grains which are broken may even be used. INDUSTRIAL APPLICATION OF MALTASE. 235 In Cochin-China the manufacture of Chinese yeast is car- ried on everywhere in the same way. In Cambodia and China the rice is sometimes replaced by the flour of beans or maize. Native Distilleries. — The native distilleries do not de- mand a complicated outfit any more than do the yeast manu- factories. The plant is composed of a shed covered with a tiled roof. Under this roof are ranged furnaces in parallel lines separated by spaces containing basins full of water, in which the receivers serving to condense the alcoholic vapors are plunged. The furnaces measure 60 centimetres in height, 1.2 m. in width and 4 metres in length. They are used for the heating of two stills and a boiler devoted to cooking the rice. The furnaces are heated by a fire of mangle-wood. The rice used for making the mash is in part hulled and mixed with a certain quantity of warm water. It is placed in the boilers, which are covered with a mat and a sheet-iron cover. In each boiler are placed 18 kilograms of grains and 22 kilograms of water, and they are cooked for 2 hours. The rice is at this time completely steeped. It is then spread on mats where it receives the Chinese yeast in a fine powdery state, after which it is placed in pots of about 20 litres capac- ity, which are half filled. The pots are closed and saccharifi- cation allowed to occur. When the starch is transformed, that is, at the end of about 3 days, the vessels are filled with water; fermentation at once begins and at the end of 48 hours the action is finished. The contents are then distilled. These stills are formed of a sheet-iron vat, a wooden dome, and a terra cotta head. A bamboo tube, 2.5 m. long and inclined at 45 , joins the still to the condenser, into which it conducts the alcoholic vapors. The stills are placed directly over the fire. The residues of the distillation are used as food for cattle. With 100 kilograms of rice and 1.5 kilograms of Chinese yeast, there is usually obtained 60 litres of 36 per cent al- cohol, or 18 litres at 100 per cent. The richness of the first 23 6 THE ENZYMES AND THEIR APPLICATIONS. distillate varies according to the distillery; it is never less than 34 or over 42 . Use of Moulds in Fermentation Industries in Non- Asiatic Countries. — From the point of view of the utilization of primary materials, Chinese yeast, as well as Japanese yeast, furnish very meagre results in the countries of their origin. According to Atkinson, the yield in alcohol in the manu- facture of sake reaches only 50 to 56 per cent of the theoretical yield. Chinese yeast does a still less satisfactory work. Accord- ing to Calmette, 100 kilograms of hulled rice, having from 81 to 84 per cent of starch, furnishes about 18 litres of alcohoL in the distilleries of Cochin-China. This unsatisfactory result must be attributed in great part to the inefficiency of the plants as well as to slovenliness in. the labor. At first sight, it must be admitted that the work by the aid of moulds is capable of being improved and of giving in- dustrial results similar, and perhaps superior, to those of the ordinary method. Moreover, the use of moulds presents great advantages. The work appears to be much more simple ; the yeast and. the malt are done away with and replaced by a mould which is very easily cultivated and less sensitive than malt and yeast to the action of heat and of the medium. But to render practicable the use of moulds it is first necessary to give up Oriental methods, adapt oneself to the conditions of European distilleries, and try to develop a practical process. This question has been studied by the Japanese chemist Takamine, and also by Collette and Boidin. Takamine has been engaged in the application of moulds to the fermentation industry for ten years. At first he par- ticularly sought a medium suitable for the development of Aspergillus orizce, which, in Japan, is cultivated exclusively on steamed hulled rice. To furnish the mineral element to the plant, a certain: INDUSTRIAL APPLICATION OF MALTASE. 237 quantity of the ash of Cornelia Japonica is generally added. Takamine replaced the ash by an addition of 1 to 4 per cent of the weight of the grains of a mixture of salts in which are ammonium tartrate and phosphate, potassium sulphate, and magnesium sulphate. According to the author, this addition of salts consider- ably increases the yield and has the further advantage of per- mitting the rice to be replaced by other cereals. To prepare, industrially, cultures of Aspergillus orizce, Takamine proposes the following process : Steam the grains until the starch-cells are swollen, cool, sprinkle with the solution of salts; mix the grains well and sow with Aspergillus orizce. The cereals thus sown are left at a temperature of 30 for 24 to 36 hours. The lumps formed are broken up and the grains placed on plates, which are left in a damp atmosphere until the com- plete maturity of the moulds. The mouldy mass then dried at a low temperature is sifted. Thus the spores are sepa- rated, which, again dried at a moderate temperature and then mixed with inactive materials, serve as agents of fermenta- tion. Takamine also makes a kind of malt which he calls talca- koji. For the preparation of this substance he prefers to use bran or brewery or distillery malts, and proceeds as follows: The raw materials are sterilized by steam and sown with the spores of Aspergillus orizce at a temperature of 30 . One gram of spores is used for 50 kilograms of raw material. The development of the mould takes place in a very damp malt-house at a temperature of 20°-30°. After 24 hours' stay in the malt-house, the mass is spread in thin layers and the plant is allowed to grow. Generally its development is sufficient after 4 or 5 days. Then the material is dried at a temperature not exceeding 50 . Takamine also recommends for taka-koji to separate the spores from the material by sifting in a silk sieve. These spores act, according to him, as agents of alcoholic fermenta- 23 8 THE ENZYMES AND THEIR APPLICATIONS. tion, while the taka-koji itself acts as agent of saccharifica- tion. Takamine also proposes, with much reason, to use for saccharification a clear infusion prepared with taka-koji. For this purpose he makes a cold extraction of the active matter and decants the liquid, which is used as saccharifying agent, while the solid part is submitted to a cooking which allows of utilizing the starch. Taka-koji serves in the manufacture of a ferment useful in the distillery and the bakery. To prepare it, bran of corn or other cereals is mixed with 3 to 10 per cent of taka-koji and 4 to 8 volumes of water. The mass is kept at 65 ° for 15 to 30 minutes and then brought to a boil. Then it is cooled to 6o°; a new portion (3 to 10 per cent) of taka-koji is added and a second saccharification allowed to go on. This ended, the liquid is separated from the solid matter by nitration or decantation, the mash is sterilized and sown with the spores of the moulds. A fermentation is produced which lasts from 12 to 16 hours. When it is ended, the ferment is deposited at the bottom of the vats in the form of a pasty material which is pressed and used in various industries. In distillery work, according to Takamine's system, the following is the manner of procedure: The raw materials, grains, potatoes, etc., are cooked un- der pressure. The starch is then saccharified by means of taka-koji. Saccharification goes on for an hour at a tem- perature of 65°-70° and the quantity of taka-koji used is from 3 to 20 per cent of the quantity of grain used, according to the amount of diastase the koji contains. Saccharification accomplished, the mash is cooled to 19 and leaven is added. For the preparation of the leaven, a mash of cereals cooked under pressure and saccharified by taka-koji is used. The saccharification of this mash is accomplished in two stages. First it is saccharified at 6o° for an hour and then slowly cooled to 19 . A new portion of taka-koji is added, INDUSTRIAL APPLICATION OF MALTASE. 239 as well as a little leaven from a previous operation, and it is al- lowed to ferment. Greatly attenuated mashes as well as yeast-mother are used for leaven in succeeding operations. Generally 2 to 10 litres of leaven are used for 100 litres of mash submitted to fermentation. Under a patent taken out in 1894, Takamine proposes to use industrially the active substances of moulds by precipitat- ing them in solid state from their solutions. To cultivate the moulds, malts, bran, or other amylaceous substances are used. The culture made, these substances are reduced to powder and soaked in cold water to extract the maltase. The liquid, separated from insoluble sub- stances, is filtered and precipitated with 1 to 3 volumes of alcohol. The product thus obtained is placed on a filter, washed with alcohol, then with ether, and dried at a moder- ate temperature. According to Takamine, the active sub- stance obtained by this process can advantageously replace malt in the distillery and the brewery. Takamine also advises (and this he holds to be im- portant) the addition to the active infusion, before the ad- dition of alcohol, of an infusion of raw materials, as bran, malts, raw grains, etc. According to him, the activity of the precipitated enzymes is considerably increased by this opera- tion. From the description of the process, it appears attractive, and we have hastened to repeat the experiment of Taka- mine; but the results we have obtained are not very encour- aging. Aspergillus orizee contains a peptonizing ferment which acts strongly on albuminoid materials. The infusion ob- tained is very viscid, refuses to filter, and the precipitate re- sulting from treatment with alcohol does not show very ac- tive properties. The addition of infusions of bran, of malts or of raw grains increases the saccharifying power of the diastases of Asper- 240 THE ENZYMES AND THEIR APPLICATIONS. gillus oriscB, as we demonstrated before Takamine, but it does not heighten the liquefying power. In other words, the in- crease is apparent rather than real.* Under a more recent patent, Takamine proposes a system for the cultivation of moulds and the preparation of active liquid which deserves mention. To secure a great surface for the culture, without wasting the nutritive materials, he places porous objects (fragments of pumice-stone, etc.) in a nutritive solution and allows the moulds to develop on these foundations. This very ingen- ious idea has since been taken up by Collette and Boidin. To produce industrially a growth of Amylomyces Rouxii straws are saturated with the nutritive liquid, sterilized, and then sown with the mould. To favor its development he keeps up a strong current of air through the mass. By this means and with relatively little nutritive sub- stance, an abundant vegetation is obtained. The fermenting of grain by Amylomyces is accomplished, according to the method of Collette and Boidin, in the follow- ing manner : The amylaceous materials, added to twice their weight of water, are cooked for 3 hours under a pressure of 3! to 4 atmospheres. The cooked mass is placed in contact with fresh crushed malt, at a temperature not exceeding 70 . The weight of the malt, estimated in barley, is from 1^ to 2 per cent of the total weight of the amylaceous materials used in the work. Liquefaction by the malt continues for about an hour. The mash is then sterilized in a great diges- ter where a pressure of 2 atmospheres is maintained, after which it is inoculated and allowed to ferment. This fermentation goes on in special vats furnished with agitators and injectors of air and steam. The boiling mash, coming from the sterilizer, is introduced into vats, con- structed in such a way that all infection can be avoided. The cooling of the mash takes place in the fermentation vats, *See Comptes Rendus de l'Academie des Sciences, 1892, vol. CIX, page 1324. INDUSTRIAL APPLICATION OF MALTASE. 241 where it falls to a temperature of 38 and is neutralized. Neutrality of medium is really indispensable to the normal development of Amylomyces. The vats are then sown with cultures of Amylomyces, grown on a small quantity of amy- laceous materials, after which sterilized air is injected and the agitator is used. This agitation is to prevent the mould from developing on the surface, because if it grows in that way it will consume the sugar of the mash. After 20 hours, the development of the mould is at a maximum. Then it is cooled to 38°-33° and sowed a second time with a pure yeast culture. This yeast produces the al- coholic fermentation. The mould, to yield the same result, would take much longer. At the end of three days the fer- mentation is sufficiently advanced, and the mash ready for distillation. Critical Examination of Oriental Processes. — The at- tempts made by Takamine, Collette, and Boidin to introduce the use of moulds into the fermentation industries has called forth many critical articles which have appeared in different reviews of distilling and brewing. The technical journals seem, on the whole, very reserved as to the value of the new method. In general, when we consider an industrial pro- cess, the only criterion which we can admit is the practical results to which it leads. Now, as regards the use of moulds in distilling, results of this kind are lacking, at present. With the exception of a few establishments where the inventors have been experimenting with their process, not a distillery is known which is run entirely by the new method. Under these circumstances, it would be premature to pronounce definitely on the value of this method of manu- facture. We have nevertheless sought to compare the dif- ferent patents of Takamine with those of Collette and Boidin, since they have been considered to stand for different pro- cesses. This comparison has not led us to any definite con- clusions. 242 THE ENZYMES AND THEIR APPLICATIONS. -Takamine's method, although seven years earlier than that of Collette and Boidin, has certain points of resemblance to the latter which may easily lead to confusion of the two. A reading of the patents relative to the use of moulds re- veals in a striking way the extravagant hopes of the inven- tors and the illusions which possessed them as to the scope of their discovery. Takamine, in his patents of 1891, claims, as his exclusive right, the use in fermentation industries of any mould capable of producing saccharification and fermentation of amylaceous materials or even one of these transformations alone. Seven years later, Collette and Boidin also aspired to this exclusive right. They claim, as the result of their re- searches, the use of all moulds which are both saccharifying- and fermenting. One might, possibly, condone the simplicity of the Japanese chemist, who is evidently little acquainted with our literature, but the same excuse does not exist for the French chemists. The use of moulds, as well as yeasts, has for a long time been public property. It is allowable to patent a special method of working with moulds, but not the principle of their use. In reading the patent it is very hard to see in what the invention of Collette and Boidin really consists. One might, perhaps, characterize their process by the sterilization of the mashes and the development in them of pure cultures. Takamine, indeed, sterilizes only the leaven and then produces fermentation in the mashes saccharified at high temperature. But, by an incomprehensible misconception, Collette and Boidin return, in additional patents, to their method of work and claim that sterilization of the mashes can be omitted. So this is not the distinctive principle of their process. One might also get the impression by studying some of the patents of the inventors, that Takamine used Aspergillus INDUSTRIAL APPLICATION OF MALTASE. 243 orizce exclusively, and that Collette and Boidin used only Amylomyces. But when all the works which they have pub- lished are reviewed, this is found not to be so. It is to be regretted that Takamine did not limit his pre- tensions to Aspergillus orizce, which would have rendered his process unquestionably superior to that of his competitors. On the whole, the practical interest of moulds centers in their saccharifying powers. By reducing the quantity of malt for the preparation of leaven, it has been possible to reduce the cost of the yeast so low that there remains little or nothing to be done in this direction. On the contrary, an economy in the malt used for saccharification of the mashes would afford a real advantage. Aspergillus orizce is unquestionably a more active pro- ducer of diastase than Amylomyces Rouxii, and from this point of view it is of much more interest to distillers. Japanese yeast affords still other advantages over Amylomyces Rouxii. Aspergillus orizce secretes not only maltase but also sucrase. It can consequently be used in molasses and beet-sugar dis- tilleries where Amylomyces Rouxii would be of no use. The process of Collette and Boidin does not yield mashes containing more than 4 to 5 per cent of alcohol, while Asper- gillus orizce produces alcohol up to 12 per cent or more. Furthermore, with Aspergillus orizce there is no need of a special equipment, while the Amylomyces Rouxii system cannot be adopted without a complete and costly refitting of the plant. These faults are peculiar to the Collette and Boidin pro- cess, but there are others common to the two methods. 1. Moulds are oxidizing agents and, as such, always cause great losses of carbohydrates. 2. Alcohol produced with moulds has a peculiar taste and contains many more impurities than that resulting from the use of good yeasts. 3. Moulds generally furnish very limited quantities of dia- stase, and to obtain a satisfactory result an abundant culture 244 THE ENZYMES AND THEIR APPLICATIONS. must be allowed to develop in the mashes, which necessarily influences the yield of alcohol. One may conclude from the preceding observations first of all that the activity of workers is not at all restrained by patents taken, and, further, that great improvements must be introduced in processes using moulds for them to become practicable. It will be necessary to study thor- oughly the conditions of development of the moulds in ques- tion and it will be necessary, also, by a systematic acclima- tization, to make them produce a diastatic secretion which shall be more active and less sensitive to the conditions of the medium. Up to the present time it has been the capitalists in par- ticular who have been occupied with the question; it is to be hoped that disinterested investigators will apply themselves to it in their turn. BIBLIOGRAPHY. Atkinson. — Sur la diastase du koji. Monit. scientifique, 1882. The Chemistry of Sake Brewing in Japan, Tokio, 1881; Nature, 1878; Chemical News, April, 1880. Eijkmann. — Mikrobiologisches tiber die Arrakfabrikation in Batavia. Centralblatt fur Bakt. und Paras., 1894. Went und Geerligs. — Uber Zucker und Alcoholbildung durch Organis- men bei der Verarbeitung der Nebenprodukte der Rohrzucker- fabrikation. Wochensch. fur Brauerei, 1894. Hofmann. — Mittheilungen der deutschen Gesellschaft fur Natur- und M. O. Korschelt. — Memoires de la Societe asiatique. Berlin, 1878. See Volkerkunde Ostasiens. Heft, 6. Dinglers Polytech. Journal, 1878. Ahlburg. — Mittheilungen der deutsch. Gesellschaft fur Natur- und Vol- kerkunde Ostasiens. December, 1878. Ikula. — Sakefabrikation. Chemik. Zeitung, 1890. Kellner. — Chemik. Zeitung, 1895. A. Calmette. — La fabrication des alcools de riz en Extreme-Orient, Saigon, Imprimerie coloniale, 1892. Mori Nagaoka. — Beitrag zur Kentniss der invertirenden Fermente. Zeit. fur physiol. Chemie, 1890. Juhler.— Centralbl. fur Bakter., 1895. Jorgensen— Centralbl. fur Bakter., 1895. Wehmer.— Centralbl. fur Bakter., 1895. INDUSTRIAL APPLICATION OF MALTASE. 245 Klocker und Schionning.— Centralbl. fur Bakter., 1895. Dr. Liebscher.— Ueber die Benutzung des Gahrungspilzes Eurot. orizae. Zeitschrift fur Spiritus Indust., 1881. Kosai Tabe. — Centralbl. fur Bakter., n, p. 619. Bodin et Rolants.— Contribution a l'etude de l'utilisation de l'Amylomyces Rouxii. La biere et les boissons fermentees, 1897. Petit— Quelques procedes nouveaux en Distillerie. Moniteur scien- tifique, 1898. Sorel— Comptes rendus de deux congres de chimie appliquee. Paris 1897. Comptes Rendus, 1895. Nititenski. — Moisissures saccharificant l'amidon. Technitscheski sbornick. La biere et les boissons ferm., 1898. Takamine.— Brevet No. 216840, 19 octobre 1891. Perfect, dans la produc- tion des ferments alcooliques. Brevet No. 214033, 3 av. 1891. Brevet No. 241322, 11 sept. 1894. Conversion des matieres amy- lacees en sucre. Brevet No. 241321, 11 sept. 1894. Perf. dans la preparation des mouts fermentes. Brevet No. 241323, 11 sept. Fabric, du Tako Koji. Collette et Boidin. — Brevets Nos. 258084, 265245, 130172 en 1896. Pro- cede d'utilisation des moisissures pour l'extraction des residus de l'alcool. France, 15 juillet 1896, No. 125722, certif. d'additus. 11 janv. 1897. CHAPTER XX. ENZYMES ACTING ON CARBOHYDRATES. TREHALASE. Trehalase is an enzyme which acts on trehalose, an isomeric sugar corresponding to the formula C 12 H 22 1X + 2H0O. This sugar plays the part in plants of a reserve substance.. Wigers and Mitscherlich have found it in spurred rye and Berthelot in the Trehala of Syria. It is frequently found in great quantity in fresh fungi, from whence it almost en- tirely -disappears during drying. For example, it consti- tutes 10 per cent of the dry matter of Agaricus muscarius. Trehalose does not reduce Fehling's solution, and is trans- formed into glucose by the action of acids. A similar hydra- tion may be obtained by the use of an enzyme, trehalase, discovered by Bourquelot. This worker discovered the presence of the enzyme in ■Aspergillus niger and Penicillium glaucum, as well as in other fungi. This enzyme is also found in malt, and in the small intestine. The transformation of trehalose into glucose may be expressed by the following equation: C 12 H 22 1]L + H 2 = 2C 6 H 12 6 . The diastatic action may be followed by the change of rotatory and reducing powers of the liquid. Trfehalose has a rotatory power of (00*198°, while the rotatory power of glucose is only ( a ) d 52.4 . 246 ENZYMES ACTING ON CARBOHYDRATES. 247 Experiments with trehalase may be made in a 2 per cent solution of trehalose, at a temperature of 33°-35°. Trehalase is much more sensitive to the action of heat than maltase. At 54 its action is checked and at 64 the enzyme is completely destroyed. The reactions of the medium also have a very great influence on trehalose. An acidity corresponding to 2 to 4 milligrams of sulphuric acid seems to favor the transformation of trehalose bv the en- zyme, but if the amount of acid is increased, the activity dim- ishes, and with 0.2 grams the action of the enzyme is almost stopped. According to Fischer, an infusion of malt may produce the decomposition of trehalose, while the salivary diastase, ptyalin, has not this property. Amylase, precipitated and purified according to the method of Lintner, acts energetically on trehalose. By leav- ing, at a temperature of 35 , 10 cubic centimetres of a 10 per cent solution of trehalose with a half-gram of amylase, the formation of 0.5 grams of glucose has been observed. Emil Fischer recognized trehalase in Frohberg yeast. This enzyme is retained in the cells of this yeast and with difficulty passes into the surrounding medium. For this reason an aqueous extract of yeast does not possess the property of transforming trehalose, while in the cells of yeast trehalose is transformed into glucose. By adding 5 grams of yeast to 1 gram of trehalose dis- solved in 10 cubic centimetres of water, Fischer was able to find, after 40 hours action at a temperature of 33 , the form- ation of 0.2 grams of reducing sugar. According to this author, the existence of trehalase is to be doubted and he believes that it is amylase which produces the transformation of trehalose into glucose. According to Fischer, therefore, amylase must have the property of acting on starch, giving maltose, and on an isomer of maltose, giving glucose. 248 THE ENZYMES AND THEIR APPLICATIONS. To prove the existence of trehalase we have made the following experiment: Equal quantities of yeast, cultivated in sterilized wort, are added, under like conditions, to a solution of dextrins and to a solution of trehalose. The 2 solutions are left for 2 days at 30 and then tested. For these experiments 2 grams of yeast are used, 25 cubic centimetres of a 1 per cent solution of soluble starch and 20 cubic centimetres of a 10 per cent solution of trehalase. The action of the yeast takes place in the presence of chloroform. The solution of trehalase yields, under these conditions^ 0.34 grams of glucose, while in the solution of soluble starch no traces of sugar are found. The enzyme secreted by the yeast is therefore not amylase, and the fact that the diastase of Lintner acts on trehalose proves only that this diastase contains other enzymes than amylase. LACTASE. Pasteur has demonstrated that sugar of milk treated with mineral acids is transformed into galactose and dextrose ac- cording to the equation: C12H22O11 + H 2 = C 6 H 12 6 + C 6 H 12 6 . Lactose. Dextrose. Galactose. In living cells, the transformation of lactose is carried on by means of an enzyme which causes the same action as the acid. The existence of this ferment was doubted for a long time and the transformation of the sugar of milk in the or- ganism attributed to vital activity. Beijerinck was the first to discover the presence of lactase in certain yeasts found in cheese and kephir. Since then Duclaux, De Kayser, and Adametz have found other species of yeasts which secrete the same diastase. Emil Fischer, repeating the experiment of Beijerinck, has confirmed the fact that the filtered in- fusion of kephir acts upon lactose. ENZYMES ACTING ON CARBOHYDRATES. 249 As in kephir the Saccharomyces act in symbiosis with other micro-organisms, it was of interest to find out whether the enzyme is secreted by the yeast or by the accompanying- bacteria. The experiments which Fischer made with this object have established the following facts: 1 st. Certain alcoholic yeasts are capable of fermenting lactose; 2nd. The action of a yeast on milk-sugar depends solely on its power to secrete lactase. The enzyme acting on the lactose is retained inside the cells and passes with great difficulty into the surrounding medium. Even when the cells of yeast are crushed with powdered glass, it is difficult to extract the active substance. The diffusion of the diastase of the cells is accelerated by chloroform. Lactase can be precipitated from its solutions by alcohol without completely losing its activity. The action of lactase on lactose can be determined by the aid of the polariscope ; by the transformation of the lactose into dextrose and galactose the rotation of the solution in- creases about a third. Lactose and dextrose have a rotatory power of {a) d -f- 52.5, while that of galactose is (a) d + 83. INULASE. Certain plants contain, as reserve substance, a carbo- hydrate called inulin. These plants generally contain at the same time an ac- tive principle which transforms this carbohydrate into an as- similable sugar. This enzyme was discovered by J. R. Green, who named it '• inulase." The presence of inulase has been observed in the tubers of Jerusalem artichokes during their development, in Asper- gillus niger, in Penicillium glaucum, and in the tubers of dahlias. According to Bourquelot, it is supposed that this enzyme is also found in chicory, garlic, and onions, as well as in many other vegetables. 250 THE ENZYMES AND THEIR APPLICATIONS. By the action of inulase, inulin is hydrated and trans- iormed into levulose according to the formula: (C 6 H 10 O 5 ) 18 + 18H0O = i8C 6 H 12 6 . Inulin. Levulose. According to Green, this transformation is accomplished • by a progressive hydration of the inulin, with formation of intermediate substances. Granting that the molecule of inulin is very complex, one may assume that, during hydra- tion, there are formed, besides levulose, different inulins hav- ing different molecular weights. However, inulin, after having undergone a partial hydra- tion, possesses the same rotatory power as before under- going the action of the enzyme. The existence of intermediate substances is somewhat problematical, as the formation of these bodies by the action of acids has not been determined. The optimum tempera- ture of inulase is found between 50 and 6o°. The action of the enzyme is influenced by the reaction of the medium. In a neutral liquid, or with 0.005 P art °f hydrochloric acid, hydration progresses regularly. In the presence of increas- ing amounts of acid, the activity of the enzyme decreases. With 0.2 of acid or 1.5 of sodium carbonate, the diastase is destroyed. The influence of the reaction of the medium is more strongly shown at 40 than at io°-i5°. The transformation of inulin into levulose may be fol- lowed either by observation of the rotatory power or by that of the reducing power. Inulin has a rotatory power of (a) d — 36, while levulose gives a rotation to the left which is almost double. In the distilleries which use Jerusalem artichokes as a raw material, the inulin has to be inverted when a satisfac- tory yield in alcohol is desired. To effect this transforma- tion, the use of barley malt is advised. ENZYMES ACTING ON CARBOHYDRATES. 251 This method is very poor indeed, for amylase is without action on inulin and malt does not contain inulase. The transformation of inulin into levulose can be brought about very easily: it is sufficient to cook the raw materials under low pressure to effect a complete inversion. PECTASE. In the pulp of carrots and beets, and also in the soft parts of fruits, Fremy found a reserve substance to which he gave the name of pectose. This substance is insoluble in water and in alcohol. It very much resembles cellulose. Pectose undergoes a succession of transformations dur- ing the ripening of fruits: it is transformed into pectin and finally into pectates. Pectin is a neutral substance which gives with water a viscous solution from which it is precipitable by alcohol. The transformation of pectose into pectin is very prob- ably produced by an enzyme which, however, has not yet been isolated. The transformation of pectin into pectates is better known, and the intervention of an enzyme is here definitely established. This active substance is called pectase. This name ought to belong to the substance acting on pectose and not to the enzyme transforming pectin. This latter enzyme, according to the correct nomenclature, should rather be called " pec- tinase." The composition of pectin is not definitely established. According to Fremy it has. the formula: and according to Chandnew: The mechanism of the reaction produced by the pectase is little known. 25 2 THE ENZYMES AND THEIR APPLICATIONS. It is not even clearly established that the reaction is caused by hydration, and it may well be that the mechanism of the reaction consists in a molecular change of the same nature as that found in the transformation of sugar into lactic acid. The action of pectase on a solution of pectin is shown by the gelatinization of the solution, and by the formation of a reducing substance. Bertrand and Mallevre have shown that the reaction takes place only in the presence of certain salts. A solution of pure pectin, with the addition of pectase, free from cal- cium salts, never becomes gelatinous. The solidification of the solution takes place instantly if to the mixture are added several drops of a solution of cal- cium chloride, a substance, which, without pectase, could not produce gelatinization. The calcium salt may be replaced by salts of barium or strontium, which play just the same part. To obtain a solution of pectase, carrots gathered in the process of growth are used, because it is then that these plants contain the most diastase. It is well to pare the carrots and use only the central part, as the skin contains, but little pectase. The substance is reduced to a pulp and the juice ex- tracted by pressure; in this way 70 to 80 per cent of a turbid liquid is obtained, which is filtered after addition of a little chloroform. This liquid is very active in a solution of pure pectin. To preserve the filtered solution of pectase, precipitate the salts of lime and magnesium by the addition of alkaline oxalate, previously determining by analyses the amount of oxalate to use. The quantity of salts contained in the juice varies slightly with the species of carrots; for three different samples Bertrand obtained the following figures: Lime 0.016% 0.018% 0.013% Magnesia 0.029 0.021 ENZYMES ACTING ON CARBOHYDRATES. 253 It is well, in practice, to use a third of alkaline oxalate in excess of the amount calculated for the salts present. The solution of pectase to which oxalate has been added rapidly becomes clear and, after filtration, gives a trans- parent solution. This product can be kept for a long time if chloroform is added and it is put fresh into full bottles sheltered from the light. Jelly is not produced in a solution of pectin free from salts. To prepare pectase in a pure state, clover is used. The plants are crushed in an iron mortar; the mass is then pressed, and the juice, with chloroform added, is placed away from the light. At the end of 24 hours, a coagulum is pro- duced in the liquid which permits of filtration. In the filtered liquid the diastase is precipitated by alcohol as has been done with the other enzymes. Measurement of Pectase.— Pectase is very widely dis- tributed in the vegetable kingdom. It is found in stems, flowers, and leaves of different plants. Bertrand and Mallevre propose the following method for measuring it: To one volume of a 2 per cent pectin solution add one vol- ume of the juice under examination and measure the dia- static power by the time required for the liquid to become gelatinized. The results obtained with the juices of different plants are given below: Tomatoes 48 hours. Grape-vine 2 4 Carrots 2 Maize (leaves) 8 Q over 10 minutes. By using this method, Bertrand and Mallevre studied the influence of the medium on pectase. Different samples of the same solution of pectin were 254 THE ENZYMES AND THEIR APPLICATIONS. acidified in different degrees and the same quantity of pec- tase was added: Hydrochloric Acid. Coagulation Per cent. at the end of o | hour. 0.02 I 0.06 3! hours. 0.1 20 " Pectase is unfavorably influenced by the acid 'reaction of the medium ; 0.06 per cent of acid in the liquid produces a delay of three hours in coagulation. However, acid does not easily destroy this active substance. By neutralizing the acid solutions, which have become weak and slightly active, new liquids are obtained which act very rapidly. This resistance of pectase to the acid reaction explains why the action of pectase is weak in green fruits: before ripening the enzyme is in the presence of a large amount of acid and does not act, or acts only very feebly, while during ripening the acidity disappears and the action of the pectase is shown with much more intensity. CYTASE. Cellulose is often assimilated by vegetable cells. This assimilation is preceded by liquefaction and a more or less complete transformation. The agent which produces the change is cytase. As celluloses exist whose properties differ considerably, it is necessary, at the outset, to assume also the existence of different cellulose-dissolving enzymes. Sachs was the first to discover that, during the germina- tion of the stones of dates, the cellulose of the endosperm is gradually dissolved and that the products formed are ab- sorbed by, the young plants which, with the cellulose, pro- duce the transitory starch. Green, by treating germinated date-seeds with glycerin, ENZYMES ACTING ON CARBOHYDRATES. 255 obtained an active solution which causes swelling as well as partial solution of certain celluloses. The destruction of vegetable tissues by moulds must also be attributed to a secretion of cytases; however, the isolation of these enzymes is accomplished with much difficulty and their existence was doubted for a long time. The difficulty met with, when it is desired to isolate this diastase, comes probably from its decomposition. It is probable that these enzymes are destroyed as rapidly as they appear, and that it is for this reason that they are not found accumulated in the cells. A *more stable cytase was discovered by Brown and Morris in malt dried in the air. To obtain this enzyme in a solid state, an infusion of malt is precipitated with alcohol, and the precipitate dried in vacuo. The product obtained contains, besides amylase, a cellu- lose-dissolving enzyme. The activity of this ferment is shown by its property of dissolving the cellulose envelope of grains of starch. This may be verified by causing it to act on the endosperm of barley. For this, very thin shavings of barley endosperm are put into an infusion of' malt and it is found that the cel- lular walls soften and then enter partially into solution. Cytase is present from the beginning of germination of cereals, appearing even before amylase. The dissolving action of cytase during malting is exer- cised upon the whole of the endosperm and, as a result, the germinated grain becomes friable and mealy. This transformation may also be produced artificially by placing a grain of barley from which the embryo has been removed in a malt infusion. By a prolonged stay, the en- dosperm changes its appearance completely; it becomes mealy and friable, while if the infusion, i-s previously heated to 6o°, the same effect is not obtained. At this temperature, the amylase of the solution does not lose its power to hydro- lyze starch, while the cytase is destroyed. 256 THE ENZYMES AND THEIR APPLICATIONS. The transformation which cytase produces during germ- ination has been studied from a chemical point of view. It is very probable that the cellulose is transformed into sugar, but it may also be that the action of the cytase is less com- plete. During germination, according to J. Gruss, the cel- lular walls are only partially liquefied, and the action of cytase is reduced to freeing the amylaceous cells and in- directly facilitating the action of amylase. By cultivating the germ of cereals in different media, Brown and Morris found that the presence of an assimilable hydrocarbon influences unfavorably the secretion of cytase. They also found that a slight acidity of the medium is, on the contrary, very favorable to the secretion. In general, all the conditions which favor the secretion of amylase are equally favorable to that of cytase. CAROUBINASE. Caroubinase is an enzyme acting on a carbohydrate isolated from grains of the Ceratonia siliqua to which we have given the name of caroubin. This enzyme causes a liquefying and saccharifying action on the endosperm of carob-seeds and plays a very important part during the first period of the development of this plant. The endosperm of the seeds of Ceratonia siliqua is found to be partially composed of a carbohydrate which occurs in the form of a homogeneous and horny mass, not colored by iodine, and possessing some properties like those of agar- agar. To prepare this carbohydrate in a pure state, the seeds are freed of their exterior envelope as well as their embryo, and the endosperm is dissolved in warm water. The solution is then precipitated by alcohol. The operation is carried out in the following manner: The seeds are allowed to soak for five or six days, the liquid being renewed three or four times a day. The grains swell a great deal and absorb three times their weight of water. In ENZYMES ACTING ON CARBOHYDRATES. 257 this state it is easy to separate the endosperm from the testa and the embryo. One hundred grams of dry seeds furnish 53 grams of albumen. The swelling of the grain during soaking is due almost entirely to the mucilaginous substance which they contain and which constitutes an elastic and re- sisting mass. By submitting the endosperm to the action of warm water, in a water-bath, a transparent jelly is obtained which can be filtered through a silk filter. It is well to use enough water to obtain a thick syrup. To precipitate the caroubin, add to the cooled syrup twice its volume of 98 per cent alcohol. The carbohydrate is thrown down in long filaments which are collected on a cloth. The first precipitate thus obtained contains 2 to 3 per cent of albuminoid materials and salts which are easily elim- inated by redissolving the product in water and again precip- itating it with alcohol. By treating the endosperm eight to ten times successively with warm water, there is obtained an almost complete extraction of the carbohydrate it contains. The product, purified and dried at ioo°, is a white sub- stance, which is spongy, very friable, and having the chemical formula of celluloses. Instead of alcohol, one may just as well use barium hydrate, which precipitates the carbohydrate in a pure state. Caroubin is easily hydrated by acids as well as by a special diastase, caroubinase. To isolate this enzyme, we used an infusion of germinated carob-beans. A hundred grams of germinated seeds reduced to a paste were put to soak in water, at a temperature of 30 , for twelve hours. To the filtered liquid 3 volumes of alcohol were added ; the precipitate was washed with alcohol, then with ether, then dried in vacuo. The active substance obtained by this method dissolves easily in water and gives a reaction with guaiacum and hydro- gen peroxide. Caroubinase acts energetically at 40 , and its activity in- creases with the temperature up to 50 , which is its optimum 258 THE ENZYMES AND THEIR APPLICATIONS. temperature; at 70 , the action becomes very weak and at 8o° the enzyme is destroyed. Caroubinase acts very slightly in a neutral medium. An addition of 0.01 to 0.03 per cent by volume of formic acid favors the action of the enzyme. To determine the diastatic power of caroubinase, the de- gree of fluidity produced in a jelly of caroubin is taken as a starting point. The diastatic power may also be estimated by the greater or less facility with which the liquid may be filtered. The solution of caroubin not transformed by the enzymes does not pass through the filter, while the solution of carou- bin with a sufficient quantity of diastase added passes through very rapidly. The process employed is as follows : Pour into test-tubes 50 cubic centimetres of water; add 0.1 c.c. of normal formic acid and 1 gram of pulverized carou- bin. Mix and add to different tubes 2, 5, 7, 10, 15 cubic cen- timetres of the liquid to be examined. If there is room, bring the volume up to 65 cubic centimetres and leave it for three hours at 45 °. All the samples receive the same amount of chloroform, and the experiments are conducted in duplicate : on the one hand with fresh infusion, on the other with this same infu- sion previously kept for a half-hour at 90 . The tubes which have not received any infusion, or in which the substance has been destroyed by heating, may be turned over without the liquid running out, while the tubes which have received a sufficient quantity of enzyme contain a very fluid substance which easily passes through the filter. To study the secretion of caroubinase, we allowed the embryos of Ceratonia siliqua to grow in varied conditions and followed the transformation of the nutritive materials as well as the quantity of diastase formed. The embryo, separated from the endosperm and culti- vated in the dark, develops very slowly and gives, after eight ENZYMES ACTING ON CARBOHYDRATES. 259 to ten days, a rootlet of the same length as itself. Then placed in calcareous earth and in the light, the germ develops into a puny plant which generally dies at the end of three to four weeks. The progress of growth is quite different when the isolated embryo is cultivated in hydrated caroubin ; the ger- mination is more rapid ; a rootlet of the length of the seed is obtained, and the embryo, set out in the earth, rapidly develops into a plant of several branches. During germination away from the light, the caroubin used swells a great deal and is partly liquefied, but the quan- tity of carbohydrate absorbed is inconsiderable. The liquefaction and absorption of the caroubin pro- gresses much more rapidly as soon as chlorophyll appears in the plantlet. The embryo, developed in the dark and trans- planted in a calcareous soil, absorbs in three or four days a quantity of caroubin equal to its own weight. By taking specimens at different stages of germination, we found that the active substance appears abundantly when the plantlets are completely developed and that the enzyme becomes more active when the chlorophyll begins to appear. Caroubinase is both a liquefying and a saccharifying agent. When jelly of caroubin is analyzed immediately after liquefaction, the liquid is found to contain no trace of reduc- ing-sugar. Caroubin liquefied by the enzyme is easily pre- cipitated by alcohol, but the precipitate no longer has the properties of the caroubin. It is strongly dextro-rotatory and easily dissolves in water. By a prolonged action of caroubinase on caroubin, a so- lution is obtained in which alcohol no longer produces pre- cipitate and a reducing-sugar easily fermenting under the in- fluence of beer-yeast is produced. BIBLIOGRAPHY. E. Bourquelot. — Sur un ferment soluble nouveau dedoublant le trehalose en glucose. Comptes Rendus, 1893. 26o THE ENZYMES AND THEIR APPLICATIONS. E. Bourquelot. — Remarques sur le ferment soluble seer, par l'aspergillus et le penicillium. Soc. biol., 1893, juin. Digestion du trehalose. Soc. biol., 1895. Transformation du trehalose en glucose. Bui. de la Soc. chim. de Paris, 1893, p. 192. Emil Fischer. — Spaltung von Trehalose. Berichte der deutsch. chem. Gesellschaft, 1895, p. 1433. Einfluss der Configuration auf die Wirkung der Enzyme. Berichte der deutsch. chemisch. Gesellschaft, 1895, 2, p. 1429. Beijerinck. — Centralbl. fur Bakt. und Parasitenkunde. Zweite Ab- theilung, 1898. E. Bourquelot. — Inulase et fermentation alcoolique indirecte de l'inuline. Soc. biol., Paris, 1893. G. Dulle. — Ueber die Einwirkung von Oxalsaure auf Inulin. Chem. Zeit., 1895 J. R. Green. — Annals of Botany, 1888, 1893. Bourquelot and H. Herissey. — Sur la matiere gelatineuse (pectine) de la racine de gentiane. Journ. de chim. et de pharm., 1898, p. 473. Sur l'existence, dans l'orge germee, d'un ferment soluble agissant sur la pectine. Comptes Rendus, 1898, p. 191. Fremy. — Memoire sur la maturation des fruits. Ann. de chim. et phys., 1848, XXIV, p. 5. Schreibler. — Berichte der deut. chem. Gesellschaft, B. I. p. 59 Chandnew. — Liebigs Annalen, LI, p. 355. Frid Reintzer. — Ueber die wahre Natur der Gumifermente. Zeit. fiir phys. Chemie, 1890, XIV. Wiesner. — Ueber das Gumiferment: ein neues diastasisches Ferment. Berichte, 1885, p. 619. Cross. — Bull, de Soc. chim. de Paris, 1896. Bertrand and Mallevre. — Recherches sur la pectase et sur la fermentation pectique. Bull, de la Soc. chim. de Paris, 1895, XIII, pp. 77, 252. Nouvelles recherches sur la pectase et sur la fermentation pectique. Comptes Rendus, 1895, i er semestre, p. no. Sur la diffusion de la pectase dans le regne vegetal et sur la prepara- tion de cette diastase. Comptes Rendus, 1895, CXXI, p. 727. Reintzer. — Sur la diastase qui dissout les enveloppes cellulosiques. Zeit. fiir physiol. Chem., XXIII, p. 175, 1897. Tromp. de Haas and B. Tollens. — Recherches sur les matieres pectiques. Bull, de la Soc. chim. de Paris, 1895, p. 1246. Brown and Morris. — Untersuchung iiber der Keimung einiger Graser. Zeit. fur das gesammte Brauwesen, 1890. De Bary. — Ueber einige Sclerotinen und Sclerotinenkrankheiten. Bot. Zeit., 1886. Schmulewitsche. — Ueber das Verhalten der Verdauungstoffe zu Rohfasser der Nahrungsmittel. Bull. Acad, des sciences Saint Petersburg, t. XL ENZYMES ACTING ON CARBOHYDRATES. 261 * Sr trt!:\z nouve ' hydra,e de carbone ' ia caroubine - c ° m *- Sur la caroubinase. Comptes Rendus, IX, p. 764. CHAPTER XXI. FERMENTS OF GLYCERIDES AND GLUCOSIDES. Saponifying ferments. — Ferments of glycerides. — Serolipase and pancrea- tolipase. — Measurement of lipase. — Influence of temperature and alkalinity of the medium. — Differences between lipases of different origins. — Ferments of glucosides. — Myrosin, Emulsin, Rhamnase, Erythrozyme, Betulase. FERMENTS OF GLYCERIDES. LIPASE. The pancreatic juice has the property of splitting fats into fatty acids and glycerin. This property is due to the pres- ence of a soluble ferment to which has been given the name of steapsin or lipase. The reaction which steapsin causes may be represented by the following equation : C 3 H 5 (C 18 H 35 2 ) 3 + 3 H 2 = C 3 H 5 (OH) 3 + 3 C 18 H 86 O a . Stearin. Glycerin. Stearic acid. To obtain steapsin in solution, the pancreas is macerated in a solution of sodium or potassium carbonate. It can then be extracted from the pancreas by glycerin. The pancreatic juice acts upon fats as a saponifying and emulsifying agent. The emulsion is produced by the pan- creatic juice, owing to the alkaline reaction and to the vis- cosity of the liquid, and not by the action of the enzyme con- tained therein. The pancreatic juice as well as the products of maceration of the pancreas contain relatively little of the enzyme and the saponification of the fatty substances is always incomplete. The enzyme of the pancreatic juice also acts upon other substances than the fats ; it attacks the lecithins, decompos- 262 FERMENTS OF GLYCERIDES AND GLU COS IDES. 263- ing them into glycero-phosphoric acid, choline, glycerin, and free fatty acids. Steapsin acts also on some other ethers : on the benzoic ether of glycerin, and on phenyl succinate, as well as on salol. It decomposes this latter body into salicylic acid and phenol. The ferment of the glycerides is very abundant in the vegetable kingdom. Its presence has been observed in the poppy, hemp, maize, rape-seeds, as well as many other plants. To obtain an active liquid containing lipase, Green ground the germinated seeds of the castor-oil plant in a 5 per cent solution of sodium chloride, with the addition of a small quantity of potassium cyanide. He then dialysed the liquid to separate the salts from it. This solution, mixed with an, emulsion of castor-oil, soon began to decompose the oil, set- ting free the fatty acid. An active substance showing all the properties of lipase is met with in Peiiicillium glaucum. The presence of a similar substance called seroiipase is also found in the blood. It plays an important part in the assimilation of fats. Hanriot, who studied this enzyme with much care, pointed out a method for measuring the active substance and determined the influence of temperature, and of the reaction of the medium upon this enzyme. Accord- ing to him there is a difference between the lipase of the pan- creatic juice and the lipase of the blood. Measurement of Lipase. — To measure the lipase, Hanriot and Camus make use of a solution of monobutyrin. They take 1 cubic centimetre of the liquid containing the lipase to be measured, add to it 10 cubic centimetres of a 1 per cent solution of monobutyrin. The solution is carefully neutralized with sodium carbonate, then heated to 25 for 20 minutes. Under the influence of lipase, the liquid becomes acid, and this acidity is estimated by again neutralizing the solution with sodium carbonate: the number of drops used serves to measure the diastatic activity. 264 THE ENZYMES AND THEIR APPLICATIONS. The solution of sodium carbonate used for the saturation is prepared in such a way that each drop of the alkaline liquid neutralizes 0.000001 of a gram-molecule of acid. The dia- static power is expressed by the number of millionths of a gram-molecule of acid freed during 20 minutes at 25 ; 1 cubic centimetre of serum, for example, is said to possess a diastatic force of 33 if, in 20 minutes at 25 °, it frees a quan- 33 X88 tity of butyric acid, molecular weight 88, equal to - 1,000,000 Influence of Temperature and the Reaction of the Me- dium. — Heat exerts a considerable influence on the activity of lipase. Between o° and 50 it acts with an increasing en- ergy, but beyond that point the diastatic activity begins to diminish and the enzyme is soon destroyed. Temperature Quantities saponified, of the reaction. In 10 minutes. In I hour. o° 4-5 J 3-5 10 20 6.7 29.3 25 io-i 35- 37 13-5 39-5 40 16.9 56.5 50 22.6 71.2 60 27.1 36.1 70 22.6 22.6 The temperature of 6o° appears favorable at the begin- ning, but in the end destroys the diastase. The influence of temperature on lipase may be shown by warming the serum to different temperatures and causing it to act then on monobutyrin at 37 . Serum heated. Diastatic activity. 5o°-55° 4i-5 6o°-62° , 0.7 65°-66° Extremely slight. 7o°-72° '... o FERMENTS OF GLYCERIDES AND GLUCOS1DES. 265 The action of lipase is proportional to the quantity of enzyme used, at least at the beginning of the action. The following table shows this fact : Duration of the Action. Quantity of Lipase. 0.5 c.c. 6 12.5 20 30 I c.c. 1.5 c.c. 2 c.c. 20 minutes 1 hour. 1 h. 20 m. 2 hours II 25 36 54 16 37 53 73 22 48 62 66 Cessation of the proportionality is noticed, in the case of lipase as well as in that of other diastases, when the action is prolonged or takes place at high temperatures. The glycerin and the sodium butyrate formed during the action have no influence at all on the diastatic activity; the presence of monobutyrin is also almost without effect on the saponification. The alkalinity of the medium influences considerably the course of saponification by serolipase. Hanriot, to show this action, made the following ex- periment: To indentical mixtures of serum, monobutyrin, and water (10 cubic centimetres) were added varying amounts of sodium carbonate. After twenty minutes he determined the quantity of monobutyrin saponified by neutralizing with sodium carbonate. He obtained the following results: Excess of carbonate of sodium in milli- grams o 2 Activity of the lipase 22 33 4 6 8 10 15 2» 40 44 46 52 74 86 Difference between Lipases of Different Origin. — Hanriot, having remarked that the ablation of the pancreas in the or- ganism does not prevent the secretion of lipase, attributed to the blood the property of secreting a lipase different from that of the pancreas. He called it serolipase, in distinction from the first, which he called pancreatolipase. 266 THE ENZYMES AND THEIR APPLICATIONS. But the ablation of the pancreas is a very delicate opera- tion and impossible to perform without leaving active frag- ments of the gland. Lipase, on the other hand, may be pre- served in the blood. The existence of two lipases needs, therefore, to be clearly established. Hanriot sought to differentiate the two enzymes by their mode of action and their sensitiveness to physical and chem- ical agents. For that, he prepared two solutions having the same activity, that is, producing the same quantity of butyric acid by acting on monobutyrin during the same time. These two solutions should, therefore, if there exists merely a single lipase, contain the same quantity. Now, when the action of the serolipase and pancreatolipase is pro- longed for 20 minutes, it is observed that the serum enzyme produces a quantity of butyric acid double that obtained by the pancreatolipase. On the other hand, the enzyme of the pancreas acts with great difficulty in an acid medium, while serolipase produces a very energetic transformation under the same conditions. Pancreatic juice. Serum. Activity in alkaline medium (excess of sodium carbonate, per litre 0.2 gr.) 23 22 Activity in acid medium 9 16 The serolipase and the pancreatolipase act differently at the same temperatures: two solutions of these enzymes pos- sessing the same activity at 14 gave the following figures at other temperatures : c- ,. Pancrea- Serohpase. , ,. c tohpase. At 15° ti 10 "30° '• 15 10 " 42 ° 21 11 It is seen by this table that the action of pancreatolipase is, up to a certain limit, independent of temperature, while serolipase produces a much more energetic action at 42 than at 15 . FERMENTS OF GLYCERIDES AND GLUCOSIDES. 267 Finally, the two enzymes' differ as regards stability. In fact, serolipase remains unchanged during whole months, while the pancreas enzyme becomes inactive at the end of a few days. The lipases of the pancreas and the serum act differently, therefore, at the same temperatures and are influenced dif- ferently by the reaction of the medium. Furthermore, they present different characteristics of stability. These proper- ties, however, are not enough to demonstrate that seroli- pase and pancreatolipase are two very distinct chemical substances. In the case of lipase, as we have seen for amylase and glucase, the conditions of the medium produce varia- tions in the properties of the diastase. The foreign sub- stances found in the blood, as well as the extractive materials of the pancreas, give different characteristics to the two dia- static extracts, but the enzyme is really the same in the two cases. FERMENTS OF GLUCOSIDES. Glucosides are combinations of sugars and organic sub- stances containing one or more hydroxyls. Glucosides exist in which the sugar is found combined with alcohols, phenols, aldehydes or organic acids. These ethers are frequently found in plants, especially in the bark and roots. The manner of formation of glucosides in living cells is as yet little known. It is very probable that their formation is due to a molecular concentration followed by a dehydration which is produced by special enzymes. According to Gautier, the formation of certain glucosides may be explained by a transformation of formic aldehyde : i2CH 2 + H 2 = C 12 H 1(i 7 + 5H 2 0. Formic aldehyde. Arbutin. i3CH 2 + 2H0 = C,,H 18 7 + 6H 2 0. Formic aldehyde. Salicin. 268 THE ENZYMES AND THEIR APPLICATIONS. The part played by glucosides in the cells is also little known at the present time. In some cases they evidently # play the part of reserve materials. In other cases the assimilation of the products of cleavage of glucosides appears of little probability. In fact, these bodies contain, besides sugar, poisonous substances which must act unfavorably on the cells. In the parts of plants where the presence of glucosides is observed, there are almost always found enzymes under the influence of which these ethers are hydrated, then split, re- generating the sugar. The enzymes of glucosides are gen- erally enclosed in special cells which separate them from the substances on which they can act. The glucoside-splitting enzymes have this peculiarity that they act, not on a single body, as is the case with sucrase, for example, but on a whole series of bodies. Their action may be exerted on numerous ethers result- ing from the combination of glucose with bodies belonging" either to the fatty series or the aromatic series. Emulsin. — By treating bitter almonds, reduced to powder, with water, an aromatic oil is produced which did not exist in the almonds before the treatment. This reaction is caused by an enzyme, emulsin, on a special substance contained in the almond : amygdalin. The reaction may be represented by the following equation: G M H, T NO n + 2H 2 = 2C 6 H 12 6 + C 7 H e O + HCN. Amygdalin. Glucose. Benzoic aldehyde. Hydrocyanic acid. Emulsin and also amygdalin were discovered by Robiquet and Boutron. This diastase is found in the leaves of cherry-laurel as well as in sweet almonds. With the latter, oil of bitter almonds is not obtained on account of the absence of amygdalin. Bourquelot discovered the presence of emulsin in fungi. Fungi parasitic on trees, especially, contain great quantities FERMENTS OF GLYCERIDES AND GLUCOSIDES. 269 of this substance; thus he discovered the presence of this enzyme in Poly poms sulfurcus, in Armillaria mellea, and in Polypoms fomcntarhis. Emulsin has also been met with in Penicillinm glaucum, in Aspergillus niger, as well as in other moulds. Emulsin acts on a great number of glucosides, causing the reactions expressed by the following equations : C 12 H 16 7 + H 2 = C 6 H 12 6 -f C 6 H 6 2 . Arbutin. Glucose. Hydroquinone. With helicin, a product of oxidation of salicin : Ci3Hi 6 7 -f- H 2 = C 6 H 12 O e + C 7 H 6 2 . Helicin. Glucose. Salicylic aldehyde. With salicin, extracted from the bark of poplar or the flowers of Spirea ulmaria : Ci3H 18 7 + H 2 = C 6 H 12 6 + C 7 H 8 2 . Salicin. Glucose. Saligenin. With phloridzin, extracted from the bark of the apple- tree : C 21 H 24 O 10 + H 2 = C 6 H 12 O e + C 15 H 14 3 . Phloridrin. Glucose. Phloretin. With daphnin, extracted from the Daphne gnidium : C 15 H 16 9 + H 2 = C 6 H 12 6 + C 9 H 6 4 . Daphnin. Glucose. Daphnetin. With coniferin, extracted from the Larix Europe?: C 1C H^ 2 8 + H 2 = C 6 H 12 6 + C 10 H 12 O 3 . Coniferin. Glucose. Coniferic alcohol. With esculin of 2Esculus hippocastannm, which certain authors consider as an isomer of daphnin, glucose and esculi- tin are obtained : QoHicOj, -f- H 2 = C 6 H 12 O g + C 9 H 6 4 . Esculin. Glucose. Esculitin. 2 7° THE ENZYMES AND THEIR APPLICATIONS. Emulsin acts also on the chlorinated and brominated de- rivatives of the glucosides. According to Fischer emulsin can also transform lactose into galactose and dextrose. But this assertion needs to be verified, for it is very probable that the emulsin having served for these experiments contained a certain proportion of lac- tase. Emulsin, which acts on bodies very differently from a chemical point of view, acts differently on the various mono- saccharids, according to their configuration. Thus, while it acts on the /3-methyldextro-glucoside, it is without action on the <*-methyldextro-glucoside. In living plants, amygdalin is not transformed because it is localized in special cells, and is thus separated from the glucosides. Mechanical action is necessary to bring the two bodies into contact. Thus the transformation of amygdalin into oil of bitter almonds and hydrocyanic acid occurs very rapidly when the plants containing the glucoside and the enzyme are macer- ated in water. According to Guignard, the emulsin cells are located in the cotyledons. In the cherry-laurel the enzyme is local- ized in the cells of the endodermis. Emulsin gives characteristic reactions with the solution •of orcin as well as with Millon's reagent. With the latter, the vegetable cells containing emulsin are colored an orange- red. When the cells containing emulsin are carefully heated with a solution of orcin, a violet coloration is obtained. This solution is prepared by adding 2 cubic centimetres of hydro- chloric acid to a 1 : 10 solution of orcin. The physical and chemical conditions of the action of emulsin are but little known. Chloral, up to 3.5 per cent, does not influence the course of hydration by emulsin, but the enzyme is sensitive to the action of 8 per cent alcohol. Neutral salts do not appear to influence the course of the FERMENTS OF GLYCE RIDES AND GLUCOSIDES. 271 hydration. Alkaline salts, on the contrary, have a retarding influence. Emulsin plays an important part in the manufacture of the oil of bitter almonds as well as in the manufacture of laurel-water. To manufacture the oil of bitter almonds, the almonds are reduced to powder, the oil extracted, water added, and the mass left at the ordinary temperature for the reaction to take place. Fermentation ended, they are distilled with steam. To obtain a good yield one must avoid beginning to distill before the fermentation is finished. For the manufacture of laurel-water, the fresh leaves of the plant are used. They are crushed, cold water is added, and finally distilled. It is necessary to leave cold water for some time in con- tact with the leaves before heating. Emulsin is used in pharmacy, where it is prepared in the following manner : Sweet almonds are blanched, powdered, and submitted to a strong pressure which presses out the oils. The press- cakes are put to soak in three times their volume of water; the mass is again pressed and thus is obtained an oily liquid which is clarified by leaving it for some time at a temperature of 30 . Then the upper layer of the liquid, which consists of oil, is removed and the enzyme in the clear liquid is precipitated by alcohol. The precipitate is collected on a filter, washed in 95 per cent alcohol, and dried in vacuo. Thus a yellowish powder is obtained, very rich in phos- phates and mineral salts. Completely dried, it can be heated to ioo° without losing its activity. Emulsin is soluble in water and in a dry state remains un- changed for a long time. 272 THE ENZYMES AND THEIR APPLICATIONS. MYROSIN. Myrosin was discovered by Bussy in mustard-seed. The characteristic odor of black mustard-seed, when ground with water, is due to the presence and action of this enzyme. Myrosin is very widely distributed in the vegetable king- dom ; it is frequently found in plants of the family Cmcifercc. This diastase, like emulsin, is located in special cells scattered in the different organs of the plant, but chiefly in the root and the leaves. It acts on sinigrin or potassium myronate, which is de- composed by hydration. This chemical reaction is generally considered as taking place according to the equation : C 10 H 18 NKS 2 O 10 = C 6 H 12 6 + C 3 H 5 NSC + KHS0 4 . Sinigrin. Glucose.' Allyl iso-thiocyanate. Potassium bisulpbate. According to this equation, the decomposition would be produced without hydration. But free myronic acid has not yet been studied; it is very probable that potassium myro- nate has the formula: C 10 H 16 KNS 2 O 9 + H 2 0, and it is, therefore, probable that the diastase produces a hydration and not a simple decomposition. In the seeds of white mustard myrosin is also found, but the sinigrin is replaced by another glucoside, sinalbin. The reaction produced may be expressed by the following equa- tion: C 30 H 44 N 2 S 2 16 = Sinalbin. C 6 H 12 6 + C 7 H 7 0-NCS + C 16 H 24 N0 5 — HS0 4 . Glucose. Oxy-benzyl-thiocyanate. Sinapine sulphate. FERMENTS OF GLYCE RIDES AND GLU COS IDES. 273 Myrosin may also act upon many other glucosides. To this diastase is attributed the formation of essential oils of different plants such as: water-cress, Reseda odorata, and Cochlioria officinalis. RHAMNASE. This enzyme is found in the fruits of the Avignon berry (Rhamnus infectoria). It acts on a yellow coloring matter having the characteristics of a glucoside, xanthorhamnin, and transforms it into rhamnetin and isodulcite : C 24 H 32 14 + 3 H 2 = C 12 H 10 O 3 + 2C 6 H 14 O e . Xanthorhamnin. Rhamnetin. Isodulcite. ERYTHROZYME. This diastase is secreted by the root of the madder. It acts on a glucoside of alizarin : rubian, which is also found in fresh madder-root. The reaction probably occurs according to the following equation : C 26 H 28 14 +2H 2 - 2C 6 H 12 6 + C 14 H 8 4 . Rubian. Glucose. Alizarin. BETULASE. Betulase is met with in the bark of Betula lenta. This enzyme acts on gaultherin and the reaction may be expressed by the following equation: Ci4 H i 8 8 + H 2 = C H 12 O 6 + C 6 H 4 / q oqw Gaultherin. Glucose. Methyl salicylate. To prepare this enzyme, take the bark of the Betula lenta and reduce it to powder, treat it with 4 volumes of-glycerin and leave it at the ordinary temperature for 30 days. 274 THE ENZYMES AND THEIR APPLICATIONS. The mass is then pressed, and the enzyme precipitated from the solution with 5 volumes of alcohol. The deposit is filtered off, washed, and dried. A kilogram of bark gives with this treatment nearly a gram of enzyme. Betulase does not color with tincture of guaiacum and does not act on other glucosides than gaultherin. BIBLIOGRAPHY. CI. Bernard. — Legons de physiologie experimentale. Dobelle. — Actions du pancreas sur les grains et l'amidon. Proceed, of the Royal Soc, t. XIV. Duclaux. — Diastase du pancreas. Microbiolog. Encyclop. Chim., 1883, p. 153- Sur la digestion des matieres grasses et des celluloses. Comptes Rendus, 1882. Sigmundi. — Ueber die Fettspaltenden Fermente in Pflanzen. Akad. der Wissen. Wien, 1890-1891. Hanriot. — Sur un nouveau ferment du sang. Comptes Rendus, 1896, p. 753- Sur la repartition de la lipase dans l'organisme. Comptes Rendus, 1896, p. 833 ■ Sur la dosage de la lipase. Comptes Rendus, 1897, p. 235. Sur la non-identite des lipases d'origines differentes. Comptes Ren- dus, 1897, p. 778. Gerard.— Sur une lipase vegetale extraite du penicillium glaucum. Comptes Rendus, 1897, p. 37°. Robiquet. — Journal de pharmacie, 2 mai, 1838. Robiquet et Boutroux. — Nouvelles experiences sur les amandes ameres. Ann. de chim. et de phys., 1830. ■ Journal de pharmacie, XXIV, p. 326. Bussy. — Note sur la fermentation de l'huile essentielle de moutarde. Comptes Rendus, 1839, p. 815. H. Will. — Ueber einen neuen Bestandtheil des weissen Senfsamens. Akad. Sitzung. Wien, 1870, p. 178. Thomson et Richardson.— Ann. de chimie et de pharmacie, XXIX, p. 180. Hofmann— Synthese der atherischen Oele. Berichte der deutschen chem. Gesellschaft, 1874, p. 508/520, 1293. Portis.— Recherches sur les amandes ameres. Journ. de pharm. et chim., 1877, XXVI, p. 410. Emil. Fischer.— Einfluss der configuration auf Wirkung der Enzyme. Berichte der deut. chem. Gesellschaft, 1895, 2031. FERMENTS OF GLYCE RIDES AND CLUCOSIDES. 275 Procter. — Betulase. Zeit. fur phys. Chem., 1892, XVI, 271; Berichte der deut. chem. Gesellschaft, 1894, p. 864. Armand Gautier. — Legons de chimie biologique, p. 33. Journ. de ph. et chim., 1896, 6 e seri, t. Ill, p. 117. Johonson. — Sur la localisation de l'emulsine dans les amandes. Ann. des So. nat. Boton, 1887, p. 118. Ward and Dunlop. — Annals of Botany, 1887. Spatzier. — Ueber das Auftreten und die psychologische Bedeutung des Myrosins in die Pflanzen. Journal fur Wiss. Bot. 1893, XXVI, p. 55. Bulle. — Ann. de chim. et pharm., LXIX, p. 145. Ortloff.— Archiv. de pharm., XLVIII, p. 16. L. Guignard. — Recherches sur la localisation du principe actif des Cruci- feres. Journ. de Botan., 1890. Sur la localisation des principes actifs chez les Capparidees. Comptes Rendus, 1893, 587. Sur la localisation des principes actifs chez les Tropeolees. Comptes Rendus, 1893, 587. Sur la localisation des principes actifs chez les Resedacees. Comptes Rendus, 1893, p. 861. J. Effront. — Influence des antiseptiques sur les ferments. Moniteur scien- tifique, 1894. E. Bourquelot and Herissey. — Sur les proprietes de l'emulsine des. champignons. Comptes Rendus, 1895, CXXI, p. 693. Tieman and Harmann. — Ueber das Coniferin. Berichte der deutschen chem. Gesellschaft, 1874, p. 608. E. Bourquelot and Herissey. — Note concernant Taction de l'emulsine de 1'aspergillus niger sur quelques glucosides. Societe de Biologie, 1895. CHAPTER XXII. ZYMASE. Zymase or alcoholic diastase. — Preparation of the sap of yeasts and its properties. — Determination of the fermenting power of zymase. — Chemical and physical conditions of the action of zymase. — Experi- ment of Effront on intracellular fermentation. — Industrial applications of zymase. Zymase or Alcoholic Diastase.— The phenomena ob- served in alcoholic fermentation have for a long time occu- pied the scientific world and given rise to numerous theories and hypotheses. In 1858, Traube sought to explain the decomposition of sugar into alcohol and carbonic acid by the intervention of a diastase secreted by the yeast. This opinion was accepted by Berthelot as well as by some other scientists. None of them, however, brought experimental proofs to show that alcoholic fermentation constitutes a chemical reaction cap- able of being produced outside of living cells. The first attempts in this direction were made in 1871, by Mme. Manisseim, who found that the cells of dead yeast can still produce, under certain conditions, a decomposition of sugar into alcohol and carbonic acid. The experiments of Mme. Manassein were, however, far from convincing, and did not clearly establish the non-inter- vention of cells. It was Buchner who, in 1897, clearly showed the exist- ence, in the cells of yeast, of an enzyme causing alcoholic fer- mentation. By submitting yeast to a strong pressure he suc- ceeded in obtaining a very active liquid causing alcoholic fer- 276 ZYMASE. 277 mentation in the absence of any cells. He gave to the en- zyme contained in this extract the name of " zymase." This discovery gives a definite explanation of alcoholic fermentation ; it will certainly have a great influence on the study of similar phenomena, and will lead to the discovery of many other enzymes. Once established that alcoholic fer- mentation is caused by a chemical substance, there is reason to assume that other similar phenomena, such as butyric, vis- cous, and acetic fermentation, are likewise due to diastases secreted by the bacteria producing these fermentations. The isolation of these diastases seems to be only a matter of time. Preparation and Properties of the Sap of Yeast. — Buch- ner advises the following method for the preparation of the extract of yeast. Take 1 kilogram, of yeast, to which add 1 kilogram of quartz sand and 250 grams of infusorial earth. Crush the mass to make it plastic and pasty. This operation requires much care. The crushing must be done with a special machine and lasts about two hours per kilogram of yeast. The crushed mass is then submitted to a pressure of 500 at- mospheres. For this purpose a hydraulic press is used and the pressure should be produced slowly and gradually. In this way about 320 cubic centimetres of liquid are ob- tained. The mass, from which the sap has been extracted, is ground with 140 cubic centimetres of water and then pressed again very slowly at 500 atmospheres. Thus there is ob- tained, after 2 hours, 180 cubic centimetres of an extract, which is added to the liquid produced by the first pressure. By this means, 1 kilogram of yeast furnishes 500 cubic cen- timetres of extract. The liquid is stirred with 4 grams of infusorial earth, filtered through paper, and poured into a cooled receptacle. The extract obtained by Buchner's process is clear, light yellow, and has a characteristic odor. According to the origin of the yeast which has been used in its preparation, the liquid contains from 7 to 10 per cent of dry substance. 278 THE ENZYMES AND THEIR APPLICATIONS. The analysis of the liquid shows the following figures : Dry substance . . . . 6.7 Ash 1. 15 Albuminoid substances 3.7 In this analysis the albuminoid substances are calculated according to the richness of the liquid in nitrogen. The extract of yeast is saturated with carbonic acid and when it is brought to the boiling-point, one observes an abun- dant liberation of this gas and a strong coagulation which gives the liquid a semi-solid aspect. This liquid acts differently towards different sugars ; lac- tose and mannite remain intact in presence of the extract as in presence of yeast-cells ; saccharose, dextrose, levulose, and maltose, mixed with an equal amount of the yeast ex- tract, at the end of a quarter of an hour disengage carbonic acid, an action which sometimes lasts several days. The fermenting power of the liquid persists after it has been through a Berkefeld filter ; the activity of the liquid is not destroyed by passage through the Chamberland filter, but the fermenting power weakens, however, to a greater degree than in passing through the Berkefeld filter. The fermentation is retarded by these operations : the extract fil- tered in a Berkefeld filter produced fermentation only at the end of a day. The active substance contained in the extract is capable of diffusing through dialyser paper ; in fact, when a dialyser containing a certain quantity of yeast extract is placed in a 37 per cent solution of saccharose, numerous bubbles of car- bonic acd are seen to appear at the surface of the sugar solu- tion. Yeast extract can be dried at 30°-35° without losing its activity. By drying in vacuo, a hard product is obtained pre- senting the appearance of white of egg. A filtered solu- tion of this product possesses the same properties as yeast extract; in a dry state it keeps several months. ZYMASE. 279-. For the preparation of concentrated extract of yeast, one proceeds in the following manner : 500 cubic centimetres of sap are evaporated in vacuo at 20 or 25 ° to a syrupy con- sistency. The evaporation must be done very rapidly and lasts about half an hour. The syrup obtained is then spread in thin layers on glass plates and replaced in vacuo, or else left in the air at a temperature of 30° or 35 ° so that it can evaporate. After 24 hours the dried substance is scraped from the glass, reduced to powder and completely dried over sulphuric acid. Five hundred grams of yeast extract furnish 70 grams of a very soluble powder which shows great ac- tivity. It is to be observed that the concentrated extract of the juice keeps much better than the diluted extract. The solu- tion of diluted extract is rapidly destroyed in the presence of oxygen, while this same solution, brought to a syrupy con- sistency, keeps for a very long time, even at a temperature of 30 and in the air. Buchner succeeded in separating the diastase from the yeast sap by adding to the latter 12 times its volume of abso- lute alcohol. The precipitate thus obtained, and dried, is a white powder having the same properties as the extract but possessing a very slight fermenting power. The zymase enclosed in the cells resists quite high tem- peratures. A yeast dried in the air at a temperature of 37 and then heated to ioo° for 6 hours is still capable of pro- ducing alcoholic fermentation in a solution of saccharose. The cells of yeast which could not resist this temperature are killed and no longer reproduce. If, instead of bringing it to ioo°, the yeast is brought to a temperature of I40°-I45°, the cells lose all fermenting power. Zymase is then more resistant to the action of heat than the cells which secrete it. Determination of the Fermenting Power of Zymase. — The fermenting power cf yeast is measured by a method 280 THE ENZYMES AND THEIR APPLICATIONS. recommended by Meissel for the determination of the alcohol in fermented solutions. This method is based on the meas- urement of the carbonic acid formed during the fermentation. Forty cubic centimetres of extract are introduced into a flask of 1 20 cubic centimetres capacity, and a quantity of powdered saccharose sufficient to obtain a 12 to 15 per cent sugar solution is added. The flask is left alone for a few minutes, after which it is shaken and closed with a rubber stopper through which two tubes are passed. One of them is furnished with a tap on the outside, and descends to the surface of the liquid. The other tube is open and communi- cates with a washing-bottle containing 2 cubic centimetres of sulphuric acid; the open end is provided with a rubber Bunsen valve. At the end of the experiment the tap is opened, air is al- lowed to pass into the apparatus so as to drive out the carbonic acid and the apparatus is placed on the balance. The difference in weight shows the quantity of carbonic acid disengaged. Method of Decomposition of Sugar by Zymase. — A solu- tion of cane-sugar with arseniate of potassium added and fer- mented with 450 cubic centimetres of yeast extract at 12 for 40 hours furnished 6.67 gr. of carbonic acid and J.J2 gr. of alcohol, the alcohol which was in the extract at the beginning being deducted. In fermentation produced by yeast-cells there is theoret- ically obtained 48.89 parts of carbonic acid and 51.1 1 parts of alcohol. By comparing these figures with the preceding, Buchner found that the relation between the quantities of carbonic acid and alcohol is practically the same in the two cases, and that the decomposition of sugar by yeast extract is accomplished in the same way as by the cells. It appears from these data that the fermenting power of yeast extract can be measured by the carbonic acid liberated during its action. These experiments of Buchner show us only the general lines of the course of decomposition of sugar ZYMASE. by zymase. The method he adopted for the determination of alcohol and carbonic acid is far from being exact and from giving information on the degree of purity of the fermenta- tion obtained with zymase. Pasteur demonstrated that the entire amount of sugar which disappears during fermentation is not transformed quite in accordance with the following equation : C 6 H 12 O e = 2C0 2 + 2C 2 H 5 OH. There is always a little sugar which is broken up in a dif- ferent way, and furnishes glycerin and succinic acid. It is very probable that zymase acts quite differently from beer- yeasts, and that the fermentation brought about by its use may give much purer products than those obtained by yeasts. Still, the zymase isolated from the cells is relatively weak one hundred cubic centimetres of this extract, representing 200 grams of yeast, produce, by acting on a sugar solution less carbonic acid than a gram of yeast. Probably there exists in the yeast-cells only a small quantity of zymase which, further, undergoes a change during extraction. Influence of Physical and Chemical Conditions.— Zymase is very sensitive to the action of the temperature A 27 per cent solution of sugar, with yeast sap added, produces very different quantities of carbonic acid according to the temperature of fermentation. Temperature. Carbonic acid (in grams) formed after 6hours - 21 hours. 2 4 ~hours. 40 hours.' 14 °-43 1. 11 1. 14 1.27 22 ° °76 I.OI 1.02 1.00 Q At the beginning of the experiment the temperature of 22 is very favorable to the action of the enzyme; after 6 hours of fermentation there is found, at this temperature a liberation of 0.76 grams of carbonic acid, while at 12 to i> there is formed only 0.43 gr. of this gas. But if the opera- tion is prolonged at a temperature of 22°, the action 282 THE ENZYMES AND THEIR APPLICATIONS. slackens; this slackening is evidently because of a partial destruction of the diastase. The course of fermentation is also influenced by the con- centration of the sugar solutions. CO» (in grams) after Saccharose, , per cent. i6 hours> 2 ^ hours. 40 hours. l6 1-33 I.46 I.48 27 O.7O O.80 O.82 37 O.60 O.72 O.74 In liquids containing 16 per cent of sugar the fermenta- tion is more active than in those which contain 27 per cent, but the enzyme still acts on solutions containing 37 per cent of sugar. In the solutions containing from 40 to 50 per cent of sugar the fermentation is almost completely stopped, but the enzyme is not at all changed, for, by diluting the solution, fermentation can be brought about anew. The diastatic activity of zymase diminishes in proportion as its action is prolonged ; if we compare the quantity of car- bonic acid formed by the action of the enzymes during the first 16 hours with the quantity of gas freed during the fol- lowing 16 hours, we find a rapid decrease of the fermenting power. By calculating this power per 100 cubic centimetres of extract per hour, Buchner found that zymase furnishes, on the average, the following quantities of carbonic acid: From 1 to 16 h. 16 to 24 h. 24 to 40 h. 40 to 60 h. Average of 3 experiments. . . 0.17 gr. 0.060 gr. 0.020 gr. 0.002 gr. " "2 " ■-. 0.11 " 0.010 " 0.002 " " "2 " ... 0.08 " 0.016 " 0.004 " Yeast extract, like all diastatic solutions, produces in "hydrogen peroxide a liberation of oxygen. When hydrocyanic acid has been added to the extract it loses this characteristic property. But if the extract with hy- drocyanic acid added is then submitted to prolonged action of the air, the reaction with hydrogen peroxide reappears. ZYMASE. 283 It is, therefore, probable that hydrocyanic acid combines with the diastases, forming a combination which impedes their activity, and that this combination is destroyed by con- tact with the oxygen which regenerates the enzyme. An experiment of Buchner relative to the action of hy- drocyanic acid and air on zymase may be given : Four cubic centimetres of extract were mixed with 6 cubic centimetres of 2 per cent hydrocyanic acid; to one half (A) of the mixture were added 3 grams of cane-sugar; the other half (B) was submitted to the action of the air for 1 hour, then 3 grams of saccharose were added. The liquids were placed in U tubes closed at one end. In experiment A not a trace of carbonic acid was produced, while in experi- ment B the closed part of the tube was filled with this gas at the end of 20 hours, the liberation having begun at the end of 5 hours. Zymase is influenced by the chemical conditions of the medium. Neutral salts, like ammonium sulphate, calcium chloride, etc., have a retarding influence on fermentation. A direct relation is also found to exist between the ac- tivity of the yeast extract and the presence of coagulable albumen in the extract. Yeast extract, kept for some time at a temperature of 35 to 40 , becomes turbid, contains flocculent masses, and loses its activity. On the other hand, it has been observed that when an extract becomes inactive for any reason, it scarcely coagulates at all at a temperature of 40 to 50 . This rela- tion between the presence of coagulable materials and the diastatic activity is explained by Buchner in the following way. According to him, zymase is an albuminoid substance which is coagulated by heat, but this coagulation does not occur when the diastatic substance is transformed. Buchner also interprets the great instability of zymase by the presence of a peptonizing diastase in the yeast extract; this enzyme would act on zymase rendering it inactive. It 284 THE ENZYMES AND THEIR APPLICATIONS. is the simultaneous presence of these two ferments, the one peptonizing and the other producing the decomposition of sugar, which explains the activity of yeast extract at rela- tively low temperatures. At 22 peptase acts with more energy than zymase, while at a low temperature the peptonizing action is not complete and zymase can produce greater quantities of alcohol. The favorable influence of sugars on the preservation of zymase also speaks in favor of the hypothesis of the digestion of zymase by the peptonizing enzyme. It is known that in concentrated solutions of saccharose the digestion of fibrin by pepsin is retarded. Now, if one volume of yeast extract is mixed with one volume of a 75 per cent solution of saccharose, a solution is obtained which keeps for a week at the ordinary temperature and for 15 days in a refrigerator. Sugar, therefore, has a very favorable ac- tion on the preservation of zymase. The activity of yeast extract varies noticeably according to the variety of yeast used in its preparation. Generally bottom yeasts give a very active extract, while bakery yeasts contain hardly any zymase. A great difference is also ob- served between extracts of fresh yeasts and those of yeasts which have remained in the air for some time. The latter give less active extracts. However, not all brewery yeasts give an extract of the same activity. The origin of the yeast plays an important part here. The differences observable ac- cording to the origin and age of the yeast bring up an argu- ment for the hypothesis explaining the alteration of zymase by the action of another enzyme. Buchner divided a certain quantity of yeast into two parts A and B. The extract was pressed out from A immediately, while the portion B was left to itself for 3 days at 7°-8° be- fore the sap was expressed. The extract of A possessed a strong activity, while the liquid coming from B gave only a small quantity of carbonic acid. Buchner attributed the diminution of activity in the second case to a peptonization. ZYMASE. 285 produced during the 3 days' standing at 7°-8°. The action of pepsin would also, according to him, be the cause of altera- tion of the diastase contained in compressed yeast. This opinion is also reinforced by an experiment of Hohn, who quite recently demonstrated the presence of the pro- teolytic enzyme in the sap of yeasts. Buchner has, however, shown directly the peptonization of zymase by the enzyme by the following experiment: He placed on ice 3 tubes containing 3 cubic centimetres of yeast extract. To two of these tubes were added 0.1 gr. of trypsin, the third serving as the control. After 12 hours each tube received 2 grams of pulverized saccharose. The experiments made with trypsin remained absolutely inactive after the addition of cane-sugar; on the other hand, in the control tube a very active fermentation was produced. The discovery of Buchner has had numerous opponents, who have tried to show that yeast extract always contains either cells or ferments and who have attributed the decom- position of sugar to the intervention of living cells. Buchner successfully refuted all these objections by means of conclusive experiments. The existence of zymase is demonstrated by the following facts : 1st. An alcoholic fermentation can be obtained with the solid substance obtained by the precipitation of the sap by alcohol ; 2nd. With yeast extract an almost instantaneous fermen- tation may be obtained, whose intensity diminishes with time. If it is a question of living cells a contrary phenomenon is observed : the fermentation increases in intensity as the cells develop ; 3rd. Yeast extract produces a fermentation in the pres- ence of amounts of antiseptics which check the activity of living cells ; 4th. By passage through a porcelain filter, active liquids 2 86 THE ENZYMES AND THEIR APPLICATIONS. are obtained without it being possible to discern the pres- ence of organisms. To sum up, alcoholic fermentation is produced by chem- ical agents, without, and in the absence of, living cells. It is true that the material which produces this trans- formation is elaborated by the vital activity, and that its formation is intimately related to the growth and multiplica- tion of the cells. The fermenting power of the cells is, there- fore, reduced to their ability to produce zymase. Intracellular Fermentation. — Zymase should be found in many other living cells. The fermenting power which can develop in certain fungi should, it seems to us, be attributed to a secretion of zymase which occurs under special con- ditions. There is also reason to believe that, in the phenomena of intracellular fermentation, it is zymase again which plays an •active part. Pasteur found that fruits plunged in carbon- dioxide gas enter into fermentation and transform sugar into alcohol and carbonic anhydride. Muntz, by replacing air by nitrogen, found the same phenomenon for leafy plants. Under these conditions al- cohol is formed in the leaves of the plant. This phenomenon is explained by vital activity and it is assumed that the changes in the work are due to changes in the conditions of nutrition. It is easier, we believe, to suppose that the absence of oxygen is, under these conditions, favorable to the secretion of zymase. The fermentation observed in this case is similar to that produced by the action of alcoholic yeast; in both cases the zymase is the cause. The action of zymase in fruits protected from the air has furnished us with the subject of interesting researches which we are at present pursuing, and which are still far from being completed; but we can even now give some information which will find its complete development in a later work. ZYMASE. 287 The numerous experiments we have made have confirmed, in our opinion, the presence of zymase in fruits, especially cherries and plums, in peas, and in barley. The first experiments were made with cherries in the fol- lowing way : The fresh fruit was washed in a dilute solution of formic aldehyde to destroy the micro-organisms, then carefully wiped and submerged in flasks containing olive oil. At the end of 3 days the cherries were covered with little bubbles of gas and there was then found, above the oil which covered the fruits, a liberation of carbonic acid which increased after the 5th day. Fermentation continued very slowly for 20 days at a tem- perature of io°. After this time the oil was poured off, the cherries, and also the stones, were crushed in a mortar and the juice pressed out by squeezing the mass in a cloth. The residue was removed from the cloth, treated cold with ether to free it from the oil, then dried in vacuo, and re- duced to a fine powder, which was soaked in 2 volumes of water with a small amount of ether added. It was left in a corked bottle at 5 for 12 hours, after which the mass was submitted to strong pressure. Thus a liquid was obtained which, filtered through filter-paper, w r as a viscous, trans- parent solution, of slightly acid reaction, and giving the re- actions of guaiacum and hydrogen peroxide. The presence of zymase in such a liquid may be found by the following experiments : To 50 cubic centimetres of the liquid add 7 grams of pow- dered cane-sugar and leave for 6 hours at 22 in a small flask furnished with a delivery-tube. After 2 hours, the formation of carbonic acid is observed, and after 6 hours a diminution in weight of 3 decigrams. A parallel experiment is made with the same liquid pre- viously kept at 40 for an hour in a closed flask, then cooled to 22° and left at this temperature for 5 hours, leaving the delivery-tube open. In this second experiment neither liber- 288 THE ENZYMES AND THEIR APPLICATIONS. ation of gas nor diminution of weight is found. The alco- holic diastase has therefore been destroyed by heat. . The analysis of the unfermented sugar solution shows that a change has occured in its chemical composition. We have, for example, found in one of our experiments that 3.4 grams of saccharose had been transformed into invert- sugar. This transformation cannot be attributed to the acidity of the medium, for by heating the active liquid in a closed vessel for 10 minutes at 8o° before the addition of sugar, and by then keeping the liquid with sugar added for an hour at 40 , then for 5 hours at 22 ; we obtained only 0.15 gr. of invert-sugar instead of 3.4 gr. The active liquid evidently contains zymase and sucrase, and while the zymase is de- stroyed by the heat, the sucrase is not changed. The existence of zymase in the juice of cherries has been confirmed by other experiments in which it has been pos- sible to measure the alcohol produced. The following method of procedure was employed: To 200 cubic centimetres of active juice were added sugar and 2 grams of chloroform. In a parallel experiment, a little chloroform and 2 grams of yeast were added to a 15 per cent solution of sugar. The two liquids, left 5 days at io°, gave .different results. In the solution containing yeasts fermentation did not take place, while in the juice of the cherries 0.8 gr. of alcohol was found. The non-existence of yeasts in the fermented solution was confirmed by microscopic analysis as well as by cultiva- tion on plates. Experiments made with a view to precipitating the dia- stase by alcohol have not furnished satisfactory results. The active liquid loses its properties in passing through porce- lain bougies, and the zymase which we obtained differs in this respect from the enzyme isolated by Buchner. In the course of our experiments we found, furthermore, ZYMASE. 289 that fresh peas, as well as barley, furnish quite considerable quantities of alcohol by intracellular fermentation. Sugar- peas, left in oil, gave on analysis 2 per cent of alcohol. Bar- ley, previously soaked, dried, and put in oil, gave 1.6 per cent of alcohol. By treating these seeds by processes similar to those used for cherries, we were able to discover the presence of zymase. Industrial Applications of Zymase. — Zymase, while very interesting from a theoretical point of view, will per- haps also have in the future numerous industrial applica- tions. The fermentation produced by the enzyme, without direct intervention of yeasts, presents theoretically a great advantage. In this way much more rapid fermentations can be obtained, and purer and better products. At the present time distillers and brewers cultivate their own yeasts and seek to adapt them to their kind of work. However, even starting with pure yeasts, good results are not always obtained; often infection takes place and con- sequently a degeneration of the yeast. It is to be hoped that in the fuUire the cultivation of yeasts and the subsequent preparation of zymase will be done in special manufactories, where the brewers and dis- tillers will produce preparations of great activity and pro- ducing an immediate action. It is true that in the brewery yeast plays also an im- portant part from the point of view of the elimination of nitrogenous materials, a work which zymase alone could not do. It would be necessary, therefore, in working with en- zymes alone, to change completely the technique and invent new processes. The discovery of zymase is too recent to have caused great industrial changes immediately; however, the first at- tempts at practical application have already been made by Buchner, who has invented a new process having for its aim 290 THE ENZYMES AND THEIR APPLICATIONS. the preparation of a durable yeast intended to replace pressed yeast in bread-making. This process consists in first drying the yeast at low tem- perature, then heating to 50 , then to ioo°, and then, when the yeast is completely dried, grinding it to powder. It is then ready for the market. This process has many advantages. The yeast pre- pared in this way keeps much better than compressed yeast, dead cells being less subject to change than living cells. Moreover, the greater drying of this yeast recommends it from a hygienic point of view, for the micro-organisms which yeast breads always contain, are here destroyed and can no longer affect the dough, even when it is insufficiently steri- lized. Buchner's yeast is called enduring yeast (Dauerhefe) ; it is used in the bakery in the same way as the ordinary compressed yeasts. According to the author of the patent, experiments have proved that the addition of 5 to 10 per cent of enduring yeast are sufficient to produce a satisfactory dough. BIBLIOGRAPHY. Berthelot. — Chimie organique fondee par la synthese. E. Buchner. — Alkoholische Gahrung ohne Hefezellen. Berichte der deutsch. chem. Gesellschaft, XXX, 1897, p. 117. Ueber zellenfreie Gahrung. Berichte der deutsch. chem. Gesell- schaft, XXX, 1 1 10. E. Buchner. — Procede pour la fabrication des levures d'attente. Brevet, Allemagne, 1897, 2668, n° 97240. Bechamp. — Sur la presence de l'alcool dans les tissus animaux pendant la vie et apres la mort, dans les cas de putrefaction, aux points de vue physiologique et toxicologique. Comptes Rendus, 1879, p. 573. Hahn. — Berichte der deutsch. chem. Gesellschaft, 1898. Geret and Hahn. — Berichte der deutsch. chem. Gesellschaft, 1898. Pasteur. — Etude sur la biere. Comptes Rendus, 1876, LXXVII, p. 1140. Etude sur la biere, 1876. Paris, Gauthier-Villars. Memoires sur la fermentation alcoolique. Comptes Rendus, 1859, P- 1149, XIV, III. Bull, de la Soc. de chim. Paris, 1861. Sur la production de l'alcool par les fruits. Comptes Rendus, 1872,. p. 1054, LXXXV. ZYMASE. 291 Pasteur. — Sur la theorie de la fermentation. Comptes Rendus, LXXV. Liebig. — Sur les phenomenes de fermentation et de putrefaction. Ann. de chim. et de phys., 1889, t. LXXI, p. 147. A. Munz. — De la matiere sucree contenue dans les champignons. Comptes Rendus, 1874, t. LXXIV. Recherches sur la fermentation alcoolique intracellulaire dans les vegetaux. Comptes Rendus, 1878, t. LXXXVI, p. 49. Recherches sur la fermentation intracellulaire des vegetaux. Ann. de chim. et de phys., 1878, 5 e serie, t. XIII, p. 543. Alcools du sol. Comptes Rendus, XCII, p. 499. Neumeister. — Ber. der deutschen chemischen Gesellschaft, XXX, p. 2963. Stavenhagen. — Ber. der deutschen chemisch. Gesellschaft, XXX, p. 2422. Marie Manassein. — Ber. der deutschen chem. Gesellschaft, XXX, p. 3061. E. Buchner and Rapp. — Alkoholische Gahrung ohne Hefezellen. Berichte der deutsch. chem. Gesellschaft, 1897, 3, 2670; 1898, 1, 209; 1898, i, 1084; 1898, 1, 109. CHAPTER XXIII. OXIDASES. Presence of oxidases in vegetable and animal cells. — General properties. — Laccase. — Tyrosinase. — Influence of the medium. — Action of oxidases on phenols insoluble in water. — The "breaking" of wines: cenoxi- dase. — Oxidin. — Olease. Soluble ferments have for a long time been considered as substances acting- only as hydrolyzing agents, that is causing the fixation of one or more molecules of water at the same time with a molecular decomposition. Oxidation and dehydration, molecular change without fixation of water, all these chemical phenomena were attributed to the direct ac- tion of vital energy without any diastatic intervention. This entirely erroneous theory has lately been success- fully refuted by a series of discoveries accomplished in the domain of biological chemistry by Bertrand, Bourquelot, Hikorokuro Yoshida, Cazeneuve, Martinand, etc., whose works we shall have occasion to examine. The studies of these scientists have demonstrated the existence of a series of substances having characteristics of true oxidizing agents, and causing oxygen to unite with certain bodies. These substances, secreted by living cells, have received the name of oxidases. These enzymes facili- tate the oxidation of certain substances, either by dehydrat- ing them or by enlarging their molecule by the addition of oxygen. Certain vegetable juices, such as wine, the latex of the lac-tree, the juice of pears, plums, and other fruits, as well as 292 OXIDASES. 293 oertain fungi, change when exposed for some time to the air. This phenomenon, which is generally shown by a change of color or, in case of solid bodies, by an increase in tem- perature, does not occur in vacuo. It therefore possesses all the characteristics of oxidation, because the intervention of air, and consequently of oxygen, is indispensable to its production. The direct cause of this oxidation remained for a long time unknown. It was discovered in 1883 by a Japanese chemist, Hikorokuro Yoshida, who, by making experiments on the oxidation of the latex of the lac-tree, discovered in this phenomenon the intervention of a diastase. This discovery gave a new impetus to studies upon en- zymes ; the question was taken up in different laboratories, and discoveries bearing on the most varied subjects were soon numerous and conclusive. There were successively studied vegetable tissues, muscles, organic secretions, and each investigation carried on in this field brought new proofs of the existence of oxidases. The oxidation of the latex of the lac-tree and its transfor- mation into a black varnish was clearly recognized as a phenomenon of the diastatic order. A transformation pre- senting some similar characteristics and occurring in the juices of numerous vegetables, such as mushrooms, pota- toes, beets, and the rhizomes of Canna.indica, was attributed by Bourquelot, Lindet, Bertrand, etc., to another oxidizing enzyme. The decoloration of wine and the deposition of the color- ing matter were recognized as being phenomena of the same order, and were attributed to the action of a diastase which certain authors regard as pre-existent in the must, while others consider it as the product of a mould : Botrytis cinerea. In the animal kingdom the experimenters had occasion to make quite as numerous and quite as interesting dis- coveries. An oxidizing ferment was found in the saliva as well 294 THE ENZYMES AND THEIR APPLICATIONS. as in other secretions — nasal mucus, tears, semen — while the urine, bile, and intestinal secretions were found to be free from any ferment of the kind. Jacquet, in 1882, made experiments on the oxidation of benzyl alcohol and salicylic aldehyde with pieces of lungs, loins, and muscles of the horse, previously treated with car- bolised water, then frozen and reduced to pulp. These frag- ments of organs caused an oxidation which was no longer produced when they had been cooked in boiling water. Even at that time Jacquet realized that the oxidation did not come solely from the cells, because the aqueous extract of these tissues, as well as the cells themselves, produced a fixation of oxygen on benzyl alcohol and salicylic aldehyde. Aleloos and Brauwer confirmed these results by collect- ing a substance, extracted from a horse's liver, which, pre- cipitated from its aqueous solution by alcohol, oxidized formic aldehyde and transformed it into acid with liberation of carbon dioxide. This substance lost, moreover, all oxidizing action after having been heated to ioo°. Spitzer and Rhomann found this substance in the blood and in the organs of several mammals. Finally, the phenomena of internal destruction which we have had occasion to observe in yeasts, can be attributed to oxidizing diastatic actions. We have found that by reducing a certain quantity of compressed yeast to minute fragments and then heaping them up, an increase in temperature is soon manifest, which may reach 40 at the end of 2 hours. This temperature may, for example, be obtained with 2 kilograms of fresh yeast ground and massed in heaps 20 centimetres in height at a temperature of 20 . The same experiment, made in vacuo, does not result in the least elevation of temperature. The experiment may be made in the following manner: In a half-litre flask, provided with three tubes, dispose layers of yeast reduced to small fragments and alternating with layers of pumice-stone, which prevents the yeast from OXIDASES. 295 settling. A thermometer is introduced in the center tube and a current of air established with the other two. As soon as the air enters the flask the temperature rises, and if the tap is closed to the air it is immediately found to decrease. By allowing the experiment to continue for several hours, it can be renewed several times with the same yeast, for 3 or 4 consecutive days ; and it will be observed that at each entry of the air in the bottle the temperature rises.* When, on the contrary, air is allowed to pass into the bottle for 5 or 6 consecutive hours, the yeast liquefies and is completely exhausted. By crushing the yeast with pumice-stone in a powerful crusher a paste is obtained which, allowed to stand with cold water and filtered, gives a liquid free from cells and yet offer- ing, from the point of view of oxidation, the same properties as the yeast itself. The fragments of pumice-stone, impregnated with liquid, put in a mass of glycogen in the air produce there an eleva- tion of temperature of from 4 to 6 degrees. This extract is less active than the yeast itself, but a series of experiments have shown us that it possesses, like yeast, an oxidizing dia- static power. In view of all these facts it is unquestionable that the phenomena of respiration and oxidation of vegetables and animals must be generally attributed to oxidases. It is seen, after this short exposition, that the discovery of oxidases was of considerable importance, because it has thrown some light on phenomena still unexplained, or which were explained by erroneous theories. The study of oxidizing enzymes has also much interest from a chemical point of view for they constitute very sensi- tive reagents for many organic substances. * This experiment is specially interesting from the point of view of gas analysis. We have, in fact, observed that one can in this way dis- tinguish 1 per cent of oxygen when mixed with inactive gases. 296 THE ENZYMES AND THEIR APPLICATIONS. General Properties of Oxidases- — Like all diastases, oxi- dases are extremely unstable bodies. They are destroyed by heat above 6o°. Antiseptics, in general, appear capable of simply retard- ing the oxidation produced by these agents. This retarding action of antiseptics has not, however, been generally estab- lished. We think, on the contrary, that the different dia- stases belonging to this class are more or less sensitive to the action of antiseptics, and that to this fact must be attributed the negative results of a number of investigations carried on with bodies which certainly contain oxidases. Alcohol, when sufficiently dilute, does not appear to hin- der the action of enzymes of this class. The diastase of latex, laccase, still produces an oxidation in a 50 per cent alcoholic solution. Soluble oxidizing ferments give a strong blue color to tincture of guaiacum to which hydrogen peroxide is not added, guaiaconic acid being formed with the oxygen ab- sorbed from the air. Temperature, as well as the reaction of the medium, in- fluences the action of oxidases. Finally, the greater number of 'oxidases act especially on bodies of the aromatic series : phenols, amines, and their sub- stitution products. The oxidation products brought about by diastases are as yet poorly defined. The oxidation of bodies of the aro- matic series is produced either by an elimination of the hy- drogen or by direct addition of oxygen. This oxidation is never complete. The oxidation of fatty substances is much more energetic ; it leads to a complete destruction and to the formation of carbonic acid. The action of oxidases is not at all specific. Laccase, for example, transforms hydroquinone (diatomic phenol) just as well as pyrogallol (triatomic phenol). The position of the groups appears, however, to play a OXIDASES. 297 principal part ; the para position, for example, seems to in- fluence the reaction favorably. Among the diastases producing hydrolysis the individu- ality is more strongly marked; sucrase, for example, can only decompose saccharose and is incapable of acting on very closely related bodies which differ only by their molecular configuration. The quantity of oxygen absorbed under the action of oxidizing enzymes may serve, in most cases, to measure the intensity of oxidation. Preparation of Oxidases. — Oxidases are extracted from bodies which contain them by the methods generally -used for the extraction of soluble hydrolyzing ferments. The bodies serving in the preparation are ground and then extracted in the presence of chloroform. The use of the latter body constitutes a danger, however, for it is not known whether this antiseptic, which leaves most hydrolyz- ing diastases intact, is also without action on all the oxidases. It is, therefore, to be recommended, in the preparation of oxidases, to make two triturations, one with water and chloroform, the other with water containing ether. In cer- tain cases the oxidases will be found in the water and ether, while the chloroform infusion will not contain a trace of active substances. The infusion is then precipitated by alcohol ; the precip- itate formed is redissolved and reprecipitated several times to purify it. The method of extraction with glycerin is also applicable to the preparation of oxidases. LACCASE. Laccase is a soluble ferment producing the oxidation of the latex of the lac-tree and transforming it into a very beau- tiful varnish which the Japanese, the inhabitants of Tong- 298 THE ENZYMES AND THEIR APPLICATIONS. "king, and the Chinese use for varnishing their furniture. The latex is a clear liquid presenting the appearance and the con- sistency of honey. It is collected in eastern Asia by making incisions in the bark of certain resinous trees of the order Anacardiaceas (Rhus vermicifera). The odor of the latex is very slight and somewhat re- sembles that of butyric acid; it has an acid reaction. The latex changes with extraordinary rapidity. Ex- posed to oxygen it turns brown and its surface becomes covered with a resistant film of a beautiful black color and absolutely insoluble in ordinary solvents. In vacuo, change does not occur and the. latex can be kept for a very long time. The first data on laccase were obtained by the Japanese chemist Hikorokuro Yoshida. The study of the oxidation •of the latex revealed to him the presence of a body which lie called uruschic acid (C 14 H 19 2 ), a body which by oxida- tion changes into oxyuruschic acid, as is shown by the fol- lowing equation: 2C 14 H 19 2 + 3 = 2C 14 H 18 3 + H 2 0. Uruschic acid. Oxyuruschic acid. Bertrand, by diluting the latex in a great quantity of al- cohol, discovered in it two products, one which enters into solution, while the other is precipitated. This precipitate, separated from the liquid, is a sort of ;§um. It is carefully washed with alcohol, taken up again with distilled water, then precipitated again with 10 volumes of alcohol. Then it can be collected in the form of flakes and dried in vacuo. The product obtained by this method resembles ordinary gums and is, like them, transformed by "hydration into a mixture of galactose and arabinose. This body possesses a diastatic power. The alcoholic solution, after the gummy precipitate has been removed, is quickly distilled in vacuo. The residue is OXIDASES. 299 shaken in water, then in ether; the water retains the glucose, mineral salts, etc., and the ether dissolves the resinous ex- tract of the latex. The ether is then decanted, and evapor- ated in an atmosphere of hydrogen. The product obtained by this method is laccol; it is an oily liquid, with a high density, not dissolving in water but entirely soluble in alcohol, ether, chloroform, and benzol. The manipulation of this product presents certain dangers : traces of laccol may act in an injurious way on the skin. In the air, it turns a reddish-brown color, becomes somewhat viscous, and is finally converted into resin. Oxidation, favored by potash and soda, is produced in dif- ferent stages. The liquid becomes warm, turns green, then inky black, and absorbes a great quantity of oxygen. Lac- col gives with ferric chloride and lead acetate reactions much resembling the reactions which the polyatomic phenols pro- duce with the same agents. . In the presence of lacca.se, the oxidation of laccol is much more pronounced, much more rapid, and finally gives a black insoluble substance which is not obtained in the absence of the enzyme. Bertrand, at the beginning of his studies, thought that the addition of oxygen was effected by simple chemical affin- ity, and that laccase then acted on the oxidized bodies in the manner of a hydrating agent. In the course of his experiments, the French chemist suc- ceeded in determining the true mechanism of oxidation. He observed that the quantity of oxygen absorbed by the laccol in contact with the air increases with the amount of laccase used, which can only be explained by a direct oxidiz- ing action of laccase. Conclusive proofs were afterwards furnished by Bertrand. He caused a certain quantity of laccase to act on bodies nearly related to laccol, principally on hydroquinone and py- rogallol, and found that in the presence of laccase all the polyatomic phenols absorbed a certain quantity of oxygen, 3°° THE ENZYMES AND THEIR APPLICATIONS. liberating carbonic acid. In the absence of the enzyme, on the contrary, or even with a diastatic solution heated to ioo°, no oxidation occurred. The oxidizing action of laccase is therefore well demonstrated. Bertrand then discovered a very sensitive reaction for discerning the presence of oxidases in plants. He found that the alcoholic tincture of guaiacum takes, in the pres- ence of laccase, a deep blue color by the action of air alone, while to obtain the same result with hydrolyzing diastases, hydrogen peroxide must be used. The same reaction takes place also when cuttings of or- gans containing an oxidizing diastase are treated with tinc- ture of guaiacum. The sensitiveness of this reaction allowed Bertrand to recognize the presence of laccase in a great number of vege- tables, and to evolve the hypothesis, which is moreover quite justifiable, that laccase is distributed all through the vegetable kingdom. This diastase has been found in the following list of plants: Beets, carrots, turnips (roots), dahlias (roots, tubers),, potatoes (tubers), asparagus (yellow stem), lucerne, clover, ray grass (stems and leaves), Jerusalem artichokes, apples, pears, chestnuts, gardenias (flowers), lac-tree (latex). For the extraction of laccase, secreted by the vegetables, we have just named, Bertrand made use of a method slightly different from that used for the latex. The juice ex- tracted from the parenchymatous organs of the rhizomes, or tubers is precipitated immediately after its extraction. As to the liquid extracted from the green parts of the plant, chloroform is added to it and it is allowed to stand at the ordinary temperature for 24 hours; then a coagulum forms which is separated from the rest of the liquid, and in the filtered liquid the precipitation by alcohol is accomplished. This precipitation is made in the same way as for the latex o£ the lac-tree. OXIDASES. 3 QI Bertrand observed that the greatest quantity of laccase is secreted by the organs in the course of development. Emile Bourquelot and Bertrand sought the presence of laccase in mushrooms, plants which, as we know, cause energetic phenomena of oxidation. Schonbein, in 1856, had already made the curious obser- vation, which, moreover, remained as a simple observation, that the juice of two mushrooms, Boletus luridus and Aga- ricus sanguineus, colored blue tincture of guaiacum with- out addition of hydrogen peroxide and lost this faculty when it was heated to ioo°. The presence of oxidases was sought for by the French scientists in more than two hundred kinds of cryptogams and the reaction of guaiacum was tried in the various organs of these plants. They examined especially the Basidio- mycetes, some Ascomycetes, a Myxomycete, — Reticularia maxima, the Polypori, and the Agaricines. Russula f ceteris, Persoon, was studied particularly on account of the pecu- liarity which all its parts have of coloring blue with a solution of guaiacum. The investigators cut and crushed 125 grams of Russula, then soaked it in water with chloroform added. The filtered liquid took in the course of an hour pale yel- low, then dirty red tints; it presented all the characteristics of a solution of laccase. The oxidizing diastase of these different plants is soluble, at least in part, in alcohol, for when an excess of this reagent is added to the diastatic solution, even when the latter is very active, only a very weak precipitate is obtained. Below is the table which Bourquelot and Bertrand give as a summing up of their experiments, from which it is seen that the oxidizing enzyme is found in plants destitute of chlorophyll. In the mushrooms it is distributed throughout the whole reproductive portion ; it is found localized in the lamellae of certain hymenomycetes, or at the base of the stipe. 302 THE ENZYMES AND THEIR APPLICATIONS. „ xt u r Species. Genus Number of \__^ s P ecieS , With Withou? sub-genus. examined. laccasei laccase- Russula 18 18 o Lactarius 20 18 2 Psalliota 5 4 1 Boletus 18 10 8 Clitocybe 9 5 4 Marasmius 6 o 6 Hygrophorus 6 o 6 Cortinarius 12 1 11 Inocybe 6 1 5 Amanita 7 2 5 Manner of Action of Laccase. — Laccase acts on a large number of substances. Added to a solution of hydroquinone in an open vessel, it produces comparatively rapid oxida- tion. The solution takes a deep color and at the end of some time crystalline plates of a green color are formed. The oxidized liquid has the characteristic odor of quinone, and the reaction may be expressed by the following equation: 2C 6 H 4 <(g^ + 3 =2C 6 H 4 <° Hydroquinone. Quinone. The diastase also acts on gallic acid, but the product of the reaction has so far been little studied. By causing a certain quantity of laccase extracted from Russula to react on gallic acid, Bourquelot and Bertrand ob- tained the following results: Quantities used: Gallic acid 1 gr. Water 100 c.c. Laccase solution 5 c.c. After one hour: Oxygen absorbed 15.9 c.c. Carbonic acid freed 13.9 c.c. oxidases: 303 After four hours: Oxygen absorbed 17.6 c.c. Carbonic acid freed 1 1. 1 c.c. CO. After an hour the ratio equals 0.874 and after four hours 0.630. These quite high ratios show that the oxidiz- ing power of laccase is very great. By trying the action of laccase on three isomeric poly- phenols : on hydroquinone, pyrocatechin, and resorcin, the following figures have been obtained which give an idea of the rapidity of the oxidation: Oxygen absorbed. C0 2 freed. Hydroquinone (para-diphenol). After 4 h 32.0 c.c. 1.7 c.c. Pyrocatechin (ortho-diphenol). " 4 b. 17.4 " 2.8 " Resorcin (meta-diphenol) " 15 h 0.6 " 0.0 " It is seen that the quantity of oxygen absorbed is almost nought for the meta-diphenol, while the para-diphenol oxi- dizes very strongly. These facts are reproduced in all Bertrand's experiments; phloroglucin, where all the hydroxyls are in the meta po- sition, refuses, so to speak, all oxidation, while its isomer, pyrogallol, absorbs oxygen with rapidity. The different polyphenols examined by Bertrand have shown that their oxidizability is in direct proportion to the facility with which they are transformed into quinones. The whole or a part of the hydroxyls of polyphenols may be replaced by amido radicals (NH 2 ), without the progress of oxidation being modified. The paramidophenol : CH< OH(i) 6 4 NH 2 ( 4 ) is easily oxidized; metamidophenol, on the contrary, CH< OH(i) 6 4 NH 2 ( 3 ) takes up only the smallest quantities of oxygen. According to J. de Rey Pailhade, laccase exists in germi- nating grains. The enzyme acts on an oxidizable material, 304 THE ENZYMES AND THEIR APPLICATIONS. philothion, also contained in these grains. Laccase conse- quently would play a part in the respiration of vegetable cells. Still, he has not shown at all that the oxidizing en- zyme found in the grains is laccase. It is quite possible to believe that it is some other oxidase. We can now define in a more general way the manner of action of laccase. Laccase is a soluble ferment producing the oxidation of bodies of the benzene series, which possess at least two groups, OH or NH 2 , when these groups occupy the para- or ortho position. Here the observations specially applicable to laccase cease. The later works of Bourquelot and Bertrand relate to another diastase, or rather to a mixture of laccase and another enzyme, tyrosinase, whose presence investigators have recognized in a great number of vegetables. TYROSINASE. The juices extracted from beets and some other plants, when put in contact with the air, become red in color, then black. This phenomenon is due to the oxidation of the tyrosin which is found in these plants, and which is caused by the action of a diastase. The rational formula of tyrosin or oxyphenyl-amidopro- pionic acid is : rH .COOH - V 2 3 NH, HC CH (I I HC CH C I OH OXIDASES. 3° 5 It is seen by this formula that tyrosin does not belong- wholly to the class of bodies we have recognized as being surely oxidizable by laccase, that is, to the class of poly- phenols whose hydroxyls are in the para- or ortho-position. Tyrosin, in fact, when submitted to the action of laccase does not absorb oxygen. On the other hand, Bertrand found by various experi- ments that the oxidation of tyrosin did not occur when the juice extracted from the plants had been heated to ioo°. This fact indicated the intervention of a diastase. The oxidation of tyrosin may be explained by the action of laccase on a product of decomposition of tyrosin, previ- ously elaborated by another non-oxidizing enzyme contained in the liquid. To test this hypothesis, Bertrand placed in a flask a cer- tain amount of an extract of Russula and a few grams of tyrosin. After 24 hours, the whole was heated to ioo°; the addition of laccase to the liquid and exposure to the air did not then cause any oxidation. It was therefore demonstrated by this experiment that laccase is absolutely without action on tyrosin, and that there is not produced in the liquid a previous diastatic action which permits the oxidation by laccase. In reality, the absorption of oxygen is caused by the mediation of tyrosinase, an enzyme similar to laccase but acting on other bodies. Tyrosinase has been isolated by Bertrand from many plants. Extracted from potatoes, dahlias, etc., it possesses only a very feeble oxidizing power; but extracted from fungi it oxidizes tyrosin very rapidly. Bourquelot prepared tyrosinase with Russula nigricans, which he crushed in water to which chloroform had been added. The liquid, after filtration, constituted the diastatic solution. To demonstrate the action of tyrosinase, 5 cubic centi- metres of the diastatic solution are put in reaction tubes, 306 THE ENZYMES AND THEIR APPLICATIONS. then 5 cubic centimetres of a solution of tyrosin, and the mixture is shaken from time to time to introduce the air. The liquid becomes first red, then black. By this reaction and that of guaiacum, Bourquelot dem- onstrated the existence of tyrosinase in the following fungi: Boletus, Russula, Lactarius, Parillus, Psalliota, Hebeloma, Amanita, Scleroderma. Certain fungi give no reaction, either with guaiacum or with tyrosin, and it may be concluded from this that they contain no oxidizing enzyme. Tyrosinase is not so widely distributed in nature as lac- case ; but it is very often met with, simultaneously with the latter, in the same vegetable juice. Certain diastatic solu- tions extracted from plants really transform tyrosin as well as polyphenols. In a series of experiments made at 50 , 6o°, and yo Q Ber- trand observed that the faculty which vegetable juices pos- sess of transforming tyrosin disappear at relatively low tem- peratures, while the similar properties of laccase still persist in the liquid at higher temperatures. This difference in re- sistance of the two diastases allows the separation of one from the other in the following manner: One thousand five hundred grams of fresh Russula delica are reduced to a pulp and macerated in an equal weight of cold water to which chloroform has been added. By pressing out the juice of the paste thus obtained, about 2 litres of a mucilaginous liquid is obtained, to which are added 3 litres of 95 per cent alcohol ; a precipitate is formed which is separated by filtration. The alcoholic liquid, from which the precip- itate has been separated, is reduced, by distillation in vacuo at 50 , to about half a litre. The product thus obtained oxi- dizes hydroquinone and pyrogallol very rapidly, leaving the tyrosin perfectly intact. The precipitation with alcohol and the heating to 50 have destroyed every trace of tyrosinase. This latter diastase is found in the precipitate which has been separated from the alcoholic liquid. This precipitate is OXIDASES. 307 purified by diluting with water containing chloroform ; it is again precipitated with 2 volumes of alcohol and separated from the liquid. The product, after a second similar treat- ment, is dried at 35 °. It reacts with difficulty on polyphenols, but causes a very rapid oxidation of tyrosin. The individuality of the two enzymes is then well proved. Influence of the Medium on Oxidation.— Bourquelot, in a very complete work, has shown the relation existing be- tween the composition of the medium and the diastatic ac- tivity of the oxidizing ferment of fungi, a ferment composed, as we have seen, of at least two oxidizing enzymes : laccase and tyrosinase. A solution of anilin, in the presence of an infusion of fungi rich in oxidase, oxidizes very slowly, for only a slight change of color is observed. Bourquelot was then led to inquire if the alkalinity which anilin gives to the medium did not exercise an unfavorable in- fluence on the oxidizing action of the enzyme, and he studied the oxidation of anilin in the presence of increasing amounts of acetic acid. The fungus chosen for these experiments was the Russitla dclica, because the filtered juice obtained from its maceration gives a clear aqueous solution, which consequently makes it easy to observe the changes in color. It was soaked by tak- ing 5 parts of water for one part of fungus, and thus was ob- tained by filtration a liquid but slightly colored yellow. This extract, with the addition of glacial acetic acid in amounts varying from 1 to 50 parts per thousand, was tested with tincture of guaiacum. Bourquelot then saw the blue coloration appear with the same intensity and the same speed in all the experiments which he made. Therefore, the reagent is not influenced by great amounts of acetic acid and, under these conditions, the influence of the acid on the action of oxidase may be studied. This action is shown, for different amounts of acid, in the fol- lowing table: 3°8 THE ENZYMES AND THEIR APPLICATIONS. Control Test. Exper. i. Exper. a. Exper. 3. Exper. 4. Exper. 5. Exper. 6. Solution of saturated aniline. 5 c.c. 5 c.c. 5 c.c. 5 c.c. 5 c.c. 5 c.c. 5 c.c. Water. 8 c.c. 8 c.c. 8 c.c. 8 c.c. 8 c.c. 8 c.c. 8 c.c. Acetic acid, % o 5 c.c. 0.1 0.2 0.4 1 2 5 Diastatic solution. 5 c.c. 5 c.c. 5 c.c. 5 c.c. 5 c.c. 5 c.c. Result. Slight oxida- tion. A little stronger oxida- tion. Strong oxida- tion. Very strong oxida- tion. Strong oxida- tion. Very slight oxida- tion. No oxida- tion. Oxidation hardly appears in the control tube, which takes a dirty yellow tint; it increases with extraordinary rapidity in experiments 1, 2, 3, 4, where the solution is immediately colored a dirty yellow, with the formation of a brownish-yel- low precipitate, soluble in ether. As to experiments 5 and 6 containing, respectively, 2 and 5 per cent of acetic acid, the first furnishes a slight oxidation, while in the second there is absolutely no oxidation. Therefore, 2 per cent acetic acid is unfavorable to oxidation. With orthotoluidin and paratoluidin, tried under the same conditions, with the same quantities of acid, the same reactions occur, although giving different colorations. Orthotoluidin gives a transparent violet color, becoming opaque at the end of several hours. An aqueous solution of phenol takes a brown tint in the presence of the diastatic solution. This reaction, which takes place very slowly, is wholly prevented by acetic acid and favored by amounts of 0.1 to 0.4 per cent of carbonate of sodium. In general, the oxidation of substances of basic nature is favored by the acidity of the medium, while sub- stances of acid nature oxidize more readily in an alkaline medium. This influence of the medium on the progress of oxidation is very considerable. OXIDASES. 309 Action of Oxidase on Phenols Insoluble in Water. — Bour- -quelot first occupied himself with the action of oxidase on phenols which are soluble in water. He then turned his at- tention to the action of oxidase on phenols insoluble in water but soluble in ethyl alcohol or methyl alcohol. He previously assured himself that the alcohols used as solvents and suit- ably diluted produced no change in the oxidase and that the phenomenon of oxidation occurred there in the same way as in the watery solutions. Assured of this, Bourquelot made various experiments on phenols soluble in these reagents. The results of his re- searches is here given : The action of oxidase was tried on three solutions of dif- ferent xylenols containing 0.50 gr. of xylenol, 100 grams of absolute alcohol, and 50 cubic centimetres of water. The orthoxylenol (1, 2, 4), a body melting at 55 to 6o°, produced a white precipitate which then became salmon pink and soluble in ether. Metaxylenol (1, 3, 4), a liquid whose alcoholic solution becomes green under the action of ferric chloride, was im- mediately oxidized and gave a white precipitate which then became dirty pink, and is soluble in ether. Paraxylenol, melting at 74 or 75 °, was made slightly turbid, and gave a pure rose-colored precipitate insoluble in ether. Experiments upon the oxidation of thymol were made in a solution having the following composition : Thymol 0.50 gr. Water 40 c.c. Alcohol 10 c.c. Solution of carbonate of sodium (2%) . . 5 c.c. Diastatic solution 50 c.c. The solution absorbs 19 cubic centimetres of oxygen and a white precipitate is formed in the liquid. Carvacrol, tried under the same conditions, gives rise 3io THE ENZYMES AND THEIR APPLICATIONS. little by little to a turbidity, then to a white precipitate, ab- sorbing 27.5 c.c. of oxygen. " BROWNING " OF WINES. The " browning " of wines is a disease characterized by the oxidation of the coloring matter of the wine and the pre- cipitation of this material, while the entire liquid becomes yellow. In 1895, Gairaud recognized that this phenomenon was due to the action of a diastase, yet without clearly attributing it to an oxidase. P. Martinand, in a work published later, identified the diastase producing oxidation of the coloring matter of wine with the laccase recently discovered by Bertrand. This iden- tification was entirely erroneous. Indeed, it was later recog- nized that the oxidase of wine transforms polyphenols, while laccase, though hastening the oxidation of browning wines, is incapable, of itself, of producing it in wines. Cazeneuve, having added to wines a certain quantity of laccase, observed only an imperceptible alteration, although the diastatic solution used was very active and strongly colored blue the tincture of guaiacum. The diastase causing the oxidation of the coloring matter is then a well-deter- mined enzyme. Cazeneuve gave to it the name of cenoxi- dase. Preparation of (Enoxidase. — Cazeneuve observed the phenomenon of oxidation in Beaujolais wine, which was very sensitive to the action of the air; he isolated the diastase from it by the following process : The wine is submitted to the action of an excess of alcohol, which precipitates a substance having the appearance of a gum. This precipitate is taken up again with distilled water, in which it dissolves, giving an opaline, uncolored solution. The liquid obtained is again precipitated ; the new precipitate is dried in vacuo and then obtained under the form of a gum impregnated with oxidase. OXIDASES. 3 1 r Secretion of (Enoxidase. — The various reactions which characterize the diastase of the browning of wines are iden- tical with those of all oxidases. Like other soluble ferments they color blue the tincture of guaiacum. The reaction of guaiacum was tried by Martinand on ripe grapes, and it revealed the presence of oxidases. With the juice of the grape he succeeded in transforming hydro- quinone and pyrogallol. Ripe grapes secrete a greater quantity of cenoxidase than green grapes, and raisins are completely destitute of it. The fermented juices of pears, plums, and apples are richer in cenoxidase than wine. The secretion of cenoxidase was attributed by Laborde to the presence, at the root of the vine, of the mould Botrytis cincrca (" sweet rot "). Measurement and Properties of (Enoxidase. — The meas- urement of oxidases presents great difficulties. In fact these enzymes do not always exercise their action with liberation of carbonic acid, which is easy to measure ; oxygen is some- times combined with hydrogen to form water or is directly combined with the oxidizable materials. Under these con- ditions, the analysis of the products of oxidation becomes very difficult. Laborde has based a method of measurement on the coloration which a diastatic liquid assumes in the presence of tincture of guaiacum. He takes as unit the coloration which is acquired by 20 cubic centimetres of alcoholic solu- tion of guaiacum with the addition of 0.5 gr. of iodine, and he compares the coloration obtained in the same tincture by oxidase with this unit in a Dubosc colorimeter. (Enoxidase oxidizes the coloring matter of French and Italian wines; Spanish and Turkish wines undergo its action with more difficulty. Cazeneuve found that the coloring matter of wine is a phenol-like body. It is transformed by oxidation, likewise 312 THE ENZYMES AND THEIR APPLICATIONS. the ethers, alcohols, essences, etc., to which is due the bouquet of the wine. When wine is shaken with ether, it yields to this reagent a substance having the characteristics of a tannin. After oxidation of the wine, only the smallest quantities of this substance are found, often indeed not a trace is to be dis- covered. Now, neutral wine after having been treated with ether undergoes no alteration under the action of oxidases. The browning of wines, according to this experiment, therefore, appears to be due to the oxidation of a particular substance. CEnoxidase is weakened in proportion as it acts, for the quantity of oxygen absorbed at the beginning is greater than that absorbed at the end of oxidation. By introducing air into half a litre of wine, Laborde found that absorption occurred during the first eight days and that at the. end of this time a sudden check occurred. The meas- urement of the gas absorbed gave the following figures for three different wines: Oxygen absorbed CO s set free „ f ; CO a per litre. per litre. o ist experiment. . . 50.8 c.c. 32.4 c.c. 0.63 2nd experiment. . . 81.0 c.c. 38 c.c. 0.47 3rd experiment. . . 110.2 c.c. 63.8 c.c. 0.58 This table shows that there is not only oxidation of the coloring matter, but combustion of this matter and produc- tion of carbonic acid. Lagati observed that by the addition of ferrous salts the wines oxidize just as under the action of a diastase. The pre- cipitate which he thus obtained is identical with the precipi- tate of browned wines ; it is not produced if protected from the air, nor in the presence of sulphurous anhydride. This author attributed oxidation to the action of ferrous salts alone, but this opinion was refuted by Laborde in a con- OXIDASES. 313 elusive manner. Indeed, the greatest amount of iron in the ferrous state contained in a wine can absorb only 10 cubic centimetres of oxygen, while browning wine absorbs as much as no cubic centimetres per litre. Therefore, besides the action of the ferrous salt, that of a diastase is also exercised. Action of Temperature. — According to Cazeneuve, cen- oxidase is but slightly sensitive to low temperatures: at o° and even below oxidation still occurs. At 65 ° the diastase is not entirely destroyed; for the destruction to be complete, the temperature must be raised to yo°-y2°. Martinand fixed the temperature of destruction at 72 for 4 minutes or at 35° for an hour. Bouffard made interesting experiments on this subject. In 3 tubes, A, B, C, he put in A, an aqueous solution of the enzyme; in B, the same solution, with a certain quantity of io° alcohol added; in C, a solution of the same diastase with 0.5 gr. of tartaric acid added. The temperature of destruc- tion was determined for each experiment and the following results obtained: Temperature of destruction. Neutral aqueous solution 7 2 -5° Solution + alcohol at io° 6o° Solution + tartaric acid 5 2 -5° It is seen that the presence of alcohol and tartaric acid lower the temperature of destruction. When 20 per cent of alcohol is added, the temperature of destruction is lowered by 5 more. At 6o°, according to the same author, the ac- tivity lasts for 2 minutes, then decreases and disappears com- pletely at the end of 20 minutes. Laborde studied the action of temperature in an acid diastatic liquid containing 5 parts of oxidase. He brought these liquids to different temperatures and, after cooling, he determined the quantity of active substance remaining. These experiments gave the following figures : 314 THE ENZYMES AND THEIR APPLICATIONS. Oxidase. Temperature. , * — % Active. Destroyed. 6o° 2.30 2.70 65 i-5o 3-5 70 O.9O 4. 1 75 0.75 4.25 80 0.45 4.55 85 :........ o 5 The temperature of destruction of oenoxidase is, there- fore, situated between 70 and 75 °, but the activity of the enzyme diminishes considerably at 6o°. Action of Chemical Agents. — According- to Martinand, an addition of acid retards oxidation and an addition of alkali, on the contrary, is favorable to the combination of oxygen. However, when the wine already possesses a quite large natural acidity of itself, oxidation occurs, even without the addition of diastase. Concentrated alcohol decomposes the diastase, but dilute alcohol and wine containing up to 9 per cent leave it abso- lutely intact. Tricalcium phosphate and tartaric acid are without action, either accelerating or retarding, on oxidation. Formol (formic aldehyde) is also without action. Gallic, pyrocatechuic, and salicylic acids hinder oxidation. Sulphurous acid, in an amount of 0.01 to 0.08 parts per litre, checks the action of oenoxidase and causes its destruc- tion. This fact was demonstrated by Boufrard and Caze- neuve. Cazeneuve, by adding to a certain quantity of wine •0.004 grams of sulphurous acid, precipitated the diastase of this wine by the ordinary methods, washed the precipitate in alcohol, and collected it. After some time the precipitate, redissolved in water, no longer gave coloration with tincture of guaiacum. The sulphurous acid, therefore, acted directly •on the diastase. CEnoxidase is extremely unstable. In the air it is rapidly OXIDASES. 3 1 5 destroyed by absorption of oxygen. By exposing a solution of oxidase to the air, Laborde obtained the following figures : ~ . Oxidase Duration „ of aeration. D • • , Remaining. Lost. 2 days 3.5 2.0 4 " 2.8 2.7 6 " 2.4 3.1 12 " O.8 4.7 It will be noticed that the destruction, which is rapid at the beginning, slackens very perceptibly after the second day. Other Oxidations of Wine. — According to Martinand, oxidase plays an important part in the improvement of wines with age. He was able, in fact, to produce artificially, by the addition of oxidase, an ageing of a Burgundy wine. The wine, with oxidase added, and exposed to the air for 48 hours, took on a yellower color and the perfume of an old wine. The coloration of this wine corresponds to red-violet 354 of the Salleron wine colorimeter before oxidation ; after being exposed to the air, in the presence of oxidase, the tint corresponds to the third red 404. The oxidation of the sugar and tartaric acid of the wine must, according to Martinand, be attributed to a cause of the same kind. A special action of oxidase has been found in certain American grapes. These grapes have a disagreeable taste which is lost by aeration; but when they are kept at a temperature of ioo°, they retain the special flavor which disappears by the ad- dition of oxidizing diastase. OXIDIN. Boutroux, in studying the cause of the coloration of brown bread, discovered in the bran an active substance re- sembling laccase which he called oxidin. When the bran is left to soak for a half-hour with its 3i 6 THE ENZYMES AND THEIR APPLICATIONS. volume of water, there is obtained by filtration through a. porcelain filter a clear light-colored liquid, which, protected from the air, keeps without changing its color. Put in contact with the air, this liquid takes on a brown tint which deepens with age and finally becomes black. This coloration does not occur in an infusion brought up to ioo°. Boutroux succeeded in separating from the infusion the oxidizing enzyme, and the substance which undergoes oxida- tion. By adding alcohol to the filtered infusion, the oxidase precipitates without carrying with it the oxidizable sub- stance. In this way two solutions may be obtained which sepa- rately do not change color in the air and which, mixed, grow brown under the influence of oxygen. To prepare oxidin the bran is soaked in an atmosphere of carbonic acid gas, and filtered under the same conditions. To the filtered liquid is added 3 volumes of 95 per cent alcohol and the precipitate is washed with 82 per cent alcohol on. a paper filter. The filter is impregnated with an amor- phous substance, which is brown and difficult to detach. The filter is cut in pieces and dried in a vacuum. This paper, im- pregnated with active substance, acts energetically on the sterilized infusion of bran ; it also oxidizes hydroquinone like laccase. Oxidin is also precipitated by sodium chloride. An in- fusion of bran saturated with this salt does not color in the air. The enzyme is evidently precipitated, but the precipitate is not active. Oxidin plays a very important part in the coloration of brown bread, but in this phenomenon amylase also is con- cerned. The two enzymes contained in the bran act in dif- ferent ways. The intervention of oxidase is manifested during the preparation of the dough and in the first stages of panary fer- mentation. The oxidizable material of the bran is at this point trans- OXIDASES. 3 1 7 formed into coloring matter. The oxidation which oxidin produces is checked by the acidity, and when panary fermen- tation has become more active, oxidin ceases to act. The color of the dough becomes still deeper by cooking. In this stage of the work, amylase intervenes. The starch, which is in suspension in the dough before cooking, is par- tially liquefied through the influence of the amylase of the bran. An intimate mixture is brought about between the parts not yet liquefied. The mass changes in structure and this change causes coloration. The coloration of the flour may be also influenced by a substance found in the germ of the wheat. According to a verbal communication made to me by Albiana, Jr., of Barcelona, who is very expert on ques- tions of milling, the flour obtained with grain deprived of the germ is white and unchangeable, while the presence of the germ, even in relatively small quantity, furnishes a dough which colors very rapidly. It is probable that the germ contains an oxidase or some similar diastase. OLEASE. Fresh olives, when in heaps, easily undergo fermentation. One finds an increase in temperature, a liberation of carbonic anhydride with formation of acetic acid and other fatty acids. Talomei showed that this fermentation was caused by an enzyme which he called olease. This agent is sometimes found in olive oil, in which it causes a very great change. Under its action, the oil be- comes rancid, on account of the formation of fatty acids, and discolors on account of the precipitation of the coloring mat- ter. This discoloration is favored by light. Olease is isolated from the oil by stirring with water. Thus a watery solution of the enzyme is obtained and the oil remains unchanged. The optimum temperature for the action of olease is be- low 35 . The acidity of the medium checks the diastatic ac- 318 THE ENZYMES AND THEIR. APPLICATIONS. tion and it is owing to this circumstance that the change in the oil is often not very extensive, the fatty acid formed hin- dering the action of the olease. BIBLIOGRAPHY. G. Bertrand. — Sur la laccase de l'arbre a laque. Comptes Rendus, i er semestre, 1894, p. 1215. Sur la laccase et le pouvoir oxydant de cette diastase. Comptes Rendus, i er semestre, 1895, p. 266. Sur la recherche et la presence de la laccase dans les vegetaux. , Comptes Rendus, 2^ semestre, 1895, p. 166. ■ Sur les rapports qui existent entre la constitution chimique des com- poses organiques et leur oxydabilite sous l'influence de la laccase. Comptes Rendus, i er semestre, 1896, p. 1132. Sur une nouvelle oxydase ou ferment soluble oxydant d'origine vegetale. Comptes Rendus, i er semestre, 1896, p. 1215. -. Presence simultanee de la laccase et de la tyrosinase dans le sue de quelques champignons. Comptes Rendus, 2« semestre, 1896, p. 463. Bouffard. — Observations sur quelques proprietes de l'oxydase des vins. Comptes Rendus, i er semestre, 1897, p. 706. • Rappel d'une note precedente. Comptes Rendus, i er semestre, p. 1053- Em. Bourquelot and Bertrand. — La laccase dans les champignons. Comptes Rendus, 2 e semestre, 1895, p. 788. Em. Bourquelot. — Influence de la reaction du milieu sur l'activite du fer- ment oxydant des champignons. Comptes Rendus, 2 e semestre, 1896, p. 260. ■ Action du ferment soluble oxydant des champignons sur les phenols insolubles dans l'eau. Comptes Rendus, 2^ semestre, 1896, p. 423. L. Boutroux. — Le pain. Bailliere et fils, Paris. Cazeneuve. — Sur quelques proprietes du ferment soluble oxydant de la casse des vins. Comptes Rendus, i er semestre, 1897, p. 781. Sur le ferment soluble oxydant de la casse des vins. Comptes Rendus, i er semestre, 1897, p. 406. Laborde. — Sur l'absorption de l'oxygene dans la casse des vins. Comptes Rendus, 2 e semestre, 1897, p. 248. Sur l'oxydase du botrytis cinerea. Comptes Rendus, i er semestre, 1898, p. 536. Sur la casse des vins. Comptes Rendus, 1896, p. 1074. E. Bourquelot and Bertrand. — Le bleuissement et le noircissement des champignons. Soc. de Biologie de Paris, 1895. Lagati. — Sur la casse des vins; role du fer. Comptes Rendus, i ef semestre, 1897, p. 1461. V. Martinand. — Sur l'oxydation et la casse des vins. Comptes Rendus, i er semestre, 1897, p. 5 12 - OXIDASES. 319 Talomei. — Olease. Atti. Ace. di Lincei. Rnd. 1896. Berichte der deutsche chem. Gesellschaft, 1896. J. de Rey Pailhade. — Etude sur les proprietes chimiques de l'extrait al- coolique de levure de biere; formation d'acide carbonique et absorp- tion d'oxygene. J. de Rey Pailhade. — Roles respectifs du philothion et de la laccase dans les grains en germination. Comptes Rendus, 1895, p. 1162. L. Lindet.- — Sur l'oxydation des tannins des pommes a cidre. Bulletin de la Soc. chim., Paris, 1895; Comptes Rendus, 1895. Hikorokuro Yoshida. — Journal of the Chem. Society, 1883. J. Eft'ront. — Action de l'oxygene sur les levures de biere. Comptes Ren- dus, CXXVII, p. 326, 1898. Martinand. — Action de l'air sur le mout de raisin et sur le vin. Comptes Rendus, 1895, p. 502. INDEX. Amygdalin, 268. Amylase, 100. alteration of, 103. analysis of, 139. industrial application of, 147. influence of antiseptics on, 117. influence of chemical agents on, 112. influence of lactic acid on, 114. influence of sulphuric acid on, 112. influence of temperature on, 109. in moulds, 102. in saliva, 102. liquefying power, 115. preparation of, 102. properties of, 106. Amylo-dextrins, 173. Bacillus glutinis, 170. Bacillus levans, 171. Bacillus panificans, 170. Betulase, 273. Bread, fermentation, 168. Brewing, 155. Caroubin, 256. Caroubinase, 256. Cerealin, 170. Cerealose, 220. Chinese yeast, 223, 230. manufacture of, 234. Choum-choum, 223. Classification, 48-49. Cytase, 148, 255. Deterioration, 129. Dextrins. 126. Dextrins of Duclaux, 126. Dextrin syrup, 163. Diastase, estimation, 143. Diastase of Reichler, 136. Diastase: precipitation by tannin. Distillation: preparation of the mash, 179. Distilleries, East Asian, 235. Emulsin, 268. in fungi, 268. Enzymes: action of heat on, 16. chemical composition, SI. definition, 5. heat producers, 8. manner of action, 24. mechanical precipitation, 13. nomenclature, 46. properties, 12, 13. reaction with guaiacum, 17. variability, 15. Erythrozyme, 273. Fermentation, panary, 168. Floor malting, 151. Galactose, 248. Glucase, 208. Glucosides: enzyme action on, 43, 44. ferments of, 267. Glycerides, ferments of, 262. Guaiacum, 17. Infusion process, 187. Inulase, 249. Inulin, 249. Invertin (see Sucrase). Japanese yeast, 223. Keplin, 248. Koji, 223. preparation of, 224. 321 3 22 INDEX. Laccase, 297. Laccol, 299. Lactase, 248. Lactose, 248. Leaven, 171. Levulose, 250. Lipase, 262. Malt, 195. bacteria in, 180. Pilsen type, 153. Malt-sugar syrup, 163. Maltase, 208. in moulds, 212. Maltose, manufacture of, 161. Malting, 149. Malt, Munich type, 153. Malto-dextrins, 158. Migen, 223. ' Molasses, fermentation of, 88 Monobutyrin, 265. Moto, 227. Myrosin, 272. Nefrozymase, 208. CEnoxidase, 310. Olease, 317. Oxidasis, 174, 292. preparation of, 297. properties of, 296. Oxidin, 315. Panary fermentation, 168. bacteria in, 170. Pancreato-lipase, 265. Pectase, 251. Pectin, 251. Pectinase, 251. Pectose, 251. Papain, 30. Pepsin, 30. Peptase, 148. Pneumatic malting, 151. Ptyalin, 132. Rhamnase, 273. Saccharification: color changes in, 107. for distillation, 179. influence of temperature on, no.. Sake, 223. manufacture of, 229. Secretion diastase, 134. Sero-lipase, 265. Starches, action of amylase on, 127, Steapsin, 262. Sucrase, 50. deterioration, 76. estimation, 71. Symbiosis, 249. Taka-Koji, 237. Translocation diastase, 134. Trehalase, 246. Trehalose, 246. Tyrosinase, 304. Zymase, 276. conditions of action, 281. fermenting power of, 279. industrial application, 289. preparation of, 277. Zymogens, 24, Zymogenesis, 23. Zymolysis, 24. SHORT-TITLE CATALOGUE OF THE PUBLICATIONS OF JOHN WILEY & SONS, New York. London: CHAPMAN & HALL, Limited. ARRANGED UNDER SUBJECTS. 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DATE BORROWED DATE DUE DATE BORROWED DATE DUE • J ^ 25 n :, jp ;\j ^ 6 I349 C2BI1 1 40) M lOO a> eoi ^f* on / CIO, Vv JUN 17 19 A; A '//^VL f JAN 25 1943 /r^/ U-»