^La» bought with the income » " FROM THE SAGE ENDOWMENT FUND THE GIFT OF 1891 .k.\.s:j3&.i v.i4.5....\. ..« .«?m s 3081 Cornell University Library QP 601.E27 1904 Enzymes and their applications. 3 1924 003 867 011 Cornell University Library The original of tiiis book is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924003867011 ENZYMES AND THEIR APPLICATIONS. DR. JEAN EFFRONT, PROFESSOR IN THE NEW UNIVERSITY IN BRUSSe£s AND DIRECTOR OF THE FERMENTATION INSTITUTE. ENGLISH TRANSLATION BY SAMUEL C. PRESCOTT, S.B., INSTRUCTOR IN INDUSTRIAL BIOLOGY, MASSACHUSETTS INSTITUTE OF TECHNOLOGY, BOSTON. THE ENZYMES OF THE CARBOHYDRATES. THE OXIDASES. FIRST EDITION. FIRST THOUSAND. NEW YORK : JOHN Wn.EY & SONS. London: CHAPMAN & HALL, Limited. 1904. Copyrig-ht, 1902, BY JOHN WILEY & SONS. ROBERT DRUMHOND PRINTER NEW YORK. 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 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 iii 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, KirchoflF, 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 lo 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 vili T/IBLE OF CONTENTS. PAGB 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 and the structure of the bodies on which they act. — Nomenclature of enzymes. — Classification , 40 CHAPTER V. Extraction of sucrase from yeast. — Secretion by Aspergillus niger. — Preparation of sucrase in the dry state. — Influence of the quan- tity and of time. — Influence of temperature.— DifTerence between the properties of sucrases of different origin. — Effect of acidity or alkalinity of the medium. — Action of oxygen and of light. — Action of chemical substances. — Mode of secretion of sucrase in the cells. — Measurement of sucrase. — Fernbach's method. — Ef- front's method 50 CHAPTER VI. SUCRASE (continued). Retarding factors, and their explanation. — Deterioration and altera- tion of sucrase. — Experiments of Ef?ront on the influence exerted by the invert-sugar in the medium in which inversion is pro- duced. — Hypothesis of O'SuUivan and Tompson. — Arguments for and against this hypothesis. Theory of EfTront on the decom- position of cane-sugar, and experiments on the manner of action of acids in the inversion of saccharose 75 CHAPTER VII. FERMENTATION OF MOLASSES. Industrial applications of sucrase.— Fermentation of molasses 88 T/IBLE OF CONTENTS. ix CHAPTER VIII. AMYLASE. PAGS Presence of amylase in vegetable and animal cells. — Preparation. — Cohnheim's method. — Lintner's method. — Effront's method. — Wroblewsky's method. — .Properties. — Influence of quantity, time, and temperature. — Influence of chemical agents: acids, alkalies, salts. — Substances hastening diastatic action lOO CHAPTER IX. CHEMICAL WORK OF AMYLASE. Chemical work of amylase. — Theories of Payen and Musculus. — Existence of different dextrins. — Theory of Duclaux on the na- ture of different dextrins. — Preservation of diastases during sac- charification. — Experiments of Eflfront 121 CHAPTER X. AMYLASE OF DIFFERENT SOURCES. Different amylases.— Ptyalin. — Diastase of raw grains and diastase of germinated grains. — Action of translocation diastase on starch. — Reichler's diastase. — Mode of action of diastase brought to a tem- perature of 70°. — Conditions of secretion of amylase. — Quantita- tive analysis of amylase. — Comparative value. — Absolute value. — Methods of Eflfront 132 CHAPTER XL INDUSTRIAL APPLICATIONS OF AMYLASE. Malting. — Chemical transformations which accompany germination. — Methods of malting, sorting, steeping, germination, brewing. . . . 147 CHAPTER XII. SdLE OF AMYLASE IN THE BREWERY 155 CHAPTER XIII. MANUFACTURE OF MALTOSE l6l CHAPTER XIV. PANARY FERMENTATION. Of Dumas theory of panary fermentation. — Cerealine of Mege- Mouries. — Role of bacteria in panary fermentation. — Origin of sugar in flour i68 X T/tBLE OF CONTENTS. CHAPTER XV. e6i-e of amylase in the distillery. PAGE Treatment of grains by acid and by malt. — Influence of heating on saccharification. — Clioice of temperatures of saccharification. — Principal and secondary saccharification. — Experiments of Ef- front on the alteration of diastases during saccharification. — The infusion process. — Alteration of diastases during the successive stages of the work. — Control of the work in the distillery 175 CHAPTER XVI. QUANTITATIVE STUDY OF MALT. Determination of the diastatic power of malt and mashes according to the methods of Efifront. — Determination of saccharifying and liquefying powers. — Determination of the diastatic power of sweet and fermented mashes 195 CHAPTER XVII. MALTASE. Glucase of Cusenier. — Maltase of yeast. — Properties. — Differences between the optimum temperatures of the different glucases. — Maltase of moulds. — Mode of action on starch. — Processes of secretion. — Influence of nitrogenous food. — Different amylo-malt- ases of Laborde 208 CHAPTER XVIII. INDUSTRIAL APPLICATIONS OF MALTASE. Cerealose 220 CHAPTER XIX. INDUSTRIAL APPLICATIONS OF MALTASE. Japanese and Chinese yeasts in general. — Manufacture of Japanese yeast. — Preparation of "koji." — Changes produced in the rice. — Composition of "koji." — Action of salts. — Manufacture of "moto" leaven. — Manufacture of the beer "sake." — Composition of " moto." — ■ Composition of " sake." — Manufacture of Chinese yeast. — Properties of Chinese yeast. — Diastase of Chinese yeast. — Influence of temperature and chemical agents. — Native distilleries of the far East. — Utilization of Oriental processes in thi> distil- leries of western countries. — Works of Takamine, Collette, and Boidin 223 TABLE OF CONTENTS. xi CHAPTER XX. ENZYMES OF CARBOHYDRATES. PAGE Trehalase, lactase, inulase, pectase, cytase, caroubinase 246 CHAPTER XXI. FERMENTS OF GLYCERIDES AND GLUCOSIDES. Saponifying ferments. — Ferments of glycerides. — Serolipase and pan- creatolipase. — Measurement of lipase. — Influence of temperature and alkalinity of the medium. — Differences between the lipases of different origins. — Ferments of glucosides. — Myrosin, emulsin, rhamnase, erythrozyme, betulase 262 CHAPTER XXII. ZYMASE. Zymase or alcoholic diastase. — Preparation of the extract of yeast and its properties. — Determination of the fermenting power of zymase. — Chemical and physical conditions of the action of zymase. — Experiments of Effront on intracellular fermentation. — Industrial applications of zymase 276 CHAPTER XXIII. . OXIDASES. , Presence of oxidases in vegetable and animal cells. — General proper- ties. — Laccase. — Tyrosinase. — Influence of the medium. — Action of oxidases on phenols insoluble in water. — " Breaking " of wines. — CEn oxidase. — Oxidin. — O lease 292 THE ENZYMES AND THEIR APPLICATIONS. CHAPTER I. GENERAL REMARKS. Synthetical and analytical work of the living cell. — Synchronism of the two phenomena. — Difference between the chemical work and the phy- siological work. — Chemical agents and physiological agents. — Inter- vention of vital energy. — Necessity of studying the physical and chem- ical conditions of the media. — Definition of enzymes. — Their part in assimilation and disassimilation. — Enzymes as producers of heat. The activity of living cells gives rise to a series of chem- ical reactions very complex and various. A limited observa- tion of phenomena will show that, aside from purely syntheti- cal activity, the cell always carries on analytical work: in other words, that the organic substance, in the presence of the living cell, is built up and broken down. The synthetical activity is more apparent in the transformation of substances which are not very complicated, which, submitted to the ac- tion of living cells, become hydrated, oxidized and trans- formed into complex chemical compounds. In the trans- formation of complex substances, on the contrary, it is the analytical work which predominates, and the complex sub- stance is reduced to more and more simple bodies. . For ex- ample, if one submits a sweetened must, containing nitrates or some ammoniacal salts, to the action of yeasts, one notices the appearance of new cells and consequently the formation 2 THE ENZYMES AND THEIR APPLICATIONS. of protoplasmic substances, which are, from a chemical point of view, very complex substances. When, on the contrary, one subjects albuminous matters to the action of certain ferments, they undergo a putrefactive fermentation, and it is seen that they pass through a series of transformations. The albuminous matters change first into proteoses, then into peptones, into amides, and finally into ammonia, hydro- gen sulphide, oxalic and carbonic acids. In the first of the two examples which we have just given, we apparently see a work exclusively synthetic, characterized by the formation of protoplasm at the expense of sugars and nitrates. In the second example an activity absolutely op- posed to the preceding one seems to be exhibited. However, the phenomena are much more complex. In the first example the formation of new cells of yeast is ac- companied by that of a protein matter, the protoplasm ; but that substance does not persist in an unchangeable state : on the contrary, it is constantly destroyed, hydrated, and trans- formed by the living cells. When, therefore, new cells are formed, organized matter is decomposed. In the second ex- ample, the ferments which decompose the albuminoid mat- ters and transform them into various products of cleavage, multiply, grow, and thus carry on an extensive synthetical process accompanying the process of decomposition. We may conclude from these observations that the living cell works constantly in two different ways, analytically and synthetically, and one of these actions is more apparent than the other according to the particular case. Since Wohler accompHshed the synthesis of urea, it has been possible to produce artificially a great number of or- ganic substances. Emil Fischer has shown us the course to ' follow for carbohydrates, and it has been possible to repro- duce artificially almost all the natural sugars which are found in plants, according to the methods which he has indicated. While we do not actually know how to reproduce albu- GENERAL REMARKS. 3 minoid matters synthetically, the works of Schiitzenberger have at least made known the manner of decomposition of all these substances, as well as their cleavage products. Although these dififerent works have made possible many syntheses, and have indicated the course to follow to accomplish others, one must admit that there is a great dif- ference between the chemical work and the physiological work of cells. In order to promote chemical reactions, very violent means are often employed in the laboratory. Strongly acid or alkahne solutions are used; pressure or high tem- peratures are employed. To produce, for example, the phe- nomena of oxidation, use is made of reagents such as nitric acid, chromic acid, or permanganate^. As means of dehy- drating are used concentrated sulphuric acid, anhydrous phosphoric acid, zinc chloride, etc., substances which destroy cells. In the case of living cells, on the contrary, the re- actions occur in a medium either neutral or weakly acid or alkaline; the temperature is always very moderate and al- most constant. The difference between these two condi- tions is striking. In the living cell bodies react which, ac- cording to our ideas, have only very feeble affinities; one observes at the same time that substances which we regard as very stable are readily decomposed in the interior of cells. The affinity of chemical bodies appears then stronger when they are in the presence of living matter, and seems to diminish when the cells are destroyed. The increase of potential of the molecules in the interior of living cells is generally explained by the intervention of vital energy. The reactions, it is said, are produced more easily, owing to the intervention of a special force*, the vital energy, which increases the interior energy as well as the aptitude for combinations and decompositions, as do elec- tricity, magnetism, light, etc. The explanation of the phe- nomena by vital energy throws little enough light on the subject. In brief, it reduces itself to saying that reactions 4 THE ENZYMES AND THEIR APPLICATIONS. are favored in living cells by physical and chemical conditions peculiar to the medium. This proposition cannot be con- sidered as an explanation of intercellular phenomena. We cannot really understand them except by a thorough study of the media in which they are produced. A careful study of these conditions shows that the ready occurrence of all inter- cellular reactions is not to be ascribed to a single cause ; that the affinity is sometimes favored by a purely physical con- dition and sometimes by a purely chemical one. Certainly, we are far from knowing all the conditions which favor inter- cellular reactions, but it has been possible to study some among them, and from the acquired knowledge, one may conclude that whenever one observes an increase of cellular energy, this is produced, not by a single condition common to all such phenomena, but by a cause strictly determinable and differing in different cases. We know certain reactions in which chemical affinity in- creases because of purely physical conditions, such as osmosis, which continually occurs through cell membranes. In other cases we find that reactions are favored in the cells by the presence of mineral substances. The decomposition of sodium chloride, for example, and the formation of hydrochloric acid in certain cells is one of those phenomena which do not accord at all with the general ideas we have of the stability of certain substances. We know, in fact, that sodium chloride is a very stable substance, and that its decomposition in the cold, in a medium slightly acidified by weak acids, is impossible. Thus the de- composition of sodium chloride in the cells was formerly explained by the intervention of vital energy, which was said to render the body less stable and more readily decomposed. To-day a more rational explanation is given to the phenom- enon: we recognize that the decomposition of sodium chloride is caused simply by osmosis and independently of vital energy, because the salt, in a very dilute solution, is dissociated. In the cells an analogous phenomenon of dis- CENTRAL REMARKS. S sociation must be produced, and, by osmosis, the acid must pass through the cell membrane and accumulate in a certain quantity. Thus the acidity is seen to be a result of the dis- sociation of the very dilute salt solution and of its passage, by osmosis, through the cell membrane. This is a very striking example of a physical condition favoring reaction. The most convincing example of the intervention of min- eral substances is furnished by the results obtained in agricul- ture by the use of chemical fertilizers. When one places at the disposal of the cells relatively small quantities of phos- phate, the quantity of protein matter produced in the plants increases considerably. The plant-cell needs, then, to carry on its synthetic work, the presence of mineral substances, which form organo-metallic combinations; combinations which, having more affinity than the organic substance un- combined with the mineral substance, enter more easily into reaction. But we also know another extensive series of bio-chem- ical reactions which are produced without intervention of physical factors or mineral substances, and are due to the presence of chemical substances of a particular nature which we call enzymes. The study of these substances, of their manner of secretion, and of their method of action will form the object of the present work. The enzymes, soluble ferments, zymases, or diastases are active organic substances, secreted by cells, and have the property, under certain conditions, of facilitating chemical reactions between certain bodies, without entering into the composition of the definite products which result. These substances play a very important part in the phenomena of assimilation and of disassimilation of foods. In fact, most of the foods which occur in nature at the disposition of men, lower animals, or plants, are not directly assimilable; they require the intervention of a diastase in order to be trans- formed into substances assimilable and suitable for the formation of new tissues. 6 THE ENZYMES AND THEIR APPLICATIONS. Starch, which serves in the nutrition of almost all living^ creatures, is not directly assimilable, and in the higher or- ganisms it undergoes various transformations before it can be absorbed. First of all, it encounters the enzymes in the saliva, then others in the pancreatic juice, and thus it is trans- formed into maltose and glucose, foods directly suitable for the construction of tissues. Meat, milk, and white of egg must also be transformed under the influence of the diastases before becoming assimilable. These substances find the enzymes which can act upon them in the gastric and pan- creatic juices. These phenomena which are observed in the higher or- ganisms are also met with in the vegetable kingdom. During germination and florescence, the reserve substances, like starch, cellulose, fatty substances, and proteid matter, are in part consumed by the developing plant. But this utilization of reserve food is not done directly: these substances must be previously transformed by the diastases into assimilable products. Let us examine, for example the phenomenon of germination. A grain of barley left for ten or fifteen days in darkness loses thirty to forty per cent of its weight. If one determines the hydrogen and oxygen in the grain before and after germination, one finds that the loss of these two ele- ments is in the ratio of one to eight. One may conclude from this that oxygen has combined with hydrogen to form water. On the other hand, if one determines the quantity of carbonic acid formed, it is found that it corresponds almost exactly to the quantity of carbon that has disappeared. There would be, then, combustion of carbon and formation of carbonic acid on one hand, formation of water on the other hand, and the phenomenon would appear to be a simple oxi- dation. If one analyzes the reactions more closely, it is seen that germination is not a phenomenon of simple oxidation, and that during its course there occurs a series of secondary reactions. First of all, there appear in the grain diastases which act on the starch and the cellulose in such a way that GENERAL REMARKS. 7 little by little these two substances change their nature as well as their chemical composition. The cellulose is dis- solved, the starch is transformed into maltose, part of which is. oxidized, and part changed into cane-sugar by the tissue of the seed. All these transformations, as well as the oxidation itself, are produced by the diastases secreted during germination. One can follow the course of most of these transforma- tions; for example, the solution and the transformation of starch. For this purpose an embryo is separated from the grain and made to develop on a gelatinized must in which starch has been placed in suspension. By observing the phenomenon very closely and by ex- amining the starch under the microscope, one can see that the grain of starch loses its original form, that it is corroded in several places, that it then liquefies and disappears. In the culture liquid one finds substances which did not exist be- fore : a sugar, and a nitrogenous substance, the diastase, which is soluble, capable of precipitation by alcohol, and can itself produce a transformation of starch. In the assimilation of albuminoid matter by cells, there occurs a phenomenon quite analogous to the assimilation of carbohydrates. The albuminoid substances are gradually transformed by the active substances of the cells into pro- teids, peptones, and finally into amides. ' ' ■ . ■ We have said above that the diastases play an extremely important part in the phenomena of disassimilation. The molecules of albuminoid substances, hydrated, decomposed, and transformed by the enzymes, are regenerated in the pres- ence of the protoplasm of the cells, by a process of dehydra- tion and molecular condensation. The reconstructed mole- cules undergo new changes ; they are again hydrated, decom- posed, and at the same time gradually oxidized. In this phase of the transformation the albuminoid molecule is de- composed into urea, glycogen, fatty, substances, and amides. These transformations are also in great part due to the active substances secreted by the cells. 5 THE ENZYMES AND THEIR APPLICATIONS. ^' Finally, the enzymes are powerful producers of heat ; the reactions caused by the diastases are exothermic reactions. Thus a molecule of urea transformed into ammonium car- bonate yields 8 calories. A molecule of glucose, in being transformed into carbonic acid and alcohol, sets free 71 calories. Tripalmitin, in splitting into fatty acid and glycerin, yields 30 calories. A gram of albuminoid matter trans- formed into urea furnishes 4.6 Calories. It is seen that the role of the enzymes as producers of heat in living organisms is of considerable importance. This heat, set free by exothermic reactions, is then utilized by. the cells for maintenance as well as for the construction, of their new tissues. When yeast is put in a solution of saccharose, there is first secreted a diastase which makes the medium assimilable, be- cause the saccharose cannot be directly assimilated by the yeast. There is produced in the yeast a sucrase which trans- forms the sugar into invert-sugar, that is to say, into dex- trose and levulose. The cell is then in a medium favorable for its development: it can utilize the nutritive substances and transform them into tissues, but this transformation in- volves an absorption of energy. On the one hand then, the yeast has need of energy for the maintenance of its tissues ; on the other, the heat set free by the transformation of cane-sugar into invert-sugar is not very considerable and entirely insufficient to produde the re- quisite energy. The yeast-cell, therefore, secretes a second diastase acting on the invert-sugar much more powerfully, and transforming it into alcohol and carbon dioxide. These two substances, the alcohol and the carbon dioxide, are not of use to the yeast ; but the transformation which has produced them is an exothermic reaction which furnishes to the cell the energy which it needs for its maintenance. An example, perhaps still more striking, is the trans- formation of urea into carbonate of ammonium by special fer- ments. GENERAL REMARKS. 9 If these ferments are cultivated in a medium containing urea and peptones, it is found that the cells select the pep- tones as tissue-building materials; at the same time the urea is attacked and transformed into ammonium carbonate. The aim of this second transformation is to furnish the energy necessary to the cells for the construction and the maintenance of their tissues. We observe again the same phenomenon in the vegetable kingdom. In the green parts of plants, under the influence of the sun's rays, the carbonic acid is constantly decomposed ; formic aldehyde is produced, which is polymerized, and trans- formed into different carbohydrates. Through diffusion, these carbohydrates are distributed to dififerent parts of the plants, where they undergo the action of diastases which hy- drate them and decompose them. The carbohydrates are then sources of heat, which is set free by their decomposi- tion. The products of decomposition or hydration are re- turned by diffusion to the green parts, where they can again become synthesized and consequently store up heat. Thus are explained the migration, hydrations, and dehy- drations of the carbohydrates which one observes in these plants. We may conclude from all these facts that the diastases are substances absolutely iiidispensable to the life of organ- isms, for they make possible the construction of the cellular tissues, by rendering the materials assimilable and by fur- nishing the necessary energy. BIBLIOGRAPHY. CI. Bernard. — Lemons sur les digestions. Paris. V. Kuhne. — Erfahrungen und Bemerkungen iiber Enzyme und Fermente, Physiologisches Institut, Heidelberg, 1878. Ad. Mayer. — Die Lehre von den chemischen Fermente, 1882. Duclaux. — Microbiologie. Dunod, editeur. Paris, 1883, p. 134. Armand Gautier.— Legons de chimie biologique. Paris, 189-'. J. Effront. — Actions des substances minerales at des diastases sur les cellules. Moniteur scientifique, 1894, p. 562. CHAPTER II. GENERAL PROPERTIES. History of the knowledge of enzymes. — Works of Reaumur and Spallan- zani, Kirchofif, Dubrunfaut and Payen. — General properties of dia- stases. — Means of distinguishing a diastatic action from a purely chemical action. — Test by tincture of guaiacum. — Law of proportion- ality in diastatic action. — Means of distinguishing the work of or- ganized ferments from diastatic action. — Means of isolating the dia- stase from the medium which contains it. — Chemical composition of enzymes. — Zymogenesis. — Method of action of diastases. The first ideas concerning the existence, as well as the action, of enzymes belong to a very remote period. As early as the beginning of the sixteenth century the phe- nomena of digestion claimed much attention from students. Opinion on this subject was very much divided; some held that digestion was a purely mechanical work, a trituration of substances by the walls of the stomach ; others, on the con- trary, explained digestion as due to a dissolving and trans- forming activity of the juices of the stomach. Reaumur and the Abbe Spallanzani upheld the second hy- pothesis and performed very conclusive experiments to con- firm their theory. Reaumur, in order to take account of the influence of secretions of the stomach, caused hawks to swallow little metallic tubes pierced with holes and filled with meat, grain, or albumen. He examined the contents of the tubes after these had been cast out and found that the albuminoid substances alone were liquefied and transformed by the gastric juice, while the starchy substances had not un- dergone any change. Abbe Spallanzani devoted himself to the study of the gas- GENERAL PROPERTIES. ir trie juice and its action. To procure active secretions he made birds of prey swallow little sponges attached to strings. He then drew back the sponges saturated with gastric juice. Spallanzani first succeeded in producing artificial digestion by placing meat in contact with the liquid squeezed from the sponges. He found that it became liquefied and changed. These conclusive experiments which throw so much light on the phenomena of digestion, as well as on the role of the diastases, unfortunately were not appreciated at their true value. They did not succeed in convincing the scientific world, and at the beginning of the nineteenth century the phenomena of digestion were still interpreted in dififerent ways. Certain scholars maintained that the gastric juice had no such constant character, and that the nature and properties of the secretion depended especially on the foods absorbed. These differences in the interpretation of the phenomena of digestion retarded the study of enzymes, al- though it was already greatly advanced by the works of Reaumur and Spallanzani. It was not until nearly two cen- turies after their publication that the question of active sub- stances secreted by the cells again became prominent. It is very curious to find that it is the study of brewing which has led to the greatest discoveries of this century. It , was by the study of the beer-yeasts that Pasteur estabUshed definite arguments against the theory of spontaneous genera- tion. It is also by the study of the raw materials of the brewery, notably malt, that Dubrunfaut laid the foundation for the study of enzymes. The work of Dubrunfaut is connected with an observa- tion made by Kirchofif in 1814. This distinguished scholar, who was the first to study the transformation of starch by acids, had noticed that fresh gluten can act under certain conditions on starch, dissolve it, and transform it into a sac- charine substance. This experiment was taken up again by Dubrunfaut, who, in a long and masterly study, demon- strated that the activity of gluten is due to the presence of a 12 THE ENZYMES AND THEIR APPLICATIONS. small quantity of active substance originating in the raw grain. He demonstrated that this diastase is soluble in water, that the ungerminated grain contains very little of it, and that germination develops it. He explained the mode of action of this substance and the conditions under which the maximum effect is produced ; he proved finally that the sugar prepared from starch by the aid of this substance is not identical with the glucose which KirchofT obtained by the action of the acid. It is in the works of Dubrunfaut, pub- lished in 1822, that we find for the first time a scientific study of the diastases as well as precise data concerning their mode of action. Payen took up again the work of Dubrunfaut, from whom he unjustly withheld the credit for the discovery of the dia- stases of malt. In studying the properties of an infusion of malt, Payen recognized that the active substance can be pre- cipitated from its solution by alcohol, and the precipitate thus obtained exhibits all the properties which the liquid itself possessed. This experiment has played a considerable part in the discovery of enzymes, because Payen had thus found, at once, a general property of diastases and a general means of isolating them. By this method have been isolated the active substances of the gastric juice and of the pancreatic juice, as well as the enzymes acting on fatty substances and glucosides. GENERAL PROPERTIES OF ENZYMES. We have just seen that the enzymes are precipitated from their solutions by alcohol, but it is necessary to add that, while all the enzymes are precipitated by very concentrated alcohol, they are more or less soluble in dilute alcohol. Since the discoveries of Payen, there have been recognized a certain number of other properties more or less characteris- tic of enzymes. Enzymes are soluble in water, are thrown down from their solutions by indifferent precipitates, become GENERAL PROPERTIES. 13 fixed on different substances as silk and fibrin, and are somewhat resistant to poisonous substances. Enzymes lose their activity at a temperature in the neighborhood of 100°. The greater part of them decompose hydrogen peroxide. They are also characterized by the fact that, under given conditions, the action which they produce is proportional to their quantity. All these properties are, however, far from being distinctive ; many other substances than diastases possess one or another of them. The most characteristic property of an enzyme is its special mode of operation. Let us consider each of the properties which we have just cited. We have seen, first of all, that enzymes are precipi- tated by alcohol; as they are more or less soluble in dilute alcohol, the quantity of alcohol which is necessary to precipi- tate the enzymes from an aqueous solution will not always be the same. For certain diastases, the fibrin ferment for ex- ample, it will be sufficient to put in the solution 10 to 15 per cent of alcohol, which constitutes a minimum proportion. For others, as the coagulative ferment of milk, it is necessary to add a large proportion of alcohol, in order to have a liquid containing 80 to 90 per cent. But if all the enzymes are precipitated by alcohol, they are likewise all destroyed by this same agent. By a pro- longed contact of the diastase with the alcohol, the active substance is transformed, becomes insoluble and inactive. So, if one precipitates an enzyme by concentrated alcohol, the action must be stopped as quickly as possible. Enzymes, from the point of view of solubility in water, present noteworthy differences. We know of diastases which dissolve very easily and of others, on the contrary, which re- quire for their solution a large quantity of water. Moreover, remembering that active substances are fixed with ease on different bodies, it is easy to understand that the same sub- stance can be presented in soluble or insoluble form. The precipitation of enzymes by " dragging down " (mechanical precipitation) can be easily effected. One adds 14 THE ENZYMES /IND THEIR /IPPLICATIONS. to a filtered and clear infusion of malt a very dilute solution of sodium phosphate, then a solution of a calcium. salt; there is produced in the liquid a precipitate of calcium phosphate which finally settles at the bottom of the vessel; the clear liquid is decanted, the precipitate placed on a filter, washed with a little water,, and a. powder is obtained which possesses all the properties of the infusion of malt. This powder, for instance, acts on starch and produces maltose, as does the malt from which it has been extracted. This method makes it possible to obtain all the enzymes contained in an infusion. Only one condition is necessary, — that the substances em- ployed for the precipitation shall be harmless to the diastase. Good results are obtained, for iiistan'ce, in making use of magnesium carbonate or aluminmm hydrate. We have seen that the diastases become fixed on different substances. Thus a piece of fibrin placed in a solution of gastric juice becomes impregnated with the active substance in such a way that the diastase cannot any longer be removed by washing. If, after having withdrawn this fibrin from the infusion and washed it to remove as far as possible every trace of ac- tive substance, it is placed in water at a suitable temperature, the piece of fibrin dissolves. It is evident that this transfor- mation of the protein matter is due to the fact that the active substance is fixed on the fibrin like a dye. It is not more- over necessary for the diastase to act on a substance to become fixed on it. For example, if one places some pieces of silk in gastric juice, they are impregnated with active sub- stance, although the diastase does not act at all on the silk. Most diastases are insensible to the action of certain sub- stances, such as hydrocyanic acid and chloroform, which paralyze the vital activity of cells. If, for example, yeast is put in a solution of cane-sugar in the presence of chloroform, the yeast remains quiescent and does not reproduce ; how- ever, the cane-sugar is still transformed into invert-sugar. The diastase continues, then, to be secreted by the cells and GENERAL PROPERTIES. 15 to do chemical work, while the cellular activity, properly speaking, is paralyzed by the* chloroform. From' these experiments it appears that enzymes are not sensitive, either to the action of antiseptics, or to that of sub- stances which are opposed to the vital action. But the rule is not universal. We know, in fact, of several enzymes which are extremely sensitive to chloroform, ether, and thymol, as well as to hydrocyanic acid. In reality, the various diastases differ considerably among themselves as to their, nature, and as to their sensitiveness towards the different reagents. The diastases of malt as well as the active substances of the yeasts transforming the cane-sugar into invert-sugar, are very resistant enzymes, and much less sensitive than the cells which elaborate them. These same diastases sometimes occur in other more re- sistant living cells, becayse there exists among cells, as among these diastases, considerable differences in sensitive- ness to reagents. There are, then, cases where antiseptics attack enzymes before acting on the cells, and other cases where the reverse is the case. The yeast which we have just cited furnishes an example of th.e relative sensitiveness of diastases to antiseptics. As a matter of fact it is known that beer-yeast contains, in addition to the diastase changing cane-sugar into irivert-sugar, a second enzyme which transforms the invert-sugar into alcohol. The absence of fermentation in presence of chloroform proves that, of the two diastases contained in the yeast, one is destroyed by the antiseptic while the other resists it. The greater or less sensitiveness of diastases to the action of antiseptics, and to those substances paralyzing the vital activity, can be utilized to exclude the activity of micro-or- ganisms during diastatic action. When one studies, for example, the saccharification of starch or the transformation of meats by enzymes, one may often be led into error by the invasion of organisms which 1 6 THE ENZYMES AND THEIR APPLICATIONS. produce the same effect as the diastase whose action is being studied. In this case, a small amount of antiseptic, for ex- ample, a few drops of chloroform, is put in the liquid which is kept under observation, and interference from* ierments is prevented. Only, in the case of certain enzymes more sen- sitive than others, it is necessary to use other means for pre- venting the action of organized ferments, because the en- zyme itself would be destroyed by the antiseptic. This' pre- caution is especially necessary when one is studying the ac- tion of enzymes yet unknown; in this case a negative result may be due to the presence of an antiseptic. The action of heat on the enzymes is an ex- tremely important point and one which, better than any other, may serve to characterize a diastatic ac- tion. In general, with a certain number of exceptions, enzymes exert their action slowly at a temperature of o°; often at this temperature the effect they produce is impercep- tible. If one gradually increases the temperature to 40° the reaction is intensified ; from 40° to 50° there is a marked increase in intensity, — it is generally at this temperature that the diastase attains its maximum activity, — above 50° the activity diminishes ; at 80° a considerable weakening is pro- duced, and finally, above 90° the diastase is wholly destroyed. The different diastases are characterized by their optimum temperatures, that is, by the temperature at which they give their maximum of action. This temperature varies quite considerably in different enzymes, and this variation consti- tutes a property which admits of differentiation. But the property of enzymes which is most useful in studying them, is the facility with which they are destroyed from 90° to 100° in the presence of water. Some diastases, when they are in a completely dry state, can stand a tempera- ture of 90° and even more ; but all enzymes, without excep- tion, lose their activity when their aqueous solution is brought to the neighborhood of 100°. This property is utilized for distinguishing diastatic from purely chemical action. GENERAL PROPERTIES. 17 When one puts an infusion of yeast in a solution of sac- charose, the transformation of saccharose into invert-sugar takes place. But it is not to be inferred that this is neces- sarily diastatic action, for the transformation may be due, either to the acidity of the must, or to some other chemical agent. To prove that there exists in a yeast an active substance, a double experiment is necessary. One must treat equal quantities of sugar equally diluted, during the same length of time and at the same temperature, on the one hand with a certain quantity of infusion of yeast, and on the other hand with an equal quantity of the same infusion, which has been previously heated to ioo° for several minutes, and then cooled again. If the same result is obtained in the two experim.ents, one may conclude that the transformation is not due to an active substance contained in the infusion under examination. On the contrary, the activity of a diastase becomes evident, if in the experiment with the heated infusion inversion is not ob- tained, while from the action of the infusion not heated a transformation is observed. The fact that diastases are destroyed at ioo° relates them in a striking manner to living organic matter. We have said above that when an enzyme is put in Solu- tion in the presence of hydrogen peroxide, the latter is de- composed. To demonstrate this reaction, one makes use of an alcoholic solution of guaiacum. Generally 2 or 3 cubic centimetres of tincture of guaiacum are taken ; a few drops of hydrogen peroxfde are added and then, drop by drop, the liquid in which an enzyme is supposed to exist. In the pres- ence of an enzyme the red liquid becomes a very intense blue. This coloring is due to the transformation of the guaia- conic acids into guaiacosonide, a dye. The reaction with tincture of guaiacum is extremely sensitive : one can produce it with exceedingly small quantities of active substance. It must not be forgotten that tincttire of guaiacum loses i8 THE ENZYMES AND THEIR APPLICATIONS. in time the property of giving color, and that the best way is to make the tincture before the experiment by grinding powder of guaiacum with alcohol ; moreover, the use of this reagent always offers certain difficulties : it must be noted that the coloring matter formed is not very stable, that it is decomposed by heat as well as by different chemical re- agents. A slight alkalinity or even a slight acidity is suf- ficient to prevent the production of coloration, and it fol- lows that it is necessary to take some precautions when using this reagent. It is well first to neutralize exactly the hydro- gen peroxide used, for this is generally very acid; it is also well to measure the degree of acidity or alkalinity of the liquid containing the diastase to be studied, then to neu- tralize it when it is clearly alkaline or clearly acid. The coloration produced by guaiaconic acid is not destroyed by acetic acid and it is often of advantage, when working with an absolutely neutral solution, to acidify it with a drop of dilute acetic acid. The reaction of the tincture of guaiacum is of much use in the investigation of enzymes. However, this reagent is not absolutely reliable, for the coloration observed in a solu- tion may be due to other bodies than enzymes. Moreover, if a reaction is not obtained, one must not conclude that the liquid does not contain an active substance, for the coloration may be prevented by different substances which may be pres- ent with the enzymes in the liquid tested. Moreover, enzymes are known which do not give colora- tion with guaiacum and, on the other hand, diastases which, after having been submitted to certain influences, lose this property without always losing their activity. Thus, at a high temperature, certain enzymes no longer give coloration with guaiacum, although the active substance is not destroyed. With other diastases the property of coloring tincture of guaiacum disappears in a prolonged contact with hydrogen peroxide, contact which has no influence on the activity of the diastase. However, no enzymes are known GENERAL PROPERTIES. 19 which, after having lost their activity by the action of chem- ical or physical agents, still give a coloration with tincture of guaiacum. It follows that, to use the reaction of guaiacum, one must, as with experiments on the efifect of heat, make double ex- periments with the fresh infusion on the one hand, and with the infusion heated to 100° on the other hand. When the fresh infusion produces a coloration and the infusion after being heated does not, one may be confident of the presence of a diastase. Further, tincture of guaiacum gives a blue coloration, with a whole group of diastases, without hydrogen peroxide. In this case, the guaiacum justifies not only the inference that a diastase is present in a solution, but gives further informa- tion; the reaction of guaiacum without hydrogen peroxide being possible only with an oxidizing enzyme. Tincture of guaiacum can also be of great service when plant enzymes are being studied. It often happens that diastases contained in vegetable cells are changed or destroyed as the result of maceration in water, on account of the dissolving out, from the cells, of extractive substances which destroy the enzymes. In this case one must look for the diastases, not in the solution, but in the cells themselves. For this, very small sections are made which are intro- duced either in the pure tincture of guaiacum or in a solution of guaiacum added to hydrogen peroxide. The cells contain- ing the active substance are colored blue. It is often very difficult to distinguish a diastatic from a strictly cellular action. If it is noticed that a certain liquid is capable of producing chemical changes in certain substances, one is led to think that an enzyme is present if the same liquid after boiling has not the same power. But in reality there is nothing to prove in this case that the action observed is really a strict diastatic phenomenon, for certain organized ferments may have caused it. 20 THE ENZYMES y4ND THEIR /tPPLICATIONS. To determine exactly whether organized or soluble fer- ments are present, recourse may be had in certain cases to a filtration by means of a porous filter which is capable of re- taining the organisms. If the filtered liquid is still active, one may conclude that the transformations noticed are really diastatic phenomena. But the contrary does not prove the absence of an enzyme in the solution, for all enzymes are more or less retained by the porous substance of the filter, and certain among them do not pass through at all. It is in the proportionality which exists between the quan- tity of diastases employed and the quantity of substance transformed by these diastases that a certain proof is found of the existence of a diastase. The law of proportionality is not, however, an absolute law. With an infinitely small quantity- of enzyme one can transform a very considerable quantity of substance on condition that the action is allowed to be continued for a long time under such conditions that the enzyme is not destroyed by the physical and chemical agents of the medium. However, at the beginning of the action, especially if one employs a very small quantity of active substance and a large quantity of passive substance, one notes a fixed ratio between the quantities of enzyme em- ployed and of substance transformed. It is under these conditions only that the law of propor- . tionality can be verified. If one adds, for example, to lOO, cubic centimetres of a ten per cent sugar solution a slight quantity of sucrase, for example a cubic centimetre of an in- fusion of yeast, and if the action is stopped after one hour, it is found that a part of the sugar has been transformed. If in a similar liquid under the same conditions of dilution and temperature, -J cubic centimetre of the same solution of su- crase be added, one finds that the quantity of sugar inverted is very nearly half of the quantity transformed in the preced- ing experiment. If instead of diastases one employs organized ferments capable of effecting the same transformation, one never ob- GENERAL PROPERTIES. 2i serves a proportionality between the quantity employed and the result obtained. A double quantity of organized fer- ments does not transform twice as much sugar. There is evidently in' thck second case a larger quantity of sugar in- verted, but this quantity is not double. The proportionality between the-quantities of diastase employed and of substance transformed is of great use, especially when one suspects the presence of organized ferments in an active, liquid. Chemical Composition of Enzymes. — Now that we know the means of recognizing the presence of diastases in a liquid, let us study closely the chemical composition of enzymes. The elementary analysis of enzymes gives discordant figures for the different known kinds, and sometimes even for the same diastase dififerent authors have found very different results. This fact may arise because the materials submitted to analysis are never pure, but mixtures of different sub- stances. It may be also that the enzymes really differ in their composition, and this should not surprise us since they are bodies which act in various ways and upon very different substances. The composition of some enzymes is here given : Diastase from malt Ptyalin Invertin Emulsin Pancreatin .. .. Trypsin Pepsin Wliite of egg uncoag.ulated ■ Carbon. Experimenterf. Krauch. Zulkowski, Lintner. Wroblewslci. Hufner. Mayer. Brauth. Donath. Brucklau. Schmid. Hiifnefy Loenid. Sclimid. Dumas. 2 2 THE ENZYMES AND THEIR APPLICATIONS. By examining the percentage of nitrogen of the several enzymes whose composition we give in J;he table above, we notice that certain diastases, as pepsin, contain great quanti- ties, and approach albuminoid substances in composition. We see, on the contrary, that other enzymes as invertin have a much smaller nitrogen content. In the group of the oxi- dases there are also enzymes, quite recently discovered, which appear to be absolutely lacking in nitrogen. These latter substances are analogous rather to the gums. We have just said that the non-agreement of results may be due to impurity of substances analyzed. In reality, the methods which are followed for separating the enzymes from the media which contain them cannot furnish pure sub- stances. Usually the diastase is obtained from the cells by ex- tracting with water and then precipitating with alcohol the infusion obtained. In liquids which have been in the pres- ence of protoplasmic substances, there is always a large quantity -of matter which can be precipitated by alcohol, and the results obtained are of necessity mixtures of different bodies. When it is proposed to purify the precipitates by dissolving them and reprecipitating, one obtains many bodies of a stable composition, but almost entirely destitute of all active power. In enzymes one always finds a great quantityof inorganic salts, particularly calcium phosphate, in very varied propor- tions. If the method of mechanical precipitation is em- ployed to isolate the diastase, the result is the same: after the precipitation bodies containing many impurities are found. Furthermore, in precipitating a diastase in an active liquid, one always runs the risk of obtaining a mixture of dif- ferent diastases and not one alone. It then becomes abso- lutely impossible to separate them from each other, because their insolubility in alcohol is not such that they can be separated by precipitation. Thus, when barley-malt is steeped in water, there is obtained in the liquid a whole series GENERAL PROPERTIES. 23 of active substances which are precipitated together by alcohol or by other substances which can drag them down. The diastases which, according to the analyses, most nearly approach proteids in composition, still differ consider- ably from these substances. Enzymes do not give all the color reactions of proteids. Bodies of this class cannot diffuse through a parchment mem- brane, while diastases are capable of doing it, although with some difificulty. Diastases act differently from proteids. These latter bodies can be assimilated by cells, while the dia- stases cannot be. The salivaty and pancreatic enzymes never serve as reserve substances. Though stored in the cells during the period of normal nutrition, these substances are rejected in time of starvation. According to Beijerinck, amylase cannot replace in a nutritive medium either carbohy- drates or nitrogenous substances, the yeasts and the bacteria absolutely refusing to be nourished by it. Zymogenesis. — Enzymes are produced by certain special cells. According to Hiifner they are formed by the oxida- tion of albuminoid .substances. This theory is attacked by Wroblewsky, who considers diastases as proteoses. There are as yet very few data on the manner of forma- tion of enzymes. In most cases one can only observe their presence when they have acquired all their properties; in some isolated instances the presence of a non-active sub- stance capable of becoming a ferment by suitable treatment has been observed. Thus the gastric mucous membrane, when soaked in water, yields a liquid which does not coagulate milk ; but this liquid acquires that property when one adds i per cent of hydrochloric acid, and preserves its activity even after neutralization. Fresh pancreatic tissue yields in water a substance acting very slowly in the presence of a slight quantity of acid. The activity of this liquid can be acceler- • ated by passing through it a current of oxygen, or by intro- ducing into it hydrogen peroxide. 24 THE ENZYMES /tND THEIR /tPPLICATIONS. These substances, capable of becoming active, are called zymogens (proferments, proenzymases), and the transforma- tion of the zymogen into the ferment is called according to Arthus.: zymogenesis. It is very probable that most en- zymes come from zymogens, and that the phenomena of zymogenesis are as frequent as the phenomena of destruction of the diastase, called zymolysis. Manner of Action of Diastases.— The chemical analysis of an enzyme is not sufficient to characterize it. To deter- mine exactly the characteristics belonging to a diastase, one must observe its manner of action, the chemical change it can produce, and especially the substances on which it can act. Diastases can induce, according to their nature, very dif- ferent chemical reactions. Some have a hydrating action, that is to say, they can cause one or more molecules of water to unite with the substances on which they act. We can cite for example the transformation of saccharose into dextrose and levulose. C12H22O10 + H2O = CgHijOg + CgHigO^. Saccharose. Water. Dextrose, Levulose. Another series of enzymes acts on the contrary like oxidizing agents. An example of this class is the transformation of hydroquinone into quinone. CeH, < g^ + O - H,0 + C,H, 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. IS9 According to Petit, there is obtained, with the same malt successively saccharified at 60°, 65°, and 69°, the following respective quantities of malto-dextrins : Temperature 60° 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 60" 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. " 234 Diastatic power 47 45 34 17 Per cent of malto-dextrins 4.25 7.9 14.9 22.4 Type of malto-dextrins obtained 1:0.5 1:1.5 1:2 i:z 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 dift'erent methods of brewing and we prefer to refer the reader to special works. Let us only remark that by modifying the manner of hydration of the starch, beers of different kinds i6o 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 efifect 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 I'industrie de la brasserie. Paris, 1896. Wilhelm Windisch. — Das chemisclie Laboratorium des Brauers. Berlin. Paul Parey. Paul Lindner. — Mikroskopische BetriebskontroU 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 i 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 dififerent industries. He believed that pure maltose could replace with advantage cane-sugar 161 1 62 THE ENZYMES AND THEIR /tPPLlC/ITIONS. 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 reaHzed. 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 afiford 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-fiour 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: 1st. 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 7o°-75°, and 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 i 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 throiigh 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-efifect 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 i\o°-/\2° 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 riesnlts 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 18.9 Maltose 80.6 Dextrin 0.2 WHITE SYRUP (fECULa). ^ Dry substances 77.1 Maltose 59.2 Dextrin 17.4 SACCHARIFIED MAIZE. Water 20.2 Maltose "45. 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 18.8 Maltose 71. Dextrin 2.4 Foreign substances 8.2 i66 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 fiour an elastic and homogeneous dough. To this end a Httle yeast is diluted in warm water, flour is added little by Httle, 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 up 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 100°. The yeast then, according to him, would act by the car- 17° 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 he considers is the agent of panary fermentation. Laurent, in his later works, has described the Bacillus paniftcans. 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 alTjuminoid 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. PAN/IRY FERMENTATION. 171 According to some, the bacteria alone cause fermenta- tion ; according to others, the bacteria act in sy;nbiosis with the yeast: the former, by the aid of their diastase, would furnish sugar to the yeasts. Wolfifiin succeeded in producing normal bread by replac- ing the leaven by a culture of Bacillus levans. Analogous ex- periments have been made by Popofif with the same success. Boutroux, who has taken up these experiments again, and carefully studied bakery yeast, has reached the following conclusions : 1st. AlcohoHc 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 :n 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 /IND THEIR /IPPLICMTIONS. 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 i 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 insufificient 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 lipens. 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 PANARY FERMENTATION. I73 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 80°. 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 difiference in solubility comes from the differ- 174 THE ENZYMES AND THEIR. /tPPLIC/tTlONS. 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. JLeon 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 I'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. fiir 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 Efiront 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. 175 176 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 OP AMYLASE IN THE DISTILLERY. i77 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 f hour at z atmospheres loses 0.85 of sugar. " " o " 17 " " " 4 " 34 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 th^t 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 178 THE ENZYMES /IND THEIR /fPPLIC/ITIONS. 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 .? i 1 Diastatic Balling. Alcohol. p^^^^ 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 tlie 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 saccharitication. 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 work consists in making a very fine meal of the grains, and in cooking this meal for i^ 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 dififerent 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 l8o THE ENZYMES yiND THEIR yiPPLIC/tTIONS. 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 OF AMYLASE IN THE DISTILLERY. i8i 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 sacchariiication. 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 efifect 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 60° 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 dififerent 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 i82 THE ENZYMES AND THEIR /tPPLICATlONS. 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 indisperisable 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 i-aising 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- RdLE OF yIMYLASE 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 64°-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 suiifi- 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 1 hour. After 12 hours. 12 hours at 30° C 2.4 9.6 our at 50° C. and 11 hours at 30° C 8.3 10.2 ^(I2h ( I h g ( 12 hours at 30° C 2.2 9.8 ( I hour at 55° C. and 11 hours at 30° C 9.1 11. 6 P ( 12 hours at 30° C 2.2 g.g ' I hour at 59° C. and II hours at 30° C 9.5 9.7 The mahose, in all these experiments, was measured after I 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 i 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 1 1 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 i 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, Duraiion of Sacchari6catiou. Starch transformed, 1 12 hours at 30° 85% 2 I hour at 45" and 11 hours at 30°. 97 3 " 50° 96 4 " 64° 68 By repeating the same experiments with mashes of dif- ferent concentrations and containing dififerent 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 dififerent 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. " 60° " 8% " 55" " 42% " 50° " 73% Hence in the choice of a temperature of saccharification 1 86 THE ENZYMES y4ND 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 60° 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 60°. 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 yIMYLASE IN THE DISTILLERY. 107 reality, an increase of several degrees in temperature has not much inliuence 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 mat'-.rials 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 60° 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 loS 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 diiBcult ; 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. Diastatic Power. Experiments. After 8 hours. 17 hours. 26 hours. 47 hours. 52 hours. Liquid A 33 45 48 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 dififerent 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. " '* " from 55° to 59" " " *' " 3 hours. ** " '* '^ 60" to 65" " " '* ** 5^ 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. . . 31 6o 44 56 51 46 55 36 20 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, ''^'^'infu^hln^ °' After labour. 3 hours. 8 hours. 17 hours. 25 hours. 30 .. .. 31 60 49 45 55 65 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. 190 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 m'ethod 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. 19^ Besides richness in active materials, ottier factors must be taken into consideration in estimating the value of a malt. The origin of dififerences 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 dififerent temperatures, one for 8 days at i9°-22°, the other for 9 days at I2°-I5°, we have obtained malts which dififered in their resistance at a temperature of 60°. 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. 192 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. 193 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 60°. 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 ihe 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. Efifront. — Sur les conditions chimiques de I'action des diastases. Coraptes. Rendus, 1892, t. 115, p. 1524. Sur certaines conditions chimiques de Taction des levures de biere. Comptes Rendus, 1893, t. 117, p. 559. Sur la formation de I'acide 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 I'influence des composes du fluor sur les levures de biere.. Comptes Rendus, 1894, t. 118, p. 1420. Etude sur les levures lactiques. Annales de I'lnst. Pasteur, 1896, P- 524. De I'influence des fluorures sur I'accroissement et le developpement des cellules de la levure alcoholique. Moniteur scientifique, 1891,. P-^ 254. Etude sur les levures. Monit. scientifique, XI, p. 1138, 1891. Des conditions auxquelles doivent satisfaire les solutions fermen- tescibles pour que les fluorures y produisent un maximum d'efifet. Monit. scientifique, 1892, t. VI, p. 81. Maercker. — Spiritusfabrikation. Paul Parey, Berlin, 1894. Max Biicheler. Die Branntwein Industrie. Zweite vollstandig umgear- bcitete Auflage des Lehrbuches der Branntweinbrennerei von Stam- mer. Braunschweig. Leitfaden fiir 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 Eflfront. — 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 saccharification 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 /tPPLIC/ITIONS. 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 saccharifiication is made at the same temperature in all the experiments. Number of the Experiment. Fresh Infusion. Boiled Infusion. Maltose formed. I 2 3 4 5 6 1 C.C. 2 " 6 " " 1 " I " 0.37 g- 0.65 " 0.85 " 0.6 " 0.72 " infusion of barley - 5 c.c. t " 2 " Infusion of malt - 7 8 9 10 0.5 c.c. 0.5 '• 0.5 " 2 C.C. 0.5" I " 0.07 g. 0.095" 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 100°; 0.5 cubis 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 QU/1NTIT/ITI(^E STUDY OF ATALT. 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 dififerent 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 dififerent 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- fication 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 po\yer and its liquefying power. In the preceding chapter we have seen that malts differ much in their resistance to a temperature of 60°. 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. 198 THE ENZYMES AND THEIR /tPPLICATIONS. A malt of high diastatic power, but of Httle resistance to high temperatures, gives a less satisfactory resuh 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 60° 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 of 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 60° ; keep the flask in a water-bath for an hour at a temperature of 60". During saccharification, shake the flask from time to time ; the sac- charification 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 60° 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. i99 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 17I to 18 per cent of- water and is completely dissolved in warm water. By submitting to this operation dififerent potato-flours of the same origin, the same product is constantly obtained, but the result diflfers 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 cf 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 80° ; 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 80° 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 100°, 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 15", 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 on 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 20 2 THE ENZYMES AND THEIR APPLICATIONS. considered poor. The difference between tne 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 have 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 80° ; so rice-starches may 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 first 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 80° 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 it? 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 Hquefying 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 /tPPLIC/ITlONS. 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 18°, 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 dififerent 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 Lengrth of the Grains. Malt with Plumule not longer than Grains. 0.6 0.585. 0.53 Saccharifying power 17. c.c. 9.5 c.c. 20.7 c.c. Liquefying power ■< 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 ■• QUy4NTlT/lTll^E 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 liq-uefying 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 BerHn, 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°; maximurn, 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. T . r ■ (25 c.c. = liquid, l^iquefymg power < '^ ,,?.., (2 c.c. = /4 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- 2o6 THE ENZYMES /tND 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 hialt 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 dond 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 wkh a pipette divided into tenths of a cubic centimetre .25 c.c, .50 c.c, .75 c.c, I 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 vi^ater-bath at 60° 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 suiificient 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 i cubic QUANTlTATiyE STUDY OF MALT. 207 centimetre of mash shows a sufficient quantity of diastase if the hquefying 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 ysAth. 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 i, that is, with 0.75 to i 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 I'etude de I'amylase. Monit. scientifique, tome VIII, p. S4I, et tome X, p. 711. CHAPTER XVII. MALTASE. Glucase of Cusenier. — Maltase of yeast. — Properties. — Diflferences 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 tem- 203 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 60° ; 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 60°. Above 60° 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- 210 THE ENZYMES AND THEIR /tPPLIC/ITIONS. lowing method is employed : Three kilograms and a half ot maize thus prepared are treated with 5 htres 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^ 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 certaia 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 M/ILT/ISE. 211 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 possess 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. of fhrAcdon. Glucose formed. 35° 2 hours 2.90 gr. 40 " 309 45 " 2.08 The optimum temperature, according to these experi- ments, would be 40°, while Cusenier's glucase possesses an optimum temperature of 56° to 60°. 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 50° 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 arises, 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 ot Aspergillus oriza 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 riiger, 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 oriscu 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 Enrotiopsis Gayoni. To demonstrate the presence of glucase in an active liquid, a 2 per cent solution of maltose is added to a certain MALTESE. 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 + 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 dififusion 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 observed that 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- 2 14 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 Pfefifer 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 orisce is the most active, and it is this which really secretes the greatest quan- tity of maltase. Mucor alternens and Amylomyces Roux'ii 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 nliger, 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 dififerent 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: MALTESE. 215 Origin of the Diastatic Liquids. Aspergillus niger Penicillium glaucum Eurotiopsis Gayoni Duration in Polariscopic Glucose. Hours of the Rotation of Action. ttie Liquids. % gr- 12 17-5 1. 31 48 14.0 I.61 96 14.0 1.66 12 12.5 i-3i 48 12.0 i.6t 96 12.0 1.72 12 7.0 0.80 48 9.0 1. 61 96 9-3 1.92 Dextrins. V- 0.56 0.31 0.30 0.31 0.21 0.18 u. 16 0.06 0.00 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 60° 80° Penicilliiun glmicinn 45 yo 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. 2i6 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 orizcc, Mucoi' alternans and Amyloinyces 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 MALTASE. 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 100° Total acidity, H,S04 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 0.127 16.67 Aspergillus orizse. gr- 2.081 5.20 0.670 2.77 1.30 2.25 Mucor alternans. 0.667 6.27 0.980 1.58 Traces 2.99 46^ Amylo- myces. gr- 2.080 4-50 o.65o 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 orizce for 14.42 of sugar transformed, 2.77 alcohol.. " Mucor alternans " 13.68 " " " 1.58 " " Amylomyces Rouxii " 12.92 " " " 3.96 " Aspergillus orizce 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 Amylomyces 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 orizcs; 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. Bodin and Rolants have studied the action of oxygen and of 2l8 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. Residue with Acidity equivalent to 3,4 gr. of Sulphuric Acid. 3-4C.C. 0.36 4-83 10.23 10.23 5-5C.C. 0.83 2.33 7-3 4.6 gr. 3 c.c. 0.4 1.64 5-57 8.15 gr 3 c-c. 2.69 4.31 17.71 17.71 i.Sc.c. 3-13 3-.'i 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. TDubourg. — Recherches sur I'araylase de I'urine. These, P.iris, 1889. Bourquelol.— RechercUes sur les proprietes physiologiquc 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. Lintnerund 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 I'Inst. Pasteur, 1898. Sanguinetti. — Contrib. a I'etude de Tamylomyces Rouxii. Ann. de I'Inst. Pastpur, 1897. Bodin et Rolants. — Contrib. a I'etude de I'utilisation de I'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, 189s, I, p. 984- Beijerinck. — Centralblatt fur Bakteriologie, 11. 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 60° 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- INDUSTRIAL APPLICATIONS OF MALTASE. 22r 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 sUght pressure. The starch obtained by this operation is saccharified at a temperature of 63°, by the aid of a small quantity of malt (i 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. 22 2 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 MAL.TASE.— (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, CoUette, and Boidin. Japanese 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 migen 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 Amylomyces Rouxii. Kofi owes its activity to Eurotium orizce. Korschelt and Atkinson published the first data on the preparation and utilization of Japanese yeasts. 283 2 24 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 orizcB. 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 eacTi 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 ic heats again to 2y°. 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 27° 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 Part soluble in water: 37.76^... Part insoluble in water: 62.24^. - 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 : Dextrose 25.02 Dextrin (by difference) 3.88 Soluble ash 0.52 Soluble albuminoids 8.34 Insoluble albuminoids 1.50 Insoluble ash 0.09 Fatty bodies 0.45 Cellulose 4.20 Starch (by difference) 56.00 The fresh koji contains 25.82 per cent of water. The growth of Eurotium orisce 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 100°, 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 o.i per cent possesses a re- tarding action. The diastase is equally sensitive to the action of sodium chloride. 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 I 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 o£ the solution. Common salt Reducing power Specific rotatory per 100 of starch, (on copper oxide). power. o 30.8 173-8° 10 28.6 1793 30 25.1 182.6 50 23.8 187.6 75 20.9 190.3 100 20.1 189.1 150 19.1 190.2 200 18.0 192.2 300 16.9 I94-I 500 144 197-5 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." Tbe 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 moio 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 I 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 15-46 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 o.ii 0.05 0.03 Water by difference 80.80 84.42 84.67 85.47 Undissolved starch. 10.68 12.46 11.55 12.05 INDUSTRIAL APPLICATION OF MALTASE. 229 Manufacture of Sak6. — 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 13-23 Dextrose Dextrin 0.41 Glycerin 1.99 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 oriccc 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. Amylomyces 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 Saccharomyces. Diastase of Chinese Yeast. — The diastase contained in the cells of Amylomyces presents, according to Calmette, all the characteristics of the amylase of malt. This diastase is secreted by the hyphae. Calmette also attributes to the Amylomyces 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 2 32 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 Hquid 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 Ainylomyces. The ■diastatic solution is divided into several portions of 30 cubic centimetres each, which are added to a i 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 I 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 Ainylomyces ; 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. 233, 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 ar^ 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 23°, 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 80°. The presence of salts appears to be of little disadvantage to the diastase. Calmette has determined the amounts of 2 34 THE ENZYMES AND THEIR APPLICATIONS. different substances which do not influence the diastase ; he has found: i.io% of phenol. 0.05 " of silver nitrate. o.io" of copper sulphate. o. 10 " of iron sulphate. 0.10" of zinc sulphate. Oil of mustard, used in small quantities, has no influei^ce on the development of the plant. Five per cent of glycerin produces a favorable effect. Oil of garhc in very small quan- tity and mercuric chloride in 0.005 per 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 kinds of aromatic plants which give a special perfume to the alcohol formed and which, furthermore, undoubtedly act as antiseptics. These plants are exceedingly nmnerous ; the best known are the Sinapis alba, Caryophyllns 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 fcondenser, 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 236 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 a,ttributed 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 ordinai^y 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 nioulds to the fermentation industry for ten years. At first he par- ticularly sought a medium suitable for the development of Aspergillus orirjcc, 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 Camelia Japonica is generally added. Takamine replaced the ash by an addition of i 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 oiizcz, 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 ai-e 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 taka- koji. For the preparation of this substance he pi?efers to use bran or brewery or distillery malts, and proceeds as follows : Ihe raw materials are sterilized by steam and sown with the spores of Aspergillus orisce 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 o,f 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- 238 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 sohd 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 60°; 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 filtration 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 60° 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 i 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 oriza 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- 24° THE ENZYMES AND THEIR APPLICATIONS. gillus orizcB, as we demonstrated before Takamine, but it does not heighten the Uquefying power. In otlier 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 i^ 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 I'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 distilUng 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 Cohette 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 sterihzation 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 INDUSTRUL 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 orizcs, 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 orizcB is unquestionably a more active pro- ducer of diastase t\i2.n Amylomyces Rouxii, and from this point of view it is of much more interest to distillers. Japanese yeast affords still other advantages over Amylomyces Roiixii. 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 Uber die Arrakfabrikation in Batavia. Centralblatt fiir Bakt. und Paras., 1894. Went und Geerligs. — Uber Zucker und Alcoholbildung durch Organis- men bei der Verarbeitung der Nebenprodukte der Rohrzucker- fabrikation. Wochensch. fiir Brauerei, 1894. Hofmann. — Mittheilungen der deutschen Gesellschaft fiir 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 fiir 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. fiir physiol. Chemie, i8go. Juhler.— Centralbl. fiir Bakter., 1895. Jorgensen.— Centralbl. fiir Bakter., 1895. Wehmer.— Centralbl. fiir Bakter., 1895. INDUSTRIAL APPLICATION OF MALTASE. 245 Klocker und Schionning. — Centralbl. fur Bakter., 1895. Dr. Liebscher. — Ueber die Bentitzung des Gahrungspilzes Eurot. orizae. Zeitschrift fur Spiritiis Indust., 1881. Kosai Tabe. — Centralbl. fiir Bakter., 11, p. 619. Bodin et Rolants. — Contribution a I'etude de I'utilisation de I'Amylomyces Rouxii. La biere et les boissons fermentees, 1897. Petit. — Quelques precedes nouveaux en DistiUerie. Moniteur scien- tifique, 1898. Sorel. — Comptes rendus de deux congres de chimie appliquee. Paris, 1897. Comptes Rendus, 1895. Nititenski. — Moisissures saccharificant I'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 moiits 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 I'extraction des residus de I'alcool. France, IS 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 C12H22O11 + 2H2O. 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 nigcr and Penicillium glaiicum, 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: CiaH^aOn + H^O = 2C6Hi20e. The diastatic action may be followed by the change of rotatory and reducing powers of the liquid. Trfehalose has a rotatory power of (a)^i98°, while the rotatory power of glucose is only («),; S^A°- 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 iniluence on trehalose. An acidity corresponding to 2 to 4 milligrams of sulphuric acid seems to favor the transformation of trehalose by 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 i 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 beheves 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 Hke 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 i 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 + H2O = CeHi20e + CeHj20o. 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 estabHshed the following facts: 1st. 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 difificult 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)^ +- 52.5, while that of galactose is (a)^ + 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. 25° THE ENZYMES AND THEIR APPLIC/ITIONS. By the action of inulase, inulin is hydrated and trarts- iormed into levulose according to the formula: (CeH,oO,)i3 + i8H,0 = i8CeHi,0«. Inulin. Levulose. According to Green, this transformation is accompHshed 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 60°. The action of the enzyme is influenced by the reaction of the medium. In a neutral liquid, or with 0.005 P'^'^t of 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)^ — 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, tl'.e 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: ^32J^48'-'32- and according to Chandnew : ^28J^42*-'24- The mechanism of the reaction produced by the pectase is little known. 252 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 24 Carrots 2 Maize (leaves) 8 Clover 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 2 54 THE ENZYMES AND THEIR 'APPLICATIONS. acidified in different degrees and tiie same quantity of pec- tase was added: Hydrochloric Acid. Coagulation Per cent. at the end of o I hour. 0.02 I " 0.06 3f hours. O.I 20 " Pectase is unfavorably influenced by the aoid 'reaction of the medium ; 0.06 per cent of acid in tlie 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 ofthe 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 is previously heated to 60°, the same elifect 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 Ceraionia 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 J)art 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 y4CTlNG ON CARBOHYDRATES. 257 this State it is easy to separate the endosperm from the testa and the embryo. One hundred gi'ams of dry seeds furnish 53 grams of albumen. The swelHng 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 100°, 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 80" the enzyme is destroyed. Caroubinase acts very sUghtly in a neutral medium. An addition of o.oi 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 sui^cient 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 i 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 siUqua 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 gr6wth 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 I'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. fiir B,akt. und Parasitenkunde. Zweite Ab- theilung, 1898. E. Bourquelot. — Inulase et fermentation alcoolique indirecte de I'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 I'existence, dans I'orge germee, d'un ferment soluble agissant sur la pectine. Comptes Rendus, 1898, p. 191. Freray. — Memoire sur la maturation des fruits. Ann. de chim. et phys., 1848, XXIV, p. S- 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. fiiV 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" 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. ToUens. — Recherches sur les matieres pectiques. Bull, de la Soc. chim. de Paris, 1895, p. 1246. Brown and Morris. — Untersuchung fiber der Keimung einiger Graser. Zeit. fiir das gesammte Brauwesen, 1890. De Bary. — Ueber einige Sclerotinen und Sclerotinenkrankheiten. Bot. Zeit., 1886. Schmulewitsche. — Ueber das Verhalten der VerdauungstofTe zu Rohfasser der Nahrungsmittel. Bull. Acad, des sciences Saint Petersburg, t. XI. ENZYMES ACTING ON CARBOHYDRATES. 261 Effront. — Sur un nouvel hydrate de carbone, la caro'ubine. Comptes Rendus, 1897, Sur un nouvel enzyme hydrolytique, la caroubinase. Comptes Rendus, 1897, p. 116. f Sur la caroubinase. Comptes Rendus, IX, p. 764. CHAPTER XXI. FERMENTS OF GLYCERIDES AND GLUCOSIDES. Saponifying ferments. — Ferments of glycerides. — Seroiipase 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 spHtting 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,Il,(C,siis,0,), + 3H2O = C3H,(OH)3 -fsCisHaeO^. 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 GLUCOSIDES. 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 PenicilUum glaucum. The presence of a similar substance called s^rolipase 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 difiference 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 i cubic centimetre of the liquid containing the lipase to be measured, add to it 10 cubic centimetres of a i 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 /IND THEIR APPLICATIONS. The solution of sodium carbonate used for the saturation is prepared in such a way that each drop of the alkahne Uquid neutrahzes 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° ; i 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 X 88 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 0° 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 lo minutes. In i hour. 0° 4-5 13-5 10 20 6.7 29.3 25 •■■ V lo.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 60° 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'' 41-5 6o°-62° 0.7 65"-66° Extremely slight. 7o°-72° o FERMENTS OF GLYCERIDES /tND GLUCOSIDES. 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. I c.c. 1,5 c.c. 2 c.c. 20 minutes I hour. 1 h. 20 m. 2 hours 6 12.5 20 30 11 25 36 54 16' 37 53 73 22 48 62 66 Cessation of the proportionality is noticed, in the case of Hpase 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 efifect 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 20 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 secretiqn of lipase, attributed to the blood the property of secreting a lipase diiiferent 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 dehcate 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 : Serolipase. ^^""==1- ^ tolipase. At 15" II ID "30° 15 10 "42° 21 II 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 OP 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 GLtrCOSIDES. 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 : 12CH2O + H2 = C12H16O, + 5H2O. Formic aldehyde. Arbutin. 13CH2O + 2H2 = QaHigO, + 6H2O. Formic aldehyde. Salicin. 268 THE ENZYMES /IhID THEIR APPLIC/ITIONS. 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 svibstances 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: C^oH^vNOii + 2H2O = 2C6Hi20e + C.HeO + HCN. Amygdalin. Glucose. Benzoic aldehyde. Hydrocyanic acid. Emulsin and also amygdahn 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 Polyporus sulfureus, in Armillaria mellea, and in Polyporus fomenta/rius. Emulsin has also been met with in Penicillium 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 : C12H16O7 + H2O = CgHiaOe 4 CeHgOa. Arbutin. Glucose. HydroquinonCi With helicin, a product of oxidation of salicin : C13H16O7 + H2O = CgHiaOg + C7H6O2. Helicin. Glucose. Salicylic aldehyde. With salicin, extracted from the bark of poplar or the flowers of Spirea ulmaria : 'C13H18O7 + H2O = CgHiaOg + CyHgOa. Salicia. Glucose. Saligenin. With phloridzin, extracted from the bark of the apple- tree: C21H24O10 + H2O = C6Hi20e + C15H14O3. Phloridzin. Glucose. Phloretin. With daphnin, extracted from the Daphne gnidium : CisHieOc, + H2O = CeHj^Oe + C9He04. Daphnin. Glucose. Daphnetin. With coniferin, extracted from the Larix Europce : C16H22O8 + H2O = C6H12O6 + CioHi,03. Coniferin. Glucose. Coniferic alcohol. With esculin of ^sculus hippocastanum, which certain authors consider as an isomer of daphnin, glucose and esculi- tin are obtained : C15H16O9 + H2O = CeHiaOs + QHgO^. Esculin. Glucose. Esculitin. 270 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 ^-methyldextro-glucoside, it is without action on the a-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 I : 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 GLYCERIDES 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 100" 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 CrucifercB. 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 : C10H13NKS2O10 = QHi.Oe + CsH^NSC + KHSO4. Sinigrin. Glucose. Allyl iso-thiocyanate. Potassium bisulphate. 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: CjoHieKNSaOs + H2O, 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: sinalbin. CeHijOo + QH7O-NCS + C16H24NO5 — HSO4. Glucose. Oxy-benzyl-thiocyanate. Sinapine sulphate. FERMENTS OF GLYCERIDES AND GLUCOSIDES. 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 oMcinalis. 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 : Q4H32O1, + 3H2O = Q2H10O3 + 2C,ll,,0,. 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 : C26H28O14 -h2H20 = 2CeHi206 + Ci,H804. 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 : C14H18O8 + H2O = CgHiaOs + C6H4<'^Q OCH 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. 2 74 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 ofif, 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. — LeQons de physiologie experimentale. Dobelle. — Actions du pancreas sur les grains et I'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 I'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. 370. 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 I'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 GLYCERIDES AND GLUCOSIDES. 275 Procter. — Betulase. Zeit. fiir 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, 6e seri, t. Ill, p. 117. Johonson. — Sur la localisation de I'emulsine dans les amandes. Ann. des Sc. 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 fiir Wiss. Bot. 1893, XXVI, p. 55. Bulle. — Ann. de chim. et pharra., LXIX, p. 145. Ortlofif.— Archiv. de pharm., XLVIII, p. 16. L. Guignard. — Recherches sur la locaHsation 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 I'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 I'emulsine de I'aspergillus niger sur quelques glucosides. Societe de Biologic, 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 alcohoUc 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 Hving 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 alcohohc 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 i kilogram of yeast, to which add i 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, i 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 Hquid 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 &gg. 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 100° 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 100°, 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 of yeast is measured by a method 28o 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 bala^ce. The difference in weight shows the c[uantity 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 7.72 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. 11 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. 281 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 : CgHiaOe = 2CO2 + 2C2H5OH. 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. Carbonic acid (in grams) formed after Temperature. 6 hours. 21 hours. 24 hours. 40 hours. 12-14° 0.43 I. II 1. 14 1.27 22° 0.76 I.OI 1.02 I.OO 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 tempjerature, a liberation of 0.76 grams of carbonic acid, while at 12° to 14° 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. ^^ hours. 24 hours. 40 hours, 16 1.33 1.46 1.48 27 0.70 0.80 0.82 37 0.60 0.72 0.74 In liquids containing i6 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 ^y 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 pow.r. B}' 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 I 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 " ... O.II " o.oio " 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 i 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 temperaturfe'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 /IND 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 o.i 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 286 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 10°. After this time the oil was poured ofif, 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 Hquid was obtained which, filtered through filter-paper, was a viscous, trans- parent solution, of sHghtly 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 80° 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 10°, 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 apphca- 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 future 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 y6ast 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, thejirst 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 100°, 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, mo. E. Buchner. — Precede pour la fabrication des levures d'attente. Brevet, Allemagne, 1897, 2668, n° 97240. Bechamp. — Sur la presence de I'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. — fetude sur la biere. Comptes Rendus, 1876, LXXVII, p. 1140. — — Etude sur la biere, 1876. Paris, Gauthier-Villars. Meraoir-js sur la fermentation alcoolique. Comptes Rendus, 1859, p. 1149, XIV, III. Bull, de la Soc. de chim. Paris, 1861. Sur la production de I'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, s^ 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, I, 209; 1898, I, 1084; 1898, I, 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: oenoxi- 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 sertain 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- mg 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 100°. 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 oiTer- 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 I 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 60°. 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. \A'e 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- ahty is more strongly marked; sucrase, for example, can only decompose saccharose and is incapable of acting on very closely related bodies which dififer 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 Anacardiace^ (Rhus vcrinicifera). 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 ^Im 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 he called uruschic acid (C14H19O2), a body which by oxida- tion changes into oxyuruschic acid, as is shown by the fol- lowing equation: 2Q,Hi902 + 30 = 2Q,Hi303 + H2O. 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 gum. 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 alcohoHc solution, after the gummy precipitate has teen removed, is quickly distilled ;';( 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 laccase, 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, 300 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 of the lac-tree. > OXIDASES. 3QI 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- riciis sanguineus, colored blue tincture of guaiacum with- out addition of hydrogen peroxide and lost this faculty when it was heated to 100°. 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 Myxomyoete, — Reticulario- maxima, the Polypori, and the Agaricines. Russula fcetens, 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 dififerent 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. Genus Number of _i£.!i^!___ o"" species ^TTt-.u i»7-.u T" sub-genus. examined. , W"*" Without ° laccase. laccase. Russula i8 i8 o Lactarius 20 18 2 Psalliota 5 4 i Boletus 18 10 8 Clitocybe 9 5 4 Marasmius 6 o 6 Hygrophorus 6 o 6 Cortinarius 12 i 11 Inocybe 6 i 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: Hydroquinone. Quinone. The diastase also acts on galhc 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 i 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: Oxyg'en absorbed 17.6 ex. Carbonic acid freed 11. i 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. COa*freed. Hydroquinone (para-diphenol). After 4 h 32.0 c.c. 1.7 c.c. Pyrocatechin (ortho-diphenol). " 4h 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 (NHg), without the progress of oxidation being modified. The paramidophenol: C H Active. Destroyed. 60° 2.30 2.70 65 I-50 3-5 70 0.90 4. 1 75 075 425 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 60°. Action of Chemical Agents. — Accord^ing 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 o.oi to 0.08 parts per litre, checks the action of oenoxidase and causes its destruc- tion. This fact was demonstrated by Bouffard 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. 315 destroyed by absorption of oxygen. By exposing a solution of oxidase to the air, Laborde obtained the following figures : T-» .' Oxidase Duration " of aeration. C' . , T P Remaining. Lost. 2 days 3.5 2.0 4 " 2.8 2.7 6 " 2.4 3-1 12 " 0.8 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 100°, 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 31 6 THE ENZYMES AND THEIR /IPPUCATIONS. 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 brpught 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: 317 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- 3i8 THE ENZYMES /IND THEIR APPLIC/tTIONS. 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 I'arbre a laque. Comptes Rendus, i" semestre, 1894, p. 1215. Sur la laccase et le pouvoir oxydant de cette diastase. Comptes Rendus, i'^'' 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 corli- poses organiques et leur oxydabilite sous I'influence de la laccase. Comptes Rendus, i^r semestre, 1896, p. 1132. Sur une nouvelle oxydase ou ferment soluble oxydant d'origine vegetale. Comptes Rendus, i^'' semestre, 1896, p. 1215. Presence simultanee de la laccase et de la tyrosinase dans le sue de quelques champignons. Comptes Rendus, 2e semestre, 1896, p. 463. BoufEard. — Observations sur quelques proprietes de 1' oxydase des vins. Comptes Rendus, i^r semestre, 1897, p. 706. Rappel d'une note precedente. Comptes Rendus, 1" semestre, p. 1053- Em. Bourquelot and Bertrand. — La laccase dans les champignons. Comptes Rendus, 2^ semestre, 1895, p. 788. Em, Bourquelot. — Influence de la reaction du milieu sur I'activite du fer- ment oxydant des champignons. Comptes Rendus, 2^ semestre, 1896, p. 260. Action du ferment soluble oxydant des champignons sur les phenols insolubles dans I'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^r semestre, 1897, p. 781. Sur le ferment soluble oxydant de la casse des vins. Comptes Rendus, i^ semestre, 1897, p. 406. Laborde. — Sur I'absorption de I'oxygene dans la casse des vins. Comptes Rendus, 2^ semestre, 1897, p. 248. Sur I'oxydase du botrytis cinerea. Comptes Rendus, i^r semestre, 1898, p. S36. 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" semestre, 1897, p. 1461. V. Martinand. — Sur I'oxydation et la casse des vins. Comptes Rendus, ler semestre, 1897, p. S12. , OXIDASES. 3^9 Talomei. — Olease. Atti. Ace. di Lincei. Rnd. 1896. Berichte der deutsche chem. Gesellschaft, 1896. J. de Rey Pailhade. — £tude sur les proprietes chimiques de I'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. 1 162. L. Lindet. — Sur I'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. Eflront. — Action de Toxygene sur les levUres de biere. Comptes Ren- dus, CXXVII, p. 326, 1898. Martinand. — ^Action de I'air sur le mout de raisin «t 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, 142. Distillation: preparation of the mash, 179. Distilleries, East Asian, 235. Emulsin, 268. in fungi, 268. Enzymes: action of heat on, 16. , chemical composition, 21. 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' 321 322 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. j'ancreato-lipase, 265. Pectase, 251. Pectin, 251. Pectinase, 251. Pectose, 251.- Papaln, 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. Sak6, 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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