CHAPTER II.

ZYMASE AND ITS PROPERTIES.

DISCOVERY OF ZYMASE.

The history of Buchner's discovery is of great interest [Gruber, 1908; Hahn, 1908]. As early as 1893 Hans and Eduard Buchner found that the cells of even the smallest micro-organism could be broken by being ground with sand [Buchner, E. and H., and Hahn, 1903, p. 20], and in 1896 the same process was applied by these two investigators to yeast, with the object of obtaining a preparation for therapeutic purposes. Difficulties arose in the separation of the cell contents from the ground-up mixture of cell membranes, unbroken cells, and sand, but these were overcome by carrying out the suggestion of Martin Hahn (at that time assistant to Hans Buchner) that kieselguhr should be added and the liquid squeezed out by means of a hydraulic press [Buchner, E. and H., and Hahn, 1903, p. 58]. The yeast-juice thus obtained was, in the first instance, employed for animal experiments, but underwent change very rapidly. The ordinary antiseptics were found to be unsuitable, and hence sugar was added as a preservative, and it was the marked action of the juice upon this added cane sugar that drew Eduard Buchner's attention to the fact that fermentation was proceeding in the absence of yeast-cells.

As in the case of so many discoveries, the new phenomenon was brought to light, apparently by chance, as the result of an investigation directed to quite other ends, but fortunately fell under the eye of an observer possessed of the genius which enabled him to realise its importance and give to it the true interpretation.

In his first papers [1897, 1, 2; 1898], Buchner established the following facts: (1) yeast-juice free from cells is capable of producing the alcoholic fermentation of glucose, fructose, cane sugar, and maltose; (2) the fermenting power of the juice is neither destroyed by the addition of chloroform, benzene, or sodium arsenite [Hans Buchner, 1897], by filtration through a Berkefeld filter, by evaporation to dryness at 30° to 35°, nor by precipitation with alcohol; (3) the fermenting power is completely destroyed when the liquid is heated to 50°. [p019]

From these facts he drew the conclusion "that the production of alcoholic fermentation does not require so complicated an apparatus as the yeast cell, and that the fermentative power of yeast-juice is due to the presence of a dissolved substance". To this active substance he gave the name of zymase.

Buchner's discovery was not received without some hesitation. A number of investigators prepared yeast-juice, but failed to obtain an active product [Will, 1897; Delbrück, 1897; Martin and Chapman, 1898; Reynolds Green, 1897; Lintner, 1899]. A more accurate knowledge of the necessary conditions and of the properties of yeast-juice, however, led to more successful results [Will, 1898; Reynolds Green, 1898; Lange, 1898], and it was soon established that, given suitable yeast, an active preparation could be readily procured by Buchner's method. Criticism was then directed to the effect of the admitted presence of a certain number of micro-organisms in yeast-juice [Stavenhagen, 1897], but Buchner [Buchner and Rapp, 1897] was able to show by experiments in the presence of antiseptics and with juice filtered through a Chamberland candle that the fermentation was not due to living organisms of any kind.

The most weighty criticism of Buchner's conclusion consisted in an attempt to show that the properties of yeast-juice might be due to the presence, suspended in it, of fragments of living protoplasm, which, although severed from their original surroundings in the cell, might retain for some time the power of producing alcoholic fermentation. This, it will be seen, was an endeavour to extend Nägeli's theory to include in it the newly discovered fact.

In favour of this view were adduced the similarity between the effects of many antiseptics on living yeast and on the juice, the ephemeral nature of the fermenting agent present in the juice, the effect of dilution with water, and the phenomenon of autofermentation which is exhibited by the juice in the absence of added sugar [Abeles, 1898; v. Kupffer, 1897; v. Voit, 1897; Wehmer, 1898; Neumeister, 1897; Macfadyen, Morris, and Rowland, 1900; Bokorny, 1906; Fischer, 1903; Beijerinck, 1897, 1900; Wroblewski, 1899, 1901].

A brief general description of the actual properties of yeast-juice and of the phenomena of fermentation by its means is sufficient to show the great improbability of this view.

The juice prepared by Buchner's method forms a somewhat viscous opalescent brownish-yellow liquid, which is usually faintly acid in reaction [compare Ahrens, 1900] and almost optically inactive. It has a specific gravity of 1·03 to 1·06, contains 8·5 to 14 per cent. [p020] of dissolved solids, and leaves an ash amounting to 1·4 to 2 per cent. About 0·7 to 1·7 per cent. of nitrogen is present, nearly all in the form of protein, which coagulates to a thick white mass when the juice is heated.

A powerful digestive enzyme of the type of trypsin is also present, so that when the juice is preserved its albumin undergoes digestion at a rate which depends on the temperature [Hahn, 1898; Geret and Hahn, 1898, 1, 2; 1900; Buchner, E. and H., and Hahn, 1903, pp. 287-340], and is converted into a mixture of bases and amino-acids. After about six days at 37°, or 10 to 14 days at the ordinary temperature, the digestion is so complete that no coagulation occurs when the juice is boiled. As this proteoclastic enzyme, like the alcoholic enzyme, cannot be extracted from the living cells, it is termed yeast endotrypsin or endotryptase. Fresh yeast-juice produces a slow fermentation of sugar, which lasts for forty-eight to ninety-six hours at 25° to 30°, about a week at the ordinary temperature, and then ceases, owing, not to exhaustion of the sugar, but to the disappearance of the fermenting agent. When the juice is preserved or incubated in the absence of a fermentable sugar this disappearance occurs considerably sooner, so that even after standing for a single day at room temperature, or two days at 0°, no fermentation may occur when sugar is added. The reason for this behaviour has not been definitely ascertained. As will be seen later on (p. 64) the phenomenon is a complex one, but the disappearance of the enzyme was originally ascribed by Buchner to the digestive action upon it of the endotrypsin of the juice [1897, 2], and no better explanation has yet been found. Confirmation of this view is afforded by the fact that the addition of a tryptic enzyme of animal origin greatly hastens the disappearance of the alcoholic enzyme [Buchner, E. and H., and Hahn, 1903, p. 126], and that some substances which hinder the tryptic action favour fermentation [Harden, 1903]. The amount of fermentation produced is almost unaffected by the presence of such antiseptics as chloroform or toluene, although some others, such as arsenites and fluorides, decrease it when added in comparatively high concentrations, and it is only slightly diminished by dilution with three or four volumes of sugar solution, somewhat more considerably by dilution with water. When it is filtered through a Chamberland filter the first portions of the filtrate are capable of bringing about fermentation, but the fermenting power diminishes in the succeeding portions and finally disappears. The juice can be spun in a centrifugal machine without being in any way altered, and no separation into more or less active layers takes place under these conditions. [p021]

The amorphous powder obtained by drying the precipitate produced when the juice is added to a mixture of alcohol and ether is also capable of producing fermentation, and the process of precipitation may be repeated without seriously diminishing the fermenting power of the product.

These facts clearly show that the various phenomena adduced by the supporters of the theory of protoplasmic fragments are quite consistent with the presence of a dissolved enzyme as the active agent of the juice, and at the same time that the properties demanded of the living fragments of protoplasm to which fermentation is ascribed are such as cannot be reconciled with our knowledge of living matter. If living protoplasm is the cause of alcoholic fermentation by yeast-juice, a new conception of life will be necessary; the properties of the postulated fragments of protoplasm must be so different from those which the protoplasm of the living cell possesses as to deprive the theory of all real value [Buchner, 1900, 2; Buchner, E. and H., and Hahn, 1903, p. 33].

Further and very convincing evidence against the protoplasm theory is afforded by the behaviour of yeast towards various desiccating agents. When yeast is dried at the ordinary temperature it retains its vitality for a considerable period. If, however, the dried yeast be heated for six hours at 100° it loses the power of growth and reproduction but still retains that of fermenting sugar, and when ground with sand, kieselguhr and 10 per cent. glycerol solution yields an active juice [Buchner, 1897, 2; 1900, 1]. Preparations (known as zymin) obtained by treating yeast with a mixture of alcohol and ether [Albert, 1900, 1901, 1], or with acetone and ether [Albert, Buchner, and Rapp, 1902], show precisely similar properties (p. 38). The proof in this case has been carried a step further, for the active juice obtained by grinding such acetone-yeast, when precipitated with alcohol and ether, yields an amorphous powder, still capable of fermenting sugar.

THE PREPARATION OF YEAST-JUICE.

Buchner's process for the preparation of active yeast-juice is characterised by extreme simplicity. The yeast employed, which should be fresh brewery yeast, is washed two or three times by being suspended in a large amount of water and allowed to settle in deep vessels. It is then collected on a filter cloth, wrapped in a press cloth, and submitted to a pressure of about 50 kilos, per sq. cm. for five minutes. The resulting friable mass contains about 70 per cent. of water and is free from adhering wort. The washed yeast is then [p022] mixed with an equal weight of silver sand and 0·2 to 0·3 parts of kieselguhr, care being taken that this is free from acid. The correct amount of kieselguhr to be added can only be ascertained by experience, and varies with different samples of yeast. The dry powder thus obtained is brought in portions of 300 to 400 grams into a large porcelain mortar and ground by hand by means of a porcelain pestle fastened to a long iron rod which passes through a ring fixed in the wall (Fig. 1). The mortar used by Buchner has a diameter of 40 cm. and the pestle and rod together weigh 8 kilos.

As the grinding proceeds the light-coloured powder gradually darkens and becomes brown, and the mass becomes moist and adheres to the pestle, until finally, after two to three minutes' grinding, it takes the consistency of dough, at which stage the process is stopped. The mass is next enveloped in a press cloth and submitted to a pressure of 90 kilos, per sq. cm. in a hydraulic hand press, the pressure being very gradually raised in order to avoid rupture of the cloth. The cloth required for 1000 grams of yeast measures 60 by 75 cm. and is previously soaked in water and then submitted to a pressure of 50 kilos, per sq. cm., retaining about 35 to 40 c.c. of water.

The juice runs from the press on to a folded filter paper, to remove kieselguhr and yeast cells, and passes into a vessel standing in ice water.

The yield of juice obtained by Buchner in an operation of this kind from 1 kilo. of yeast amounts to 320 to 460 c.c. It may be increased by re-grinding the press cake and again submitting it to pressure, and then amounts on the average to 450 to 500 c.c.

Since the cell membranes constitute about 20 per cent. of the weight of the dry yeast, this yield corresponds to more than 60 per cent. of the total cell contents of the yeast. It has been computed by Will [quoted by Buchner, E. and H., and Hahn, 1903, p. 66] that [p023] only about 20 per cent. of the cells are left unaltered by one grinding and pressing, and only 4 per cent. after a repetition of the process, at least 57 per cent. of the cells being actually ruptured by the double process, and the remainder to some extent altered. It seems probable from these figures that a certain amount of the juice may be derived from the unbroken cells, and Will expressly states that many unbroken cells have lost their vacuoles.

If the yeast be submitted to a process of regeneration, which consists in exposure to a well-aerated solution of sugar and mineral salts until fermentation is complete, the juice subsequently obtained [p024] is more active than that yielded by the original yeast [Albert, 1899, 1].

A modified method of grinding yeast was introduced by Macfadyen, Morris, and Rowland [1900], who placed a mixture of yeast and sand in a jacketed and cooled vessel, in which a spindle carrying brass flanges was rapidly rotated [Rowland, 1901]. One kilo. of yeast ground in this way for 3·5 hours yielded 350 c.c. of juice.

This grinding process was at first adopted by Harden and Young in their experiments but was afterwards abandoned in favour of Buchner's hand-grinding process, as it was found liable to yield juices of low fermenting power, probably on account of inefficient cooling during the grinding process. A slight modification of Buchner's process has, however, been introduced, the hand-ground mass being mixed with a further quantity of kieselguhr until a nearly dry powder is formed, and the mass packed between two layers of chain cloth in steel filter plates and pressed out in a hydraulic press at about 2 tons to the square inch (300 kilos. per sq. cm.). The press and plates are shown in section in Fig. 2. It has also been found convenient to remove yeast cells and kieselguhr from the freshly pressed juice by centrifugalisation instead of by filtration through paper, and to wash the yeast before grinding by means of a filter-press.

Working with English top yeasts Harden and Young have found the yield of juice extremely variable, the general rule being that the amount of juice obtainable from freshly skimmed yeast is smaller than that yielded by the same yeast after standing for a day or two after being skimmed. The yield for 1000 grams of pressed brewer's yeast varies from 150 to 375 c.c., and is on the average about 250 c.c.

Very fresh yeast occasionally presents the peculiar phenomenon that scarcely any juice can be expressed from the ground mass, although the latter does not differ in appearance or consistency from a mass which gives a good yield.

EXTRACTION OF ZYMASE FROM UNGROUND YEAST.

/1. Maceration of Dried Yeast./

A valuable addition to the methods of obtaining an active solution of zymase was made in 1911 by Lebedeff [1911, 2; 1912, 2; see also 1911, 3, 7, and 1912, 1]. This investigator had been in the habit of grinding dried yeast with water for preparing samples of yeast-juice of uniform character and observed that when the dried yeast was digested with sugar solution and the mixture heated, coagulation [p025] took place throughout the whole liquid, the proteins of the yeast having passed out of the cells. Further examination revealed the interesting fact that dried yeast readily yielded an active extract when macerated in water for some time. The quality of the resulting "maceration extract" depends on a considerable number of factors, the chief of which are: (1) the temperature of drying of the yeast; (2) the temperature of maceration; (3) the duration of maceration; and (4) the nature of the yeast, as well as, of course, the amount of water added in maceration.

In general the yeast should be dried at 25°-30° and then macerated with 3 parts of water for 2 hours at 35°.

The temperature of maceration may as a rule be varied, without detriment to the product provided that the time of maceration is also suitably altered; thus with dried Munich yeast, maceration for 4·5 hours at 25° is about as effective as 2 hours at 35°, whereas treatment for a shorter time at 25° or a longer time at 35° produces in general a less efficacious extract. Yeast dried at a lower temperature than 25° tends to yield an extract poor in co-enzyme (p. 59) and hence of low fermenting power, this being especially marked at air temperature.

The subsequent treatment of the yeast during maceration may, however, be of great influence in such cases. Thus a yeast dried at 15° gave by maceration at 25° for 4·5 hours a weak extract (yielding with excess of sugar 0·33g. CO{2}), whereas when macerated at 35° for 2 hours it yielded a normal extract (1·36g. CO{2}).

The nature of the yeast is of paramount importance. Thus while Munich (bottom) yeast usually gives a good result, a top yeast from a Paris brewery was found to yield extracts containing neither zymase nor its co-enzyme in whatever way the preparation was conducted. The existence of such yeasts is of great interest, and it was probably due to the unfortunate selection of such a yeast for his experiments that Pasteur was unable to prepare active fermenting extracts and therefore failed to anticipate Buchner by more than 30 years (see p. 15). The English top yeasts as a rule give poor results [see Dixon and Atkins, 1913] and sometimes yield totally inactive maceration extract. It is not understood why the enzyme passes out of the cell during the process of maceration and the whole method gives rise to a number of extremely interesting problems.

/Method./--A suitable yeast is washed by decantation, filtered through a cloth, lightly pressed by means of a hand press, and then passed through a sieve of 5mm. mesh, spread out in a layer 1-1·5cm. thick and left at 25°-35° for two days. Fifty grams of the dried yeast is [Pg 026] thoroughly and carefully mixed with 150 c.c. of water in a basin by means of a spatula and the whole digested for two hours at 35°. The mass often froths considerably. It is then filtered through ordinary folded filter paper, preferably in two portions, and collected in a vessel cooled by ice. The separation may also be effected by centrifuging or pressing out the mass, and the maceration may be conveniently conducted in a flask immersed in the water of a thermostat. It is not advisable to macerate more than 50 grams in one operation. Under these conditions 25-30 c.c. of extract are obtained after 20 minutes' filtration, 70-80 c.c. in twelve hours. Dried Munich yeast can be bought from Messrs. Schroder of Munich and serves as a convenient source of the extract.[1]

[1] The material supplied is occasionally found to yield an inactive extract and every sample should be tested.

This extract closely resembles in properties the juice obtained by grinding the same yeast, but it is usually more active and contains more inorganic phosphate (see p. 46).

/2. Other Methods./

Attempts to prepare active extracts from undried yeast in an analogous manner have so far not been very successful. Thus Rinckleben [1911] found that plasmolysis by glycerol (8 per cent.) or sodium phosphate (5 per cent.) sometimes yielded an active juice and sometimes a juice which contained enzyme but no co-enzyme, but more often an inactive juice incapable of activation (p. 64) [see also Kayser, 1911].

Giglioli [1911] by the addition of chloroform also obtained an active liquid. It appears in fact as though almost any method of plasmolysing the yeast cell may yield a certain proportion of zymase in the exudate.

An ingenious process has been devised by Dixon and Atkins [1913] who applied the method of freezing in liquid air which they had found efficacious for obtaining the sap from various plant organs. They thus succeeded in obtaining from yeast, derived from Guinness' brewery in Dublin, liquids capable of fermenting sugar and of about the same efficacy as the maceration extracts prepared by Lebedeff's method from the same yeast. The results were, however, in both cases very low, the maximum total production of CO{2} by 25 c.c. of liquid from excess of sugar being 32·5 c.c. (air temperature) or about 0·06g. Munich yeast on the other hand yields, either by maceration or grinding, a liquid giving as much as 1·5-2g. of CO{2} per 25 c.c., whilst [p027] English yeast-juice prepared by grinding often gives as much as 0·5-0·7g. of CO{2}.

No direct comparison with the juice prepared by grinding was made by Dixon and Atkins, but it may be concluded from their results that the best method of obtaining an active preparation from the top yeasts used in this country is that of grinding. Maceration, freezing and plasmolysis alike yield poor results. With Munich yeast on the other hand the maceration process yields excellent results, whilst the liquid air process has not so far been tried.

PRACTICAL METHODS FOR THE ESTIMATION OF THE FERMENTING POWER OF YEAST-JUICE.

In order to estimate the amount of carbon dioxide evolved in a given time and the total amount evolved by the action of yeast-juice on sugar, Buchner adopted an extremely simple method, which consisted in carrying out the fermentation in an Erlenmeyer flask provided with a small wash-bottle, which contained sulphuric acid and was closed by a Bunsen valve, and ascertaining the loss of weight during the experiment. Corrections are necessary for the carbon dioxide present in the original juice and retained in the liquid at the close of the experiment and for that present in the air space of the apparatus, but it was found that for most purposes these could be neglected. In cases in which greater accuracy was desired, the carbon dioxide was displaced by air before the weighings were made. A typical experiment of this kind, without displacement of carbon dioxide, is the following:--

March 22, 1899, Berlin bottom yeast V. 20 c.c. juice + 8 grams cane sugar + 0·2 c.c. toluene as antiseptic at 16°. Grams of carbon dioxide after

24 48 72 96 hours. 0·40 0·64 0·99 1·11

The total weight of carbon dioxide evolved under these conditions is termed the fermenting power of the juice (Buchner).

A more accurate method [Macfadyen, Morris, and Rowland, 1900] consists in passing the carbon dioxide into caustic soda solution and estimating it by titration. The yeast-juice, sugar, and antiseptic are placed in an Erlenmeyer flask provided with a straight glass tube, through which air can be passed over the surface of the liquid, and a conducting tube leading into a second flask which contains 50 c.c. of 10 per cent. caustic soda solution and is connected with the air by a guard tube containing soda lime. The juice can be freed from carbon dioxide by agitation in a current of air before the flask is connected to [p028] that containing the caustic soda solution, and at the end of the period of incubation air is passed through the apparatus, the liquid being boiled out if great accuracy is required. The absorption flask is then disconnected and the amount of absorbed carbon dioxide estimated by titration. This is carried out by making up the contents of the flask to 200 c.c., taking out an aliquot portion, rendering this exactly neutral to phenophthalein by the addition first of normal and finally of decinormal acid, adding methyl orange and titrating with decinormal acid to exact neutrality. Each c.c. of decinormal acid used in this last titration represents 0·0044 gram of carbon dioxide in the quantity of solution titrated.

These methods are only suitable for observations at considerable intervals of time. For the continuous observation of the course of fermentation Harden, Thompson and Young [1910] connect the fermentation flask with a Schiff's azotometer filled with mercury and measure the volume of gas evolved, the liquid having been previously saturated with carbon dioxide (Fig. 3). The level of the mercury in the reservoir is kept constant by a syphon overflow, as shown in the figure, or, according to a modification introduced by S. G. Paine, by a specially constructed bottle provided with two tubulures near the bottom. This ensures that no change in the pressure in the flask occurs, and the volume of gas observed is reduced to normal pressure by means of a table. Before making a reading it is necessary to shake the fermenting mixture thoroughly, as the albuminous liquid very readily becomes greatly supersaturated with carbon dioxide, so much so in fact that very little gas is evolved in the intervals between the shakings. The exact procedure in making an observation consists in shaking the flask [p029] thoroughly, replacing in the thermostat, allowing to remain for one minute, and then reading the level of the mercury in the azotometer. After the required time, say five minutes, has elapsed from the time at which the flask was first shaken, it is again removed from the bath, shaken as before, replaced, allowed to remain for one minute and the reading then taken. In this way readings can be conveniently made at intervals of three or five minutes or even less, and much more detailed information obtained about the course of the reaction than is possible by means of observations made at intervals of several hours.

Another form of volumetric apparatus, designed by Walton [1904], has been used by Lebedeff [1909].

An apparatus on a different principle has been designed by Slator [1906] for use with living yeast, but is equally applicable to yeast-juice, and a very similar form has been more recently employed by Iwanoff [1909, 2]. In this apparatus the change of pressure produced by the evolution of carbon dioxide is measured at constant volume, and comparative rates of evolution can be obtained with considerable accuracy, although the method has the disadvantage that the absolute volume of gas evolved is not measured. The apparatus consists of a bottle or flask connected with a mercury manometer. The fermenting mixture is placed in the bottle along with glass beads to facilitate agitation, the pressure is reduced to a small amount by the water-pump, and the rise of pressure is then observed at intervals, this being proportional to the volume of gas produced. As in the preceding case, the liquid must be well shaken before a reading is made.

THE ALCOHOLIC FERMENTATION OF THE SUGARS BY YEAST-JUICE.

Yeast-juice brings about a slow fermentation of those sugars which are fermented by the yeast from which it is prepared as well as of dextrin, and of starch and glycogen, which are not fermented by living yeast.

(/a/) /Relation to Fermentation by living Yeast./

Both in rate of fermentation and in the total fermentation produced, yeast-juice stands far behind the equivalent amount of living yeast. Taking 25 c.c. of yeast-juice to be equivalent to at least 36 grams of pressed yeast containing 70 per cent. of moisture, it is found that whereas the yeast-juice (from English top yeast) gives with glucose a maximum rate of fermentation of about 3 c.c. in five minutes, the living yeast ferments the sugar at the rate of about 126 c.c. in the same time, or [p030] about forty times as quickly. The total carbon dioxide obtainable from the yeast-juice, moreover, corresponds to the fermentation of only 2 to 3 grams of sugar, whilst the living yeast will readily ferment a much larger quantity, although the exact limit in this respect has not been accurately determined. The reasons for this great difference in behaviour will be discussed later on, after the various factors concerned in fermentation have been considered (p. 123).

(/b/) /Relation of Alcohol to Carbon Dioxide./

In all cases of fermentation by yeast-juice and zymin, the relative amounts of carbon dioxide and alcohol produced are substantially in the ratio of the molecular weights of the compounds, that is as 44: 46, so that for 1 part of carbon dioxide 1·04 of alcohol are formed. This has been shown for the juice and zymin from bottom yeasts by Buchner [Buchner, E. and H., and Hahn, 1903, pp. 210, 211], who obtained the ratios 1·01, 0·98, 1·01, and 0·99 from experiments in which from 8 to 15 grams of alcohol were produced. Similar numbers, 0·90, 1·12, 0·95, 0·91 and 0·92, have been obtained for the juice from top yeasts by Harden and Young [1904], who worked with much smaller quantities. The variable results obtained with juice from top yeast by Macfadyen, Morris and Rowland [1900], have not been confirmed.

(/c/) /Relation of Carbon Dioxide and Alcohol Produced to the Amount of Sugar Fermented./

The construction of a balance-sheet between the sugar fermented and the products formed is of special interest in the case of alcoholic fermentation by yeast-juice, because, there being no cell growth as in the case of living yeast, an opportunity appears to be afforded of ascertaining whether the whole of the sugar is converted into alcohol and carbon dioxide, or whether some fraction of the sugar passes into any of the well-known subsidiary products of alcoholic fermentation by yeast, such as glycerol, fusel oil, or succinic acid. Unfortunately the question cannot be settled in this way. When the loss of sugar during the fermentation is estimated directly, it is usually found to be considerably greater than the sum of the alcohol and carbon dioxide produced from it. This fact was first observed by Macfadyen, Morris and Rowland [1900], and was then confirmed by Buchner [Buchner, E. and H., and Hahn, 1903, p. 212], in one instance, the excess of sugar lost over products being in this case about 15 per cent. of the total sugar which had disappeared. The matter was then more thoroughly investigated by Harden and Young [1904]. [p031]

The conditions under which the experiment must be carried out are not very favourable to the attainment of extreme accuracy. Yeast-juice contains glycogen and a diastatic enzyme which converts this into dextrins and finally into sugar. This process goes on throughout fermentation, tending to increase the sugar present and to make the apparent loss of sugar less than the sum of the products. In spite of this it was found that a certain amount of sugar invariably disappeared without being accounted for as alcohol or carbon dioxide, and this whether the fermentation lasted sixty or a hundred and eight hours, and independently of the dilution of the juice. This disappearing sugar amounted in some cases to 44 per cent. of the total loss of sugar, and on the average of twenty-five experiments was 38 per cent. Further information was sought by converting all the sugar-yielding constituents of the juice into sugar by hydrolysis before and after the fermentation. This process revealed the fact that when the glucose equivalent of the juice before and after fermentation was determined after hydrolysis with three times normal acid for three hours (and a correction made for the loss of reducing power experienced by glucose itself when submitted to this treatment), the difference was almost exactly equal to the alcohol and carbon dioxide produced. In other words, accompanying fermentation, a change proceeds by which sugar is converted into a less reducing substance, reconvertible into sugar by hydrolysis with acids. Similar results were subsequently obtained by Buchner and Meisenheimer [1906], who employed 1·5 normal acid and observed a small nett loss of sugar. Still more recently Lebedeff [1909, 1910, see also 1913, 2] has carried out similar estimations with the same result. It is doubtful whether the experiments which have so far been made on this point are sufficiently accurate to decide with certainty whether or not the loss of sugar is exactly equal to the sum of the carbon dioxide and alcohol produced. It has been shown by Buchner and Meisenheimer [1906] that glycerol is a constant product of alcoholic fermentation by yeast-juice (p. 95), and no other source for this than the sugar has yet been found, so that it is not improbable that a small amount of sugar is converted into non-carbohydrate substances other than carbon dioxide and alcohol.

It has also been shown [Harden and Young, 1913] that the deficit of sugar is not due to the formation of hexosephosphate (p. 47), which has a lower reduction than glucose, and that the solution from which the sugar (either glucose or fructose) has disappeared actually contains some substance of relatively high dextrorotation and of low reducing power. [p032]

However this may be, it may be considered as established that during alcoholic fermentation sugar is converted by an enzyme into some compound of less reducing power, which again yields sugar on hydrolysis with acids. The exact nature of this substance has not been ascertained, but it appears likely that the process is a synthetical one resulting in the formation of some polysaccharide, possibly intermediate between the hexoses and glycogen.

A similar phenomenon has been observed with living yeast by Euler and Johansson [1912, 1], and Euler and Berggren [1912], whose interpretation of the observation is discussed later on (p. 57).

(/d/) /Fermentation of Different Carbohydrates. Autofermentation./

Yeast-juice and zymin ferment all the sugars which are fermented by the yeast from which they are prepared, and, in addition, a number of colloidal substances which cannot pass through the membrane of the living yeast cell, but which are hydrolysed by enzymes in the juice and thus converted into simpler sugars capable of fermentation [Buchner and Rapp, 1898, 3; 1899, 2]. Of the simple sugars which have been examined, glucose, fructose, and mannose are freely fermented, l-arabinose not at all, whilst the case of galactose is doubtful. Galactose is, however, fermented by juice prepared from a yeast which has been "trained" to ferment galactose [Harden and Norris, 1910]. As regards both the rate of fermentation and the total amount of carbon dioxide evolved from glucose and fructose by the action of a definite amount of yeast-juice, Buchner and Rapp obtained practically identical numbers. Harden and Young [1909], using juice from top yeast, found that fructose was slightly more rapidly fermented and gave a somewhat larger total than glucose, whilst mannose was initially fermented at almost the same rate as glucose, but gave a decidedly lower total, the following being the average result:--

Sugar. Relative Rates. Relative Totals. Glucose 1 1 Fructose 1·29 1·15 Mannose 1·04 0·67

Among the disaccharides, cane sugar and maltose are freely fermented, and the juice can be shown like living yeast to contain invertase and maltase. The extent of fermentation does not differ materially from that attained with glucose. Lactose is not fermented.

Of the higher sugars raffinose is fermented by juice from bottom yeast, but more slowly than cane sugar or maltose. No experiments seem to have been made with juice from top yeast. [p033]

As regards the fermentation of the higher carbohydrates, very little experimental work has been carried out. Buchner and Rapp found that the fermentation of starch paste was doubtful, but that soluble starch and commercial dextrin were fermented with some freedom. No special study has been made of the diastatic enzymes which bring about the hydrolysis of these substances.

The fermentation of glycogen by yeast-juice is of considerable interest, since it is known that the characteristic reserve carbohydrate of the yeast cell is glycogen [see Harden and Young, 1902, where the literature is cited], and moreover that in living yeast the intracellular fermentation of glycogen proceeds readily, whereas glycogen added to a solution in which yeast is suspended is not affected. Yeast-juice contains a diastatic enzyme which hydrolyses glycogen to a reducing and fermentable sugar, so that in a juice poor in zymase to which glycogen has been added, the amount of sugar is found to increase, the hydrolysis of the glycogen proceeding more quickly than the fermentation of the resulting sugar [Harden and Young, 1904], but the course of this enzymic hydrolysis of glycogen by yeast-juice has not yet been studied. As a rule, it is found both with juices from top and bottom yeast that the evolution of carbon dioxide from glycogen proceeds less rapidly and reaches a lower total than from an equivalent amount of glucose.

Since nearly all samples of yeast contain glycogen, yeast-juice and also zymin usually contain this substance as well as the products of its hydrolysis. These provide a source of sugar which enters into alcoholic fermentation, so that a slow spontaneous production of carbon dioxide and alcohol proceeds when yeast-juice is preserved without any addition of sugar. The extent of this autofermentation varies considerably, as might be expected, with the nature of the yeast employed or the preparation of the material, but is generally confined within the limits of 0·06 to 0·5 gram of carbon dioxide for 25 c.c. of juice.

In juice from bottom yeast it amounts to about 5 to 10 per cent. of the total fermentation obtainable with glucose [Buchner, 1900, 2], whereas in juice from top yeasts, which gives a smaller total fermentation with glucose, it may occasionally equal, or even exceed, the glucose fermentation, and frequently amounts to 30 to 50 per cent. of it. It is therefore generally advisable in studying the effect of yeast-juice on any particular substance to ascertain the extent of autofermentation by means of a parallel experiment.

The maceration extract of Lebedeff (p. 24) is usually, but not invariably [Oppenheimer, 1914, 2], free from glycogen, which is hydrolysed [p034] and fermented during the processes of drying and macerating, and therefore as a rule shows no appreciable autofermentation.

(/e/) /Effect of Concentration of Sugar on the Total Amount of Fermentation./

The kinetics of fermentation by zymase will be considered later on (p. 120), but the effect on the total fermentation of different concentrations of sugar, this substance being present throughout in considerable excess, may be advantageously discussed at this stage. The subject has been investigated by Buchner [Buchner, E. and H., and Hahn, 1903, pp. 150-8; Buchner and Rapp, 1897] using cane sugar, and he has found both for yeast-juice and for dried yeast-juice dissolved in water that (/a/) the total amount of fermentation increases with the concentration of the sugar; (/b/) the initial rate of fermentation decreases with the concentration of the sugar. The following are the results of a typical experiment, 20 c.c. of yeast-juice being employed in presence of toluene at 22°:--

Cane Sugar. │ CO{2} in grams after ─────────┬───────────┼──────────┬──────────┬────────── Weight. │ Per cent. │ 6 hours. │24 hours. │ 96 hours. ─────────┼───────────┼──────────┼──────────┼────────── 2·2 │ 10 │ 0·17 │ 0·50 │ 0·55 3·52 │ 15 │ 0·14 │ 0·53 │ 0·64 5 │ 20 │ 0·13 │ 0·54 │ 0·73 6·66 │ 25 │ 0·13 │ 0·52 │ 0·80 8·56 │ 30 │ 0·12 │ 0·46 │ 0·81 10·76 │ 35 │ 0·12 │ 0·40 │ 0·82 13·33 │ 40 │ 0·11 │ 0·36 │ 0·82 10·76 │ 35 │ 0·12 │ 0·40 │ 0·82 13·33 │ 40 │ 0·11 │ 0·36 │ 0·82

The results as to the total fermentations in experiments of this kind are liable to be vitiated by the circumstance that when a low initial concentration of sugar is employed, the supply of sugar may be so greatly exhausted before the close of the experiment as to cause a marked diminution in the rate of fermentation and hence an unduly low total. Even allowing, however, for any effect of this kind, the foregoing table clearly shows the increase in total fermentation and the decrease in initial rate accompanying the increase of sugar concentration from 10 to 40 per cent. Working with a greater range of concentrations (3·3-53·3 grm. per 100 c.c.) Lebedeff has obtained similar results with maceration extract [1911, 4], but has found that the total amount fermented diminishes after a certain optimum concentration (about 33·3 grm. per 100 c.c.) is reached.

A practical conclusion from these experiments is that a high [p035] concentration of sugar tends to preserve the enzyme in an active state for a longer time. Simultaneously it prevents the development of bacteria and yeast cells.

(/f/) /Effect of Varying Concentration of Yeast-Juice./

This subject, which is of considerable importance with reference to the question of the protoplasmic or enzymic nature of the active agent in yeast-juice, has been examined in some detail by Buchner [Buchner, E. and H., and Hahn, 1903, pp. 158-65] and by Meisenheimer [1903] for juices from bottom yeast, by Harden and Young [1904] for those from top yeast, and by Lebedeff [1911, 4] for maceration extract, the results obtained being in substantial agreement.

Dilution of yeast-juice with sugar solution, so that the concentration of the sugar remains constant, produces a small progressive diminution in the total fermentation, which only becomes marked when more than 2 volumes are added, and this independently of the actual concentration of the sugar. Dilution with water produces a somewhat more decided diminution, which, however, does not exceed 50 per cent. of the total for the addition of 3 volumes of water. The effect on maceration extract is somewhat greater but of the same kind. The autofermentation of juice from top yeast is scarcely affected by dilution with 4 volumes of water.

┌───────────────────────────────────────────────────────────────────┐ │ Nature of Per cent. Volumes of Volumes of Total │ │ Juice of Sugar Sugar Water Added. Fermentation │ │ (1,2,3) Employed Solution in g. │ │ by Weight Added. of CO{2}. │ │ Bottom Yeast──────────────────────────────────────────────────────┤ │ 1 29 0 -- 0·99 │ │ 1 -- 1·13 │ │ 2 -- 0·92 │ │ 4 -- 0·79 │ │ 2 9 0 -- 0·43 │ │ 1 -- 0·60 │ │ 2 -- 0·53 │ │ 4 -- 0·41 │ │ 3 9 -- 0 0·46 │ │ -- 1 0·32 │ │ -- 2 0·33 │ │ -- 3 0·36 │ │ Top Yeast─────────────────────────────────────────────────────────┤ │ 1 0 -- 0 0·29 │ │ (Autofermentation) -- 2 0·29 │ │ -- 3 0·28 │ │ 2 29 0 -- 0·31 │ │ 1 -- 0·34 │ │ 2 -- 0·31 │ │ 4 -- 0·35 │ │ 6 -- 0·28 │ │ 3 7·4 -- 0 0·44 │ │ -- 1 0·35 │ │ -- 2 0·30 │ │ -- 3 0·28 │ └───────────────────────────────────────────────────────────────────┘

[p036]

On the whole, therefore, yeast-juice may be said to be only slightly affected by dilution even with pure water, and the effect of the latter can in no way be regarded as comparable with the poisonous effect which it exerts on living protoplasm, as suggested by Macfadyen, Morris, and Rowland [1900].

(/g/) /The Effect of Antiseptics on the Fermentation of Sugars by Yeast-Juice./

Buchner has paid special attention to the effect of antiseptics on the course of fermentation by yeast-juice [Buchner and Rapp, 1897; 1898, 2, 3; 1899, 1; Buchner and Antoni, 1905, 1; Buchner and Hoffmann, 1907; Buchner, E. and H., and Hahn, 1903, pp. 169-205; see also Albert, 1899, 2; Gromoff and Grigorieff, 1904; Duchaček, 1909] in order (1) to obtain evidence as to the possibility of the active agent in yeast-juice consisting of fragments of protoplasm and not of a soluble enzyme, and (2) also to provide a safe method of avoiding contamination, by the growth of bacteria or yeasts, of the liquids used which were often kept at 25° for several days. The results of these experiments are briefly summarised in the following table, in which the effect of each substance on the total fermentation produced is noted:--

Substance. Effect on Total Fermentation.

Concentrated solution of glycerol Slight diminution " " " sugar " increase Toluene (to saturation or excess) Less than 10 per cent. diminution Chloroform 0·5 per cent. Slight increase " 0·8 per cent. (saturation) No change " Large excess (17 per cent.) 64 per cent. diminution Chloral hydrate 0·7 per cent. Increase up to 27 per cent. " 3·5-5·4 per cent. Completely destroyed Phenol 0·1 per cent. No change " 0·5 " 40 per cent. diminution " 1·2 " Completely destroyed Thymol 1 " Slight diminution " 5 " Marked " Benzoic acid 0·1 " 7 per cent. diminution " 0·25 " 26 " " Salicylic acid 0·1 " 10 " " " 0·27 " 35 " " Formaldehyde 0·12 " 20 " " " 0·24 " 30-60 " " Acetone 6 " 20 " " " 14 " 80 " " Alcohol 6 " 0-20 " " " 14 " 75 " " Sodium fluoride 0·5 " 90 " " " 2 " Almost completely destroyed Ammonium fluoride 0·55 per cent. Completely destroyed Sodium azoimide, NaN{3}, 0·36 per cent. Slight diminution " " 0·71 " Marked " Quinine hydrochloride 1 " Slight increase Ozone 10·4-34·8 milligrams per 20 c.c. Marked diminution Hydrocyanic acid 1·2 per cent. Completely destroyed

[p037]

The general result of these experiments is to show that quantities of antiseptics which are sufficient to inhibit the characteristic action of living cells have only a slight effect on the fermentative activity of yeast-juice. A large excess of the antiseptic in many cases produces a very decided diminution or total destruction of the fermenting power, and accompanying this a precipitation of the constituents of the juice. The decided increase of activity produced by small quantities of chloral hydrate, and to a less marked extent by chloroform and a few other substances, is of considerable interest. It is ascribed by Duchaček to a selective action on the proteoclastic enzyme, but without satisfactory evidence.

Hydrocyanic acid, even in dilute solution, completely suspends the fermenting power of the juice, without, however, producing any permanent change in the fermenting complex, as is shown by the fact that when the hydrocyanic acid is removed by a current of air, the juice regains its fermenting power. In this respect hydrocyanic acid behaves precisely as with many other enzymes and with colloidal platinum [Bredig, 1901]. Sodium arsenite is a pronounced protoplasmic poison, which rapidly destroys the power of growth and reproduction in living cells, and was therefore applied to yeast-juice to differentiate between protoplasmic and enzymic action. It was, however, found that the action of this substance was complicated by some unknown factor and very irregular results were obtained [Buchner, E. and H., and Hahn, 1903, pp. 193 ff.]. These phenomena appear to be of the same order as those produced by the addition of arsenates to yeast-juice [Harden and Young, 1906, 3], and will be discussed along with the latter (p. 77).

PERMANENT PREPARATIONS CONTAINING ACTIVE ZYMASE.

A considerable number of preparations have been obtained in the dry state which retain some proportion of the fermenting power of yeast or yeast-juice.

Starting with yeast-juice, it is possible to arrive at this result either by evaporation or precipitation. When the juice is very rapidly evaporated to a syrup at 20° to 25° and then further dried at 35°, either in the air or in a vacuum and finally exposed over sulphuric acid in a vacuum desiccator, a dry brittle mass is obtained which is soluble in water and retains practically the whole of the fermenting power of the juice. The success of the preparation depends on the nature of the yeast from which the juice is derived, Berlin yeasts V and S yielding much less satisfactory results than Munich yeast. The powder when [p038] thoroughly dry is found to retain its properties almost unimpaired for at least a year, and can be heated to 85° for eight hours without undergoing any serious loss of fermenting power [Buchner and Rapp, 1898, 4; 1901; Buchner, E. and H., and Hahn, 1903, pp. 132-9].

Active powders can also be obtained by precipitating yeast-juice with alcohol, alcohol and ether, or acetone. The preparation is best effected by bringing the juice into 10 volumes of acetone, centrifuging at once and as rapidly as possible, washing, first with acetone and then with ether, and finally drying over sulphuric acid. The white powder thus obtained is not completely soluble in water but is almost entirely dissolved by aqueous glycerol (2·5 to 20 per cent.), forming a solution which has practically the same fermenting power as the original juice. The precipitation can be repeated without any serious loss of fermenting power. Prolonged contact of the precipitate with the supernatant liquid, especially when alcohol or alcohol and ether are used, causes a rapid loss of the characteristic property [Albert and Buchner, 1900, 1, 2; Buchner, E. and H., and Hahn, 1903, pp. 228-246; Buchner and Duchaček, 1909].

Dry preparations capable of fermenting sugar can also be readily obtained from yeast without any preliminary rupture of the cells. Heat alone (yielding a product known as hefanol) or treatment with dehydrating agents may be used for this purpose, and a brief allusion has already been made (p. 21) to the different varieties of permanent yeast (Dauerhefe) obtainable in these ways. The most important of these products are the dried Munich yeast (Lebedeff, see p. 25), and the material known as zymin, which is now made under patent rights for medicinal purposes by Schroder of Munich. The latter has proved of value in the investigation of the production of zymase in the yeast cell [Buchner and Spitta, 1902], and of many other problems concerned with alcoholic fermentation. In order to prepare it 500 grams of finely divided pressed brewer's yeast, containing about 70 per cent. of water, are brought into 3 litres of acetone, stirred for ten minutes, and filtered and drained at the pump. The mass is then well mixed with 1 litre of acetone for two minutes and again filtered and drained. The residue is roughly powdered, well kneaded with 250 c.c. of ether for three minutes, filtered, drained, and spread on filter paper or porous plates. After standing for an hour in the air it is dried at 45° for twenty-four hours. About 150 grams of an almost white powder containing only 5·5 to 6·5 per cent. of water are obtained. This is quite incapable of growth or reproduction but produces a very considerable amount of alcoholic fermentation, far greater indeed than a corresponding [p039] quantity of yeast-juice. Two grams of the powder corresponding to 6 grams of yeast and about 3·5 to 4 c.c. of yeast-juice, are capable of fermenting about 2 grams of sugar, whereas the 4 c.c. of yeast-juice would on the average only ferment from one-quarter to one-sixth of this amount of sugar. The rate produced by this amount of zymin is about one-eighth of that given by the corresponding amount of living yeast [Albert, 1900; Albert, Buchner, and Rapp, 1902]. The proteoclastic ferment is still present in zymin, which undergoes autolysis in presence of water in a similar manner to yeast-juice [Albert, 1901, 2].

As already mentioned an active juice can be prepared by grinding acetone-yeast with water, sand, and kieselguhr, and this process presents the advantage that samples of yeast-juice of approximately constant composition can be prepared at intervals from successive portions of a uniform supply of acetone-yeast.

Preparations of acetone-yeast, made from yeast freed from glycogen by exposure in a thin layer to the air for three or four hours at 35° to 45°, or eight hours at the ordinary temperature [Buchner and Mitscherlich, 1904], show practically no autofermentation and may be used analytically for the estimation of fermentable sugars.

All the foregoing preparations exhibit the same general properties as yeast-juice, as regards their behaviour towards the various sugars, antiseptics, etc.

When zymin is mixed with sugar solution without being previously ground, it exhibits a peculiarity which is of some practical interest. The time which elapses before the normal rate of fermentation is attained and the total fermentation obtainable vary with the amount of sugar solution added, the time increasing and the total diminishing as the quantity of this increases. This phenomenon appears to have been noticed by Trommsdorff [1902], and a single experiment of Buchner shows the influence of the same conditions [Buchner, E. and H., and Hahn, 1903, p. 265, Nos. 700-1]. Harden and Young have found that when 2 grams of zymin are mixed with varying quantities of 10 per cent. sugar solution the following results are obtained:--

───────────┬────────────────────────────────────────── Volumes of │ Total Gas Evolved in Sugar │ 1 2 3 4 22·5 hours. Solution │ ───────────┼────────────────────────────────────────── 5 c.c. │ 15·7 31·6 44·8 56·5 233·3 10 " │ 2·2 10·5 23 31·8 202·3 20 " │ 0·9 2·4 13·6 23·7 125·5 40 " │ 1·4 1·7 2·3 2·9 56·3 ───────────┴──────────────────────────────────────────

[p040]

This behaviour appears to be due to the removal of soluble matter essential for fermentation from the cell, which is discussed later on. It follows that when zymin is being tested for fermenting power, a uniform method should be adopted, and all comparative tests should be made with the same volumes of added sugar solution. Ground zymin appears to begin to ferment somewhat more slowly than unground (2 grm. to 12·4 c.c. of sugar solution in each case), but eventually produces the same total volume of gas [Buchner and Antoni, 1905, 1]. [p041]