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. | CO2 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 |
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 Juice. | Per cent. of Sugar Employed by Weight. | Volumes of Sugar Solution Added. | Volumes of Water Added. | Total Fermentation in g. of CO2. | |
|---|---|---|---|---|---|
| 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 (Auto- ferment- ation) | — | 0 | 0·29 |
| — | 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 | |||
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 | |
| Concentrated solution of sugar | Slight 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, NaN3, | 0·36 per cent. | Slight diminution |
| 0·71 " | Marked " | |
| Quinine hydrochloride | 1 " | Slight increase |
| Ozone | 10·4–34·8 mgs. per 20 c.c. | Marked diminution |
| Hydrocyanic acid | 1·2 per cent. | Completely destroyed |
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]).