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 Sugar Solution | Total Gas Evolved in | ||||
|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 22·5 hours. | |
| 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 |
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]].
CHAPTER III. THE FUNCTION OF PHOSPHATES IN ALCOHOLIC FERMENTATION.
In the course of some preliminary experiments (commenced by the late Allan Macfadyen, but subsequently abandoned) on the production of anti-ferments by the injection of yeast-juice into animals, the serum of the treated animals was tested for the presence of such antibodies both for the alcoholic and proteoclastic enzymes of yeast-juice, and it was then observed that the serum of normal and of treated animals alike greatly diminished the autolysis of yeast-juice.
As the explanation of the comparatively rapid disappearance of the fermenting power from yeast-juice had been sought, as already mentioned (p. [20]), in the hydrolytic action of the tryptic enzyme which always accompanies zymase, the experiment was made of carrying out the fermentation in the presence of serum, with the result that about 60 to 80 per cent. more sugar was fermented than in the absence of the serum [Harden, [1903]].
This fact was the starting-point of a series of attempts to obtain a similar effect by different means, in the course of which a boiled and filtered solution of autolysed yeast-juice was used, in the hope that the products formed by the action of the tryptic enzyme on the proteins of the juice would, in accordance with the general rule, prove to be an effective inhibitant of that enzyme. This solution was, in fact, found to produce a very marked increase in the total fermentation effected by yeast-juice, the addition of a volume of boiled juice equal to that of the yeast-juice doubling the amount of carbon dioxide evolved [Harden and Young, [1905, 1]]. This effect was found to be common to the filtrates from boiled fresh yeast-juice and from boiled autolysed yeast-juice, and was ultimately traced in the main, not to the antitryptic effect which had been surmised, but to two independent factors, either of which was capable in some degree of bringing about the observed result.
Boiled yeast-juice was indeed found to possess a decided anti-autolytic effect, as determined by a comparison of the amounts of nitrogen rendered non-precipitable by tannic acid in yeast-juice alone [p042] and in a mixture of yeast-juice and boiled juice on preservation [Harden, [1905]]. The anti-autolytic effect, however, appeared to vary independently of the effect on the fermentation, and the conclusion was drawn, as stated above, that the increase in the alcoholic fermentation was not directly dependent on the decrease in the action of the proteoclastic enzyme but was due to some independent cause. The property possessed by boiled yeast-juice of diminishing the autolysis of yeast-juice has now been carefully examined by Buchner and Haehn [[1910, 2]] and ascribed by them to a soluble antiprotease (p. [65]).
The two factors to which the increase in fermentation produced by the addition of boiled juice were ultimately traced were (1) the presence of phosphates in the liquid, and (2) the existence in boiled fresh yeast-juice of a co-ferment or co-enzyme, the presence of which is indispensable for fermentation [Harden and Young, [1905, 1], [2]].
The former of these factors will be here discussed and the co-enzyme will form the subject of the following chapter.
The general fact that sodium phosphate increases the total fermentation produced by a given volume of yeast juice was observed on several occasions by Wroblewski [[1901]] and also by Buchner [Buchner, E. and H., and Hahn, [1903], pp. 141–2], who ascribed the action of this salt to its alkalinity, comparing it in this respect with potassium carbonate and remarking that the increase in both cases took place chiefly in the first twenty hours of fermentation. The increased amount of fermentation following the addition of boiled yeast-juice was also noted by Buchner and Rapp [[1899, 2], No. 265, p. 2093] in a single experiment.
Observations made at intervals of a few minutes instead of twenty hours have, however, revealed the fact that phosphates play a part of fundamental importance in alcoholic fermentation and that their presence is absolutely essential for the production of the phenomenon.