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.