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.
Fig. 1.
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.