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.
Effect of the Addition of Phosphate to a Fermenting Mixture of Yeast-Juice and Sugar.
When a suitable quantity[2] of a soluble phosphate is added to a fermenting mixture of glucose, fructose, or mannose with yeast-juice, the rate of fermentation rapidly rises, sometimes increasing as much as twenty-fold, continues at this high value for a certain period and then falls again to a value approximately equal to, but generally [p043] somewhat higher than, that which it originally had. Careful experiments have shown that during this period of enhanced fermentation the amounts of carbon dioxide and alcohol produced exceed those which would have been formed in the absence of added phosphate by a quantity exactly equivalent to the phosphate added in the ratio CO2 or C2H6O:R′2HPO4 [Harden and Young, [1906, 1]].
[2] The effect of an excess of phosphate is discussed later on, p. [71].
This result is of fundamental importance, and the evidence on which it rests deserves some consideration. Quantitative experiments on this subject require certain preliminary precautions. The acid phosphates are too acid to permit of any extended fermentation and the phosphates of the formula R′2HPO4 absorb a considerable volume of carbon dioxide with production of a bicarbonate, according to the reaction:—
R2HPO4 + H2CO3 ⇌ RHCO3 + RH2PO4.