(2) When yeast extract has been heated for 30 minutes to 40° it effects the esterification of phosphoric acid at a much greater rate than the unheated extract (2–10 times). Heating at 50° for 30 minutes however completely inactivates the extract. The cause of the activation is as yet unknown. The temperature coefficient for the unheated extract (17·5°–30°) is 1·4–1·5 for 10° rise of temperature [Euler and Ohlsén, [1911]].
(3) Yeasts which in the dried state all produce rapid esterification of phosphoric acid, yield extracts of very unequal powers in this respect [Euler, [1912, 1]].
CHAPTER IV. THE CO-ENZYME OF YEAST-JUICE.
In the previous chapter reference was made to the fact that the addition of boiled yeast-juice greatly increases the amounts of carbon dioxide and alcohol formed from sugar by the action of a given volume of yeast-juice.
Fig. 5.
When the boiled juice is dialysed the substance or substances to which this effect is due pass into the dialysate, the residue being quite inactive. In order to ascertain the effect on the process of alcoholic fermentation of the complete removal of these unknown substances from yeast-juice itself, dialysis experiments were instituted with fresh yeast-juice, capable of bringing about an active production of carbon dioxide and alcohol from sugar. It was already known from the experiments of Buchner and Rapp [[1898, 1]] that dialysis in parchment paper for seventeen hours at 0° against water or physiological salt solution only produced a diminution of about 20 per cent. in the total amount of fermentation obtainable, and in view of the less permanent character of the juice from top yeasts a more rapid method of dialysis was sought. This was found in the process of filtration under pressure through a film of gelatin, supported in the pores of a Chamberland filter candle, which had been introduced by Martin [[1896]].
In this way it was found possible to divide the juice into a residue and a filtrate, each of which was itself incapable of setting up the alcoholic fermentation of glucose, whereas, when they were reunited, the mixture produced almost as active a fermentation as the original juice [Harden and Young, [1905, 1]; [1906, 2]].
The apparatus employed for this purpose consists of a brass tube provided with a flange in which the gelatinised candle is held by a compressed india-rubber ring, and is shown in section in Fig. 5. Two such apparatus are used, each capable of holding about 70 c.c. of the liquid to be filtered. The tubes, after being filled with the yeast-juice, are connected by means of a screw joint with a cylinder of compressed air and the filtration carried out under a pressure of 50 atmospheres, [p060] the arrangement employed being shown in Fig. 6. In the earlier experiments 25 to 50 c.c. of yeast-juice were placed in each tube and the filtration continued until no more liquid passed through. The residue was then washed several times in situ by adding water and forcing it through the candle. The time occupied in this process varied from six to twelve hours with different preparations of yeast-juice. The candle was then removed from the brass casing and the thick, brown-coloured residue scraped off, dissolved in water, and at once examined. It was subsequently found to be possible to dry this residue in vacuo over sulphuric acid without seriously altering the fermenting power, and this led to a slight modification of the method, which is now conducted as follows. Two quantities of 50 c.c. each of yeast-juice are filtered, without washing, and the residues spread on watch-glasses and dried in vacuo. Two fresh quantities of 50 c.c. are then filtered through the same candles and the residues also dried. The 200 c.c. of juice treated in this way give a dry residue of 17 to 24 grams. The residue is then dissolved in 100 c.c. of water and filtered in quantities of 50 c.c. through two fresh gelatinised candles and the residue again dried. A considerable diminution in weight occurs, partly owing to incomplete removal from the candle and brass casing, and the final residue only amounts to about 9 to 12 grams. Occasionally it is necessary to repeat the processes of dissolving in water, filtering, and drying, but a considerable loss both of material and fermenting power attends each such operation.