Alcoholic fermentation / by Arthur Harden.

  • Arthur Harden
Date:
1932
Catalogue details

Licence: In copyright

Credit: Alcoholic fermentation / by Arthur Harden. Source: Wellcome Collection.

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    danger of hydrolysis of the hexosediphosphate during this process [Robison and Morgan, 1930] leading to the formation of the hexose- monophosphate of Neuberg (p. 62). Robison and Morgan [1930] therefore recommend the precipitation of the protein by trichloroacetic acid. In a typical preparation according to this method, which is recommended as the best at present available, 30 g. of glucose or fructose are added to 300 c.c. of yeast-juice or maceration extract, or to 50 g. of dried yeast or zymin mixed with 180 c.c. of water and brought to 28° to 30°. Dried yeast or zymin should be used for the preparation of hexosediphos- phate, dried yeast for trehalosemonophosphate, and yeast-juice or maceration extract for hexosemonophosphate. The dried yeast or zymin is left in contact with the sugar solution for one hour before the addition of phosphate. To the fermenting mixture at 28° to 30° about 350 c.c. of a 20 per cent, solution of Na2HP04 . i2H20(o‘5b M) con- taining also 20 per cent, of the sugar, or a corresponding solution of K2HP04 which is more easily soluble (35 g. in 200 c.c. solution with 60 g. sugar), is either added in five or six equal portions, the rate of fermentation being allowed to fall to the normal before each addition, or is run in continuously at a controlled rate. Potassium phosphate, however, gives a smaller proportion of hexosemonophosphate. It is convenient to collect the gas evolved over brine in a large nitrometer (500 c.c. capacity) as the rate of evolution of C02 affords a good indication of the progress of the reaction. The fermentation can also conveniently be carried out at room temperature. At the conclusion of the fermentation sufficient 25 per cent, solution of trichloroacetic acid is added to bring the trichloroacetic acid content to 4 per cent., and the mixture is then well shaken, allowed to stand for two hours, preferably at 0°, and filtered after the addition of a few drops of capryl alcohol. In the protein-free filtrate the barium salts are precipitated by adding an amount of barium acetate equal in weight to the crystalline sodium phosphate added, and then bringing the reaction to _pH 8*4 by the addition of baryta, the bulk of which is added in hot saturated solution, and the final adjustment made with cold saturated solution. The precipitate is filtered off, washed with small quantities of water, pressed out, washed with absolute alcohol and dried in vacuo. This precipitate contains the bulk of the hexose- diphosphate along with barium phosphate resulting from any excess of inorganic phosphate, whilst the hexosemonophosphate and tre- halosephosphate are present in the filtrate. The separation is not a sharp one because (a) the hexosediphosphate is slightly soluble in
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