Zymin (p. [38]) yields precisely similar results to yeast-juice, but in this case the rate of fermentation is not so largely increased. This has the effect that the extra amount of carbon dioxide cannot be quite so accurately estimated for zymin, because a slight error in the determination of the final rate of fermentation has a greater influence on the result. The equivalence between the extra amount of carbon dioxide evolved and the phosphate added is, however, unmistakable, as is shown by the following results of an experiment with zymin, in which 6 grams of zymin (Schroder) + 3 grams of fructose (Schering) + 25 c.c. of water were incubated at 25° in presence of toluene until a steady rate had been attained. Five c.c. of a solution of sodium phosphate equivalent to 32·2 c.c. carbon dioxide at N.T.P. were then added.
| Maximum rate attained, c.c. per five minutes | 14·1 |
| Final rate of fermentation | 6·2 |
| Total evolved by fermentation in eighty minutes after addition of phosphate | 131 |
| Correction for evolution in absence of phosphate in eighty minutes | 99·2 |
| Extra carbon dioxide at 16° and 767·1 mm | 31·8 |
| Extra carbon dioxide at N.T.P | 29·8 |
Considering the small proportional rise in rate and the long period of accelerated fermentation, the agreement between the volume observed, 29·8 c.c., and that calculated from the phosphate, 32·2, is quite satisfactory [Harden and Young, [1910, 1].] Precisely the same relations hold for maceration extract, but in this case it must be remembered that a large amount of free phosphate is present in the extract, as much as 0·3129 grm. Mg2P2O7 being obtained from 20 c.c. in one preparation, so that the original extract had the concentration of a 0·14 molar solution of sodium phosphate. It is in fact not improbable that the delay in the onset of fermentation sometimes observed with maceration extract (see Lebedeff, 1912, 2; Neuberg and Rosenthal, 1913) may be due to the presence of phosphate in so great an excess of the amount which can be rapidly esterified by the enzymes that the rate of fermentation is at first greatly lowered (see p. [71]). When this phosphate is removed by incubation with glucose or fructose, the subsequent addition of phosphate produces the characteristic action and the extra carbon dioxide evolved is, as with other yeast preparations, equivalent to the phosphate added. An actual estimation carried out in this way gave 35 c.c. of CO2 for an addition of phosphate equivalent to 32·9 c.c. [Harden and Young, [1912]]. [p047]
Within the limits imposed by the experimental conditions, then, the fact is well established that the addition of a soluble phosphate to a fermenting mixture of a hexose with yeast-juice, maceration extract, dried yeast, or zymin causes the production of an equivalent amount of carbon dioxide and alcohol.
This fact indicates that a definite chemical reaction occurs in which sugar and phosphate are concerned, and this conclusion is confirmed when the fate of the added phosphate is investigated. If an experiment, such as one of those described above, be interrupted as soon as the rate of fermentation has again become normal, and the liquid be boiled and filtered, it is found that nearly the whole of the phosphorus present passes into the filtrate, but that only a small proportion of this exists as mineral phosphate, whilst the remainder, including that added in the form of a soluble phosphate, is no longer precipitable by magnesium citrate mixture [Harden and Young, [1905, 2]].
A similar observation was made at a later date by Iwanoff [[1907]], who had previously observed [[1905]] that living yeast, like many other vegetable organisms, converted mineral phosphates into organic derivatives. Iwanoff employed zymin and hefanol (p. [38]) instead of yeast-juice, and found that phosphates were thereby rendered non-precipitable by uranium acetate solution, but did not observe the accelerated fermentation caused by their addition.
The foregoing conclusions have been strikingly confirmed by experiments with maceration extract carried out by Euler and Johansson [[1913]], in which both the carbon dioxide evolved and the phosphate rendered non-precipitable by magnesia were determined at intervals. When dried yeast is employed as the fermenting agent, the amount of phosphate esterified in the earlier stages is greater than would be expected, but ultimately becomes exactly equivalent to the carbon dioxide evolved.
Nature of the Phospho-organic Compound formed by Yeast-Juice and Zymin from the Hexoses and Phosphate.
The formation and properties of the compound produced from phosphates in the manner just described have been investigated by Harden and Young [[1905, 2]; [1908, 1]; [1909]; [1911, 2]], Young [[1909]; [1911]], Iwanoff [[1907]; [1909, 1]], Lebedeff [[1909]; [1910]; [1911, 5], [6]; [1912, 3]; [1913, 1]]; and Euler [[1912, 1]; Euler and Fodor, [1911]; Euler and Kullberg, [1911, 3]; Euler and Ohlsén, [1911]; [1912]; Euler and Johansson, [1912, 4]; Euler and Bäckström, [1912]], but its exact constitution cannot as yet be regarded as definitely known. [p048]
Phosphates undergo this characteristic change when the sugar undergoing fermentation is glucose, mannose, or fructose, and it may be said at once that no distinction can be established between the products formed from these various hexoses; they all appear to be identical. The compound produced is, as already mentioned, not precipitated by ammoniacal magnesium citrate mixture, nor by uranium acetate solution. It can, however, be precipitated by copper acetate (Iwanoff) and by lead acetate (Young). The preparation of the pure lead salt from the liquid obtained by fermenting a sugar with yeast-juice or zymin in presence of phosphate is commenced by boiling and filtering the liquid. Magnesium nitrate solution and a small quantity of caustic soda solution are then added to precipitate any free phosphate, and the liquid well stirred and allowed to stand over night. To the neutralised filtrate lead acetate is then added together with sufficient caustic soda solution to maintain the reaction neutral to litmus, until no further precipitate is formed. The liquid is then filtered or, better, centrifugalised, and the precipitate repeatedly washed with water until a portion of the clear filtrate gives no reduction when boiled with Fehling's solution. It is essential that this washing should be thorough as evidence has recently been obtained of the formation under certain conditions of a hexosephosphate, the lead salt of which is not so sparingly soluble as that of the hexosediphosphate [Harden and Robison, [1914]]. The lead precipitate is then suspended in water, decomposed by a current of sulphuretted hydrogen, the clear filtrate freed from sulphuretted hydrogen by a current of air, and finally neutralised with caustic soda. The removal of phosphate and conversion into lead salt are repeated twice, and the resulting lead salt is then found to be free from nitrogen and to have a composition represented by the formula C6H10O4(PO4Pb)2. Lebedeff carries out the preparation in a somewhat different manner. The fermentation is effected by means of air-dried yeast (150 grams to 1 litre of water, 210 grams cane-sugar and 105 grams of a mixture of 2 parts Na2HPO4 and 1 part NaH2PO4) and the liquid (about 700 c.c.) after boiling and filtering, is treated with an equal volume of acetone. About 300 c.c. of a thick liquid is precipitated and this is redissolved in water and precipitated by an equal volume of acetone two or three times. The final liquid is then precipitated with warm lead acetate solution and filtered and washed with dilute lead acetate solution until the filtrate is clear and no longer reduces Fehling's solution after removal of the lead [[1910]]. Euler and Fodor [[1911]] on the other hand precipitate the free phosphate with magnesia mixture and then add acetone, dissolve the syrup thus precipitated in water and add copper [p049] acetate solution. A blue copper salt is precipitated which is thoroughly washed with water and used for the preparation of solutions of the acid. A solution of the free acid can readily be prepared by the action of sulphuretted hydrogen on the lead salt suspended in water. It forms a strongly acid liquid, which requires exactly two equivalents of base for each atom of phosphorus present to render it neutral to phenolphthalein. It decomposes when evaporated, leaving a charred mass containing free phosphoric acid. The acid is slightly optically active, and has [aD] = + 3·4°. A number of amorphous salts have been prepared by precipitation from a solution of the sodium salt, and of these the silver, barium, and calcium salts have been analysed with results agreeing with the general formula C6H10O4(PO4R′2)2. The magnesium, calcium, barium, and manganese salts, which are only sparingly soluble, are all precipitated when their solutions are boiled but re-dissolve on cooling, and this property can be utilised for their purification. The alkali salts have only been obtained as viscid residues.