| C6H4 | ||||
| ╱ | │ | |||
| HN | │ | |||
| ╲ | │ | |||
| H | C═══ | C·CH2·CH2OH |
which was also first prepared in this way [Ehrlich, [1912]] and has a very faintly bitter, somewhat biting taste.
The extent to which the amino-acids of a medium in which yeast is producing fermentation are decomposed in this sense depends on the amount of the available nitrogen and on the form in which it is present. Thus the addition of ammonium carbonate to a mixture of yeast and sugar was found to lower the production of fusel oil from 0·7 to 0·33 per cent. of the alcohol produced. The addition of leucine alone raised the percentage from 0·7 to 2·78, but the addition of both leucine and ammonium carbonate resulted in the formation of only 0·78 per cent. of fusel oil, The production of fusel oil therefore and the character of the constituents of the fusel oil alike depend on the composition of the medium in which fermentation occurs. This affords a ready explanation of the fact that molasses, which contains almost equal amounts of leucine and isoleucine, yields a fusel oil also containing approximately equal amounts of isoamyl alcohol and d-amyl alcohol [Marckwald, [1902]], whilst corn and potatoes, in which leucine preponderates over isoleucine, yield fusel oils containing a relatively large amount of the inactive alcohol. The subject is, in fact, one of great interest to the technologist, for as Ehrlich points out "the great variety of the bouquets of wine and aromas of brandy, cognac, arrak, rum, etc., may be very simply referred to the manifold variety of the proteins of the raw materials (grapes, corn, rice, sugar cane, etc.) from which they are derived".
Yeast can also form fusel oil at the expense of its own protein, but this only occurs to any considerable extent when the external [p089] supply of nitrogen is insufficient. Under these circumstances the amino-acids formed by autolysis may be decomposed and their nitrogen employed over again for the construction of the protein of the cell.
The yield is also influenced by the condition of the yeast employed with regard to nitrogen, a yeast poor in nitrogen being more efficacious in decomposing amino-acids than one which is already well supplied with nitrogenous materials. The nature of the carbonaceous nutriment and finally the species of yeast are also of great importance [see Ehrlich, [1911, 2]; Ehrlich and Jacobsen, [1911]].
A very important characteristic of the action of yeast on the amino-acids is that the two stereo-isomerides of these optically active compounds are fermented at different rates. When inactive, racemic leucine is treated with yeast and sugar, the naturally occurring component, the l-leucine, is more rapidly attacked, so that if the experiment be interrupted at the proper moment the other component, the d-leucine, alone is present and may be isolated in the pure state. In an actual experiment 3·8 grams of this component were obtained in the pure state from 10 grams of dl-leucine [Ehrlich, [1906, 1]], so that the whole of the l-leucine (5 grams) had been decomposed but only 1·2 grams of the d-leucine. This mode of action has been found to be characteristic of the alcoholic fermentation of the amino-acids by yeast. In all the instances so far observed, both components of the inactive amino-acid are attacked, but usually the naturally occurring isomeride is the more rapidly decomposed, although in the case of β-aminobutyric acid both components disappear at the same rate [Ehrlich and Wendel, [1908, 1]]. This reaction therefore must be classed along with the action of moulds on hydroxy-acids [McKenzie and Harden, [1903]], and the action of lipase on inactive esters [Dakin, [1903], [1905]], in which both isomerides are attacked but at unequal rates, and differs sharply from the action of yeast itself on sugars [Fischer and Thierfelder, [1894]], and of emulsin, maltase, etc., which only act on one isomeride and leave the other entirely untouched [see Bayliss, [1914], pp. 55, 77, 117].
Succinic Acid.
The origin of the succinic acid formed in fermentation has also been traced by Ehrlich [[1909]] to the alcoholic fermentation of the amino-acids. It was shown by Buchner and by Kunz [[1906]] that succinic acid like fusel oil is not formed during fermentation by yeast-juice or zymin, and, in the light of Ehrlich's work on fusel oil, several [p090] modes of formation appeared possible for this substance [Ehrlich, [1906, 3]]. The dibasic amino-acids might, for example, undergo simple reduction, the NH2 group being removed as ammonia and replaced by hydrogen. Aspartic acid would thus pass into succinic acid:—
COOH·CH2·CH(NH2)·COOH + 2 H = COOH·CH2·CH2·COOH + NH3.
This change can be effected in the laboratory only by heating with hydriodic acid. Biologically it has been observed [E. and H. Salkowski, [1879]] when aspartic acid is submitted to the action of putrefactive bacteria, and almost quantitatively when Bacillus coli communis is cultivated in a mixture of aspartic acid and glucose [Harden, [1901]]. In this case a well-defined source of hydrogen exists in the glucose, which when acted on by this bacillus yields a large volume of gaseous hydrogen, which is not evolved in the presence of aspartic acid. Some such source is also available in the case of yeast, although it cannot be chemically defined, for this organism is known to produce many reducing actions, which are usually ascribed to the presence of reducing ferments or reductases in the cell.