Fusel Oil.
All forms of alcohol prepared by fermentation contain a fraction of high boiling-point, which is termed fusel oil, and amounts to about [p086] 0·1 to 0·7 per cent. of the crude spirit obtained by distillation. This material is not an individual substance, but consists of a mixture of very varied compounds, all occurring in small amount relatively to the ethyl alcohol from which they have been separated. The chief constituents of the mixture are the two amyl alcohols, isoamyl alcohol,
(CH3)2·CH·CH2·CH2·OH,
and d-amyl alcohol,
CH3·CH(C2H5)·CH2·OH,
which contains an asymmetric carbon atom and is optically active. In addition to these, much smaller amounts of propyl alcohol and isobutyl alcohol are present, together with traces of fatty acids, aldehydes, and other substances.
The origin of these purely non-nitrogenous compounds was usually sought in the sugar of the liquid fermented, from which they were thought to be formed by the yeast itself or by the agency of bacteria [Emmerling, [1904], [1905]; Pringsheim, [1905], [1907], [1908], [1909]], whilst others traced their formation to the direct reduction of fatty acids. Felix Ehrlich has, however, conclusively shown in a series of masterly researches that the alcohols, and probably also the aldehydes, contained in fusel oil are in reality derived from the amino-acids which are formed by the hydrolysis of the proteins.
The close relationship between the composition of leucine,
(CH3)2·CH·CH2·CH(NH2)·COOH,
and isoamyl alcohol,
(CH3)2·CH·CH2·CH2·OH,
had previously led to the surmise that a genetic relation might exist between these substances, but the idea had not been experimentally confirmed. In 1903 Ehrlich discovered [[1903]; [1904, 1], [2]; [1907, 2]; [1908]; Ehrlich and Wendel, [1908, 2]] that proteins also yield on hydrolysis an isomeride of leucine known as isoleucine, which has the constitution
CH3·CH(C2H5)·CH(NH2)·COOH,
and therefore stands to d-amyl alcohol,
CH3·CH(C2H5)·CH2·OH,
in precisely the same relation as leucine to isoamyl alcohol. This suggestive fact at once directed his attention to the problem of the origin of the amyl alcohols in alcoholic fermentation. Using a pure culture of yeast, and thus excluding the participation of bacteria in the change, he found that leucine readily yielded isoamyl alcohol, and isoleucine d-amyl alcohol when these amino-acids were added in the pure state [p087] to a solution of sugar and treated with a considerable proportion of yeast [[1905]; [1906, 2], [3]; [1907, 1], [3]]. The chemical reactions involved are simple ones and are represented by the following equations:—
(1)
(CH3)2·CH·CH2·CH(NH2)·COOH
Leucine
+ H2O =
(CH3)2CH·CH2CH2·OH
Isoamyl alcohol
+ CO2 + NH3
(2)
CH3·CH(C2H5)·CH(NH2)·COOH
Isoleucine
+ H2O =
CH3·CH(C2H5)·CH2·OH
d-Amyl alcohol
+ CO2 + NH3
The experiments by which these important changes were demonstrated were of a very simple and convincing character [Ehrlich, [1907, 1]]. Two hundred grams of sugar and 3 to 10 grams of the nitrogenous substance to be examined were dissolved in 2 to 2·5 litres of tap water in a 3 to 4 litre flask, the liquid was sterilised by being boiled for several hours, and after cooling 40 to 60 grams of fresh yeast were added and the flask allowed to stand at room temperature until the whole of the sugar had been decomposed by fermentation. In the earlier experiments the amyl alcohols were isolated and identified by conversion into the corresponding valerianic acids, but as a rule the fusel oil as a whole was quantitatively estimated in the filtrate by the Röse-Herzfeld method [Lunge, [1905], p. 571].
The following are typical results. (1) An experiment carried out as above without any addition of leucine gave 97·32 grams of alcohol containing 0·40 per cent. of fusel oil. (2) When 6 grams of synthetic, optically inactive leucine were added, 97·26 grams of alcohol were obtained, containing 2·11 per cent. of fusel oil, which was also optically inactive; 2·5 grams of leucine were recovered, so that 87 per cent. of the theoretical yield of isoamyl alcohol was obtained from the 3·5 grams of leucine decomposed. (3) In the presence of 2·5 grams of d-isoleucine (prepared from molasses residues), 200 grams of sugar gave 93·99 grams of alcohol, containing 1·44 per cent. of fusel oil, which was lævo-rotatory. This corresponds with 80 per cent. of the theoretical yield of d-amyl alcohol from the isoleucine added.
This change, which Ehrlich has termed the alcoholic fermentation of the amino-acids, although brought about by living yeast, does not appear to occur at all when zymin [Ehrlich, [1906, 4]; Pringsheim, [1906]] or yeast-juice [Buchner and Meisenheimer, [1906]] is substituted for the intact organism, nor is it effected even by living yeast in the absence of a fermentable sugar [Ehrlich, [1907, 1]]. The reaction appears indeed to be intimately connected with the nitrogenous metabolism of the cell, and the whole of the ammonia produced is at once assimilated and does not appear in the fermented liquid. Other amino-acids [p088] undergo a corresponding change, and the reaction appears to be a general one. Thus tyrosine, OH·C6H4·CH2·CH(NH2)·COOH, yields p-hydroxyphenylethyl alcohol, or tyrosol [Ehrlich, [1911, 1]; Ehrlich and Pistschimucka, [1912, 2]], OH·C6H4·CH2·CH2OH, a substance of intensely bitter taste, which was first prepared in this way and is probably one of the most important factors in determining the flavour of beers, etc. Phenylalanine, C6H5·CH2·CH(NH2)·COOH, in a similar way yields phenylethyl alcohol, C6H5·CH2·CH2OH, one of the constituents of oil of roses, whilst tryptophane,
| C6H4 | ||||
| ╱ | │ | |||
| HN | │ | |||
| ╲ | │ | |||
| H | C═══ | C·CH2·CH(NH2)·COOH |
yields tryptophol,
| 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].