[advances in applied microbiology] advances in applied microbiology volume 5 volume 5 || fusel oil

Fusel Oil

A. DINSMOOR WEBB AND JOHN L. INCRAHAM Department of Viticulture and Enology and the Department of Bacteriology,

University of California, Davis, California

I. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Characteristics of Fusel Oil Components ....................

111. Analytical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Distillation Methods ................................. B. Chromatographic Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Chemical Methods .................................. D. Other Analytical Methods ............................

IV. Results of Fusel Oil Analysis ............................. V. Biosynthesis of Fusel Oil Components ......................

A. Fusel Oil Formation from Amino Acids . . . . . . . . . . . . . . . . . B. Fusel Oil Formation from Glucose .................... C. n-Propyl and n-Butyl Alcohols ........................ D. Factors Affecting Fusel Oil Formation . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

317 318 322 323 325 329 330 331 338 338 341 343 346 350

All yeast-fermented aqueous alcoholic mixtures contain small amounts of materials which will separate into a second, oily layer upon removal of most of the ethanol. This oily phase is usually obtained by adding water to a small side stream drawn from a point below the ethanol draw point of the rectification column of a continuous distillation unit. It may also be obtained from residues of simple distillations and from wines or beers by extractions with solvents which are immiscible with the aqueous phase. The separated mixture is designated fusel oil, from the old German word, fousel, meaning bad spirit.

1. History

Scheele (1785) described the separation of a second phase from a grain spirit of low alcohol content during severely cold weather which he characterized as being “nauseous-smelling” and white in color. This is perhaps the earliest recorded occurrence of fusel oil, although the term “fusel” was not used in his description. He reported that the addition of this material to a fine brandy gave it the unpleasant smell of ordinary grain spirit.

J. B. Dumas (1834) analyzed a sample of fousel obtained from 317

318 A. DINSMOOR WEBB AXD JOHN L. INGRAHAM

the distillate of fermented potatoes. After repeated water washings and fractional distillations, he obtained a sample boiling from 130" to 132°C. which had the empirical formula, C5H120. But it was Cahours (1839) who recognized that Dumas' compound was "hydrate of amylene" or amyl alcohol, a heavier member of the homologous series which includes methanol and ethanol. Dumas and Stas ( 1840) confirmed Cahours' identification.

Pasteur (1855) credits J. B. Biot with the discovery that the fusel oil boiling from 130" to 132°C. is optically active. Since the optical activity of fusel oils from different sources varied, Pasteur con- cluded that at least two substances were present, one optically active and the other inactive, i.e., fusel oils of differing activities merely reflect the difference in proportions of the two substances. The determination of the structure of the amyl alcohols present in fusel oils resulted from the researches of many workers. Pedler ( 1868), Erlenmeyer and Hell (1871), and Kramer and Pinner (1870) all contributed to characterizing the fusel oil alcohols as isoamyl ( 3-methyl-l-butanol) and active amyl ( 2-methyl-l-butanol ) . Le Be1 (1873, 1876) developed methods for obtaining relatively pure active amyl fractions from the crude fusel oils. He also studied methods for synthesis of related compounds from active amyl alcohol.

Somewhat earlier Wurtz (1852) became interested in the alcoholic substances present in fusel oils with boiling points between those of ethyl alcohol and the amyl alcohols. He isolated isobutyl alcohol and prepared a number of derivatives from it. One year later, Chancel (1853) isolated a fourth alcohol, normal propyl, from the fusel oil mixture thus completing the list of primary alcohols which are known today as the principal components of the mixture.

II. Characteristics of Fuse1 Oil Components

Dumas (1834) observed that repeated water washings and fractional distillation yielded a fusel oil boiling from 130" to 132°C. with the empirical formula corresponding to amyl alcohol. Pasteur (1855) separated active amyl alcohol from isoamyl alcohol by fractional crystallization of the barium salts of their acid esters of sulfuric acid and reported the boiling point of active amyl alcohol to be between 127" and 128°C. at atmospheric pressure. The boiling

FUSEL OIL 319

point of the inactive isoamyl alcohol was reported to be 129°C. under the same conditions. Marckwald and McKenzie (1901) reported active amyl alcohol to boil at 128°C. More recent work by Terry (1960) establishes the boiling point of active amyl alcohol as 128.5"C. and that of isoamyl alcohol as 132.0"C. at atmospheric pressure.

Of the other two principal alcohols of fusel oil, n-propyl is listed by Timmermans (1950) as boiling at 97.2"C., while isobutyl alcohol boils at 108.0"C.

As Schiipphaus (1892) pointed out, separation of the four principal alcohols from a crude fusel oil by fractional distillation is more difficult than the properties of the pure compounds would indicate because water-alcohol azeotropes with similar boiling points are formed. Horsley (1952) recorded the alcohol-water binary azeotropes of interest in fuse1 oil distillation (Table I ) . Informa-

TABLE I WATER AZEOTROPES OF CERTAIN FUSEL OIL COMPONENTS

Alcohol

Ethyl Isopropyl n-Propyl sec-Butyl Isobutyl

Isoamyl n-Hexyl

n-Butyl

Azeotrope

B.P., "C.

78.17 80.3 87 87.5 89.8 92.7 95.2 97.8

Wt. % water

4.0 12.6 28.3 27.3 33 42.5 49.6 75

tion concerning the active amyl alcohol-water azeotrope is not available but it is likely that the properties of this system are similar to those of the isoamyl alcohol-water system. Horsley lists no ternary azeotropes of water with two different alcohols.

Bukala et al. (1961) extensively studied azeotropic systems which might be applicable to recovery of higher alcohols from the crude fusel oils obtained in fermented sulfite liquors from paper and pulp mills. A system was developed in which the benzene-ethanol azeotrope is used to isolate the higher aliphatic alcohols in anhydrous condition from the crude fusel oil. No separation of active from isoamyl alcohol is obtained.

The formation of binary azeotropes between either isoamyl alco-

320 A. DINSMOOR WEBB AND JOHN L. INGRAHAM

hol or active amyl alcohol and other substances was investigated by Terry (1960) in an attempt to find a system which would permit ready separation of the two amyl alcohols by fractional distillation. The attempt was not successful since in all cases investigated the boiling points of the azeotropes were closer than were the boiling points of the pure alcohols (Table 11) .

TABLE I1 AZEOTROPES OF ISOAMYL AND AcavE AMYL ALCOHOLS

System

Binary azeotropic mixtures B.P. of B.P.

component ( "C. )

n-Octane Active amyl alcohol Isoamyl alcohol

Active amyl alcohol Isoamyl alcohol



2,2,5-Trimethylhexane Active amyl alcohof Isoamyl alcohol


Diisopropyl ketone Active amyl alcohol Isoamyl alcohol


2,6-Dimethylpiperidine Active amyl alcohol Isoamyl alcohol

1,2-Dimethylpiperidine Active amyl alcohol Isoamyl alcohol

Chlorobenzene

E thylbenzene

Toluene

o-Fluorotoluene

2-Picoline

126 138.5 132.0 132

136

111

124

114

125

129

128

128

( 760 mm. ) Alcohol Alcohol ( "C.) (wt. % ) (mole %)

117.0 34 40 117.0 30 36

124.4 43 49 123.9 38 44

125.0 53 57 125.7 49 53

109.9 12 12 109.7 10 10

115.5 29 37 116.0 26 34

112.0 16 19 112.1 14 17

124.1 21 26 124.5 8 10

132.8 49 50 132.8 61 62

130.7 54 60 132.6 76 80

130.3 Undetermined - 132.5 81 85

FUSEL OIL 32 1

The densities and refractive indexes of the alcohols of interest in fuse1 oils are shown in Table 1II.I

TABLE 111 DENSITIES AND REFRACTIVE INDEXES OF FUSEL OIL ALCOHOLS

Alcohol Density (gm./ml., 20°C. ) Refractive index (D, 20°C. )

Ethyl Isoprop yl n-Propyl sec-Butyl Isobutyl n-Butyl Is o a m y 1 Active amyl n-Hexvl

0.7893 1.3614 0.7851 1.3775 He y 0.8035 1 .38402,,, 4.8109,,, 1.3995,,. 0.8020 1.3959 0.8096 1.3970,,. 0.8059, 1.4048,,. 0.8154,,. 1.4088,,. 0.8225,,. -

Optical rotary dispersion (rotation of plane-polarized light as a function of wave length) of purified active amyl alcohol was determined by Marckwald and McKenzie ( 1901 ) . Timmermans (1950) lists values obtained by others.

The physical properties of mixtures of isoamyl and active amyl alcohols have been extensively studied. Hafslund and Lovell (1946) found that the density-composition plot was linear; however, Ikeda et al. (1956) found deviations from linearity, particularly in the mixtures of over 70% active amyl alcohol content. The refractive index-composition curve was also found to deviate from linearity for mixtures rich in active amyl alcohol ( Ikeda et d., 1956). Optical rotation has a linear relationship to composition of the two alcohols according to Marckwald and McKenzie ( 1901). Hafslund and Lovell (1946) were also able to fit their data for specific rotation as a function of composition to a straight line, as did Ikeda et al. (1956), using eleven different mixtures. These data have proven quite useful for rapid analyses of mixtures of the two alcohols, e.g., when following the progress of a distillation. The curves for density, optical rotation, and refractive index as a function of the composition of the active amyl-isoamyl mixture are shown in Fig. 1.

1 It is of historical interest that Pasteur (1855) detected the significant difference in density between isoamyl and active amyl alcohols and stated that the difference was nearly 0.01. Actually, the difference is 0.0095 g./ml. or 1,165-hundredths of the value, Pasteur’s alcohols must have been very nearly pure.

3 2 A. DINSMOOR WEBB AND JOHN L. INGRAHAM

0m50m 0 I0 20 30 40 50 60 70 80 90 100 !I % Active omyl

FIG. 1. Per cent active amyl alcohol in isoamyl alcohol plotted against refractive index, density, and observed optical rotation. (From Ikeda et al., 1956).

111. Analytical Methods

Methods which have been developed for analyses of fusel oil are in reality directed toward two different objectives. In one case the desired end is measurement of the total quantity of fusel oil present in an aqueous alcoholic mixture while in the second case the primary interest is in determining the relative amounts of the various components of the fusel oil mixture itself. In some cases, of course, the newer analytical methods have permitted determination of the exact amounts of each of the several fusel oil components actually present in a beverage, thus combining the two objectives.

The earliest methods of fusel oil analysis depended upon the fact that the amyl alcohols, and to a lesser extent isobutyl alcohol, have limited solubility in cold aqueous solutions of relatively low ethanol content. The fusel oils were separated from the nonvolatile materials of beer or wine by a simple distillation followed by fractional distillation to remove the bulk of the ethanol. On cooling the fractional distillation residue, fusel oil separates as a second phase. Water solubility of the higher alcohols is decreased by the dissolu- tion of salt in the aqueous phase, and this technique is employed in some of the earlier analytical methods. As the separated fusel

FUSEL OIL 323

oil phase was measured either volumetrically or gravimetrically, relatively large amounts of the wine or beer were required to obtain meaningful results. The solubility of the fusel oil alcohols in water-immiscible solvents such as carbon tetrachloride, chloroform, or petroleum ether has also been the basis for analytical procedures. In most methods of this type, fusel oil and ethanol are extracted from the preIiminary distillate and then the ethanol is removed by a subsequent extraction with saturated aqueous salt solutions. Since the partition coefficients of proply and isobutyl alcohols are similar to that of ethanol, this method, like those described above, tends to miss the lighter alcohols. The measurement thus becomes primarily a determination of the amyl alcohols.

Variations of the basic method of volumetric determination of separated fusel oil are numerous. In one case the alcohols are oxidized to acids and estimated by titration while in another a quantitative acetylation is employed. Both methods have difficulties. Quantitative oxidation of alcohols to acids is not practically attain- able. Determination of alcohols by acetylation is a rather com- plicated procedure. The uncertainties in both the oxidation and acetylation techniques can be lessened if the procedures are rigor- ously standardized. Calibration curves must be run and the composition of the fusel oils being measured cannot be widely variable. There are variations in the completeness of oxidation among the alcohols found in fusel oil mixtures as Lafon and Baraud (1960) have shown. Kepner and Webb (1954) have pointed out the necessity of careful control in the acetylation procedure. The secondary alcohol, 2-butanol, which is present in some fusel oils in minor amounts presents a problem in both procedures. It does not, of course, yield an equivalent amount of acid on oxidation, and it may not behave normally under the acetylation conditions employed for primary alcohols.

A. DISTILLATION METHODS

Simple distillation is employed in the isolation of the fusel oil from the wine or beer mixture in nearly every analytical method. In the very earliest analyses, crude fractional distillations combined with water washings and chemical drying were used to isolate and determine the fusel oil. The technique employed could only approximate the quantities of amyl alcohols present, and the lower


molecular weight alcohols were completely lost. With the development of more efficient fractional distillation equipment good separations of propyl alcohol from isobutyl and isobutyl from the amyls has become possible. However, the problem of isolating small amounts of fusel oil from the large quantity of aqueous ethanol remains.

There is no simple, quick method for isolating the fusel oil mixture from a wine or beer; neither distillation nor extraction is satisfactory in all respects. Perhaps the process which offers the best compromise between speed and completeness of fusel oil recovery is the following: Four or five liters of wine or beer are distilled slowly in an apparatus fitted with a Vigreaux column to obtain some fractionation. The portion distilling below the boiling point of ethanol is discarded since it contains esters, aldehydes, ethanol, and water only. The alcohol-water-higher alcohol fraction which distills next is collected; distillation is stopped only after the temperature at the head of the column has been at the boiling point of water for some time. This assures that all of the higher alcohol-water azeotropes have been collected. This fraction containing ethanol, water, and fusel oil is then redistilled using a fractionating column of fifty or more theoretical plates. The ethanol fraction is discarded and the distillation is continued until all of the higher alcohol-water azeotropes have been collected. The alcohols are extracted from the aqueous phase of the distillate with diethylether and the ether extract is dried over anhydrous mag- nesium sulfate which reduces the water content to less than 0.2% by weight (Webb et al., 1952). One part, by weight, of purified anhydrous ethanol is added to ten parts of the dry fusel oil-ether mixture.

The resulting mixture is distilled through a high efficiency fractionating column to effect the analysis. The low boiling fractions ( ether, ethanol-water azeotrope, and ethanol ) are discarded, and the propyl, isobutyl, and mixed isoamyl and active amyl fractions are collected separately and weighed. The weights of the smaI1 fractions collected while the head temperature is changing between pure fractions may be divided evenly between the adjacent pure components. Good separation of the active amyl alcohol from isoamyl alcohol is not possible. Therefore, the two alcohols are collected and determined together. Their relative proportions may

FUSEL OIL 325

be determined by optical activity, density, or the refractive index measurements with reference to the curves of Ikeda et al. (Fig. 1 ) .

In certain fusel oils small amounts of isopropyl alcohol, 2-butanol, n-butanol, n-pentanol, n-hexanol, and high boiling esters are found. In general, no one of these substances is present in amounts greater than about 5% by weight. The lower boiling alcohols are usually indicated by inflections in the temperature vs. quantity distillation curve while the n-amyl, n-hexyl, and ester portion remain in the distillation pot.

Jensen and Rinne (1952) analyzed fusel oils from fifteen Finnish sulfite mills by means of fractional distillation through spinning band columns. Compositions were calculated from the distillation curves after an aliquot of each constant boiling fraction had been positively identified by means of its other physical properties. Ogata and Matsubara (1953) determined the composition of a fusel oil from fermented sweet potatoes by fractional distillation using the Widmer column. The separated fractions were identified by physical measurements and by formation of derivatives.

B. CHROMATOGRAPHIC METHODS Recently various chromatographic techniques have been used in

fusel oil analysis. Liquid-liquid column partition chromatography using silicic acid as the stationary phase was adapted by White and Dryden ( 1948) to the analysis of the 3,5-dinitrobenzoate derivatives of alcohols. A fluorescent dye, adsorbed on the silicic acid provided a yellow background when viewed under UV radiation. Against this background the zones of 3,5-dinitrobenzoate appeared as black bands thus allowing ready separation of the various fractions. The White and Dryden method has certain intrinsic difficulties: Forma- tion of the 3,5-dinitrobenzoate is very probably not quantitative, and isolation of the alcohols in an anhydrous condition prior to analysis, is required. Also, active amyl-3,5-dinitrobenzoate is not separable from isoamyl-3,5-dinitrobenzoate by this method. How- ever, the technique is applicable to the estimation of relative amounts of the components of a mixture of primary alcohols since the degree of reaction of the various alcohols and 3,5-dinitrobenzoyl chloride is approximateIy the same.

It is likely that liquid-liquid partition systems could be developed for a number of solid derivatives of alcohols such as the p-phen-


ylazophenylurethanes. Derivatives carrying the azo linkage have the advantage of being colored and hence more easily visible on columns than are the 3,5-dinitrobenzoates under UV light. Also the alcohols could be oxidized to acids and separated as the p- phenylazophenacyl derivatives on silicic acid columns. Such techniques however multiply the possibilities of error, since the number of operations is increased.

It is also possible that a mixture of fusel oil alcohols could be separated directly on a silicic acid column carrying a polyol as absorbing phase by using a less polar moving phase. An instru- mental detection system (e.g., UV absorption or refractive index, etc.) would be required to monitor the effluent stream.

Paper chromatographic techniques have been applied to the determination of the alcohols by several investigators but in every case there are reasons for preferring other techniques. Daghetta (1956), for instance, extracted the fusel oil with chloroform, oxidized the alcohols to acids and chromatographed the acids as their ammonium salts using butanol. Sundt and Winter (1957) prepared the 3,5-dinitrobenzoates of the alcohols and chromatographed these on paper. Again paper chromatography systems of the various colored alcohol derivatives such as the 3,5-dinitrobenzoate-a-naphthylamine addition compounds, the p-phenylazo- phenylurethanes, and the p-phenylazobenzoates appear to hold promise. Also, there are colored acid derivatives which can be separated by paper chromatography. In any case, however, as with the column chromatographic methods, a number of preparative steps are required and methods of measuring the amounts of the separated alcohols or derivatives are imprecise. The authors know of no system of column or paper chromatography which is capable of resolving the active amyl-isoamyl alcohol or derivative systems.

The rapid development of the technique of gas-liquid partition chromatography has resulted in many applications to fusel oil analyses. Analyses both to determine the total quantity of higher alcohols in an alcoholic beverage and to measure the relative amounts of various compounds in previously isolated fusel oils have been developed. The problem of determination of the amounts of active amyl and isoamyl alcohols in fusel oil mixtures, so difficult by distillation and as yet unsolved by paper or column chromatography, has proved to be relatively simple by gas-liquid partition chromatography, as will be discussed later.

FUSEL OIL 327

As a method for routine analysis of wine or other alcoholic beverage for fusel oil content, gas-liquid partition chromatography is also quite useful. Since nonvolatile solids are present and the higher alcohols occur only in trace amounts a preliminary distillation is frequently required. For this purpose Bouthilet and Lowrey (1959) described a simple still by which a 20:l concentration of the higher alcohols in brandy samples can be attained. Harold d at. (1961) used steam distillation to separate the fusel oil of beer from the solids and then extracted with ether to separate the alcohols from the large volumes of water. Mecke and de Vries (1959) employed a mixture of two parts ether and one part pentane as extractant and made one back-extraction with water to remove ethanol. The higher alcohols were almost quantitatively recovered and contamination with ethanol was minimized. Webb and Kepner (1961) removed the ethanol and fusel oils from wines in a simple still equipped with a Vigreaux column. The distillate was saturated with salt and the alcohols were extracted with diethyl ether. Then the ether and most of the ethanol were removed by distillation through a Micropodbielniak column leaving an anhydrous fusel oil mixture as pot residue.

The presence of relatively large amounts of water and ethanol in samples of higher alcohols to be analyzed by gas chromatography presents a problem. Bouthilet and Lowrey (1959) used a column packed with Flexol and detected the higher alcohols as humps on the trailing side of the large ethanol peak. Zarembo and Lysyj (1959) found that columns packed with Armeen SD, a mixture of straight-chain saturated amines of about 16 carbon atoms chain length, permitted water to pass through the column rapidly, followed by the alcohols approximately in order of increasing molecular weights. Diglycerol and glycerol columns, on the other hand, have high retention times for water. Repeated routine analysis is possible with these columns by back-flushing the water from the fore part of the column between determinations. For most accurate estimates of the quantities of the various alcohols in the mixtures, however, the ethanol content must be reduced to the same order of magnitude as that of the other alcohols to prevent the ethanol peak from obscuring adjacent peaks.

The results provided by gas-liquid partition chromatographic analysis of fusel oil mixtures present problems of interpretation. If, on one hand, one is interested simply in the relative amounts


of the higher alcohols and esters present, these ratios may readily be obtained from the ratios of the areas of the appropriate peaks on the chromatographic chart. If, on the other hand, one wishes to determine the total concentration of fusel oils in the original sample one must decide how many of the higher boiling components separated by the chromatograph are considered to be fusel oil. For example, Harold et al. (1961) report a beer analysis in terms of individual compounds but apparently consider the fusel oil to con- sist of butyl and amyl acetates as well as the higher alcohols, but most published research describing fusel oil analyses by gas-liquid partition chromatography consider only relative amounts of the four principal alcohols, n-propyl, isobutyl, and the amyl alcohols.

Also, in the determination of total fusel oil by gas-liquid phase chromatography, losses during the concentration operations must be kept to a minimum and the volume of the injected sample must be accurately known or some unnatural component must be added in known concentration to serve as an internal standard.

After years of experience with gas-liquid partition chromatography Corse and Dimick (1958) stated that there was no packing known which could allow separation of active amyl from isoamyl alcohol. Shortly afterward, Van der Kloot and co-workers ( 1958) discovered that the two alcohols could be readily resolved on columns packed with glycerol. Webb and Kepner (1961) demon- strated that, in addition to glycerol, 1,4-butanediol, 1,2,4-butanetriol, i-erythritol, sorbitol, and diglycerol were capable of resolving the two amyl alcohols, and Prabucki and Pfenninger (1961) report that separation is also possible on columns packed with diethyl-D-tar- trate. Baraud ( 1961 ) obtained good separations using triethanola- mine. The detergent Tide was used by Porcaro and Johnston ( 1961 ) to separate the three amyl alcohols, active amyl, isoamyl, and n-amyl. Kambayashi et al. (1960) using columns of tetraethylene glycol dimethyl ether, polyethylene glycol, or dibenzyl ether and Kuffner and Kallina (1959) using Carbowax 300 were unable to resolve active amyl and isoamyl alcohols. Ingraham and Guymon (1960) used UCON-LB385 for separation of n-propyl, isobutyl, and fermentation amyl alcohols. Using a second aliquot of the sample and a glycerol column, the ratio of active amyl alcohol to isoamyl alcohol was determined, thus permitting a complete determination of the relative amounts of the four main fusel oil alcohols.

FUSEL OIL 329

C. CHEMICAL METHODS

As Guymon and Nakagiri (1952) pointed out in their review of fusel oil analytical methods, the colorimetric method based upon the Komarowsky reaction (1903), i.e., the reaction of isobutyl and isoamyl alcohols with aromatic aldehydes in concentrated sulfuric acid solution, is particularly satisfactory for determining fusel oil in alcoholic beverages. As compared with other nonchromatographic techniques it has the great advantages of speed and accuracy. Mathers and Schoeneman ( 1955) suggest that 4-hydroxy-benzalde- hyde-3-sulfonic acid sodium salt is the aromatic aldehyde of preference owing to the increased stability of the resulting color complexes. Methods using this aldehyde are less sensitive to minor variations in procedure. The method determines isoamyl, active amyl, tertiary amyl, 1%-amyl, and isobutyl alcohols and is not affected by ethyl, isopropyl, and n-propyl alcohols. The response to 2- butanol and to n-butanol, alcohols occasionally present in fusel oils, has not been determined. One would expect, however, that they react similarly to isobutyl alcohol although Stevens (1960) states that, “In general, straight chain alcohols give little or no colour . . . .” Mathers and Schoeneman (1955) showed that the visible spectrum of the color complex formed with n-amyl alcohol is similar to that formed with active amyl alcohol.

Both Guymon and Mathers have discussed the feasibility of determining the relative amounts of isobutyl alcohol in the fusel oil mixture by means of absorbance measurements a t two wave lengths, the absorbance-wave length curves for the color complex of isobutyl alcohol being significantly different from that of isoamyl alcohol. Ingraham and Guymon (19sO) used this method, as well as gas- liquid partition chromatography, in their studies of fusel oil production by mutant yeasts. However, the ratio of the absorbancies of the active amyl alcohol complex at 445 mp and at 560 mp approaches one-half the corresponding ratio for isobutyl alcohol complex ( Mathers and Schoeneman, 1955). Variations in concentration of active amyl alcohol in a fusel oil would therefore inter- fere with estimations of isobutyl alcohol by this technique, a t least in cases where 4-hydroxybenzaldehyde-3-sulfonic acid is the color reagent. An independent measurement of the optical rotatory power of the fusel oil can be made to estimate the active amyl alcohol. However, 2-butanol which is occasionally present interferes.


Measurement of the color developed upon heating a sample of spirit containing 50% ethyl alcohol with an equal volume of concentrated sulfuric acid is the basis of another analytical method for fusel oil. Aldehydes are removed in a preliminary step, and the color developed after heating is compared with that developed in solutions containing known amounts of isobutyl alcohol. The apparent results are divided by 0.6. Lafon and Couillaud (1955) consider this method to be arbitrary and indicate a preference for techniques using an aromatic aldehyde.

Genevois and Lafon (1958) developed a method for the estimation of secondary alcohols in mixtures of alcohols by oxidation to the corresponding ketone and measurement of the ketone by the iodoform reaction. The desirability of prior demonstration of the absence of methyl ketones in the mixture is obvious.

While the Rose-Herzfeld (Ehrlich, 1907) method for fusel oil analysis, depending as it does upon measurement of the increase in volume of chloroform as the higher alcohols are extracted into it, is a strictly physical method, the Allen-Marquardt ( A.O.A.C., 1950), Schichtanz and Etienne (1939), Schichtanz et al. (1940) techniques all possess certain chemical phases. In each of the latter methods physical techniques-extractions-are used to isolate the higher alcohols from the wine or spirit. The separated higher alcohols (mainly isoamyl and active amyl ) are determined chemically in the Allen-Marquardt procedure by chromic acid oxidation followed by acidimetric titration and in the Schichtanz-Etienne method by acetylation with acetylchloride followed by titration of the liberated hydrochloric acid.

D. OTHER ANALYTICAL METHODS The mass spectrometer was used by Webb and co-workers

(1952) to determine active amyl alcohol in mixtures containing isoamyl alcohol as the only other component. It is possible that the technique could be extended to the analysis of four or five component mixtures. With fusel oils as usually isolated, however, the mixture is too complex to permit interpretation of the mass spectrogram.

Batsin (1955) has used the polarimeter as a means of estimat- ing fusel oil quantities. The method depends upon there being a constant proportion of active amyl alcohol and 2-butanol (and other optically active substances) in the samples. Batsin collected

FUSEL OIL 331

data from a number of different distilleries and for various distillation fractions. Tables were prepared from which it was possible to approximate fusel oil concentrations closely by polarimeter readings. The specific rotation for active amyl alcohol was assumed to be 4 . 9 0 " at 20°C. and using sodium light. The presence of 2-butanol or other optically active compounds was not considered.

IV. Results of Fuse1 Oil Analysis

Numerous researchers have reviewed various phases of production and analyses of fusel oils. Primarily of historical interest are those of Schoen (1937), Schupphaus (1892), and Penniman et al. ( 1937). More recently Brau (1957), Genevois and Lafon ( 1957), Peynaud and Guimberteau (1959), Thoukis (1958a), Baraud (1961), and Stevens (1960) reviewed the field drawing on the findings provided by recent analytical advances. Baraud has made the valuable point that one cannot gain much knowledge about the influence of different yeasts, substrates, or fermentation conditions on fusel oil composition from analysis of fusel oils of industrial origin, because the conditions of isolation and treatment of the fusel oil which vary widely with location and time, have a great influence on composition. Design and operating conditions of the rectification column, for instance, are very probably responsible for the fact that isopropyl alcohol has not been found in all fuel oils. In cases of insufficient plates or of forced operation the isopropyl alcohol does not separate with the other higher alcohols but appears in the product stream. Similarly, it is common industrial practice to minimize loss of ethyl alcohol into the fusel oil fraction by repeated water washes or by adding salt or quicklime to induce separation of a second alcohol-rich phase. Such treatments markedly change the ratios of the propyl and butyl to amyl alcohols. Ikeda et aZ. (1956) consider this problem at length and conclude that it is preferable to work with fusel oil samples from laboratory fermentations in which conditions of isolation and concentration are known and controlled.

The number of published fusel oil analyses which truly represent the content of the wine, beer, or distilled beverage is relatively small. Some typical representative analyses are summarized in Table IV. The two analyses of brandies are not comparable with the wine and beer analyses because in the former fusel oil was

w w tQ

TABLE IV CONTENT OF VARIOUS BEVERAGES IN CONGENERS

(WT. % OF COMPOUNDS REPORTED) Baraud ( 1961 )

Webb & Kepner (1961) Cognac Cognac grande Burgundy Montrachet Jerez *

Compound fin bois champagne yeast yeast yeast EI - 5 Methanol 0.80 0.40 - -

Acetal 3.15 trace - - - 5 1-Propanol 1.65 1.70 18.2 2.6 20.2 m

4 8 Active amyl alcohol 78.05" 79.35" 12.0 16.5 4.5

3 8

0

- - - 2-Butanol 0.90 0.85 Isobutyl alcohol 15.45 17.70 12.4 2.7 8.4

Isoamyl alcohol 57.4 78.2 66.9 >

c-(

Harold& al. Van der Kloot Hudson & Stevens (1960) ( 1961 ) Enebo and Wilcox Z

(1957) (1959) P Unhopped Australian 8 Compound Pale ale Strong ale Stout beer beer Beer Beer Methanol - - - - - - - Acetal 1-Propanol - trace 9 1.6 7 4 2-Butanol - Isobutyl alcohol 25 24.2 21 23 7 8 Active amyl alcohol 750 75.80 70" 10.9 790 88" 25.3

P - - - - - - - 5: -

- - - - - 7 -

Isoamyl alcohol 64.1 74.7 a Sum of active amyl and isoamyl alcohols.

TABLE V COMPOSITIONS OF FUSEL OILS FROM VARIOUS SOURCES

(WT. % OF COMPOUNDS REPORTED)

Isobutyl Active amyl Isoamyl Reference Source 1-Propanol alcohol alcohol alcohol

Baraud (1961) Apples - 2.6 18.2 79.2 Molasses 15.0 15.7 28.3 41.0 Barley 7.6 33.5 18.2 40.7 SuKte liquor 15.4 37.5 8.5 38.6

Ikeda ( 1956) Thompson Seedless 0.8 7.4 16.7 75.1 Emperor 5.6 10.8 15.4 68.2 w

9 Muscat Alexandria 1.2 5.5 16.0 77.3 Mixed 4.9 21.3 11.1 62.7 r

Webb and Kepner Muscat raisin 0.7 4.9 19.3 75.1 F

M

0

(1961a) Zinfandel 2.3 15.6 16.8 65.3 Kumamoto (1932) Kaoliang 6.8 0.7 19.3 73.2

Molasses 2.8 0 19.1 78.1 Sweet potato 0 0 86.1 13.9

Ogata (1953) Sweet potato 8.4 6.8 46.2 38.6 Enders ( 1938) Fermented wood sugar 0.3 21.0 34.2 44.5

~~

Boswell and Beet molasses 0 Gooderham (1912)

8 56 36

Hellstrom (1943) Sulfite liquor 2 23 13 62 0 0 w

334 A. DINSMOOR WEBB AXD JOHN L. INGRAHAM

certainly lost in the “heads” and “tails” cuts during distillation. The three wine analyses probably reflect the influence of yeast strains as they were aliquots of the same grape juice fermented under identical conditions. The variations in amounts of n-propyl alcohol produced are striking. The beer analyses also show large variations in the leveI of propyl alcohol.

Numerous analyses of fusel oils from different sources have been published, These are of interest only in that they do illus- trate the variability of fusel oils as they are available from different distilleries. In Table V some typical analyses are listed. Only those in which both the active and isoamyl alcohols were determined are tabulated. The large variation in the ratio of isoamyl to active amyl alcohols is of particular interest. Since these two alcohols have nearly identical properties except for optical activity, they should be influenced to the same degree by the distillation and subsequent washing treatments. Consequently, the variations in quantities of the two must reflect significant influences of substrate or fermentation conditions. The variations in amounts of propyl and isobutyl alcohols can, of course, reflect substrate or fermentation differences but it is much more likely that rectification and washing treatment variations cause the observed differences.

Depending on the method of collection, all or part of the ethanol- soluble, water-insoluble compounds with boiling points near to and higher than that of ethanol of the material to be distilled may be found in the fusel oil. A very large number of such compounds have been found. Stevens (1960) listed a number of these compounds according to chemical type. Table VI summarizes litera- ture references in which these various compounds have been definitely identified. The various source materials are tabulated. It is recognized that certain of the compounds listed may have been extracted from wooden containers during aging or may have been produced during distillation; n-propyl, isobutyI, active amyl, and isoamyl alcohols are not listed since they are common to all yeast- fermented beverages. Indeed, it is likely that these four alcohols are present in all plant systems.

The wide range in types of compounds and the great number of compounds of any particular type present in fusel oil is striking (Table V I ) . As analytical techniques have improved the list of compounds has grown; certainly, it will lengthen in the future.

FUSEL OIL 335

TABLE VI COMPOUNDS OTHER THAN 1-PROPANOL, ISOBUTYL ALCOHOL, ACTIVE AMYL

ALCOHOL, AND ISOAMYL ALCOHOL REPORTED PRESENT IN F u s n Ous FROM VARIOUS SOURCES"

Compound Isopropyl alcohol

I-Butanol

( - ) -2-Butanol tert-Butyl alcohol 1-Pentanol

3-Pentanol 3-Methylbutan-2-01 1-Hexanol

1-Heptanol

2-Heptanol 1-Octanol 1-Nonanol 2-Nonanol 1-Decanol 2-Phenethyl alcohol

Borneo1 Fenchyl alcohol

Fermented material found in:a Wine-brandy (3); beer (19); cane molasses (11);

beet molasses (3); wood alcohol (12); sweet potato (25)

Wine-brandy (38); beer ( 19); cane molasses (27 ); beet molasses ( 5 ) ; sulfite alcohol ( 17 )

Wine-brandy (10) Beer ( 4 ) Wine-brandy (38); beer ( 19); cane molasses

Beet molasses (3) Wine-brandy (3); cane molasses (3) Wine-brandy (26); cane molasses (11); beet mo-

lasses (5); sweet potato (25); potato (29); sulfite alcohol (17)

potato (29)

(27); sweet potato (24)

Wine-brandy (26) ; cane molasses (32) ;

Cane molasses (32); sweet potato (25) Cane molasses (32); potato (29) Cane molasses (32); potato (29) Cane molasses (33) Cane molasses (32) Wine-brandy ( 18) ; beer ( 1 ) ; cane molasses ( 34 ) ;

sweet potato (35); sake (33); synthetic medium (31)

Sulfite alcohol (13) Sulfite alcohol (23)

a The numbers in parentheses indicate the source of data: (1) Ayrapaa, 1961; ( 2 ) Baraud, 1961; ( 3 ) Baraud and Genevois, 1958; ( 4 ) Bavisotto et al., 1961; (5) Boswell and Gooderham, 1912; (6) Braus and Miller, 1958; ( 7 ) Carrol and O'Brien, 1958; (8) Chapman and Hatch, 1929; (9) Duhaux and Belien, 1959; (10) Durodie and Roelens, 1942; (11) Dutt, 1938; (12) Enders and Kambach, 1938; (13) Ekstrom, 1932; (14) Enebo, 1957; (15) Gryaznof, 1959; (16) Harold et al., 1961; (17) Hellstrom, 1943; (17a) Hellstrom, 1944; (18) Ikeda et al., 1956; ( 19) Jenard, 1960; (20) Jensen, 1950; (21) Kepner and Webb, 1956; (22) Kepner and Webb, 1961; (23) Komppa and Toluitie, 1931; (24) Kumamoto, 1932; (25) Ogata and Matsu- bara, 1953; (26) Ordonneau, 1886; (27) Rao, 1938; (28) Rao, 1943; (29) Shoruigin et al., 1933; (30) Siefker and Pollock, 1956; (31) Smith and Coff- man, 1960; (32) Swenerton, 1929; (33) Taira, 1933; (34) Taira, 1936; (35) Taira and Masujima, 1934; (36) Ubeda, 1941; (37) Van der Kloot et nl., 1958; (38) Webb et al., 1953; (39) Webb and Kepner, 1962.


TABLE VI (Continued)

Compound Fermented material found in:a Guaiacol

d-Citronellol dl-a-Terpeniol Formic acid Acetic acid Butyric acid Isobutyric acid Isovaleric acid Caproic acid Enanthic acid Caprylic acid

Capric acid

Pelnrgonic acid Lauric acid Salicylic acid Methyl salicylate Ethyl formate Ethyl acetate

Ethyl propionate Ethyl isobutyrate Ethyl caproate

Ethyl enanthate Ethyl caprylate

Ethyl pelargonate Ethyl caprate

Ethyl laurate

Ethyl myristate Ethyl pentadecanoate Ethyl palmitate Ethyl lactate Ethyl succinate Ethyl malate Propyl valerate Butyl acetate Butyl valerate Isobutvl acetate

Whiskey ( 6 ) ; wood alcohol (12); sulfite alcohol ( 1 7 )

Sweet potato (25) Sweet potato ( 2 5 ) Wood alcohol (12) Wine-brandy (22); whiskey ( 12) Wine-brandy (38); wood alcohol (12) Wine-brandy ( 2 2 ) Wine-brandy ( 2 2 ) Wine-brandy ( 22); cane molasses (28) Wine-brandy ( 22 ) Wine-brandy ( 22 ) ; cane molasses ( 28 ) ; wood

alcohol (12) Wine-brandy ( 22) ; cane molasses ( 28) ; wood

alcohol ( 12) Wine-brandy ( 9 ) ; cane molasses (28) Cane molasses (28) Cane molasses (28) Wine-brandy (38) Beer (14) ; whiskey (7) Wine-brandy (39) ; beer ( 14) ; whiskey (7) ;

\Vine-brandy ( 2 ) Wine-brandy (39); cane molasses ( 11 ) Wine-brandy (38); cane molasses ( 2 8 ) ;

potato ( 2 9 ) Wine-brandy ( 2 ) Wine-brandy ( 22) ; cane molasses ( 28 ) ;

potato (29) Wine-brandy ( 22 ); cane molasses (28) Wine-brandy ( 22 ) ; cane molasses ( 28 ) ;

Wine-brandy ( 22) ; cane molasses ( 28 ) ;

Wine-brandy ( 22 ) ; potato ( 29 ) Wine-brandy (22) Wine-brandy (22) ; potato ( 2 9 ) Wine-brandy (39); synthetic medium (31 ) Wine-brandy (39) Wine-brandy (39) Kaoliang ( 24 ) Beer (19) Kaoliang (24)

raw spirit (15)

potato (29)

potato (29)

Wine-brandy ( 2 ) ; beer (16) a For sources, see footnote on page 335.

FUSEL OIL 337

TABLE VI f Continued)

Compound Fermented material found in: a

Isobutyl caprylate Isobutyl caprate sec-Butyl acetate Isoamyl acetate Isoamyl valerate Isoamyl isovalerate Isoamyl caproate Isoamyl caprylate Isoamyl caprate Isoamyl laurate Isoamyl lactate Isoamyl palmitate Active amyl caproate Active amyl caprylate Active amyl caprate Active amyl laurate Hexyl acetate Hexyl valerate 2-Phenethyl acetate 2-Phenethyl caproate y-Butyrolactone Diacetyl Dimethyl sulfide Pyridine Trimethylpyrazine Tetramethylpyrazine Diethylpyrazine Methyltriethylpyrazine Formaldehyde Acetaldehyde Isobutyraldehyde Hexanol 2-Hexenal Acetal Acetone Furfural p-Methylguaiacol p-Ethylguaiacol Vanillin Phenol 4-OH-3-CH,O-1-

propylbenzene 2-Butanone Limonene Camphene

Wine-brandy ( 38) Wine-brandy (38) Beer (16) Wine-brandy ( 2 ) ; beer (19); raw spirit (15) Kaoliang (24) Wine-brandy (39) Wine-brandy (38) Wine-brandy ( 38) Wine-brandy (38); kaoliang ( 12) Wine-brandy ( 38 ) Wine-brandy (39) Kaoliang (12) Wine-brandy (38) Wine-brandy (38) Wine-brandy (38) Wine-brandy (38) Wine-brandy (39) Kaoliang (24) Wine-brandy (22 ) Wine-brandy (39) Wine-brandy (39); synthetic medium (31) Wine-brandy (21 ); beer ( 4 ) Beer ( 4 ) Beet molasses (34) Beet molasses (8 ) ; potato (29) Beet molasses (8) ; potato (29) Beet molasses ( 8 ) ; potato (29) Potato (29) Beer (30) ; whiskey ( 7 ) Beer ( 37); whiskey (7 ) ; synthetic medium (31) Wine-brandy (21) Wine-brandy (21 ) Wine-brandy (21) Wine-brandy (38); cane molasses (11) Beer (37) ; whiskey ( 7 ) Wine-brandy (36); beer (30); cane molasses ( 11) Whiskey ( 6 ) Whiskey (6) Whiskey ( 6 ) Whiskey ( 6 ) Sulfite alcohol (20)

Wine-brandy (4 ) Sulfite alcohol (17a) Sulfite alcohol (23)

a For sources, see footnote on page 335.


V. Biosynthesis of Fuse1 Oil Components

In the introduction to the chemical section of this review fusel oil was defined in an operational sense, i.e., that liquid which separates as a second phase from distillates of yeast-fermented media. In this section we will discuss all the monohydric alcohols, other than ethanol, which yeast produce. This latter definition is more restrictive in that only alcohols are considered and less restrictive in that nonvolatile alcohols are included.

A. FUEL OIL FORMATION FROM AMINO ACIDS

Speculations concerning the possible origin of fusel oil components began late in the 19th century when it was proposed that these compounds might result from bacterial contamination or from the reduction of fatty acids (Emmerling, 1905; Pierre and Puchot, 1872; Pringsheim, 1905a, by 1906, 1907, 1908). But the first experimental approach to the problem was that of Felix Ehrlich, who in a series of carefully planned experiments, estab- lished that amino acids can serve as precursors of fusel oil. Ehrlich was probably led to investigate this possibility as a result of his studies on the “leucines.” In 1904 (Ehrlich, 1904) he isolated and characterized isoleucine. The structural similarity between leucine and isoamyl alcohol on the one hand and between isoleucine and active amyl alcohol on the other suggested a meta- bolic relationship between these amino acids and aliphatic alcohols. Accordingly, Ehrlich ( 1906a, 1907) carried out resting-cell fermentations of glucose by yeast to which leucine and isoleucine had been added, and showed that their addition increased the amount of fusel oil 7- to 8-fold, and that about two-thirds of the leucine dis- appearing could be accounted for by the fusel oil produced. On the basis of these experiments, Ehrlich proposed that leucine and isoleucine are split by a “hydrating” enzyme to form isoamyl and active amyl alcohols, respectively, in addition to COZ and ammonia, i.e.,

CH, C H w CH- CH,- CH(NH,)-COOK i- H,O -

CHf CHf CH- CH,- CH,OH + CO, + NH,

Leucine Isoamyl alcohol

and

CH,-CH,-CH-CH(NH,)-COOH + H,O-CH,-CH,- H CH,OH + CO, + NH, I 7 - CH, CHS

Isoleucine Active amyl alcohol

FUSEL OIL 339

However, Ehrlich was not able to detect the presence of free ammonia during the fermentation, an observation which lead him to conclude that the ammonia was immediately incorporated into the yeast protein (Ehrlich, 1907). This viewpoint was strengthened when he‘ was unable to obtain fusel oil formation by acetone-dried powders and pressed yeast juice, which were able to catalyze alcoholic fermentation, i.e., a condition in which protein synthesis did not occur (Ehrlich, 1906a). Ehrlich also showed that addition of ammonium salts and asparagine inhibited the formation of fusel oil. Together these observations of Ehrlich form the basis for the concept that higher molecular weight aliphatic alcohols are by- products of “alcoholic fermentation of amino acids” (Ehrlich, 1907) by yeasts. If readily utilizable forms of nitrogen such as asparagine or ammonium are present, they are preferentially used, but if leucine, isoleucine, and valine must be metabolized to satisfy nitrogen requirements for growth, fusel oil results.

Ehrlich was disturbed by his observation that yeast cells fermenting sucrose without added nitrogen produce about 20% as much fusel oil as is produced in a fermentation to which 0.6% leucine is added. But he suggested that autolysis of yeast protein supplies the amino acids to produce fusel oil under these conditions, and he supported this hypothesis by citing the observation that slow fermentations, in which there is more time for autolysis, produce more fusel oil than rapid fermentations.

Ehrlich also showed that yeasts produced tyrosol (p-hydroxy- phenyl ethanol) if tyrosine was added to the fermenting mixture (Ehrlich, 1907, 1911) and tryptophol was produced if tryptophan was added to the fermentation (Ehrlich, 1912).

Ehrlich’s contributions to our understanding of fusel oil formation remain, after almost 60 years, the most significant that have been made. One is particularly impressed by these contributions if he considers the techniques available to Ehrlich. In his early experiments he concentrated the amyl alcohols by fractional distillation, oxidized them to the corresponding carboxylic acids and made their silver salts. In his later experiments he determined the increase in volume of a chloroform layer after extracting the fusel oil from a specifically diluted alcohol solution.

Neubauer and Fromherz (1911) investigated the pathway of fusel oil formation from amino acids in greater detail. They set about to prove that a-keto acids were intermediates in the process


by showing that ( 1 ) a-keto acids can be produced from amino acids and that ( 2 ) added a-keto acids can be converted to alcohols with one less carbon atom. Since a-keto acids could not be isolated when naturally occurring amino acids were added to yeast fermentation, they added the unnatural phenylaminoacetic acid and were able to isolate the corresponding keto acid, phenylglyoxylic acid, in addition to benzyl alcohol and mandelic acid. Also p-hydroxy- phenylethyl alcohol could be isolated from fermentations to which p-hydroxyphenylpyruvic acid was added, but it was present in only trace amounts in fermentations to which p-hydroxyphenyllactic acid was added. On the basis of these observations they modified Ehrlichs scheme as follows:

R

CH,OH

R R I I c=o L

~ = CHO - I R I NH, y N H , I COOH COOH +

CO,

The essence of the observation of Ehrlich and Neubauer and Fromherz were reconfirmed by numerous investigators over the next several decades (Buchner and Meisenheimer, 1906; Lampitt, 1919; Houssian, 1937; Thorne, 1937; Zalesskaya, 1940; Yamada 1932; Genevois, 1952; Vogt, 1952; Ribkreau-Gayon et al., 1955; Spanyer and Thomas, 19%; Antoniani et at., 1958; Yoshizawa et al., 1961): but nothing basically new was added until 1958 when SentheShanmuganathan and Elsden ( 1958) reinvestigated the problem in the light of modern knowledge using modern techniques. SentheShanmuganathan and Elsden studied tyrosol formation because they found that this product can be easily estimated by the Folin and Ciocalteu (1927) reagent after separation from interfering materials by an ether extraction under alkaline conditions. They obtained a cell-free system which catalyzed the conversion of tyrosine to tyrosol if supplemented with pyridoxal phos- phate and a-ketoglutarate. Tyramine was eliminated as an intermediate since it could not be converted to tyrosol either by intact cells or by the cell-free system, while p-hydroxyphenylpyruvic acid and p-hydroxyphenylacetaldehyde were converted to tyrosol if DPNH is made available. On the basis of their experiments, it seems clear that the pathway of tyrosol formation by Saccharomyces cerevisiae is:

FUSEL OIL 341

transaminase ( 1 ) tyrosine + a-ketoglutarate glutamic acid + p-hydroxy-

phenylpyruvic acid

carboxylase ( 2 ) p-hydroxyphenylpymvic acid , p-hydroxyphenylacetaldehyde

+ co, alcohol dehydrogenase

( 3 ) p-hydroxyphenylacetaldehyde + DPKH , tyrosol + DPN

In a later paper SentheShanmuganathan (1960a) showed that crude preparations of S . cerevisiae were similarly capable of trans- ferring the amino groups of aspartic acid, leucine, norleucine, isoleucine, valine, norvaline, methionine, phenylalanine, and tryptophan to a-ketoglutaric acid. He ( SentheShanmuganathan, 1960b ) purified the tyrosine transaminase 100-fold, and showed it to be specific for a-ketoglutarate. The crude preparations were also capable of decarboxylating the resulting a-keto acids. It appears from the results of SentheShanmuganathan that isobutyl, isoamyl, and active amyl alcohol can be formed from valine, leucine, and isoleucine by the scheme suggested above for tyrosol formation, and from his results and others (Neuberg and Kariczag, 1911) that yeast carboylase catalyzes the decarboxylation step. SentheShan- muganathan suggests that from his observations and those of Bar- ron and Levine (1952) and Ebisuzaki and Barron (1957) that the third step in the formation of fusel oil from amino acids is catalyzed by either the classic alcohol dehydrogenase or by alcohol dehydrogenase I1 ( Barron and Levine, 1952), or both.

B. FUSEL OIL FORMATION FROM GLUCOSE

From the foregoing research it is clear that the major fusel oil components can be synthesized from amino acids by transami- nation, decarboxylation, and reduction, and the results strongly suggest that the components of fusel oil are by-products of the anaerobic nitrogen metabolism of yeast. However a number of observations are not satisfactorily explained by this theory, namely: (1) Significant levels of fusel oil including phenylethyl alcohol and tyrosol (Stevens, 1960) are formed by resting yeast cells in the absence of added amino acids. ( 2 ) In media containing low levels of amino acids, there is not a good correlation between the amino acid composition of the medium and the composition of the resulting fusel oil. ( 3 ) The kinetics of amino acid utilization and fusel


oil formation during alcohol fermentation in complex media are not complementary, i.e., amino acids are quickly removed from the medium, but the rate of fusel oil formation is no greater during the period of rapid amino acid uptake than it is later when the medium is essentially free of amino acids (Fig. 2). In fact,

200) 1

I Time in hours I

i f

25 50 75 100 125 150 175 200 Time in hours

FIG. 2. The course of amino acid disappearance, yeast cell multiplication, and formation of ethanol and fusel oil during fermentation of grape juice (Castor and Guymon, 1952). (Reproduced from Science by permission.)

fusel oil formation appears to parallel ethanol production not amino acid utilization. (4) Certain fusel oil components, e.g., n-propanol, a major component, and n-butanol, a minor component, do not correspond to any natural amino acids.

As stated earlier, Ehrlich suggests that autolysis of yeast protein accounted for fusel oil formation in the absence of exogenous amino acids. The inadequacy of this explanation was proven by Thoukis ( 1958a) who carried out nine successive resting-cell fermentations of sucrose with the same batch of yeast cells and obtained a cumulative yield of about 0.5 gm. of fusel oil from 7 gm. of yeast (dry weight). Clearly, the fusel oil was formed from sucrose and not from yeast protein.

Genevois and Lafon (1956) showed that C14-labeled acetate is

FWSEL OLL 343

incorporated into isoamyl alcohol formed during fermentation in the presence of unlabeled amino acids thus establishing beyond any doubt that not all of this alcohol is formed from exogenous leucine.

Since Saccharomyces cerevisiae has the ability to synthesize all amino acids, one might expect that fusel oil which is not derived from exogenous amino acids is synthesized at least to the keto acid stage by the same route as that by which the corresponding amino acid is synthesized. Ingraham and Guymon (1960) produced strong evidence to support this hypothesis by their study of amino acid auxotrophic yeasts. Resting-cell fermentations by yeast strains incapable of synthesizing a particular amino acid were found to be completely incapable of synthesizing the corresponding component of fusel oil, i.e., strains requiring leucine, isoleucine, or valine for growth did not produce isoamyl, active amyl, or isobutyl alcohols, respectively.

C. ~-PROPYL AND ~ -BUTYL ALCOHOLS The pathway of synthesis of n-propyl and n-butyl alcohols by

yeasts have been elucidated only recently. The first attempts were those of Kepner et al. (1954) who reasoned that n-propyl alcohol might be formed from a-aminobutyric acid by the conventional Ehrlich pathway. However, when they added this compound to a resting-cell fermentation of glucose by yeast, they obtained only a 0.03% yield of propyl alcohol from this nonprotein amino acid in contrast to a nearly 100% conversion of leucine to isoamyl alcohol.

A likely precursor of n-propyl was a-ketobutyric acid, a known intermediate in the synthesis of isoleucine (Willson and Adelberg, 1957) and hence also an intermediate in the synthesis of active amyl alcohol. Guymon et al. (1961a) showed this to be the case. They added gradated levels of a-aminobutyric acid, which is known to serve as a source of a-ketobutyric acid in yeasts, to nitrogen-free fermentations of glucose. The amounts of n-propyl and active amyl alcohols which were produced by these fermentations increased linearly with the amount of a-aminobutyric acid added to the fermentation, while the amounts of isobutyl alcohol remained essentially constant and that of isoamyl alcohol decreased slightly at high concentrations of the added amino acid (Fig. 3) . These authors further showed that in the presence of a-aminobutyric acid- l-C14 there was no significant labeling of any fusel oil component,


while the addition of a-aminobutyric a ~ i d - 2 - C ~ ~ brought about the selective labeling of n-propyl and active amyl alcohols. As expected from the proposed scheme the n-propyl alcohol produced contained essentially all of the labels of the carbinol carbon atom. Yamada c?t al. (1962) have also shown that the addition of a-aminobutyric acid stimulates n-propyl alcohol formation.

..;-/-; lsoomyl alcohol

OL= 004 006 008 % o-omino butyric acid

10

FIG. 3. The effect of addition of a-aminobutyric acid to resting-cell fermentation of glucose by Saccharomyces cereoisiae on the production of isoamyl, active amyl, isobutyl, and n-propyl alcohols (Guymon et al., 1961a). (Re- produced from Archives of Biochemistry and Biophysics by permission. )

Among the amino acid auxotrophic mutants studied by Ingraham et al. (1961) one required both isoleucine and valine for growth. Since leucine is synthesized from the immediate precursor of valine, this mutant is incapable of synthesizing all three branched chain amino acids. As expected, resting-cell fermentations of glucose by this mutant produced no isoamyl, active amyl, or isobutyl alcohols. But, quite unexpectedly large amounts of n-butyl and higher than normal amounts of n-propyl alcohols were produced.

Tracer experiments indicated that n-butyl alcohol is synthesized by this mutant from a-keto-n-valeric acid which in turn is synthesized from a-ketobutyric acid by a sequence of reactions analogous to those known to be responsible for the conversion of keto valine to keto leucine (Strassman et at?., 1956) (see Fig. 4 ) . That is, addition of 2-labeled acetate results in predomi-

I rz-Propyl Alcohol] 1 Active Amy1 Alcohol]

t t it -Propanaldehyde + CO,

t Methylbutyraldehyde + CO,

t a -Acetohydroxy- - a ~ ~ ~ ~ ~ ~ : ~ ~ ~ ~ ” - -a -Keto-B-rn&hylvalerate + Isoleucine Threonine _c a -Ketobutyrate - I

butyrate

Active Acetaldehyde _- a-Ketoisovalerate Valine a , O-Dihydroxy- isovalerate I I

t L Pyruvate - ci -Acetolactate _t.

1 [Acetyl-CoA

[ 4-Carboxy-0-hydroxyvalerate]

a -Hydroxy-8-carboxyvalerate t

+ CoA

w {Acetyl-CoA 3

F

Isobutyraldehyde, r-7 P isocaproate -Carboxy 4-hydroxy- + CoA 8

a -Hydro- -p -carboxy - Isobutyl Alcohol isocaproate a -Ketovalerate - n-Butyraldehyde -1 n -Butyl Alcohol I Alcohol - Isovaleraldehyde - a -Ketoisocaproate + CO,

14 +

1 F A c e t y l = CoA 0-Carboxy-0-hydroxycaproate

I I

a -Hydroxy-8-carboxycaproate I

I I co, Leucine

t ci-Hydroxycaproate - n -Valeraldehyde - n -Amy1 Alcohol

FIG. 4. Pathways of formation of major fuse1 oil components and n-butyl and n-amyl alcohols.

W A cn

346 A. DINSMOOR WEBB AND JOHN L. WGRAHAM

nate l-labeling of n-butyl alcohol; 2-labeled a-ketobutyrate results in %labeling of n-butyl alcohol, while l-labeled a-ketobutyrate and l-labeled acetate does not result in significant activity in n-butyl alcohol.

Since a-keto-n-valeric acid was a suspected intermediate in the production of n-butyl alcohol, norvaline was added as a source of the keto acid to resting-cell fermentations of both the auxotrophic mutant and its wild type parent. As predicted, the production of n-butyl alcohol by both strains was stimulated by the addition of the amino acid. Surprisingly, however, under these conditions, the mutant was found to produce n-amyl alcohol in addition to n-butyl and n-propyl alcohols. Tracer experiments indicated that n-amyl alcohol is synthesized by the mutant from a-ketovaleric acid by a pathway analogous to that by which n-butyl alcohol is synthesized from a-ketobutyric acid, i.e., by way of a-ketocaproic acid (Fig. 4 ) . The enzymes of this reaction pathway which has the net result of converting an a-keto acid to an alcohol with the same number of carbon atoms, appear to be nonspecific since a-ketoiso- valeric, a-keto-n-butyric, and a-keto-n-valeric acids can serve as primary substrates for isoamyl, n-butyl, and n-amyl alcohols respectively. It was suggested by Guymon et al. (1961a) that some n-propyl alcohol may be made by this pathway from pyruvate since threonine auxotrophs which cannot make a-ketobutyric acid were found to produce this alcohol. There are limits, however. Norleucine is known to be converted to a-keto-n-caproic acid by Saccharomyces cerevisiae. Yet addition of this amino acid to a resting-cell fermentation of glucose by the mutant, does not bring about the production of n-hexyl alcohol.

It now appears that the pathways of formation of the various fusel oil components are known. The major components, isoamyl, active amyl, isobutyl, and n-propyl alcohols and the minor component, n-butyl alcohol, are formed from intermediates in the synthesis of the three branched chain aliphatic amino acids : leucine, isoleucine, and valine (Fig. 4) .

D. FACTORS AFFECTING FUSEL OIL FORMATION The major components of fusel oil are formed by a number of

yeasts under a wide variety of physiological conditions. Guymon et al. (1961b) showed that aerobic conditions do not inhibit the formation of fusel oil; in fact, more fusel oil is formed under aerobic

FUSEL OIL 347

conditions by a factor of about 4 in spite of the fact that under these conditions yeasts are capable of reutilizing fusel oil components (Mertz and Ingraham, 1961). The fusel oil mixture produced aerobically contains up to three times as much isobutyl alcohol as the mixture produced anaerobically, although it is not known whether this enrichment is caused by greater production of this alcohol or by lesser reutilization.

Naturally occurring strains of wine yeasts do not vary greatly in ability to produce fusel oil (Castor, 1954), although there is con- siderable variation among different genera of yeast. In a study (Guymon et al., 1961a) of nine yeast species including fermenta- tive and nonfermentative types, all were found to produce fusel oil (Table VII) regardless of their ability to produce ethanol. Fusel oil production appears to be a general property of yeasts.

TABLE VII PRODUCTION OF FUSEL OIL BY YEASTS OF DIFFERENT FERMENTATION

CHARACTER UNDER AERATED AND NONAERATED CONDITIONS

Fusel oil

Ethyl alcohol mg./100 gm. formed (vol. % ) mg./100 ml. (sugar used)

Aer- Anaer- Aer- Anaer- Aer- Anaer- Yeast obic obic obic obic obic obic

Pichia kluyveri 1.2 4.9 50.2 6.4 437 72

Hansenulu anomala 0.2 4.3 16.8 4.3 142 57 Schwanniomyces

Candida albicans 1.9 2.9 67.5 3.8 614 109 Candida rugosa Tr. Tr. 7.6 0.3 84 12 Kloeckera magnu 0.2 4.2 4.4 3.2 52 53 Debaryomyces kloeckeri Tr. 0.2 35.0 1.1 350 31 Saccharomyces beticus 10.8 12.3 20.8 4.7 95 26

Pichia membranafaciens 0.4 0.1 3.1 0.3 27 7

occidentalis 3.5 0.8 21.9 3.6 219 -

The effect of temperature of fermentation on fusel oil formation was studied by Lindet in 1888 (Lindet, 1888). He found that about 10% less fusel oil was formed in the range of 8"-1OoC. than in the range of 25"-27"C. More recently, similar results were obtained by Castor (1954). The effect of a decrease in fusel oil formation at low temperature is too small to be of industrial significance.

It is commonly observed that the presence of suspended solids during fermentation increase the yield of fusel oil. The evidence


indicates that this effect has a physical rather than a chemical basis, since the addition of animal charcoal has been reported to increase fusel oil production 2.5 times ( Dietrich and Klammerth, 1941). It is possible that suspended solids exert their effect by creating aerobic conditions during the early stages of fermentation. Fusel oil produced under such conditions would not be reutilized during the later anaerobic stages.

The effect of the addition of amino acids which are not direct precursors of fusel oil components on the formation of fusel oil has been studied extensively (Yamada et al., 1962; Antoniani et d., 1958; Peynaud and Guimberteau, 1958). Certain observations are easily explained on the basis of present knowledge; for example, the effect of addition of leucine, isoleucine, and valine on the stimulation of production of the corresponding higher alcohols. Also the stimulation of active amyl alcohol production by the addition of threonine (Yoshizawa et al., 1961) is to be expected since this amino acid is an intermediate in the formation of active amyl alcohol ( Fig. 4 ) . Similarly, the observed stimulation of isobutyl alcohol production by the addition of alanine (Yamada et aE., 1962) might be expected since pyruvate is a precursor of valine.

Asparagine undoubtedly exerts its depressive effect on higher alcohol production (Ehrlich, 1907) by serving as a readily available nitrogen source as does ammonium ion (Ehrlich, 1907). Addition of other amino acids has led to conflicting results (Antoniani et al., 1958; Peynaud and Guimberteau, 1958).

The essentiality of glucose to fusel oil formation from amino acids by intact yeast cells has been known since Ehrlich and considered by him to substantiate his view that fusel oil is a by-product of the process by which the cell obtains nitrogen for growth. Senthe- Shanmuganathan and Elsden (1958) have pointed out two obvious requirements for glucose and suggested a third. Glucose metabolism is required to supply a-ketoglutarate to accept the amino group and to supply DPNH to reduce the aldehyde precursor of the particular fusel oil component in question. They also showed that sodium azide and 2,4-dinitrophenol inhibit tyrosol formation from tyrosine indicating that the process requires energy. They suggest that the energy-requiring step might well be the entry of tyrosine into the cell.

Fusel oil production is a normal activity of yeast. All strains examined carry out the process. Production occurs under aerobic

FUSEL OIL 349

as well as anaerobic conditions; in complex media in which exogenous amino acids are plentiful and in minimal media in which they are absent; in media in which glucose concentration is high and in media in which glucose concentration is low. Fuse1 oil is produced in glucose-limited chemostat culture ( Mertz and Ingraham, 1961), a condition of minimal glucose concentration. In media high in fermentable carbohydrate, fusel oil is produced during the period of active growth as well as the period of metabolism following the cessation of growth.

Competition in the biological world permits only the most efficient organisms to survive. Consequently we are able to find “purpose” in most biochemical events. One cannot help but wonder, in this vein, about the significance of fusel oil formation by yeasts. Its significance under anaerobic conditions in certain complex media is probably just as Ehrlich and others have suggested, namely: fusel oil formation is an inevitable consequence of the cell’s use of amino acids as a source of nitrogen.

But this explanation cannot account for the process under other conditions. According to SentheShanmuganathan ( 1960) the last two steps in the formation of fusel oil components are catalyzed by the same enzymes as those which catalyze the last two steps in ethanol production. If keto acids are present, therefore, it seems inevitable that they be converted to the homologous alcohol. Pos- sibly this is the answer.

It is interesting that the major components of fusel oil are by- products of isoleucine-valine-leucine metabolism. Since it is known that the synthesis of these amino acids is controlled by multivalent repression in Escherichia coli (Freundlich et al., 1962) it is tempt- ing to speculate that control is inadequate in yeasts thus permitting higher alcohols to be produced by a process of “shunt metabolism” (Foster, 1949). However, it is also possible that the formation of propyl, amyl, and butyl alcohols is favored merely as a result of the specificity of carboxylase.

The energy which is apparently wasted in the formation of fusel oil is not great. Utilization of DPNH in the last step of the process cannot be considered to be a loss of energy under anaerobic conditions; the only energy loss is the expenditure of one acetyl-CoA in the synthesis of isoamyl alcohol. Considering the very small amounts of fusel oil formed the net energy loss is insignificant.


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