Ethyl Esters Of Higher Fatty Acids Of Rhums

Parfait A., Namory M., Dubois P., 1972. Les esters éthyliques des acides gras supérieurs des rhums. Annales de Technologie Agricoles 21, 2, 199–210.

ETHYL ESTERS OF HIGHER FATTY ACIDS OF RHUMS

A. PARFAIT, M. NAMORY and P. DUBOIS *

Station de Technologie végétale, I. N. R. A.,
Petit-Bourg (Guadeloupe)
*Station de Technologic des Produite vegetaux, I. N. R. A.,
21034 Dijon Cedes

Summary

Rums of good quality, especially in ethyl esters, generally have high fatty acids with a high content of volatile esters, and particularly of ethyl esters of higher fatty acids (from C8 to C16). These esters are by-products of alcoholic fermentation, such as higher alcohols, and behave like them during continuous distillation.

The levels of ethyl esters of the higher fatty acids are higher when the distillation is done on the lees, when cane wax is added to the must before fermentation and by selection of a species, or even of a strain, of yeast.

The ethyl ester contents of the distillate fatty acids were three times higher with Saccharomyces cerevisiae S. 132 than with Saccharomyces cerevisiae Berlin II. The highest content was obtained with a strain of Schizosaccharomyces Pombe yeast from the sugar cane regions.

It has not been possible to establish definitively a correlation between the fatty acid composition of the lipids of the yeast cell and the ethyl esters produced.

Key words: rums, ethyl esters, higher fatty acids, distillation.

I- INTRODUCTION

The eaux-de-vie can be considered as hydroalcoholic solutions of a “non alcohol” which characterizes them and that the chemical analysis makes it possible to separate in dry extract, acids, aldehydes, esters and higher alcohols, These analyzes can be completed by olfactory observations on isolated fractions by distillation. Finally, much finer separations can be obtained by gas chromatography for the determination of volatile constituents and by other chromatographic methods for the study of nonvolatile compounds.

In the case of rums, there may be a relationship between their quality and their ester content (KERVEGANT, 1946) and, in spite of many exceptions, it seems that high ester levels characterize the most aromatic rums. Observations made after fractional distillation even suggest that heavy esters have the greatest influence on the aroma of these eaux-de-vie, as is the case with whiskeys (SALO et al., 1972). The technology of rums should therefore be able to take advantage of recent studies on the formation of esters by yeasts during the fermentation of wines and beers.

A.—Mechanism for the formation of esters

PEYNAUD (1956) has shown that the levels of ethyl acetate in fermented media depend on the yeast species, and that they are always higher than those predicted by the calculation from chemical equilibrium reactions.

According to NORDSTRÖM (1964), the esters are formed by alcoholysis of the acyl-coenzymes A according to the reaction

and their formation depends on the contents of acyl-Co A and alcohols (RAINBOW, 1970) The alcohols react all the better as they are primary, with a linear chain and of lower molecular weight. Since ethyl alcohol is the most abundant, the esters formed are mainly ethyl esters.
On their side, acyl-Co A have three modes of formation.

Activation of fatty acids in the presence of ATP.

In view of the very small quantities of free fatty acids in fermented media, it seems that this reaction can play only a minor role.

Oxidative decarboxylation of α-cetanol acids.

This reaction is thought to be responsible for most of the acetyl-Co A, by oxidative decarboxylation of pyruvic acid. The other α-keto acids, that is to say certain intermediates of the metabolism of sugars and of amino acids, are present in much smaller quantities than pyruvic acid, and very few esters of the corresponding acids are formed. (propionic, isobutyric, methyl-2 and 3-methyl-butyric).

Reaction between an acyt-Co A and malonyl-Co A.

This reaction, which leads to the formation of the fatty acids of the lipids of constitution of the yeast, is also at the origin of the ethyl esters of linear fatty acids with an even number of carbon atoms.

Ester formation is related to yeast growth, as is that of higher alcohols and the formation of higher fatty acid esters is more particularly related to lipid metabolism.

It is therefore, like that of lipids, favored by the presence of pantothenic acid, as constituent of coenzyme A, and of the biotin which participates in the carboxylation of acetyl-Co A in malonyl-Co A, and which therefore, competes with the formation of ethyl acetate.

Factors that limit the development of yeasts have an inhibitory role. This is particularly the case of linear fatty acids having 6 to 10 carbon atoms which are toxic to yeasts.

B.— Fermentations in rhummeries

We know imperfectly the flora that develops in the environments put in fermentation to produce the different types of rums. HARRISSON and GRAHAM (1970) point out, in a development, that the budding yeasts gradually replaced, in Jamaica, a fission yeast, Schizosaccharomyces melacei, which was dominant at the beginning of the century.

Overall, fission yeasts are preferable to budding yeasts, in part because they promote the development of butyric bacteria (Clostridium saccharobulyricum, in particular) that produce very large quantities of esters. These fission yeasts are particularly abundant in the flora used for the elaboration of “grand arôme” rums.

It is also possible that yeasts of the genus Torulopsis play an important role in the formation of esters from sugars.

In the French Antilles, Saccharomyces cerevisiae is the main agent of fermentations. Other yeasts are present, including Pichia, Hansenula, Candida and Schizosaccharomyces. Their role is difficult to assess in practice.

C.— Role of distillation

Esters are poorly soluble in water and behave like head products in low alcohol environments. They behave, at the same time, as bottoms in the distillation columns when the alcoholic degree reaches values ​​of the order of 50 to 60 ° GL. Only ethyl acetate is always in the heads. Butyrate and ethyl hexanoate distill substantially at the same time as ethanol. The ethyl esters of acids that have 8 carbon atoms and more distillate after ethanol and it is these acids that we consider here as superior.

The contents of the rums in the ethyl esters of the higher fatty acids are therefore related to the alcoholic degrees to which they are distilled in the columns. The higher this degree, the poorer the rum obtained in these esters. It is interesting to note that the behavior of higher alcohols is quite similar to that of heavy esters.

In any case, the analytical results obtained on rums, and in particular the quantitative results obtained by LIEBICH et al. (1970) (Table I) shows that the main esters are the ethyl esters of fatty acids number by carbon atoms. Ethyl acetate and butyrate dominate among the light esters, caprate and palmitate among the heavy esters. Certain unsaturated acid esters are also present, but in smaller proportions.

Table I
Ethyl esters reported in rums

After having measured the ethyl esters of higher fatty acids in commercial rums, various factors have been studied that may affect the levels of these constituents in rums: presence of yeasts during the distillation fermentation in the presence of cane wax fermentation by various species of yeast. These different points were completed by the analysis of lipids of yeast constitution.

II- MATERIAL AND METHODS

A.— Culture media

Two media were used, one is based on cane molasses, the other synthetic.

These solutions are brought to pH = 5 with sulfuric acid and sterilized by treatment at 110 ° C. for 35 minutes.

B.— Yeast uses

Apart from Schizosaccharomyces pombe, which was isolated from a fermentation of cane juice medium, the other species came from the Dijon Plant Products Technology Station collection: Saccharomyces cerevisiae strains Berlin II and S. 132, Pichia membranaefaciene, Hansenula anomala and Candida krusei. These yeasts are those which have been reported as participating in the fermentation of musts in rhummeries.

With a young culture of yeasts, flasks containing 100 ml of liquid Wickerham medium are inoculated with malt. After about 24 hours at 28 ° C, in stirred medium, the yeasts are collected by centrifugation, then they are rinsed twice with saline water. All media are inoculated so that they initially contain 5 · 10 ^ 6 yeasts per ml. A magnetic bar agitates the first day and fermentation lasts 3 to 4 days at 28 ° C.

C.— Analysis made on distillates

The apparatus to be distilled is made of glass and includes a balloon, an electric heater, a column of Vigreux of 50 cm of height and a refrigerant.

Ethyl esters of fatty acids of 8 to 16 carbon atoms are not very polar and their most selective solvent must itself be polar. We chose pentane.

A test portion of 100 ml of distillate at 50 ° GL is stirred vigorously with 50 ml of pentane. The addition of 100 ml of water causes an immediate demixtion without emulsion formation. The organic phase is dried over anhydrous sodium sulphate and brought to a tenth of a milliliter by distillation. Two microliters are injected into a Perkin-Elmer model 880 chromatograph under the following conditions: column filled with Chromosorb G 80/100 mesh impregnated with OV 17 at 5 p. 100, length 4 meters, diameter 3 mm. Nitrogen flow 25 ml / min. Temperature program of the injection from 160 to 240 ° C at 2 ° C per minute. Since the rums contain only traces of ethyl pelargonate, this analysis is made quantitative by the addition of one milliliter of an alcoholic solution of this ester at 1 mg / ml to the 100 ml of distillate used. Previous tests performed on synthetic solutions showed that the relative errors were less than 5 percent. The exact nature of the esters was verified in the chromatographic conditions, but in coupling with a Varian *** type CH 5 mass spectrometer (250° C source, 70 eV electron energy). All these ethyl esters are characterized by a rearrangement peak for m / e = 88,

and by their molecular peaks. Mass spectrometry also makes it possible to observe the absence of important interferences.

D.— Analyses faitee sur les levures

After fermentation, the yeasts are separated by centrifugation (10,000 g). They are twice washed with physiological water, and then harvested at the bottom of the tubes using a spatula. At weighing of the wet mass, the dry matter content is determined on an aliquot part. [aliquot was a guess. The scanning is cutting off the edge for this page].

The total lipids of the yeasts are determined by the method of KAHANE and ROUS (1961) and then saponified by the alcoholic potash. Unsaponifiable matter is extracted with petroleum ether. After acidification, the fatty acids are extracted with the same solvent, then they are esterified with hydrochloric methanol and analyzed by gas chromatography on an impregnated column [guess] of diethylene glycol succinate (impregnation rate 15%). 3 meters, inside diameter 3 mm, Chromosorb G 80/1000 mesh, temperature 190 ° C.

E.— Autres dosages

The yeasts are counted with a cell count on a possible dilution of the medium. The reducing sugars are dosed, after acid hydrolysis, by the method of Bertrand. Finally, the volatile acids are determined after entrainment by steam. [I think I translated that last part correctly.]

III. — RESULTS

A.— Levels of ethyl esters of the higher fatty acids of commercial rums

The contents of these esters of five rums were compared: two agricultural rums obtained from cane juice in a traditional factory where fermentations are most often spontaneous, and three rums obtained from industrial rum distillates at 64 ° GL, a light rum distilled at 94 ° GL, and a rum “grand arôme” distilled at 63 ° GL as industrial rum, but obtained by a fermentation procedure that uses a starter with yeasts of the genus Schizosaccharomyces and bacteria.

The results obtained are shown in Table 2.
The first three rums have very similar contents in these esters, light rum contains only traces, the most abundant being the *** of ethyl whose content is of the order of 0.05 mg / liter of pure alcohol. The numbers obtained for the rum “grand arome” are much weaker than were expected. This rum has a very low content in total esters (about 2 g / liter of pure alcohol).

Table 2

Ethyl esters of various commercial rums
(in mg per liter of pure alcohol)

B.— Role of yeasts during distillation

The synthetic medium and the molasses-based medium were used with yeast Saccharomyces cerevisiae Berlin II. In each test, four liters of medium are prepared and fermented, then they are divided into two equal parts, one of which is centrifuged before distillation to eliminate the yeasts.

The results reported in Table 3 show that distillation in the presence of yeasts leads to a significant gain in esters. These compounds are poorly soluble in low alcohol environments and are likely to be absorbed on the surface of yeasts and suspended particles.

TABLE 3

Role des levures et de la cire de canne sur les teneurs des distillats en esters ethyliques des acides gras superieure
(n-C8 a n-C16 en mg par litre d’alcool pur)

C.— Role of cane wax

The wax is the richest part of the higher fatty acids of the cane (*** according to MARTIN and JUNIPER, 1970), and it was interesting to see what could be its influence.

It was added in the form of an emulsion at a dose of 0.300 g per 4 liters of medium. The results (Table 3) show that this addition of wax allows a medium based on cane molasses to double the levels of distilled ethyl esters of fatty acids with 8 and 10 carbon atoms which are among the interesting ones on the olfactory plane ( SALO et al., 1972).

The synthetic media are incompletely fermented and the results obtained are unusable.

D.- Role of the yeast species

EL SHEHATA (1960) has shown that molasses musts are fermented, in practice, by the following yeast species: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hansenula anomala. Pichia membranaefaciene and Candida krusei. The first two ferment well in anaerobic environment, the last two do not ferment sucrose.

TABLE 4

Ethyl esters of distillates obtained with different yeast species
(in mg per liter of pure alcohol)

Yeast species used:
Pichia membranacfaciens (P. m.), Hansenula anomala (H. a.), Schizosaccharomyces pombe (S. p), Candida krusei (C. k.), Saccharomyces cerevisiae S. 132 (S. c. 132).

We have fermented, under the conditions defined above, five mediums based on molasses with respectively each of the following yeasts:

– Pichia membranaefaciens (P. m.)
– Hansenula anomala (H. a.)
– Schizosaccharomyces pombe (S. p.)
– Candida krusei (C. k.)
– Saccharomyces cerevisiae S. 132 (S. c. 132).

After fermentation, the media are distilled in the presence of yeasts. The results are summarized in Table 4.

Some media have been incompletely fermented because Pichia and Candida do not ferment sucrose.

E.- Fatty acid content of yeasts

The determinations were made on yeasts derived from fermented media based on molasses similar to the preceding ones, but, to promote the multiplication of yeasts, the stirring period was increased to 48 hours instead of 24. Tables 5 and 6 report on the results obtained during various determinations.

TABLE 5

Data on the fermentation by different species of yeasts in molasses medium

TABLE 6

Comparative composition of lipids of various yeast species

There appears to be some correlation between the amount of ethyl esters of the higher fatty acids produced by these yeasts and their levels of saturated fatty acids. However, the methods used do not make it possible to know if the fatty acids doses were glycerol related in the lipids of constitution, or if they were in the form of ethyl esters and associated with the yeast walls.

IV.— DISCUSSION

The levels of ethyl esters of the higher fatty acids of commercial rums are close to those we were expecting to find except for the “grand arôme” rum. On rum of this type Liebich et al. (1970) indicate contents of the order of 170 mg per liter of pure alcohol, thus of much higher contents and which are similar to those obtained when working with Schizosaccharomyces pombe. The poorly defined origin of these products makes it possible to make only findings.

The presence of yeasts during the distillation makes it possible to obtain richer eaux-de-vie in esters. This confirms the work of GUYMON and CROWELL (1969) and partially explains the preference of practitioners for the lees distillation method.

GUYMON and CROWELL (1969) assume that, during continuous distillations, the fatty acids released by the yeasts are esterified with ethanol on the first plats of the column. The results reported here do not seem to be explained in this way since the distillations were performed, in the laboratory, discontinuously and that therefore the higher fatty acids could not be found in the free state presence of a high concentration of ethanol. It seems more probable that esters were linked to yeasts, either on their wall or in their cells and that they were released by heating.

The results concerning the addition of wax are more difficult to interpret since its composition is poorly known. It is known to contain a small amount of free fatty acids with a very high carbon number, which are yeast growth activators, but its biotin and pantothenic acid contents are not known in industrial practice. It will be interesting to know if the simple addition of palmitic acid would not have effect, palmitic acid being the main fatty acid of the lipids of yeast.

Finally, there were important differences between one yeast species and another, even from one strain to another within the same species. Under the same conditions, Saccharomyces cerevisiae Berlin II produced 36.5 mg of these esters per liter of pure alcohol (Table 3) while Saccharomyces cerevisiae S. produced 114 (Table 4).

The highest content was obtained with Schizosaccharomyces pombe, which is the dominant species in the flora of wines prepared for the production of “grand arôme” rum.

V.— CONCLUSION

Although their mixture can not be at the origin of the characteristic aroma of the rums, it seems probable that the ethyl esters of the volatile fatty acids participate in their qualities. Therefore, it was useful to specify the conditions allows rums rich in these constituents.

The most important factor is doubtless the distillation and alcoholic degree to which the alcohols are obtained continuously. The higher the rectification rate, the lower the ester and higher alcohol contents.

On the other hand, three factors seem to be able to be used to increase the levels of esters while keeping low contents of higher alcohols: the addition of wax, the distillation of the turbid substances and the selection of a species, or even of a yeast strain.

Received for publication in October 1972.

SUMMARY

ETHYL ESTERS OF THE RUM HIGHER FATTY ACIDS

Good quality rums have generally a high content of volatile esters and especially of ethyl esters of higher fatty acids (n-C8, to n-C16). These esters are secondary products of alcoholic fermentation, like higher alcohols,and behave like them during a continuous distillation process.

The quality of rums should be improved using our recent knowledges on esters production by yeasts in beer and wine.

Higher contents of ethyl esters of higher fatty acids can be obtained when the yeasts are not removed from the wines before distillation, when sugar cane wax is added to the must before fermentation and when a yeast species, and even a yeast strain, is selected.

The higher fatty acid ethyl ester contents of distillates were three times higher with Saccharomyces cerevisiae S. 132 than with Saccharomyces cerevisiae Berlin II. The highest content was obtained with a strain of Schizosaccharomyces pombe, a native yeast of sugar cane growing Countries.

A correlation between the fatty acids composition of the lipids of the yeasts cells and the ethyl esters produced could not be conclusively established.

BIBLIOGRAPHY

BARAUD J.,MAURICE A., 1963. Les alcools et esters des eaux-de-vie de canne et de pomme. Ind. aliment. agric., (1), 3-7.

EL SHEHATA A. M., 1960. Yeasts isolated from sugar cane and its juice during the production of Aguardente de Cana. Appl. Microbial., (8), 73-75.

GUYMON J. F., CROWELL E. A., 1969. Gas chromatographic determination of ethyl esters of fatty acids in brandy or wine distillates. Amer. J. Enol. Vitic., 20 (2), 76-85.

HARRISSON J. S., GRAHAM J. C. J., 1970. In The Yeasts, vol. 3, Acad. Press, London.

KAHANE E., Rous S., 1961. Nouvelle methode d’extraction des lipides, in Enzymes of lipid metabolism 82-90, Pergamon Press, Oxford.

KERVEGANT D., 1946. Rhums et eaux-de-vie de canne. Les Editions du Golfe, Vannes.

LIEBICH H. M., KOENIG W. A., BAYER E. 1970. Analysis of the flavor of rum by gas liquid chromatography and mass spectrometry. J. Chromatogr. Sci., 8 (9), 527-533.

MAARSE H., ten NOEVER DE BRAUW M. C., 1966. The analysis of volatile components of Jamaica rum. J. Food Sci., 31, 951-955.

MARTIN J. T., JUNIPER B. E, 1970. The cuticles of plants. Ed. Arnold Publishers Ltd, Edinburgh.

NORDSTRÖM K., 1964. Studies on the formation of volatile esters in fermentation with brewer’s yeast. Svensk Kemisk Tidskrft, 76 (9), 510-543.

PEYNAUD E., 1956. Sur la formation d’acetate d’ethyle par les levures du vin. Industr. aliment. agric. 73 (4), 253-256.

RAINBOW C., 1970. Brewer’s yeasts, in The yeasts, vol. 3, Acad. Press, London.

STEVENS  R., 1965. Gas chromatographic identification of ethyl ester of fatty acids in domestic and imported rums. J. Ass. off. agric. Chem., 48 (4), 802-805.

SALO P., NYKANEN L., SUOMALAINEN H., 1972. Odor thresholds and relative intensities of volatile aroma components in an artificial beverage imitating whisky. J. Food Sci., 37 (3), 394-398.

SUOMALAINEN H., PUPUTTI E., NYKANEN L., 1968. Composition of the aroma in some brands of whisky and rum analyzed by customary methods and by gas chromatography. Kemian Teollisuus 25 (5), 399-404.