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This article was referenced by Problems Posed by the use of Schizosaccharomyces Pombe in the Making of Rums which is well worth a read.
Ann. Technol. agric., 1975, 24 (3-4), 421-436.
[I do not provide the original because I only had images and not a PDF. The images were also quite rough and it took a lot of effort to become legible.]
FORMATION OF HIGHER ALCOHOLS IN RUMS
A. PARFAIT et C. JOURET *
Station de Technologie,
Centre de Recherches agronomiques des Antilles et de la Guyane,
97170 Petit-Bourg (Guadeloupe)
Laboratoire de Technologie végétale,
Centre de Recherches de Toulouse,
Auzeville, 31.320 Castanet-Tolosan, B.P. 12
Rum is an eau-de-vie obtained after fermentation of molasses or sugar cane juice. The formation of higher alcohols obeys the same rules as those encountered in other fermented drinks.
Fermentation conditions may explain the relative importance of each higher alcohol content. Laboratory tests reported here were deliberately focused on various fermentation parameters easily transposable to the industrial level. It thus appears that the choice of yeast species and strain remain essential means of controlling higher alcohol formation.
Perspectives are also offered by the study of the influence of the nitrogen supplement of the fermentation medium on this production. The first tests show that higher alcohols quantities formed can be determined within a certain limit by the nature of the nitrogen source and by quantities added. At the same time, the key role that keto-butyric acid can play has been highlighted.
Higher alcohols are formed during fermentation of molasses and cane juice. They constitute part of the impurities that contribute to the taste of rums and cane eau-de-vie. Reduction in levels of these compounds is currently considered to be a factor in improving the organoleptic and nutritional quality of these products. For this, industry has above all endeavored to explore the possibilities offered by the development of distillation techniques. However, eaux-le-vie obtained under these distilling conditions are too neutral, because certain very aromatic compounds (heavy esters for example) are also largely eliminated. [cut off sentence…] progressed considerably. Not all of them have led to industrial applications because of the current fermentation technology for molasses and cane juice. In this review, we will recall the data on this problem and examine the resulting prospects for rum technology.
A. — Mechanism of higher alcohol formation.
It was chemists who were the first to take an interest in the “fusel oil” of eaux-de-vie. They found that alcohols other than ethanol were the largest fraction.
In 1906, Ehrlich was struck by the structural resemblance between higher alcohols and amino acids. NEUBAUER and FROMHERZ give in 1911 a more complete explanation of the Ehrlich reaction. SENTHE-SHANAUGNATHAN in 1958 showed that three enzymes catalysed this transformation: a transaminase, a carboxylase and an alcohol dehydrogenase. A little later, several authors, including GUYMON (1960), demonstrated that ketonic acids were common intermediates in the synthesis of higher alcohols and that of amino acids; they therefore derived, in part, from the metabolism of sugars. WEBB and INGRAHAM (1963) presented the main reactions in the biosynthesis of higher alcohols which are reported in Figure 1. The work of ÄYRÄPÄÄ (1971) currently constitutes a very interesting contribution in the search for control of the formation mechanisms.
During the oxidative degradation of lipids, aldehydes may appear which subsequently lead to the formation of higher alcohols. The latter are in very low quantity in rums and their biosynthesis will not be considered here. STEVENS (1960) showed that higher alcohols could also be formed during the synthesis of terpenes and sterols. This metabolic origin even for 3-methyl-1-butanol remains minor.
The Ehrlich reaction does not concern all amino acids. Ketoglutaric acid is the acceptor of the amino group in the biochemical reaction. Transaminase and carboxylase require cofactors which are pyridoxal-phosphate and thiamine-pyrophosphate respectively. In fact, there are two enzyme complexes, the first concerns the transamination, the second the rest of the reaction. The 2-keto-acid dehydrogenases constitute a system of three enzymes which can be dissociated: a keto-acid decarboxylase (coenzyme: thiamine-pyrophosphate, a lipoyl-reductase-transacylase (containing lipoic acid) and a dihydrolipoyl dehydrogenase (containing flavin-adenine-dinucleotide and nicotinamide-adenine-dinucleotide) The relative role of this mechanism in the total production of higher alcohols seems minor in all cases.
Several remarks can be made on the production of higher alcohols from compounds resulting from the metabolism of sugars. 3-Methyl-1-butanol, 2-methyl-1-butanol, 12-propanol and isobutanol constitute the bulk of the higher alcohols formed by this mechanism. The formation of higher alcohols follows a curve parallel to that of ethanol, but the relationship is not close with the use of amino acids. We refer to the ÄYRÄPÄÄ diagram to explain the essential role played by the pool of α-ketonic acids in these transformations (fig. 2).
The α-keto acids are formed inside the yeast cell, the protoplasmic membrane regulates their transport to the medium. Among these acids, α-keto butyric acid plays an essential role. The following amino acids: valine, isoleucine and leucine can exert a repressive action on the biosynthesis of the enzymes which lead to their formation. Concentration of assimilable nitrogen in fermentation media influences the final higher alcohol content. We have reported below results that we found on a synthetic medium with ammonium sulphate (fig. 3) and urea (fig. 4) as a nitrogen source. Other authors, including ÄYRÄPÄÄ, have arrived, using different nitrogen sources, at similar conclusions with these alcohols and with phenylethanol. The variation curve for this alcohol is similar to that of the isopentanols. Corresponding amino acids are not all found in fermentation media; even if we do not consider the case of n-propanol, we cannot accept the sole hypothesis of the repressive action by amino acids to explain the shape of the curve of higher alcohols variation.
Sugar concentration intervenes not only on the production of α-keto acids, but also on general yeast metabolism. It is currently difficult to give a complete explanation of its influence on the production of higher alcohols. ÄYRÄPÄÄ (1971) also studied variations in the quantities of higher alcohols obtained in relation to sugars consumed. When assimilable nitrogen has disappeared from the medium, synthesis of higher alcohols increases, except that of propanol (fig. 5). With molasses and cane juice, the fermentation media are most often supplemented with ammonium sulphate and urea which are used fairly quickly. In the technology of rums, one can wonder whether the use of slowly assimilated nitrogenous substances (delayed nitrogen) would not make it possible to control the formation of higher alcohols.
We have in a previous work (PARFAIT et al., 1972) made an inventory of ethyl esters of higher fatty acids in rums (table 1). These compounds have an important role in the formation of aroma. They have the same behavior during distillation as higher alcohols, which explains why, in certain rums having undergone a serious rectification, they are only found in trace amounts. Distillation does not seem to be able to easily solve this problem of the elimination of a significant part of the higher alcohols in the distillate without a parallel elimination of the heavy esters.
These are the considerations that led us to consider the use of delayed nitrogen in fermentation media. We will first review the old data on the possibilities of controlling higher alcohol content before considering some new hypotheses.
B. — Old Data.
YOKOTA and FAGERSON (1971) identified among the compounds of molasses 2-methyl-2-butanol, 2-methyl-1-propanol, n-propanol and 3-methyl-1-butanol. We do not know the origin and quality of the molasses he studied. Several authors and in particular LIEBICH et al. (1970) found the following higher alcohols in rum: n-propanol, n-butanol, 2-butanol, 2-methyl-1-propanol, n-pentanol, 3-methyl-1-butanol, 2-methyl-2 -butanol, 1-pentanol, 1-hexanol, 2-hexanol, phenylethanol. During distillation, and depending on the procedure, certain higher alcohols formed in the wine must not pass into the distillate. To control the contents of higher alcohols in cane spirits and rums, one can think of using well-known data on the general conditions of fermentation: temperature, pH, yeast strain, nitrogen, inoculation rate...
In the case of rum yeasts, optimum fermentation temperature is slightly below 30°C. In the range of 25 to 35°C, n-propanol varies little. The same is true for 2-methyl-1-butanol. On the other hand, reductions are a little greater for 2-methyl-1-propanol and 3-methyl-1-butanol (fig. 6). For molasses,the chosen temperature range is narrow. Industrial applications that can be drawn from these results are limited by the influence of temperature on the general metabolism of yeast. We worked on small volumes. Although industrial vats can be maintained at temperatures close to 30°C, it seems quite difficult to carry out fermentations on large volumes at a temperature of 35°C without affecting the fermentative power of the yeasts (MERRITT, 1966).
There is a maximum overall production of higher alcohols around pH = 4.5. For reasons similar to the previous ones, fermentations cannot be carried out at too low pH values. In industry, values of the order of 5 are adopted.
ARROYO (1945) had already demonstrated the advantage of choosing a given strain to control the higher alcohol content in wines. We have, from fermentation media in the French West Indies, isolated and operated a selection of yeast strains (PARFAIT and SABIN. 1975). Among the yeasts with good rum qualities, Schizosaccharomyces pombe are those which produce the least higher alcohols. They have the disadvantage of having a low growth rate. They have moreover been supplanted in fermentation media by Saccharomyces. For the latter yeasts, the following observations can be made: depending on the strain, the level of isobutanol varies little, the level of n-propanol can be multiplied by 4, that of the isopentanols increases by a factor of two. Some strains have low overall production; in our laboratory tests, it is around 120 g/hl of pure alcohol.
Molasses and cane juice must be supplemented by an external source of nitrogen in the fermentation media. FIG. 7 relates to tests with or without complementation. Nitroform is made up of several more or less soluble polymers of urea and formaldehyde. Urea and ammonium sulphate are good nitrogen supplements. They lead to a decrease in isopentanols, isobutanol varies little, n-propanol increases significantly. The results with addition of nitroform or without complementary addition of nitrogen are practically similar. Nitroform, according to our observations, is used up very slowly by yeast. [missing an entire line] quality of the additional nitrogen it must be able to be used by the yeast in the first hours of fermentation with sufficient speed.
The seeding rate leads to variable conclusions according to the authors. ENGAN (1972) thinks it is meaningless in most cases. GRATCHEVA et al. (1973) working at concentrations of 0.2 to 50 g/L find that the maximum quantity of higher alcohols corresponds to the greatest increase in absolute value of the yeasts for an initial quantity introduced. There is also a good correlation with the increase in biomass. In our opinion, the nature and the quantity of the raw material can explain these different results.
DUBOIS et al. (1968) compared grape wines produced using continuous and discontinuous fermentation techniques. In the first case, there is a decrease in higher alcohol content. ENGAN (1972) arrives with opposite results from beers. As in the previous case, it is difficult to draw general conclusions. There are certain relationships between the contents of musts in ketonic acids, higher alcohols and amino acids. These relationships are only one aspect of yeast activities during fermentation.
The need for rapid establishment of anaerobic conditions and presence of a sufficient quantity of vitamins can also be explained by the very nature of the fermentation phenomenon. To ensure its aerobic growth, the yeast is led to increase synthesis of amino acids and it can be expected, for this reason, that more higher alcohols will appear from these amino acids and their intermediates, α-keto acids. Finally, many growth factors seem to be involved in the mechanisms of formation of higher alcohols.
The nature of the sugars in the media based on molasses and cane juice does not appear to influence the formation of higher alcohols. We found identical results with sucrose, glucose and fructose.
In general, three data are used to control the contents of higher alcohols in fermentation media based on molasses and cane juice. Yeast strain choice is essential. In addition, media must be supplemented with a source of quality nitrogen so as to correctly ensure yeast growth with a sufficient initial quantity of nitrogen in the first hours of fermentation. Other factors, without being decisive, can allow the various yeast activities to proceed smoothly; starting from this, it is possible to maintain, within certain limits, production of higher alcohols.
C. — New Hypothesis.
All the previous results have one thing in common. There are, in general, no clear modifications of the mechanisms of formation of the higher alcohols. The use of mutants could lead to more characteristic results. The problems created by maintaining these strains in fermentation media have not been solved in all cases. Other solutions must be sought.
ÄYRÄPÄÄ (1971) showed that at the start of fermentation, in the case where assimilable nitrogen is not yet exhausted, quantities of higher alcohols formed for given consumptions of sugars are practically the same. After exhaustion of available nitrogen (fig. 5) this is no longer the case. In discontinuous fermentations on molasses and on cane juice, musts must be supplemented with an external source of nitrogen. All nitrogenous forms of the raw material are not equally assimilable under the operating conditions. Ammonium sulphate and urea which are usually added are fairly quickly used by yeast. This shows the potential interest in researching consequences of an additional supplement of delayed nitrogen in fermentation media.
We conducted tests under these conditions on a simplifed medium and on a molasses-based medium. The results are shown in Figures 8 and 9. On the simple medium, for urea concentrations below 300 mg/L, addition of a delayed nitrogen can lead to modifications in the production of certain higher alcohols. Isobutanol contents vary little, those of 1-propanol increase, those of isopentanol decrease. For contents greater than or equal to 300 mg/L in urea, it seems that the addition of delayed nitrogen does not have a specific character in comparison with that of urea for example. While in the first case total production of higher alcohols varies little, in the other cases it may increase due to increases brought by n-propanol and isobutanol. In the molasses medium supplemented with 200 mg/l of nitrogen provided by ammonium sulphate, results similar to those obtained in the first part of the preceding tests are found. The n-propanol increases and the isopentanols decrease.
All previous tests were conducted with a single strain of yeast which is a good producer of higher alcohols. At first sight, one can think that we arrive at conclusions close to those which figure 7 brings: increase of propanol and reduction of isopentanols. This is not exactly the case. High amounts of ammonium sulphate and urea lead to a drop in pH which has an effect on general yeast metabolism (stopping of fermentation). There can therefore be a decrease in higher alcohol production due to a lower fermentation efficiency. A similar phenomenon is not observed for nitroform; it even seems that for a certain level of nitroform concentration there is an increase in fermentation efficiency. Results are too fragmentary to go further in the discussion. Indeed, for a molasses medium, raw material composition in different nitrogenous forms and corresponding contents can be significant. For all yeast strains, the shapes of the higher alcohol formation curves are similar. Concentrations of higher alcohols from the same procedure are not the same. It is therefore necessary to better specify the methods according to which the nitrogen sources are added.
In view of these results, the role played by α-ketobutyric acid is highlighted. It is a common intermediate in the biosynthesis of isopentanols (especially 2-methyl-1-butanol and n-propanol). If we refer to figure 10, between threonine, isoleucine and higher alcohols, there are several possible links GUYMON et al. (1961) had studied higher alcohol formation from three mutants deficient in threonine, one produced neither n-propanol nor 2-methyl-1-butanol, the other two produced it. Existence of metabolic pathways A and B explain these behaviors. One can wonder if the different productions in n-propanol that we observed in our strains cannot be explained by a variable functioning of these pathways A and B. In our next tests, we will use in parallel yeasts having productions varied in n-propanol. We will determine, in each case, the final contents of higher alcohols in the fermentation media of known composition where a delayed nitrogen will have been introduced.
Further work may lead to interesting results. This is the case for the determination during fermentation of the respective concentrations of higher alcohols, ketonic acids and amino acids ÄYRÄPÄÄ (1971) found that the depletion of nitrogen supplied in the fermentation medium did not modify the rate of formation of the whole [leucine + 3-methyl-1-butanol] unlike what happened for the sets [isoleucine + 2-methyl-1butanol] and [valine + isobutanol] where there was a reduction. However, α-ketoisovaleric acid is a common precursor in the synthesis of isobutanol and isoamyl alcohols. Formation of these latter alcohols also involves the supply of an acetyl CoA. Necessary energy and acetyl CoA available for the course of the reactions could explain certain facts. ARROYO (1945) had noted that co-inoculation of certain Clostridia in fermenting media with Saccharomyces cerevisae or Schizosaccharomyces pombe caused a reduction in the quantities of higher alcohols formed. We noted under these conditions that there was an increase in the contents of volatile acids and ethyl acetate in particular.
The work of HARVALIA et al. (1974) indicate existence of a relationship between higher alcohol content in wines and content of nitrogenous substances, in particular amino acids, in grape must. MONTREAU and DUFRENOT (1974) obtained by fermentation from the juice of six cane varieties different productions in each higher alcohol. Information we have on the amino acid content of canes is insufficient to draw any other conclusions. It can however be said that the contents molasses in different nitrogenous fractions and in particular in amino acids must also be related to the final quantities of higher alcohols. The source of supplemental nitrogen, as we have seen above, can modify this relationship which should not be simple.
Mechanisms of higher alcohol formation are known. Some intermediate steps and the possibilities of regulation of different pathways are incompletely elucidated. A number of data relating to yeast and fermentation conditions have so far made it possible to partially control concentrations of higher alcohols in the media at the end of the operation. Beneficial aromatic compounds exhibit the same behavior during distillation as higher alcohols. There is therefore an interest in seeking to reduce in significant proportions formation of higher alcohols during fermentation. New hypotheses are the subject of multiple verifications. They are based for the most part on the influence of the nitrogenous diet (nitrogen of the raw material, plus nitrogen supplements) on the metabolism of the higher alcohol, ketonic acid, amino acid combination.
SUMMARY [supplied in English]
FORMATION OF HIGHER ALCOHOLS IN RUMS
Rum is a brandy obtained after fermentation of molasses or sugar-cane juice. The formation of higher alcohols is submitted to the same rules as the ones observed for other fermented beverages.
The fermentation conditions may explain the relative importance of the percentages of each higher alcohol. The laboratory trials described here have been deliberately limited to the different fermentation parameters which can be easily transposed at the industrial level. The choice of the species and of the strain of yeast seem to be the main factors for controlling the formation of higher alcohols.
New prospects are also opened up by the study of the influence of the nitrogen complement added to the fermentation medium on this production. The first trials show that the amounts of higher alcohols formed can be determined under certain conditions by the nature of the nitrogen source and by the quantities added. The key role that may play the ketobutyric acid was also stressed.
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