Problems Posed by the use of Schizosaccharomyces Pombe in the Making of Rums

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Ganou-Parfait B., Parfait A., 1980. Problèmes posés par l’utilisation de Schizosaccharomyces pombe dans la fabrication des rhums. Industries alimentaires et Agricoles 97, 575-580.

Problems posed by the use of Schizosaccharomyces pombe in the making of rums

by B. GANOU-PARFAIT and A. PARFAIT
Station de Technologie des Produits Végétaux
Centre de Recherches des Antilles et de la Guyane, INRA
97.17O PETIT-BOURG -Guadeloupe 

SUMMARY

Schizosaccharomyces pombe can be used like Saccharomyces cerevisiae in rum technology. Strains of S. pombe have been selected for microbiological and biochemical studies. A medium with cane juice is proposed. The rates of the fermentation can be increased with yeast concentration. According to the formation of the major volatile components S. pombe seems better than Saccharomyces; nevertheless other studies are necessary to confirm potentialities.

[this was supplied in English the French which I translate below comes out noticeably different.]

Summary
In the production of rums, strains of Schizosaccharomyces pombe and those of Saccharomyces cerevisiae are used. A selection was conducted to have a collection of Schizosaccharomyces pombe on which microbiological, biochemical and technological studies were conducted. A culture medium based on cane juice is proposed. Fermentation rates are generally low, but we want to accelerate the fermentations using significant seeding rates. The level of formation of the major components among the volatiles should give preference to Schizosaccharomyces pombe. It turns out that further work is needed to allow the reintroduction of Schizosaccharomyces pombe under the best conditions in fermentation media.

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In general, the use of the selected yeasts have several advantages in fermentations leading to alcoholic beverages. The choice of corresponding yeast species and strains obeys a certain number of criteria which are fixed, but it can also be the consequence of a given industrial situation. These particular considerations are for molasses, syrups and cane juices that are raw materials in fermentations leading to rums.

Kervégant (1946) collected a series of observations on schizosaccharomyces pombe in rum. This yeast was present in fermentations, especially molasses and syrup. Several species and several strains were known. In the production of rums, budding yeasts of the Saccharomyces cerevisiae type have been preferred to Schizosaccharomyces pombe because the former are in general faster.

The use of gas chromatography, alone or in combination with other techniques, makes it possible more and more to make a complete analysis of rums under conditions that are generally quite easy. It is therefore possible to propose quality criteria for rums: absence or presence of certain compounds at given concentrations. To meet these requirements it is sometimes necessary to resort to technological innovation.

Following our observations in the French West Indies, Parfait et al. 1975 and in view of current techniques used elsewhere in the world – Kampen (1975) – it is likely that the rum industry will experience such a situation. It is therefore reasonable to envisage the reintroduction of Schizosaccharomyces pombe in fermentative media.

SELECTION OF STRAINS OF SCHIZOSACCHAROMYCES POMBE

These yeasts are common in tropical environments. Several authors have reported them in fermentations of derivatives of sugar cane. Like all Schizosaccharomyces, the essential physiological characteristic is division by fission. The spherical to cylindrical cells are often larger than those of other yeasts and in particular those of Saccharomyces cerevisiae. We first made microscopic observations. The identification of colonies obtained after plating a colony on Petri dishes is done by the method of Lodder and Van Rij. The Schizosaccharomyces pombe cells are practically absent in fermentative environments in the French West Indies, except in the case where heave-flavored rums are manufactured. These same cells are found in certain soils where sugar cane is cultivated, but there they are in very small numbers. They are much larger in the fermented musts of small distilleries in Haiti. For decades, they have not changed their manufacturing conditions, and they are often isolated in the middle of the countryside. We can therefore estimate that the modifications of the flora have been practically nil. In all cases, to facilitate the selection of strains of Shizosaccharomyces, different properties are used Ganou-Parfait (1979).

Some are mentioned below:

Table 1

Use of citric acid by S. pombe. (+) low growth, (-) no growth The concentration of citric acid is 0.5%

Table 2

The influence of butyric acid on a mixture of yeasts. The seeding rate, 1 × 10 6 / ml for each yeast, Count of revivifiable cells after 76 hours of culture at 30° C. in a medium with malt extract containing 150% of sucrose.

Sensitivity to Acids.

In rums, in general, acetic acid is the most important constituent of the acid fraction, of which it accounts for nearly 80% of the total. For a type of rum represented by the large aroma rums, the butyric acid fraction is also significant. We compared the behavior of Schizosaccharomyces pombe and Saccharomyces cerevisiae strains in the presence of varying amounts of different acids. The comparison was made either aerobically or anaerobically, and the determination of the number of total germs by the Malassez cell made it possible to measure the sensitivity of yeasts to acids.

In the case of citric acid, 10 ml of medium are placed in test tubes. In each case, citric acid is added at a concentration of 0.5%. The results are obtained on strains of Schizosaccharomyces pombe, they are shown in Table 1, and are not better if the citric acid is replaced by malic acid. Note that some authors have found that in the case of the latter acid, there is a sharp reduction in the growth of Saccharomyces cerevisiae for concentrations ranging from 0.2 to 0.4% malic acid.

The influence of increasing amounts of acetic acid on yeast growth is well known. There is a slowdown in the fermentation rate and a decrease in the amount of sugar used. The results are identical with butyric acid.

To compare the influence of the latter on a mixture of Saccharomyces cerevisiae and Schizosaccharomyces pombe, we made a count of total germs after sixteen hours. The medium used is the following: 10 ml of malt extract supplemented with sucrose at a rate of 150 g/l. The seeding rate is 1 X 10 6/ml for each yeast. The butyric acid slows down according to the concentration used, the growth of Saccharomyces cerevisiae and has a variable effect on that of Schizosaccharomyces pombe. In case we want to make a selective medium to isolate Schizosaccharomyces pombe in view of previous results the addition of 0.25% butyric acid could be a formula. However, experience shows that the results obtained on a liquid medium are not transposable in a solid medium. In all our experiments, the growth of Saccharomyces cerevisiae has always superseded that of Schizosaccharomyces pombe.

Search for a favorable environment for Schizosaccharomyces pombe

During microscopic observations, it was found that in natural samples, Schizosaccharomyces pombe cells had a granular appearance which disappeared after multiplication of the cells in a favorable medium. We have sorted in a large number of culture media used for yeasts. During this operation the following conclusions were reached:

Peptones represent a better source of nitrogen than ammonium salts. In fact, when comparing the ammonium salts with each other, it is found that the acidity induced by the anion is an essential factor. The optimum pH is of the order of 5, but the pH range is from 4 to 6. Sucrose is better assimilated than glucose.

We compared several synthetic media: Wickerham malt, Czapek, Dox Agar, Davis Yeast Salt Agar, malt extract. This last medium supplemented with sucrose gives the best results. We made different media formulas from cane juice and molasses. It is with cane juice that we have the best results. We therefore propose the following medium:

— Peptone = 1 g
— Ammonium Sulfate = 2 g
— Cane Juice = 1.000 ml.

The pH is adjusted to 5, sterilized at 120 ° C for 15 minutes. In order to reduce the importance of flocculation during sterilization, peeled sugar canes are used.

Selection Results

Of all the samples we have studied, we have extracted a strain of yeast to make rums of great aroma, and sixty strains from different media collected in Haiti. Beside these last strains, we also found about ten strains of Schizosaccharomyces malidevorans. This species is easily distinguished because it is the only Schizosaccharomyces that does not use maltose. It should be noted that Schizosaccharomyces sporulate with difficulty, whatever the medium used.

GROWTH OF STRAINS OF SCHIZOSACCHAROMYCES POMBE

Generally, Schizosaccharomyces pombe is considered to have a low growth rate. In order to get closer to the industrial criteria, we used the procedure below to compare the strains.

The cells proliferate for 72 hours at 30° C on agitated Wickerham malt medium. They are recovered by centrifugation 3,000 tr/15 minutes, and then washed. After counting, a molasses-based medium is inoculated at 1 X 10 6 yeast / ml. The fermentation is carried out in 125 ml flasks closed with a rubber stopper crossed by a tapered glass tube at one end, and plugged at the other end with carded cotton.

The environment is as follows:

— Molasses 300 g
— Ammonium Sulfate = 1 g
— Water q.s.p. = 1.000 m)
— pH = 5.2, sterilization 15 minutes at 110°C.

The fermentation curves are plotted in Figure 1. From this examination it appears that the lag phase is longer for Schizosaccharomyces pombe and that overall the fermentation rate is lower than that of Saccharomyces cerevisiae.

But fermentation rates can be varied by increasing seeding rates. The tests are conducted under the same conditions as above, with different seeding rates determined by the dry matter. Fermentation rates are conventionally represented by the mass losses of each vial after 24 hours.

Table III Fermentation speed for increasing rates of seeding with Schizosaccharomyces pombe

We find in Table III results similar to those we found with S. cerevisiae. It can therefore be estimated that for large seeding rates (2 to 5 g/L) the behavior of these two yeast species is close.

Use in Industrial Fermentation

For twenty years, there has been an interest in the use of Schizosaccharomyces pombe to deacidify wines, Bidan (1974). During this operation, the malic acid is converted into ethanol.

In his important work on rums, Arroyo found that both species Schizosaccharomyces pombe and Saccharomyces cerevisiae could both provide good products. In making rums, he advocated the second because it fermented faster. Recently, Rose (1976) has selected S. pombe strains from yeasts that can produce from molasses musts an alcohol content of 11° to 12° GL. Such a concentration of ethanol makes it possible, compared with conventional methods, to reduce the quantities of energy required during distillation relative to wines of 4-5 ° GL.

Today, S. pombe is a fermentative agent of cane molasses next to several Clostridia including Clostridium acetobutylicum in the manufacture of grand arôme. This type of rum in the French West Indies is characterized by a high level of non-alcohol (800-1,800 g/hl pure alcohol). In detail, there is a significant fraction of ethyl acetate and acetic acid, about 300 grams for each term and a small amount of higher alcohols — less than 100 g — with a abundance of n-propanol. The musts are composed with vinasses [stillage or dunder] that have surely undergone the phenomena of pre-fermentation. They are rich in volatile acids and in fixed acids. Fermentations are slow and must involve different metabolic pathways that have not yet been fully elucidated.

Products of Fermentation

Among the compounds found in rums, some are already present in molasses as a result of various more or less advanced prefermentations. But yeast is mainly responsible for their formation during the alcoholic fermentation. In various previous works, Parfait (1977-79), we have studied certain products of the molasses fermentation by S. pombe, which can be referred to for the various procedures.

a. Ethyl esters of higher fatty acids

Pombe produces more of these compounds than most baker’s yeasts of the species Saccharomyces cerevisiae, but some good yeasts in our collection belonging to this species have equivalent productions to S. pombe. This production is related (FIG. 2) with the cell growth yield that can be appreciated by the ratio of final yeast to initial yeast. For seeding rates between 0.1 and 5 g /L yeast dry matter, there is a correlation between the amount of ester produced and the cell yield. This proportionality is also checked for each ester in the series.

b. Ethyl Acetate

In quantity, ethyl acetate is the main ester of rums made with Saccharomyces cerevisiae. Under the same conditions of fermentation and distillation S. pombe brings a double production, 100 g/hl of pure alcohol instead of 50 g/hl pure alcohol. In industrial rums of high aroma type, the production is very strong, more than 300 g/ hl pure alcohol without the production of ethyl esters of higher fatty acids is affected. Esterification is primarily a biochemical phenomenon, distillation in the presence of yeasts can increase the levels of ethyl esters of rums, but their formation involves acetyl CO A. If we are inoculating a must of molasses with a mixed culture of Schizosaccharomyces pombe and Clostridium acetobutylicum, the presence of the bacterium has the effect of increasing the amount of ethyl acetate formed. One may wonder if under certain culture conditions, especially in musts leading to rums of high aroma type where the medium is already rich in acetic acid, there is no different functioning of esterase.

c. Higher Fatty Acids

Caprylic and capric acids are the major constituents of this fraction of rums made with Saccharomyces cerevisiae or Schizosaccharomyces pombe. Temperature and pH affect the total production of higher fatty acids, just as they affect the general activity of yeast. Depending on the sugar concentration, fatty acid concentrations increase in rums made from Schizosaccharomyces pombe, except for caprylic (Table IV).

Table IV: Influence of molasses concentration on the formation of higher fatty acids. The results for the fatty acids are expressed in mg/l of pure alcohol. The initial seeding rate is 3 g/l

d. Acetic Acid

In pure culture, the productions of acetic acid are comparable for Schizosaccharomyces pombe and Saccharomyces cerevisiae. The high levels of acetic acid found in the aroma of rums originate from the vinasses which enter into the composition of the must and the further extraction during the distillation.

e. The Higher Alcohols

We have already reviewed the mechanisms of formation of higher alcohols, Parfait (1975). The Schizosaccharomyces pombe strains generally provide fewer higher alcohols than those of Saccharomyces cerevisiae, and this with a predominance for n-propanol.

DISCUSSION

A number of questions arise when using Schizosaccharomyces pombe in the production of rums.

The growth of yeast is lagging at low seeding rates. It is possible to accelerate the fermentations by increasing this rate. This technological device must not obscure the different physiological behaviors of Saccharomyces cerevisiae and Schizosaccharomyces pombe. In the study of a suitable medium for the culture of this last yeast, we found that sucrose was better than glucose. This result was explained by Hayashibe (1973). The growth curves are not the same for glucose and mannose on the one hand, and sucrose on the other hand; but fermentation rates are the same when using cell extracts. It can therefore be linked to sugar transport phenomena. Billon-Grand (1977) demonstrated the existence of intracellular enzymes 1α and β glucosidases and invertase or β fructofuranosidase, capable of degrading these sugars. As often in this case, the transport of sugars is facilitated by the addition of NH4 + ions in the medium. It should be noted that Schizosaccharomyces pombe does not use glycerol and ethanol. This difference with Saccharomyces may partly explain the high levels of glycerol. Many studies have been done on the influence of oleic acid and sterols in the anaerobic metabolism of several yeasts. Very little data has been established on the fatty acid and lipid composition of yeasts of the genus Schizosaccharomyces. Their obtaining will thus explain the importance of the lag phase for these microorganisms. Bush (1977) has confirmed the absence of mannan which plays a role in the budding process of several yeasts, but the presence of galactomannan raises the question of the nature of the compounds that play a role in the fission process. Similarly, there is a difference in the plasticity of the cell wall and its protective role vis-à-vis the cellular content. Ultimately, the composition of the cell membrane and its impact on Schizosaccharomyces pombe metabolism are important enough to explain the differences in physiological behavior with Saccharomyces.

Of the volatile compounds produced during fermentation by Schizosaccharomyces pombe, special mention must be made of ethyl acetate and higher alcohols. Part of the ethyl acetate arises as a result of the oxidative decarboxylation of pyruvic acid and an alcoholysis reaction:
(1) CH3CO COOH — NAD — CoA v SH –———>

CH3CO v SCoA + NADH2 + CO2

[not sure about this notation and what the italicized “v” stands for]

(2) CH3CO SCOA + CH3CH2OH ——->
CH3 COOC2H5 + HS v CoA

But the high concentration of acetic acid that exists in some musts may explain the formation of ethyl acetate by shifting the equilibrium during the reaction.

(3) CH3 COOH + CH3CH2OH ⇔ CH3 COOCH2
CH2 + H2O

We will undertake enzymatic and kinetic studies of these three reactions to justify the different levels of ethyl acetate found in rums.

The amounts of each higher alcohol manufactured by Schizosaccharomyces pombe are quite remarkable: low levels of methyl-3-butanol. 1, methyl 2 – butanol 1, and isobutanol, against a higher propanol content. The latter is manufactured in the following way:

Thréonine –» amino acid – 2 butenoïque –» acide
thréonine déhydratase
deaminase σ cétobutyrique –> CO2 — τn propanaldéhyde –>
n propano
décarboxylation déhydrogénase

[I’m insecure about translating this section. Any help? and background on it?]

This route for propanol is specific, even though it contains σ ketobutyric acid, which is a key intermediate in the biosynthetic formation of other higher alcohols. We have done a nearly complete study of the formation of higher alcohols in rums. It appears necessary, in the case of Schizosaccharomyces pombe, to determine the variations of the amino acid pool, taking into account the ambient factors and in particular the nitrogen diet during the fermentation.

Without waiting for its results, our first observations – Pafait, (1977) – showed that by means of the acceleration of fermentations can ferment with Schizosaccharomyces pombe molasses musts containing 150 to 180 g/l of sugar under conditions as well as can Saccharomyces cerevisiae. For this last yeast, it appears that the choice of the strain is determining in the level of formation of the volatile products and in the fermentative efficiency. This is also the case for Schizosaccharomyces pombe and some strains show, in particular, a very low fermentative efficiency. The properties of these yeasts begin to be explained through different biochemical studies.

Here we have specified a number of pathways (cell membrane formation and metabolite transport, kinetics of ethyl acetate formation, composition and amino acid pool variation) that are promising. Besides this, an organoleptic study of rums is needed. We chose the technique of Micko – Parfait, (1979) – for the tasting of rums and cane spirits. Equivalent fractions may have a different flavor depending on the yeast and the strain that served as the fermentation agent. The perception thresholds of each constituent are not the same depending on whether they are used alone or in association with other bodies. Nowadays, the distillates obtained from Schizosaccharomyces pombe and Saccharomyces cerevisiae have different contents, at least for the main constituents: higher alcohols, aldehyde and ethyl acetate. Finally, it is as a result of various technological operations, fermentation, distillation, assembly, maturation that the rums obtained from Saccharomyces cerevisiae present a composition and a favor given. The reintroduction of Schizosaccharomyces pombe in fermentation media will allow these different operations to be carried out under other conditions to obtain products equivalent to those which now exist.

CONCLUSION

Different studies have shown that the current compositional criteria for rums can be more easily achieved with Schizosaccharomyces pombe as a fermentative agent, rather than Saccharomyces cerevisiae. The selection of strains of this first yeast, even in favorable ecological environments, has only been made possible by a study of some of its microbiological and physiological properties. The use of Schizosaccharomyces pombe in musts made from molasses and sugar cane juice poses a series of biochemical, technological and organoleptic problems whose solution lies in a better knowledge of the metabolic pathways. This preliminary work made it possible to determine the axes that will be the subject of future research.

BBLOGRAPHE

G. BILLON-GRAND (1977). – Recherche d’enzymes intracellulaires dans le genre Schizosaccharomyces, lmplications systématiques. Mycopathology, 61 (2), 111-115 

P. Bidan (1974). — Les Schizosaccharomyces en CEnologie.
Bull. OIV, 47 (523), 682-706.

D.A. BUSH, M. HORISBERGER, I. HORMAN, P. WURSCH, 1977. – The wall structure of Schizosaccharomyces pombe. Nestlé research News, 73-77.

M. HAYASHIBE, N. SANDO, Y. OHBA, K. NAKAMURA, K. DKA., K. KONNO, M. GOYO (1973). — Utilisation of hexoses in fission yeast. Proceedings of the 3rd international specialized symposium on yeast OTANIEN. Helsinki Part, II, 91-102.

B. GANOU, PARFAIT, 1979. — Les microorganismes des fermentations de mélasse et de jus de cannne. 1979 (en préparation).

D. KAMPEN (1975). – Technology of the rum industry. Sugar y azucar, 70 (8), 36-43.

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

A. PARFAIT (1972). — Les esters éthyliques des acides gras supérieurs de rhums. Ann. Technol. Agric.., 21 (2), 199-210.

A. PARFAIT (1975). – Formation des alcools supérieurs dans les rhums. Ann. Technol. Agric., 24 (3-4), 421-436.

A. PARFAIT et G. SABIN (1975). — Les fermentations traditionnelles de mélasses et de jus de canne aux Antilles françaises. Industries alimentaires et agricoles, 2 (1) 27-30.

A. PARFAIT (1977). — La fabrication des rhums. Rapport d’un contrat DGRST, No 74-7-09-06.

A. PARFAIT (1979). — Suite de l’étude sur la fabrication des rhums. Rapport d’un contrat DGRST, No 77-7-03-55.

D. ROSE (1976). – Yeasts for Molasses alcohol. Process Biochemistry, 12 (2), 10-16.

Fermentation Properties of Rhumerie Yeasts

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Fahrasmane L., Parfait A., Galzy P., 1986. Propriétés fermentaires des levures de fermentation. Industries alimentaires et Agricoles 103, 125-127.

Fermentation Properties of Rhumerie Yeasts
by L. Fahrasmane*, A. Parfait*, P. Galzy**

* Station de Technologie INRA-Antilles – Domaine Duclos 97170 Petit-Bourg
** Laboratoire de la chaire génétique ENSAM, Place Viala, 34060 Montpellier

Introduction
Fermentations of molasses and sugar cane juice take place in the West Indies, in a non-sterile environment (Parfait and Sabin, 1975). Formerly, the dominant yeast species was Schizosaccharomyces pombe Lindner. This yeast is osmophilic and often gives rums of quality in association with an abundant bacterial flora. She was most often supplanted by Saccharomyces cerevisiae Hansen. The latter species, baker’s yeast, was commercially available in bulk and at low prices. It was therefore tempting for manufacturers to regulate and accelerate fermentations by massive sowing of baker’s yeast.

The purpose of this note is to compare the fermentative properties of these two species which are still the pivot of rum fermentations. We will not present here the result of a particular experiment, but rather a synthesis of several independent studies carried out on laboratory strains in sterile medium (Parfait et al., Perfect et Jouret, 1975, 1979, Fahrasmane, 1983, Fahrasmane et al., 1985); these results are discussed in the light of numerous industry observations and long experience in making rums acquired by some of us.

Material and Methods

1. Biological Materials
Most of the works summarized or cited here have been done with a large number of Strains. However, to simplify the presentation we have limited ourselves voluntarily to give results of a strain of each species considered representative. These two strains are:

– Saccharomyces cerevisiae listed 493,
– Schizosaccharomyces pombe listed G.

2. Culture Media
We used a cane juice (vesou) from natural and health canes, diluted to 100 g/l of sugar; a molasses-based medium also reduced to 100 g/l of sugar and a synthetic medium according to Oura (1974) supplemented with the main organic acids of cane juice according to Fahrasmane (1983).

3. Analysis Techniques
We used the Classic methods of rums study, including:

-the official method of assaying the higher alcohols in the eaux de vie (Fraud Control, Anonymous, 1973).
-Jouret’s method for the determination of short chain fatty acids described by Fahrasmane et al. (1983).
-The method described by Parfait et al. (1972) for the determination of ethyl esters of higher fatty acids.

Experimental Results

I. Biomass and ethanol production

Schizosaccharomyces pombe generally gives slow growth and a relatively small amount of biomass, much lower than that obtained with Saccharomyce cerevisiae (table 1). The difference between the two species fades in the case of a mixed culture. It Seems that Schizosaccharomyces pombe has special nutritional requirements that it does not find on synthetic medium or on cane juice (Vesou); on the contrary, it finds them in the much richer environment constituted by molasses. This result suggests difficulties in all industrial uses of Schizosaccharomyces pombe. The addition in a synthetic medium of the organic acids of the cane juice, in particular of cis-Aconitic acid, causes an abundant cell multiplication. This result suggests that these acids activate cell multiplication by probably intervening in the Krebs Cycle. He also explains that the yeast populations observed in crops on cane products are still exceptionally abundant. Correlatively, the yield of ethanol is not very good in rum fermentation.

The yield of ethanol expressed as a percentage of the Pasteur yield is always higher, in pure culture, for Schizosaccharomyces pombe than for Saccharomyces cerevisiae. This observation very largely explains the current craze of certain distillers who recommend the use of Schizosaccharomyces pombe.

The fermentation times are always very long for Schizosaccharomyces pombe. As a result, the fermentation medium is always more sensitive to bacterial contamination. The duration of the fermentation become extremely long on synthetic medium; the use of Schizosaccharomyces pombe for fermenting new substrates in relatively poor environments certainly has an indisputable randomness.

It should be noted that Schizosaccharomyces pombe produces significant amounts of glycerol (8 to 10 g/l per 100 grams of fermented sugar); under the same conditions, Saccharomyces cerevisiae produces only 2-3 g/l (Parfait and Jouret, 1980). Given the large bacterial flora able to attack glycerol in rum fermentation, this character is certainly a serious problem for the use of Schizosaccharomyces роmbe.

II. Formation of Higher Alcohols

Using the same culture media we studied the higher alcohols produced by both strains (Table II).

Schizosaccharomyces pombe produces far fewer higher alcohols than Saccharomyces cerevisiae. However, it appears again here that Schizosaccharomyces pombe is more sensitive to environmental conditions than Saccharomyces cerevisiae. While the latter species gives total higher alcohol concentrations substantially independent of the culture conditions, Schizosaccaromyces pombe produces twice as much higher alcohols in molasses culture than in the other Crop Conditions tested.

III. Formation of Volatile Fatty Acids

Again (Table 3), Schizosaccharomyces pombe produces much less short-chain fatty acids, important constituents of the aroma of rums, than Saccharomyces cerevisiae. It is worth mentioning that both species produce propionic acid on cane juice medium. Only Schizosaccharomyces pombe produces acrylic acid; it is probable that propionic acid is the precursor of acrylic acid. It is also likely that sugarcane media contain a propionic acid precursor for use by both yeasts.

In cultures on product derived from sugar cane (molasses) it also appears in the medium of long chain fatty acids C8 to C16 as well as the corresponding ethyl esters. Fermentation of 100 g of sugar yields about 80 to 100 mg/l of these esters regardless of the yeast species used (Parfait et al., 1972).

Conclusion
Schizosaccharomyces pombe presents in the laboratory the considerable advantage of giving a high yield of ethanol; it also has the advantage of giving relatively few higher alcohols and fatty acids. In fact, it seems obvious that these two advantages are related. Low cell growth, partly indirectly responsible for good ethanol yield, is not only beneficial; a slow and slow growth of the yeasts largely leaves room for bacterial developments. The abundant production of glycerol is also a favorable factor for the development of many germs, some aroma beneficial, other sources of manufacturing flaws. These general properties should make Schizosaccharomyces pombe a good strain of rum fermentation: it is able to give very aromatic rums with a good bacterial flora; it could give very light rums, particularly sought after, as long as one manages to control the flora; unfortunately manufacturing flaws can occur.

In recent years, it has been sought by industrialists for new substrates for the production of ethanol. Schizosaccharomyces pombe could a priori be suitable for the production of alcohol for chemical use or rectified alcohol as only a few secondary products are formed. The results we have presented show that this species is very demanding from the point of view of growth needs. This can result in a significant over-cost related to the need to complement the new fermentation media. The relative fragility of the fermentative medium with respect to bacterial contamination is also a disadvantage that should not be underestimated.

Saccharomyces cerevisiae gives higher amounts of higher alcohols and fatty acids; the yield of ethanol is a little lower than that observed in Schizosaccharomyces pombe. But the growth is fast and abundant, the occupation of the ground is good, the danger of serious bacterial accidents is reduced. This species ultimately makes it possible to obtain relatively light rums. For fermentations of new products, this species has definite advantages, provided that the substrate to be fermented is accessible (hexose, sucrose or maltose).

The characteristics of these two species explain fairly well the evolution of the rum fermentation technique. In the past, rum was prepared almost exclusively from molasses.

Vinasses [dunder or stillage] were recycled as a means of diluting molasses. Thus the fermentation medium was rich in mineral salts, nitrogenous matter. Osmotic pressure was important. This medium was favorable to Schizosaccharomyces pombe which was naturally selected. This system also favored the preferential proliferation of heat-resistant, sporulated, anaerobic bacteria. This resulted in a very particular type of rum. The sugar crisis helped, it was made more and more of direct fermentation of Vesou [fresh cane juice]. The osmotic pressure became much weaker here. The medium was poorer in biotic elements and lacked nitrogen feed for the yeast. Under these conditions, it was inevitable that Saccharomyces cerevisiae would replace Schizosaccharomyces pombe. In the same way, the dominant bacteria flora became naturally present on sugar cane: aerobic Coryneform bacteria, aerobic Bacillus, and lactic flora. The Yeast defend better against this type of flora, it resulted in a lighter rum and better suited to current consumption. It seems clear to us that the lessons learned from a reflection on the rum industry are not without interest for other ethanol manufacturing industries be it alcohol, alcohol for industrial use or alcohol fuel.

It would be useful to better understand the nutritional requirements and the general metabolism of the fermentation strains of these two species. This work becomes more and more necessary as the variety of used substrates expands. Let’s mention in the case of rum the range of raw materials: vesou, juice defecated, syrup and molasses at various stages including molasses final.

[The vesou here as opposed to defecated juice may refer to what Cape Verde uses which isn’t centrifuged and strained.]

Bibliography

FAHRASMANE L. – 1983 – Contribution à l’étude de la formation des acides gras Courts et des alcools supérieurs par des levures de rhumerie. Thèse de 3° cycle. USTL Montpellier.

FAHRASMANEL, PARFAITA., JOURETC., GALZY P. – Production of higher alcohols and short chain fatty acids by different yeats used in rum fermentation. Accepte pour publication le 22 avril 1985 par Journal of Food Science.

OURA E. – 1974 – Some aspects of aeration intensity on the biochemical composition of baker’s yeast. 1. – Factors affecting the type of metabolism. Biotechnol-Bioeng. 16, 9, 1197.

PARFAITA., NAMORY M., DUBOIS P. — 1972 — Les esters éthyliques des acides gras supérieurs des rhums. Ann. Technol. Agric., 21, 2, 199-210.

PARFAITA., SABIN G. — 1975 – Les fermentations traditionnelles de mélasse et de jus de canne aux Antilles Françaises. Ind. Agric. Alim., 92, 1, 27-34.

PARFAITA, JOURET C. — 1979 – Rapport fin de Contrat DGRST. Décision d’aide n° 74 70906 et 74 7O 907.

PARFAITA., JOURETC. – 1980 – Le glycérol dans la fermentation alcoolique des mélasses et des jus de canne à sucre. Industries alimentaires et agricoles, 7-8, 721-724.

Répression des fraudes – 1973 – Méthodes officielles d’analyse des alcools et eauxde-vie. J.O. de la République Française du 2.10, no 73-231.

F. I Scard, The Chemistry of Rum

The name F. I. Scard has come up before in a drab paper, Scientific Control of a Rum Distillery. That idea turned out to be slightly more exciting in our recent reframing of Bourbon where we saw that scientific control was something that was significantly aided by onsite excise officers which the West Indies didn’t seem to have in those days. Better control made the collecting of tax revenue much more predictable.

Scard returns with another short paper, The Chemistry of Rum, from 1920. There is some great language in there and some unique factoids.

What might be called the beneficient bacteria of rum, which cause the distinctive flavour, are the acetic acid organism, which produces acetic acid from the alcohol, and the butyric acid organism, which gives from the presence of organic matter peculiar to sugar cane molasses, butyric acid—the same body which gives the characteristic flavour to rancid butter.

We use that rancid butter factoid as common trivia these days, but I’ve never seen it stated that far back.

During distillation the acids mentioned above combine with the alcohol, forming what are known as “esters” or compound ether, and it is these esters which impart the flavour to rum and give it stimulating properties.

I highlight this because Scard mentions stimulating properties. I posited stimulating properties in rum back in my infamous Mezan XO spirits review that ended up with the Mezan XO challenge! Scard was writing before the wide recognition of rum oil as a congener category, to which I attribute the mysterious stimulation rather than esters. Does the logic of his language imply pharmacological stimulation, apart from ethanol, or am I grasping? We have only seen real rum re-enter the market recently so I suggest you drink more to make a better educated decision.

The object of adding sulphuric acid to wash is the produce a certain acidity, thus putting an obstacle in the way of the putrifactive bacteria, which feed on yeast cells, at the same time helping the development of the butyric ferment,  which requires an acid condition for its development. It is the ester formed from this acid which gives the “pineapple” flavour to Jamaica rum. Its presence is essential to all rums, as without this ester the spirit ceases to be rum.

A strong aesthetic pronouncement! Those are rare.

And here we go…

The reason why Jamaica rum contains so much of this body, and is consequentially so valuable, is as follows: The yeast which provides the fermentation in sugar-cane distilleries is derived from the cane itself. The ordinary variety consists of round cellular bodies which grow by budding—that is, one cell buds out from another. This variety, unfortunately, will not flourish when the acidity gets beyond a certain point. When this point is reached—and the production of acetic acid soon brings it about if the fermentation is slow—alcohol production ceases. But in Jamaica there is an especial yeast which will grow in a highly acid medium. Unlike the other yeast, it is rod-shaped, and multiplies by splitting up. The presence of this yeast, therefore, enables the fermentation to be prolonged, and substances such as bottoms, dunder, &c., to be used in the wash, which are favourable to the development of butyric acid.

Here we see the return of our especial hero, Schizosaccharomyces Pombe, which is still not widely recognized in contemporary rum connoisseurship. We don’t exactly know who is using it currently and who isn’t and who was and who stopped. The first person to bring a Pombe rum to the U.S. will have a lot of success. And I’d be happy to help them. There are ways to achieve great ends without a Pombe ferment, but they do not tell such an archaic story of questing Victorian geniuses. They will not be as dank, concentrated, or brick house powerful.

In this connection it may be remarked that the writer on one occasion added butyric ether (ester) to a puncheon of rum in Demerara, which was reported upon in Mincing-lane as “resembling Jamaica”.

There is a lot here besides the admission of fraud. First off, Scard is an island hopper which shows yet again how ideas and know how easily spread between the islands. Everyone was following everyone. Therefore the forces that created style were largely economics, risk tolerance, and responsibility (to process mountains of molasses or not). Mincing-lane was a market for rum and other articles from the West Indies. Lots of tasting descriptors were developed in these markets.

The cane-juice itself is an important factor. Different kinds of canes give a different quality of rum, due, partly, to the case itself and partly to variations in chemical treatment necessitated there in the sugar manufacture. Even the different conditions of the same variety of cane will affect the flavour of the rum. On one occasion some Demerara rum made from very rank Bourbon canes were reported upon as being “green and stalky.” There is therefore outside the ethers specified some bodies present in excessive proportions which come down from the cane itself.

Scard here is arriving at a notion of proto-terroir. He isn’t exactly celebrating variation, but he is noting that variations exist. I’m a little confused by the “rank” canes. These could be moldy rum canes which were prized or be something else. Distilling them could also have been an experiment, and if they were fermented and distilled as a fresh juice rum, they may have had that character on account of not being centrifuged like the fresh juice rhums we know of today.

His closing remarks are nice:

Another agent in flavour is the nature of the still.

Spirits Review: Mezan XO Jamaica Rum

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The Mezan XO Jamaica rum is likely the greatest deal in all of spirits at the moment, yet it has been slow to catch on. Even in this unprecedented era of spirits education buyers seem slow to discover anything. The product is a very smart blend likely assembled by E & A Sheer, who has unparalleled access to blending stocks. The product forgoes traditional coloring and subtle sugaring giving it a very sleek modern truth seeking quality.

Despite a righteous flavor and probable noble E & A Sheer heritage, the branding comes across as a vodka startup like veneer that may irk some. Don’t fall into that trap, the gates to MGP whiskey may be wide open, but access to the lost rums of the world is elusive and I recommend taking it any way you can get it.

This rum from Mezan has that je ne sais quoi, and that is appreciable quantities of rum oil, the most noble (if not divine!) of all the congeners. The new generation of spirits connoisseurs is slowly digesting the concept of esters, but the king congener class is the fairly high boiling point terpenes that are the product of glycoside hydrolysis (these are different from gin botanical terpenes). This is absolutely at the forefront of distillation research, being led by Cognac and also finds itself at the forefront of theoretical oenology where researchers are pointing to the same congener class as a significant layer of the terroir phenomenon.

You can fake esters, but you cannot fake rum oil. If you target esters in your production you will produce some rum oil, but if you target rum oil you maximize your potential and you get all the esters you want at the same time. This is easier said that done and was the dogged pursuit of the 1940’s rum researcher, Rafael Arroyo (it is pretty much what his 1945 book is all about). Production ends up requiring a virtuosic attention to detail or wild amounts of divine chance. It is hard to say how the producers behind Mezan XO do it.

Two distilleries can start with the same substrate and thus the same amount of glycosides yet end up with wildly different amounts of rum oil. This congener class can be seen as silent or bound aroma that needs to be unlocked with care. Glycosides are typically split via enzymes produced by yeast. Alt, non-sacharomyces yeasts produce far more enzymes than typical sacharomyces (think budding bakers or brewers yeasts). This is where our hero from other posts, Schizosacharomyces Pombe, comes in (as well as a few others).

Catalysts, like acidity, also act to increase rum oil production as well as that expensive ingredient of time. Longer fermentations (and resting periods) yield more opportunity for glycoside hydrolysis, but at the risk of aroma-detrimental bacterial infections. Risk is worth money and that is why we should prize this congener class. Authenticity is also worth money, and unlike esters, this congener class is something that cannot be faked. There is no easy road to rum oil.

We are building up to the Mezan XO challenge, but first we need to go a little bit further.

Many spirits of great repute have lost this congener class as their production has been scaled upwards because no one really knew where it originated. The main loss comes from migration to low risk pure culture fermentations adopted by many formerly traditional distilleries because typical sacharomyces yeast produce less of the enzymes needed to split glycosides. Besides spirits, this has profound implications for wine. Pure culture fermentations forgo a lot of this aroma because they result in a much narrower microbial community. For spirits, tequila may have been the most negatively affected by yeast changes as production scaled up.

Devastating changes to a spirit often happen when a distillery changes physical buildings as result of increasing production because so much of the microbial community is held in the architecture. Hampden estates, with some production areas covered in aroma-beneficial molds, is the perfect nth degree case study while others like the cult beer producer Cantillion are also notable.

So little basic science has been done on architecture embedded microbial communities that we don’t even know how they start or get balanced forming a SCOBY (I have a collection of anecdotes!). Aroma-beneficial molds are often over looked in Jamaican rum production in favor of aroma-beneficial ester producing bacteria, but they likely have their origins in the long forgotten “rum canes”. When Jamaican rum wash bills used percentages of fresh sugar cane juice, it likely came from Rum Canes which were canes infected with molds (also rat eaten or infested with boring insects). These could be analogous to the noble rot in wine grapes, but definitely different in the finer points. They might not even exist anymore having been eradicated by modern cultivation methods and pesticides and thus only available through the physical buildings we take for granted.

We’re getting closer to the Mezan XO challenge, but first we have to look at the end of rum science history in the 1990’s and how and why Cognac took over. Rum science seems to end in the 1990’s with a call to explore alt yeasts but never directly pointing the finger at aroma from glycosides as the most significant source of rum quality. Cognac picks up where rum leaves off for some really interesting reasons. This means that if we want to advance rum further we have to look to Cognac and some of the ideas at the forefront of oenology research.

Bon vivants will note that there is a lot of overlapping character between the finest rums and the finest Cognacs. Many rums historically were designed to mimic Cognac. Grapes used for Cognac production are also high in glycosides. Cognac production also has a few other properties overlapping with rum we could go into, but I’ll spare you.

Cognac oil as a congener class, just like rum oil, has been recognized for over a hundred years, but the big driving force behind why the torch was passed to Cognac is because they have their back up against a wall. Everyone else focuses on expansion instead of quality improvement, but Cognac is a small region and their product has been legendary for centuries. They have cultivated near all viable area. They cannot expand, they can only improve so that is where they spend their energies and do it quite well.

We can only hope the new American distilleries end up similarly with their back up against a wall. Right now they are all trying to expand rapidly, forgoing quality. If new American distilleries balloon from 600 to 3000, the focus will likely go from expansion to quality improvement as a way of staying competitive.

Cognac researchers are also notably in tune with their heritage and they bring us from an era of traditional practices to guided traditional practices. Chaotic diversified microbial communities are the hallmark of traditional practices and science is starting to recognize the importance of minority community member’s role of producing the rarest most extraordinary aroma. Tradition alone, in this context, is associated with ignorance and ideology best exemplified in the sloppy natural wines flooding the market. While guided tradition recognizes the science behind the chaos, does not seek to master it so much as frame careful windows around it to reign in the risk. The resultant products are consistently extraordinary (In wine, I would single out Randall Grahm immediately, but so many deserve cognition).

Before the Mezan XO challenge I’d quickly like to note that certain Armagnacs are very high in aroma from glycosides and they can be very hard to tell apart from Jamaica rums. Certain tequilas are notably high, but fewer than there used to be. Older rums from cult producers had it and lost it. Use your nose and keep track (there are also a few amazing chemical tests taught by Arroyo*). If we highlight exemplary producers they will become stronger guided traditionalists and be mindful as they scale up to global demands.

(*The most basic test is to take a 2 oz. sample and add sulfuric acid which will destroy all the esters and aldehydes subtracting their aroma. If strong residual aroma remains, it can be attributed to the rum oil congener class. This sample is now undrinkable!)

Rum oil, Cognac oil, and aroma derived from glycosides may have pharmacological effects, that is what the challenge is about. If you drink spirits high in these congeners you may feel significantly less dehydrated by the ethanol. Your buzz may seem to hang broadly in a really lovely way. It is a different drunk with lots of anecdotal evidence to support it. Search your recollections, have you ever experienced something like it? Is rum oil the pattern behind mysterious lack of hangover after significant consumption? Are wines of terroir more gentle?

Most all congener classes have been widely studied and ruled out as specifically contributing to hangovers in broad populations. Rum oil has not been studied because of near no awareness and that it is appreciable in less than 1% of all spirits. It is the product of very specific microbial communities just like so many drugs, there is no scientific reason to immediately dismiss its unique potential power.

Remember, I am the guy perceptive enough to have identified all of the olfactory illusions in the wild categorized by Richard Stevenson. When wallowing through subjectivity, my track record of acuteness rivals a neurologist.

I encourage any devoted bon vivant to take the Mezan XO challenge and consume appreciable amounts of the spirit (safely) and note the effects. Do this especially if you are aging and your tolerance for alcohol is changing negatively hangover wise. Who can afford to crush eight ounces of Martel Cordon Bleu, but anyone can afford Mezan XO. Sacrifice your body for speculative science. Design controlled drinking experiments. Supply of truly fine spirits will not come without demand and here I am unraveling the chemical pattern. No hangover research has been focused enough to look at a mythic congener class that is barely acknowledged and not widely available on the market. Maybe we can inspire researchers to pursue it. What comes before the science? This.

Take the Mezan XO challenge and/or search your recollections then please leave a comment!

[A vodka company is validating some of the ideas. Spirit gentleness on the body is not so much about what a spirit doesn’t have, i.e. vodka, but possibly what it does have. Congeners matter.]