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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
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.]
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.
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:
Use of citric acid by S. pombe. (+) low growth, (-) no growth The concentration of citric acid is 0.5%
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.
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.
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.
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
deaminase σ cétobutyrique –> CO2 — τn propanaldéhyde –>
[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.
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.
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