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.