Can Inst. Food Sci. Technol. J. Vol. 21, No. 5, pp. 555-557, 1988
Effect of steroids from sugar cane on yeasts in alcoholic fermentation
Laboratoire de Chimie des Substances Naturelles,
BP 592, 97167 Pointe a Pitre Cedex,
French West Indies.
Station de technologie,
Institut National de la Recherche Agronomique (I.N.R.A.),
BP 1232, 97184 Pointe a Pitre Cedex,
French West Indies.
Effect of cane sugar steroid on yeast in alcoholic fermentation. Filter muds of cane sugar factories were analysed. Steroid components, mainly stigmasterol and sitosterol were isolated from the wax fraction. These two components have been added to alcoholic fermentation media in order to determine their effect on yeast behaviour. Their presence had a definite favorable effect on ethanol production. Baker’s yeast which is relatively rich in sterol is much less sensitive to the adjunction of cane sugar steroid than “wild yeast” isolated from unseeded media in rum factories.
A “survival factor” effect on anaerobic yeast, of certain sterols in the cuticle of grapes (ergosterol, oleanic acid) has been shown by some authors in oenology (Brechot et al, 1971; Radler, 1978; Lafon , 1978). Rose (1978) has shown that in Saccharomyces cerevisiae enriched in unsaturated side chain sterols, such as stigmasterol, viability in the presence of ethanol increases. More recently, Nes et al. (1986) have shown that “wild yeasts” of the species Saccharomyces cerevisiae actively absorb anaerobically various steroids including stigmasterol, sitosterol, campesterol and brassicasterol. Examination of alkaline defecation sludge from sugar cane factories has shown a relative abundance of a wax fraction itself containing significant quantities of sterol-type molecules. Thus extraction, purification, and isolation of a steroid fraction constituted in majority of stigmasterol and sitosterol and provided quantities necessary for a study of their action on the behavior of yeasts in alcoholic fermentation.
Material and methods
Steroids were obtained from conventional methods used in the chemistry of organic compounds of natural origin. The fraction used for this study is a mixture of stigmasterol and sitosterol (60/40), identified from the examination of the mass, NMR and infrared spectra of the crude product. Their identity was confirmed by an analysis in HPLC which allowed their separation. Note that during this work another steroid compound was spotted more oxidized than the previous ones, its identification is currently in progress.
The experiments were carried out with “wild” yeast strains, isolated from rum fermentation media and preserved at the INRA-CRAAG technology station, it involves:
—Saccharomyces cerevisiae listed 493 and 390
—Saccharomyces chevalieri listed 377
and a strain of baker’s yeast, Saccharomyces cerevisiae, listed LB, widely used in local rum.
The starters corresponding to the yeast strains are produced on Malt Wickerham medium:
Yeast extract 3g;
Malt extract 3g;
Distilled water q.s.p .: 1 liter (pH 5-6, sterilized for 18 min at 120° C).
The yeast cells are recovered by centrifugation, rinsed in distilled water and seeded at the rate of 100 mg/L in the fermentation medium.
Fermentation medium is the “G” medium developed by Galzy (1964), containing as the only carbon source of glucose at concentrations of 180 g/L (S 180) or 230 g/L (S 230). The purified cane sterols are added to it dissolved in Tween 80 at a dose of 40 mg/L (SS 180 and SS 230). S mediums serve as a witness compared to SS mediums. [I was not sure if witness should be better translated as control.]
The progress of the fermentation is followed by measuring the mass loss of the medium due to the transformation of glucose into ethanol and to the release of carbon dioxide.
Biomass content is evaluated by weighing: after centrifugation and washing of the cells with distilled water, the yeast pellet is taken up in distilled water and then subjected to drying at 108° C., to constant mass.
Determination of ethanol
Gas chromatography with flame ionization detector is the technique used. Measurement conditions are as follows: porapak columns (length 50 cm, diameter 1/8 inch); injector temperature 175° C; column temperature 140° C; detector temperature 285° C; hydrogen flow 25 ml/min; air flow 350 ml/min; nitrogen flow 30 ml/min; volume injects 2μl; internal standard: propanol-1 (0.1% (v / v) final).
The sludge (1 kg) is air dried and then treated in a Soxhlet extractor with cyclohexane; after evaporation of the solvent, the extracted residue (60 g) is washed twice with 250 ml of methanol. The residual solid consists mainly of long-chain hydrocarbon waxes; the fraction extracted with methanol contains inter alia the steroids. These are isolated by chromatography on silica gel and then purified by crystallization from methanol (F: 148).
Results and discussions
In Table 1 we report the mass losses after 200 h of fermentation, according to the strains and comparatively on S and SS media, this for the two glucose concentrations. Examining the values leads to the following remarks:
1°) In medium S 180 “wild” yeasts have an activity almost comparable, however inferior to that of baker’s yeast. In the SS 180 medium, on the other hand, we note that the fermentation by “wild” yeasts is clearly enhanced and becomes comparable to that of Lb.
The addition of sterol results in an additional mass loss of 1g for the “wild” strains whereas it is only only 0.4 g for Lb.
2 °) When the fermentation medium is more concentrated in glucose (230 g/L) there are two trends:
— a decrease in the activity whatever the strain when operating with an S 230 medium compared to S 180
—an increase in this activity in the case of the SS 230 medium.
This slight change for Lb, is notable for strains 377 and 493.
It can therefore be seen that the addition of cane sterols makes fermentation by so-called “wild” strains more efficient compared to baker’s yeast. The gain after 200 h of fermentation observed on SS medium 230 g is to be emphasized since with a strain such as 493 we exceed the activity of baker’s yeast.
This analysis which concerns the situation of fermentations after 200 h is in agreement with the kinetics of the phenomenon in the different cases. FIG. 1 gives an example of the curves relating to the loss of mass as a function of time, for two strains Lb and 493 each working on medium S 230 and SS 230. We note without ambiguity the difference in behavior of the two strains.
Curves of the same type were made in the other cases, we find the results indicated above.
In Table 2 we have reported the quantities of ethanol and biomass produced at the end of fermentation. Generally there is a gain in ethanol production when fermentation takes place on SS medium, whatever the strain. Again, we find the trend noted from the other parameters: the intake of sterols enhances the activity of “wild” yeasts rather than that of baker’s yeast.
This comment should be clarified. In the current state of our work, we do not have sufficient data capable of supporting a discussion. However, two comments can be made which point to a lower sensitivity of Lb to the supply of sterols:
—It is known that baker’s yeast and brewer’s yeast are relatively rich in steroids (Maugenet, 1964)
—The LB starter, which was used for seeding the fermentation media during this work, is produced in aerobic conditions, a condition generally favorable to the synthesis of sterols by this yeast (Maugenet, 1964). This argument is not significant, however, since the starters prepared from other strains are also produced aerobically.
The results that we have presented relate to fermentations carried out on media rich in glucose. We also used poorer environments. At 100 g/L the addition of sterols practically does not modify fermentation process. At 130 g/L, there is a slight improvement in the activity of strain 493.
The addition of sugar cane steroids in alcoholic fermentation media has a favorable effect on ethanol productivity. “Wild” rhummerie yeasts are particularly sensitive to this contribution in high concentration media (> 150 g/L of glucose); they are then more productive than baker’s yeast. Light rum production technology could take advantage of this. Indeed, the kinetic approach indicates that the added sterols seem to prolong the efficiency of the yeast, thus creating conditions more favorable to fermentation metabolisms.
Furthermore, the satisfactory activity of wild yeasts, even in media rich in sugar, a condition generally limiting the activity of bacteria in fermentation media, could possibly be exploited by our local rum industry to improve the quality of its products.
Brechet, P., Chauvet, J., Dupuy, P., Croson, M. et Rabattu, A. 1971. Acide oleanique, facteur croissance anaerobie de la levure de vin. Ann. Technol. Agric. 206: 103.
Galzy, P., 1964. Etude genetique et physiologique de metabolisme de l’acide lactique chez Saccharomyces cerevisae HAN SEN., Ann., Technol. Agric. 13: 109.
Lafon, S. 1978. Les steroides “facteur de survie” des levures au cours de la vinification. Ann. Technol. Agric, 27: 215
Maugenet, P., Dupuy, P. 1964. Synthese des sterols par la levure. Ann. TechnoI. Agric. 13: 329.
Nes, W., Dhanuka, I., Pinto, W, 1986. Evidence for facilitated transport in the absorption of sterols by Saccharomyces cerevisiae. Lipids. 21: 102.
Radler, F. 1978. Les activateurs du developpement anaerobie de la levure. Ann. TechnoI. Agric. 27: 203.
Rose, A. 1978. Composition-function relationships in the yeast envelope. In: Biochemistry and genetics of yeast p. 197, Academic press Inc., New York, NY.
Submitted May 5, 1988
Revised August 8, 1988
Accepted August 23, 1988