Glycerol in the alcoholic fermentation of molasses and sugar cane juice

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Parfait A., Jouret C., 1980. Le glycérol dans la fermentation alcoolique des mélasses et des jus de canne à sucre. Industries alimentaires et Agricoles 97, 721-724.

Le glycérol dans la fermentation alcoolique des mélasses et des jus de cannes à sucre
by PARFAIT A. *, JOURET C. **
with technical collaboration from G. SABIN and Madame G. MIGLIORI

(*) C.R.A.A.G. Station de Technologie, Guadeloupe.
(**) C.R.A. de Toulouse, Laboratoire de Technologie des Produits Végétaux, Auzeville.

Summary

Authors have specified the influence of various factors (mode of conduct, pH, seeding rate, sugar content, yeast species) on the amount of glycerol found in rums.

They have also shown that glycerol can serve as a carbon substrate for various bacteria and give, as a result, derivatives having a negative role on the organoleptic qualities of this eau-de-vie.

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Glycerol is a secondary product formed by the metabolism of sugars during alcoholic fermentation.

Depending on the conditions, a more or less important fraction of the sugars is thus transformed into glycerol. This results in different values of the fermentation yield.

Genevois (1936) proposed an equation between various byproducts of alcoholic fermentation

2A + B + 2M + H + 5S = ε = G

A, B, M, H, S and G being respectively the molar concentrations of acetic acid, 2-3 butane diol, acetoin, acetaldehyde, succinic acid and glycerol. This equation has been the subject of much work and has been confirmed by Lafon (1955). It can be considered valid in 90% of fermentations.

Subsequently, Nordstrom (1968) and Oura (1977) showed that there was a correlation between the redox potential of the fermenting medium and the formation of glycerol.

According to Oura, the formation of succinic acid is related to the production of glycerol and has the same purpose: to balance the excess of reduced nucleotides.

Glycerol, whose physiological interest for yeast appears to be small, seems however to play a significant role in the regulation of compounds such as pyruvic and succinic acids that enter, instead, in the formation of constituents of the cell. Similarly, glycerol, via its phosphoric ester, L-α glycerophosphate, combines with activated fatty acids to give phosphatidic acid. This last body leads to lipids. This same ester allows the use of glycerol by many breeds of yeasts as a source of carbon. Although the low volatility of glycerol explains its absence in rums, it has been possible to determine various compounds from its degradation. These generally have a negative effect on the organoleptic qualities of eaux-de-vie.

Since the work of Warcollier and Le Moal (1932) on ciders and those of Serjak et al. (1954), we attribute to the action of lactic acid bacteria the appearance of acrolein in spirits.

Dubois et al. (1973) identified two acrolein derivatives in an abnormal taste rum: ethoxy-3-propanol and ethoxypropane. These compounds are not directly responsible for the unpleasant flavor of the rum studied but can serve as indicators.

Smedt and Liddle (1976) have correlated the presence of allylic alcohol (2propene 1 ol [I think that is correct nomenclature]) with some bad tastes in various types of spirits. They also showed a relationship between the contents of this alcohol and those of ethoxypropane.

Thus, the glycerol which is at the origin of acrolein (and products derived from this aldehyde) following metabolic pathways not completely elucidated, can therefore be degraded in fermenting media based on cane juice and molasses.

Given these biochemical and technological considerations, it seemed interesting to specify some parameters of the production of glycerol in the fermentation of the basic products of the different types of rums.

Experimental Protocol

Glycerol was determined according to the enzymatic technique of Eggstein and Kuhlmann (1974) after defecation of natural media with lead acetate.

Samples of fermented media were taken from industrial plants previously described by Sabin and Parfait (1975). Just remember that the following raw materials cane juice, syrup and molasses, are used respectively for the development of agricultural rums, syrup and industrial.

The results are reported in Table 1.

Table 1.
Glycerol content expressed in g / l in fermentation media of different types of rums

The seeding was carried out using yeast creams in order to eliminate the glycerol fraction that could be brought by the starter.

The yeast strain saccharomycès cerevisiae used is the No. 493 of our collection, isolated from a natural fermentative medium of agricultural rum.

Influence of pH:
Initial pHs were set at 3.5 – 4.0 – 4.5 – 5.0 – 5.3.

The evolution of the glycerol level during the fermentation was regularly monitored.

For example, for the medium at pH 40, the following figures were noted:

These figures vary very little according to the different pHs tested.

Influence of molasses richness:

The molasses concentrations of 150 g/L, 200 g/L, 250 g/L and 300 g/L were varied, the seeding rate was 1 g/L, the fermentation temperature 30° C and the initial pH set to 5.

The glycerol levels found were in order: 1.5 g/L, 1.7 g/L, 2.3 g/L and 2.5 g/L. They follow the same progression as that of sugars.

Influence of seeding rate:

By changing the seeding rate from 0.25 g/L, 0.50 g/L, 1.0 g/L, 2.7 g/L and 2.5 g/L, 4.0 g/L, 5.0 g/L in a medium similar to the previous one, does not find a statistically valid variation in the final glycerol contents.

Influence of yeast species isolated from natural fermentation media: Hansenula anomala, Saccharomyces cerevisiae, Saccharomyces aceti, Schizzosaccharomyces pombe.

Only these last yeasts have glycerol production curves very different from those of the other yeasts tested.

Influence of the mode of conduct of industrial fermentations:

By examining the manufacturing process generally followed in the industrial production of rum, we realized that glycerol appears during the aerobic growth phase of yeasts. Given the reduced richness of the medium, often less than 200 g/L of molasses or about 100 g/L of fermentable sugars, and the low rate of seeding practiced, it can be said that currently in the French West Indies, a part not negligible sugar is consumed to develop glycerol.

On the other hand, the operation of industrial installations is discontinuous. The canes brought to the distillery may be subject to pre-fermentation, with consequent production of glycerol. The glycerol level reaches 0.8 g/L on average in pipes that are poorly emptied.

Glycerol derivatives

Yeasts and bacteria can use glycerol. For these latter microorganisms, a review was conducted by Lin (1976).

Ganou and Parfait (1980) determined many species of bacteria in the flora of fermentation media leading to the various qualities of rums. As can be seen from Table II, cane juice and molasses, even when preserved, contain few germs. From the first hours of fermentation, a flora of varied origin (installations, water, environment) develops. During the course of fermentation, anaerobiosis causes a reduction in the number of species present in the medium. Lactic acid bacteria and Clostridia are mainly found. Acetobacters may appear at the end of fermentation and degrade the ethanol formed.

If sometimes the intervention is beneficial (some strains of clostridia, among others, for the production of rum aroma) most often it is detrimental to the organoleptic qualities. Acetobacters cause, for example, a detrimental increase in the level of acetic acid and ethyl acetate.

The appearance of volatile derivatives of glycerol is due, for a large part, to the activity of lactic acid bacteria, as we have observed in some distilleries. We searched in the lab, among the species of lactic acid bacteria that we had isolated, those that degraded glycerol. The tests were done aerobically and anaerobically.

Three culture media were used: M1, M2, M3.

TABLE 2

The kinds of bacteria found in fermentative environments. (+) = present, (-) = absent. The number of signs indicates the frequency. A, B and C represent the environments leading to agricultural rum, molasses rum and rum grand arôme

Under the conditions of our tests, some lactic acid bacteria use glycerol as a carbon source. Surely we could identify among them strains of leuconestoc mesenteroid. Other species also having a metabolic activity from glycerol are being identified.

Acrolein and 2propene ol 1 were found among the products formed by gas chromatography using a Tracor 560 with flame ionization detectors. The phase for the 50 foot Scotch column and 0.2 inch diameter used is carbowax 1540; the flow rate of nitrogen, carrier gas, is 3 ml/min. Temperature programming was carried out: 6 minutes at 58° C. and then an increase of 8°C. per minute up to 120°C.

The injection of 1 μL of rum can detect 1 ppm acrolein or 2propene ol 1.

A typical chromatogram is given in Figure 1.

Conclusions

In the production of rum, glycerol may be found in greater or lesser quantities depending on the mode of conduct of fermentation operations.

If the raw material (fresh juice or molasses) has not been the subject of microbial activity, in particular by yeasts, the glycerol contents will be very low.

The use of a leavening tank causes a significant concentration of glycerol from the beginning of the anaerobic phase. Schizzosaccharomyces cause the appearance of significant amounts of glycerol, which can cause problems when there is a risk of contamination by bacteria degrading this substance.

Indeed, lactic acid bacteria (leuconostoc mesenteroid type) can metabolize glycerol to lead in particular to acrolein and 2propene ol 1 found in rums with other products of negative organoleptic character.

These observations should guide the process of fermentation of raw materials to obtain a good quality rum.

BIBLIOGRAPHY

DUBOIS P., PARFAIT A., DE KIMPE J. (Mme), 1973. – Présence de dérivés de l’acroléine dans un rhum à goût anormal. Ann. Technol. Agric., 22, 131-135.

EGGSTEIN M., KUHLMANN E., 1974. – In methods of enzymatic analysis (Bergmeyer H.L.), Vol. 4, 1825-1835, « Verlag Chemie Weinheim ».

GANOU B. (Mme), PARFAIT A., 1980. – Les microorganismes de fermentation de mélasse et de jus de canne (en préparation).

LAFON M. (Mme), 1955. – Contribution à l’étude de la formation des produits secondaires de la fermentation alcoolique. Thèse de Docteur en sciences physiques, Bordeaux, Ann. Techn. Agri., 198 p.

LIN E.C., 1976, – Catabolisme du glycérol et sa régulation chez certaines bactéries. Annal Review of microbiology, vol. 30.

NORDSTROM K., 1968. – Yeast growth and glycerol formation II carbon and redox balances. J. Inst. of brewing, 74, 429.432.

OURA. E., 1977. – Reaction products of yeasts fermentations. Process Biochemistry, 12, 19-35.

SABIN G., PARFAIT A., 1975. — Les fermentations traditionnelles de mélasse et de jus de canne aux Antilles Françaises, lnd. Agric. Alim., 92, 27-34.

SER JAK W.C., DAY W.H., VANLANEN J. M., BORUFF C.S., 1954. – Acrolein production by bacteria found in distillary grain mashes, Applied Microbiology, 2, 14-20.

DE SMEDT P., LIDDLE P.A.P., 1976. – Présence d’alcool allylique (2propène  ol 1) et dérivés dans les eaux-de-vie. Ind. Alim. Agric., 93, .41-43.

WARCOLLIER G., LE MOAL. A., 1932. – Présence accidentelle d’acroléine dans l’eau-de-vie de cidre. C.R. Acad. Science, 194, 394.