Fahrasmane L., Ganou-Parfait B., Bazile F., 1989. Le métabolisme du soufre dans la rhumerie. Mircen Journal 5, 239-245.
Sulfur Metabolism in the Rhummerie
L. Fahrasmane*, Berthe Gandu-Parfait et F. Bazile
I.N.R.A. Station de Technologie des Produits végétaux, B.P. 1232, 97184 Pointe-a-Pitre Cedex, French West Indies
Received as revised 1 November 1988; accepted 12 December 1988
* Address for all correspondence.
Rum products and effluents are distinguished by their content of sulfur compounds.
Rums are generally made from the byproduct of cane refining: molasses which gives ‘industrial’ rum. In Guadeloupe, Martinique and Haiti, there is a large production of rum from the pressing of sugar cane juice, which gives ‘agricultural’ rum.
Sugar cane is one of the ‘entry points’ for sulfur in the distillery. Indeed, this raw material of the sugar factory and the distillery is particularly rich in sulfur compounds. According to Bravo (1986) it contains 0.26% of sulfur compared to dry matter.
We found in the French West Indies, that during the preparation of must based on sugar cane juice, significant quantities of sulfur-containing compounds (sulfuric acid and ammonium sulfate) are added (0.01 mol / L in sulfate ion) in order to acidify the must and to supplement it with nitrogen. At this level, there is the second point of sulfur supply in the distillery.
Compared to other spirits, it is in rum, a product of the distillery, that the highest concentrations of certain volatile sulfur compounds are found.
Downstream of the rum factory, the bio-methanisation of vinasse, for energy and purification purposes is an industrial reality (Bolivar 1983; Szendrey 1983; Bories et al. 1988). Molasses vinasses are relatively rich in sulphate (3.5 to 4.0 g/L; Bories et al. 1988). This sulphate is a substrate for sulphate-reducing bacteria which, by metabolizing it, generate foul-smelling hydrogen sulphide. In addition, in anaerobic digestion plants, rich in sulphate, the development of sulphate-reducing germs can under certain conditions generate or prevent the activity of methanogenic bacteria (Isa et al., 1986; Hansen 1988).
Sulfur metabolism and microorganisms of rum fermentation
In the French West Indies, rum must is neither pasteurized nor sterilized. Yeasts and bacteria are in mixed culture with specific activities.
Sulfur is an essential element for the growth of yeasts. It presents a primordial biochemical interest. Present in small quantities in cells, it is however involved in many reactions (Bidan and Collon 1985).
In Saccharomyces cerevisiae, sulfur represents from 0.2 to 0.9% of the dry weight, or on average 0.4%. The sulfur requirements of yeast are covered by inorganic and organic sulfur sources. An assimilable source of sulfur is essential for yeast growth. Sulfates can play this role (McCready and Din 1974).
The formation of H2S is under the control of the enzymes involved in the reduction of sulfates and in the biosynthesis of methionine. The composition of the medium in amino acids, more specifically in methionine, influences the formation of hydrogen sulfide.
The effect of sulfate concentration on growth seems simple. Below 30 mg/L, growth is significantly limited, while beyond this concentration, sulfate is no longer an important limiting factor (Jordan and Slaughter 1986).
The relationship between sulfate concentration and H2S production seems more complicated. It is possible to discern three zones. From 0 to 20 mg/L of sulphate, the production of hydrogen sulphide remains low. From 20 to 50 mg/L, this production is very sensitive to the sulfate concentration. Between 50 and 250 mg/L, the synthesis of H2S varies little. In metabolic terms, these data suggest that when the sulphate concentration increases, the efficiency of its incorporation in amino acids decreases towards 30 mg/L, that is to say about 0.3 mmol/L. Above 30 mg/L and up to 100 mg/L, the additional quantity of sulphate is transformed into sulphide. Above 100 mg/L, an increase in the sulphate concentration only very slightly increases the production of sulphide (Jordan and Slaughter 1986).
Working at high temperatures promotes the production of H2S. The ions Mg 2+ and Mn 2+ have an activating role in the reaction to form phospho-adenylsulfate (P-APS). Heavy metals are generally quite harmful. The formation of H2S in the presence of mercury or copper is a mechanism of detoxification of yeast vis-à-vis these metals. The phenomenon appears to be very amplified when the strains are classified as resistant to metals, whereas the production of H2S is low for the strains which are not very resistant (Desbordes 1970).
Dimethyl sulfide (DMS) is one of the sulfur compounds in rum. It is the only eau-de-vie to contain it (Leppanen et al. 1979). It is also found in beer. A lot of work has been done on its origin in the brewery. One of the origins of DMS is linked to the metabolism of yeasts by reduction of dimethylsulfoxide (DMSO). Since the reduction of DMSO is linked to the metabolism of sulfates and sulfites, the production of DMS should be influenced by the use of these sulfur sources by yeasts. Another known precursor of DMS in beer is S-methyl-methionine (SMM). DMS is beneficial to the taste and aroma of beer at concentrations between 30 μg/L and 100 μg/L (Anness and Bamforth 1982). Above 100 μ/L, DMS gives an aroma usually described as grilled onion.
The metabolism of sulfur by bacteria during the manufacture of beverages has been little studied. On the other hand, it has been the subject of numerous studies on anaerobic bacteria in the methanisation processes of industrial effluents and ecosystems rich in sulphate.
Assimilative reduction of sulfates
As with yeast, sulfates are the main source of sulfur for bacteria. The pathways of assimilative metabolism are identical and the L-cysteine formed constitutes the main precursor of sulfur compounds in the cell.
Some bacteria have a strictly anaerobic metabolism of sulfide fermentation or the sulfate is used as the final electron acceptor. The hydrogen sulfide formed is excreted.
Sulfur compounds in the rum sector
Sulfur in sugar cane and its derivatives
The determination of the elements of the mineral fraction of sugar cane was carried out on two varieties by Bravo (1986). He notes that the sulfur content is 0.26% compared to the dry matter, for a water content of 77.5%. In addition, it shows that sulfur is a highly diluted element in water (Γs (H20) = 0.8). Most of the sulfur in sugar cane would therefore be found in the juice. Taking these elements into account, the sulfur content of cane juice can be estimated at approximately 0.02 mol / L, expressed as sulfate.
Molasses, due to the concentration of dry matter in cane juice during the process which leads to their production, are rich in sulfur. Meade and Chen (1977) give 1.10% SO2-/3 compared to the dry matter.
Analysis of cane juice shows that sulfur amino acids are quite often absent or in the form of traces (Meade and Chen 1977). In the molasses, we measured methionine: it represents from 0.01 to 0.03% of the dry weight.
Godshall et al. (1978) identified dimethyl sulfide (DMS) as a volatile constituent of molasses, responsible for their herbaceous aromatic note. A solution of a few ppm in water has an odor close to molasses. In the cane leaves, these same authors also identified a precursor of DMS. Data on tomatoes (Wong and Carson 1966) and corn (Bills and Keenan 1968), which is a taxonomically similar plant to sugar cane, have shown the presence of S-methyl-methionine which is one of the precursors known to DMS. After heating this compound, homoserine and DMS are obtained. Apparently this reaction occurs when the cane leaves are heated. Indeed, after heating the leaves, the amount of DMS detected is multiplied by a hundred, based on the integration unit compared to an unheated sample. This compound appears at this stage, passes into the juice and then throughout the process, and is found in the raw sugar (Godshall et al. 1978).
Sulfur compounds in distillery
In the rum industry, during the preparation of musts, the following sulfur compounds are added:
(i) ammonium sulphate which constitutes a nitrogen supply for the yeast;
(ii) sulfuric acid so as to lower the pH of the must approximately 4.5 and to limit the activity of the bacterial flora.
The use of ammonium sulphate, and to a lesser extent ammonium phosphates, has taken on a certain importance in rum. Ammonium sulphate was already commonly used in the French West Indies at the end of the last century, generally at a dose of 4 to 5 kg/100hL of must. In some islands like Jamaica, this salt was very little used (Kervegant 1946). We have noted, in the French Antilles, its use in mother tanks and in fermentation at concentrations of up to 10 kg/100hL and 5 kg/100hL respectively in molasses-based media. These doses are reduced by 20% in media based on cane juice. For economic reasons, other ammoniacal salts are little or not used.
The addition of sulfuric acid to molasses and vesou must has been in use for quite a long time. Pairault (1903) reports that “at the end of the last century, a large number of distillers from the French West Indies added small quantities of sulfuric acid to their composition in order to obtain purer fermentations”. At present, the use of sulfuric acid is widespread in the manufacture of ordinary rums. On the other hand, when one wants to obtain grand arôme rum, one carefully avoids using it, the degree of acidity necessary for the yeast being obtained by the addition of vinasses or scum.
The quantities of sulfuric acid added to musts can reach 5 liters / 100 hL in a mother tank for molasses and 4 liters / 100 hL for cane juice.
The distillery contributions can be estimated between 0.010 M and 0.013 M, expressed in sulfate ion.
The conduct of fermentations in the French West Indies is such that we obtain low-grade wines (4 to 5 degrees Gay-Lassac, °GL). The distillation of these wines leads to the implementation of high operating temperatures as well as relatively long column residence times which, according to Nikanen and Suomalainen (1983), are favorable for the formation of mercaptans. The use of columns with few trays in concentration promotes ‘harsh’ thermal conditions.
Sulfur compounds in rum
The sulfur compounds identified in the rum are shown in Table 1.
It is interesting to note that rum contains a greater variety and greater quantities of organo-sulfur compounds than other spirits. According to Leppanen et al. 1979): “rum is the only spirit containing dimethyl sulfide (Table 2). The low sulfur content of bourbon and Canadian whiskey clearly differentiates them from Scotch and Irish whiskey. Vodkas have low levels of sulfur compounds. In addition, the contents of methyl polysulfide and ethyl sulfide are relatively high compared to vodka, whiskey, brandy, cognac and armagnac.” These same authors observed that the contents of dimethyl sulfide and dimethyltrisulfide decrease during the aging of the spirits in barrels.
For grain-based spirits, most of the sulfur compounds are formed during the preparation of the wort and the kilning of the grain (Leppanen et al. 1979; Anness and Bamforth 1982). For rum, these compounds appear during distillation, their content increasing with temperature and the duration of distillation (Nykanen and Suomalainen 1983).
Methanization of vinasses
The industrial methanization of rum vinasse is relatively recent (Bolivar 1983; Szendrey 1983; Bories et al. 1988). The high sulfur content of vinasses, partly linked to the significant inputs upstream of the sector, can pose the delicate problem of the balance, in digestors, of methanisation, in particular between methanigenic flora and sulphate-reducing bacteria.
Reducing the microbial load of raw materials by heat treatment and operations, such as clarification, aimed at reducing the buffering capacity, would be elements which should make it possible to reduce the intake of sulfur-containing compounds. Fermentation processes with a yeast content must allow us to go in the same direction.
Several elements of rum technology are favorable for obtaining a product with a high content of sulfur compounds: the composition of the raw material, the intake of sulfur compounds, the composition of the flora and its activity, fermentation temperature (34°C) as well as distillation conditions. The contribution of each of these elements in the organo-sulfur fraction is not known. This fraction deserves a thorough and systematic study because it presents an analytical interest as to the characterization of rums and organoleptic interest given the high favorable aromatic power or not of these compounds.
The fact of using the juice of a plant in the case of sugar cane poses problems of composition of the raw material which are not found in the other agricultural raw materials.
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Le rhum, comparativement aux autres eaux-de-vie, se singularise par la variété et les teneurs relativement 61ev6es de composés organo-soufrés qu’il contient. La composition de la canne sucre ainsi que les apports de sulfate et d’acide sulfurique tout au long de la fili~re industrielle font que les milieux de fermentations sont riches en dérivés soufrés. En aval de la distillerie, la méthanisation des effluents pose le probléme d’un équilibre précaire entre les flores méthanigéne et sulfato-réductrice. La fraction organo-soufrée mérite une étude approfondie et systématique, car elle présente un intér~t analytique et organoleptique pour la caractérisation des rhums.
The metabolism of sulphur during the manufacture of rum Rum distinguishes itself from other brandies by the variety and the relatively high content of organic sulphur compounds. Composition of sugar-cane as well as additions of sulphate and sulphuric acid along the industrial process gives fermentation media that are rich in sulphur- compounds. Downstream from the distillery, biomethanation of effluents is difficult because of a precarious equilibrium between the methanogenic and the sulphate-reducing bacterial floras. The organic sulphur-fraction requires a systematic study because the latter are involved in the organoleptic properties of rums.