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1 THE ROLE OF MICROBIAL GUMS IN THE COLOR AND CONSISTENCY OF PALM WINE S.V.A. UZOCHUKWU',4. P.O. NGODDY' and E. BALOGH3 'Bioclzernistry Division, NIFOR P.M.B. 1030, Benin City, Nigeria 'Department of Food Science and Technology University of Nigeria, Nsukka, Nigeria 'Department of Food Science and Technology, Obafemi Awolowo University, Ile-lfe, Nigeria Accepted for Publication December 15, 1993 ABSTRACT Viscosity and soluble color changes in palm wine during fermentation were found to show an early increase followed by a gradual decline. Addition of absolute alcohol to palm wine precipitated gums that were absent in the palm sap, indicating production during fermentation. Precipitation of the gum was accompanied by loss in coloration and a signifcant reduction in viscosity. These properties were regained on reintroduction of the gum showing that the gums were responsible for the properties. Yeasts and bacteria isolated from palm wine were employed in pure culture fermentations of sterile palm sap to determine the origin of the gums. The results showed that the gums were produced mainly by leuconostocs, while two other bacteria not filly identified and a homofermenting Lactobacillus spp, also produced some. Analysis showed the Lactobateriaceae gums to be a11 glucans, while the gums of the other bacteria were fructans. Palm wine was found to contain both types of gum (80% glucans, 20 % fructans), showing that many bacteria produce gums in palm wine concomitantly. INTRODUCTION Palm wine is the alcoholic beverage obtained by the spontaneous yeamactic fermentation of the sap of palms (family: Palmae). The unfermented sap is a 4To whom correspondence should be sent. Journal of Food Quality 17 (1994) All Rights Reserved. Copyright I994 by Food & Nurrition Press, Inc., Trutnbull, Connecticut. 393

2 394 S.V.A. UZOCHUKWU, P.O. NGODDY and E. BALOGH transparent gold-tinged liquid, while the fermented product, is whitish and efferverscent. The white color was usually attributed to the yeast and bacteria cells present (Okafor 1977). This is only partially the case, since a distinct white coloration remains after microbial cells might settled out or been removed by centrifugation, indicating that a soluble white coloring component is produced during fermentation. Some workers (Uloa 1981; Samarajeewa ef al. 1985), have mentioned the possibility that Leuconostocs may produce gums in palm wine. If bacteria produce gums in palm wine, then there should be a gradual increase in both viscosity and optical density (color) during fermentation, and it should be possible to obtain such gums from palm wine using the correct extraction procedures. Leuconostocs are known to produce both dextrans (Jeanes 1965) and levans (Lewis et al. 1967) in media containing sucrose. The sugar in palm wine is mainly sucrose. Such gums, being hydrocolloids, would affect both the color and consistency of palm wine. It is the aim of this study to determine: (1) if gums are produced during fermentation of palm sap, (2) whether there are any viscosity and color changes during fermentation of palm sap to wine (3) the organisms responsible for the production of the gum and (4) characteristics of the gums. Palm Sap Fermentation MATERIALS AND METHODS Palm sap was collected from a 3-day old incision at the base of the male inflorescence of the oil palm (EZaeis guineensis) using tapping implements and receiving bottles sterilized by autoclaving. One liter of sap was allowed to ferment spontaneously at 28C for 120 h. At regular intervals, 10 ml portions were removed asceptically to determine viscosity, ph, color and sugar content. Viscosity was determined using a No. 2 Ubbelohde Viscometer tube in a Gallenkamp water bath regulated at 30C. Reference standards were glycerol and distilled water. ph was determined with the aid of a Pye Unicam model 291 ph meter. Sugar was determined by the Anthrone method (Hodge and Hofreiter 1962) after precipitating polysaccharides using absolute ethanol. Color was estimated by determining optical density of centrifuged samples on a pye Unicam sp 500 series 2 spectrophotometer at 720nm.

3 GUMS IN PALM WINE 395 Gum Extraction and Analysis. Gums were extracted from palm wine using increasing strengths of ethanol according to the method of Jeanes (1965). Extraction was usually at a predetermined maximal viscosity during fermentation. Maximum viscosity achieved during fermentation was determined in several preliminary experiments so that an average value was obtained and was used as a guide for gum extractions. All gums prepared were divided into two sets. One set was hydrolyzed by heating with 10% HCI under reflux for 4 h. The second set was hydrolyzed for one hour only with 0.5% oxalic acid. The hydrolyzates were neutralized with CaCO, and characterized by thin-layer chromatography. The thin layer plates were coated with 0.25 mm layer of silica gel, G. and developed with either benzene: glacial acetic acid: methanol (20:20:60) or butanol: acetone: water (4050: 10). For visualization, benzidine [OS g in glacial acetic acid ethanol (20:80)], was used for general sugar detection and anthrone reagent for ketoses (0.3 g anthrone in 10 ml glacial acetic acid + 20 ml 90% ethanol + 3 ml phosphoric acid + 1 ml water). The viscosity of 10% solutions of the gums, their texture, solubility in water and yield were also determined. Properties of Degummed Palm Wine. Palm wine from which gum had been extracted as above was concentrated to its original volume by vacuum evaporation with a Gucci rotary evaporator. The viscosity of the resulting fluid was measured. The optical density was also determined The presence or absence of typical palm wine color was also visually assessed. Pure Culture Fermentations. All the yeasts and bacteria shown to be relevant to palm wine fermentation (Uzochukwu ef al. 1991) were employed in pure culture fermentations using sterile palm sap as the medium to determine the roles they play in gum production in palm wine. The organisms had been isolated and identified earlier in these series of studies on different aspects of palm wine by the present authors. Palm sap was centrifuged at 3,000 rpm (1 108 x g) for 15 min to obtain clear gold-colored sap, which was distributed in 250 ml portions in cotton-wool stoppered flasks. These were then steamed for 30 min for three consecutive days and incubated at 30C in between. The sap was cooled quickly after the last steaming and immediately inoculated. Each 250 ml of sap was inoculated with 5 ml of inoculum containing about lo4 cells per ml. Each flask was inoculated with only one of the microorganisms isolated from palm wine. The microorganisms were Succharomyces cerevisiae, S. chevalieri, Leuconostoc mesenteroides, L. dextranicum, Lactobacillus casei, S. casei, an unidentified Lactobacillus sp. and 2 incompletely identified bacteria numbered 2 and 3, respectively. The unidentified Lactobacillus sp. consisted of very long

4 396 S.V.A. UZOCHUKWU, P.O. NGODDY and E. BALOGH thin chains of short gram-positive, catalase negative, non-spore-forming rods, which produced no gas from glucose. It could not be further identified due mainly to its unusual morphology. The unidentified levan producer, No. 2, consisted of large oval, grain, positive, catalase positive, non-spore-producing diplococci. They were oxidase negative and grew on both nutrient and lactate agar. The unidentified levan producer, No. 3, had the same characteristics, but the cocci were round and smaller. The morphology and chemical reactions of the two isolates did not fit those of any of the known genera of levan producers viz: Aerobacter, Bacillus, Colynebacterium, Pseudomonas, Acetobacter, Streptococcus, Xanthomonas and Leuconostoc (Lewis et al. 1967). Changes in viscosity during fermentation were monitored by viscosity determinations at regular intervals for all the pure culture fermentations as was done for palm wine fermentation. The fully fermented individual palm sap samples were centrifuged and those with persistent white color in the supernatant were subjected to ethanol extraction and subsequent analysis of gum obtained, as described for palm wine. Another set of experiments was carried out with the same organisms under the same fermentation conditions but using enriched sucrose solutions (Jeanes 1965) to determine if the gums elaborated in palm sap and the sucrose solution were similar in quality and quantity. All experiments were carried out in triplicate and all analyses in duplicate. RESULTS AND DISCUSSION Viscosity and Color Changes in Palm Wine The pattern of change in viscosity during palm wine fermentation is shown in Fig. I, together with the corresponding sugar and ph values. Within the first 24 h, the viscosity increased rapidly from a value of about 1.08 cps to about 1.76 cps and then decreased again almost to the starting value by the 6th day. The viscosity peaked when the sugar concentration was between 9 and lo%, ph about 3.7. The viscocity curve also showed an initial drop before the rapid increase. This is probably due to a phenomenon reported by Pour and Kadlec (1983) in which a dextran concentration of about 0.05% tended to decrease viscosity in sucrose solutions and at 0.1 % and above began to increase it. The sharp drop in viscosity after 1.76 cps or 9% sugar is not as easily accounted for. Jeanes et al. (1954, 1957) observed a drop in viscosity of a sucrose medium after completion of dextran synthesis when they employed one of their Leuconostoc strains in dextran synthesis. They were able to show that this type of viscosity decrease was due to degradation of high molecular weight

5 GUMS IN PALM WINE 397 dextrans to low molecular weight fragments by endocellular enzymes during autolytic changes in the cells. All gum producers isolated in the present study were cultured in palm sap to check for such autolytic activity on gums produced. None of the cultures showed any decrease in viscosity after the initial increases. The viscosity patterns in such inoculated palm saps are shown in Fig. 2. Therefore, either the viscosity decrease in palm wine is mediated by autocatalytic enzymes from gum-producers not isolated in this study, or one or more of the

6 398 S.V.A. UZOCHUKWU, P.O. NGODDY and E. BALOGH L TIME (DAY 5) FIG. 2. CHANGES IN VISCOSITY OF STERILE PALM SAP INOCULATED WITH SOME GUM-PRODUCTING BACTERIA ISOLATED FROM PALM WINE Key: 34 = Leuconostoc dextranicum; 44 = Leuconostoc dextranicum; 2 and 3 = unidentified fructan producers. other bacteria usually found in palm wine produce a dextranase and/or levanase but do not themselves produce gums. In this respect, the yeasts and Lactobacillus cusei spp. isolated from palm wine were also each incubated with palm sap in which dextran had been produced by Leuconostoc dextranicum and no viscosity decrease was observed. Incubation of dextran with appropriate solutions of ethanol, lactic acid and acetic acid, which are the major metabolites in palm wine, also caused no decrease in viscosity. Exodetranases, which can catalyze a stepwise hydrolysis of the dextran molecule, have been isolated from species of Bacillus (Zevenhuizen 1968) and Lactobacillus (Sidebotham et ul. 1971). Various species of these bacteria occur regularly in palm wine (Okafor 1978; Van Pee and Swings 1971) and may be responsible for breaking up the gums.

7 GUMS IN PALM WINE 399 Outside palm sap, however, it was found that the viscosity of sucrose broth inoculated with Leuconostoc mesenteroides attained a maximum viscosity of 403 centipoises after 3 days, but this gradually decreased to 134 centipoises by the 7th day. Similar changes were not observed in sucrose broth inoculated with the other gum-producing organisms. This observation suggests that L. mesenteroides does produce a dextranase and may therefore be partly or wholly responsible for the viscosity decreases observed in palm wine fermentation. The viscosity and color (optical density) changes indicated in Fig. 1 show that as palm wine ferments its viscosity increases and its color changes rapidly to white. As it gets to the souring stage (below 7% sugar), it gradually acquires a watery consistency as a result of a decrease in viscosity. This means that palm wine is most viscose or full bodied between 9.5% and 8.5% sugar content stage, i.e., when it is still relatively very sweet. It is also noteworthy that the viscosity and color peaks coincide with the end of the ph drop, thus making this perhaps the best point at which to bottle sweet palm wine. At this stage, the palm wine is at about maximum viscosity and color intensity, a safe ph (from a processing point of view), and is not too sweet (8% sugar). There appears to be very close correlation between viscosity and color intensity after centrifugation (depicted by O.D. at 720 nm and also shown in Fig. 1). This suggests that the two properties may be controlled by the same factors and that a component is synthesized in palm wine during fermentation, which increases its viscosity and imparts soluble white coloration. Microbial gums in palm wine could be responsible for both. Degumming of Palm Wine Addition of absolute ethanol to palm wine to give an ethanol concentration of 60% yielded a white gummy precipitate. Removing ethanol in VQCUO from the supernatant, produced a colorless and much less viscous liquid still possessing palm wine odor. Table 1 shows the values for viscosity and color in palm wine before and after degumming with ethanol and those for reconstituted palm wine gum. The reconstituted gum is the extracted gum dissolved in distilled water of the same volume as the original palm wine from which the gum was extracted. The experiment confirms that color and viscosity are imparted to palm wine by ethanol insoluble gums in it. Organisms Producing Gums in Palm Wine When organisms isolated from palm wine were grown in pure culture in sterile palm sap; it was found that when cells had settled at the end of fermen-

8 400 S.V.A. UZOCHUKWU, P.O. NGODDY and E. BALOGH T I 9 T- cu c. 0 +I 03? c- r 9 0 +I =t I T- 9 0 aj C.r( 3 e 4 5 a a ho aj n a 5 rn e.-i bd pc

9 GUMS IN PALM WINE 40 1 tation, the saps fermented by the yeasts and Lacrobacillus casei remained as transparent as the unfermented sap, with no increase in viscosity. On the other hand, those fermented by the Leuconosrocs, the unidentified Lactobacillus spp. (AW) and organisms No. 2 and 3, showed a color change towards white or cream and marked viscosity increases. Table 2 shows a summary of the viscosity and color of pure culture fermentations of sterile palm sap by individual palm wine microorganisms. The indication once again is that color and consistency are linked. The saps fermented by the yeasts and those fermented by Leuconosrocs are shown in Plate 1 to illustrate the color difference between the saps fermented by nongum producers on one hand and by gum producers on the other. These samples, respectively, look like unfermented and fermented saps shown in Plate 2. Among the gum producers, the color of the sap fermented by organisms 2 and 3 is about midway between the transparency of the yeast-fermented saps and the deep turbidity of the Leuconostoc ones. That of the gum producing Lczctobacillus species also looked like the Leuconosroc fermented saps. What these results suggest is that color and consistency are governed in palm wine by gums produced by bacteria, especially the Leuconostocs, The reduced solubility of the Lactobaclllus gum and the high viscosity of the palm sap when gum is produced by this organism and by Leuconostoc rnesenteroides, respectively, in it (Table 2) show that these organisms cannot be important gum producers in palm wine; otherwise palm wine would be very thick and viscous. The viscosity of the sap fermented by L. dexfranicum is closest to that of palm wine and suggests that this organism, as well as the organisms No. 2 and 3, which also produce low viscosity palm sap ferments, may be the most important organisms for gum production in palm wine (Ref. Table 2). The yeasts do not seem to play any role in this. They have been shown however, to influence the aroma and part of the taste of palm wine (Mmegwa 1987). Preparation and Some Properties of Palm Wine Gums and Those of Gum-Producing Bacteria Found in Palm Wine Two main gum fractions were obtained from palm wine, one group which precipitated at an ethanol concentration of 50% and another which precipitated at 60-65%. The precipitate at 50% was a moderate gum and could easily be prepared as a white fluffy powder following the procedure already referred to (Jeanes 1965). The 60% ethanol-insoluble gum, on the other hand, did hot yield a powder but a tough ball. Freeze drying would probably be the best method for obtaining a powder of this gum. The gum fraction precipitating at 50% ethanol concentration made up about 80% by weight of the total gum in palm wine, while that precipitating at 60 % ethanol concentration made up about 20%.

10 TABLE 2. PALM SAP CHARACTERISTIC AFTER 24 H FERMENTATION BY PURE CULTURES OF MICROORGANISMS ISOLATED FROM PALM WINE ~ ~~ - Color on settlement of microorganisms Creamy White lkansparent Transparent Creamy White White Transparent Transparent Faint white Transparent but slimy Creamy White P 0 N Sample/organism in palm sap -~ Viscosity (cps) Color at 72Onm Fresh Palm sap (0-h ) wansparent with brownish tinge -do- Pasteurized palm sap (0-h ) Naturally fermented palm sap(pa1m Wine) - S. cerevisiae - S. chevalieri Leuconostoc mesenteroides Leuconostoc dextranicum Lactobacillus & _--- L. casei SS casei Organism No. 2 Organism No. 3 Unidentified Lactobacillus (AW) 1.021, , , % , , , ,

11 GUMS IN PALM WINE 403 PLATE 1. PALM SAP AFTER FERMENTATION BY SACCHAROMYCES CEREWSIAE (10) SACCHAROMYCES CHEVALJERE (14), LEUCONOSTOC DEXTRAhTCCJM (34) AND LEUCONOSTOC MESEEROIDES (35) PLATE 2. PALM WINE (A AND B) AND PALM SAP (C AND D) IN CENTRIFUGED (A AND D) AND NATIVE (B AND C) FORMS

12 TABLE 3. PROPERTIES OF GUMS FOUND IN PALM WINE AND GUMS PRODUCED BY GUM-FORMING BACTERIA ISOLATED FROM PALM WINE Solubi. lity in water (-I + Const i tuent sugar Gliicose Glucose Fructose Pruc tose Glucose Glucose Fructose 0 P Gum Producer/gum type Leuconostoc mesenteroides Leuconostoc dextranicum Fructan producer No. 2 Grown in Palm Sap Yield g/ioomls ' 1.01 f 0.0: O.OE Viscosity of 10% so lu t ion (cps) ,+ 0.2 Grown in sucrose Yield g/?ocmls 2.5 ;I Viscosity of ICb so 1 u t ion (cps) TI Nature of gullla Touch cohesive Short cohesive Fluid Fructan Producer No. 3 PW 50 Lactobacillus spp. PW ' : 3.22 f Stringy Short crumbly Short crumbly Stringy PW 50 = 50$ ethanol-insoluble palm wine gum; + = soluble +(-) = sparingly soluble PW 60 = 6% ethanol-inxclulle palm wine cum a = observed w1.e~ precipitated from aquebus ethanol solution,

13 GUMS IN PALM WINE 405 Each gum-producing bacterium was grown in both palm sap and standard sucrose medium. The characteristic features such as yield, viscosities, constituent sugars and physical nature of all the gums prepared are shown in Table 3. The gums produced by Lactobacteriaceae and the 50% ethanol insoluble palm wine gum required refluxing in 10% HC1 for 4 h or more for complete hydrolysis. The gums of organisms No. 2 and 3 and the 60% ethanol-insoluble palm wine gum, required refluxing in % oxalic acid for 30 min only for complete hydrolysis. On thin-layer chromatography of the hydrolysates, it was found that the gums that required HCl for hydrolysis contained glucose as the only sugar, while those easily hydrolyzed with oxalic acid contained only fructose. It is a characteristic of fructose polymers such as levans to be easily hydrolyzed. The results show great similarity between the Lactobacteriaceae gums and the 50% ethanol-insoluble palm wine gum. The 60% ethanol-insoluble palm wine gum and the gums of the organisms No. 2 and 3 are also comparable. This suggests that in natural palm wine fermentation, the Lactobacteriaceae produce the palm wine glucans, while such organisms as No. 2 and 3 suspected to be non-spore-forming Bacillus produce the fructans in the beverage. Leuconostocs can also produce fructans (Sidebotham el al. 1971), but there was no evidence (from the pure culture studies) that those encountered in the present study produced any. These gums, which are soluble in water, form gel-like structures in cold water, and contain either exclusively glucose or exclusively fructose, are likely to be dextrans and levans, respectively. However, only a determination of the types of linkages involved can confirm this. Apart from color and consistency, gums in palm wine may further influence other organoleptic properties in the beverage. Several workers (Koikini 1985; Moskowitz and Arabie 1970; Pangborn et al. 1973) have shown that gums or hydrocolloids depress perceived sweetness and sourness in fluid foods. It is conceivable that the gums in palm wine may play this role, thus removing the sharp edge from sweetness and sourness and leaving a smoother taste. Thus, it could be recommended that the 8.5% sugar concentration stage is the best point at which to bottle palm wine, for the relatively high viscosity at that stage may mean a smoother taste. CONCLUSION All microorganisms in palm wine contribute to its turbidity, but the white coloration that remains when these might have settled to the bottom or after centrifugation is due to a soluble material absent before fermentation. Results

14 406 S.V.A. UZOCHUKWU, P.O. NGODDY and E. BALOGH from this study suggest that the viscosity and stable white coloration of palm wine change during fermentation and that these two characteristics are controlled in the beverage by gums produced during fermentation by various gum-producing bacteria. Of the gum producers isolated from palm wine, Leuconostoc dextranicum and the fructan producers thought to be Bacillus spp. appear to be the most important. The presence of only glucose and fructose, respectively, in the gums as well as their other characteristics indicate that the gums are likely to be dextrans and levans, respectively. Results suggest that palm wine is at its best at the % sugar stage and should be bottled at that point for sweet palm wine. REFERENCES HODGE, J.E. and HOFREITER, B.T Determination of reducing sugars and carbohydrates. Meth. Carbohyd. Chem. I, 116. JEANES, A Preparation of dextran from growing Leuconostoc cultures. In Methods in Carbohydrate Chemistry, Vol. 5, (R.L. Whistler, ed.) pp , Academic Press, New York. JEANES, A. et al Characterisation and classification of dextrans from 96 strains of bacteria. J. Am. Chem. SOC. 76, JEANES, A., WILHAM, C.A., TSUCHIYA, H.M. and HAYNESS, W.C., Properties of dextrans isolated from whole cultures at various stages of incubation. Arch. Biochem. Biophis. 71, KOKINI, J.L Fluid and semifluid food texture and texture-taste interactions. Food Technol. 39, LEWIS, B.A., St. CYR, M.J. and SMITH, F.S The constitution of the fructan produced by Leuconostoc rnesenteroides Strain C. Carbohyd. Res. 5, MMEGWA, S.V.A Factors controlling organoleptic properties in palm wine fermentation. Ph.D. Thesis. University of Nigeria, Nsukka. MOSKOWITZ, H.R. and ARABIE, P Taste intensity as a function of stimulus concentration and solvent viscosity. J. Texture Studies I, OKAFOR, N The microbiological basis for a method for palm wine preservation. J. Appl. Bacteriol. 43, OKAFOR, N Microbiology and biochemistry of oil palm wine. Adv. Appl. Microbiol. 24, PANGBORN R.M., TRABUE, I.M. and SZCEZESNIAK, A.S Effect of hydrocolloids on oral viscosity and basic taste intensities. J. Texture Studies 4, POUR, V. and KADLEC, F Effect of dextran on the viscosity of sugar solutions. Sb. Vys. Sk. Chem. Technol. Praze. E. 56, 9-25.

15 GUMS IN PALM WINE 407 SAMARAJEEWA, V., MATHES, D.T., WIJERATNE, M.C.P. and WARNA- KULA, T Effect of sodium metabisulfite on ethanol production in coconut inflorescence sap. Food Microbiol. 2, SIDEBOTHAM, R.R., WEIGEL, H. and BOWEN, W.H Studies on dextrans and dextranases. Part IX. Dextrans elaborated by cariogenic organisms. Carbohy. Res. 19, ULOA, M. 1981, Indigenous fermented beverages of Mexico. In Global Impacts of Applied Microbiology, (S.O. Emejuaiwe, 0. Gumbi and S.O. Sanni, eds.), Academic Press, New York. UZOCHUKWU, S.V.A., BALOGH, E. and NGODDY, P.O Standard pure culture inoculum for natural and formulated palm sap fermentation. Niger. Food. J. 9, VAN PEE, W. and SWINGS, J.G Chemical and microbiological studies on Congolese palm wine (Elaeis guineensis) East Afr. Agric. For. J. 36, ZEVENHUIZEN, L.P.T.M Cell-bound exodextranases of Bacillus species. Carbohyd. Res. 6,