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Growth of yeasts, lactic and acetic acid bacteria in palm wine during tapping and fermentation from felled oil palm (Elaeis guineensis) in Ghana
W.K. Amoa-Awua1, E. Sampson2 and K. Tano-Debrah3
1 Food Research Institute, Council for Scientiﬁc and Industrial Research, Accra, Ghana 2 Nestle Ghana Ltd., Tema, Ghana 3 Department of Nutrition and Food Science, University of Ghana, Legon, Ghana
Keywords acetic acid bacteria, lactic acid bacteria, palm wine, Saccharomyces cerevisiae, yeasts. Correspondence W.K. Amoa-Awua, Food Research Institute, Council for Scientiﬁc and Industrial Research, P.O. Box M.20, Accra, Ghana. E-mail: firstname.lastname@example.org
Abstract Aim: To investigate the microbiological and biochemical changes which occur in palm wine during the tapping of felled oil palm trees. Methods and Results: Microbiological and biochemical contents of palm wine were determined during the tapping of felled oil palm trees for 5 weeks and also during the storage. Saccharomyces cerevisiae dominated the yeast biota and was the only species isolated in the mature samples. Lactobacillus plantarum and Leuconostoc mesenteroides were the dominated lactic acid bacteria, whilst acetic acid bacteria were isolated only after the third day when levels of alcohol had become substantial. The pH, lactic and acetic acid concentrations during the tapping were among 3Æ5–4Æ0%, 0Æ1–0Æ3% and 0Æ2–0Æ4% respectively, whilst the alcohol contents of samples collected within the day were between 1Æ4% and 2Æ82%; palm wine which had accumulated over night, 3Æ24% to 4Æ75%; and palm wine held for 24 h, over 7Æ0%. Conclusion: Accumulation of alcohol in palm wine occurs in three stages during the tapping and marketing with the concurrent lactic and acetic acid fermentation taking place as well. Signiﬁcance and Impact of the study: Yeasts, lactic and acetic acid bacteria are all important in the fermentation of palm wine and inﬂuence the composition of the product.
2006/0063: received 18 January 2006, revised 15 April 2006 and accepted 27 April 2006
Introduction The sap of the oil palm tree (Elaeis guinneesis) serves as a rich substrate for various types of micro-organisms to grow. However, it is as a source for producing the traditional wine, palm wine, that the substrate is popular. In various African countries and beyond, the sap of the palm tree is tapped and allowed to undergo spontaneous fermentation, which allows the proliferation of yeasts species to covert the sweet substrate into an alcoholic beverage. In various traditional African societies, the palm wine played a signiﬁcant role in customary practices, especially the distilled product from the palm wine, a potent gin called by various names in West Africa, for example
akpeteshie in Ghana. Because of the central role that the alcoholic beverage has played in the traditional society, it is important that the microbiology and biochemistry of the fermentation process are well understood. However, there is a limit to the number of studies carried out in this context, especially information available in the international literature on the microbiology of palm wine tapped from felled palm trees. The publications on palm wine have dealt with the product processed from the palm tree whilst it is still left intact and standing upright as a growing tree. In this instance, the palm sap is obtained from either the immature male inﬂorescence (inﬂorescence tapping) or from the stem (stem tapping). This is commonly practised in Nigeria, Benin and
ª 2006 The Authors Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 599–606
Merck KGaA. Aurora.. lactose.. An incision is made in the ﬂoor of the well through which a bamboo tube is inserted to deliver the sap into a receiver. Tapping involves the removal of the fronds in the meristematic region and cutting out a deep rectangular well of about 7Æ5 cm in the vegetative core of the tree. Merck 10660. Aerobic mesophiles were enumerated by pour plate on Plate Count Agar (Difco 0479–17–3. samples collected from different trees on a particular day were pooled together in a plastic container. galactose. Amoa-Awua et al. 600 Enumeration of yeasts and bacteria The palm wine sample was shaken by hand in the stomacher bag and 1Æ0 ml pipetted into 9 ml sterile Salt Peptone Solution containing 0Æ1% peptone (Difco 0118–17. trehalose.0010. On GYC. USA) incubated at 30°C for 4 days. 25.118–17). Hartley 1984). Darmstadt. kept at ambient temperature (Ca 30°C) either for 24 h or 6 days and sampled either every 2 h or daily respectively. Palm wine sample was collected every other day from each tree during the 5 weeks of tapping into a stomacher bag and taken immediately to the laboratory for analysis. Oxoid CM545. Becton Dickinson & Co. Oxoid Ltd. Acetic acid bacteria were enumerated by spread plate on GYC and YPM Agar at 30°C for up to 2 weeks for GYC plates. characterization and tentative identiﬁcation of isolates About 15 colonies from a segment of the highest dilution or suitable OGYEA. Isolates which were gram-positive catalase- ª 2006 The Authors Journal compilation ª 2006 The Society for Applied Microbiology. MI.08342) and 20. After serial dilution. MRS. tapping from the mature felled palm tree (down wine) results in a different composition of palm sap than that obtained from the live tree. the development of various microbial species in palm wine during the tapping of felled palm trees was investigated with emphasis on the yeasts and lactic acid bacteria responsible for the alcoholic fermentation and souring of the product. The role of acetic acid bacteria in the development of vinegary taste in palm wine was also investigated.Growth of yeasts. All isolates were examined by colony and cell morphology. Isolates on MRS were examined by Gram reaction and catalase test. According to Ayernor and Matthews (1971).. Journal of Applied Microbiology 102 (2007) 599–606 . The storage experiments were carried out in three replicates. in Ghana. The GYC medium contained g l)1 distilled water. 50. only colonies which were able to produce clearing zones around their colonies at one stage or other were isolated.03753). xylose and meliobiose according to Kurtzman and Fell (1998) and also utilization of various carbo´ hydrates in ID 32 C galleries (Bio Merieux S. the process of tapping palm wine involves ﬁrst felling or cutting down the tree. 3. 10 mg ml)1 of cycloheximide (ICN 100183) made up in 50% ethanol and 20 ml l)1 of penicillin (AppliChem A1837. respectively. Palm wine samples undergoing the further fermentation for distillation were collected from three traditional distillers near Accra for analysis.. CaCO3 (Merck 1. To both GYC and YPM. Sparks. Basingstoke. followed by tapping for up to 8 weeks. USA) to suppress yeast growth and incubated in an anaerobic jar with anaerocult A (Merck) at 30°C for 5 days. it begins to develop a vinegary taste.A. However. yeast extract (Merck 1. Hartley 1984). Germany) prepared from a 0Æ25% stock solution were added to inhibit the growth of yeasts and lactic acid bacteria. Market samples of palm wine were also obtained from the Eastern and Ashanti Regions for analysis. USA) and 0Æ85% NaCl with pH adjusted to 7Æ2 as the 1 : 10 dilution. Palm wine may also be allowed to undergo further alcoholic fermentation by local distillers. which may be a clay pot or a plastic container (Okraku-Offei 1968. who add sugar to the product and allow it to ferment for a further 4–7 days and then distill it into the local gin. yeast extract (Merck 1. ICN Biomedicals Inc. La Cote d’Ivoire (Okafor 1972.118–17). maltose. Lactic acid bacteria were enumerated by pour plate on DeMan. sucrose. GYC and YPM plates were subcultured by streaking repeatedly on agar till pure cultures were obtained. glucose (Merck 1.02066). AppliChem GmbH. If the collected palm wine is not consumed within a few days. France). Materials and methods Materials Palm wine samples were obtained during the tapping of 15 palm trees in ﬁve different locations in the Greater Accra Region of Ghana over a period of 6 months. OH. Isolation.03753).05982) and 12. which was carried out in replicates. peptone (Difco 0. which is unacceptable to consumers. Marcyl’Etoile.K. Rogosa and Sharpe Agar (MRS. yeasts were enumerated by pour plate on Oxytetracycline-Glucose Yeast Extract Agar (OGYEA. Difco Laboratories. For elucidating the microbiological and chemical changes which occur during the storage of palm wine. MD. agar (Merck 1. 10. leaving the felled tree for a period of about 2 weeks for the sap to concentrate. Detroit.01614). cellobiose. rafﬁnose. 30. mannitol (Merck 1. UK) incubated at 30°C for 5 days. 5. Hampshire. Yeast isolates were tentatively identiﬁed by determining their pattern of fermentation and assimilation of glucose. lactic and acetic acid bacteria in palm wine W. agar (Difco 0. Germany) containing 10 mg ml)1 cycloheximide (ICN 100183. YPM agar contained g l)1 distilled water. In this work. Darmstadt. and that down wine contains some methanol and propanol in addition to ethanol.
A.W. palm wine samples were not collected beyond 5 weeks as most of the trees had ceased to produce palm wine. the aerobic mesophiles from Day 4 onwards reduced to concentrations of about 108 CFU ml)1 and maintained this concentration throughout the tapping. The species of lactic acid bacteria were tentatively identiﬁed by determining their pattern of carbohydrate fermenta´ tion in API 50 CHL galleries (Bio Merieux S. by the fourth day. Similarly. cessation of the ﬂow of palm wine varied from tree to tree.03753). Growth of yeasts. lactic and acetic acid bacteria in palm wine negative regular rods. their ability to grow on differential agar including GYC. Acetic acid bacteria were isolated in almost all samples only after the third day and maintained a concentration of 107 CFU g)1 throughout the tapping. lactic acid bacteria counts ranging between 5Æ1 · 108 and 1Æ3 · 109 CFU ml)1. occurred in yeast numbers after the initial increase and from Day 2 onwards were encountered at concentrations of 107 CFU ml)1. Radiometer Analytical A/S. and aerobic mesophiles from concentrations of 107 to about 109 CFU g)1 during the ﬁrst 24 h of tapping (Fig. The Modiﬁed Carr Medium contained g l)1 distilled water. Amoa-Awua et al. the shortest duration being 2 weeks and the longest 8 weeks. Bagsvaerd. Carr 1968. and Yeast Extract-Ethanol broth containing bromocresol purple for over-oxidation of ethanol (Frateur 1950. growth at pH 4Æ4 and 9Æ6. For over-oxidation of ethanol. however. a drastic increase was observed in yeast numbers from average values of 105 to 107 CFU ml)1. The gradual buildup in the concentration of alcohol during the initial stages of tapping was considered to be responsible for the increase in the numbers of the acetic acid bacteria to detectable concentrations by the fourth day as they were able to use the ethanol produced as carbon source for growth. Hundred millilitres of palm wine was diluted with 50 ml of water. Microbiological examination of four market samples of palm wine obtained from the Ashanti and Eastern Regions recorded aerobic mesophilic counts ranging between 8Æ4 · 108 and 5Æ2 · 109 CFU ml)1. 20. 1a). and Hugh and Leifson test (Hugh and Leifson 1953).K. Drysdale and Fleet 1988. The duration of tapping i. growth at 10°C and 45°C. however. yeast counts between 3Æ5 · 107 and 1Æ5 · 108 CFU ml)1 and acetic acid bacteria counts of between 1Æ2 · 108 and 6Æ0 · 108 CFU ml)1. 30. Journal of Applied Microbiology 102 (2007) 599–606 . However.e. its relative speciﬁc gravity was determined by dividing the weight of 25 ml of the distillate by the weight of an equal volume of water using a 25-ml speciﬁc gravity bottle and referring to the reference table. Chemical analysis Determination of pH and titratable acidity The pH of palm wine samples was determined directly using a pH meter (Radiometer PHM 92. samples collected within 3 h of tapping had alcoholic content of about 1Æ5%. Percentage of acetic acid was obtained by multiplying the titre by 0Æ3 (Kirk and Sawyer 1991). 30. Results Microbial population in palm wine The products obtained during the ﬁrst day of tapping were very sugary and did not contain substantial concentrations of alcohol. Determination of total sugars The percentage of total sugars in palm wine samples was determined by the Lane and Eynon method according to Kirk and Sawyer (1991). yeast extract (Merck 1. ethanol. For determination of titratable acidity. Determination of alcohol content from speciﬁc gravity The percentage of alcohol by volume from speciﬁc gravity was determined according to AOAC (1990). After the initial increase in numbers. One millilitre of 0Æ1 mol l)1 NaOH was taken as equivalent to 0Æ009 g lactic acid. Determination of acetic acid concentration Ten millilitres of sample was diluted with distilled water and titrated against 0Æ5 mol l)1 NaOH using the phenolphthalein as indicator. the broth medium contained g l)1 distilled water. and 0Æ022 g of bromocresol blue with an inverted Durham tube to trap the CO2 liberated from over-oxidation of ethanol. Isolates of acetic acid bacteria were examined by gram and catalase reaction. After collecting the 100 ml of distillate. YPM. and distilled. Palm wine samples being further fermented by traditional distillers for the production of the local gin. Du Toit and Lambrechts 2002). No reduction. the lactic acid bacteria population dipped slightly to a concentration of about 108 CFU g)1 and remained at this concentration throughout the tapping. Denmark) after calibration using the standard buffers. lactic acid bacteria from concentrations of 105 to 109 CFU ml)1. 601 ª 2006 The Authors Journal compilation ª 2006 The Society for Applied Microbiology. Modiﬁed Carr Medium. With respect to the microbial population. ethanol.). coccoid or cocci were assumed to be lactic acid bacteria and further examined by gas production in MRS broth with Durham tube and also in MRS broth in which glucose was replaced with gluconate as sole carbon source. growth in 6Æ5 and 18% (w/v) NaCl.03753) and 20. yeast extract (Merck 1. 10 ml of sample was made up to 200 ml with distilled water and 80 ml titrated against 0Æ1 mol l)1 NaOH using 1% phenolphthalein as indicator.
Of these isolates. pH. (b) Mean pH and percentage total sugars in ﬁfteen samples of palm wine during the tapping for 5 weeks (SD for various mean pH values between 0Æ07 and 0Æ45).Growth of yeasts. as acetic acid was present in all the mature palm wine samples at concentrations of 0Æ2 to 0Æ4%. Yeasts. lactic and acetic acids. palm wine samples collected within the day were found to contain between 1Æ4% and 2Æ82% alcohol. whilst the percentage of acetic acid after the sixth day was between 0Æ2 and 0Æ4%. They were tentatively identiﬁed as S. lactic and acetic acid bacteria in palm wine W. Palm wine samples undergoing further fermentation for distillation into the local gin had pH values ranging between 3Æ73 and 3Æ86. respectively. The average percentage of total sugars in the palm wine samples dropped from initial concentrations of about 14% to about 11% by the fourth day and subsequently to between 8% and 12%. and acetic acid bacteria counts at the concentrations of 107 CFU ml)1. Journal of Applied Microbiology 102 (2007) 599–606 . and percentage acetic acid between 0Æ46% and 0Æ55%. Also. lactic acid. four different types of yeasts were recognized in the palm wine samples.K. two were identiﬁed as ª 2006 The Authors Journal compilation ª 2006 The Society for Applied Microbiology. percentage lactic acid between 0Æ24% and 0Æ28%. SD of various mean values for aerobic mesopiles between 0Æ16 and 0Æ85. they were suspected to be Saccharomyces cerevisiae. Chemical composition of palm wine The results of pH conﬁrmed the importance of lactic acid bacteria in the fermentation of palm wine. After the fourth day of tapping. cerevisiae. showed aerobic mesophilic and yeast counts increasing from the concentrations of 107 to 109 and 107 to 108 CFU ml)1. whilst samples which had accumulated overnight had much higher levels of alcohol. lactic acid bacteria ranging between 8Æ0 · 107 and 2Æ2 · 108. Aerobic mesopiles. According to Faparusi (1973). Only ﬁve out of the 188 isolates identiﬁed were not S. trehalose and rafﬁnose. The percentage of lactic acid after the ﬁrst few days of tapping was between 0Æ1 and 0Æ3%. all the yeast isolates fermented glucose. They assimilated mainly glucose. between 3Æ24% and 4Æ75% and even over 6% in a few cases. lactose. Amoa-Awua et al. lactic acid bacteria between 0Æ05 and 0Æ56. acetic acid was considered as part of the aroma of palm wine. acetic acid between 0Æ11% and 0Æ13% and alcohol content between 7Æ47% and 10Æ58%. isolated in the palm wine samples on the ﬁrst 3 days of tapping. This concentration was never exceeded during the 36 days of sample collection in any of the samples. Lactic acid bacteria. From near neutral values on the ﬁrst day. Based on the colony and cell morphology including the growth of isolates in liquid medium as well as the assimilation and fermentation of 10 carbohydrates. Total sugars. ( Acetic acid bacteria. By reference to Kurtzman and Fell (1998). rafﬁnose. Lactic acid bacteria were considered to be responsible for the rapid acidiﬁcation of the product as the acetic acid bacteria were not 602 A total of 188 yeast isolates were identiﬁed. representative isolates utilized mainly galactose. saccharose. sucrose. acetic acid bacteria between 0Æ12 and 0Æ43. cellubiose. In ID 32 C galleries. Nearly. but not arabinose. palm wine generally becomes unacceptable to consumers when the concentration of acetic acid exceeds 0Æ6%. Yeasts present in palm wine akpeteshie. but not lactose and trehalose. (a) Microbial counts in log10 CFU ml–1 10 9 8 7 6 5 4 3 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 Day of tapping palm wine 8 7 6 5 4 3 2 1 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 Day of tapping palm wine 0 pH (b) 16 Percentage total sugars 14 12 10 8 6 4 2 0 Figure 1 (a) Mean values of the population of various micro-organisms in palm wine during the tapping of the ﬁfteen felled oil palm trees. sucrose and rafﬁnose. lactic acid concentration between 0Æ63% and 0Æ75%. cerevisiae based on the API database. 1b). The market samples of palm wine from the Eastern and Ashanti Regions had pH values of between 3Æ3 and 3Æ6. maltose. the pH dropped to values between pH 4Æ5 and 4Æ0 within 24 h and subsequently to between 3Æ5 and 4Æ0 for most of the trees throughout the tapping (Fig. This concentration of sugars was maintained because of the continual oozing of the sweet sap into the fermenting palm wine despite the conversion of the fermentable sugars into alcohol. maltose and glucose. yeasts between 0Æ08 and 0Æ80. ribose and xylose.
N-acetyl glucosamine. the following factor appears important. however. as high numbers of aerobic mesophiles.e. d-rafﬁnose. rafﬁnose. Growth of yeasts. respectively. They were able to grow at 15°C but not at 45°C. The drop in the alcohol content in all cases coincided with an increase in the concentration of acetic acid from a mean value of 0Æ51% on the third day and had exceeded 0Æ6% by the next day. catalase-positive. d-mannose. nor in 6Æ5% (w/v) NaCl. sorbitol. This was very revealing as it showed that the development of alcohol in palm wine was more pronounced during the handling of the product than during the tapping. The presence of Gluconobacter species were also recognized as they were unable to re-deposit calcium carbonate at the bottom of GYC Standard Medium. b-gentiobiose and d-turanose. hence facultatively heterofermentative. cellobiose. saccharose. i. They also produced gas in the Durham tubes. Growth of these isolates on Malt Extract Agar containing bromocresol blue resulted in a change in colour from purple to yellow and on further incubation back to purple. one as Candida krusei and two could not be not identiﬁed with the API kit. trehalose. lactic acid bacteria. a-methyl-d-mannoside. Journal of Applied Microbiology 102 (2007) 599–606 . The dominant species could grow at 15°C but not at 45°C. mannitol. d-mannose. d-fructose. Acetic acid bacteria present in palm wine Acetic acid bacteria isolated from palm wine during the tapping were gram-negative. arbutin. salicin. They fermented mostly ribose. ribose. lactose. cerevisiae were isolated. increased slightly during the ﬁrst 2 days and then remained at that concentration for the rest of the 6 days. d-glucose. salicin. cellobiose. However. In the market samples from the Eastern and Ashanti Regions as well as further fermenting palm wine obtained from distillers. galactose. which metabolized the glucose fermentatively in Hugh and Leifson medium. Longer storage of palm wine eventually led to the accumulation of acetic acid to concentrations that made the product unacceptable. they fermented mostly l-arabinose. only the S. d-xylose. they also caused a change in colour from purple to yellow. 2). The acetic acid bacteria population. nor in 6Æ5% (w/v) NaCl. trehalose. l-arabinose. In discussing the population of the various micro-organisms in palm wine. melezitose. 3). These results showed that the isolates were able to oxidize ethanol to acetic acid and then further to CO2 and H2O showing that they were Acetobacter species. Lactic acid bacteria present in palm wine Lactic acid bacteria isolated from palm wine during the tapping were dominated by regular rods.W. maltose. yet showed a steady increase in the average alcohol content of three replicate samples from 3Æ42% to 8Æ16% (Fig. Amoa-Awua et al. d-glucose. whilst the mean values for both lactic and acetic acids remained fairly constant at the concentrations of 0Æ27% to 0Æ32% and 0Æ42% to 0Æ48%. melibiose. esculin. oxidasenegative. were obligately heterofermentative. maltose. In Modiﬁed Carr Medium containing bromocresol blue. each of the products had developed a distinctly vinegary character with very high acetic acid content averaging 2Æ03%. Discussion Growth of micro-organisms in palm wine during fermentation The sap of the palm tree has been shown to be a rich medium capable of supporting the growth of various types of micro-oganisms. produced pigments which dissolved in water and were able to dissolve the calcium carbonate at the bottom of GYC Standard Medium plates. d-fructose. a-methyl-d-glucoside. The yeasts and lactic acid bacteria population in three replicate samples remained fairly constant for 4 days and then began to drop (Fig. The average values of the total sugars dropped steadily from 11Æ02% to 3Æ32% in the holding time of 24 h. d-turanose and gluconate and were tentatively identiﬁed as Lactobacillus plantarum. lactose. They were tentatively identiﬁed as Leuconostoc mesenteroides. yeasts and acetic acid bacteria were found in palm wine during the tapping of felled palm trees for up to 5 weeks. esculin. A second type of lactic acid bacteria identiﬁed was lenticular to coccoid in shape and metabolized glucose fermentatively in Hugh and Leifson medium. amygdaline. and produced gas from glucose but not gluconate. A drastic increase occurred in the average alcohol content of the samples during the ﬁrst day to about 7% and remained high for the next 3 days after which it began to drop. isolates which were considered to be Acetobacter species redeposited the calcium carbonate after a few days. but on further incubation reverted to purple. were nonoxidative. In the API galleries. By the sixth day. b-gentiobiose. N-acetyl glucosamine. saccharose. Holding/storage of palm wine Holding/storing palm wine for 24 h produced little changes in the population of the various micro-oganisms present. melibiose. amygdaline. galactose. they did not produce gas in the Durham tubes and changed the colour of the indicator from purple to yellow without reversion to the purple colour on further incubation. arbutin. In Modiﬁed Carr Medium with bromocresol blue and Durham tube.K. lactic and acetic acid bacteria in palm wine Kloechera apiculata. That to prevent insect 603 ª 2006 The Authors Journal compilation ª 2006 The Society for Applied Microbiology.
Palm wine tappers collect their wine in the morning and evening. the tapper cuts a thin slice off the walls of the receptacle daily to expose a fresh layer. The nature/system of palm wine fermentation and production of alcohol The concentration of alcohol in palm wine samples collected during the tapping were found to be low and dependent on several factors including the nature/system of fermentation. % acetic acid. lactic and acetic acid bacteria in palm wine W. the average alcohol content of palm wine samples was found to have increased from about 3% to over 7%. the fermentation of palm wine can be described on three levels or as a threestage fermentation. and larvae infestation and also facilitate the oozing of the sap. Lactic acid bacteria. 2. Yeasts. pH. however. Lactic acid bacteria. Despite this. appears to occur after the product has been collected by the tapper i. This could be explained by the results presented in Fig. (b) Changes in the chemical composition of palm wine during the holding for 24 h. · Lactic acid. Acetic acid. during the handling and marketing. · % lactic acid. The ﬁrst level occurs in the receptacle cut out in the palm tree itself. The highest build of alcohol in palm wine. Based on the above observations. Such samples were found to contain much higher concentrations of alcohol ranging from 3% to 6Æ5% and this is in agreement with the alcohol concentrations reported by Herzog et al. Figure 3 (a) Mean values of the population of various micro-organisms in three samples of palm wine during the storage/holding for Aerobic mesophiles. but wine collected in the morning would have accumulated throughout the night. Samples collected within 8 h of tapping 604 did not contain more than 3% alcohol. During this period. lactic and acetic bacteria during the 24 h storage.K. (b) Changes in the chemical composition of palm wine during the storage/holding for 6 days Alcohol. (1995) in La Cote d’Ivoire. (a) 9 Microbial counts in log10 CFU ml–1 8 7 6 5 4 3 2 1 0 0 2 4 6 8 10 12 14 16 18 20 22 24 Holding time (h) Microbial counts in log10 CFU ml–1 (a) 9 8 7 6 5 4 3 2 1 0 0 1 2 3 4 5 Storage/holding time (days) 6 (b) 8 (b) 12 10 Percentage 8 6 4 2 0 0 2 4 6 4·5 4·3 4·1 3·9 3·7 3·5 3·3 3·1 2·9 2·7 2·5 8 10 12 14 16 18 20 22 24 Holding time (h) 7 6 Percentage 5 4 3 2 1 0 0 1 2 3 4 Storage/holding time (days) 5 6 4·5 4·3 4·1 3·9 3·7 3·5 3·3 3·1 2·9 2·7 2·5 pH Figure 2 (a) Mean values of the population of various micro-organisms in three samples of palm wine during the holding for 24 h Aerobic mesopiles. aerobic mesophiles. This physically removed the microbiota that had colonized the walls of the receptacle reducing the microbial load in the chamber. pH. the microbial population found in the palm wine samples appeared to be fairly stable. ( 6 days Acetic acid bacteria.Growth of yeasts. % total sugars. In 24 h. Yeasts. which showed that very little variations occurred in the populations of yeasts. the concentration of acetic acid did not change and it was only after 4 days that the acetic acid buildup reached the unacceptable concentrations. % alcohol. Amoa-Awua et al.e. Journal of Applied Microbiology 102 (2007) 599–606 pH . This occurs as a continu- ª 2006 The Authors Journal compilation ª 2006 The Society for Applied Microbiology. ( Acetic acid bacteria.
The presence of lactobacilli and leuconostoc in palm wine samples in Nigeria have also been reported by Faparusi and Bassir (1971) and Okafor (1975). In mature palm wine samples. During the holding/marketing of palm wine. from palm wine samples in Nigeria. Pichia. cerevisiae in down wine. This third stage of fermentation occurs as a batch process under more anaerobic conditions. The highest buildup in alcohol level occurs during the distribution and marketing. Microbial species responsible for the fermentation of palm wine Saccharomyces cerevisiae has been conﬁrmed in the present work as the dominant yeast species responsible for the fermentation of palm wine tapped from the felled palm trees. Earlier. the concentration of alcohol increased from 3% to over 7% in 24 h. whilst Fahwehinmi (1981) also reported the presence of Saccharomyces chevalieri and Pichia membrabefaciens. Faparusi (1973) and Okafor (1972) had reported S. Lactobacillus plantarum and Leuconostoc mesenteriodes have been identiﬁed in the present work as the dominant lactic acid bacteria responsible for the sour taste of palm wine tapped from the felled palm trees in Ghana. Journal of Applied Microbiology 102 (2007) 599–606 . cerevisiae and Schizosaccharomyces spp. This is in agreement with other studies carried out in Ghana (Okraku-Offei 1968. Saccharomycoides and Schizosacchromyces in palm wine tapped from oil and rafﬁa palms. Dieng et al. and samples which had accumulated overnight had alcohol contents of over 3%. mesenteriodes as being responsible for the souring of palm wine tapped from the live upright palm tree. plantarum and L. reported the presence of several genera of yeasts including Saccharomyces. It is possible that differences in the chemical composition of palm wine tapped from the felled and upright trees favour the complete domination of the yeast biota by S. The buildup of alcohol in the container is faster than in the tree trunk because there is no loss of the product from this chamber although there is continual dilution of the contents with the fermenting juice trickling in. Acknowledgement This work was facilitated by the ﬁnancial support from DANIDA (The Danish International Development Assistance) under the collaborative research project of the CSIR-Food Research Institute. Amoa-Awua et al. mesenteroides. whilst Ezeronye and Okerentugba (2000) reported the presence of S. Brown (1994) used restriction fragment length polymorphism to identify the yeast isolates from the market samples of palm wine from several towns in southern Ghana and reported the presence of only S. In Nigeria. Earlier. Enwefa et al. ‘Capability Building for Research Into Traditional Fermented Food Processing in West Africa’. lactic and acetic acid bacteria in palm wine ous culture fermentation although the palm wine tapper periodically disturbs the microbial population in the biological fermentor (a semi-continuous). (1992).W. remained high for the next 3 days and began to drop. Uzochukwu et al. Kleoclera. apiculata from the palm wine. The three other yeasts encountered in the present work including K.K. Hansenula. cerevisiae were isolated and this species appear to completely dominate the fermentation of palm wine in the felled palm trees. (1998) (unpublished results) identiﬁed S. The second level occurs as palm wine accumulates in the container placed under the tree. cerevisiae and K. krusei were isolated during the ﬁrst 3 days of tapping when the sap collected was basically nonalcoholic. Accra. (1994) isolated L. Increases in the alcohol level of palm wine were faster in the container into which the palm wine accumulated during the tapping than in the receptacle cut out in the tree trunk. Growth of yeasts. Conclusion A concurrent alcoholic. other yeast species have been reported to be important in the fermentation of palm wine in addition to S. Owuana and Saunders (1990) isolated both S. cerevisiae. cerevisae and Schizosacchoromyces pombe as the dominant yeasts in palm wine. The authors are grateful to Messrs 605 ª 2006 The Authors Journal compilation ª 2006 The Society for Applied Microbiology. According to Ayernor and Matthews (1971). 1994). lactic acid and acetic acid fermentation occurred during the tapping of palm wine from felled oil palm trees. only S. cerevisiae. Van Pee and Swing (1971) reported S. cerevisiae and Saccharomyces pastorianus as the dominant yeasts in palm wine samples in Congo. Lactic acid bacteria dominated by L. as the dominant yeasts in palm wine samples in Senegal. Brown 1990. In the palm wine samples tapped from the upright trees in neighbouring countries. cerevisiae. Yeast growth dominated by S. leuconostoc dextranicum and Lactobacillus spp. Owusu 1982. plantarum and L. apiculata and C. cerevisiae started immediately after tapping began and alcohol concentrations became substantial in the product after the third day. The concentration of acetic acid increased slowly from a concentration of about 0Æ42–0Æ48% and after 4 days had exceeded the acceptable level of 0Æ6%. which favour alcoholic fermentation by the yeasts. the methods of palm wine tapping and collection of palm sap inﬂuence the microbial content of the sap. mesenteriodes were responsible for a rapid acidiﬁcation of the product during the ﬁrst 24 h of tapping whilst the growth of acetic acid bacteria involving both Acetobacter and Gluconobacter species became pronounced after the buildup in alcohol concentrations on the third day. however. Bassir (1968) had reported L. Candida. Endomycopsis.
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