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Isolation, characterisation and identification of lactic acid bacteria from bushera: A Ugandan traditional fermented beverage Article in International Journal of Food Microbiology · March 2003 DOI: 10.1016/S0168-1605(02)00148-4 · Source: PubMed

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International Journal of Food Microbiology 80 (2003) 201 – 210 www.elsevier.com/locate/ijfoodmicro

Isolation, characterisation and identification of lactic acid bacteria from bushera: a Ugandan traditional fermented beverage C.M.B.K. Muyanja a,b, J.A. Narvhus b,*, J. Treimo b, T. Langsrud b a

Department of Food Science and Technology, Makerere University, P.O. Box 7062, Kampala, Uganda Department of Food Science, Agricultural University of Norway, P.O. Box 5036, N-1432, A˚s, Norway

b

Received 14 October 2001; accepted 21 March 2002

Abstract One hundred and thirteen strains of lactic acid bacteria (LAB) were selected from 351 isolates from 15 samples of traditionally fermented household bushera from Uganda and also from laboratory-prepared bushera. Isolates were phenotypically characterised by their ability to ferment 49 carbohydrates using API 50 CHL kits and additional biochemical tests. Coliforms, yeasts and LAB were enumerated in bushera. The pH, volatile organic compounds and organic acids were also determined. The LAB counts in household bushera varied between 7.1 and 9.4 log cfu ml 1. The coliform counts varied between < 1 and 5.2 log cfu ml 1. The pH of bushera ranged from 3.7 to 4.5. Ethanol (max, 0.27%) was the major volatile organic compound while lactic acid (max, 0.52%) was identified as the dominant organic acid in household bushera. The initial numbers of LAB and coliforms in laboratory-fermented bushera were similar; however, the LAB numbers increased faster during the first 24 h. LAB counts increased from 5.5 to 9.0 log cfu ml 1 during the laboratory fermentation. Coliform counts increased from 5.9 to 7.8 log cfu ml 1 at 24 h, but after 48 h, counts were less 4 log cfu ml 1. Yeasts increased from 4.3 to 7.7 log cfu ml 1 at 48 h, but thereafter decreased slightly. The pH declined from 7.0 to around 4.0. Lactic acid and ethanol increased from zero to 0.75% and 0.20%, respectively. Lactic acid bacteria isolated from household bushera belonged to Lactobacillus, Streptococcus and Enterococcus genera. Tentatively, Lactobacillus isolates were identified as Lactobacillus plantarum, L. paracasei subsp. paracasei, L. fermentum, L. brevis and L. delbrueckii subsp. delbrueckii. Streptococcus thermophilus strains were also identified in household bushera. LAB isolated from bushera produced in the laboratory belonged to five genera (Lactococcus, Leuconostoc, Lactobacillus, Weissella and Enterococcus. Eight isolates were able to produce acid from starch and were identified as Lactococcus lactis subsp. lactis (four strains), Leuconostoc mesenteroides subsp. mesenteroides (one strain), Leuconostoc mesenteroides subsp. dextranicum (one strain), Weissella confusa (one strain) and L. plantarum (one strain). D 2002 Elsevier Science B.V. All rights reserved. Keywords: Lactic acid bacteria; Fermentation; Bushera; Sorghum; Millet

1. Introduction

*

Corresponding author. Tel.: +47-6494-8550; fax: +47-64943789. E-mail address: [email protected] (J.A. Narvhus).

Sorghum [Sorghum bicolor (L.) Moench] and finger millet (Eleusine coracana) are important food crops in arid and semi-arid regions of the world (Sulma et al., 1991; Mukuru, 1992; Usha et al., 1996; Owuama,

0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 ( 0 2 ) 0 0 1 4 8 - 4

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C.M.B.K. Muyanja et al. / International Journal of Food Microbiology 80 (2003) 201–210

1997). Fermented sorghum or millet-based foods, alcoholic and non-alcoholic drinks or beverages are prepared in many African countries for human consumption (Ekundayo, 1969; Ahmed et al., 1988; Chavan and Kadam, 1989; Steinkraus, 1996; Odunfa et al., 1996; Usha et al., 1996). The potential of sorghum as an alternative substrate for lager beer brewing has been described by Owuama (1997). Alcoholic beverages prepared from sorghum and millet in Uganda have been surveyed by Mwesigye and Okurut (1995). Sorghum is mainly fermented to produce products called muramba (alcoholic beverage) and bushera (non-alcoholic beverage). Muramba is prepared by mixing sorghum flour with water and heating to form a thin porridge that is allowed to ferment in a pot for a week (Mukuru, 1992). Sugar or honey may be added during fermentation. In brief, to prepare bushera, sorghum or millet flour from germinated sorghum and millet grains is mixed with boiling water and left to cool to ambient temperature (unpublished information). Germinated millet or sorghum flour is then added and the mixture is left to ferment at ambient temperature for 1 – 6 days (unpublished information). Bushera is the most common traditional beverage prepared in the Western highlands of Uganda (Kabale and Rukungiri districts). Low-income women at village level produce bushera for home consumption and sale. The product is consumed by both young children (only 1-day fermented bushera) and adults. Although sorghum and millet are important staple and commercial crops of the majority of people in Western Uganda, there is no documentation of the traditional bushera fermentation process from sorghum and millet. This study was undertaken to isolate, identify and characterise the lactic acid bacteria (LAB) present during bushera fermentation. This information can contribute to the development of starter cultures with predictable characteristics, for use in small-scale and commercial production of bushera with stable and consistent quality.

2. Materials and methods 2.1. Materials Fifteen samples of traditionally fermented bushera made from germinated sorghum, millet and mixtures

of the two flours were obtained from local households and markets in Western Uganda (Kabale and Rukungiri district). The age of bushera ranged from 1 to 3 days. Samples were transported in a cooling box to the Department of Food Science and Technology at Makerere University, Kampala, Uganda. The samples were kept under refrigeration (4 jC) until the next day when microbiological analyses were carried out. Fermentation of the household samples was prolonged up to 8 days during which isolations were carried out. Germinated sorghum flour was also purchased from the local market and used for the laboratory production of bushera. 2.2. Laboratory preparation and fermentation of bushera In the laboratory, sorghum bushera was prepared under sterile conditions in 1500 ml screw-capped plastic containers with sterile water according to the traditional procedure (unpublished information). Bushera was prepared by mixing germinated sorghum flour (120 g) with 500 ml of hot water. The mixture was then boiled while stirring to avoid lump formation for 2 min. This was cooled to approximately 30 jC and then an additional 500 ml of cooled boiled water was added to obtain a desired consistency. Germinated sorghum flour (100 g) was added to initiate the fermentation. The mixture was fermented at ambient temperature for 5 days. During fermentation, samples were aseptically withdrawn each day for pH determination and microbiological analysis. The experiment was conducted four independent times. 2.3. Enumeration of LAB, coliforms and yeasts in bushera Numbers of LAB were determined on the elective media MRS and M17 agar (Merck, Darmstadt, Germany) with glucose as a source of energy. Appropriate dilutions were plated on MRS and M17 agars and incubated at 30 jC for 48 h. Yeasts were enumerated by surface plating on Rose Bengal Chloramphenicol agar (RBCA) (Oxoid, Basingstoke, Hampshire, UK) and incubated aerobically at 25 jC for 3– 6 days. Coliforms were enumerated by pour plating on Violet Red Bile agar (VRBA) (Oxoid). After solidification,


the VRBA plates were placed in plastic bags and incubated aerobically at 37 jC for 24 h. 2.4. Isolation of LAB from bushera Lactic acid bacteria were isolated from bushera obtained from the households and from laboratoryprepared bushera. MRS agar was used for isolation of LAB such as Lactobacillus, Leuconostoc, and Pediococcus, whereas M17 agar was used for isolation of Lactococcus and Enterococcus. Surface-plated MRS and M17 agars were incubated anaerobically (BBL Gas Pak Plus Anaerobic System, Beckon Dickinson Microbiology System, Cockeysville, MD, USA) at 30 jC for 48 h. After counting, colonies on different plates were randomly picked from plates at higher dilution (10 6) and transferred into 10-ml test tubes with sterile MRS or M17 broth. The isolates were purified by successive streaking on the appropriate agar media before being subjected to characterisation. The isolates were Gram-stained and tested for catalase reaction (Harrigan and McCance, 1990). A total of 264 isolates were obtained from samples collected from households and 87 isolates were obtained from laboratory-prepared bushera. Presumptive LAB were selected based on the morphology, Gram reaction and the catalase test. Out of the 351 isolates, 42 isolates were catalase-positive and 2 isolates Gram-negative. The remaining isolates were then characterised by their growth at various temperatures (10, 15, 45 jC), tolerance of different salt levels (2%, 4%, and 6.5% NaCl), production of gas from glucose, dextran from sucrose and hydrolysis of arginine (Harrigan and McCance, 1990). Sixty seven (67) isolates from household bushera and 46 isolates from laboratory-prepared bushera were then selected based on the above tests for further identification. The isolates were stored at 40 jC in MRS or M17 broth containing 10% glycerol and transported frozen to Norway for further analysis. 2.5. Characterisation and identification of isolated LAB to species level The carbohydrate fermentation profiles of the selected 67 isolates from household bushera and 46 isolates from laboratory prepared bushera were investigated using API 50 CH strips and API CHL medium

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according to manufacturer’s instructions (API system, Bio-Merieux, France). Of the 67 household isolates, 23 were isolated from millet bushera, 29 from sorghum bushera and 15 from mixed bushera. Strains were tentatively designated to species using APILAB PLUS (Version 3.33, Bio-Merieux) and standard taxonomic descriptions from Wood and Holzapfel (1995). 2.6. Determination of pH, volatile organic compounds and organic acids in bushera The pH of household bushera was determined using a digital pH meter (CKI Digi-sense Model NO 607) with a combined electrode (pHC 2005-8 < RedRod > Radiometer analytical, Copenhagen, Denmark) in Uganda. For the laboratory-prepared bushera, pH was determined by Lab pH meter (PHM92, Lab pH meter, Radiometer analytical) combined with pH electrode (Radiometer analytical) in Norway. The pH meters were calibrated using commercial buffers (Merck) pH 4 and 7. The pH of the bushera was monitored at intervals of 1 day during fermentation. Volatile organic compounds were determined by automatic headspace gas chromatography (HS-GC) according to Narvhus et al. (1998) and Mugula (2001). Organic acids were determined by HPLC according to Marsili et al. (1981), incorporating modifications described by Narvhus et al. (1998) and Mugula (2001).

3. Results 3.1. Enumeration of LAB, coliforms and yeasts Table 1 shows the numbers of microorganisms in the household bushera samples. The microbial counts differed among the samples of same type of bushera and the different types of bushera. Lactic acid bacteria numbers in household bushera varied between 8.4 and 9.0 log cfu ml 1 whereas coliforms varied between 2.0 and 3.1 log cfu ml 1 (Table 1). The changes in microbial numbers during laboratory fermentation of bushera are shown in Table 2. The LAB counts on MRS and M17 increased from 5.5 to 9.1 log cfu ml 1 and from 5.9 to 9.1 log cfu ml 1, respectively. The largest increase in the numbers of LAB was noted during the first 24 h of fermentation and further incubation led to a slight decrease. The

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Table 1 The pH and microbial counts (log cfu ml Product

Millet Sorghum Mixture a b

1

) of different bushera types obtained from households

Number of samples

pH

Counts (log cfu/ml) b

5 5 5

4.1 F 0.42 4.3 F 0.35 3.8 F 0.07

3.2. Characterization of LAB from bushera Table 3 shows which species were tentatively identified in bushera according to phenotypic characterisation. The LAB isolated comprised of five genera, Lactobacillus, Lactococcus, Leuconostoc, Enterococcus and Streptococcus. Lactobacillus brevis was more frequently isolated than other species in household bushera. L. fermentum, L. paracasei subsp. paracasei and L. plantarum predominated the fermentation of bushera in the later stages, although they were also found in the 2-day-old household bushera. The number of Lactobacillus strains (70% of isolates) (L. fermentum, L. brevis, L. plantarum, L. paracasei subsp. paracasei and L. delbrueckii subsp. delbrueckii) suggests that lactobacilli predominated in the household fermented bushera. Likewise, lactoba-

Table 2 Changes in pH and microbial counts (log cfu ml Fermentation time (days)

pH

0 1 2 3 4

7.0 F 0.46c 4.6 F 0.69 4.0 F 0.22 3.7 F 0.03 3.6 F 0.13 b c

M17

VRBAa

8.4 F 1.14 8.4 F 1.84 8.9 F 0.70

8.1 F 1.48 8.4 F 1.48 9.0 F 0.70

2.0 F 1.41 3.1 F 2.96 2.0 F 1.14

Violet Red Bile agar. Results given as averages F S.D.

yeast counts increased from 4.3 to 7.7 log cfu ml 1 (Table 2). The highest numbers of yeasts were observed on the fourth day. Coliforms increased during laboratory fermentation by about 2 log cycles after 24 h. However, after 3 days, the coliform counts had decreased to less than 4. 0 log cfu ml 1 and were not detectable after the fourth day.

a

MRS

1

cilli were found to predominate during laboratory fermentation of bushera. Enterococci (24%) were also frequently isolated from household bushera in the later stages. Eight isolates were able to produce acid from starch: Lactococcus lactis subsp. lactis (four strains), Leuconostoc mesenteroides subsp. mesenteroides (one strain), Leuconostoc mesenteroides subsp. dextranicun (one strain) Weisella confusa (one strain) and L. plantarum (one strain). 3.3. pH, organic acids and volatile organic compounds in bushera The pH ranges of bushera obtained from households are shown in Table 1. Bushera prepared from a mixture of millet and sorghum flour had the lowest pH values between 3.7 and 3.8, whereas sorghum bushera had the highest pH value between 4.0 and 4.5. During laboratory fermentation, the pH of bushera decreased from 7.0 to 4.0 within 48 h of fermentation (Table 2). After 48 h, the pH remained relatively stable at pH 3.7. Organic acids identified in household bushera included citric, pyruvic, succinic, acetic, lactic and pyroglutamic acids (Table 4). The results show that lactic and acetic acid are the dominant organic acids.

) during laboratory fermentation of bushera from germinated sorghum flour Counts (log cfu ml

1

)

MRS

M17

VRBAa

RBCA-Yeastb

5.5 F 0.21 9.1 F 0.48 9.0 F 0.20 8.9 F 0.40 8.8 F 0.49

5.9 F 0.44 8.9 F 0.40 9.0 F 0.16 9.1 F 0.35 8.7 F 0.22

5.9 F 0.71 7.8 F 0.34 6.3 F 0.74 3.8 F 1.27 0.00 F 0.00

4.3 F 0.28 5.8 F 0.57 6.5 F 1.30 7.4 F 0.33 7.7 F 0.38

Violet Red Bile agar. Rose Bengal Chloramphenicol agar. Results given as averages of duplicate determinations F S.D.


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Table 3 Tentative identification of lactic acid bacteria isolated from household and laboratory-fermented bushera according to phenotypic characterisation Species identified

Household bushera Lactobacillus brevis Lactobacillus fermentum Lactobacillus plantarum Lactobacillus paracasei subsp. paracasei Lactobacillus delbrueckii subsp. delbrueckii Enterococcus faecium Streptococcus thermophilus Laboratory-fermented bushera Lactobacillus plantarum Lactobacillus brevis Lactobacillus paracasei subsp. paracasei Weissella confusa Leuconostoc citreum Leuconostoc mesenteroides subsp. dextranicum Leuconostoc mesenteroides subsp. mesenteroides Lactococcus raffinolactis Lactococcus lactis subsp. hordniae Lactococcus lactis subsp. lactis Enterococcus mundtii Enterococcus faecium a

Number of isolates

Days of isolation (age of the product) 0

1

2

3

4

5

6

7

8

24 7 7 7 2 16 4

NDa ND ND ND 0 ND 0

ND ND ND ND 2 ND 4

5 1 2 2 ND 3 0

1 1 2 1 ND 5 0

1 0 1 ND 2 0

6 2 0 1 ND 4 ND

6 1 3 2 ND 2 ND

2 0 0 0 ND 0 ND

3 2 0 0 ND 0 ND

1 0 0 0 0 0 0 1 0 1 0 1

0 2 0 2 1 0 5 0 0 2 1 0

2 0 0 1 2 0 0 0 1 1 0 0

2 0 0 1 0 3 2 0 0 0 0 3

0 0 1 0 1 3 1 0 0 1 0 4

ND ND ND ND ND ND ND ND ND ND ND ND




4 2 1 5 4 6 8 1 1 5 1 8

ND refers to no isolation done.

However, large variations in concentration of organic acids between household bushera samples were observed. Table 4 The organic acids and volatile organic compounds (mg kg Product

Number of samples

During laboratory fermentation of sorghum bushera, the most rapid production of lactic acid was observed between 12 and 60 h (Fig. 1). Lactic

1

) in different bushera types obtained from households

Organic acids and volatile organic compoundsa Citric b

Succinic

Lactic

Acetic

DL-Pyrogl

Pyruvic 58 F 40.62 169 F 45.40 132 F 84.08

Millet Sorghum Mixture

3 4 4

1187 F 1030 857 F 579 1244 F 595

127 F 28.92 311 F 240.55 82 F 68.77

3557 F 2131 4280 F 1692 3747 F 1165

945 F 189 846 F 154 1087 F 463

3.3 F 3.05 34 F 3.54 16 F 13.89

Acetald

Ethanol

Acetone

Diacetyl

Acetoin

Millet Sorghum Mixture

3 3 3

29.00 F 7.07 3.09 F 1.92 0.71 F 0.21

326 F 12.02 1670 F 1492 244 F 175.93

0.07 F 0.01 0.30 F 0.10 0.54 F 0.38

0.55 F 0.07 0.61 F 0.59 1.28 F 0.19

11.20 F 1.48 41.63 F 28.12 31.77 F 17.51

2-ME-pro-al

2-ME-pro-ol

3-ME-but-al

2-ME-but-al

3-ME-but-ol

2-ME-but-ol

0.02 F 0.01 0.07 F 0.04 0.03 F 0.03

0.35 F 0.16 0.56 F 0.50 0.34 F 0.01

0.02 F 0.01 0.02 F 0.01 0.01 F 0.00

0.02 F 0.01 0.01 F 0.00 0.03 F 0.01

0.74 F 0.01 1.11 F 1.50 0.69 F 0.01

0.16 F 0.02 0.27 F 0.26 0.23 F 0.01

Millet Sorghum Mixture a

3 3 3

DL-Pyrogl: pyroglutamic acid, Acetald: acetaldehyde, 2-ME-pro-al: 2-methyl-propanal, 2-ME-pro-ol: 2-methyl-propanol, 3-ME-but-al: 3methyl-butanal, 2-ME-but-al: 2-methyl-butanal, 3-ME-but-ol: 3-methyl-butanol, and 2-ME-but-ol: 2-methyl-butanol. b Results given as averages F S.D.

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Fig. 1. Changes in lactic acid and ethanol content (%) during laboratory fermentation of bushera. Results given as average and standard deviation indicated by bars.

acid increased from 0 to 0.75% during the fermentation. Volatile organic compounds identified in household bushera included acetaldehyde, acetone, acetoin, diacetyl, 2-methyl-propanol, 2-methyl-propanal, 2methyl-butanal, 3-methyl-butanal, 2-methyl-butanol, and 3-methyl-butanol (Table 4). Large variations in volatile organic compounds were also observed between individual household bushera samples. Ethanol was the major volatile organic compound in bushera. The highest concentration of ethanol and lactic acid was measured in one of the household sorghum bushera samples. Unidentified volatile organic compounds were also detected which might have some importance for the sensory acceptance of the product. The ethanol content in laboratory-prepared bushera increased during fermentation (Fig. 1). The main production of ethanol took place after 24 h. The highest concentration of ethanol measured in laboratory-prepared sorghum bushera was 0.2%.

4. Discussion The number of LAB and yeasts differed among household samples and increased during laboratory fermentation. Gobbetti et al. (1994) and Steinkraus

(1996) proposed that lactic acid bacteria create an acidic environment conducive to yeast proliferation while the yeasts provide vitamins and other growth factors such as amino acids for the lactic acid bacteria. The simultaneous increase in numbers of both LAB and yeasts may therefore be attributed to their symbiotic association. The results are in agreement with those reported by other authors (Mbugua, 1984; Odunfa and Adeyele, 1985; Sulma et al., 1991; Nche et al., 1994). Melaku and Faulks (1988) also indicated that numbers of LAB increased during the first stages of the natural fermentation with a slight reduction in number during the later stages of fermentation. The decrease in number of coliforms to undetectable levels during fermentation may be explained by the high acidity (low pH) as a result of acid production by lactic acid bacteria. The lower coliform counts observed in household bushera as compared to laboratory-prepared bushera might be explained by the age of the samples. Coliforms are known to be acid intolerant (Steinkraus, 1996). Similar results have also been reported for other fermented products (Mbugua, 1984; Nche et al., 1994). A rapid decline in numbers of Enterobacteriaceae during the production of kenkey, a Ghanian fermented maize dough, was observed (Nche et al., 1994). A pH of 3.5– 4.0 has been reported to inhibit Enterobacteriaceae and other Gram-negative bacteria (Mbugua, 1985; Chavan and Kadam, 1989; Nout, 1991; Steinkraus, 1996). Other antimicrobial substances produced by the dominating lactic acid bacteria (Helander et al., 1997) in addition to lactic acid may also contribute to inhibition of Enterobacteriaceae (Mensah et al., 1991; Nche et al., 1994). From the microbial counts, it can be suggested that the initial stages of fermentation of bushera is dominated by LAB and coliforms but in the late stages by LAB and yeasts. In contrast, Mbugua (1984) indicated that a spontaneous fermentation of uji (a Kenyan sorghum –maize-based porridge) was dominated by only coliforms in the early stages of the fermentation (16 –24 h). The decrease in pH and increase in lactic acid followed the same trend as reported for other natural fermented foods (Sulma et al., 1991; Choi et al., 1994; Dziedzoaze et al., 1996). The high levels of lactic acid might be attributed to the predominance of LAB. A slight decrease in lactic acid concentration was observed in the late stages of fermentation. Akinrele


(1970) attributed the decrease in lactic acid concentration to the utilisation of lactic acid by yeasts. The organic acids identified in bushera have also been reported in other fermented cereal-based foods. Banigo and Muller (1972) reported the main organic acids in ogi as lactic, butyric, acetic and formic acid. Lactic and acetic acids have been reported to be flavour enhancers (Gobbetti and Corsetti, 1997). Akinrele (1970) indicated that lactobacilli are responsible for acid formation and flavour enhancement. The late increase in ethanol concentration in bushera may be attributed to the participation of yeasts in the fermentation process. Other compounds like acetoin and diacetyl (butter aroma) have been indicated to result from utilisation of citrate and pyruvate by LAB (Martinez-Anaya, 1996; Narvhus et al., 1998; Axelsson, 1998). The presence of acetoin-like compounds in cereal products has been reported to be associated with bacterial rather than yeast-dominated flora (Halm et al., 1993). Some of the other volatile compounds may be formed by degradation of amino acids to aldehydes, which later may be oxidised to acids or reduced to alcohols (Gobbetti et al., 1994; Narvhus et al., 1998). The resulting methyl alcohols are reported to be flavour enhancers (Gobbetti and Corsetti, 1997). Different groups of lactic acid bacteria were isolated from household and laboratory-prepared bushera. The biochemical properties of species identified in bushera were similar to those reported by Morishita and Shiromizu (1986), Hounhouigan et al. (1993), and Wood and Holzapfel (1995). However, there have been some discussions of the classification of two of the species, L. paracasei subsp. paracasei and L. confusus. Some authors (Mori et al., 1997; Klein et al., 1998; Roy et al., 1999) have suggested a reclassification of L. paracasei subsp. paracasei into the restored species of L. casei. However, in this study, it was decided to retain the old names until the change is internationally accepted. Collins et al. (1993) and Stiles and Holzapfel, (1997) suggested that L. confusus belongs to the genus Weissella. We decided to use the recent species name of W. confusa. W. confusa was earlier classified as L. confusus or L. coprophilus (Sulma et al., 1991; Hounhouigan et al., 1993; Johansson et al., 1995). The lactic acid bacteria identified in bushera have been reported in other fermented foods. L. plantarum has been isolated from the raw material, sorghum

207

powder and also from corresponding fermented and cooked fermented samples (Kunene et al., 2000). L. plantarum has been shown to be the dominant organism at the end of several natural cereal fermentations (Nout, 1980; Mbugua, 1984; Olasupo et al., 1997) as for instance in maize-derived products like ogi (Akinrele, 1970; Odunfa and Adeyele, 1985; Johansson et al., 1995; Steinkraus, 1996). L. plantarum has also been identified as the predominant species in most vegetable fermentations (Oyewole and Odunfa, 1990). The dominance of L. plantarum at the late stages of fermentation has been attributed to its high acid tolerance (Akinrele, 1970; Mbugua, 1984; Oyewole and Odunfa, 1990; Hounhouigan et al., 1993). One of the strains of L. plantarum isolated from laboratory-fermented bushera was able to produce acid from starch, L. plantarum MINF19. Starchdegrading L. plantarum strains have also been isolated from cassava and other fermented foods (Johansson et al., 1995) but are not common. However, no starchdegrading strains of L. plantarum were isolated from household bushera. A starch-degrading strain of W. confusa MINF8 was also isolated. Among the facultative species isolated, L. paracasei subsp. paracasei has not been previously reported in African indigenous fermented foods (Steinkraus, 1996), but it has been isolated from plant materials and fermented foods (Winter et al., 1998; Paludan-Muller et al., 1999) from other continents. L. brevis and L. fermentum have been suggested to be the predominating microorganisms during the fermentation of fufu and ogi, two Nigerian foods (Adegoke and Babalola, 1988), kisra a Sudanese sorghum fermented flat bread (Sulma et al., 1991; Abd Elmoniem et al., 1994), kenkey, a Ghananian fermented maize dough (Halm et al., 1993), mawe, a Benin fermented maize dough (Hounhouigan et al., 1993), and agbelima, a Ghananian cassava dough (Kofi et al., 1996). These species have also been reported to occur in fermenting plant materials and sourdough (Wood and Holzapfel, 1995; Corsetti et al., 2001), in sorghum beer (Van Walt, 1956; von Holdt and Brand, 1960), and in togwa (Steinkraus, 1996; Mugula, 2001). W. confusa has also been isolated from ogi (Odunfa and Adeyele, 1985), kisra (Sulma et al., 1991), mawe (Hounhouigan et al., 1993), togwa, (Mugula, 2001) and wheat sourdough (Corsetti et al., 2001).

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During the early stages of bushera fermentation, a number of strains of Lactococcus lactis subsp. lactis, Leuconostoc mesenteroides subsp. mesenteroides, Leuconostoc mesenteroides subsp. dextranicum and Leuconostoc citreum were identified. Leuconostocs have been reported to be common in indigenous fermented foods (Mbugua, 1985; Steinkraus, 1996) and are known to initiate the acidification in vegetable products such as uji (Mbugua, 1985). Mbugua (1985) indicated that addition of a pure culture of Leuconostoc mesenteroides, in addition to the native starter, ensured a more stable flavour of uji by retarding or inhibiting Gram-negative bacteria through its rapid acidification. A few strains of Leuconostoc mesenteroides subsp. mesenteroides have been reported to ferment starch (Johansson et al., 1995). In our study, strains of starch-degrading Leuconostoc mesenteroides and Lactococcus lactis subsp. lactis were also isolated. Leuconostoc mesenteroides has also been isolated from fufu (Oyewole and Odunfa, 1990) and from mawe (Hounhouigan et al., 1993). Enterococcus faecium was isolated from bushera, and has been reported to occur in plants (Reuter, 1985). E. faecium strains have been found and used in the sourdough process (Gobbetti, 1998) and are associated with the fermentation of a number of southern European cheeses (Stiles and Holzapfel, 1997). E. faecium has also been isolated from kisra (Sulma et al., 1991). The role of E. faecium in bushera may warrant further investigation despite the question of its potential pathogenicity. The occurrence of Streptococcus thermophilus and L. delbrueckii subsp. delbrueckii in household bushera samples may indicate different fermentation conditions. The growth of these species has been shown to be favoured by high temperatures and they have been isolated from food materials fermented at higher temperatures (Wood and Holzapfel, 1995). L. delbrueckii subsp. delbrueckii has been isolated from sourdough (Corsetti et al., 2001). S. thermophilus was also found to be effective in acidifying wet maize slurry, but it is usually not acid tolerant and is quickly inhibited by low pH values (Mbugua, 1987). L. delbrueckii has been used as added starter in the fermentation of mahewu, a Bantu beer (South Africa) equivalent to ogi and the fermentation time of mahewu was then reduced from 36 to 6 h (Akinrele, 1970).

5. Conclusion The difference in microbial composition observed between the household bushera and laboratory-fermented bushera may be attributed to differences in microorganisms on the flour used in the laboratory, preparation and processing techniques and also to the duration of fermentation (age of the product). Differing microbial composition may also be due to competitive differences between the different microorganisms and the inability of, for example, Lactococcus and Leuconostoc, to grow at lower pH values. The succession observed among the lactic acid bacteria may be attributed to the increasing acidity of the fermenting bushera. The observed variation in the amounts of organic acids and volatile compounds in the different household bushera was expected as the age of product differed, the processing techniques may have differed slightly from one household to another and also the differences in the fermenting microorganisms. The study has demonstrated the effect of acid production in the suppression of coliforms during spontaneous fermentation of bushera. Furthermore, the results of this study have indicated that several different species of lactic acid bacteria can be implicated in the fermentation of bushera. Therefore, there is a need for investigation into the selection of the most suitable strains for controlled fermentation of bushera. The starch-fermenting strains might be important in the development of the starter cultures and for use in the development of small-scale commercial production of bushera. The potential use of strains from our isolates as starter cultures will depend on further studies on selected isolates, and assessment of their effect on the quality of bushera.

Acknowledgements The authors thank the Norwegian Universities’ Committee for Research, Development and Education (NUFU, Project 26/96) for financial support. The authors are also grateful to the Agricultural University of Norway for provision of technical facilities. Thanks to technical staff at the Department of Food Science, Agricultural University of


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