International Journal of Food Microbiology 80 (2003) 187 199 www.elsevier.

com/locate/ijfoodmicro

Microbiological and fermentation characteristics of togwa, a Tanzanian fermented food

J.K. Mugula a,b,*, S.A.M. Nnko a, J.A. Narvhus b, T. Srhaug b
a

Sokoine University of Agriculture, Department of Food Science and Technology, PO Box 3006, Morogoro, Tanzania b Agricultural University of Norway, Department of Food Science, PO Box 5036, N-1432, As, Norway Received 13 July 2001; received in revised form 21 December 2001; accepted 21 March 2002

Abstract Selected microbiological and metabolic characteristics of sorghum, maize, millet and maize sorghum togwa were investigated during natural fermentation for 24 h. The process was predominated by lactic acid bacteria (LAB) and yeasts. The mesophiles, lactic acid bacteria, and yeasts increased and the Enterobacteriaceae decreased to undetectable levels within 24 h. The isolated microorganisms were tentatively identified as Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus cellobiosus, Pediococcus pentosaceus, Weissella confusa, Issatchenkia orientalis, Saccharomyces cerevisiae, Candida pelliculosa and Candida tropicalis. The pH decreased from 5.24 5.52 to 3.10 3.34. Maltose increased initially and then decreased, fructose decreased and glucose levels increased during the first 12 h of fermentation. The organic acids detected during fermentation included DL-lactic, succinic, formic, pyruvic, citric, pyroglutamic and uric acid. Lactate was the predominant acid and increased significantly with time. The volatile organic compounds (VOC) detected included acetaldehyde, 2-methyl-propanal, 2-methyl-butanal, 3-methyl-butanal, ethanol, 2-methyl-1-propanol, 2-methyl-1-butanol, 3methyl-1-butanol, diacetyl and acetoin. Ethanol was the predominant VOC and it increased significantly with time. D 2002 Elsevier Science B.V. All rights reserved.
Keywords: Cereal fermentation; Togwa; Lactic acid bacteria; Yeasts; Organic acids; Volatile organic compounds

1. Introduction Fermented foods and beverages constitute a major portion of peoples diets in Africa (Sanni, 1993; Oyewole, 1997). Cereal grains including sorghum, maize and millet are common substrates for lactic acid-fermented gruels and beverages known by differ* Corresponding author. Sokoine University of Agriculture, Department of Food Science and Technology, PO Box 3006, Morogoro, Tanzania. Tel./fax: +255-23-260-4402. E-mail address: jmugula@yahoo.com (J.K. Mugula).

ent names (Odunfa and Adeyele, 1985), such as togwa in Tanzania (Lorri and Svanberg, 1995; Mugula et al., 2001). Togwa is widely produced in Tanzanian homes for use directly as a weaning food or diluted for use as refreshment. Fermentation is spontaneous and uncontrolled thus resulting in a product of variable quality. The consumption of togwa, like that of many African traditional fermented foods, is declining, the product being now associated with low-income groups, and its popularity is being undermined by its poor shelf-life and unhygienic preparation techniques (Mugula et al., 2001). On the other hand, lactic acid-fermented por-

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 1 - 1

188

J.K. Mugula et al. / International Journal of Food Microbiology 80 (2003) 187199

ridge has attracted attention due to its microbial stability, improved nutritional and organoleptic qualities (Cooke et al., 1987; Mokhoro and Jackson, 1995), and probiotic potential (Kingamkono et al., 1994; Willumsen et al., 1997). At present, there is no adequate information on the spectrum of microorganisms associated with the fermentation of cereals to produce togwa and the development of flavour compounds during the process. This knowledge is essential for the development of the product with improved quality for increased consumption and commercial production and marketing. The present study reports on the identification of lactic acid bacteria and yeasts associated with the natural fermentation and the development of metabolic compounds during the preparation of cerealbased togwa.

2. Materials and methods 2.1. Samples Nine samples each of maize, sorghum, fingermillet and, maize sorghum (1/1, w/w) togwa were collected in 250-ml crew-capped bottles from producers in Morogoro municipality, Tanzania and transported in cooler boxes to the laboratory for microbiological analyses during fermentation. The samples were prepared according to the traditional method (Fig. 1) by mixing cereal flour with water (about 10%, w/v). The slurry was boiled for about 10 20 min while stirring to avoid formation of lumps, cooled to around 30 jC, started with 5% (w/v) sorghum malt flour and backslopped with up to 10% (v/v) of togwa, then fermented at 30 jC for up to 24 h. Samples were

Fig. 1. Flowchart for the traditional preparation of cereal-based togwa.

J.K. Mugula et al. / International Journal of Food Microbiology 80 (2003) 187199

189

withdrawn at 0, 4, 8, 12 and 24 h of fermentation for determination of microbial counts and isolation of LAB and yeasts, pH, organic acids, sugars and volatile organic compounds. 2.2. Characterization of the dominating microorganisms 2.2.1. Enumeration and isolation Duplicate samples of togwa (10 ml) were homogenized with 90 ml sterile peptone physiological saline solution (5 g peptone, 8.5 g NaCl, 1000 ml distilled water, pH 7.0 F 0.2). The homogenate was decimal diluted and the relevant dilutions surface plated. M17 agar (Merck, Darmstadt, Germany) plates containing 0.1% (w/v) glucose were incubated aerobically and MRS agar (Merck) plates containing 0.1% (w/v) natamycin (Delvocid, Delft, The Netherlands) were incubated anaerobically (BBL Gas Pak, H2 and CO2; Becton Dickinson, Cockeysville, MD, USA) for 48 h at 30 jC for the enumeration and isolation of lactic acid bacteria (LAB). A total of 120 representative colonies were randomly picked from higher

dilution plates of various fermentation stages and confirmed to be Gram-positive and catalase-negative. For subsequent purification and sub-culturing M17 and MRS agar and broths were used. The pure bacterial cultures were inoculated into appropriate broth, incubated for 24 h at 30 jC, centrifuged (Kubota 2010, Kubota, Tokyo, Japan) at 3000 rpm for 15 min and the supernatant decanted. The cell pellets were re-suspended either in sterile MRS or M17 broth containing 10% (v/v) glycerol. The suspension was asceptically transferred into sterile cryotubes containing acid-washed glass beads and stored at 80 jC until required for identification. Aerobic mesophilic bacteria in togwa were enumerated on plate count agar (PCA, Merck) after incubation for 2 days at 30 jC and Enterobacteriaceae on violet red bile glucose agar (VRBGA, Oxoid) after incubation for 24 h at 37 jC. Yeasts were enumerated and isolated after incubation for 3 5 days at 25 jC on wort agar (WA, Merck) containing 0.01% (w/v) sterile oxytetracyline (Merck) or on Rose Bengal Chloramphenicol agar (RBCA, Oxoid) containing 0.01% (w/v) chloramphenicol (selective supplement, Oxoid). Puri-

Fig. 2. PCA of characterization tests on lactic acid bacteria isolated from togwa. Key to bacteria isolates, API and other testsgroup 1: L. brevis (B, E, e, f, r, v, x, y), L. cellobiosus (A, C, k,), L. fermentum (b); group 2: W. confusa (a, c, f, g, h, n, p, q, s, t); group 3: P. pentosaceus (d); group 4: L. plantarum (D, F, j, m, u, w, z). 4, L-arabinose; 5, ribose; 6, D-xylose; 10, galactose; 11, glucose; 12, fructose; 13, mannose; 14, sorbose; 15, rhamnose; 18, mannitol; 19, sorbitol; 22, N-acetyl-glucosamine; 23, amylin; 24, arbutin; 25, esculin; 26 salicin; 27, celibiose; 28, maltose; 29, lactose; 30, melibiose; 31, sucrose; 32, trehalose; 34, melezitose; 35, rafinnose; 36, starch; 37, glycogen; 39, gentibiose; 40, D-turanose; 44, Lfucose; 46, L-arabitol; 47, gluconate; 49, 5-keto-gluconate; CO2, carbon dioxide production; NH3, ammonium production; 15 or 45 jC, growth at 15 or 45 jC; 4 or 6.5%, growth in 4 or 6.5% NaCl.

190

J.K. Mugula et al. / International Journal of Food Microbiology 80 (2003) 187199

fication and sub-culturing was done using potato dextrose agar (PDA, Oxoid) and yeast extract-malt extract (YM) broth. The purified yeast cultures were stored on PDA slants at 4 jC until required for identification. 2.2.2. Characteristics of isolates The bacteria were characterized by microscopic examination and by conventional biochemical and physiological tests. The cultures were examined for colony and cell morphology; motility, cell arrangement, Gram reaction; catalase reaction; growth in broth at 10, 15, 40 and 45 jC; growth in presence of 2%, 4%, and 6.5% (w/v) NaCl; production of ammonia from arginine; production of dextran from sucrose; and production of carbon dioxide from glucose using Gibsons litmus milk. These tests were done according to procedures described by Harrigan and McCance (1990). The production of carbon dioxide was also determined in MRS and M17 broth, after incubation at 30 jC for 24 h, using an infrared gas analyzer (ADC 225 MK3, The Analytical Development Hertfordshire,

UK) connected to a Chromatopac (C-R3A, Shimadzu Analytical Instruments, Kyoto, Japan) according to Narvhus et al. (1992). Preliminary grouping for selection of 30 isolates for API tests was based on the above-mentioned morphological, physiological and biochemical characteristics. The fermentation pattern among carbohydrates was determined by using the API 50 CH gallery with the API 50 CHL medium (Bio Merieux, Marcy-lEtoile, France). Anaerobiosis in the inoculated tubes was obtained by overlaying with sterile paraffin oil. The inoculated galleries were incubated at 30 jC and the observations were made after 24 and 48 h. The Principal Component Analysis was used for grouping organisms with identical physiological and biochemical characteristics. The identification of the isolates was facilitated by the use of a computer programme, APILAB PLUS, version 3.2.2. (Bio Merieux) and reference to Bergeys Manual of Systematic Bacteriology (Sneath et al., 1986) and Wood and Holzapfel (1995). The yeast isolates were identified by using the Simplified Identification Method (SIM) described by

Fig. 3. PCA of characterization tests on yeasts isolated from togwa. Key to yeast isolates, API and other testsgroup 1: I. orientalis (A, a, B, b, d, h, q, r, s, t, v, y); group 2: S. cerevisiae (C, c, k, m, w, x, z, w); group 3: C. pelliculosa (e, f, g), and group 4: C. tropicalis (j, n, p); 1, galactose; 2, actidione; 3, saccharose; 4, N-acetyl-glucosamine; 5, DL-lactate; 7, cellobiose; 8, raffinose; 9, maltose; 10, trehalose; 11, 2-keto-gluconate; 12, a-methyl-D-glucoside; 13, sorbitol; 14, D-xylose; 15, ribose: 16, glycerol; 18, palatinose; 19, erythritol; 22, melezitose; 23, gluconate; 25, mannose; 28, glucose; 30, glucosamine, 31, esculine; Stass, starch assimilation; Lys, lysine; Cad, cadavarine; KNO3, nitrate assimilation; 40C, growth at 40 jC; Benz, growth in presence of benzoate; spore, spore formation; pellicle; pellicle formation; pshyp, formation of pseudohyphae.

J.K. Mugula et al. / International Journal of Food Microbiology 80 (2003) 187199

191

Deak and Beuchat (1996), with additional standard taxonomical methods (Kurtzman, 1998; Meyer et al., 1998; Vaughani-Martini and Martini,- 1998), the use of ID32C diagnostic kits (Bio Merieux), assisted by a computer software (API LAB PLUS version 3.2.2, Bio Merieux) and by the Principal Component Analysis. For the SIM, the tests were done as described by Yarrow (1998) including the fermentation patterns among D-glucose, fructose, raffinose, maltose, D-galactose (Merck), lactose, sucrose (BDH, Poole, England); and the assimilation patterns among xylose, melibiose, rhamnose, trehalose, mannitol, arabinose, citrate, soluble starch, cellobiose, D-ribose, melezitose, DL-lactate, L-sorbose, lactose, sucrose, galactose, and raffinose (Merck), erythritol, 2-ketogluconate, amethyl-D-glucoside (Sigma, St. Louis, MO, USA). Other tests included starch formation, cycloheximide (Sigma) resistance, urease activity, assimilation of potassium nitrate (Merck), L-lysine and cadavarine (Sigma); growth at 37 and 40 jC; growth in 60% glucose yeast extract agar, growth in presence of 16% NaCl, growth in vitamin-free medium, growth in media containing 1% acetic acid, potassium sorbate or benzoate. The formation of mycelium and pseudohyphae was examined by microscopy of Dalmau plates; ascospore formation on Gorodkova agar, acetate agar and YM agar, and the cell morphology on YM broth culture wet mounts. The preliminary grouping of the representative 30 isolates used for API tests was based on the above-mentioned morphological, physiological and biochemical characteristics. 2.3. pH The pH was determined by using a pH meter (PHM61, Radiometer, Copenhagen, Denmark) with an Orion 9102 glass electrode (Orion Research, Boston, MA, USA). The pH meter was calibrated using standard buffer solutions (Merck) at pH 4.0 and 7.0. 2.4. Determination of organic acids and sugars Organic acids were analyzed by the high-performance liquid chromatography (HPLC) method according to Marsili et al. (1981) as modified by Narvhus et al. (1998). Five grams of sample were taken at 0 h and after 4, 8, 12 and 24 h during fermentation, added to 0.7 ml of 0.5 M H2SO4 and 20 ml of acetonitrile

(Rathburn Chemicals, Walkerburn, Scotland) in screw-capped tubes, mixed (Multifix M80, Nerlien, Oslo, Norway) for 30 min and then centrifuged (Funke-Gerber, Berlin, Germany) for 15 min at 7000 rpm/min. The supernatant was filtered into an HPLC sample vial through a 0.2-Am filter (MFS-13, MFS, California, USA). The separation of organic acids was achieved with an Aminex HPX-87H HPLC column (Bio-Rad Labs., Richmond, CA, USA) held at 45 jC, using 9 mM H2SO4 as a mobile phase at a flow rate of 0.4 ml/min. The detector response was monitored by Turbochrom software (Perkin-Elmer, Norwark, CT, USA). The acids were identified and quantified by comparison of their retention times with those of standard solutions of the following acids: DL-lactic, citric, pyruvic, succinic, formic, propionic, orotic, uric and pyroglutamic acid (Sigma). The analysis was externally calibrated using mixed standard solutions in deionised water, prepared as for the samples. Glucose, fructose and maltose were analyzed by HPLC as for acids, but using a refractive index detector (Perkin-Elmer) in series with the UV detector and calibrated using standard sugar solutions (Sigma). 2.5. Determination of volatile organic compounds (VOC) The VOC in togwa were determined by automatic static headspace gas chromatography according to Narvhus et al. (1998). A 10-g sample was put into a headspace vial (20-CV Chromacol, Welwyn Garden City, UK) and sealed with a PTFE-coated septum and aluminium ring (20-CBT3 Chromacol and 20-ACB). The samples were analysed using a DANI HSS 3950 automatic headspace sampler (Dani, Monza, Italy) connected to a Carlo Erba HRGC 5300 gas chromatograph (Carlo Erba Instruments, Milan, Italy), fitted with a flame ionisation detector. Samples were equilibrated for 45 min at 50 jC before the headspace sample (1.0 ml) was automatically injected into the GC. Nitrogen was used as carrier at a flow rate of 5.0 ml/min. The GC temperature programme was set at 53 jC, 1 min; increased at 15 jC/min to 70 jC, 2 min; increased at 22 jC/min to 130 jC, 3 min. The injector and detector temperatures were 180 and 200 jC, respectively. Volatile compounds were separated on a Supelco SPC-1 GC column: 30 m 0.53 mm I.D., film thickness 5.0 Am (Supelco, Bellefonte, PA,

192

J.K. Mugula et al. / International Journal of Food Microbiology 80 (2003) 187199

USA). Detector response was monitored by PerkinElmer Turbochrom chromatography software (PerkinElmer). The analysis was externally calibrated using standard solutions of the following compounds: acetaldehyde (Fluka, Buchs, Switzerland), ethanol (Vinmonopolet, Oslo, Norway), diacetyl (Sigma), acetoin (Merck), 3-methyl butanal, 3-methyl butanol, 2-

methyl butanal, 2-methyl butanol, 2-methyl propanal, 2-methyl-1-propanol (Aldrich, Steinheim, Germany). 2.6. Statistical analyses The Principal Component Analysis (PCA) was performed on data using the Unscrambler 6 computer

Fig. 4. Changes in pH, organic acids and sugar content in togwa during natural fermentation.

J.K. Mugula et al. / International Journal of Food Microbiology 80 (2003) 187199

193

Fig. 4 (continued ).

software (Camo, Trondheim, Norway) to group the LAB and yeasts based on the morphological, physiological and biochemical characteristics. The data obtained were subjected to analysis of variance (SAS/stat, 1996), and mean differences determined by Duncans multiple range test ( P < 0.05).

3. Results 3.1. Microbiological characteristics The microbial population was predominated by LAB and yeasts, which increased from about 106 to 109 and from 105 to 107 CFU/ml, respectively. There was an increase in the number of mesophiles, LAB and yeasts with fermentation time, although the latter decreased slightly at the end. The LAB counts on M17 agar were comparable to those on MRS agar. There was a decrease in the number of Enterobacteriaceae counts to undetectable levels after 24 h. There was no significant difference ( P < 0.05) in LAB and yeasts counts between the samples. Among the LAB isolates, rods accounted for 90%, cocci 10%, dextran producers 36%, CO2 producers 70%, while 62% of the isolates were able to grow at 45 jC, and 34% tolerated 6.5% NaCl. The cocci were homofermentative, grew at 10 to 45 jC and hydrolysed arginine. The isolates were grouped into four main clusters by PCA (Fig. 2) and tentatively identified as :(1) Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus cellobiosus, (2) Weissella

confusa, (3) Pediococcus pentosaceus, and (4) Lactobacillus plantarum. The species were isolated from all types of togwa and at all culture stages and L. plantarum dominated the final stages of fermentation. Yeasts were grouped by PCA into four main clusters (Fig. 3) and tentatively identified as: (1) Issatchenkia orientalis (50%), (2) Saccharomyces cerevisiae (23%), (3) Candida tropicalis (10%) and (4) Candida pelliculosa (17%). The species were isolated from all types of togwa and at all stages of fermentation. 3.2. Chemical characteristics The pH decreased from 5.24 5.52 to 3.10 3.34 during fermentation for 24 h (Fig. 4). The organic acids detected during fermentation included lactate, succinate, pyruvate, DL-pyroglutamate, formate, citrate and uric acid. Propionic and orotic acid were not detected. Citrate disappeared within the first 4 h of fermentation, except in millet-based togwa, which contained higher amounts of the acid (Fig. 4). Lactate increased throughout and formate increased towards the end, while succinate decreased during the first 4 h and then increased. Pyroglutamate decreased (0.03 to 0 mg/kg) while there was no significant change in the content of pyruvate (0.2 to 0.1 mg/kg) and uric acid (0.01 to 0 mg/kg). Maltose increased initially and then decreased towards the end of fermentation while fructose decreased, and glucose increased during the first 12 h (Fig. 4). Three isolates of LAB were able to hydrolyse starch and were identified as L. plantarum.

194

J.K. Mugula et al. / International Journal of Food Microbiology 80 (2003) 187199

The volatile compounds identified in togwa were acetaldehyde, 2-methyl-propanal, 2-methyl-butanal, 3-methyl-butanal, ethanol, 2-methyl-1-propanol, 2methyl-1-butanol, 3-methyl-1-butanol, diacetyl and

acetoin (Fig. 5). The concentration of alcohols increased with time. There was an increased level of 2-methyl propanal and 2-methyl butanal after 24 h, except in millet-based togwa. There was a relatively

Fig. 5. Changes in content of volatile organic compounds in togwa during natural fermentation.

J.K. Mugula et al. / International Journal of Food Microbiology 80 (2003) 187199

195

Fig. 5 (continued ).

lower concentration of volatile compounds in the millet-based product.

4. Discussion LAB and yeasts are common in a wide range of African traditional food and beverage fermentations (Adegoke and Babaola, 1988; Steinkraus, 1996). The bacteria isolated from togwa included L. plantarum, L. brevis, L. fermentum, L. cellobiosus, W. confusa and P. pentosaceus. All of them were present from the beginning to the end of fermentation. The majority of the bacteria belonged to the L. plantarum group, which dominated at the end of fermentation followed by L. brevis, L. cellobiosus, L. fermentum, W. confusa and P. pentosaceus. L. brevis, L. cellobiosus, L. fermentum and L. plantarum have been isolated from several indigenous fermented foods including fufu

(fermented cassava), iru (fermented African locust bean), kenkey and ogi (fermented maize), kukun-zaki (fermented millet), ugba (fermented African oil bean) and wara (fermented skimmed cows milk). The species most commonly isolated was L. plantarum (Olasupo et al., 1997). L. plantarum has been identified as the dominant organism at the end of several natural lactic acid fermentations (Nout, 1980; Mbugua, 1984; Brauman et al., 1996; Olasupo et al., 1997, Kunene et al., 2000), probably due to its acid tolerance (Fleming and McFeters, 1981) and superior ability to utilize the substrates (Oyewole and Odunfa, 1990), including dextrins (Akinrele, 1970). Some of the L. plantarum isolates from togwa were able to hydrolyse starch. Giraud et al. (1994), isolated an amylolytic L. plantarum strain with an ability to break down cassava raw starch that has not been subjected to preliminary physicochemical treatment. P. pentosaceus and L. plantarum have also been isolated from

196

J.K. Mugula et al. / International Journal of Food Microbiology 80 (2003) 187199

sorghum powder and fermented sorghum porridge samples (Kunene et al., 2000). L. fermentum and L. brevis have been reported to dominate in the intermediate and final stages of the fermentation of fufu and to produce the flavour typical of the product (Adegoke and Babaola, 1988). Among yeast isolates I. orientalis occurred in highest numbers in togwa, followed by S. cerevisiae, C. pelliculosa and C. tropicalis. Halm et al. (1993) reported the dominance of Candida spp. followed by Saccharomyces spp. in fermented maize dough. S. cerevisiae and I. orientalis have been isolated frequently from acidic fermentation of plant substrates (Nout, 1980; Hounhouigan et al., 1993; Gobbetti et al., 1994; Nago et al., 1998). A co-metabolism between yeasts and lactic acid bacteria has been suggested, whereby the bacteria provide the acid environment, which selects for the growth of yeasts and, the yeasts provide vitamins and other growth factors to the bacteria (Gobbetti et al., 1994; Steinkraus, 1996). Yeasts have also been reported to make a useful contribution to the improvement of flavour and acceptability of fermented cereal gruels (Banigo et al., 1974; Odunfa and Adeyele, 1985), and Akinrele (1970) reported the contribution of S. cerevisiae and Candida mycoderma to the flavour acceptability of ogi. S. cerevisiae proliferated at the beginning while the latter was predominant at the end of fermentation. Brauman et al. (1996) observed that although yeasts (mostly Candida spp.) did not seem to play a significant role in the fermentation of foo-foo, their increasing numbers in the last stage of the process might influence the flavour and the preservation of the end products. Saccharomyces and Candida species are capable of proliferating at low pH in porridge (Akinrele, 1970; Nout et al., 1989). An initial increase followed by a decrease in the number of Enterobacteriaceae observed in togwa is in accordance with their death kinetics reported in similar natural fermented plant materials (Nout et al., 1989; Masha et al., 1998). Nout (1991) and Masha et al. (1998) reported their disappearance as pH comes decreases below 4.5, although Mensah et al. (1991) suggested that the anti-microbial effect of fermented maize dough porridge was not due to pH per se but probably to the presence of other antimicrobial compounds. Kingamkono et al. (1994, 1995) reported on the anti-microbial effect of togwa against several

enteropathogens and its potential for decreasing the incidence of diarrhoea in children (Willumsen et al., 1997). Enterobacteriaceae are usually active in the early stages of fermentation of cereal-based slurries and their activity is eliminated when an enriched culture is used (Mbugua, 1987). Coliforms have been reported to be associated with the spoilage of fermented gruels (Mbugua, 1984). The acidity increased and the pH decreased during fermentation (Fig. 4). Onyango et al. (2000) also reported a drop in pH from 5.5 to 3.7 4.1 and an increase in total titratable acidity from 0.22 0.36% to 3.26 4.54% lactic acid equivalent (w/w, on dry matter basis) in back-slopped (7.5% w/w) maize, fingermillet, cassava, maize fingermillet (1:1) and cassava fingermillet (1:1) uji fermented for 24 h. Nche et al. (1994), reported an increase in acidity and a decrease in pH in kenkey to be associated with an increase in LAB counts. The pH of different types of togwa was reported to vary from 3.1 to 4.2 (Lorri and Svanberg, 1995; Kingamkono et al., 1999). The variation was attributed to differences in formulation, duration of fermentation and use of malt or backslopping. In the present work, the acid found in highest concentrations in togwa was lactate. Banigo and Muller (1972) identified the main acids in ogi as lactate, butyrate, acetate and formate. The production of these acids during the fermentation of maize dough porridge and their ability to inhibit a variety of organisms has been reported (Mensah et al., 1991). The increase followed by a decrease in maltose and glucose may be related to the action of enzymes both from malt and fermentation organisms. Lactobacilli have been reported to be responsible for acid production and flavour development in ogi (Akinrele, 1970) and gari (Ngaba and Lee, 1979). Lactic, acetic, malic, succinic and formic acid have been reported in cerealbased fermented traditional beverages in Nigeria (Sanni et al., 1999). In addition to these acids, butyric and propionic acids have been reported during the fermentation of maize dough, and all were reported to inhibit a variety of microorganisms (Mensah et al., 1991). LAB in cereal fermentations can also metabolize substrates such as citrate and pyruvate, producing flavour-related compounds like acetoin and diacetyl (Redler and Bohl, 1984; Martinez-Anaya, 1996). Lactate and acetate are important flavour compounds in fermented cereals (Onyango et al.,

J.K. Mugula et al. / International Journal of Food Microbiology 80 (2003) 187199

197

2000), and the latter has been reported to act as a flavour enhancer, sensitizing consumers towards other aromatic compounds in products such as sourdough, the effect being related to its actual concentration (Gobbetti and Corsetti, 1997). Pyroglutamate decreased during fermentation in togwa. Propionic acid was not detected in togwa. The results correspond to those reported by Onyango et al. (2000) in maize, fingermillet, cassava, maize fingermillet and cassava fingermillet fermented uji. The proteolytic activity of fermentation microorganisms often in combination with malt enzymes may produce precursors of flavour compounds, such as amino acids, which may be deaminated or decarboxylated to aldehydes, and these may be oxidized to acids or reduced to alcohols (Gobbetti et al., 1994; Hansen and Hansen, 1996). The production of 2-methyl-propanal and alcohols increased during fermentation. Yeasts may produce alcohol, however, Lactobacillus species have also been reported to produce ethanol (Hansen and Hansen, 1996). Masha et al. (1998) reported the production of alcohols, esters and acids in spontaneously fermented and back-slopped uji. The reported taste threshold values of ethanol and 3-methyl-butanol in water are 100 800 and 1.0 mg/kg, respectively (Imhof et al., 1994). Some of the compounds that were reported to produce malty flavour in foods, i.e., 2-methyl-propanal, 2-methyl-propanol, 2-methylbutanal, 3-methyl-butanol and 3-methyl-butanal (Narvhus et al., 1998; Hartivgsen et al., 2000) were detected in togwa. The taste thresholds of the aldehydes were reported to be 0.10, 0.13 and 0.06 mg/l, respectively, whereas the corresponding alcohols have considerably higher threshold taste values (Sheldon et al., 1971). As the threshold flavour values for methyl-aldehydes are low, these compounds may impart flavour in the product (Imhof et al., 1994). Some of the flavour compounds may originate from the unfermented substrates (Imhof et al., 1994) or malting process (Beal and Mottram, 1994). For use as a refreshment or thirst-quenching beverage, togwa is diluted by adding water (to a total soluble solids ranging from about 2 to 5 jBrix and a specific gravity of around 1.02) and sugar. The dilution would subsequently lower the concentrations of organic acids, sugars and other flavour compounds, probably to the

extent that some of them will not have an impact on the taste of the product. The results of this study indicated that togwa contains a variety of lactic acid bacteria and yeasts and several flavour compounds. It will be important to assess the performance of the organisms under controlled fermentation and their contribution to the taste and flavour of the product. The development of starter cultures is important for the potential production of togwa at a commercial, small industrial scale, and for the improvement of its acceptability, microbiological stability and hygienic safety.

Acknowledgements This study was supported by grants from the Norwegian Council of Universities Committee for Development Research and Education (NUFU, Project 26/96) through the Agricultural University of Norway and Sokoine University of Agriculture, and the Lanekassen of Norway. We are grateful to Kari Olsen for assistance with the GC and HPLC analyses. We also acknowledge the cooperation given by the processors of togwa.

References
Adegoke, G.O., Babaola, A.K., 1988. Characteristics of microorganisms of importance in the fermentation of fufu and ogitwo Nigerian foods. J. Appl. Bacteriol. 65, 449 453. Akinrele, I.A., 1970. Fermentation studies on maize during the preparation of a traditional African starch-cake food. J. Sci. Food Agric. 21, 619 625. Banigo, E.O.I., Muller, H.G., 1972. Carboxylic acid patterns in ogi fermentation. J. Sci. Food Agric. 23, 101 111. Banigo, E.O.I., deMan, J.M., Duitschaever, C.L., 1974. Utilization of high-lysine corn for the manufacture of ogi using a new improved processing system. Cereal Chem. 51, 559 572. Beal, A.D., Mottram, D.S., 1994. Compounds contributing to the characteristic aroma of malted barley. J. Agric. Food Chem. 42, 2880 2884. Brauman, A., Keleke, S., Malonga, M., Miambi, E., Ampe, F., 1996. Microbiological and biochemical characterization of cassava retting, a traditional lactic acid fermentation for foo-foo (cassava flour) production. Appl. Environ. Microbiol. 62, 2854 2858. Cooke, R.D., Twiddy, D.R., Reilly, P.J.A., 1987. Lactic acid fermentation as a low-cost means of food preservation in tropical countries. FEMS Microbiol. Rev. 46, 369 379.

198

J.K. Mugula et al. / International Journal of Food Microbiology 80 (2003) 187199 Martinez-Anaya, M., 1996. Enzymes and bread flavour. J. Agric. Food Chem. 44, 2469 2480. Masha, G.G.K., Ipsen, M.A., Petersen, M.A., Jakobsen, M., 1998. Microbiological, rheological and aromatic characteristics of fermented uji (an East African sour porridge). World J. Microbiol. Biotechnol. 14, 451 456. Mbugua, S.K., 1984. Isolation and characterisation of lactic acid bacteria during the traditional fermentation of Uji. East Afr. Agric. For. J. 50, 36 43. Mbugua, S.K., 1987. The nutritional and fermentation characteristics of uji produced from dry milled maize flour (unga baridi) and whole wet milled maize. Chem. Mikrobiol. Technol. Lebensm. 10, 154 161. Mensah, P., Tomkins, A.M., Drasar, B.S., Harrison, T.J., 1991. Antimicrobial effect of fermented Ghanaian maize dough. J. Appl. Bacteriol. 70, 203 210. Meyer, S.A, Payne, R.W., Yarrow, D., 1998. Candida Berkhout. In: Kurtzman, C.P., Fell, W.C. (Eds.), The yeasts, A Taxonomic Study, 4th edn. Elsevier, Amsterdam, pp. 454 573. Mokhoro, C.T., Jackson, D.S., 1995. Starch related changes in stored soft sorghum porridge. J. Food Sci. 60, 389 394. Mugula, J.K., Nnko, S.A.M., Srhaug, T., 2001. Changes in quality attributes during storage of togwa, a lactic acid fermented gruel. J. Food Saf. 21, 181 194. Nago, C.M., Hounhouigan, D.J., Akissoe, N., Zanou, E., Mestres, C., 1998. Characterization of the Beninese traditional ogi, a fermented maize slurry: physiological and microbiological aspects. Int. J. Food Sci. Technol. 33, 307 315. Narvhus, J.A., Hulbkdal, A., Thorvaldsen, K.R., Baugerd, M., Abrahamsen, R.K., 1992. Measurement of CO2 production and O2 metabolism by pure and mixed cultures of lactic acid bacteria growing in milk. Actes du Colloque LACTIC 91. Centre de Publications de IUniversite de Caen, Caen, France, p. 371. Narvhus, J.A., Osteraas, K., Mutukumira, T., Abrahamsen, R.K., 1998. Production of fermented milk using a malty-compound producing strain of Lactococcus lactis subsp lactis biovar diacetylactis isolated from Zimbabwean naturally fermented milk. Int. J. Food Microbiol. 14, 73 80. Nche, P.F., Nout, M.J.R., Rombouts, F.M., 1994. The effect of cowpea supplementation on the quality of kenkey, a traditional Ghanaian fermented food. J. Cereal Sci. 19, 191 197. Ngaba, P.A., Lee, J.S., 1979. Fermentation of cassava (Manihot esculenta Crantz). J. Food Sci. 44, 1570 1571. Nout, M.J.R., 1980. Microbiological aspects of the traditional manufacture of busaa, a Kenyan opaque maize beer. Chem. Mikrobiol. Technol. Lebensm. 6, 137 142. Nout, M.J.R., 1991. Ecology of accelerated natural lactic fermentation of sorghum-based infant formulas. Int. J. Food Microbiol. 12, 217 224. Nout, M.J.R., Rombouts, F.M., Havelaar, A., 1989. Effect of natural lactic fermentation of infant food ingredients on some pathogenic microorganisms. Int. J. Food Microbiol. 8, 351 361. Odunfa, S.A., Adeyele, S., 1985. Microbiological changes during the traditional production of ogi-baba, a West African fermented sorghum gruel. J. Cereal Sci. 3, 173 180. Olasupo, N.A., Olukoya, D.K., Odunfa, S.A, 1997. Identification of

Deak, T., Beuchat, L., 1996. Handbook of food spoilage yeasts CRC Press, Boca Raton. Fleming, H.P., McFeters, R.F., 1981. Use of microbial cultures: vegetable products. Food Technol. 35, 84. Giraud, E., Champailler, A., Raimbault, M., 1994. Degradation of raw starch by a wild amylolytic strain of Lactobacillus plantarum. Appl. Environ. Microbiol. 60, 4319 4323. Gobbetti, M., Corsetti, A., 1997. Lactobacillus sanfransiscoa key sourdough lactic acid bacterium: a review. Food Microbiol. 14, 175 187. Gobbetti, M., Corsetti, A., Rossi, J., 1994. The sourdough microflora. Interactions between lactic acid bacteria and yeasts: metabolism of carbohydrates. Appl. Microbiol. Biotechnol. 41, 456 460. Halm, M., Lillie, A., Sorensen, A.K., Jakobsen, M., 1993. Microbiological and aromatic characteristics of fermented maize dough for kenkey production in Ghana. Int. J. Food Microbiol. 19, 135 143. Hansen, A., Hansen, B., 1996. Flavour of sourdough wheat crumb. Z. Lebensm.-Unters. -Forsch. 202, 244 249. Harrigan, W.F., McCance, M.E., 1990. Laboratory methods in food and dairy microbiology, 8th edn. Academic Press, London. Hartvigsen, K., Lund, P., Hansen, L.F., Gunhild, H., 2000. Dynamic Headspace gas chromatography/mass spectrometry characterization of volatiles produced in fish oil enriched mayonnaise during storage. J. Agric. Food Chem. 48, 4858 4867. Hounhouigan, D.J., Nout, M.J.R., Nago, C.M., Houben, J.H., Rombouts, F.M., 1993. Microbiological changes in mawe during natural fermentation. World J. Microbiol. Biotechnol. 10, 410 413. Imhof, R., Glattli, H., Bosset, J.O., 1994. Volatile organic aroma compounds produced by thermophilic mixed strain dairy starter cultures. Lebensm.-Wiss. Technol. 27, 442 449. Kingamkono, R., Sjogren, E., Svanberg, U., Kaijser, B., 1994. pH and acidity in lactic-fermenting cereal gruels: effects on viability of enteropathogenic microorganisms. World J. Microbiol. Biotechnol. 10, 664 669. Kingamkono, R., Sjogren, E., Svanberg, U., Kaijser, B., 1995. Inhi bition of different strains of enteropathogens in a lactic-fermenting cereal gruel. World J. Microbiol. Biotechnol. 11, 299 303. Kingamkono, R., Sjogren, E., Svanberg, U., 1999. Enteropathogen ic bacteria in faecal swabs of young children fed on lactic acidfermented cereal gruels. Epidemiol. Infect. 122, 23 32. Kunene, N.F., Geornaras, I., von Holy, A., Hastings, J.W., 2000. Characterization and determination of origin of lactic acid bacteria from a sorghum-based fermented food by analysis of soluble proteins and amplified fragment length polymorphism fingerprinting. Appl. Environ. Microbiol. 66, 1084 1092. Kurtzman, C.P., 1998. Issatchenkia Kurdryavtsev amend. In: Kurtzman, C.P., Fell, W.C. (Eds.), The Yeasts, a Taxonomic Study, 4th edn. Elsevier, Amsterdam, pp. 221 226. Lorri, W., Svanberg, U., 1995. An overview of the use of fermented foods for child feeding in Tanzania. Ecol. Food Nutr. 34, 65 81. Marsili, R.T., Ostapenko, H., Simmons, R.E., Green, D.E., 1981. High performance liquid chromatographic determination of organic acids in dairy products. J. Food Sci. 46, 52 57.

J.K. Mugula et al. / International Journal of Food Microbiology 80 (2003) 187199 Lactobacillus species associated with selected African fermented foods. Z. Naturforsch. 52, 105 108. Onyango, C., Okoth, M.W., Mbugua, S.K., 2000. Effect of drying lactic fermented uji (an East African sour porridge) on some carboxylic acids. J. Sci. Food Agric. 80, 1854 1858. Oyewole, O.B., 1997. Lactic fermented foods in Africa and their benefits. Food Control 8, 289 297. Oyewole, O.B., Odunfa, S.A., 1990. Characterization and distribution of lactic acid bacteria in cassava fermentation during fufu production. J. Appl. Bacteriol. 68, 145 152. Radler, F., Bohl, K., 1984. The metabolism of several carboxylic acids by lactic acid bacteria. Z. Lebensm.-Unters. -Forsch. 179, 228 231. Sanni, A.I., 1993. The need for process optimization of African fermented foods and beverages. Int J. Food Microbiol. 18, 85 95. Sanni, A.I., Onilude, A.A., Fadahunsi, I.F., Afolabi, R.O., 1999. Microbial deterioration of traditional alcoholic beverages in Nigeria. Food Res. Int. 32, 163 167. SAS/stat, 1996. SAS/stat Users Guide. Version 6 Statistical Analysis System Institute, Cary, NC, USA. Sheldon, R.M., Lindsay, R.C., Libbey, L.M., Morgan, M.E., 1971.

199

Chemical nature of malty flavour and aroma produced by Streptococcus lactis var. maltigenes. Appl. Microbiol. 22, 263 266. Sneath, P.H.A., Mair, N.S., Sharpe, M.E., Holt, J.G., 1986. Bergeys Manual of Systematic Bacteriology, vol. 2, Williams and Wilkins, Baltimore. Steinkraus, K.H., 1996. Handbook of Indigenous Fermented Foods, 2nd edn. Marcel Dekker, New York. Vaughani-Martini, A., Martini, A., 1998. Saccharomyces Meyen ex Reess. In: Kurtzman, C.P., Fell, W.C. (Eds.), The Yeasts, A Taxonomic Study, 4th edn. Elsevier, Amsterdam, pp. 358 371. Willumsen, J.F., Darling, J.C., Kitundu, J.A., Kingamkono, R.R., Msengi, A.E., Mduma, B., Sullivan, K.R., Tomkins, A.M., 1997. Dietary management of acute diarrhoea in children: effect of fermented and amylose-digested weaning foods on intestinal permeability. J. Pediatr. Gastroenterol. Nutr. 24, 235 241. Wood, B.J.B., Holzapfel, W.H., 1995. The Genera of Lactic Acid Bacteria, vol. 2, Blackie Academic and Professional, Glasgow. Yarrow, D., 1998. Methods for the isolation, maintenance and identification of yeasts. In: Kurtzman, C.P., Fell, W.C. (Eds.), The Yeasts, A Taxonomic Study, 4th edn. Elsevier, Amsterdam, pp. 77 121.