V . L E I A N D M . J A K O B S E N . 2004.Aims: To identify and examine the diversity of predominant lactic acid bacteria (LAB) in koko and koko sour water (KSW) from different Ghanaian production sites with regard to pattern of fermentation (API 50 CHL), genotype, antimicrobial activity, and resistance to low pH and bile salts. Methods and Results: In total 215 LAB were isolated from koko and KSW. The isolates were identified using intergenic transcribed spacers (ITS)-PCR restriction fragment length polymorphism (RFLP), API 50 CHL, restriction enzyme analysis with pulsed-field gel electrophoresis (REA-PFGE) and sequencing of the 16S rRNA gene. The dominating micro-organisms in koko was found to be Weisella confusa and Lactobacillus fermentum, followed by Lact. salivarius and Pediococcus spp. Chemometric data analysis were used to link the LAB species to the different production stages and production sites. At intra-species level the isolates were found to have a great diversity. The isolates were investigated for antimicrobial activity using agar diffusion assays, and acid and bile tolerance. Most isolates showed low levels of antimicrobial activity towards the indicator strain Listeria innocua, but not towards the bacteriocin-sensitive Lact. sakei. Growth of all LAB isolates was unaffected by the presence of 0AE3% (v/v) oxgall bile. The isolates were able to survive, but were not able to grow in growth medium adjusted to pH 2AE5. Conclusions: The dominating LAB of koko and KSW were W. confusa and Lact. fermentum showing a pronounced taxonomic biodiversity at sub-species level between stages within the production as well as between production sites. Other species observed in KSW were Lact. salivarius, Ped. pentosaceus, Ped. acidilactici and Lact. paraplantarum. They occurred in levels of 10 8 CFU ml )1 in fresh KSW and showed uniform antimicrobial activity, and acid and bile tolerance. Significance and Impact of the Study: The present study gives a detailed picture of the taxonomy and diversity of LAB in an African-fermented millet product that may have potential as a probiotic product for the local population. The chemometric tools Principal Component Analysis and ANOVA ANOVA Partial Least Squares Regression were proven to be useful in the analysis of microbial groupings and associations with specific sites and stages in the production of koko and KSW.

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Aim: To quantify and identify the predominant lactic acid bacteria (LAB) in dolo and pito wort processing, and to examine their biodiversity at strain level. Materials and Results:The processing of dolo and pito wort was studied at four production sites in Burkina Faso and Ghana. The succession of dominant micro-organisms, pH and titratable acidity were determined from sorghum malt through mashing and acidification to final wort. In the sorghum malt and during mashing, the LAB counts were 5AE7-7AE5 log CFU g )1 . Similar levels of yeasts and gram-negative, catalase-positive bacteria were observed. These levels decreased to 3AE7-4AE5 log CFU g )1 and £2-3 log CFU g )1 , respectively, at the end of mashing, including a mild heat treatment. During acidification at ambient temperature (30-33°C) lasting for 12-16 h, LAB counts increased to 8AE8-9AE9 log CFU g )1 , pH decreased from 5AE55 ± 0AE12 to 3AE72 ± 0AE24, and the titratable acidity calculated as lactic acid, increased from 0AE13% to 0AE61%. The gram-negative, catalase-positive bacteria and yeasts observed in the malt and during mashing were no longer detected. A total of 556 strains of LAB were isolated and purified. The LAB isolates were characterized and identified by a polyphasic approach based on phenotypic and genotypic methods, such as carbohydrate fermentation patterns using API 50 CHL, intergenic transcribed spacers-polymerase chain reaction ⁄ restriction fragment length polymorphism (ITS-PCR ⁄ RFLP), pulsed-field gel electrophoresis (PFGE) and 16S rRNA gene sequencing. Lactobacillus fermentum was identified as the dominant LAB species in the malt during mashing and during acidification. The other species observed during acidification were Lactobacillus delbrueckii ssp. delbrueckii, Lact. delbrueckii ssp. bulgaricus and Pediococcus acidilactici. These bacteria comprised a minor fraction of the bacterial population and no distinct microbial succession was observed for the LAB. At species level, the LAB profiles were similar for the four production sites; however, a pronounced diversity was observed at strain level. For one site, which had implemented a cleaning procedure between batches only, Lact. fermentum was found. Conclusion: Lact. fermentum was found to be the dominant LAB species throughout the entire process to final dolo and pito wort, including the acidification. Lact. delbrueckii ssp. delbrueckii, Lact. delbrueckii ssp. bulgaricus and P. acidilactici occurred in low numbers. At strain level, a high diversity based on PFGE-RFLP was observed for Lact. fermentum within and between sites.

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Indigenous lactic acid fermented foods may have potential as probiotic treatment for diarrhoea, due to high levels of lactic acid bacteria. In this study the effect of a millet drink, spontaneously fermented by lactic acid bacteria, as a therapeutic agent among Ghanaian children with diarrhoea, was assessed. Children below 5 years of age coming to Northern Ghana health clinics for treatment of diarrhoea were randomised to two groups. Children of both groups received treatment for diarrhoea given at the local clinic. The intervention group in addition received up to 300 ml fermented millet drink (KSW) daily for 5 days after enrolment. The clinical outcome of diarrhoea and reported well-being were registered every day for the 5-day intervention and again 14 days after diagnosis. Among 184 children (mean age 17.4, standard deviation 11.3 months) included, no effects of the intervention were found with respect to stool frequency, stool consistency and duration of diarrhoea. However, KSW was associated with greater reported well-being 14 days after the start of the intervention (P=0.02). The fact that no effect of KSW on diarrhoea was observed could be because many children had a mild form of diarrhoea, and many were treated with antibiotics. Either this could have affected the lactic acid bacteria, or the lactic acid bacteria in KSW had no probiotic effects. It is speculated that the effect after two weeks could be due to a preventing effect of KSW on antibiotic-associated diarrhoea which could help reducing persistent diarrhoea.