Microbiota Of Fermenting Millet In Hausa Koko Production: Their Diversity, Fermentative Characteristics And Potential For Starter Culture Development

Abstract

Hausa koko is a traditional free -flowing spicy fermented pearl millet porridge produced mostly at the household level by women and sold as street food in Ghana. The fermentation is spontaneously done, not controlled, and the final product prone to contamination with potential foodborne pathogens. To standardize and control the fermentation process to achieve better product quality and safety for large-scale production, the use of starter culture containing beneficial fermenting microbes such as lactic acid bacteria (LAB) and yeast was considered in this study. Samples at different processing stages were obtained from twelve (12) different commercial processors located in six regions of Ghana. Their bacterial community were determined using the V4 variable region of the 16S rRNA gene and their metabolite profiled using 1HNuclear Magnetic Resonance (NMR) spectroscopy. Out of the 12 commercial processors, samples from five (5) were enumerated for lactic acid bacteria (LAB) and yeast. The LAB isolates were fingerprinted using (GTG)5 based rep-PCR before whole genome sequenced, while 28S rRNA genes were Sanger sequenced for yeast isolates for genetic characterisation and identification. The identified isolates were then screened for some technological and probiotic characteristics in-vitro, beneficial isolates were used to develop starter culture whose performance was evaluated in-situ and tested for consumer acceptability. Results revealed the most comprehensive bacterial community with over four hundred (400) different Gram-positive and Gram-negative organisms and profiled thirty-three (33) metabolites. The LAB isolates were made up of both homo and hetero fermentative organisms. They included Limosilactobacillus pontis (31.11 %), Pediococcus acidilactici and Limosilactobacillus fermentum (16.67 % each), Pediococcus pentosaceus (11.11 %), Limosilactobacillus reuteri (10 %), Weissella confusa (6.67 %), Schleiferilactobacillus harbinensis (3.33 %), Lactiplantibacillus plantarum and Lacticaseibacillus paracasei (2.22 % each). L. pontis, L. fermentum, P. pentosaceus and L. reuteri occurred at all the stages of Hausa koko production. Saccharomyces cf. cerevisiae/paradoxus (41.4 %), Saccharomyces cerevisiae (31.0 %), Pichia kudriavzevii (13.8 %), Clavispora lusitaniae (8.6 %) and Candida tropicalis (5.2 %) were the yeast identified. A total of 27 LAB isolates were predicted to have bacteriocin producing genes and genes related to nutritive and enzyme production. Subsequently, these isolates were selected for further testing. The majority of the selected LAB and yeast isolates exhibited good technological and potential probiotic characteristics in vitro. The LAB showed good rates of acidification, strong inhibitory activity against some foodborne indicator organisms, amylase production (66.7 %), and low production of exopolysaccharides (EPS) (33.3 %). They also exhibited good tolerance and survival in acid conditions at pH (2.5 - 6.0) and at pH 7, tolerance and survival against bile (0.3 - 1.0 %). Similar good probiotic characteristic was obtained from the yeast isolates including tolerance to low-neutral pH (2.0, 3.0, 5.5 and 7.0), bile (0.3 - 1.0 %), high temperatures (25 °C and 37 °C) and salt concentrations (4 and 6 %). LAB isolates L. reuteri LDOD-Sud, L. pontis LTAD-12g and L. fermentum LMAN-Sdb, and yeast isolates, S. cerevisiae YSUN-Sud and P. kudriavzevii YTAD-12j selected for further studies in the development of a starter culture or inoculum enrichment during millet fermentation in different combinations produced acceptable results. Reduction or total inhibition of aflatoxins B1, B2 and G2 infected millet slurries were recorded when fermented with the different combinations. The most preferred starter culture combination was Limosilactobacillus reuteri LDOD-Sud (R) + Limosilactobacillus fermentum LMAN-Sdb (F) + Saccharomyces cerevisiae YSUN-Sud (C) referred to as RFC Hausa koko Starter Culture (RFCH). Although not under a strictly controlled fermentation set up, RFCH starter culture demonstrated desirable traits including quality and safety improvement, reduced fermentation time from the normal 48 - 72 h to only 12 h during semi-industrial scale fermentation. The study presented the most comprehensive bacterial and metabolites profile, the diversity, technological and probiotic potential of microorganisms associated with Hausa koko processing and the development of a starter culture. Also, it has shown the possibility of using starter culture by commercial processors at semi-industrial scale to standardize the production process for improved quality and safety