MOLECULAR CHARACTERIZATION OF THE MICROBIAL COMMUNITIES, PROXIMATE COMPOSITION AND SENSORY PROPERTIES OF “OTIKA” AND “AGADAGIDI” FERMENTED BEVERAGES

Abstract

The aim of this study was to employ molecular methods in establishing the identities and succession of microorganisms associated with the fermentation of the Nigerian beverages, “Otika” and “Agadagidi” and determining proximate compositions and sensory characterization of the beverages. In order to achieve this goal, isolation, enumeration and identification of total bacterial and fungal loads were carried out by using standard pour plate, cell morphological and biochemical characterization method. To identify the microorganisms genetically, DNA extraction of the isolates, PCR amplification of the genes coding for the 16S rRNA and 26S rRNA conserved region of the microorganisms, gel electrophoresis, sequencing and Basic Local Alignment Search Tool were done. The genetic relatedness of the microbes was determined. The microbes were screened for class IIa bacteriocin using PCR rapid screening and specific primer encoding plantaricin, pediocin and enterocin. Antagonistic and mutualistic interactions among the microorganisms were studied using agar well diffusion method. Proximate composition and sensory characteristics of the beverages were investigated using standard methods according to Association of Official Analytical Chemist. Microbial loads increased during the production of the two beverages. In “Otika”, Total bacterial (TB), Total fungal (TF), lactic acid bacterial ( LAB) and Viable enterobacteriaceae counts (VE) increased from 1.6× 106 ± 0.33 cfu//ml, 3.4 × 105 ± 0.10 cfu/ml, 3.0 × 106 ± 0.0 cfu/ml and 1.5× 106 ± 0.15 cfu/ml to 4.6 × 107 ± 0.30 cfu/ml, 4.5 × 106 ± 0.10 cfu/ml, 9.0 × 107 ± 0.05 cfu/ml and 3.7 × 107 ± 0.2 cfu/ml respectively on the sorghum grains and at early stages of fermentation. Later, bacterial load decreased steadily along the fermentation period while enterobacteriaceae decreased until it was undetectable. In “Agadagidi”, the TB, TF and VE loads increased from the ripe plantain pulp to 5.2 × 107 ± 0.05 cfu/ml, 2.0 × 107 ± 0.05 cfu/ml and 2.8 × 107± 0.05 cfu/ml at 24 hr respectively to decrease at 48 hours of fermentation at 2.0 × 105 ± 0.11 cfu/ml, 1.03 × 105± 0.05 cfu/ml and 1.3 × 105 ±0.11cfu/ml respectively. LAB had a load of 6.7 × 106± 0.05cfu/ml on the overripe plantain pulp which reduced after 24 hr of fermentation to 4.2 × 105± 0.05 cfu/ml and thereafter increased to 8.6×107 ± 0.1cfu/ml at 48 hr. Generally conventional microbiological methods could not show the species and strain names of some of the microbes while some bacteria were not correctly named in comparism with molecular techniques used. Based on the later method, different types of bacteria and yeast associated with “Otika” and “Agadagidi” preparation were identified. The moulds obtained in “Otika” and “Agadagidi” were in both beverages. Bacillus species and LAB dominated the beverages. The phylogenetic tree showed divergence among some of the microorganisms. Staphylococcus aureus and Listeria monocytogenes, Klebsiella aerogenes and Lactobacillus paraplantarum were closely related but are distantly paired. Lactobacillus fermentum and Bacillus subtilis divergences were close. The genetic relatedness among the yeasts in “Otika” shows that Candida krusei and Saccharomyces cerevisiae were closely related compared with C. tropicalis. However, in the production of “Agadagidi”, based on the bootstrap (0.6m), the pair of Lactobacillus plantarum and L. fermentum, B. subtilis and B. cereus with Leuconostoc psuedomesenteroides and Lactococcus lactis are from the same ancessor. The genetic relatedness among the yeasts in “Agadagidi” revealed that C. utilis and Geotrichum candida is closely related compared with Saccharomyces cerevisiae. Two (20.5%) strains had a bacteriocin named plantaricin. Yeasts and the LAB exhibited positive interaction (mutualistic/commensalism) whereas all LAB and yeasts were antagonistic against other bacteria. The highest value of the antagonistic interaction was found at pH 7 and 30°C. The use of the strain of L. plantarum as single starter culture in the fermentation for ‘‘Otika’’ increased the nutritive value of the beverages. Leuconostoc mesenteriodes used as a single culture in the preparation of ‘‘Agadagidi’’ increased the moisture and protein composition. The data obtained in this study has provided vital information which should be used in the production of consistent quality of “Otika” and “Agadagidi beverages.