Bacterial Diversity Studies and probiotic Functions of Bacillus Species in Fermented Locust Bean Seeds (Parkia Biglobosa Jacq. Benth)
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Date
2016-01
Authors
Adewumi, G.A
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Abstract
Fermented vegetable protein cotyledons processed into condiments are widely consumed in many West African countries. Production of these condiments involves microbial and biochemical changes during fermentation. The process is natural, uncontrolled and results into product batch variations, safety challenges, and low shelf-life. In this study, multiphasic approaches that combined culture-independent molecular biology techniques, phenotypic microbial characterization and identification, and culture-dependent genomic fingerprints were applied to understand the bacterial diversity and functional dynamics of iru, a traditional alkaline fermented food condiment made from Parkia biglobosa (Jacq. Benth) cotyledons. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) gel profiles based on V3 variable region of amplified 16S rRNA gene, revealed similarities and variations in bacterial community structure in 16 iru samples obtained from different geographical regions in Nigeria, as determined by Dice similarity coefficient and Non-metric Multidimensional Scaling (nMDS). This also grouped the iru samples into distinct clusters, indicating that their bacterial composition is far more diverse and complex than earlier reported. DNA sequences of PCR-DGGE bands identified Bacillus subtilis as the predominant bacterial species in iru. Other bands identified were closest relatives of potential food-borne pathogens and contaminants such as Staphylococcus vitulinus, S. saprophyticus, B. thuringiensis, Morganella morganii, Tetragenococcus halophilus, Salinicoccus jeotgali, Ureibacillus thermosphaericus, Brevibacillus parabrevis, Brevibacterium sp. and uncultured bacteria clones. An average population density of 106 was enumerated in the various iru samples cultured, and mean pH of 7.2. Cultured bacterial colonies ca. 280 phenotyped were Gram-positive rods, catalase-positive, aerobic and facultative anaerobic endospore-formers, and tentatively identified as Bacillus species and its closest relatives. Amplified ribosomal DNA restriction analysis (ARDRA) genomic sub-typing of wild strains comparatively with typed and referenced strains differentiated Bacillus species into B. subtilis and B. cereus phylotypes. Combined profiles of ARDRA, internal transcribed spacer (ITS) PCR, ITS-PCR-restriction fragment length polymorphism (ITS-PCR-RFLP) and randomly amplified polymorphic DNA (RAPD-PCR) showed dominance and diversity of B. subtilis strains in iru. Phylogenetic analysis of the 16S rRNA gene established genetic divergence, evolutionary and clonal relationships among Bacillus strains isolated from Africa and similar Asian fermented condiments. Also, multi locus sequence analysis (MLSA) based on PCR amplification of gyrB, glpF and gmk housekeeping genes and sequences showed phylogenetic relatedness of common ancestral origin between B. cereus and B. thuringiensis, confirming B. cereus as the major member of the B. cereus group associated with iru. Divergent B. subtilis strains from iru, B. subtilis U170B and B. subtilis U146A, including a reference B. clausii UBB-07 (MTCC 5472) were studied for probiotic functions. Vegetative cells of bacilli analysed, generally showed resistance to acidic conditions of pH 2.0 and high tolerance to 0.3% (w/v) bile salts concentration. The two B. subtilis strains fairly inhibited food-borne pathogens, while B. clausii UBB-07 showed stronger inhibitory spectrum. Bacterial cell surface properties comparatively were highest for B. subtilis U146A, B. subtilis U170B and B. clausii UBB-07 in decreasing order. Although, B. clausii UBB-07 adhered more to both undifferentiated and differentiated HT-29 cell lines, the three Bacillus strains effectively interfered and hindered adhesion of Salmonella enterica serovar Typhimurium MBU 1047 to HT-29 cell monolayers. This study comprehensively defined molecular bacterial ecology of iru, as well as established occurrence and dominance of bacilli in this important naturally fermented food condiment in W. Africa. Diverse autochthonous B. subtilis strains and B. cereus were found, a prequisite for prediction of functions and safety. Bacilli in iru demonstrated beneficial probiotic functions. Strains such as these could be used as probiotic starter cultures in small- and large-scale industrial production of condiments to enhance human health.
Description
A Thesis Submitted to the School of Postgraduate Studies, University of Lagos
Keywords
Fermented condiments , Bacteria diversity , Bacillus subtilis , Probiotics , Research Subject Categories::NATURAL SCIENCES::Biology
Citation
Adewumi, G.A (2016). Bacterial Diversity Studies and probiotic Functions of Bacillus Species in Fermented Locust Bean Seeds (Parkia Biglobosa Jacq. Benth). A Thesis Submitted to University of Lagos School of Postgraduate Studies Phd Thesis and Dissertation, 214pp.