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Genotypic and Phenotypic Diversity Among Bacillus Species Isolated from Mbuja, a Cameroonian Traditional Fermented Condiment

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Genotypic and Phenotypic Diversity Among Bacillus Species Isolated from Mbuja, a Cameroonian Traditional Fermented Condiment African Journal of Biotechnology Vol. 12(12), pp. 1335-1343, 20 March, 2013 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB12.2715 ISSN 1684–5315 ©2013 Academic Journals Full Length Research Paper Genotypic and phenotypic diversity among Bacillus species isolated from Mbuja, a Cameroonian traditional fermented condiment Bouba-Adji Mohamadou1*, Carl Moses Mbofung2 and Georges Barbier3 1Department of Food Engineering and Quality Control, University Institute of Technology, P. O. Box 454 Ngaoundere, Cameroon. 2Department of Food Science and Nutrition, National School of Agro-Industrial Sciences of Ngaoundere P. O. Box 455, Cameroon. 3Laboratoire Universitaire de Biodiversité et Ecologie Microbienne (EA 3882), IFR 148, ESMISAB, Université Européenne de Bretagne, Technopôle Brest-Iroise, 29280 Plouzané, France. Accepted 1 February, 2013 Dichotomous keys based on morphological, cultural and biochemical tests have long been used to identify Bacillus species. The analysis of 16S rDNA is suggested to be used for identification that is more exact. The present study was carried out to compare a conventional phenotypic method with the analysis of the 16S rRNA and gyrB gene for better identification, to determine their phylogenetic relationships and to contribute to selecting starter cultures for Mbuja production. Twenty-six (26) Bacillus strains isolated from 12 samples of Mbuja formerly identified by phenotyping as representatives of eight species (licheniformis, polymyxa, laterosporus, cereus, circulans, subtilis, pumilus and brevis) were studied. Results of genotypic analyses were not concurrent with previous phenotypic identification. Bacillus from different species were able to cluster together to form phylogenetic groups. An insight of these groups revealed important genetic diversity between strains from the same species. Bacillus subtilis and close relatives were the most abundant and presented appreciable biochemical traits. This group could therefore be considered for starter selection. Key words: Mbuja, Bacillus sp., phenotyping, genotyping, starter. INTRODUCTION Mbuja is a traditional condiment produced in Cameroon (Ouoba et al., 2003a, b). The ability of Bacillus sp. to by fermenting 10 day-old cooked seeds of Hibiscus develop a variety of biochemical activities in the substrate sabdariffa in earthen ware pots. We have earlier showed contribute to the development of desirable sensory that Mbuja is of good anti-oxidant quality and could characteristics of the condiments. Given that biochemical potentially help in maintaining consumer health and sensory characteristics of fermented products are a (Mohamadou et al., 2007). The main bacteria associated direct consequence of the metabolic activity of the with the fermentation process belong to the Bacillus fermenting microflora, the characterization of the most genera (Mohamadou et al., 2009). The role of Bacillus important microorganisms appears to be not only a key species in other African condiments has been studied, for understanding these changes in the seeds but also including the production of Soumbala in West Africa identify and possibly select optimal starter cultures. Both phenotypic and molecular typing methods are used to this effect. Phenotypic methods include the use of API 50CHB (Ouoba et al., 2004) and the dichotomic *Corresponding author. E-mail: [email protected]. Tel: + 237 key of Gordon (Cavalcanti et al., 2007; Guerra-Cantera et 77 80 31 37. al., 2005) based on a set of morphological characteristics 1336 Afr. J. Biotechnol. and biochemical tests, including carbohydrates PCR amplification of the 16S rDNA fermentation and enzyme detection of species. However, due to the variety of ecosystems in which the species Bacterial 16S rDNA were amplified by PCR using bacterial universal primers. Two couples of primers (Table 1) were used: grow, conventional methods often result to ambiguous w18/po2 (used for S3, S7, S9, S10, S11, S12, S14, S16, S19, S20, identification. S21, S22, S23, SY and SAc) and PA/PH (S1, S2, S4, S5, S6, S8, Recently, different techniques have been applied for S13, S15, S17, S18 and SX). Reactions were carried out in a total the typing and identification of bacteria, among which are: volume of 27 µl consisting of 0.5 µM of each primer, 1X Taq TM amplification of the transcribed intergenic region 16S-23S Titanium DNA (BD Biosciences, Clontech Laboratories, Mountain View, USA), 200 µM of each dNTP, 1× of Taq Titanium PCR buffer, (ITS-PCR) for differentiating species (Johnson et al., sterile distilled water and 3 µl of DNA extract (80 ng/µl). After an 2000; Ouoba et al., 2010), use of restriction fragment- initial denaturation for 2 min at 94°C, 25 cycles were performed linked polymorphism analysis of the intergenic using a PTC-100 Programmable Thermal Controller (MJ Research transcribed region to differentiate between species and Inc, Waltham, USA) with the following parameters: 1 min strains (Joung and Coté, 2001), pulse field gel denaturation at 94°C, 1 min elongation at 72°C and 1 min annealing electrophoresis (PFGE) to differentiate strains (Mendo et at 55°C. The final elongation was 10 min at 72°C. The amplification products were checked on agarose gels (1% w/v, Promega, al., 2000) and 16S rDNA sequencing to describe Madison, USA) stained with ethidium bromide (0.5 mg/l). phylogenic relationship among species (El-Helow, 2001). In previous studies (Mohamadou et al., 2009), we reported the isolation and identification of 26 Bacillus PCR-amplification of gyrB strains using the dichotomous key of Gordon et al. Specific amplification was performed for presumable B. cereus (1973). Eight species were identified: Bacillus subtilis, species revealed by phenotypic methods. PCR assays were Bacillus pumilus, Bacillus brevis, Bacillus polymyxa, performed as described above using PCR primer sets designed Bacillus licheniformis, Bacillus laterosporus, Bacillus from the gyrase subunit B gene (gyrB) to differentiate between B. cereus and Bacillus circulans. The aim of the present cereus (BC1 and BC2r), B. thuringiensis (BT1 and BT2r) and B. anthracis (BA1 and BA2r) (Yamada et al., 1999) (Table 1). work was to compare identification of Bacillus sp. using Reactions were carried out in a total volume of 20 µl consisting of 1 phenotypic tests and 16S rDNA analysis in order to μl (80 ng/µl) of each Bacillus template DNA, 1 μM of each primer, achieve a complete identification and phylogenetic 1× BD TitaniumTM Taq DNA Polymerase (BD Biosciences, relationship between isolates and to screen their Clontech Laboratories, Mountain View, USA), 1× BD Titanium Taq technological properties for use as starter cultures in PCR Buffer, and sterile water (7.7 µl). An initial denaturation for 5 controlled fermentations. min at 95°C was followed by 30 cycles with the following parameters: 1 min denaturation at 94°C, 2.5 min elongation at 72°C and 1.5 min annealing at 58°C. The final elongation was 7 min at 72°C. The amplification was checked on agarose gels (1%, MATERIALS AND METHODS Promega, Madison, USA) stained with ethidium bromide (0.5 mg/l) and bands were visualised under UV (Gel Doc 2000, BIORAD, Bacteria Hercules, USA). Bacillus strains studied in this work were isolated from 12 samples of Mbuja (fermented Hibiscus sabdariffa seeds), purified and Sequencing of the 16S rDNA identified phenotypically in a previous work by Mohamadou et al. (2009). These strains were stored in liquid nitrogen in the University 16S rDNA sequencing was performed on 10 µl of PCR product (40 Laboratory of Biodiversity and Microbial Ecology (EA 3882, IFR ng/µl) using the Montage µPCR (EMD Millipore Corporation, 148, Brest, France) before use. They were identified by the Billerica, MA, USA) kit as described by Renault et al. (2007). The dichotomous key of Gordon et al. (1973) as B. laterosporus (S8, reaction of sequence was carried out by a GeneAmp® PCR System S9, S11, S14, S15, S17 and S22), B. pumilus (S2, S12, S16, S21, 9700 thermocycler (Applied Biosystem, Carlsbad, CA, USA) and S23), B. licheniformis (S4, S6, S7, and S20), B. brevis (S3, according to the following conditions: an initial denaturation for 5 S19, SX, SY), B. subtilis (S1, S13), B. cereus (S10 and SAc), B. min at 96°C; 50 cycles of amplification including a denaturation for circulans (S18) and B. polymyxa (S5). 30 s at 96°C, a hybridization for 30 s at 57°C and an extension for 4 min at 60°C; a final extension of 10 min at 72°C. Two couples of primers were used: w18/po2 and PA/PH. Each 5 µl of the reaction volume contained 1 µl of DNA; 1 µl of primer (12 ng/µl); 0.5 µl of Big Genotyping Dye Terminator V 3.1; 0.75 µl of 5× buffer and 1.75 µl of distilled water. Sequencing was completed on an automatic sequencer, Extraction of genomic DNA Applied Biosystems 3130xl Genetic Analyser (GIS Ouest Génopole- GENOMER, Roscoff, France). The total DNA was extracted from pure cultures of cells. Cell lysis was realised by heat shocks (Cavalcanti et al., 2007) with some modifications and ultrasounds. Young cultures were suspended in Sequence analysis and phylogenetic relationships between the 300 µl of sterile physiological water (9 g/l of NaCl) and frozen at – strains 22°C for 24 h before being heated to 80°C for 10 min in dry conditions using a ThermoStat plus (Eppendorf). The suspensions Sequence analysis was performed with the sequences in the NCBI obtained were subsequently treated by ultrasounds for 10 min. DNA database (www.ncbi.nlm.nih.gov/blast) using the Basic Local extracts were checked for purity and concentration
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