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Phenotypic identification
A preliminary analysis in Bacillus identification often involves one or more phenotypic methods based on morphology, growth parameters, physiological and biochemical profiles. Morphological characteristics include shape, size, surface characteristics, pigmentation, Gram–staining, sporulation characteristics, and motility. Bacillus and related genera are generally Gram positive, spore-forming, catalase-positive microorganisms, cultivable in several microbiological media. Endospore formation, found in the group of Bacillus assumed the most important survival strategy peculiar to the genera when in adverse environment (Moir, 2006). The biochemical tests use specific growth media, nutrients, chemicals, or growth conditions to elicit observable or measurable biochemical responses from the Bacillus and related genera, thereby enabling their characterization and further identification. Recognized tests include phenol red carbohydrate fermentation, catalase production, oxidase test, oxidation-fermentation, methyl red test, Voges–Proskauer reaction, nitrate reduction, starch hydrolysis, tryptophan hydrolysis, hydrogen sulphide production, citrate utilization, and litmus milk reactions.
The API 50 CHB system, Microgen™ Bacillus ID is one of the numerous miniaturized and automated commercial systems are available with well-defined quality control procedures that allow rapid characterization of Bacillus (Logan and Berkeley, 1984). Phenotypic characteristics formerly considered suitable for the typing of individual strains within a species, however, it has been recognized for a long time that the genus Bacillus is phenotypically heterogeneous (Claus and Berkeley, 1986; Priest, 1993). Misidentification of some bacteria reported in some studies indicates that the identification of bacteria based on the phenotypic properties alone may not always be suitable (Towner and Cockayne, 1993; Ouoba et al., 2004).
Molecular identification
Molecular techniques such as polymerase chain reaction (PCR)-based, DNA fingerprinting and gene sequencing methods may be applied as tools for either species identification or differentiation of strains. The major advantages of these genotyping methods are based on their discriminatory power and their universal applicability. With molecular typing methods, closely related strains with similar phenotypic features may consistently be distinguished. Methods that have been used in the identification and genotypic characterization of Bacillus and related genera include internal transcribed spacer PCR (ITS-PCR) (Liu et al., 1997); random amplification polymorphic DNA-PCR (RAPD-PCR) (Daffonchio et al., 1998); repetitive sequence-based PCR (rep-PCR) (Da Silva et al., 1999); pulsed field gel electrophoresis (PFGE) (Yamada et al., 1999); restriction fragment length polymorphism (RFLP) analysis of rRNA operons as well as sequencing of 16S rRNA, gyrA, gyrB, and rpoB genes (Chun and Bae, 2000; Herman and Heyndrickx, 2000; Mendo et al., 2000; Thorsen et al., 2011). The 16S rRNA gene based taxonomy is a clear way forward for bacterial identification (Woese, 1987). Reclassification of several species of Bacillus based on 16S rRNA sequence alignment have been achieved such as the successful identification of B. subtilis and B. pumilus in several investigations (Wang et al., 2007). However, sequencing of the 16S rRNA gene for identification of Bacillus shows limitations in differentiating closely related species, therefore, used for basic classification for species delineation. Analysis based on pair wise alignment of 16S rRNA gene sequences showed limited variation in these closely related species of B. subtilis group (e.g. B. subtilis and B. amyloliquefaciens showed more than 99% similarities), which prevented the resolution of strains and species relationship. It was not possible to distinguish B. subtilis from B. licheniformis or B. cereus from B. thuringiensis by16S rRNA sequencing (Azokpota et al., 2007; Parkouda et al., 2010). Both groups have been reported too closely related that they could not easily be distinguished by sequencing the 16S rRNA (Daffonchio et al., 1998).
The RFLP analysis of rRNA operons has been reported to discriminate the species in the genus Bacillus except closely related members of B. cereus group (B. cereus, B. thuringiensis and B. mycoides) and the B. subtilis group (B. subtilis, B. amyloliquefaciens and B. licheniformis) (Daffonchio et al., 1998). It is very difficult to differentiate these closely related members because of very high sequence homology in the ribosomal operons. The 16S-23S rRNA gene internal transcribed spacer (ITS)-RFLP analysis also not differentiated B. subtilis, B. amyloliquefaciens and B. licheniformis (Daffonchio et al., 1998). Chun and Bae (2000) demonstrated that the different strains of Bacillus could show almost identical 16S rRNA sequences, but significantly low gyrA nucleotide (NT) similarities. They concluded that the method for the amplification and sequencing of partial gyrA genes may be useful for the rapid identification of B. subtilis and allied taxa, especially organisms in these taxa, which cannot be differentiated by using conventional phenotypic tests and 16S rRNA analysis (Chun and Bae, 2000). Additionally, sequencing of other genes, such as rpoB and gyrB were reported better discriminatory for improved identification and differentiation of closely related Bacillus species (De Clerck and De Vos, 2004). Nowadays taxonomy based on multi– locus sequence typing (MLST) of housekeeping genes have been reported as a promising tool for differentiating closely related Bacillus species.
Volatile compounds of dawadawa African fermented condiments
The widespread use of African condiments is due to their peculiar odour, pleasant taste and nutritional qualities (Owens et al., 1997; Azokpota et al., 2010; Ouoba et al., 2005). The formation of volatile compounds in alkaline fermentation has been attributed to the metabolic activities of Bacillus species during fermentation (Ouoba et al., 2005; Beaumont, 2002; Omafuvbe et al., 2000). The major role of Bacillus species involves hydrolyzing proteins to peptides, amino acids and releasing ammonia thereby creating an alkaline pH, which aids the inhibition of spoilage microorganisms (Allagheny et al., 1996; Parkouda et al., 2009). Conversely, proteolytic activities of microorganisms on legume proteins and utilization of free amino acids during fermentation lead to the formation of ammonia that gives characteristic pungent or ammoniacal flavour of these condiments (Azokpota et al., 2008; Leejeerajumnean et al., 2001; Owens et al., 1997). According to Beaumont (2002), the amino acid content of dawadawa, in particular glutamate contributes to the flavour enhancement, as well as peptides and aroma volatile constituents to be responsible for the flavour of the product.
1.0 INTRODUCTION AND PROBLEM STATEMENT
2.0 LITERATURE REVIEW
2.1 Legume–based alkaline fermented food condiments of the world
2.2 Production of legume-based alkaline fermented food condiments from Africa
2.3 Production process of dawadawa African food condiment
2.4 Microorganism associated with African food condiments
2.5 Bacillus species in African food condiments
2.6 Identification and taxonomic characteristics of Bacillus species
2.7 Volatile compounds of dawadawa African fermented condiments
2.8 Sensory attributes of dawadawa condiments
2.9 Conclusions
3.0 HYPOTHESIS AND OBJECTIVES
3.1 Hypothesis
3.2 Objectives
4.0 RESEARCH
4.1 Diversity and functionality of Bacillus species associated with alkaline fermentation of Bambara
groundnut (Vigna subterranean L. Verdc) into dawadawa–like African condiment
4.2 Characterisation of volatile compounds in a dawadawa–like African food condiment produced from Bambara groundnut (Vigna subterranean (L.) Verdc) using Bacillus species starter cultures
4.3 Sensory quality of dawadawa–like African food condiments produced from alkaline fermentation of an underutilized legume: Bambara groundnut (Vigna subterranea (L.) Verdc)
5.0 GENERAL DISCUSSION
5.1 Methodological considerations
5.2 Potential of dawadawa–like African food condiment production from Bambara groundnut using Bacillus starter cultures and transfer of technology to local communities (Commercialization)
5.3 Future research
6.0 CONCLUSIONS AND RECOMMENDATIONS
7.0 REFERENCES
8.0 PUBLICATIONS AND CONFERENCE PRESENTATIONS BASED ON THIS RESEARCH
