Assessing the effects of exposure to environmental stress on some functional properties of Bifidobacterium animalis ssp. lactis.

This study assessed the effects of exposing a strain of Bifidobacterium animalis ssp. lactis to acid, bile and osmotic stresses on antagonistic properties, biofilm formation and antibiotic susceptibility/resistance profile. Exposure to each stress factor appeared to have no significant effect on the antagonism against Escherichia coli NCTC 12900 and Salmonella enterica serovar Enteritidis PT4. No suppression in biofilm formation due to exposure to stress was observed. Bile and osmotic stresses resulted in significantly higher biofilm formation. Expression of an exopolysaccharide synthesis gene, gtf 01207, was significantly higher when the B. animalis ssp. lactis strain was exposed to osmotic stress. Susceptibility of the B. animalis ssp. lactis strain to chloramphenicol, erythromycin, ampicillin and vancomycin, and resistance to tetracycline remained unchanged when exposed to each stress. The expression of a tetracycline resistance gene, tet(W), was significantly higher when exposed to each stress. These results may suggest that the potential for the B. animalis ssp. lactis strain to provide probiotic benefit, after exposure to the stressful conditions of the gastrointestinal tract, remains intact.

Molecular identification and safety of Bacillus species involved in the fermentation of African oil beans (Pentaclethra macrophylla Benth) for production of Ugba.

Molecular identification of Bacillus spp. involved in the fermentation of African oil bean seeds for production of Ugba, as well as ability of the Bacillus spp. isolated to produce toxins, were investigated. Forty-nine bacteria were isolated from Ugba produced in different areas of South Eastern Nigeria and identified by phenotyping and sequencing of 16S rRNA, gyrB and rpoB genes. Genotypic diversities at interspecies and intraspecies level of the isolates were screened by PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS-PCR) and repetitive sequence-based PCR (rep-PCR). The ability of the bacteria to produce toxins was also investigated by detection of genes encoding production of haemolysin BL (HblA, HblC, HblD), non-haemolytic enterotoxin (NheA, NheB, NheC), cytotoxin K (CytK) and emetic toxin (EM1) using PCR with specific primers. Moreover, a Bacillus cereus Enterotoxin Reverse Passive Latex Agglutination test kit (BCET-RPLA) was used to screen ability of the isolates to produce haemolysin in broth and during fermentation of African oil bean seeds. The isolates were characterized as motile, rod-shaped, endospore forming, catalase positive, Gram-positive bacteria. They were identified as Bacillus cereus sensu lato (42), Lysinibacillus xylanilyticus (3), Bacillus clausii (1), Bacillus licheniformis (1), Bacillus subtilis (1), and Bacillus safensis (1). B. cereus was the predominant Bacillus species and was present in all samples studied. Using ITS-PCR, interspecies diversity was observed among isolates, with six clusters representing each of the pre-cited species. Rep-PCR was more discriminatory (eight clusters) and allowed further differentiation at intraspecies level for the B. cereus and L. xylanilyticus isolates with two genotypes for each species. Genes encoding production of non-haemolytic enterotoxin (NheA, NheB, NheC) and cytotoxin K (CytK) genes were detected in all B. cereus isolates, while Hbl genes (HblA, HblC, HblD) were detected in only one isolate. The emetic-specific gene fragment was not detected in any of the isolates studied. None of the toxin genes screened was detected in isolates belonging to other Bacillus species. Using RPLA, haemolysin production was detected in one isolate of B. cereus, which showed positive amplicons for Hbl genes, both during cultivation in broth and during fermentation of oil bean seeds.

The microbiology of Bandji, palm wine of Borassus akeassii from Burkina Faso: identification and genotypic diversity of yeasts, lactic acid and acetic acid bacteria.

AIM: To investigate physicochemical characteristics and especially genotypic diversity of the main culturable micro-organisms involved in fermentation of sap from Borassus akeassii, a newly identified palm tree from West Africa. METHODS AND RESULTS: Physicochemical characterization was performed using conventional methods. Identification of micro-organisms included phenotyping and sequencing of: 26S rRNA gene for yeasts, 16S rRNA and gyrB genes for lactic acid bacteria (LAB) and acetic acid bacteria (AAB). Interspecies and intraspecies genotypic diversities of the micro-organisms were screened respectively by amplification of the ITS1-5.8S rDNA-ITS2/16S-23S rDNA ITS regions and repetitive sequence-based PCR (rep-PCR). The physicochemical characteristics of samples were: pH: 3.48-4.12, titratable acidity: 1.67-3.50 mg KOH g(-1), acetic acid: 0.16-0.37%, alcohol content: 0.30-2.73%, sugars (degrees Brix): 2.70-8.50. Yeast included mainly Saccharomyces cerevisiae and species of the genera Arthroascus, Issatchenkia, Candida, Trichosporon, Hanseniaspora, Kodamaea, Schizosaccharomyces, Trigonopsis and Galactomyces. Lactobacillus plantarum was the predominant LAB species. Three other species of Lactobacillus were also identified as well as isolates of Leuconostoc mesenteroides, Fructobacillus durionis and Streptococcus mitis. Acetic acid bacteria included nine species of the genus Acetobacter with Acetobacter indonesiensis as predominant species. In addition, isolates of Gluconobacter oxydans and Gluconacetobacter saccharivorans were also identified. Intraspecies diversity was observed for some species of micro-organisms including four genotypes for Acet. indonesiensis, three for Candida tropicalis and Lactobacillus fermentum and two each for S. cerevisiae, Trichosporon asahii, Candida pararugosa and Acetobacter tropicalis. CONCLUSION: fermentation of palm sap from B. akeassii involved multi-yeast-LAB-AAB cultures at genus, species and intraspecies level. SIGNIFICANCE AND IMPACT OF THE STUDY: First study describing microbiological and physicochemical characteristics of palm wine from B. akeassii. Genotypic diversity of palm wine LAB and AAB not reported before is demonstrated and this constitutes valuable information for better understanding of the fermentation which can be used to improve the product quality and develop added value by-products.

Genotypic characterization of Brochothrix spp. isolated from meat, poultry and fish.

AIM: To study genotypic diversity of isolates of Brochothrix thermosphacta recovered from meat, poultry and fish. METHODS AND RESULTS: A total of 27 bacteria isolated from 19 samples of meat, poultry and fish were identified phenotypically and genotypically using PCR amplification of 16S-23S rDNA intergenic transcribed spacer (ITS-PCR), repetitive sequence-based PCR (rep-PCR) and 16S rDNA sequencing. Using ITS-PCR, all bacteria showed the same DNA profile as the reference strains of Br. thermosphacta, allowing typing of the isolates at species level. Using 16S rDNA sequencing, all isolates were identified, at genus and species level, as Br. thermosphacta. Identification as Br. campestris was observed with a lower, but very close, level of similarity. Rep-PCR was more discriminatory than ITS-PCR and allowed differentiation of four subgroups among the isolates. CONCLUSION: Minor genotypic differences among Br. thermosphacta strains from meat, poultry and fish were observed. SIGNIFICANCE AND IMPACT OF THE STUDY: A rudimentary exploration of genotypic differences of Br. thermosphacta from meat, poultry and fish resulted in preliminary confirmation of the suitability of ITS-PCR for typing Br. thermosphacta and confirmed the value of rep-PCR fingerprinting to discriminate between Br. thermosphacta strains.

Genotypic diversity of lactic acid bacteria isolated from African traditional alkaline-fermented foods.

AIM: To identify and compare lactic acid bacteria (LAB) isolated from alkaline fermentations of cassava (Manihot esculenta Crantz) leaves, roselle (Hibiscus sabdariffa) and African locust bean (Parkia biglobosa) seeds for production of, respectively, Ntoba Mbodi, Bikalga and Soumbala. METHODS AND RESULTS: A total of 121 LAB were isolated, identified and compared by phenotyping and genotyping using PCR amplification of 16S-23S rDNA intergenic transcribed spacer (ITS-PCR), repetitive sequence-based PCR (rep-PCR) and DNA sequencing. The results revealed a diversity of genera, species and subspecies of LAB in African alkaline fermentations. The isolates were characterized as nonmotile (in most cases) Gram-positive rods, cocci or coccobacilli, catalase and oxidase negative. ITS-PCR allowed typing mainly at species level, with differentiation of a few bacteria at subspecies level. Rep-PCR permitted typing at subspecies level and revealed significant genotypic differences between the same species of bacteria from different raw materials. DNA sequencing combined with use of API 50CHL and API 20Strep systems allowed identification of bacteria as Weissella confusa, Weissella cibaria, Lactobacillus plantarum, Pediococcus pentosaceus, Enterococcus casseliflavus, Enterococcus faecium, Enterococcus faecalis, Enterococcus avium and Enterococcus hirae from Ntoba Mbodi; Ent. faecium, Ent. hirae and Pediococcus acidilactici from Bikalga and Soumbala. CONCLUSION: LAB found in African alkaline-fermented foods belong to a range of genera, species and subspecies of bacteria and vary considerably according to raw material. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study confirms that LAB survive in alkaline fermentations, a first crucial stage in determining their significance and possible value as probiotic bacteria.

Identification of Bacillus spp. from Bikalga, fermented seeds of Hibiscus sabdariffa: phenotypic and genotypic characterization.

AIMS: To identify Bacillus spp. responsible of the fermentation of Hibiscus sabdariffa for production of Bikalga, an alkaline fermented food used as a condiment in Burkina Faso. METHODS AND RESULTS: Seventy bacteria were isolated from Bikalga produced in different regions of Burkina Faso and identified by phenotyping and genotyping using PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS-PCR), repetitive sequence-based PCR (rep-PCR) and DNA sequencing. The isolates were characterized as motile, rod-shaped, endospore forming, catalase positive, Gram-positive bacteria. ITS-PCR allowed typing mainly at species level. Rep-PCR was more discriminative and allowed a typing at ssp. level. The DNA sequencing combined with the Blast search program and fermentation profiles using API 50CHB system allowed an identification of the bacteria as Bacillus subtilis, B. licheniformis, B. cereus, B. pumilus, B. badius, Brevibacillus bortelensis, B. sphaericus and B. fusiformis. B. subtilis were the predominant bacterium (42) followed by B. licheniformis (16). CONCLUSIONS: Various species and ssp. of Bacillus are involved in fermentation of H. sabdariffa for production of Bikalga. SIGNIFICANCE AND IMPACT OF THE STUDY: Selection of starter cultures of Bacillus for controlled production of Bikalga, selection of probiotic bacteria.

Antimicrobial activity of Bacillus subtilis and Bacillus pumilus during the fermentation of African locust bean (Parkia biglobosa) for Soumbala production.

AIMS: To examine predominant isolates of Bacillus subtilis and B. pumilus isolated from Soumbala for their antimicrobial activity against indicator microorganisms as Micrococcus luteus, Staphyloccocus aureus, Bacillus cereus, Enterococus facium, Listeria monocytogenes, Escherichia coli, Salmonella typhimurium, Shigella dysenteriae, Yersinia enterocolitica, Aspergillus ochraceus and Penicillium roqueforti. METHODS AND RESULTS: Growth inhibition of indicator microorganisms by cells and supernatants of three B. subtilis and two B. pumilus strains was investigated using agar diffusion tests. Inactivation of indicator microorganisms was investigated in laboratory broth and during the fermentation of African locust bean for Soumbala production. The Bacillus isolates showed variable ability of inhibition and inactivation according to the indicator microorganism. The supernatants of pure cultures of B. subtilis inhibited one strain of B. cereus, one of Staph. aureus and E. coli and caused abnormal germination of Aspergillus ochraceus. The supernatant of mixed cultures of B. subtilis and indicators inhibited all the indicators. A treatment with protease eliminated the inhibitions. Isolates of B. subtilis inactivated all the indicators organisms during the fermentation of African locust bean as well as in laboratory broth with about five to eight decimal reduction. CONCLUSION: Bacillus isolates from Soumbala inhibit and inactivate Gram-positive and Gram-negative bacteria as well as ochratoxin A producing fungi during both laboratory cultivation and natural fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: Selection of starter cultures of Bacillus spp. for controlled production of Soumbala.

Volatile compounds of Soumbala, a fermented African locust bean (Parkia biglobosa) food condiment.

AIMS: To determine the profile of volatile compounds responsible for the aroma of Soumbala produced spontaneously and with pure and mixed cultures of Bacillus subtilis and Bacillus pumilus. METHODS AND RESULTS: Traditional and controlled fermentation trials of African locust bean with pure and mixed starter cultures of B. subtilis (B7, B9 and B15) and B. pumilus (B10) were performed. Aroma volatiles were analysed using Likens-Nikerson method coupled with gas chromatography and mass spectrophotometry. Sensory analysis of Soumbala as well as rice dishes prepared with each type of Soumbala were carried out by 10 panellists. In total 116 compounds were identified. They included pyrazines, aldehydes, ketones, esters, alcohols, acids, alkanes, alkenes, amines, pyridines, benzenes, phenols, sulphurs, furans and other compounds. Using principal component analysis for comparison, the aroma profiles of the Soumbala samples could be separated into three groups. The sensory evaluation showed variable acceptability. However, it was noticed that Soumbala samples produced with starter cultures were scored higher than traditionally prepared Soumbala. CONCLUSIONS: Aroma volatiles and organoleptic properties of Soumbala vary according to the Bacillus isolates involved in the fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the selection of Bacillus starter cultures for controlled production of Soumbala.

Degradation of African locust bean oil by Bacillus subtilis and Bacillus pumilus isolated from soumbala, a fermented African locust bean condiment.

AIMS: To investigate predominant isolates of Bacillus subtilis and B. pumilus in soumbala, a fermented African locust bean condiment, for their ability to degrade African locust bean oil (ALBO). METHODS AND RESULTS: Agar diffusion test in tributyrin and ALBO agar was used for screening of the isolates for esterase and lipase activity, respectively. The quantity and the profile of free fatty acids (FFA) during 72 h of degradation of ALBO by the Bacillus isolates were studied by titration and gas chromatography. The degradation of tributyrin and ALBO was variable among the isolates. Two strains of B. subtilis and two strains of B. pumilus showed significantly higher esterase and lipolytic activities than the others. The degradation ALBO was most pronounced in enriched nutrient agar except for one isolate of B. pumilus degrading ALBO to the same extent regardless of the enrichment. The quantity of FFA released from ALBO by the most lipolytic strains of Bacillus increased mainly between 0 and 24 h and differed among the isolates. The profile of FFA was similar for the Bacillus isolates with oleic acid (C18:2) occurring as the major FFA in all the samples except in samples incubated with B. subtilis B9 where stearic acid (C18) was dominant. CONCLUSION: Bacillus isolates from soumbala showed high strain dependent lipolytic activity against ALBO. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the selection of Bacillus strains to be used as starter cultures for controlled production of soumbala.

Degradation of proteins during the fermentation of African locust bean (Parkia biglobosa) by strains of Bacillus subtilis and Bacillus pumilus for production of Soumbala.

AIMS: To examine isolates of Bacillus subtilis and B. pumilus predominant in Soumbala for their ability to degrade African locust bean proteins (ALBP). METHODS AND RESULTS: Agar diffusion test in casein and ALBP agar was used for screening of isolates. The profiles of water-soluble proteins and free amino acids (FAA) during the fermentation of ALBP by the Bacillus isolates were studied by SDS-PAGE and cation exchange chromatography. The profile of soluble proteins changed with the fermentation time and varied depending on the isolate. The quantity of total FAA and essential FAA such as lysine was increased sharply between 24 and 48 h of fermentation and differed among the isolates. Simultaneously, a pH increase was observed. Cysteine, methionine, leucine, isoleucine, tyrosine and phenylalaline appeared during fermentation. CONCLUSION: The Bacillus isolates studied degraded ALBP leading to a profile of soluble proteins and FAA specific for each isolate. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the selection of Bacillus strains to be used as starter cultures for controlled production of Soumbala.