L.I.I. Ouoba

Characterization of Lactic Acid Bacteria Isolated from Italian Bella di Cerignola Table Olives: Selection of Potential Multifunctional Starter Cultures

Lactic acid bacteria (19 isolates) from Bella di Cerignola Italian table olives were investigated for their technological and probiotic properties for the selection of multifunctional starter cultures for table olives. The bacteria were first identified by phenotyping and genotyping, then characterized for the production of biogenic amines, growth at different pH, NaCl concentrations, and temperatures. The potentiality of the bacteria to have some probiotic properties (antimicrobial activity against foodborne pathogens, survival in low pH and in the presence of bile salts, ability to adhere to the mammalian cells model IPEC-J2) was also investigated. Eighteen of the studied isolates were identified as Lactobacillus plantarum and one as Enterococcus faecalis. All bacteria were able to grow at a range of pH between 4.0 and 10.0 as well as in media supplemented with 2.5 to 7.5% of NaCl and 0.3% bile salts and survived in MRS broth acidified at pH 2.5; moreover, they inhibited significantly Escherichia coli O157:H7. The adhesion to IPEC-J2 cells was in general low to moderate (5.3 to 8.3%); however, 2 isolates of L. plantarum (c16 and c19) showed interesting higher adhesion values (up to 16%). Our results suggest that at least 3 isolates could be possible multifunctional starters for Bella di Cerignola olives: L. plantarum 16 and 19 for mainly their probiotic properties and L. plantarum 10 for mainly its technological characteristics. Practical Application: A functional starter is a microorganism exerting benefits on human health (probiotic) and able to guide a fermentation (starter). The main goal of this article was to select a functional starter for table olives.

Enterotoxins and emetic toxins production by Bacillus cereus and other species of Bacillus isolated from Soumbala and Bikalga, African alkaline fermented food condiments

The ability of various species of Bacillus from fermented seeds of Parkia biglobosa known as African locust bean (Soumbala) and fermented seeds of Hibiscus sabdariffa (Bikalga) was investigated. The study included screening of the isolates by haemolysis on blood agar, detection of toxins in broth and during the fermentation of African locust bean using the Bacillus cereus Enterotoxin Reverse Passive Latex Agglutination test kit (BCET-RPLA) and the Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (BDEVIA). Detection of genes encoding cytotoxin K (CytK), haemolysin BL (Hbl A, Hbl C, Hbl D), non-hemolytic enterotoxin (NheA, NheB, NheC) and EM1 specific of emetic toxin producers was also investigated using PCR with single pair and multiplex primers. Of 41 isolates, 29 Bacillus belonging to the species of B. cereus, Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus showed haemolysis on blood agar. Using RPLA, enterotoxin production was detected for three isolates of B. cereus in broth and all B. cereus (9) in fermented seeds. Using BDEVIA, enterotoxin production was detected in broth as well as in fermented seeds for all B. cereus isolates. None of the isolates belonging to the other Bacillus species was able to produce enterotoxins either by RPLA or BDEVIA. Nhe genes were detected in all B. cereus while Hbl and CytK genes were detected respectively in five and six B. cereus strains. A weak presence of Hbl (A, D) and CytK genes was detected in two isolates of B. subtilis and one of B. licheniformis but results were inconsistent, especially for Hbl genes. The emetic specific gene fragment EM1 was not detected in any of the isolates studied.

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.

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.

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

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.

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.

Use of molecular methods to characterize the bacterial community and to monitor different native starter cultures throughout the ripening of Galician chorizo.

The development of Lactobacillus and Staphylococcus strains used as starter cultures throughout the ripening of Galician chorizo, a traditional dry fermented sausage from the north-west of Spain, was monitored combining different molecular-based techniques. The bacterial diversity occurring in the inoculated sausages at the beginning and the end of the ripening was also studied and compared to the indigenous population in an uninoculated control batch. Real-time PCR was used to monitor the Lactobacillus and Staphylococcus community using genus and species-specific primer to quantify the occurring microbiota. The identification of isolates at genus or species level was achieved by specific PCR and 16S rRNA gene sequencing. rep-PCR using (GTG)5-PCR primer was used to characterize this bacterial community at strain level. According to the data obtained, the strains Lactobacillus sakei LS131, Staphylococcus equorum SA25 and Staphylococcus saprophyticus SB12 were dominant during the ripening process, whereas the strain Staphylococcus epidermidis SA49, that was added in order to study its behaviour with a merely scientific purpose, did not succeed in dominating ripening, since it seemed to be outcompeted by autochthonous microbiota. In conclusion, the combination of a quantitative method such as real-time PCR with the identification and typing techniques used in this study (genus and species-specific PCR, 16S rRNA gene sequencing and (GTG)5-PCR) provided accurate and complete information about the starter cultures development, assessing their growth and survival over the ripening process.

Response mechanisms of lactic acid bacteria to alkaline environments: A review

Regulation of the cytoplasmic or internal pH (pHin) is a fundamental requirement for the survival and viability of bacteria. The optimum pHin for most bacteria is near the neutral point (pH 7.0). Therefore, bacteria may have some strategies to adapt themselves to the acidity or alkalinity of cytoplasm. As other microorganisms, lactic acid bacteria (LAB) are able to maintain a neutral or near neutral cytoplasmic pH even when the pH of the external medium varies. Mechanisms facilitating survival and growth under alkaline conditions of LAB are reviewed. These mechanisms are: (i) the active potassium extrusion and the potassium-proton antiport system, (ii) the sodium-proton antiport system, (iii) the proton-translocating adenosine triphosphatase (ATPase), (iv) the formation of transmembrane proton gradients (ΔpH) in a reversed direction, and (v) the adaptation, cross-protection, and changes in protein synthesis.

Lysinibacillus louembei sp. nov., a spore-forming bacterium isolated from Ntoba Mbodi, alkaline fermented leaves of cassava from the Republic of the Congo

Investigation of the microbial diversity of Ntoba Mbodi, an African food made from the alkaline fermentation of cassava leaves, revealed the presence of a Gram-positive, catalase-positive, aerobic, motile and rod-shaped endospore-forming bacterium (NM73) with unusual phenotypic and genotypic characteristics. The analysis of the 16S rRNA gene sequence revealed that the isolate was most closely related to Lysinibacillus meyeri WS 4626T (98.93%), Lysinibacillus xylanilyticus XDB9T (96.95%) and Lysinibacillus odysseyi 34hs-1T (96.94%). The DNA-DNA relatedness of the isolate with L. meyeri LMG 26643T, L. xylanilyticus DSM 23493T and L. odysseyi DSM 18869T was 41%, 16% and 15%, respectively. The internal transcribed spacer-PCR profile of the isolate was different from those of closely related bacteria. The cell-wall peptidoglycan type was A4α, L-Lys-D-Asp and the major fatty acids were iso-C15:0, anteiso-C15:0, anteiso-C17:0 and iso-C17:0 and iso-C17:1ω10c. The polar lipids included phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphoaminolipid, aminolipid, two phospholipids and two unknown lipids. The predominant menaquinones were MK-7 and MK-6. Ribose was the only whole-cell sugar detected. The DNA G+C content was 38 mol%. Based on the results of the phenotypic and genotypic characterization, it was concluded that the isolate represents a novel species of the genus Lysinibacillus, for which the name of Lysinibacillus louembei sp. nov. is proposed. NM73T ( = DSM 25583T = LMG 26837T) represents the type strain.

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.