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 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.     

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 B last 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.     

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.     

The role of Bacillus species in the fermentation of cassava

Cassava dough inoculum is added to grated cassava in order to achieve a modification of texture during fermentation into the fermented cassava meal, agbelima. The microflora of two different types of inocula and subsequently inoculated cassava mash at 0, 24 and 48 h of fermentation were examined in order to determine the mechanism responsible for the breakdown of cassava tissue. Bacillus spp. occurred in high numbers, 10₇–10₈ cfu g_-1, in both types of inocula and persisted throughout the cassava dough fermentation. Bacillus spp. found were B. subtilis , B. mycoides , B. pumilus , B. cereus , B. amyloliquefaciens and B. licheniformis , with B. subtilis accounting for more than half of Bacillus isolates. All Bacillus isolates produced a wide spectrum of enzymes and showed similar enzymatic activities but only B. pumilus , B. licheniformis and B. amyloliquefaciens produced linamarase. Some isolates produced the tissue degrading enzymes polygalacturonase and pectin esterase and nearly all isolates hydrolysed starch. All isolates showed cellulase activity and were able to disintegrate cassava tissue. When cassava pieces were incubated in amylase, cellulase, pectin esterase and polygalacturonase solutions, only pieces in cellulase solution were dissolved revealing that the breakdown of cassava dough texture during fermentation with the inocula examined is brought about by Bacillus spp. through cellulase activity.     

A DNA microarray facilitates the diagnosis of Bacillus anthracis in environmental samples

Aims:  In order to improve the diagnosis of Bacillus anthracis in environmental samples, we established a DNA microarray based on the ArrayTube technology of Clondiag.
Methods and Results:  Total DNA of a bacterial colony is randomly biotinylated and hybridized to the array. The probes on the array target the virulence genes, the genomic marker gene rpoB , as well as the selective 16S rDNA sequence regions of B. anthracis , of the Bacillus cereus group and of Bacillus subtilis . Eight B. anthracis reference strains were tested and correctly identified. Among the analysed environmental Bacillus isolates, no virulent B. anthracis strain was detected.
Conclusions:  This array clearly differentiates B. anthracis from members of the B. cereus group and other Bacillus species in environmental samples by chromosomal ( rpoB ) and plasmid markers. Additionally, recognition of B. cereus strains harbouring the toxin genes or atypical B. anthracis strains that have lost the virulence plasmids is feasible.
Significance and Impact of the Study:  The array is applicable to the complex diagnostics for B. anthracis detection in environmental samples. Because of low costs, high security and easy handling, the microarray is applicable to routine diagnostics.     

Characterization of Bacillus probiotics available for human use

Bacillus species (Bacillus cereus, Bacillus clausii, Bacillus pumilus) carried in five commercial probiotic products consisting of bacterial spores were characterized for potential attributes (colonization, immunostimulation, and antimicrobial activity) that could account for their claimed probiotic properties. Three B. cereus strains were shown to persist in the mouse gastrointestinal tract for up to 18 days postadministration, demonstrating that these organisms have some ability to colonize. Spores of one B. cereus strain were extremely sensitive to simulated gastric conditions and simulated intestinal fluids. Spores of all strains were immunogenic when they were given orally to mice, but the B. pumilus strain was found to generate particularly high anti-spore immunoglobulin G titers. Spores of B. pumilus and of a laboratory strain of B. subtilis were found to induce the proinflammatory cytokine interleukin-6 in a cultured macrophage cell line, and in vivo, spores of B. pumilus and B. subtilis induced the proinflammatory cytokine tumor necrosis factor alpha and the Th1 cytokine gamma interferon. The B. pumilus strain and one B. cereus strain (B. cereus var. vietnami) were found to produce a bacteriocin-like activity against other Bacillus species. The results that provided evidence of colonization, immunostimulation, and antimicrobial activity support the hypothesis that the organisms have a potential probiotic effect. However, the three B. cereus strains were also found to produce the Hbl and Nhe enterotoxins, which makes them unsafe for human use.     

Phylogeny of gamma-polyglutamic acid-producing Bacillus strains isolated from a fermented locust bean product manufactured in West Africa

Twenty-five Bacillus strains capable of producing gamma-polyglutamic acid (PGA) were isolated from fermented locust bean products manufactured in the savanna area of Ghana. To clarify the phylogeny of these PGA-producing strains, phylogenetic analyses based on sequences of 16S rDNA, rpoB (RNA polymerase beta-subunit) and fus (elongation factor G) genes were performed. A phylogenetic tree based on 16S rDNA indicated that ten isolates were clustered in the same group of Bacillus subtilis. Another ten isolates were located in the cluster of B. amyloliquefaciens, and the remaining isolates were identified as B. pumilus (three isolates) and B. licheniformis (two isolates), respectively. Phylogenetic trees based on the partial sequences of rpoB and fus genes were similar to the phylogeny based on 16S rDNA sequences. Thirty-four strains in 27 species belonging to the genus Bacillus and its neighbors were also investigated for PGA production. It was found that PGA was produced by B. amyloliquefaciens NBRC 14141 and NBRC 15535(T), B. atrophaeus NBRC 15539(T), B. licheniformis NBRC 12107, B. mojavensis NBRC 15718(T), B. pumilus NBRC 12094, B. subtilis NBRC 16449, and Lysinibacillus sphaericus NBRC 3525. Except for L. sphaericus, the above Bacillus species are very closely related in phylogeny, indicating that PGA-producing Bacillus strains constitute a cluster.     

The combined effects of high pressure and nisin on germination and inactivation of Bacillus spores in milk

Aims:  The aim of this work was to investigate the germination and inactivation of spores of Bacillus species in buffer and milk subjected to high pressure (HP) and nisin.
Methods and Results:  Spores of Bacillus subtilis and Bacillus cereus suspended in milk or buffer were treated at 100 or 500 MPa at 40°C with or without 500 IU ml⁻¹ of nisin. Treatment at 500 MPa resulted in high levels of germination (4 log units) of B. subtilis spores in both milk and buffer; this increased to >6 logs by applying a second cycle of pressure. Viability of B. subtilis spores in milk and buffer was reduced by 2·5 logs by cycled HP, while the addition of nisin (500 IU ml⁻¹) prior to HP treatment resulted in log reductions of 5·7 and 5·9 in phosphate buffered saline and milk, respectively. Physical damage of spores of B. subtilis following HP was apparent using scanning electron microscopy. Treating four strains of B. cereus at 500 MPa for 5 min twice at 40°C in the presence of 500 IU ml⁻¹ nisin proved less effective at inactivating the spores of these isolates compared with B. subtilis and some strain-to-strain variability was observed.
Conclusions:  Although high levels of germination of Bacillus spores could be achieved by combining HP and nisin, complete inactivation was not achieved using the aforementioned treatments.
Significance and Impact of the Study:  Combinations of HP treatment and nisin may be an appealing alternative to heat pasteurization of milk.     

Inhibitory effects of Bacillus probionts on growth and toxin production of Vibrio harveyi pathogens of shrimp

Aim:  To investigate the effects of Bacillus subtilis , Bacillus licheniformis and Bacillus megaterium in terms of toxin and growth of pathogenic Vibrio harveyi .
Methods and Results:  Three Bacillus probionts were isolated from probiotic BZT aquaculture and identified using a 16S rDNA sequence. Growth inhibition assay showed that supernatants from the 24-h culture of three Bacillus species were able to inhibit the growth of V. harveyi (LMG 4044); B. subtilis was the most effective based on the well diffusion method. Results of a liquid culture model showed that B. subtilis was also widely effective in inhibiting three strains of V. harveyi (isolated from Thailand, the Philippines and LMG 4044), and that both B. licheniformis and B. megaterium inhibit the growth of V. harveyi isolated from the Philippines. Moreover, a haemolytic activity assay demonstrated that V. harveyi (IFO 15634) was significantly decreased by the addition of B. licheniformis or B. megaterium supernatant.
Conclusions:  Bacillus subtilis inhibited Vibrio growth, and both B. licheniformis and B. megaterium suppressed haemolytic activity in Vibrio .
Significance and Impact of the Study:  The cell-free supernatants produced by Bacillus probionts inhibit Vibrio disease, and Bacillus probionts might have an influence on Vibrio cell-to-cell communications.     

Genotyping and toxigenic potential of Bacillus subtilis and Bacillus pumilus strains occurring in industrial and artisanal cured sausages

Artisanal and industrial sausages were analyzed for their aerobic, heat-resistant microflora to assess whether new emerging pathogens could be present among Bacillus strains naturally contaminating cured meat products. Sixty-four isolates were characterized by randomly amplified polymorphic DNA (RAPD)-PCR and fluorescent amplified fragment length polymorphism (fAFLP). The biotypes, identified by partial 16S rRNA gene sequence analysis, belonged to Bacillus subtilis, Bacillus pumilus, and Bacillus amyloliquefaciens species. Both RAPD-PCR and fAFLP analyses demonstrated that a high genetic heterogeneity is present in the B. subtilis group even in strains harvested from the same source, making it possible to isolate 56 different biotypes. Moreover, fAFLP analysis made it possible to distinguish B. subtilis from B. pumilus strains. The strains were characterized for their toxigenic potential by molecular, physiological, and immunological techniques. Specific PCR analyses revealed the absence of DNA sequences related to HBL, BcET, NHE, and entFM Bacillus cereus enterotoxins and the enzymes sphingomyelinase Sph and phospholipase PI-PLC in all strains; also, the immunological analyses showed that Bacillus strains did not react with NHE- and HBL-specific antibodies. However, some isolates were found to be positive for hemolytic and lecithinase activity. The absence of toxigenic potential in Bacillus strains from the sausages analyzed indicates that these products can be considered safe under the processing conditions they were produced; however, great care should be taken when the ripening time is shortened, particularly in the case of traditional sausages, which could contain high amounts of Bacillus strains and possibly some B. cereus cells.     

Sigma 29-like protein is a common sporulation-specific element in bacteria of the genus Bacillus.

A monoclonal antibody specific for an antigenic determinant on the Bacillus subtilis sporulation-induced sigma factor sigma 29 reacted with proteins similar in size to sigma 29 in extracts of sporulating Bacillus licheniformis, Bacillus amyloliquifaciens, Bacillus cereus, Bacillus natto, and Bacillus pumilus but not in extracts prepared from vegetatively growing cultures of these bacteria. These results indicate that RNA polymerase modifications, initially described for B. subtilis, are likely to be common among sporulating Bacillus spp. and that at least some of the specific modifications that are observed in sporulating B. subtilis are conserved among members of this genus.     

Determining the source of Bacillus cereus and Bacillus licheniformis isolated from raw milk, pasteurized milk and yoghurt

Aims:  Strain-specific detection of Bacillus cereus and Bacillus licheniformi s in raw and pasteurized milk, and yoghurt during processing.
Methods and Results:  Randomly selected isolates of Bacillus spp. were subjected to PCR analysis, where single primer targeting to the repetitive sequence Box elements was used to fingerprint the species. The isolates were separated into six different fingerprint patterns. The results show that isolates clustered together at about the 57% similarity level with two main groups at the 82% and 83% similarity levels, respectively. Contamination with identical strains both of B. cereus and B . licheniformis in raw and pasteurized milk was found as well as contaminated with different strains (in the case of raw milk and yoghurt/pasteurized milk and yoghurt). Several BOX types traced in processed milk samples were not discovered in the original raw milk.
Conclusions:  BOX-PCR fingerprinting is useful for characterizing Bacillus populations in a dairy environment. It can be used to confirm environmental contamination, eventually clonal transfer of Bacillus strains during the technological processing of milk.
Significance and Impact of the Study:  Despite the limited number of strains analysed, the two Bacillus species yielded adequately detectable banding profiles, permitting differentiation of bacteria at the strain level and showing their diversity throughout dairy processing.     

A new strategy to apply Bacillus subtilis MA139 for the production of solid-state fermentation feed

Aim:  The study investigated the potential of using Bacillus subtilis MA139 in combination with Lactobacillus fermentum and Saccharomyces cerevisae to produce solid-state fermentation feed.
Methods and Results:  In a pure fermentation, B. subtilis MA139 was able to grow and synthesize antimicrobial substances at temperatures from 25 to 37°C and at a pH from 5·0 to 9·0. Subsequently, B. subtilis MA139, Lact. fermentum and S. cerevisae were used as starter strains co-inoculated in unsterilized substrate (feed-grade soybean meal and wheat bran). Following 10 days of fermentation in a newly developed plastic bag equipped with a one-way valve, lactic acid bacteria and Bacillus became the predominant strains while S. cerevisae cells decreased slightly. Enterobacteriaceae ( Escherichia coli K88 and Salmonella typhimurium ) were not detected.
Conclusions:  Use of B. subtilis MA139 as a starter strain co-inoculated with S. cerevisae and Lact. fermentum successfully controlled the growth of enterobacteriaceae.
Significance and Impact of the Study:  This study provided a facile and low-cost way to produce solid-state fermentation feed.     

Fructose and glucose mediates enterotoxin production and anaerobic metabolism of Bacillus cereus ATCC14579T

Aims:  To determine the effects of carbohydrates on Bacillus cereus ATCC14579^T anaerobic metabolism and enterotoxin production in amino acids rich medium.
Methods and Results:  Bacillus cereus anaerobic growth on different carbohydrates (glucose, fructose, sucrose or glucose–fructose mixture) was examined in synthetic mMOD medium under continuous cultures (μ = 0·2 h⁻¹). Fermentation end-products, flux partitioning at each key branch points of the mixed acid pathway and consumption or production of amino acids were determined. On both fructose and sucrose, ATP production was favoured via acetate production from acetyl-CoA. In addition, amino acids present in the growth medium showed significant variations with high consumption of serine and net production of glutamate and alanine on some or all sugars. Enterotoxins Hbl and Nhe production was high during growth on fructose (or mixtures involving a fructose moiety).
Conclusions:  Fructose was identified as a key sugar influencing anaerobic metabolism and toxin production of B. cereus .
Significance and Impact of the Study:  The physiological differences associated with the fermentation of the various carbohydrates clearly modify toxinogenesis indicating that the risk of foodborne pathogens is to some extent dependent upon the prevailing nutritional environment.     

The use of Bacillus diarrhoeal enterotoxin (BDE) detection using an ELISA technique in the confirmation of the aetiology of Bacillus-mediated diarrhoea

A commercially available ELISA kit was used for the detection of Bacillus diarrhoeal enterotoxin (BDE) in a variety of foods and faeces. The ability of isolates of Bacillus spp., including Bacillus cereus , to produce BDE in Brain Heart Infusion broth containing 0·1% glucose was also checked by use of the kit. Results show that 29 out of 31 B. cereus isolates were enterotoxigenic. Foods positive for preformed BDE were always contaminated with >10⁵ B. cereus cfu g⁻¹, but not all foods contaminated with large numbers of B. cereus were positive for BDE. Bacillus spp., other than one isolate which closely resembled B. subtilis , were negative for BDE production. Criteria for the confirmation of Bacillus -mediated diarrhoea should now include reports of symptoms and incubation periods consistent with the diarrhoeal form of food-poisoning by Bacillus spp., together with the results of tests for enterotoxigenicity of the Bacillus isolate, and detection of BDE in either the food and/or faeces.     

Evaluation of different methods to discriminate Bacillus anthracis from other bacteria of the Bacillus cereus group

Aims:  To evaluate different methods that are useful for rapid and definitive discrimination of Bacillus anthracis from other bacteria of the Bacillus cereus group in environmental samples like letters claimed to contain anthrax spores.
Methods and Results:  Characterized strains and bacteria from environmental samples were analysed by microbiological and molecular methods (PCR and restriction analysis). Environmental isolates often shared several microbiological features with B. anthracis , e.g. lack of β -haemolysis and phospholipase C activity, and only the gamma phage assay was specific for B. anthracis . PCR assays targeting markers from the virulence plasmids exclusively detected B. anthracis , but other PCR targets were also detected in nonanthrax isolates. Additionally, the restriction pattern in an Alu I restriction analysis of the SG-749 fragment is not 100% specific. The loci used for multiple-locus variable-number tandem repeat analysis of B. anthracis are also present in other members of the B. cereus group, but amplicon sizes are usually different.
Conclusions:  Environmental samples often contain borderline isolates closely related to B. anthracis both on microbiological and genetic levels. Real-time PCR targeting plasmidal and chromosomal markers should be used for rapid and definitive exclusion of a virulent strain of B. anthracis in such samples.
Significance and Impact of the Study:  This study gives an overview of the current microbiological and molecular methods used for identification of B. anthracis and shows that most assays have limits when borderline isolates present in environmental samples are analysed.     

Hemicellulases of Bacillus species: preliminary comparative studies on production and properties of mannanases and galactanases

A range of Bacillus subtilis strains and other Bacillus species were screened for mannanase, β-mannosidase and galactanase activities. Maximum mannanase activity, 106.2 units/ml, was produced by B. subtilis NRRL 356. β-Mannosidase and galactanase activities from all strains were relatively low. The effect of carbon and nitrogen source on mannanase and galactanase production by B. brevis ATCC 8186, B. licheniformis ATCC 27811, B. polymyxa NRRL 842 and B. subtilis NRRL 356 was investigated. Highest mannanase production was observed in the four strains tested when the mannan substrate, locust bean gum, was used as carbon source. Induction was most dramatic in the case of B. subtilis NRRL 356 where only basal enzyme levels were produced in the presence of other carbon sources. β-Mannosidase was induced in the four Bacillus cultures by locust bean gum. Results indicated that galactose acted as an inducer for production of galactanase. Organic and inorganic nitrogen sources resulted in induction of high mannanase titres in B. subtilis. Highest galactanase activity was produced by each organism in media containing sodium nitrate as nitrogen source. Mannanases from B. brevis, B. licheniformis, B. polymyxa and B. subtilis retained 100% residual activity after a 3 h incubation at 65°C, 65°C, 60°C and 55°C respectively. Galactanases retained more than 95% activity at 55°C after 3 h. The pH optima of mannanases ranged from 6.5–6.8 whereas galactanases ranged from 5.1 in the case of B. brevis to 7.0 for B. polymyxa.     

Degumming and characterization of ramie fibre using pectate lyase from immobilized Bacillus pumilus DKS1

Aims:  The present study was aimed at finding the optimal conditions for the production of pectate lyase using immobilized Bacillus pumilus DKS1 cells in calcium-alginate (Ca-alginate) beads and determining the efficient degumming of ramie fibre.
Methods and Results:  The active cells of B. pumilus DKS1 were immobilized in Ca-alginate and used for the production of pectate lyase. The production of enzyme increased significantly with increasing alginate concentration and reached a maximum enzyme yield of 38·5 U ml⁻¹ at 18 g l⁻¹. This was about 1·5-fold higher than that obtained by free cells. Degummed fibre using immobilized cells showed better tenacity than that prepared by using nonimmobilized cells.
Conclusions:  The Ca-alginate entrapment is a promising immobilization method of B. pumilus DKS1 for semicontinuous enzyme production. Enzyme production by immobilized cells is superior to that of free cells because it leads to higher volumetric activities within the same period of fermentation. Fibre degumming by using immobilized cells produced better quality fibre.
Significance and Impact of the Study:  This is the first report of degumming of fibre using enzyme from immobilized B. pumilus cells as per our knowledge. High-quality degummed fibre could be prepared with relatively inexpensive inputs for use in the textile and paper industry.