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Kantong is a condiment produced in Ghana by the spontaneous fermentation of kapok tree (Ceiba pentandra) seeds with cassava flour as an additive. Fermentation is over a 48h period followed by a drying and a kneading process. Although lactic acid bacteria (LAB) have previously been identified other micro-organisms may also be involved in the fermentation process. In this study we examined the occurrence of aerobic endospore-forming bacteria (AEB) in raw materials, during fermentation and in the final product at 2 production sites in Northern Ghana. Total aerobic mesophilic bacterial counts increased from 5.4±0.1log10CFU/g in the raw materials to 8.9±0.1log10CFU/g in the final products, with the AEB accounting for between 23% and 80% of the total aerobic mesophilic (TAM) counts. A total of 196 AEB were identified at a species/subspecies level by the use of phenotypic tests and genotypic methods including M13-PCR typing, 16S rRNA and gyrA gene sequencing. Bacillus subtilis subsp. subtilis (63% of the AEB), Bacillus safensis (26% of the AEB) and Bacillus amyloliquefaciens subsp. plantarum/Bacillus methylotrophicus (9% of the AEB) were the predominant Bacillus species during fermentation and in the final products. B. amyloliquefaciens/B. methylotrophicus originated from cassava flour, B. safensis from seeds and cassava flour, while the origin of B. subtilis was less clear. Brevibacillus agri and Peanibacillus spp. occurred sporadically. Further investigations are required to elucidate the role of AEB occurring in high numbers, in the fermentation of Kantong., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Bacillus spp. are commonly used as probiotic species in the feed industry, however, their benefits need to be confirmed. This study describes a high throughput screening combined with the detailed characterization of endospore-forming bacteria with the aim to identify new Bacillus spp. strains for use as probiotic additives in pig feed. A total of 245 bacterial isolates derived from African fermented food, feces and soil were identified by 16S rRNA gene sequencing and screened for antimicrobial activity and growth in the presence of antibiotics, bile salts and at pH 4.0. Thirty-three Bacillus spp. isolates with the best characteristics were identified by gyrB and rpoB gene sequencing as B. amyloliquefaciens subsp. plantarum, B. amyloliquefaciens subsp. amyloliquefaciens, B. subtilis subsp. subtilis, B. licheniformis, B. mojavensis, B. pumilus and B. megaterium. These isolates were further investigated for their activity against the pathogenic bacteria, antibiotic susceptibility, sporulation rates, biofilm formation and production of glycosyl hydrolytic enzymes. Additionally, ten selected isolates were assessed for heat resistance of spores and the effect on porcine epithelial cells IPEC-J2. Isolates of B. amyloliquefaciens, B. subtilis and B. mojavensis, showed the best overall characteristics and, therefore, potential for usage as probiotic additives in feed. A large number of taxonomically diverse strains made it possible to reveal species and subspecies-specific trends, contributing to our understanding of the probiotic potential of Bacillus species.

Aims: To identify and screen dominant Bacillus spp. strains isolated from Bikalga, fermented seeds of Hibiscus sabdariffa for their antimicrobial activities in brain heart infusion (BHI) medium and in a H. sabdariffa seed-based medium. Further, to characterize the antimicrobial substances produced., Methods and Results: The strains were identified by gyrB gene sequencing and phenotypic tests as B. amyloliquefaciens ssp. plantarum. Their antimicrobial activity was determined by the agar spot and well assay, being inhibitory to a wide range of Gram-positive and Gram-negative pathogenic bacteria and fungi. Antimicrobial activity against Bacillus cereus was produced in H. sabdariffa seed-based medium. PCR results revealed that the isolates have potential for the lipopeptides iturin, fengycin, surfactin, the polyketides difficidin, macrolactin, bacillaene and the dipeptide bacilysin production. Ultra-high-performance liquid chromatography-time of flight mass spectrometry analysis of antimicrobial substance produced in BHI broth allowed identification of iturin, fengycin and surfactin., Conclusions: The Bacillus amyloliquefaciens ssp. plantarum exhibited broad-spectrum antifungal and antibacterial properties. They produced several lipopeptide antibiotics and showed good potential for biological control of Bikalga., Significance and Impact of the Study: Pathogenic bacteria often occur in spontaneous food fermentations. This is the first report to identify indigenous B. amyloliquefaciens ssp. plantarum strains as potential protective starter cultures for safeguarding Bikalga., (Journal of Applied Microbiology © 2013 The Society for Applied Microbiology.)

Cyanogenic food crops abound in nature with important crops like cassava forming the staple food for over half a billion people. Detoxification by hydrolysis of cassava cyanogenic glycosides often involves acid fermentation, and in some of these processes Bacillus species are encountered. Forty Bacillus spp. (20 Bacillus subtilis, 11 Bacillus licheniformis, 7 Bacillus sonorensis, 2 Bacillus cereus) isolated from acid fermented primary starters to produce Gergoush, a Sudanese fermented snack, were screened for their ability to grow and to hydrolyze linamarin, the major cyanogen found in cassava at pH levels below 5.0; also the cyanogen amygdalin was assessed. The B. subtilis isolates grew in both HCl and lactic acid environments from pH 4.5-6.0 while being able to break down the cyanogenic glycosides. The B. licheniformis and B. sonorensis isolates grew and degraded cyanogens at pH 5.0 in a HCl environment, while two B. cereus isolates used in the study showed no breakdown reaction under all conditions tested. One B. subtilis isolate was observed to have substrate specificity between the breakdown of linamarin and amygdalin. We conclude that some Bacillus spp. isolates are important in the microbiological breakdown of cyanogens in cassava fermentations even at pH 4.5-5.0 though further investigations are required., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Bacillus spp. are widely used as feed additives and probiotics. However, there is limited information on their resistance to various antibiotics, and there is a growing concern over the transfer of antibiotic resistance genes. The MIC for 8 antibiotics was determined for 85 Bacillus species strains, Bacillus subtilis subsp. subtilis (n = 29), Bacillus licheniformis (n = 38), and Bacillus sonorensis (n = 18), all of which were isolated from starters for Sudanese bread production. All the strains were sensitive to tetracycline (8.0 mg/liter), vancomycin (4.0 mg/liter), and gentamicin (4.0 mg/liter) but resistant to streptomycin. Sensitivity to clindamycin, chloramphenicol, and kanamycin was species specific. The erythromycin resistance genes ermD and ermK were detected by PCR in all of the erythromycin-resistant (MIC, ≥16.0 mg/liter) B. licheniformis strains and one erythromycin-sensitive (MIC, 4.0 mg/liter) B. licheniformis strain. Several amino acid changes were present in the translated ermD and ermK nucleotide sequences of the erythromycin-sensitive strain, which could indicate ErmD and ErmK protein functionalities different from those of the resistance strains. The ermD and ermK genes were localized on an 11.4-kbp plasmid. All of the B. sonorensis strains harbored the bacitracin synthetase gene, bacA, and the transporter gene bcrA, which correlated with their observed resistance to bacitracin. Bacitracin was produced by all the investigated species strains (28%), as determined by ultra-high-definition quadrupole time-of-flight liquid chromatography-mass spectrometry (UHD-QTOF LC/MS). The present study has revealed species-specific variations in the antimicrobial susceptibilities of Bacillus spp. and provides new information on MIC values, as well as the occurrence of resistance genes in Bacillus spp., including the newly described species B. sonorensis.

Gergoush is a naturally fermented Sudanese Bread snack produced in three fermentation steps (primary starter, adapted starter and final dough), followed by three baking steps for a half to one hour at above 200 °C. This study examines the microbiota of two sets of fermentations performed at a traditional production site in Khartoum, Sudan in 2006 and 2009, respectively. In 2006 four different milk/legume based primary starters (faba bean, chick pea, lentil and white bean) were sampled in order to enumerate and identify the Bacillus at species or group level. In 2009 specific focus was on the enumeration and safety evaluation of the dominant Bacillus cereus group species occurring during various Gergoush productions (including the three fermentations steps and after baking). In 2006, the primary starters contained Bacillus spp. in the order of between 7.7 and 8.1 log(10) CFU/g. Species identifications were performed by M13-PCR typing using the Escherichia coli phage M13 derived primer PM13 combined with internally transcribed 16-23S rRNA PCR, 16S rRNA gene and gyrA or gyrB gene sequencing, and selected phenotypic tests. Depending on the legume used, 40-68% of the isolates were identified as B. cereus sensu lato, 16-27% as Bacillus licheniformis, 8-32% as Bacillus subtilis and 4-20% as Bacillus sonorensis. During the second set of fermentation trials performed in 2009, the Bacillus spp. and B. cereus occurred in numbers of between 7.7-9.9 and 6.1-7.8 log(10) CFU/g, respectively, while no bacteria were detected after baking. A total of 180 B. cereus sensu lato isolates from four different primary starters, adapted starters and final doughs were further identified as B. cereus sensu stricto (118 isolates) and Bacillus thuringiensis (62 isolates). The safety of Gergoush was evaluated based on the counts and toxin gene profiles of the dominant B. cereus species. Considering that no bacteria survived the baking process, and that the cereulide synthetase gene cesB involved in the production of the heat stable emetic toxin cereulide was not detected in any of the investigated B. cereus isolates, the results indicate, that Gergoush produced at the traditional production site is safe for human consumption. This study is the first to identify the Bacillus of Gergoush to species level, and it is the first to perform a safety evaluation of the product, based on the dominant B. cereus species., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Emetic toxin (cereulide) formation was recently identified in a psychrotolerant species, Bacillus weihenstephanensis [Thorsen, L., Hansen, B.M., Nielsen, K.F., Hendriksen, N.B., Phipps, R.K., Budde, B.B., 2006. Characterization of emetic Bacillus weihenstephanensisis, a new cereulide-producing bacterium. Applied and Environmental Microbiology, 72, 5118-5121.]. Although recent findings indicated B. weihenstephanensis as a cereulide producer only limited information is available regarding environmental conditions affecting cereulide production. In the present study a model agar system was used to compare cereulide production during surface growth of B. weihenstephanensis MC67, and two well known mesophilic cereulide producing Bacillus cereus strains, NC7401 and NS117. Cereulide production was quantified by use of Liquid-Chromatography Mass Spectrometry/Mass Spectrometry. Cereulide production of B. weihenstephanensis MC67 occurred in stationary growth phase, as previously observed for B. cereus, and biomass formation and cereulide formation showed a linear correlation. During incubation at 5 degrees C for 1, 2 and 3 weeks growth was inhibited and as a consequence no detectable cereulide production occurred for any of the three strains. Similar results were obtained for the mesophilic B. cereus strains when incubated at 8 degrees C, whereas B. weihenstephanensis MC67 grew to stationary phase and produced 0.002 microg cereulide/cm(2) agar surface in 1 week. Raising the temperature from 5 degrees C to 25 degrees C for 24 h after 1 week of incubation resulted in growth to stationary phase and production of variable levels of cereulide. B. weihenstephanensis MC67 produced 6.18 microg cereulide/cm(2), B. cereus NS117 0.91 microg cereulide/cm(2) and B. cereus NC7401 0.09 microg cereulide/cm(2). Similar levels of cereulide was produced by the mesophilic strains when raising the temperature from 8 degrees C (instead of from 5 degrees C) to 25 degrees C for 24 h, while a considerably lower level was produced by B. weihenstephanensis MC67 (0.10 microg cereulide /cm(2)). If the temperature was raised from 5 degrees C and 8 degrees C to 25 degrees C for 24 h after an increased incubation time for 2 and 3 weeks, all three strains produced considerably less cereulide. B. weihenstephanensis MC67 produced 100-6000 times less and the mesophilic B. cereus strains produced 9-40 times less cereulide. These results can partly be explained by differences in the growth at the temperature abuse. Effect of chill storage on cereulide production at temperature abuse has not been investigated previously. Results of the present study indicate that storage at 5 and 8 degrees C will not lead to emetic intoxications, however the time at, and choice of chill temperature will determine the amount of cereulide produced in a temperature abuse situation. These results are of relevance for the safety of chilled foods of extended durability.

The effect of modified atmosphere packaging (MAP) on the germination and growth of toxin producing psychrotolerant Bacillus spp is not well described. A model agar system mimicking a cooked meat product was used in initial experiments. Incubation at refrigeration temperature of 8 degrees C for 5 weeks of 26 Bacillus weihenstephanensis including two emetic toxin (cereulide) producing strains showed that B. weihenstephanensis is sensitive to MAP containing CO2. The sensitivity to 20% CO2 was dependent on strain and oxygen level, being increased when oxygen was excluded from the MAP. Growth from spores was observed at the earliest within 2 weeks when 20% CO2 was combined with 2% O2 and in 3 weeks when combined with "0"% O2 (the remaining atmosphere was made up from N2). Results were validated in a cooked meat sausage model for two non-emetic and one emetic B. weihenstephanensis strain. The packaging film oxygen transfer rates (OTR) were 1.3 and 40 ml/m(2)/24 h and the atmospheres were 2% O2/20% CO2 and "0"% O2/20% CO2. Oxygen availability had a large impact on the growth from spores in the MAP meat sausage, only the most oxygen restricted condition (OTR of 1.3 ml/m(2)/24 h and "0"% O2/20 % CO2 inhibited growth of the three strains during 4 weeks storage at 8 degrees C. Cereulide production was undetectable during storage at 8 degrees C irrespective of choice of the MAP (quantified by liquid chromatography mass spectrometry/mass spectrometry). MAP storage at 8 degrees C for 1 and 3 weeks followed by opening of packages and temperature abuse for 1.5 h daily at 20 degrees C during 1 week resulted in increased cell counts and variable cereulide production in the meat sausage. A pre-history at 8 degrees C for 1 week in MAP with OTR of 1.3 or 40 ml/m(2)/24 h and 2% O2 resulted in cereulide concentrations of 0.816-1.353 microg/g meat sausage, while a pre-history under the most oxygen restricted condition (OTR of 1.3 ml/m(2)/24 h, "0"% O2/20 % CO2 resulted in minimal cereulide production (0.004 microg/g meat sausage) at abuse condition. Extension of MAP storage at 8 degrees C for 3 weeks followed by abuse resulted in a substantially reduced cereulide production. Data demonstrates that MAP can be used to inhibit growth of a psychrotolerant toxin producing Bacillus spp. during chill storage at 8 degrees C, and substantially reduce the risk of emetic food poisoning at abuse condition. Results are of relevance for improving safety of ready to eat processed chilled foods of extended durability.

Alkaline-fermented food condiments play an important role in the diets of many people in developing and a few developed countries. The rise in pH during production of these foods is due to the ability of the dominant microorganisms, Bacillus spp., to hydrolyze proteins into amino acids and ammonia. Studies have been undertaken which have investigated a number of these products like dawadawa, ugba, bikalga, kinema, natto, and thua-nao. In this review, current knowledge about the principal microbiological activities and biochemical modifications which occur during the processing of the alkaline condiments including nutritional, antimicrobial, and probiotic aspects are discussed. The current use of molecular biology methods in microbiological research has allowed unambiguous and more reliable identification of microorganisms involved in these fermentations generating sufficient knowledge for the selection of potential starter cultures for controlled and better production procedures for alkaline-fermented seeds 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.

Cereulide production has until now been restricted to the species Bacillus cereus. Here we report on two psychrotolerant Bacillus weihenstephanensis strains, MC67 and MC118, that produce cereulide. The strains are atypical with regard to pheno- and genotypic characteristics normally used for identification of emetic B. cereus strains. MC67 and MC118 produced cereulide at temperatures of as low as 8 degrees C.