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.     

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.     

Volatile compounds produced by Lactobacillus fermentum,Saccharomyces cerevisiae and Candida krusei in single starter culture fermentations of Ghanaian maize dough

AIMS: To identify and compare the volatile compounds associated with maize dough samples prepared by spontaneous fermentation and by the use of added starter cultures in Ghana. METHODS AND RESULTS: The starter cultures examined were Lactobacillus fermentum, Saccharomyces cerevisiae and Candida krusei. For identification of aroma volatiles, extracts by the Likens-Nickerson simultaneous distillation and extraction technique were analysed by gas chromatography-mass spectrometry (GC-MS) and using a trained panel of four judges by GC-Olfactometry (GC-sniffing). Compounds identified by GC-MS in maize dough samples after 72 h of fermentation included 20 alcohols, 22 carbonyls, 11 esters, seven acids, a furan and three phenolic compounds. Of the total 64 volatile compounds, 51 were detected by GC-sniffing as contributing to the aroma of the different fermented dough samples. Spontaneously fermented maize dough was characterized by higher levels of carbonyl compounds while fermentations with added L. fermentum recorded the highest concentration of acetic acid. S. cerevisiae produced higher amounts of fusel alcohols and increasing levels of esters with fermentation time and C. krusei showed similarity to L. fermentum with lower levels of most volatiles identified. CONCLUSION: The present study has given a detailed picture of the aroma compounds in fermented maize and demonstrated that the predominant micro-organisms in fermented maize dough can be used as starter cultures to modify the aroma of fermented maize dough. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has documented the advantage of using starter cultures in African traditional food processing and provided a scientific background for introducing better controlled fermentations.     

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.     

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.     

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.     

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.     

微生物发酵对木薯渣营养成分的影响

[背景]将木薯渣作为饲料资源进行开发和利用能够减轻环境污染,实现资源就地转化,已成为国内外研究热点.微生物发酵可降低木薯渣的粗纤维含量,改善适口性,提高饲料转化率.[目的]筛选对木薯渣发酵效果较好的微生物发酵剂及其发酵时长.[方法]试验选用A(芽孢菌+乳酸菌+酿酒酵母菌)、B(戊糖片球菌+酿酒酵母菌)和C(植物乳杆菌+...     

Curie-point pyrolysis mass spectrometry applied to characterization and identification of selected Bacillus species

The use of pyrolysis mass spectrometry in the characterization and identification of Bacillus species was studied. Fifty-three strains of four closely related groups, Bacillus subtilis, B. pumilus, B. licheniformis and 'B. amyloliquefaciens', were used in a study of both sporulated and nonsporulated cultures. Pyrolysis was carried out using a Pyromass 8-80, a novel pyrolysis mass spectrometer specifically designed for fingerprinting complex samples. The pyrolysis data obtained were analysed using multivariate statistical techniques. All four groups could be differentiated using data from non-sporulated cultures but the data from sporulated cultures did not separate B. subtilis from 'B. amyloliquefaciens' or B. pumilus. In contrast, B. licheniformis was more clearly differentiated from the other three species using these data. Culture maturity affected the mass spectra obtained from non-sporulated cultures.     

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.     

The mtrB gene of Bacillus pumilus encodes a protein with sequence and functional homology to the trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis.

The mtrB gene from Bacillus pumilus encodes a 76-amino-acid polypeptide with 77% identity to the trp RNA-binding attenuation protein (TRAP) from Bacillus subtilis. B. pumilus TRAP binds trp leader RNA from either B. subtilis or B. pumilus in a tryptophan-dependent manner. Altering threonine 52 to alanine eliminated RNA-binding activity of B. pumilus TRAP.     

Bacillus pumilus SG2 isolated from saline conditions produces and secretes two chitinases

AIMS: Isolation and characterization of chitinases from a halotolerant Bacillus pumilus. METHODS AND RESULTS: Bacillus pumilus strain SG2 was isolated from saline conditions. It is able to produce chitinase activity at high salt concentration. SDS-PAGE analysis of the B. pumilus SG2 culture supernatant showed two major bands that were induced by chitin. The amino acid sequence of the two proteins, designated ChiS and ChiL, showed a high homology with the chitinase of B. subtilis CHU26, and chitinase A of B. licheniformis, respectively. N-terminal signal peptide of both proteins was also determined. The molecular weight and isoelectric point of the chitinases were determined to be 63 and 74 kDa, and 4.5 and 5.1, for ChiS and ChiL respectively. The genes encoding for both chitinases were isolated and their sequence determined. The regulation of the chitinase genes is under the control of the catabolite repression system. CONCLUSIONS: Secreted chitinase genes and their flanking region on the genome of B. pumilus SG2 have been identified and sequenced. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a multiple chitinases-producing B. pumilus halotolerant strain. We have identified two chitinases by using a reverse genetics approach. The chitinases show resistance to salt.     

Development of starter culture for improved processing of Lafun,an African fermented cassava food product

Aims: To select appropriate micro‐organisms to be used as starter culture for reliable and reproducible fermentation of Lafun. Methods and Results: A total of 22 cultures consisting of yeast, lactic acid bacteria (LAB) and Bacillus cereus strains predominant in traditionally fermented cassava during Lafun processing were tested as potential starter cultures. In an initial screening, Saccharomyces cerevisiae 2Y48P22, Lactobacillus fermentum 2L48P21, Lactobacillus plantarum 1L48P35 and B. cereus 2B24P31 were found to be the most promising of the cultures and were subsequently tested in different combinations as mixed starter cultures to ferment submerged cassava roots. Saccharomyces cerevisiae, inoculated singly or combined with B. cereus, gave the softest cassava root after 48 h of fermentation according to determination of compression profile and stress at fracture. Overall, sensory quality testing showed that Lafun obtained from S. cerevisiae‐fermented cassava gave the most preferred stiff porridge. Saccharomyces cerevisiae 2Y48P22 showed pectinase production in a model system. Conclusions: The results suggest that S. cerevisiae 2Y48P22 is the most efficient organism for cassava softening during the fermentation. Therefore, it could be combined with LAB and used as starter for Lafun processing. Significance and Impact of the Study: Starter cultures are made available for controlled fermentation of Lafun.     

Effect of temperatures during pure culture fermentation of Kinema

Kinema was prepared by fermenting whole cooked soybeans with pure culture of Bacillus subtilis KK2:B10 (MTCC 2756) strain at 35°C, 40°C and 45°C for 24h. Temperature, mesophilic plate counts, relative viscosity, water-soluble nitrogen, formal nitrogen contents and reducing sugars of fermenting soybeans were investigated during fermentation. At higher temperatures the growth rate of B. subtilis KK2:B10 was faster. A remarkable increase in the relative viscosity of kinema was observed at 40°C during fermentation. Water-soluble nitrogen and formol nitrogen to total nitrogen contents increased throughout the 24h of fermentation. Reducing sugars increased during the log phase and then decreased sharply. Kinema matured below 10°C for 1 day after the desired fermentation showed a significant increase in relative viscosity. The quality of kinema was maintained with pure culture fermentation by B. subtilis KK2:B10 at 40°C for 20h and matured at 5°C for 1 day.     

Bacterial fermentation of soya bean for 'daddawa' production

The fermentation of soya bean for 'daddawa' production in Nigeria is carried out by bacteria. Bacillus subtilis and Bacillus pumilus were consistently isolated in fermentations lasting 48 h.     

Optimization of cassava fermentation for fufu production: effects of single starter cultures

Single starter cultures were used to ferment cassava for fufu production in a process which involved steeping seeded cassava tubers in water for 96 h. The cultures used include Bacillus subtilis, Klebsiella sp., Lactobacillus plantarum and Candida krusei. Lactobacillus plantarum exhibited the highest acid producing ability, decreasing the pH of the cassava tubers from 6.0 to 3.62, with a corresponding increase in total titratable acidity (TTA) from 0.072% to 0.250% during the 96 h fermentation period. The effected changes in pH and TTA by other organisms ranged respectively from 5.03 and 0.125% for Klebsiella sp., 5.07 and 0.126% for C. krusei , to 5.20 and 0.128% for B. subtilis within the period. Lactobacillus plantarum grew better than other cultures singly inoculated. While all the cultures were found to contribute to the characteristic odour of fufu, C. krusei effected the highest perceived characteristic odour. Also, the cultures variably caused softening of the tubers, with B. subtilis showing the highest softening capability.     

Microbiological,rheological and aromaticcharacteristics of fermented Uji (an East African Sour Porridge)

Three methods for fermentation of uji were compared in laboratory trials: spontaneous, backslopping (using an inoculum from a previous fermentation) and use of a starter culture of lactic acid bacteria. Spontaneous fermentation resulted in the slowest decrease in pH, while the use of starter culture led to the lowest final pH (3.5). Coliforms were eliminated in less than 8 h using backslopping or starter culture, but increased in numbers during spontaneous fermentation. The viscosity of uji was only marginally affected by the method of fermentation. The aroma profile following spontaneous fermentation contained esters with fruity notes and ethanol and higher alcohols, while mainly organic acids was produced by fermentation with the starter culture. Backslopping led to the lowest production of almost all volatiles identified.     

Lactic acid bacteria: starters for flavour

Abstract Changing milk into other organoleptically acceptable products by fermentation requires particular attributes of the bacteria. These include rapid acid production from lactose and development of suitable quantities of the volatile compounds, diacetyl and acetaldehyde. These compounds must not be over-produced nor should they be accompanied by off-flavours. More knowledge has accumulated concerning starter cultures for milk fermentation than for any of the other cultured foods. We are approaching the time when we can tailor-make mixed cultures of known species of bacteria to provide specific flavours because we are becoming more aware of their metabolic activity within a given foodstuff. To provide and keep a good starter for a particular fermented product it is essential to know what is expected of it in terms of flavour and aroma. This knowledge is not always available and many products are poor because of this. Knowledge of the biochemical pathways leading to flavour production can help in making the right choice of starter.     

Bacteria decomposing technical oils

Fifty bacterial cultures were isolated from technical oils stored for different periods of time. Twenty out of the fifty strains decomposed technical oils. The bacteria were identified and classified as Bacillus strains. Bacillus subtilis, strains 7 and 10, and B. pumilus, strain 13, isolated from oils MK-8 + 10% Akor-1 and MT-16p as well as from AMG-10 were found to be most active and can be recommended as test cultures to study the resistance of technical oils against bacterial degradation.