Genome Subtyping of Autochthonous Bacillus Species Isolated from Iru,a Fermented Parkia biglobosa Seed

Phylogenetic relationship and strains sub-typing of Bacillus species isolated from iru, a traditional fermented condiment in Africa were studied using polyphasic genomic approaches and the profiles compared with bacilli isolated from similar Asian condiments. The 16S rRNA gene sequencing identified the strains as Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus licheniformis, Bacillus pumilus, and Brevibacillus formosus. The phylogenetic analysis conducted showed five distinct clusters with genetic relatedness among B. subtilis and B. amyloliquefaciens strains from Africa and Asia. Amplified ribosomal DNA restriction analysis (ARDRA) successfully differentiated species of B. subtilis phylogeny from B. cereus. Combined analyses of ARDRA, internal transcribed spacer-polymerase chain reaction (ITS-PCR), ITS-PCR-restriction fragment length polymorphism (ITS-PCR-RFLP) and randomly amplified polymorphic DNA (RAPD-PCR) further confirmed B. subtilis and B. amyloliquefaciens as the dominant Bacillus species associated with fermentation of iru, and revealed high strains genetic diversity, while multilocus sequence analysis (MLSA) data distinguished B. cereus from B. thuringiensis. This information is essential for selection of starter cultures with desirable functional attributes to guarantee product consistency and safety quality of traditional fermented foods.     

Isolation and Identification of Bacillus spp. and Related Genera from Different Starchy Foods

ABSTRACT: Samples of butternut squash, potatoes, rice, and wheat flour were analyzed. Bacillus spp. and related species belonging to Paenibacillus and Brevibacillus genera were found in 96% of the samples. In butternut squash, predominant species were Bacillus pumilus and Paenibacillus polymyxa together with other Bacillus spp. species (B. cereus, B. licheniformis, B. sphaericus, and B. subtilis). In all the potato samples, Bacillus species were detected (B. cereus, B. mycoides, and B. licheniformis). Also, Bacillus spp. were detected in 100% of the unhusked rice samples, while incidence in white rice samples was 83%. In total rice samples, B. pumilus, Brevibacillus brevis, and Paenibacillus macerans were the main species and B. cereus, P. polymyxa, B. subtilis, and Brevibacillus laterosporus had the lower percentage. The most important species found in wheat flour was P. polymyxa with colony forming units per gram of about 10². As the identified species were potentially causatives of foodborne diseases, attention should be given to sanitary and temperature conditions as critical factors that influence the safety and shelf life of these products. Practical Application: Foodborne illness produce by B. cereus have been associated with a wide variety of food. In addition, some other Bacillus species have been related to foodborne disease in humans. Information about the virulence mechanisms of other Bacillus spp. is scanty and their risk is underestimated. Identifying the group of food and the food processes in which Bacillus cereus or other Bacillus spp. would be hazardous for human health is vital for the prevention of foodborne outbreak. In this study, we determined the incidence of Bacillus spp. and related genera in some food items of agriculture origin from Argentina. This research is relevant to identify the presence of potentially pathogen Bacillus species and related genera in this type of food.     

Fermentation with Bacillus spp. as a bioprocess to enhance anthocyanin content,the angiotensin converting enzyme inhibitory effect,and the reducing activity of black soybeans

Food possessing anthocyanins, angiotensin converting enzyme (ACE) inhibitory activity or reducing activity show beneficial effect on human health. To develop healthy food, black soybeans were fermented with either Bacillus subtilis BCRC 14715 or Bacillus sp. CN11, or a mixture of both Bacillus spp. in the present study. The anthocyanin content, the ACE inhibitory activity and the reducing power of the fermented black soybean were then examined. It was found that the ACE inhibitory activity of the extracts of bean and viscous material from the fermented black soybeans varied with extraction solvents and starter organism, yet increased as the fermentation period was extended, regardless of starter organism. After 18 h of fermentation, the water extract of bean showed less ACE inhibitory activity than did the respective 80% ethanol extract. While the water extract of viscous material showed a higher ACE inhibitory activity than the respective ethanol extract. With respect to extraction yield, it was found that the ACE inhibitor in the fermented black soybean could be extracted more efficiently with water than 80% ethanol. Fermentation with B. subtilis BCRC 14715 was also found to increase the anthocyanin content of black soybean and the reducing activity of the extracts. Finally, the 80% ethanol extract showed a higher reducing activity than the water extract.     

Protease and γ-glutamyltranspeptidase activities of bacillus spp. isolated from rice straw

Bacillus strains were isolated from the rice straw of 7 districts in Korea, and almost all were identified as either B. amyloliquefaciens, B. pumilus, or B. subtilis. The correlation between the protease activity and γ-glutamyltranspeptidase activity produced by the identified Bacillus spp. was positively correlated (R²rF0.747). Statistically, the B. subtilis species might be the most potent producer of protease and γ-glutamyltranspeptidase of 3 identified Bacillus strains.     

Volatile compounds produced by Bacillus species alkaline fermentation of bambara groundnut (Vigna subterranean (L.) Verdc) into a dawadawa-type African food condiment using headspace solid-phase microextraction and GC × GC–TOFMS

The reports on the volatile compounds of a dawadawa-type African food condiment produced from the alkaline fermentation of bambara groundnut (Vigna subterranea (L.) Verdc.) using Bacillus starter cultures are limited. Volatile compounds were isolated from dawadawa-type condiments using headspace solid phase microextraction and analysed by comprehensive gas chromatography coupled to time of flight mass spectrometry. Acids, aldehydes and alcohols accounted for over 70% of the volatile compounds produced in the Bacillus fermented samples. B. subtilis subsp. subtilis SFBA3 produced the highest content of acids (4969.60 µg kg^?1), while the highest content of aldehydes (2811.90 µg kg^?1) and alcohols (1247.60 µg kg^?1) was detected with Bacillus cereus PALB7 and Bacillus licheniformis OALB2, respectively. Sulphur-containing compounds concentration (85.80 µg kg^?1) was highest for Bacillus amyloliquefaciens SFBA2. Maximum 2-methyl butanoic acid and 3-methyl butanoic acid concentrations, indicative of typical dawadawa aroma, were produced by B. subtilis subsp. subtilis SFBA3.     

Characterisation and profiling of Bacillus subtilis,Bacillus cereus and Bacillus licheniformis by MALDI-TOF mass fingerprinting

The Bacillus genus includes species such as Bacillus cereus, Bacillus licheniformis and Bacillus subtilis, some of which may be pathogenic or causative agents in the spoilage of food products. The main goal of this work was to apply matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass fingerprinting to the classification of these Bacillus species. Genetic analyses were also compared to phyloproteomic analyses. A collection of 57 Bacillus strains isolated from fresh and processed food and from culture collections were studied and their mass spectra compiled. The resulting mass fingerprints were compared and characteristic peaks at the strain and species levels were assigned. The results showed that MALDI-TOF was a good complementary approach to 16S rRNA sequencing and even a more powerful tool in the accurate classification of Bacillus species, especially for differentiating B. subtilis and B. cereus from Bacillus amyloliquefaciens and Bacillus thuringiensis, respectively. MALDI-TOF was also found to provide valuable information at both intra- and interspecies levels in the Bacillus species studied.     

Characterization of Soybean Kapi During Fermentation with Bacillus spp.

The changes in enzymatic activities, aroma profiles, color development, phenolic content (including isoflavones), and radical scavenging properties during fermentation to produce soybean kapi condiment using two isolates of Bacillus subtilis IS4 (IS4) and B. amyloliquefaciens RY1 (RY1) are reported. The activities of three hydrolytic enzymes (β-glucosidase, α-amylase, and protease) from both strains showed similar change patterns; the highest activities were observed during days 2–4 of fermentation. With respect to volatile compound formation, the common dominant volatile compounds found in both IS4 and RY1 fermented samples were aldehydes and N-containing compounds. However, IS4 also contained acids and esters as dominant compounds, while RY1 also contained alcohols as dominant compounds. Moreover, the change of Hunter color (increase in a*- and b*- value and decrease in L*-value) appeared to be coincidental with the development of browning and the increase in fluorescence intensity in all samples of IS4 and RY1. In addition, the fermented samples had significant increase (P<0.05) of phenolic content as well as DPPH radical scavenging activity, which was well correlated. Although the content of glucoside isoflavone (daidzin and genistin) and aglycone isoflavone (daidzein and genistein) increased in the early stage of fermentation and dramatically decreased thereafter in both strains, their derivatives may possess antioxidant activity. These results show the potential of the individual B. subtilis strains IS4 and B. amyloliquefaciens RY1 as functional starter cultures for the enrichment of the antioxidant and free-radical scavenging activity in soybean kapi.     

In vitro digestibility of Bacillus fermented soya bean

Bacillus fermented legume products include among others dawadawa and soumbala made from African locust bean, and natto and kinema made from soya bean. Bacillus subtilis is the dominant species involved in the fermentation. During Bacillus fermentation for 48 h of autoclaved soya bean the quantity of soluble and dialyzable matter increased from 22% and 6% up to 65% and 40%, respectively. Protein and carbohydrate degradation during fermentation of soya bean with several Bacillus spp. was investigated and appeared to be substantial during the first 18 h of fermentation resulting in the release of high levels of peptides and oligosaccharides. In vitro digestibility was increased from 29% up to 33–43% after Bacillus fermentation for 48 h. It was shown that Bacillus spp. were able to degrade soya bean macromolecules to a large extent resulting in water-soluble low molecular weight compounds. In vitro digestion of Bacillus fermented soya bean using gastrointestinal enzymes only slightly increased the amount of dialyzable matter, which clearly demonstrated the beneficial effect of Bacillus fermentation on food nutrient availability.     

Prevalence of Bacillus spp. in different food products collected in Argentina

The prevalence of Bacillus spp. in 279 samples of different food products collected in Argentina was studied. Bacillus spp. was confirmed in 28 out of 70 honey samples, 29 out of 29 flour samples, 15 out of 50 cheese samples, and 30 out of 30 spice samples, while Bacillus spp. was not found in fresh anchovy. Among the 70 honeys studied, Bacillus cereus, Bacillus pumilus, Bacillus laterosporus and Paenibacillus larvae subspp. larvae showed an incidence of 23%, 4%, 8% and 38%, respectively. More diversity of Bacillus species was found in rye flours than in white flours, Bacillus subtilis being the predominant species isolated from rye flour. B. cereus had an incidence of 50% in Port Salut Argentino cheeses. Meanwhile, B. pumilus was identified in both Port Salut and Quartirolo cheeses with an incidence of 50% and 25%, respectively. All the spices analysed showed Bacillus mycoides as the sole aerobic spore-forming bacilli isolated. The association of the presence of B. cereus, B. subtilis and Bacillus licheniformis with both the potential spoilage of foods and foodborne outbreaks is well known. In this study, Bacillus spp. had an incidence of 38% among all the samples analysed, therefore the monitoring of those species should be routinely done in microbiological food analyses.     

Degradation of polysaccharides and non-digestible oligosaccharides by Bacillus subtilis and Bacillus pumilus isolated from Soumbala, a fermented African locust bean (Parkia biglobosa) food Condiment

In order to select starter cultures for controlled fermentation of African locust beans, Bacillus subtilis and B. pumilus from Soumbala were investigated for their ability to degrade arabinogalactan, stachyose, raffinose and sucrose—the main polysaccharides and oligosaccharides in African locust bean. The methods used were liquefaction of gels of galactomannan for screening, pH measurement and High Performance Anion Exhange Chromatography – Pulsed Amperometric Detection (HPAEC–PAD) for detailed degradation study of the carbohydrates. For B. subtilis isolates, liquefaction of galactomannan gels was observed and HPAEC–PAD showed that arabinogalactan and stachyose were partially degraded while raffinose was fully degraded after 48 h of fermentation. Melibiose, fructose and traces of galactose were detected as residual sugars. For B. pumilus isolates much weaker degradation of the carbohydrates was observed. Two isolates of B. subtilis showed strong ability to degrade the oligosaccharides and they are seen as promising starter cultures for fermentation of African locust bean.     

Detecting single Bacillus spores by surface enhanced Raman spectroscopy

Detection of Bacillus spores is of considerable importance to the food industry since they can survive standard processing procedures and sanitation treatments. Surface enhanced Raman spectroscopy (SERS) coupled with gold SERS-active substrates was used to detect and discriminate among five Bacillus spores (B. cereus ATCC 13061, B. cereus ATCC 10876, B. cereus sp., B. subtilis sp., and B. stearothermophilus sp.). Tremendously enhanced Raman signals of Bacillus spores deposited onto the gold substrates were detected, allowing the limit of detection (LOD) of SERS to reach the level of a single spore. Distinct spectral differences were observed between different Bacillus spores. Hierarchical cluster analysis (HCA) and principle component analysis (PCA) show clear data segregations at the species level between five Bacillus spores. Principle component (PC) values indicate that the Raman shift range between 900 and 1200 cm⁻¹ contributed significantly to the total data variance in the PCA results. In particular, a prominent band of dipicolinic acid (DPA) was observed at 998 cm⁻¹ and served as a biomarker for bacterial spores. This study demonstrates that SERS coupled with gold nanosubstrates is able to detect and discriminate single Bacillus spores non-destructively and with minimum sample preparation. SERS method is a promising tool for rapid, ultra-sensitive, and selective detection of bacterial spores, potentially in foods and other complex biological matrices.     

Comparison of microbial communities between normal and swollen canned soy sauces using nested PCR‐denaturing gradient gel electrophoresis,HPLC and plate techniques

The microbial community of normal and swollen canned soy sauce was investigated using molecular biological method. The PCR‐denaturing gradient gel electrophoresis (DGGE) profiles showed that four lactic acid bacteria (LAB), including Lactobacillus acidipiscis, L. pobuzihii, L. piscium and another Lactobacillus sp., were involved in the swollen canned samples. Plate technique showed that three diverse species of Bacillus (B. subtilis, B. oleronius and B. flexus) were present in the swollen canned samples. However, much less bacterial contaminants were detected in the normal samples. According to the HPLC analysis, the lactic acid concentrations of the swollen canned samples were significantly higher than those in the normal samples. These results indicated that LAB can play a key role in contributing to the acidisation of the swollen canned soy sauce products. Our results confirmed the existence of Bacillus sp. and LAB in the packaged fermented soy sauce.     

Incidence and characterisation of aerobic spore‐forming bacteria originating from dairy milk in Tunisia

The purpose of this study was to evaluate the incidence and characterisation of aerobic spore‐forming bacteria originating from dairy milk in Tunisia. The distribution of Bacillus species in raw milk, pasteurised milk and UHT milk were 47.5%, 27.5% and 25%, respectively. Seven Bacillus species, including Bacillus pumilus (10%), Bacillus subtilis (12.5%), Brevibacillus brevis (10%), Bacillus cereus (22.5%), Bacillus sphaericus (7.5%), Bacillus licheniformis (12.5%) and Bacillus sporothermodurans (25%) were identified in different milk samples. Bacillus cereus was predominant in raw and pasteurised milk. Although B. sporothermodurans was the predominant sporogenous micro‐organism in UHT milk, B. cereus, B. sphaericus and B. licheniformis were also present. This study showed that there is a high degree of diversity, both phenotypic and genotypic, among Bacillus isolates from Tunisian milk and the persistence of spoilage risk in UHT milk.     

Application of Autochthonous Mixed Starter for Controlled Kedong Sufu Fermentation in Pilot Plant Tests

Traditional sufu is fermented by back‐slopping and back‐slopping has many defects. The objective of this study was to apply autochthonous mixed starter to control Kedong sufu fermentation. Sufu was manufactured using back‐slopping (batch A) and autochthonous mixed starter (batch B) with Kocuria kristinae F7, Micrococcus luteus KDF1, and Staphylococcus carnosus KDFR1676. Considering physicochemical properties of sufu, 150‐day sufu samples from batch A and 90‐day sufu samples from batch B met the standard requirements, respectively. Considering sensory characteristics of sufu, 150‐day sufu samples from batch A and 90‐day sufu samples from batch B showed no significant differences (P > 0.05). The maturation period of sufu was shortened by 60 d. Profiles of free amino acids and peptides partly revealed the mechanism of typical sensory quality and shorter ripening time of sufu manufactured by autochthonous mixed starter. In final products, content of total biogenic amines was reduced by 48%. Autochthonous mixed starter performed better than back‐slopping. Fermentation had a positive influence on the quality, safety, and sensory properties of sufu. The application of autochthonous mixed starter does not change the sensory characteristics of traditional fermented sufu. In addition, it reduces maturation period and improves their homogeneity and safety. It is possible to substitute autochthonous mixed starter for back‐slopping in the manufacture of sufu.     

Effect of an additionally introduced degQ gene on di-d-fructofuranosyl 2,6′:2′,6 anhydride (DFA IV) production by recombinant Bacillus subtilis in a single culture production system

Di-d-fructofuranosyl 2,6′:2′,6 anhydride (DFA IV) was produced directly from sucrose using a single culture of recombinant Bacillus subtilis 168 carrying the levan fructotransferase (lft) gene. In this study, three plasmids carrying the degQ36 gene, which is a degQ allele of B. subtilis (degQ36) with a degQ36 mutation on its promoter, were constructed to overproduce intact DegQ in B. subtilis 168. The transformant B. subtilis/pHT-D36 (with the degQ36 gene) consumed sucrose and produced levan at a higher rate than B. subtilis/pHT43 (without the degQ36 gene). The transformant B. subtilis/pLFT-GD36, carrying the lft and degQ36 genes, also consumed sucrose at a higher rate and produced more DFA IV than B. subtilis/pLFT-G, carrying the lft but without the degQ36 gene. B. subtilis/pLFT-GD36 produced 43.5 g/l of DFA IV and consumed 240 g/l of sucrose (96% of added sucrose) by 72 h of cultivation, whereas B. subtilis/pLFT-G produced 23.4 g/l of DFA IV with 76.9 g/l of sucrose still remaining in the system. Sucrose-inducible expression vectors were also constructed, which made it possible to produce DFA IV without IPTG induction. Using these vectors, sucrose consumption rates were enhanced and DFA IV production was increased upon introduction of the degQ36 gene. From these results, it can be concluded that the additionally introduced regulatory gene, degQ, was able to stimulate sucrose conversion to levan, and therefore increased DFA IV production in this system.     

Properties of Bacillus cereus and other bacilli contaminating biomaterial-based industrial processes

This paper is an overview on bacilli in industrial processes, with focus on food grade paper and paperboard production. Paperboards mainly contain sporeforming bacteria belonging to the genera Bacillus, Paenibacillus and Brevibacillus, usually found in quantities from <50 to 250 cfu g⁻¹ homogenized paperboard. Of those frequently found, Bacillus cereus group, B. licheniformis, B. subtilis and Brevibacillus brevis are important for food hygiene because of their hydrolytic activities on food components and the ability of some strains to produce food poisoning toxins or to grow at refrigerated temperatures. We found that the phenotypic properties (lecithinase activity, nitrate reduction) used in standard methods (e.g., ISO, FDA, IDF) to recognize B. cereus, were unreliable for industrial isolates. Whole cell fatty acid composition of a group of the industrial isolates deviated so much from those in a widely used commercial database that the strains were not or only poorly recognized as B. cereus. Industrial isolates, including toxigenic ones, often missed one or more of these characters, even in cases where 100% 16S rDNA identity was found with B. cereus or with B. thuringiensis. 11-Methyldodecanoic acid and trans-9-hexadecenoic acid were found without exception in over 200 industrial B. cereus group isolates and in over 30 culture collection strains. The detection of these fatty acids is a secure method for the identification of B. cereus. Negative reaction for starch hydrolysis and for BCET-RPLA test and a specific ribotype were found in all B. cereus strains producing the emetic toxin.     

Microbiological and Toxicological Aspects of Fermentation of Castor Oil Seeds for Ogiri Production

The microorganisms involved in the fermentation of castor oil bean for ogiri production were isolated and characterized. The most predominant microorganism was Bacillus subtilis. Other species were B. licheniformis, B. megaterium and B. firmus. All the Bacillus species were proteolytic and were capable of fermenting castor oil seeds and producing the characteristic ogiri aroma in pure cultures. The optimum pH for growth of the three major isolates was 7-8 while the optimal temperatures were 30^oC for B. subtilis and B. megaterium and 45^oC for B. firmus. Toxicological evaluation of the fermented product by chicken embryo bioassay showed that the initial toxicity of the beans decreased significantly but was not completely eliminated.     

ACCELERATED PROTEOLYSIS OF SOY PROTEINS DURING FERMENTATION OF THUA-NAO INOCULATED WITH BACILLUS SUBTILIS

Thua‐nao is a traditional Thai‐fermented soy product. It was prepared by the conventional method and also with starter inoculation. Inoculated soybean exhibited a higher rate of fermentation than did the natural fermentation as indicated by greater rate of pH increase, higher extent of proteolysis, ammonia‐nitrogen content and nitrogen solubility. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis patterns indicated the extensive degradation of soy proteins during fermentation. Activity staining of Thua‐nao extract suggested that proteolysis of soybean was mediated by various proteinases. Active proteinases with estimated molecular weight (MW) of 29,000, 27,000 and 19,000 Da were present during natural fermentation of soybeans. However, proteinases with MW of 40,000 and 29,000 Da were predominant in inoculated soybeans and like the activity bands observed with spent culture broth of Bacillus subtilis BIOTEC 7123. The results suggested that proteinases released by the dominant species in the inoculum, especially B. subtilis, play an important role in proteolysis of soy proteins during fermentation. Inoculation with B. subtilis resulted in an increased proteolysis, which is useful for subsequent reactions leading to development of Thua‐nao characteristics.     

Effect of Novel Starter Culture on Reduction of Biogenic Amines,Quality Improvement,and Sensory Properties of Doenjang,a Traditional Korean Soybean Fermented Sauce Variety

To select appropriate microorganisms as starter cultures for the reliable and reproducible fermentation of soybean fermented products of Korean Doenjang, various ratios of fungi (Aspergillus oryzae J, Mucor racemosus 15, M. racemosus 42) combined with Bacillus subtilis TKSP 24 were selected as either single, double, or multiple Meju strains for commercial mass production of Doenjang, followed by analysis of sensory characteristics. In the sensory evaluation, Doenjang BAM15‐1 and BAM42‐1, which were fermented with multiple strains (1:1:1), showed the highest sensory scores as compared to control. Based on sensory characteristics, 6 Doenjang samples were subjected to quantitative determination of amino acids, free sugars, and organic acids (volatile and nonvolatile) contents, followed by determination of biogenic amines. Total sweet taste amino acid contents were highest in BAM15‐1 and BAM42‐1 samples (333.7 and 295.8 mg/100 g, respectively) and similar that of control (391.1 mg/100 g). Samples BAM15‐1 and BAM42‐1 showed the relatively high volatile and nonvolatile organic acid contents (154.24, 192.26, and 71.31, 82.42 mg/100 g, respectively). In addition, BAM15‐1 and BAM42‐1 showed negligible biogenic amine formation, ranging from 0.00 to 1.02 and 0.00 to 3.92 mg/100 g, respectively. These findings indicate that determination of food components along with sensory and quality attributes using multiple microbial Meju strains as a starter culture may provide substantial results on improved quality fermented Doenjang products.     

Microbial and physiochemical properties of Cheonggukjang fermented using Bacillus strains with antibacterial or antifungal activities

Cheonggukjang was produced with improved functionalities and safety using Bacillus subtilis W42 with an antibacterial activity and B. amyloliquefaciens MJ1-4 with an antifungal activity as starters with coinoculation of B. subtilis W42 and B. amyloliquefaciens (MW Cheonggukjang). Control cheonggukjang was prepared by inoculation of B. subtilis 168 (BS cheonggukjang) or a commercial cheonggukjang prepared using traditional methods (TM cheonggukjang). Cheonggukjang samples were immediately spiked with B. cereus ATCC11778 (1×10⁵ CFU/g of dry soybean) and Penicillium sp. that produced ochratoxin (1×10⁵ spores/g of dry soybean). During 72 h of fermentation at 37°C, total Bacillus counts increased, reaching 10⁹ CFU/g in MW and BS cheonggukjang. Numbers of B. cereus and Penicillium sp. decreased. The largest reduction was observed in MW cheonggukjang. Fungi were not detected after 24 h in MW and BS cheonggukjang. Fibrinolytic activity was detected only from MW cheonggukjang and the antioxidant activity was highest in MW cheonggukjang.