Enzymatic synthesis of glycosylated puerarin using maltogenic amylase from Bacillus stearothermophilus expressed in Bacillus subtilis

BACKGROUND: The maltogenic amylase from Bacillus stearothermophilus (BSMA) is a valuable biocatalyst that has been used to transglycosylate natural glycosides to improve solubility. To ensure safety, BSMA was produced in Bacillus subtilis, using new shuttle vector‐based expression vectors. The transglycosylation of puerarin was also conducted with crude BSMA and analyzed. RESULTS: Two expression systems, each containing one of the promoters from the genes encoding Bacillus licheniformis maltogenic amylase (BLMA) and an α‐amylase from B. subtilis NA64 (amyR2), were constructed. The amyR2 promoter system was chosen as the best system; it yielded 107 mg of pure BSMA from a 2 L culture. In the transglycosylation reactions of puerarin using crude BSMA, relative amounts for maltosyl‐α‐(1 → 6)‐puerarin, glucosyl‐α‐(1 → 6)‐puerarin, glucosyl‐α‐(1 → 3)‐puerarin, and puerarin were determined as 26:18:7:49. A two‐step purification process, including gel permeation chromatography, yielded 1.7 g of the transfer products from 3 g of puerarin. CONCLUSION: The crude BSMA produced from a host generally recognized as safe (B. subtilis) can be used to transglycosylate various functional compounds. The expression system developed in this study will be helpful for the production of other food‐grade enzymes by B. subtilis. Copyright © 2010 Society of Chemical Industry     

Functional expression of recombinant sweet-tasting protein brazzein by Escherichia coli and Bacillus licheniformis

Brazzein is an attractive sweetener candidate because of its sugar-like taste, high sweetness, and good stability at high temperature and wide pH range. This study was aimed to express and purify bioactive recombinant brazzein (rBrazzein). The rBrazzein gene was synthesized according to the preferred codons of Bacillus subtilis and successfully expressed in Escherichia coli and Bacillus licheniformis. In E. coli host, lower induction temperature of 30°C increased soluble rBrazzein (Ebrazzein) at high level. In B. licheniformis host, two signal peptides (Sec type and Tat type) were evaluated for the expression of rBarzzein in B. subtilis and B. licheniformis. However, only the Sec-type signal peptide guided the secretion expression of rBrazzein in B. licheniformis. The rBrazzein was expressed steadily and the highest yield reached about 57 mg/L at 36 h by small-scale fermentation. The purification procedure of rBrazzein by B. licheniformis (Bbrazzein) was thus established. Approximately 5 mg/L purified rBrazzein was obtained and the purity was 85%. The conformational state of rBrazzeins was confirmed by circular dichroism. The bioactivities of rBrazzeins were evaluated by sweet taste testing. The Bbrazzein and Ebrazzein were 266 times and 400 times sweeter than sucrose on a weight basis, respectively. The formation of disulfide bonds were both confirmed by LC/MS/MS and MALDI-TOF. The CD analysis indicated that Ebrazzein has a similar secondary structure with natural brazzein, which explained why Ebrazzein had a higher intensity of sweetness. This study demonstrated that B. licheniformis system is useful to produce active recombinant brazzein, and has potential food industry applications.     

Effect of different Bacillus strains on the profile of organic acids in a liquid culture of Daqu

A reversed‐phase high‐performance liquid chromatography method for the analysis of malic, lactic, acetic, citric, succinic, propionic and butyric acids, during the incubation of Bacillus spp., was developed. All samples taken from cultivation were centrifuged (20 min, 11,500g at 5°C) and filtered through a 0.45 µm membrane filter before injection. A programme pumping phosphate buffer at pH 2.50 and acetonitrile was used to separate the compounds on a C₁₈ column. Various parameters affecting analysis were optimized to take less than 30 min with a good linearity (R > 0.999). Bacillus licheniformis or B. subtilis, isolated from Daqu, was inoculated to ferment a liquid culture of Daqu. Growth of bacteria and organic acid production during the fermentation were investigated. Although there were no significant differences in the production of organic acids between B. licheniformis and B. subtilis during the first 8 h, significant differences in the production rates of organic acids, except for citric acid, were observed between B. licheniformis and B. subtilis from 8 to 48 h, with the final concentration of each organic acid varying. Copyright © 2013 The Institute of Brewing & Distilling     

Acidic bacterial proteases from Bacillus species as an alternative agent for scouring of cotton fabrics

Protease production by Bacillus licheniformis and Bacillus cerus sources follows similar profiles with marginal differences in activity levels (690, 648?U/mL/min, respectively). Optimum pH values of proteases were closer to the neutral at 60°C. Lower scouring efficiency was observed in proteases produced by B. cerus compared to that of B. licheniformis, in terms of weight loss (4.0 and 3.34%), drop absorbency (1.59 and 3.28?s), extractable impurities (2.03 and 2.82%) and Ruthenium red binding (35 and 43%). Reduction in the tear strength was observed in protease-scoured samples compared to the alkali-scoured samples. FTIR results showed the elimination of nitrogen-containing compounds related to proteins, while the peaks related to wax, pectin were present in the scoured cotton samples.     

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.     

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.     

Design of multiplex PCR for simultaneous detection of rope-forming Bacillus strains in Iranian bread dough

BACKGROUND: The objective of this study was optimisation of multiplex polymerase chain reaction (PCR) by a new primer set for simultaneous detection of ropiness agents as the main bacterial spoilage of Iranian bread. After inoculation of bread dough with activated Bacillus licheniformis (ATCC 9789) and Bacillus subtilis (ATCC 6633), DNA was extracted from the dough and subjected to PCR. Then simplex and multiplex PCR tests were optimised. RESULTS: From the results obtained, the optimum PCR conditions for simultaneous detection of the target genes in Bacillus species were an annealing temperature of 59 °C and an MgCl₂ concentration of 2.5 mmol L^?1. To design primers for these two bacteria, owing to close homology and the existence of similar conserved sequences in their 16S rRNA genes, lpaL and aprE genes (497 and 744 bp target sequences) respectively were chosen. Finally, by sequencing of PCR products, accurate and specific detection of the two desired Bacillus species was confirmed. The results were registered with GenBank under accession numbers HQ877873 and HQ871154. CONCLUSION: Compared with culture‐dependent methods, this procedure offers significantly higher accuracy and speed, which are crucial criteria when it comes to food safety and high volumes of referred samples respectively. Copyright © 2012 Society of Chemical Industry     

Bacillus subtilis HJ18‐4 from Traditional Fermented Soybean Food Inhibits Bacillus cereus Growth and Toxin‐Related Genes

Bacillus subtilis HJ18‐4 isolated from buckwheat sokseongjang, a traditional Korean fermented soybean food, exhibits broad‐spectrum antimicrobial activity against foodborne pathogens, including Bacillus cereus. In this study, we investigated the antibacterial efficacy and regulation of toxin gene expression in B. cereus by B. subtilis HJ18‐4. Expression of B. cereus toxin–related genes (groEL, nheA, nheC, and entFM) was downregulated by B. subtilis HJ18‐4, which also exhibited strong antibacterial activity against B. cereus. We also found that water extracts of soy product fermented with B. subtilis HJ18‐4 significantly inhibited the growth of B. cereus and toxin expression. These results indicate that B. subtilis HJ18‐4 could be used as an antimicrobial agent to control B. cereus in the fermented soybean food industry. Our findings also provide an opportunity to develop an efficient biological control agent against B. cereus.     

Detection and quantification of spoilage and pathogenic Bacillus cereus, Bacillus subtilis and Bacillus licheniformis by real-time PCR

A new primer-probe set for the detection and quantification of Bacillus cereus, Bacillus licheniformis and Bacillus subtilis by real-time PCR (Rti-PCR) was developed. For it, forty-eight strains belonging to these species were considered. The DNA of these strains was isolated and a fragment of the 16S rRNA gene amplified. The amplicons were sequenced and the obtained sequences were aligned with reference sequences from the GenBank. For the development of the Real-Time PCR (RTi-PCR) methodology based on TaqMan probes, a primer pair and probe, specific for the studied Bacillus spp., were designed. To establish the quantification method, two RTi-PCR standard curves were constructed; one with DNA extracted from a serially-diluted B. cereus culture and a second curve with DNA extracted from a sterilised food product inoculated with serial dilutions of B. cereus. The curves exhibited R² values of 0.9969 and 0.9958 respectively. Linear correlations between the log₁₀ input DNA concentration and the threshold cycle (Ct) values were observed with a magnitude of linearity in the range of 1.65 × 10¹ CFU/mL to 1.65 × 10⁶ CFU/mL for both standard curves. The specificity of the designed primers and probe was tested with DNA extracted from B. cereus, B. licheniformis and B. subtilis strains, which gave Ct values between 14 and 15, whereas non-specific amplifications of the DNA from other microbial species of food interest exhibited a Ct value above 28.5. To our knowledge, this method represents the first study about the quantification of spoilage and/or pathogenic B. cereus, B. licheniformis and B. subtilis in food products, with the aim to prevent the presence of these undesirable species in the food chain.     

Characterization of L-Arabinose Isomerase in Bacillus subtilis,a GRAS Host,for the Production of Edible Tagatose

L-Arabinose isomerase (AI; E.C. 5.3.1.4), a commercial enzyme for the production of edible tagatose in vitro and ribulose production in vivo, has been studied using enzymes expressed in an Escherichia coli system, which might cause noxious by-products in food. To ensure food safety in the tagatose manufacturing process, we studied an AI expression system in Bacillus subtilis. The AI gene from Geobacillus stearothermophilus (GSAI) was expressed in Bacillus subtilis, a GRAS host used in the production of fermented soybean in Korea, after subcloning into a Bacillus subtilis - E. coli shuttle vector, and was characterized after purification. The activities of the crude enzyme extract and a purified sample were 0.15 U/mg protein and 2.7 U/mg protein, respectively. The optimal pH and the optimal temperature for arabinose and galactose as substrates were pH 8.0 and 60°C, respectively, the same as those for GSAI in an E. coli expression system. Substrate affinities (K_m) for arabinose and galactose were 77 mM and 279 mM, respectively, whereas in the E. coli expression system, they were 100 mM and 578 mM, respectively. Catalytic efficiencies (k_cat/K_m) for arabinose and galactose were 58.3 and 11.4 mM^?1 min^?1, respectively. The potential use of GSAI expressed in a GRAS host for the production of edible tagatose is discussed in light of these results.     

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.     

Bacilli Spoilage in Part-baked and Rebaked Brown Soda Bread

Brown soda bread had a pH of 7-9 depending on the sodium bicarbonate concentration. Part-baked bread developed ropiness after two days at room temperature. Bacillus subtilis, B. pumilus and B. licheniformis were isolated and their spores displayed D-values in bread dough of 14, 10 and 56 min at 100°C. Germination and growth was examined in broth at pH 6-10 and at 4°, 20°, 30° and 37°C. No growth was observed at 4°C and at pH 10. Rebaking of the part-baked bread heat activated particularly B. licheniformis spores.     

Use of Bacillus subtilis to enrich isoflavone aglycones in fermented natto

BACKGROUND: Natto is a food made by fermenting cooked soybeans with Bacillus subtilis. Soybean isoflavones are reported to provide many health benefits, including oestrogenic effects. However, isoflavone aglycones may be absorbed faster and in higher amounts in the human intestine than their glucosides. This study aimed to investigate the content of isoflavone components in commercial natto products as well as the use of B. subtilis strains to ferment cooked soybeans to produce a high level of isoflavone aglycones in natto. RESULTS: The content and composition of isoflavones in commercial natto products were predominantly (>76%) isoflavone glucosides. Fermentation of cooked soybeans with B. subtilis BCRC 14718 at 37 °C for 48 h was more effective in converting glucosides to aglycones than with other strains of B. subtilis, increasing the proportion of isoflavone aglycones from 12 to 68% of the total isoflavones in the fermented natto. The proportions of the isoflavone aglycones daidzein and genistein in cooked soybeans fermented with B. subtilis BCRC 14718 for 48 h increased from 6 to 54% and from 5 to 13% respectively. CONCLUSION: Bacillus subtilis BCRC 14718 incubated with cooked soybeans produces higher levels of isoflavone aglycones, which may enhance health benefits over traditional fermented natto. Copyright © 2008 Society of Chemical Industry     

Antagonistic action of Bacillus subtilis strain fmbj on the postharvest pathogen Rhizopus stolonifer

Abstract: The antagonistic activities of Bacillus subtilis fmbj against the Rhizopus stolonifer pathogen causing peach soft rot disease were studied in this paper. Bacillus subtilis fmbj exhibited a high antifungal effect on the mycelium growth, sportulation, and germ tube elongation of R. stolonifer with the inhibition rate of 86.5% and 97.42%, respectively, spore germination rate of 20% under antagonist strain concentration of 10⁸ CFU/mL. By using of scanning electron microscope (SEM) and transmission electron microscope (TEM), it was observed that B. subtilis fmbj strain strongly induced morphological abnormalities of R. stolonifer and destroyed structure of hypha and spore. When hypha cell wall of R. stolonifer was damaged, the organelles and cytoplasms inside cell would exude, which led to cell death.     

Enhancement of Recombinant Subtilisin YaB Production by Bacillus subtilis

Bacillus subtilis is an ideal host for the production of extracellular heterologous proteins. The use of a strong, constitutive promoter is a good means of optimizing the expression of heterologous proteins. In this study, high level extracellular production of subtilisin YaB, a potent meat tenderizer, was achieved by engineering the wild-type subtilisin YaB promoter into an artificial promoter and expressed in Bacillus subtilis host. This artificial promoter provides an efficient tool to produce mutant subtilisin YaB or other recombinant heterologous proteins by B. subtilis at high level.     

1H NMR‐based metabolomics approach for understanding the fermentation behaviour of Bacillus licheniformis

Bacillus licheniformis has been found to be one of the persistent dominant microorganisms in Daqu, which is a traditional fermentation starter, and it has been used to intensify certain strains. To understand the impact of B. licheniformis on Daqu, the fermentation behaviour of B. licheniformis was studied using ¹H NMR‐based non‐targeted analysis and principal component analysis. During the fermentation, 53 compounds were identified. Among them, seven compounds were largely consumed and 17 metabolites were largely accumulated. The macromolecular starch and cellulose were degraded by B. licheniformis, and then utilized to produce acetic acid, lactic acid, propionic acid, succinate acid, etc. Principal component analysis was carried out to study the variations of analytes during the fermentation. Samples collected at each time point could be clearly discriminated and the biomarkers of each time point were defined. A variety of biochemical compounds (such as acetate, ethanol, lactate, pyruvate, malate, maltose and sucrose) were changed during the fermentation of B. licheniformis. The results are useful to reveal how and why B. licheniformis becomes dominant in Daqu, and to reveal its impact on the aroma formation of Daqu and its derived products. Copyright © 2015 The Institute of Brewing & Distilling     

Effects of type of packaging material on physicochemical and sensory characteristics of deep‐fat‐fried banana chips

A storage study of deep‐fat‐fried banana chips was carried out for 8 weeks at ambient temperature (27 °C), using four types of packaging material: laminated aluminium foil (LAF), oriented polypropylene (OPP), polypropylene (PP) and low‐density polyethylene (LDPE). The physicochemical and sensory characteristics of the stored banana chips were analysed at weeks 0, 2, 4, 6 and 8. The quality parameters determined were moisture content, water activity (a_w), thiobarbituric acid‐reactive substances (TBARS), texture (breaking force), colour and sensory attributes. The moisture content, a_w, TBARS and breaking force values of all samples increased during storage. The colour also changed during storage, showing higher L and lower a and b values. Samples packed in LAF had the lowest moisture content, a_w, TBARS and breaking force values. The most notable sensory change that occurred during storage was a decrease in crispness. Samples packed in LAF had higher scores than the other three samples, whilst LDPE gave the lowest scores for crispness as well as product colour. There were no significant differences (P > 0.05) in rancid odour among samples packed in OPP, PP and LDPE. However, there were significant differences (P < 0.05) between samples packed in LAF and the other three samples, with LAF giving the lowest rancid odour. © 2002 Society of Chemical Industry     

Altered Oxygen Permeation Through Packaging Polymers in Contact with Liquids—a Mathematical Model Confirmed in a Dilute Xanthan Gum Solution

The ability of liquids to reduce oxygen permeation through low density polyethylene (LDPE), oriented polypropylene (OPP), polyethylene terepthalate (PET), and polyvinylidene chloride (PVTDC) films was mathematically modeled using a water/xanthan solution as a model medium. For low barrier films (LDPE and OPP), the medium substantially reduced oxygen permeation. In PET, an intermediate barrier, the medium produced a recognizable, but minor reductions in permeation. For the highest barrier (PVDC), the medium's influence on permeation was almost unidentifiable. The effect of the medium's oxygen solubility constant and diffusion coefficient on permeation was also examined. For all films, reduction in permeation became significant only after solubility and diffusion values fell below 40% of water values.     

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.     

Antimicrobial Activity of Monoacyl Hexose Coexistent with Lysozyme against Gram-Positive Bacilli

The antimicrobial activities of myristoyl, palmitoyl, or stearoyl hexoses, which were glucose, mannose, and galactose, coexistent with lysozyme against three Gram-positive bacteria, Bacillus coagulans, Bacillus subtilis, and Bacillus licheniformis, were measured in order to investigate the availability of monoacyl hexose as an antimicrobial co-agent. The lysozyme exhibited an antimicrobial activity against Bacillus subtilis and Bacillus licheniformis, but there was no significant difference between the dependencies of the antimicrobial activity against the two bacteria on the lysozyme concentration. However, the antimicrobial activities of the monoacyl hexoses coexistent with the lysozyme were different between those against the two bacteria. The stearoyl hexose coexistent with the lysozyme exhibited the highest antimicrobial activity against two bacteria. It was indicated that the antimicrobial action of the monoacyl hexose would be exerted parallel with the bacterial lysis of lysozyme. Stearoyl hexose, which has a high hydrophobicity, coexistent with the lysozyme could exhibit a higher antimicrobial activity than only the lysozyme.