Safety Assessment of Bacillus subtilis MB40 for Use in Foods and Dietary Supplements.

With the growing popularity of probiotics in dietary supplements, foods, and beverages, it is important to substantiate not only the health benefits and efficacy of unique strains but also safety. In the interest of consumer safety and product transparency, strain identification should include whole-genome sequencing and safety assessment should include genotypic and phenotypic studies. Bacillus subtilis MB40, a unique strain marketed for use in dietary supplements, and food and beverage, was assessed for safety and tolerability across in silico, in vitro, and in vivo studies. MB40 was assessed for the absence of undesirable genetic elements encoding toxins and mobile antibiotic resistance. Tolerability was assessed in both rats and healthy human volunteers. In silico and in vitro testing confirmed the absence of enterotoxin and mobile antibiotic resistance genes of safety concern to humans. In rats, the no-observed-adverse-effect level (NOAEL) for MB40 after repeated oral administration for 14 days was determined to be 2000 mg/kg bw/day (equivalent to 3.7 × 1011 CFU/kg bw/day). In a 28 day human tolerability trial, 10 × 109 CFU/day of MB40 was well tolerated. Based on genome sequencing, strain characterization, screening for undesirable attributes and evidence of safety by appropriately designed safety evaluation studies in rats and humans, Bacillus subtilis MB40 does not pose any human health concerns under the conditions tested.

Isolation, selection and evaluation of Bacillus spp. as potential multi-mode probiotics for poultry.

Bacillus based probiotics are becoming relevant as alternatives to antibiotics used in poultry production and in other animal husbandry. This study describes the isolation of 48 Bacillus spp. candidates, from chickens and chicken environments, for use as potential probiotics in poultry production. These isolates, plus a further 18, were tested in a comprehensive in vitro screening regime that was specifically designed to select the best isolates that satisfied multiple modes of action desirable for commercial poultry probiotics. This screening programme involved the evaluation of the ability of the isolates to survive and grow in the limiting conditions of the chicken gastrointestinal tract. Only 11 of the isolates fulfilled these criteria; hence, they were further evaluated for the ability to adhere to epithelial cells, produce extracellular enzymes, and to demonstrate antagonistic activity against selected pathogens of significant importance in poultry production. Of these, a total of 6 isolates were selected, due to their all-round probiotic capability. Identification by 16S RNA sequencing confirmed these isolates as B. subtilis and B. velezensis, identities which are generally regarded as safe. The Bacillus isolates reported in our study exhibit strong all-inclusive probiotic effects and can potentially be formulated as a probiotic preparation for poultry production.

OPTIMIZATION OF ALKALINE Α-AMYLASE PRODUCTION BY THERMOPHILIC BACILL US SUBTILIS.

BACKGROUND: Starch-degrading amylase enzyme is important in biotechnological applications as food, fermentation, textile, paper and pharmaceutical purposes. The aim of current study to isolate alkaline thermostable α-amylase bacteria and then study the composition of medium and culture conditions to optimize cells growth and a-amylase production. MATERIALS AND METHODS: Thermophilic amylase producing bacterium was isolated from local hot water-springs in Gazan city Saudi Arabia. RESULTS: Phylogenetic analysis of 16 S rRNA sequence for the strain revealed that the strain have the same sequence of Bacillus subtilis. Maximum amylase production was observed, when B. subtilis cultured in medium containing starch at concentration 0.5%, and 10 g/L peptones as nitrogen source at pH 8.5 in when it was incubated for 48 h at 45°C. CONCLUSION: An amylase-producing bacterium were isolated from hot-spring water and was identified as B. subtilis. Amylase produced from B.subtilis had optimum temperature 45°C and pH 8.5 in shaking media.

The Effects of Direct-fed Microbial Supplementation, as an Alternative to Antibiotics, on Growth Performance, Intestinal Immune Status, and Epithelial Barrier Gene Expression in Broiler Chickens.

The objective of this study was to investigate the effects of Bacillus subtilis-based probiotic supplementation in broiler chicken diets on growth performance, feed efficiency, intestinal cytokine, and tight junction (TJ) protein mRNA expression. Zero-day-old broiler chicks (n = 140) were randomly assigned to one of five dietary treatments: basal diet (CON); basal diet supplemented with either antibiotic bacitracin methylene disalicylate (BMD); or probiotics, namely, B. subtilis strain 1781 (PB1), a combination of B. subtilis strain 1104 + strain 747 (PB2), or B. subtilis strain 1781 + strain 747 (PB3). Body weight and feed intake were measured at 14 days of age, and the feed conversion ratio (FCR) was calculated. At 14 days of age, ileal samples were collected and used for intestinal cytokine, TJ protein, and mucin gene expression analysis using qRT-PCR. The chickens supplemented with antibiotic (BMD) and B. subtilis strain 1781 alone (PB1) had significantly higher body weights compared to controls of the same age. Dietary supplementation with antibiotic (BMD) or probiotics (PB1, PB2, PB3) significantly improved the feed efficiency as evidenced by decreased FCR compared to controls. No differences were observed in the expression of IL1ß, IL17F, IFNγ, and MUC2 gene among the different treatment groups. However, elevated expression of IL6 (BMD, PB1, PB2), IL8 (PB2), and TNFSF15 (PB1, PB2, PB3) compared to controls was observed in the ileum. IL2 and IL10 expression was upregulated in chicks in the PB2 and PB3 groups, and IL4 was elevated in the PB1 group. IL13 was elevated in all probiotic-fed groups (PB1, PB2, PB3). Probiotic supplementation was also shown to significantly increase the expression of TJ proteins JAM2, ZO1 (PB2, PB3), and occludin (PB1, PB2). Taken together, B. subtilis supplementation altered intestinal immune activity and influenced gut barrier integrity through increased tight junction gene expression.

Lipid composition in a strain of Bacillus subtilis, a producer of iturin A lipopeptides that are active against uropathogenic bacteria.

Urinary tract infections are a common disease in humans. Therefore, new methods are needed to destroy biofilms that are formed by uropathogens. Iturin A lipopeptides (LPs) C14 and C15 are potent biosurfactants synthetized by the Bacillus subtilis I'1a strain. The biological activity of extracted LPs was confirmed by examining extracts from I'1a cultures against uropathogenic bacteria that had been isolated from biofilms on urinary catheters. Compared with cultures of DSM 3257, which produce surfactin at a relatively low level, the extract obtained from strain I'1a exhibited a greater inhibitory effect against both planktonic and sessile forms of Escherichia coli, Serratia marcescens, Enterobacter cloacae, Proteus mirabilis, Citrobacter freundii and Enterococcus faecalis. Moreover, cyclic LP biosurfactants may disturb the integrity of cytoplasmic membranes; therefore, we investigated the effects of synthetized LPs on fatty acids and phospholipids of B. subtilis. LPs and lipids were analyzed using GC-MS, LC-MS/MS and MALDI-TOF/TOF techniques. Compared with B. subtilis DSM 3257, membranes of the I'1a strain were characterized by an increased amount of anteiso fatty acids and a ten-fold higher ratio of phosphatidylglycerol (PG)-to-phosphatidylethanolamine (PE). Interestingly, in cultures of B. subtilis DSM 3257 supplemented with LP extracts of the I'1a strain, the PG-to-PE ratio was fourfold higher, and the amount of anteiso fatty acids was also increased.

Endophytic Detection in Selected European Herbal Plants.

A total of 181 cultivable endophytic bacterial isolates were collected from stems of 13 species of herbs inhabiting Europe (Poland): Chelidonium majus L., Elymus repens L., Erigeron annuus L., Euphrasia rostkoviana Hayne, Foeniculum vulgare L., Geranium pratense L., Humulus lupulus L., Matricaria chamomilla L., Mentha arvensis L., Papaver rhoeas L., Rosmarinus officinalis L., Solidago gigantea L. and Vinca minor L. The isolates were screened for their antifungal activity and fifty three were found to inhibit fungal growth. Of these, five had strong antifungal properties. These selected isolates were identified as: Pseudomonas azotoformans, P. cedrina, Bacillus subtilis group and Erwinia persicina.

Efficacy of Bacillus subtilis V26 as a biological control agent against Rhizoctonia solani on potato.

The aim of this study is to evaluate the efficacy of the strain Bacillus subtilis V26, a local isolate from the Tunisian soil, to control potato black scurf caused by Rhizoctonia solani. The in vitro antifungal activity of V26 significantly inhibited R. solani growth compared to the untreated control. Microscopic observations revealed that V26 caused considerable morphological deformations of the fungal hyphae such as vacuolation, protoplast leakage and mycelia crack. The most effective control was achieved when strain V26 was applied 24h prior to inoculation (protective activity) in potato slices. The antagonistic bacterium V26 induced significant suppression of root canker and black scurf tuber colonization compared to untreated controls with a decrease in incidence disease of 63% and 81%, respectively, and promoted plant growth under greenhouse conditions on potato plants. Therefore, B. subtilis V26 has a great potential to be commercialized as a biocontrol agent against R. solani on potato crops.

Characterization of a Novel Fibrinolytic Enzyme, BsfA, from Bacillus subtilis ZA400 in Kimchi Reveals Its Pertinence to Thrombosis Treatment.

Recently, the cardiovascular disease has been widely problematic in humans probably due to fibrin formation via the unbalanced Western style diet. Although direct (human plasmin) and indirect methods (plasminogen activators) have been available, bacterial enzyme methods have been studied because of their cheap and mass production. To detect a novel bacterial fibrinolytic enzyme, 111 bacterial strains with fibrinolytic activity were selected from kimchi. Among them, 14 strains were selected because of their stronger activity than 0.02 U of plasmin. Their 16S rRNA sequence analysis revealed that they belong to Bacillus, Leuconostoc, Propionibacterium, Weissella, Staphylococcus, and Bifidobacterium. The strain B. subtilis ZA400, with the highest fibrinolytic activity, was selected and the gene encoding fibrinolytic enzyme (bsfA) was cloned and expressed in the E. coli overexpression system. The purified enzyme was analyzed with SDS-PAGE, western blot, and MALDI-TOF analyses, showing to be 28.4 kDa. Subsequently, the BsfA was characterized to be stable under various stress conditions such as temperature (4-40°C), metal ions (Mn(2+), Ca(2+), K(2+), and Mg(2+)), and inhibitors (EDTA and SDS), suggesting that BsfA could be a good candidate for development of a novel fibrinolytic enzyme for thrombosis treatment and may even be useful as a new bacterial starter for manufacturing functional fermented foods.

Detailed structure-function correlations of Bacillus subtilis acetolactate synthase.

Isobutanol is deemed to be a next-generation biofuel and a renewable platform chemical.1 Non-natural biosynthetic pathways for isobutanol production have been implemented in cell-based and in vitro systems with Bacillus subtilis acetolactate synthase (AlsS) as key biocatalyst.2-6 AlsS catalyzes the condensation of two pyruvate molecules to acetolactate with thiamine diphosphate and Mg(2+) as cofactors. AlsS also catalyzes the conversion of 2-ketoisovalerate into isobutyraldehyde, the immediate precursor of isobutanol. Our phylogenetic analysis suggests that the ALS enzyme family forms a distinct subgroup of ThDP-dependent enzymes. To unravel catalytically relevant structure-function relationships, we solved the AlsS crystal structure at 2.3 Å in the presence of ThDP, Mg(2+) and in a transition state with a 2-lactyl moiety bound to ThDP. We supplemented our structural data by point mutations in the active site to identify catalytically important residues.

Phylogeny and molecular taxonomy of the Bacillus subtilis species complex and description of Bacillus subtilis subsp. inaquosorum subsp. nov.

The Bacillus subtilis species complex is a tight assemblage of closely related species. For many years, it has been recognized that these species cannot be differentiated on the basis of phenotypic characteristics. Recently, it has been shown that phylogenetic analysis of the 16S rRNA gene also fails to differentiate species within the complex due to the highly conserved nature of the gene, yet DNA-DNA hybridization values fall well below 70 % for the same species comparisons. As a complementary approach, we propose that phylogenetic analysis of multiple protein-coding loci can be used as a means to detect and differentiate novel Bacillus taxa. Indeed, our phylogenetic analyses revealed the existence of a previously unknown group of strains closely related to, but distinct from, Bacillus subtilis subsp. spizizenii. Results of matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses revealed that the group produces a novel surfactin-like lipopeptide with mass m/z 1120.8 that is not produced by the other currently recognized subspecies. In addition, the group displayed differences in the total cellular content of the fatty acids C(16 : 0) and iso-C(17 : 1)omega10c that distinguish it from the closely related B. subtilis subsp. spizizenii. Consequently, the correlation of these novel phenotypic traits with the phylogenetic distinctiveness of this previously unknown subspecies group showed that phylogenetic analysis of multiple protein-coding loci can be used as a means to detect and differentiate novel Bacillus taxa. Therefore, we propose that this new group should be recognized as representing a novel taxon, Bacillus subtilis subsp. inaquosorum subsp. nov., with the type strain NRRL B-23052(T) (=KCTC 13429(T)=BGSC 3A28(T)).

Effects of lipidic carbon sources on the extracellular lipolytic activity of a newly isolated strain of Bacillus subtilis.

An isolate exhibiting high extracellular lipolytic activity was identified as Bacillus subtilis by 16S rRNA gene sequence analysis. The enzyme activity of the isolate was improved by using different concentrations of lipidic carbon sources such as vegetable oils, fatty acids and triglycerides. Lipolytic activity was assayed spectrophotometrically using p-nitrophenyl palmitate. One percent (v/v) of sesame oil provided the highest activity with 80 and 98% enhancements with respect to 1% (v/v) concentrations of linoleic acid and triolein as the favored fatty acid and triglyceride, respectively. Glucose presented a repressive effect on lipase production. Lipase secreted by B. subtilis was partially purified by ultrafiltration and anion exchange chromatography; and the purified enzyme was tested for its residual activity in the presence of EDTA, SDS, Triton X-100, Tween 20, Tween 80 and protease. The present work reports, for the first time, that the lipolytic activity of a B. subtilis strain can be improved by using inexpensive vegetable oils; and also that B. subtilis lipase is suitable for use in detergents.

[Isolation and identification of surfactin producing Bacillus subtilis strain and its effect of surfactin on crude oil].

Strains producing biological surfactants were isolated from formation water of Daqing oil field, Heilongjiang Province, China. From which a lipopeptide producing strain ZW-3 was screened out by PCR of the sfp gene. The morphology, cultural characteristics, physiological, biochemical properties and chemotaxonomy of strain ZW-3 were studied. The strain is rod shaped (0.7-0.8 microm x 2-3 microm), gram-positive, spore-forming and aerobic bacteria. Its (G+C) content was determined to be 42.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequence demonstrated that it was closely related to the genus Bacillus subtilis, and the metabolites of strain ZW-3 was analyzed by thin layer chromatography and high performance liquid chromatography, the result indicated that the biosurfactant strain ZW-3 produced was surfactin. It could reduce surface tension of bacterial fermentation culture medium and water/oil- interfacial tension from 68.82 mN/m to 24.88 mN/m and from 23.53 mN/m to 4.57 mN/m, respectively, and its mixture with 1.8% NaOH could reduce water/oil- interfacial tension to an ultra low level (1.2 x 10(-3) mN/m), Its critical micelle concentration (CMC) was tested to be 33.3 mg/L (3.24 x 10(-5) mol/L)at 25 degrees C, and it had excellent emulsifying (2.89U) and foaming property. All these results showed that this biosurfactant had great potential in pharmaceutics, environmental protection, cosmetic, oil recovery and many other application fields.

Pectate lyase C from Bacillus subtilis: a novel endo-cleaving enzyme with activity on highly methylated pectin.

The gene yvpA from Bacillus subtilis was cloned and expressed in Escherichia coli. It encoded a pectate lyase of 221 amino acids that was denominated PelC. The heterologously expressed enzyme was purified by His-tag affinity chromatography and characterized. PelC depolymerized polygalacturonate and pectins of methyl esterification degree from 22 % to 89 %, exhibiting maximum activity on 22 % esterified citrus pectin. It showed an absolute Ca2+ requirement and the optimum temperature and pH were 65 degrees C and pH 10, respectively. The deduced amino acid sequence of PelC showed 53 % identity to pectate lyase PelA from Paenibacillus barcinonensis, which was also characterized. Similarly to PelC, purified PelA showed activity on polygalacturonate and pectins with a high degree of methyl esterification. The two enzymes cleaved pectic polymers to a mixture of oligogalacturonates, indicating an endo mode of action. Analysis of activity on trigalacturonate showed that PelC cleaved it to galacturonic acid and unsaturated digalacturonate, whereas PelA did not show activity on this substrate. PelC and PelA showed high homology to a few recently identified pectate lyases of family 3 and form with them a cluster of small-sized pectate lyases from non-pathogenic micro-organisms.

Purification and characterization of a novel enantioselective hydrolase from Bacillus subtilis.

Screening of the micro-organisms from an in-house microbial culture repository, identified a bacterial strain bearing membrane-bound, inducible ester hydrolase activity. The strain designated as RRL-BB1 has been identified as Bacillus subtilis by 16 S rRNA typing. Its application in the kinetic resolution of several racemates, including drug intermediates, showed moderate to high enantioselectivity. The enzyme, designated as BBL, exhibited high enantioselectivity (ee approximately 99%) with acyl derivatives of unsubstituted and substituted 1-(phenyl)ethanols and 1-(6-methoxy-2-naphthyl)ethanols. With acyl derivatives of 2-(6-methoxy-2-naphthyl)propan-1-ol, moderate enantioselectivity was observed. The enzyme also showed moderate enantioselectivity with alkyl esters of carboxylic acids i.e. 2-bromopropanoic acid and 2-hydroxy-4-phenylbutanoic acid. The enzyme was purified to >90% purity from cell-free extract of RRL-BB1 with 26% overall yield. The purified enzyme exhibited hydrolase activity without any noticeable decrease in the rate of hydrolysis or the enantioselectivity profile. A specific activity of 450 units/mg protein resulted after at least a 200-fold purification of the crude cell-free extract. The key purification step was the irreversible adsorption of the salt-precipitated crude enzyme on hydrophobic resin, in the presence of a low salt concentration, and desorption of the enzyme with a linear gradient of 1% sodium cholate. The purified enzyme was a 45 kD monomer as shown by SDS/PAGE. The N-terminal amino acid sequence of the purified enzyme was determined as Thr-Lys-Leu-Thr-Val-Gln-Thr-Arg-Asp-Gly-Ala-Leu-Arg-Gly-Thr. The N-terminal sequence did not bear any homology with other known bacterial lipases. BBL is maximally active at 37 degrees C, pH 8.0 and fairly stable up to 40 degrees C, pH 6-10. The enzyme is insensitive to EDTA but inhibited by serine protease inhibitor PMSF. Its activity (72%) was retained in the presence of the anionic detergent SDS at a concentration of 0.2% (w/v).

Polypeptide antibiotic 26a from Bacillus subtilis. I. Taxonomy and fermentative production.

In surface cultures on NK/2-Sym's medium, the isolate No. 26a of Bacillus subtilis from the intestinal tract of Galleria mellonella larvae produced three antibacterial substances which were separated by gel filtration on Sephadex G-25 column. The major bioactive compound named 26a had a close resemblance to bacitracin family of polypeptide antibiotics. Two minor active compounds, i.e. a bacteriolytic enzyme with endo-beta-N-acetylmuramidglycanohydrolase (EC. 3. 2. 1. 17) activity and other unidentified factor were usually synthetized in trace amounts. Maximum yield of 26a generally occurred after 120 hour incubation, when the producer reached the stationary growth phase and general sporulation of the bacterial cultures was found. The basal medium of NK/2-Sym supplemented by addition of manganese ions (10(-4) M), d-glucose (1%) and inorganic nitrogen beneficially resulted in antibiotic potency of the fermentation broth. The antibiotics produced by other isolates (Nos 5AK, 15 and 92) have been also analyzed and from their properties they can be tentatively classified as members of bacitracin group polypeptides. A possible role of the antibiotics produced by intestinal Bacillus spp in the formation process of typical gut microflora of G. mellonella is discussed.

Correlation between pigment production and amino acid requirements in Bacillus subtilis.

Several Bacillus subtilis W-23 auxotrophs were unable to produce wild-type pigment normally on minimal agars supplemented sufficiently for growth. This offers a reliable means for scoring genotypes.