LTA and PGN from Bacillus siamensis can alleviate soybean meal-induced enteritis and microbiota dysbiosis in Lateolabrax maculatus.

An eight-week feeding trial was designed to assess which component of commensal Bacillus siamensis LF4 can mitigate SBM-induced enteritis and microbiota dysbiosis in spotted seabass (Lateolabrax maculatus) based on TLRs-MAPKs/NF-кB signaling pathways. Fish continuously fed low SBM (containing 16 % SBM) and high SBM (containing 40 % SBM) diets were used as positive (FM group) and negative (SBM group) control, respectively. After feeding high SBM diet for 28 days, fish were supplemented with B. siamensis LF4-derived whole cell wall (CW), cell wall protein (CWP), lipoteichoic acid (LTA) or peptidoglycan (PGN) until 56 days. The results showed that a high inclusion of SBM in the diet caused enteritis, characterized with significantly (P < 0.05) decreased muscular thickness, villus height, villus width, atrophied and loosely arranged microvillus. Moreover, high SBM inclusion induced an up-regulation of pro-inflammatory cytokines and a down-regulation of occludin, E-cadherin, anti-inflammatory cytokines, apoptosis related genes and antimicrobial peptides. However, dietary supplementation with CW, LTA, and PGN of B. siamensis LF4 could effectively alleviate enteritis caused by a high level of dietary SBM. Additionally, CWP and PGN administration increased beneficial Cetobacterium and decreased pathogenic Plesiomonas and Brevinema, while dietary LTA decreased Plesiomonas and Brevinema, suggesting that CWP, LTA and PGN positively modulated intestinal microbiota in spotted seabass. Furthermore, CW, LTA, and PGN application significantly stimulated TLR2, TLR5 and MyD88 expressions, and inhibited the downstream p38 and NF-κB signaling. Taken together, these results suggest that LTA and PGN from B. siamensis LF4 could alleviate soybean meal-induced enteritis and microbiota dysbiosis in L. maculatus, and p38 MAPK/NF-κB pathways might be involved in those processes.

Bacillus velezensis iturins inhibit the hemolytic activity of Staphylococcus aureus.

Bovine mastitis caused by S. aureus has a major economic impact on the dairy sector. With the crucial need for new therapies, anti-virulence strategies have gained attention as alternatives to antibiotics. Here we aimed to identify novel compounds that inhibit the production/activity of hemolysins, a virulence factor of S. aureus associated with mastitis severity. We screened Bacillus strains obtained from diverse sources for compounds showing anti-hemolytic activity. Our results demonstrate that lipopeptides produced by Bacillus spp. completely prevented the hemolytic activity of S. aureus at certain concentrations. Following purification, both iturins, fengycins, and surfactins were able to reduce hemolysis caused by S. aureus, with iturins showing the highest anti-hemolytic activity (up to 76% reduction). The lipopeptides showed an effect at the post-translational level. Molecular docking simulations demonstrated that these compounds can bind to hemolysin, possibly interfering with enzyme action. Lastly, molecular dynamics analysis indicated general stability of important residues for hemolysin activity as well as the presence of hydrogen bonds between iturins and these residues, with longevous interactions. Our data reveals, for the first time, an anti-hemolytic activity of lipopeptides and highlights the potential application of iturins as an anti-virulence therapy to control bovine mastitis caused by S. aureus.

Site-Directed Mutagenesis: Improving the Acid Resistance and Thermostability of Bacillus velezensis α-Amylase and Its Preliminary Feed Application.

The current study aimed to improve the acid resistance and thermostability of Bacillus velezensis α-amylase through site-directed mutagenesis, with a specific focus on its applicability to the feed industry. Four mutation sites, P546E, H572D, A614E, and K622E, were designed in the C domain of α-amylase, and three mutants, Mut1 (E), Mut2 (ED), and Mut3 (EDEE), were produced. The results showed that the specific activity of Mut3 was 50 U/mg higher than the original α-amylase (Ori) after incubation at 40 °C for 4 h. Compared to Ori, the acid resistance of Mut3 showed a twofold increase in specific activity at pH 2.0. Moreover, the results of preliminary feed hydrolysis were compared between Ori and Mut3 by designing three factors, three levels of orthogonal experiment for enzymatic hydrolysis time, feed quantity, and amount of amylase. It was observed that the enzymatic hydrolysis time and feed quantity showed an extremely significant difference (p < 0.01) in Mut3 compared to Ori. However, the amount of enzyme showed significant (p < 0.05) improvement in the enzymatic hydrolysis in Mut3 as compared to Ori. The study identified Mut3 as a promising candidate for the application of α-amylase in the feed industry.

Antipathogenic Activities of Volatile Organic Compounds Produced by Bacillus velezensis LT1 against Sclerotium rolfsii LC1, the Pathogen of Southern Blight in Coptis chinensis.

This study explores the antipathogenic properties of volatile organic compounds (VOCs) produced by Bacillus velezensis LT1, isolated from the rhizosphere soil of Coptis chinensis. The impact of these VOCs on the mycelial growth of Sclerotium rolfsii LC1, the causative agent of southern blight in C. chinensis, was evaluated using a double Petri-dish assay. The biocontrol efficacy of these VOCs was further assessed through leaf inoculation and pot experiments. Antifungal VOCs were collected using headspace solid-phase microextraction (SPME), and their components were identified via gas chromatography-mass spectrometry (GC-MS). The results revealed that the VOCs significantly inhibited the mycelial growth and sclerotia germination of S. rolfsii LC1 and disrupted the morphological integrity of fungal mycelia. Under the influence of these VOCs, genes associated with chitin synthesis were upregulated, while those related to cell wall degrading enzymes were downregulated. Notably, 2-dodecanone and 2-undecanone exhibited inhibition rates of 81.67% and 80.08%, respectively. This research provides a novel approach for the prevention and management of southern blight in C. chinensis, highlighting the potential of microbial VOCs in biocontrol strategies.

Production of Lipopeptides from Bacillus velezensis BVQ121 and Their Application in Chitosan Antibacterial Coating.

The extracellular substance of Bacillus has antibacterial effects inhibiting multiple foodborne pathogens and plays important roles in food production. This study found one Bacillus velezensis BVQ121 strain producing antibacterial lipopeptides (BVAL). After optimization of the fermentation conditions, the BVAL yield was the highest at 1.316 ± 0.03 g/L in reality with the initial pH 6.0, temperature 31 °C, and shaker speed 238 rpm when the optimal nitrogen and carbon sources were used in Landy medium for fermentation. The antibacterial components were identified as iturin, surfactin, and fengycin by HPLC and MALDI-TOF-MS. The MIC was at 2 mg/mL and MBC was at 5 mg/mL. The 6% weight ratio of nanocellulose dosage in chitosan solution could improve the tensile length and strength of the film, and the antibacterial performance was enhanced by the addition of BVAL. The addition of BVAL had no effect on the color and ductility of the film and improved its antibacterial effect. The shelf life of pigeon eggs can be extended by more than 10 days to resist bacterial infections after coating with the chitosan-nanocellulose-BVAL film solution.

Inclusion of a Bacillus-based probiotic in non-starch polysaccharides-rich broiler diets.

This study examined the effects of a 3-strain Bacillus-based probiotic (BP; Bacillus amyloliquefaciens and two Bacillus subtilis) in broiler diets with different rye levels on performance, mucus, viscosity, and nutrient digestibility. We distributed 720 one-d-old female broilers into 72 pens and designed nine diets using a 3 × 3 factorial approach, varying BP levels (0, 1.2 × 106, and 1.2 × 107 CFU/g) and rye concentrations (0, 200, 400 g/kg). On d 35, diets with 200 or 400 g/kg rye reduced broiler weight gain (BWG). Diets with 400 g/kg rye had the highest FCR, while rye-free diets had the lowest (p ≤0.05). Adding BP increased feed intake and BWG in weeks two and three (p ≤0.05). It should be noted that the overall performance fell below the goals of the breed. Including rye in diets reduced the coefficient of apparent ileal digestibility (CAID) for protein, ether extract (EE), calcium, phosphorus, and all amino acids (p ≤0.05). Rye-free diets exhibited the highest CAID for all nutrients, except for methionine, EE, and calcium, while diets with 400 g/kg of rye demonstrated the lowest CAID (p ≤0.05). BP in diets decreased phosphorus CAID (p ≤0.05). Diets containing 1.2 × 107 CFU/g (10X) of BP exhibited higher CAID of methionine than the other two diets (p ≤0.05). Diets containing 10X of BP showed higher CAID of cysteine than diets with no BP (p ≤0.05). Ileal viscosity increased as the inclusion level of rye in the diets increased (p ≤0.05). The ileal concentration of glucosamine in chickens fed diets with 400 g/kg of rye was higher than in those fed diets with no rye (p ≤0.05). Furthermore, ileal galactosamine concentrations were elevated in diets with 200 and 400 g/kg of rye when compared to rye-free diets (p ≤0.05). However, BP in diets had no impact on ileal viscosity, galactosamine, or glucosamine (p > 0.05). In conclusion, the applied Bacillus strains appeared to have a limited capacity to produce arabinoxylan-degrading enzymes and were only partially effective in mitigating the negative impacts of rye arabinoxylans on broilers.

Genomic analysis reveals genes that encode the synthesis of volatile compounds by a Bacillus velezensis-based biofungicide used in the treatment of grapes to control Aspergillus carbonarius.

Fungal control strategies based on the use of Bacillus have emerged in agriculture as eco-friendly alternatives to replace/reduce the use of synthetic pesticides. Bacillus sp. P1 was reported as a new promising strain for control of Aspergillus carbonarius, a known producer of ochratoxin A, categorized as possible human carcinogen with high nephrotoxic potential. Grape quality can be influenced by vineyard management practices, including the use of fungal control agents. The aim of this study was to evaluate, for the first time, the quality parameters of Chardonnay grapes exposed to an antifungal Bacillus-based strategy for control of A. carbonarius, supporting findings by genomic investigations. Furthermore, genomic tools were used to confirm that the strain P1 belongs to the non-pathogenic species Bacillus velezensis and also to certify its biosafety. The genome of B. velezensis P1 harbors genes that are putatively involved in the production of volatiles and hydrolytic enzymes, which are responsible for releasing the free form of aroma compounds. In addition to promote biocontrol of phytopathogenic fungi and ochratoxins, the treatment with B. velezensis P1 did not change the texture (hardness and firmness), color and pH of the grapes. Heat map and hierarchical clustering analysis (HCA) of volatiles evaluated by GC/MS revealed that Bacillus-treated grapes showed higher levels of compounds with a pleasant odor descriptions such as 3-hydroxy-2-butanone, 2,3-butanediol, 3-methyl-1-butanol, 3,4-dihydro-ß-ionone, ß-ionone, dihydroactinidiolide, linalool oxide, and ß-terpineol. The results of this study indicate that B. velezensis P1 presents desirable properties to be used as a biocontrol agent.

UV-ARTP compound mutagenesis breeding improves macrolactins production of Bacillus siamensis and reveals metabolism changes by proteomic.

Macrolactins are a type of compound with complex macrolide structure which mainly be obtained through microbiological fermentation now. They have excellent antifungal, antibacterial and antitumor activity. In order to improve macrolactins production, Bacillus siamensis YB304 was used as the research object, and a mutant Mut-K53 with stable genetic characters was selected by UV-ARTP compound mutagenesis. The yield of macrolactins was 156.46 mg/L, 3.95 times higher than original strain. The metabolic pathway changes and regulatory mechanism of macrolactins were analyzed by quantitative proteomics combined with parallel reaction monitoring. This study revealed that 1794 proteins were extracted from strain YB304 and strain Mut-K53, most of them were related to metabolism. After UV-ARTP compound mutagenesis treatment, the expression of 628 proteins were significantly changed, of which 299 proteins were significantly up-regulated. KEGG pathway analysis showed that differentially expression proteins mainly distributed in biological process, cellular component, and molecular function processing pathways. Such as utilization of carbon sources, glycolysis pathway, and amino acid metabolism pathway. Furthermore, key precursor substances such as acyl-CoA and amino acids of macrolactin biosynthesis are mostly up-regulated, which are one of the main reasons for increased production of macrolactin.This study will provide a new way to increase the yield of macrolactins through mutagenesis breeding and proteomics.

Culex quinquefasciatus membrane-bound alkaline phosphatase is a putative receptor for Lysinibacillus sphaericus Tpp49Aa1 toxin.

The binary toxin Cry48Aa1/Tpp49Aa1 produced by Lysinibacillus sphaericus exhibits potent toxicity against Culicidae larvae. Both Cry48Aa1 and Tpp49Aa1 toxins are crucial for binding to the toxin receptor in Culex quinquefasciatus larvae, albeit with different binding sites. Previous studies have identified Glu71, a membrane-bound α-glucosidase, as a putative binding protein for the Cry48Aa1 toxin, involved in the Cry48Aa1/Tpp49Aa1 toxicity. In this study, we employed pulldown assays to identify a group of Tpp49Aa1-binding proteins from C. quinquefasciatus solubilized midgut brush-border membrane proteins (BBMFs). RNA interference assays revealed that the silencing of an alkaline phosphatase gene (referred to as ALP1263) in C. quinquefasciatus resulted in a significant reduction in larval mortality upon exposure to Cry48Aa1/Tpp49Aa1 toxin in vivo. Furthermore, the ALP1263 protein exhibited specific and high-affinity binding to the Tpp49Aa1 toxin, with a dissociation constant (Kd) of approximately 57.3 nM. The dot blot analysis demonstrated that Tpp49Aa1 C-terminal region was essential for its interaction with the ALP1263 protein. In summary, our findings establish ALP1263 as a functional receptor for Tpp49Aa1 and emphasize its role in the toxicity of Cry48Aa1/Tpp49Aa1.

Two Antimicrobial Peptides Derived from Bacillus and Their Properties.

Growth promotion and disease prevention are important strategies in the modern husbandry industry, and for this reason, antibiotics are widely used as animal feed additives. However, the overuse of antibiotics has led to the serious problem of increasing resistance of pathogenic microorganisms, posing a major threat to the environment and human health. "Limiting antibiotics" and "Banning antibiotics" have become the inevitable trends in the development of the livestock feed industry, so the search for alternative antimicrobial agents has become a top priority. Antimicrobial peptides (AMPs) produced by Bacillus spp. have emerged as a promising alternative to antibiotics, due to their broad-spectrum antimicrobial activity against resistant pathogens. In this study, two strains of Bacillus velezensis 9-1 and B. inaquosorum 76-1 with good antibacterial activity were isolated from commercial feed additives, and the antimicrobial peptides produced by them were purified by ammonium sulfate precipitation, anion exchange chromatography, gel chromatography, and RP-HPLC. Finally, two small molecule peptides, named peptide-I and peptide-II, were obtained from strain 9-1 and 76-1, respectively. The molecular weight and sequences of the peptides were analyzed and identified by LC-MS/MS, which were 988.5706 Da and VFLENVLR, and 1286.6255 Da and FSGSGSGTAFTLR, respectively. The results of an antibacterial activity and stability study showed that the two peptides had good antibacterial activity against Staphylococcus aureus, B. cereus, and Salmonella enterica, and the minimum inhibitory concentrations were 64 µg/mL and 16 µg/mL, 32 µg/mL and 64 µg/mL, and 8 µg/mL and 8 µg/mL, respectively. All of them have good heat, acid, and alkali resistance and protease stability, and can be further developed as feed antibiotic substitutes.

Characterization and genomic insight of surfactin-producing Bacillus velezensis and its biocontrol potential against pathogenic contamination in lettuce hydroponics.

Due to food borne pathogen, maintaining the viability of fresh fruits and vegetable is a great concern. Several strategies including microbial and plant-based formulations to reduce their infection and maintain quality of the fresh food are in practice. Currently, Bacillus has gained significant traction as a biocontrol agent for regulating diseases affecting a variety of agricultural and horticultural crops. Food-grade citric acid and plant growth-promoting rhizobacteria (PGPR) were used as antimicrobial agent, MIC results showed that PGPR (14.87 mm) and CA (20.25 mm) exhibited notable antimicrobial activity against E. coli. Lettuce treated with PGPR showed reduction in E. coli contamination, E. coli was detected at 3.30, 3.68 in control, and 2.7 log CFU/g in random root injury lettuce inoculated with PGPR KACC 21110 respectively. Random root injury showed a trend toward increasing E. coli internalization. The strains exhibited resistance to multiple antibiotics, including Imipenem, tetracycline, ampicillin, cefotaxime, cefoxitin, and ceftriaxone. Comprehensive data analysis revealed the presence of ten putative bacteriocin or bacteriocin-like gene clusters. The structure of lipopeptide homologs was characterized by using QTOF-MS/MS. The mass ion peaks attributed to surfactin homologs, surfactin A ion at m/z 1008.66, surfactin B, C at m/z 1022.67 and 1036.69. In addition to surfactin, a polyketide oxydifficidin and lipopeptide NO were extracted and detected from the extract of B. velezensis. Both isolates are key biocontrol agents and have significant potential in combating foodborne pathogens and can be utilized to explore novel antibacterial products for preventing pathogens in fresh produce.

Glycosylated and Succinylated Macrocyclic Lactones with Amyloid-ß-Aggregation-Regulating Activity from a Marine Bacillus sp.

Two new glycosylated and succinylated macrocyclic lactones, succinyl glyco-oxydifficidin (1) and succinyl macrolactin O (2), were isolated from a Bacillus strain collected from an intertidal mudflat on Anmyeon Island in Korea. The planar structures of 1 and 2 were proposed using mass spectrometric analysis and NMR spectroscopic data. The absolute configurations of 1 and 2 were determined by optical rotation, J-based configuration analysis, chemical derivatizations, including the modified Mosher's method, and quantum-mechanics-based calculation. Biological evaluation of 1 and 2 revealed that succinyl glyco-oxydifficidin (1) inhibited/dissociated amyloid ß (Aß) aggregation, whereas succinyl macrolactin O (2) inhibited Aß aggregation, indicating their therapeutic potential for disassembling and removing Aß aggregation.

Composition and activity of antifungal lipopeptides produced by Bacillus spp. in daqu fermentation.

Daqu is a solid-state fermentation and saccharification starter for the Chinese liquor baijou. During the daqu stage, amylolytic and proteolytic enzymes are produced by Bacillus and fungi. Bacillus spp. also produce lipopeptides with a broad spectrum of antimicrobial activities but direct evidence for their impact on community assembly in daqu is lacking. This study aimed to study the interaction between Bacillus spp. and fungi in daqu models. The antifungal activity of surfactin, fengycin, and iturin A was initially assessed in vitro. Iturin A displayed the strongest antifungal activity (MIC = 10-50 mg/L). In situ antifungal activity of B. amyloliquefaciens and B. velezensis against molds was observed in a simple daqu model inoculated with single strains of Bacillus species. Formation of lipopeptides in situ was supported by quantification of mRNA encoding for enzymes for surfactin, fengycin, and iturin A biosynthesis. In situ antifungal activity of Bacillus species was also observed in a complex daqu model that was inoculated with 8 bacterial or fungal strains plus one of the three strains of Bacillus. A relationship of lipopeptides to in situ antifungal activity was further supported by detection of the lipopeptides by liquid chromatography coupled to mass spectrometry. Both results indicated that B velezensis FUA2155 had higher antifungal activity in the daqu model, and was the only strain that produced multiple iturin A congeners in situ. Taken together, this study provides evidence that production of lipopeptides by Bacillus species in daqu may impact community assembly and hence product quality.

Nematicidal lipopeptides from Bacillus paralicheniformis and Bacillus subtilis: A comparative study.

The aim of this work was to develop a comparative study between Bacillus paralicheniformis TB197 and B. subtilis ATCC 21332 strains in terms of growth, cyclic lipopeptide production, nematicidal activity, and active lipopeptide characteristics. Crude lipopeptide extracts (CLEs) from their fermentation broths were obtained, and their nematicidal activity (NA) was estimated as the mean lethal dose (LD50), employing Caenorhabditis elegans. Using a bioguided approach, CLE components were fractionated by semipreparative thin layer chromatography, and active lipopeptides were characterized by mass spectrometry. Both strains produced similar concentrations of CLEs (p ≥ 0.05) (0.99 ± 0.11 and 1.14 ± 0.15 mg/mL by TB197 and ATCC 21332, respectively). The estimated LD50 values of CLEs from the TB197 and ATCC 21332 strains were 3.88 and 8.15 mg/mL, respectively, showing that the NA of the TB197 strain CLE was 2.1-fold higher (p ≤ 0.05). Mass spectrometry revealed that strain TB197 synthesizes several families of lipopeptides, namely, fengycin A (C14-C17), fengycin B (C16-C17), surfactin (C15-C17), and lichenysin (C12, C13, C14, and C16), from which fengycins and lichenysins possess the highest NA (100 and 60% mortality in C. elegans larvae, respectively), while the ATCC 21332 strain produces mainly surfactin (C13-C17) (NA 63% mortality). The main differences found in this study were that the TB197 strain has a higher tolerance to inhibition by the product, and the lipopeptides they synthesize have a higher nematicidal activity due to the diversity of families compared to ATCC 21332. Likewise, it was shown that more polar lipopeptides (fengycins) are more effective at causing mortality in C. elegans larvae. KEY POINTS: • The nematicidal activity of lipopeptides from TB197 is higher than from ATCC 21332 • TB197 produces surfactin, lichenysin, and fengycin, while ATCC 21332 mainly produces surfactin • The most polar lipopeptides (fengycins) cause more mortality in C. elegans L2.

Seaweed-associated heterotrophic Bacillus altitudinis MTCC13046: a promising marine bacterium for use against human hepatocellular adenocarcinoma.

Since the report of the antibiotic with anticancer properties, scientists have been focusing to isolate and characterize novel anti-microbial natural products possessing anticancer activities. The current study describes the production of seaweed-associated heterotrophic Bacillus altitudinis MTCC13046 with potential anticancer properties. The bacterium was screened for its capacity to diminish the cell proliferation of the human hepatocellular adenocarcinoma (HepG2) cell line, without upsetting the normal cells. The bacterial extract showed anticancer properties in a dose-reactive form against HepG2 (IC50, half maximal inhibitory concentration ~ 29.5 µg/ml) on tetrazolium bromide analysis with less significant cytotoxicity on common fibroblast (HDF) cells (IC50 ~ 77 µg/ml). The potential antioxidant ability of the organic extract of B. altitudinis MTCC13046 (IC90 133 µg/ml) could corroborate its capacity to attenuate the pathophysiology leading to carcinogenesis. The results of the apoptosis assay showed that the crude extracts of B. altitudinis maintained 68% viability in normal cells compared to 11% in the cancer cells (IC50 76.9 µg/ml). According to the findings, B. altitudinis MTCC13046 could be used to develop prospective anticancer agents.

Antifungal potential of biosurfactants produced by strains of Bacillus spp. (Bacillales: Bacillaceae) selected by molecular screening / Potencial antifúngico de biosulfactantes producidos por cepas de Bacillus spp. (Bacillales: Bacillaceae) seleccionadas por tamizaje molecular

Introduction: Bacillus species are used as biological controllers for phytopathogenic fungi, and the mechanisms to produce controllers include biosynthesis of lipopeptide biosurfactants with antifungal activity. Objective: To evaluate the antifungal potential of the biosurfactants produced by Bacillus strains, selected by molecular screening, on Fusarium oxysporum. Methods: We selected four molecular markers, related to the biosynthesis of surfactin, fengicin, and lichenysin (srfA, spf, fenB, LichAA) in nine Bacillus strains. We used two mineral media with several culture conditions, for biosurfactant production, and a well diffusion test for antifungal potential. Results: Only the biosurfactant produced by UFAB25 inhibits the mycelial growth of F. oxysporum (44 % ± 13): this biosurfactant was positive for srfA, spf, and fenB genes involved in the synthesis of surfactin and fengicine. Antifungal activity depends on culture conditions and the strain. Conclusions: Genetic markers are useful to detect strains with antifungal potential, facilitating the selection of bio-controllers. The biosurfactant profile is influenced by the strain and by culture conditions.

Introducción: Especies de Bacillus han sido empleadas como controladores biológicos contra hongos fitopatógenos. Entre los mecanismos utilizados se destaca la biosíntesis de biosurfactantes lipopeptídicos con actividad antifúngica. Objetivo: Evaluar el potencial antifúngico de los biosurfactantes producidos por cepas Bacillus nativas, previamente seleccionadas mediante tamizaje molecular, sobre Fusarium oxysporum. Métodos: Se utilizaron cuatro marcadores moleculares, relacionados con la biosíntesis de surfactina, fengicina y liquenisina (srfA, spf, fenB, LichAA) sobre nueve cepas de Bacillus. Se utilizaron dos medios minerales con diferentes condiciones de cultivo para la producción del biosurfactante. Se evaluó el potencial antifúngico de los biosurfactantes mediante la prueba de difusión en pozos. Resultados: Se determinó que solo el biosurfactante producido por UFAB25 actúa como inhibidor del crecimiento micelial de Fusarium oxysporum (43.6 % ± 13), esta cepa es positiva para los genes srfA, spf y fenB, involucrados en la síntesis de surfactina y fengicina. La actividad antifúngica depende de las condiciones de cultivo y la cepa. Conclusiones: Los marcadores genéticos ayudan a detectar cepas con potencial antifúngico, facilitando la selección de biocontroladores. El perfil del biosurfactante está influenciado no solo por la cepa, sino también por las condiciones del cultivo.

Antibacterial activity against enterovirulent Escherichia coli strains from Bacillus amyloliquefaciens B31 and Bacillus subtilis subsp. subtilis C4: MALDI-TOF MS profiling and MALDI TOF/TOF MS structural analysis on lipopeptides mixtures.

In this work, two Bacillus strains isolated from honey (Bacillus subtilis subsp. subtilis C4; access code HQ828992) and from a waste of an artisanal tannery (Bacillus amyloliquefaciens B31; access code KP893752) were evaluated in order to determine their antibacterial activity against five enteropathogenic Escherichia coli strains. The number of viable cultivable cells of the different strains of E. coli analyzed was determined by plate count. The crude cell-free supernatants of both Bacillus strains exerted anti-E. coli activities, whereas only the lipopeptide fraction of B31 had significant E. coli inhibition. The lipopeptides produced by the Bacillus were analyzed using matrix-assisted laser desorption-ionization mass spectrometry (MALDI MS). The analysis was conducted combining the profiles (fingerprints) of the lipopeptides mixture and the individual lipopeptide fragmentation (tandem mass spectrometry [MS/MS] mode), both obtained from the same lipopeptides mixture sample, for higher output. Data obtained from C4 and B31 revealed that surfactin homologues were the most abundant lipopeptides produced by both strains studied. Additionally, kurstakin, iturin, and fengycin homologues were detected. Using the MS/MS mode, it was demonstrated that isobar compounds belonging to different families were produced by each Bacillus strain (e.g., C-16 bacillomycin D was detected in B31 samples, meanwhile C-15 iturin C was detected in C4). MS/MS analysis contributed with relevant information about the type of lipopeptides synthesized by Bacillus strains studied in this work.

Metabolomic and Lipidomic Profiling of Bacillus Using Two-Dimensional Tandem Mass Spectrometry.

Lipidomic and metabolomic profiles of sporulated and vegetative Bacillus subtilis and Bacillus thuringiensis from irradiated lysates were recorded using a quadrupole ion trap mass spectrometer modified to perform two-dimensional tandem mass spectrometry (2D MS/MS). The 2D MS/MS data domains, acquired using a 1.2 s scan of negative ions generated by nanoelectrospray ionization of microwave irradiated spores, showed the presence of dipicolinic acid (DPA) as well as various lipids. Aside from microwave radiation to extract DPA and lipids from spores, sample preparation was minimal. Characteristic lipid and metabolic profiles were observed using 107─108 cells of the two Bacillus species. Major features of the lipid profiles observed for the vegetative states included sets of phosphatidylglycerol (PG) lipids. Product ion spectra were extracted from the 2D MS/MS data, and they provided structural information on the fatty acid components of the PG lipids. The study demonstrates the flexibility, speed, and informative power of metabolomic and lipidomic fingerprinting for identifying the presence of spore-forming biological agents using 2D MS/MS as a rapid profiling screening method.

The evaluation of Bacillus-secreted polyglutamic acid as anti-scaling treatment for circulating cooling water.

Polyaspartic acid is considered a green agent for the treatment of circulating cooling water. However, its chemosynthetic process is not green, as it requires significant amounts of energy and causes water pollution. In this work, we identified an analog of polyaspartic acid, namely polyglutamic acid (γ-PGA), which could be directly produced by Bacillus spp., and we explored its performance and scale inhibition mechanism as a scale inhibitor. We found that γ-PGA secreted by B. megaterium with a molecular weight of ~ 70 kDa showed poor scale inhibition, while the γ-PGA secreted by B. licheniformis with a molecular weight of ~ 15 kDa had a 26.87% higher efficiency compared to commercially available polyaspartic acid. The scale inhibition mechanism was explored using the γ-PGA material secreted by B. licheniformis. Fourier transform spectrometer, field emission scanning electron microscopy, and X-ray photoelectron spectroscopy analysis demonstrated that the scale inhibition performance of γ-PGA was due to the combination of its functional groups and Ca2+, which affected the growth process of CaCO3 and inhibited the formation of CaCO3. This study provided deeper insight into scale inhibition performance related to the scale inhibition mechanism.

Biocontrol Ability of the Bacillus amyloliquefaciens Group, B. amyloliquefaciens, B. velezensis, B. nakamurai, and B. siamensis, for the Management of Fungal Postharvest Diseases: A Review.

The Bacillus amyloliquefaciens group, composed of B. amyloliquefaciens, B. velezensis, B. nakamurai, and B. siamensis, has recently emerged as an interesting source of biocontrol agents for the management of pathogenic fungi. In this review, all the reports regarding the ability of these species to control postharvest fungal diseases have been covered for the first time. B. amyloliquefaciens species showed various antifungal mechanisms, including production of antifungal lipopeptides and volatile organic compounds, competition for nutrients, and induction of disease resistance. Most reports discussed their use for the control of fruit diseases. Several strains were studied in combination with additives, improving their inhibitory efficacies. In addition, a few strains have been commercialized. Overall, studies showed that B. amyloliquefaciens species are a suitable environmentally friendly alternative for the control of postharvest diseases. However, there are still crucial knowledge gaps to improve their efficacy and host range.