Bioassay-Guided Identification of Natural Products for Biocontrol by Thin Layer Chromatography-Direct Bioautography.

Thin layer chromatography-direct bioautography (TLC-DB) is a well-established bioassay used to separate and identify natural products (NPs) that are antagonistic against a target pathogen. It is a rapid, inexpensive, and simple option for the bioassay-guided isolation and identification of NPs that hinges on separation by TLC coupled with the direct application of a target pathogen to examine bioactivity. It is typically used for the analysis of bioactive plant extracts, detecting inhibitory activity against bacteria, fungi, and enzymes. That being said, it has great potential in bacterial NP discovery, particularly for evaluating bacterial NPs against pertinent agricultural pathogens, which is valuable for discovering and developing novel biopesticides for the agriculture industry. Furthermore, it is a tunable protocol that could be applied to other target pathogens or sources of NPs in research programs concerning the discovery and identification of bioactive compounds. Herein, we describe a model system for discovering and identifying biopesticide NPs using TLC-DB with Bacillus spp. and the agricultural pathogen Sclerotinia sclerotiorum.

Characterization of Exopolysaccharides Having Potential Antiviral Properties from Priestia Aryabattai Strain MK1 and Bacillus sp. Strain MK2.

Viral diseases are a serious threat to humans while the most antiviral drugs have low efficiency and side effects on human health. Therefore, using microbial biopolymers as the drugs alternate to treat viral infections seems cost-effective and human friendly option. In the present study, thirty-four exopolysaccharides (EPSs) producing bacteria were isolated, and EPSs production capacity of five salt-tolerant isolates was determined under 0, 100 and 150 mM NaCl. Among these, two isolates exhibiting high anti-coliphage activity were identified through 16S rRNA gene analysis. Moreover, the EPSs were characterized by Fourier-transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) analysis, and their composition was determined. Five salt-tolerant bacteria (MK1, MK2, MK10, MK22 and MK29) exhibited higher production of EPSs at 100 mM NaCl compared to that under non-saline control. At 100 mM NaCl, the yield of EPSs ranged between 105 and 330 mg 100 mL-1 broth. The EPSs produced by the isolates MK1 and MK2 exhibited higher anti-coliphage activity (plaque forming unit decreased from 43 × 106 mL-1 to 3 × 106 and 4 × 106 mL-1, respectively), and were comprised of glucose, fructose, galactose, sucrose, lactose and xylose sugars. FTIR spectroscopy depicted that EPSs are mainly composed of hydroxyl, aliphatic, carboxyl, sulfate and phosphate functional groups, which could have bound coliphage and thus conferred higher anti-coliphage activities to the EPSs. Phylogenetic analysis revealed that MK1 and MK2 isolates formed clades within genus Priestia and Bacillus sequences, respectively. High EPSs production capacity of bacterial isolates under saline condition and high anti-coliphage activity of the EPSs implies that bacterial biopolymers could be useful in antiviral drugs therapy.

Microscopic and Transcriptomic Analyses to Elucidate Antifungal Mechanisms of Bacillus velezensis TCS001 Lipopeptides against Botrytis cinerea.

Botrytis cinerea is an important fungal pathogen that causes gray mold disease in plants. Previously, Bacillus velezensis TCS001 live culture presented broad-spectrum antifungal activity against various plant pathogenic fungi and oomycetes, particularly B. cinerea. Here, the bioactivity of lipopeptides produced by TCS001 against B. cinerea was investigated. The IC50 values of the crude lipopeptide extract (CLE) from TCS001 to suppress mycelial growth and conidial germination were 14.20 and 49.39 mg/L, respectively. SEM and TEM imaging revealed that CLE caused morphological deformities and ultrastructural changes in the mycelium. Transcriptomic analyses combined with ΔBcpsd mutant construction demonstrated that the CLE could confer antifungal activity via suppressing Bcpsd expression in the pathogen. In addition, the CLE activated the plant immune system by increasing the content of defense-related enzymes and the expression of marker genes in immunity signaling pathways in cucumber plants. Therefore, TCS001 CLE could be potentially developed into biopesticides for the biocontrol of gray mold disease.

Exploring the protective role of Bacillus velezensis BV1704-Y in zebrafish health and disease resistance against Aeromonas hydrophila infection.

Bacillus genus, particularly Bacillus velezensis, is increasingly considered as viable alternatives to antibiotics in aquaculture due to their safety and probiotic potential. However, the specific mechanisms through which probiotic B. velezensis confers protection against Aeromonas hydrophila infection in fish remain poorly understood. This study delved into the multifaceted impacts of B. velezensis BV1704-Y on diverse facets of zebrafish health, including gut barrier function, immune response, oxidative stress, gut environment, microbiome composition, and disease resistance. Our findings demonstrate that supplementation with B. velezensis BV1704-Y significantly alleviated symptoms and reduced mortality in zebrafish infected with A. hydrophila. Furthermore, a notable reduction in the expression of pivotal immune-related genes, such as IL-1ß, IL6, and TNF-α, was evident in the gut and head kidney of zebrafish upon infection. Moreover, B. velezensis BV1704-Y supplementation resulted in elevated activity levels of essential antioxidant enzymes, including SOD, CAT, and GSH, in gut tissue. Notably, B. velezensis BV1704-Y positively modulated the structure and function of the intestinal microbiome, potentially enhancing immune response and resilience in zebrafish. Specifically, supplementation with B. velezensis BV1704-Y promoted the relative abundance of beneficial bacteria, such as Cetobacterium, which showed a noteworthy negative correlation with the expression of pro-inflammatory genes and a positive correlation with gut barrier-related genes. Altogether, our study suggests that B. velezensis BV1704-Y holds promise as an effective probiotic for protecting zebrafish against A. hydrophila infection, offering potential benefits for the aquaculture industry.

Effects of branched-chain amino acids on surfactin structure and antibacterial activity in Bacillus velezensis YA215.

Antibiotics are essential for combating pathogens; however, their misuse has led to increased resistance, necessitating the search for effective, low-toxicity alternatives. Surfactin, a cyclic lipopeptide with a C12-C17 ß-hydroxy fatty acid chain, exhibits significant antibacterial activity and resists resistance, making it a research focus. Nonetheless, the effects of branched-chain amino acids (BCAAs) on surfactin's structure and activity are not well understood. This study examines the influence of BCAAs (L-valine, L-leucine, and L-isoleucine) on the lipopeptide (surfactin) produced by Bacillus velezensis YA215. Process optimization shows that adding 1 g/L of L-Leu and L-Ile, and 0.5 g/L of L-Val, maximized surfactin production to 18.59%, 19.23%, and 20.64%, respectively. Surfactin content peaked at 36 h with L-Val and L-Ile, yielding 19.72% and 11.37%. In contrast, L-Leu addition peaked at 24 h, yielding 11.33%. Notably, L-Val supplementation resulted in the highest relative surfactin content. Antimicrobial testing demonstrated that BCAAs significantly enhance the antibacterial effects of lipopeptides against Escherichia coli and Staphylococcus aureus, with Val showing the most pronounced effect. The addition of BCAAs notably altered the composition of surfactin fatty acid chains. Specifically, Val increased the proportions of iso C14 and iso C16 ß-hydroxy fatty acids from 13.3% and 4.216-23.803% and 8.31%, respectively. Additionally, the amino acid composition at the 7th position of the peptide chain changed significantly, especially with Val addition, which increased the proportion of C14 [Val 7] surfactin by 3.29 times. These structural changes are likely associated with the enhanced antibacterial activity of surfactin. These findings provide valuable insights into the roles of BCAAs in microbial fermentation, underscoring their importance in metabolic engineering to enhance the production of bioactive compounds.

Boosting resistant starch in rice: Bacterial inulin as a metabolic and glucose uptake modulator.

Bacillus stercoris PSSR12 (B. stercoris PE), an isolate from rice field soils, was identified via 16s rRNA sequencing. The synthesis of the inulin and inulin producing enzyme (IPE) in B. stercoris PE was verified using SDS-PAGE and FTIR. This study aimed to assess the impact of B. stercoris PE treatment on in vitro inhibition of α-amylase and α-glucosidase from traditional and commercial rice varieties of South India. Additionally, the study investigated enzymatic inhibition and mRNA expression of starch synthesis genes (RAmy1a, GBSSIa, SBEIIa, and SBEIIb). Glucose transporter gene expression (GLUT1 and GLUT4) patterns were analyzed in 3T3-L1 adipocytes to evaluate glucose uptake in B. stercoris PE treated rice varieties. The application of B. stercoris PE enhanced grain quality by imparting starch ultra-structural rigidity, inhibiting starch metabolizing enzymes, and inducing molecular changes in starch synthesis genes. This approach holds promise for managing type II diabetes mellitus and potentially reducing insulin dependence.

Cryo-EM Structure of the Mnx Protein Complex Reveals a Tunnel Framework for the Mechanism of Manganese Biomineralization.

The global manganese cycle relies on microbes to oxidize soluble Mn(II) to insoluble Mn(IV) oxides. Some microbes require peroxide or superoxide as oxidants, but others can use O2 directly, via multicopper oxidase (MCO) enzymes. One of these, MnxG from Bacillus sp. strain PL-12, was isolated in tight association with small accessory proteins, MnxE and MnxF. The protein complex, called Mnx, has eluded crystallization efforts, but we now report the 3D structure of a point mutant using cryo-EM single particle analysis, cross-linking mass spectrometry, and AlphaFold Multimer prediction. The ß-sheet-rich complex features MnxG enzyme, capped by a heterohexameric ring of alternating MnxE and MnxF subunits, and a tunnel that runs through MnxG and its MnxE3F3 cap. The tunnel dimensions and charges can accommodate the mechanistically inferred binuclear manganese intermediates. Comparison with the Fe(II)-oxidizing MCO, ceruloplasmin, identifies likely coordinating groups for the Mn(II) substrate, at the entrance to the tunnel. Thus, the 3D structure provides a rationale for the established manganese oxidase mechanism, and a platform for further experiments to elucidate mechanistic details of manganese biomineralization.

Dietary Bacillus velezensis KNF-209 supplementation improves growth performance, enhances immunity, and promotes gut health in broilers.

This study aimed to investigate the effects of dietary Bacillus velezensis KNF-209 (BV-KNF-209) on the growth performance, immunity, and gut health of broilers. A total of 540 one-day-old male Cobb-500 broilers were randomly divided into 5 groups of 6 replicates with 18 broilers per replicate. Dietary treatments were corn-soybean meal basal diets supplemented with 0, 50, 100, 200, and 400 mg/kg BV-KNF-209 (CON, BV 50, BV 100, BV 200, and BV 400 groups, respectively) for 42 d. Compared with the CON group, the average daily gains (ADG) at 0 to 42 d in the BV 100 and BV 200 groups were significantly increased (P < 0.01), and the feed-to-gain (F:G) ratios were significantly decreased at 0 to 21 d (P < 0.01) and 0 to 42 d (P < 0.05). The BV 200 and BV 400 groups had higher serum immunoglobulin M (IgM) levels at d 21 and 42 (P < 0.05). The serum levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) were significantly decreased in the BV 50, BV 100, and BV 200 groups at d 21 (P < 0.05), and serum IL-1ß and IL-6 levels were also reduced in the BV 100 and BV 200 groups at d 42 (P < 0.05). Meanwhile, increased interleukin-10 (IL-10) levels in the jejunal and ileal mucosa at d 42 were observed in the BV 100, BV 200, and BV 400 groups (P < 0.05), while the IL-1ß and IL-6 levels (P < 0.01) were decreased. The BV 200 and BV 400 groups showed significantly higher activities of lipase and trypsin (P < 0.05) in jejunal digesta as well as higher activities of amylase and trypsin (P < 0.01) in ileal digesta at d 42. The cecal acetic acid and propionic acid levels in the BV groups and lactic acid levels in the BV 50, BV 100, and BV 200 groups (P < 0.05) were significantly higher compared to those in the CON group. Overall, dietary BV-KNF-209 supplementation significantly improved broiler growth performance, an effect that may have been achieved by heightening immunity, increasing digestive enzyme activity, and raising intestinal short-chain fatty acids and lactic acid levels.

Unveiling novel insights into Bacillus velezensis 16B pectin lyase for improved fruit juice processing.

Microbial pectinolytic enzymes are important in various industries, particularly food processing. This study focuses on uncovering insights into a novel pectin lyase, BvPelB, from Bacillus velezensis 16B, with the aim of enhancing fruit juice processing. The study examines the structural and functional characteristics of pectinolytic enzyme, underscoring the critical nature of substrate specificity and enzymatic reaction mechanisms. BvPelB was successfully expressed and purified, exhibiting robust activity under alkaline conditions and thermal stability. Structural analysis revealed similarities with other pectin lyases, despite limited sequence identity. Biochemical characterization showed BvPelB's preference for highly methylated pectins and its endo-acting mode of cleavage. Treatment with BvPelB significantly increased juice yield and clarity without generating excessive methanol, making it a promising candidate for fruit juice processing. Overall, this study provides valuable insights into the enzymatic properties of BvPelB and its potential industrial applications in improving fruit juice processing efficiency and quality.

Screening and effects of intestinal probiotics on growth performance, gut health, immunity, and disease resistance of Nile tilapia (Oreochromis niloticus) against Streptococcus agalactiae.

In the present study, 59 autochthonous bacteria were isolated from the intestine of tilapia. Following enzyme producing activity, antagonistic ability, hemolytic activity, drug sensitivity assessments, and in vivo safety evaluation, 7 potential probiotic strains were screened out: Bacillus tequilensis BT0825-2 (BT), Bacillus aryabhattai BA0829-3 (BA1), Bacillus megaterium BM0505-6 (BM), Bacillus velezensis BV0505-11 (BV), Bacillus licheniformis BL0505-18 (BL), B. aryabhattai BA0505-19 (BA2), and Lactococcus lactis LL0306-15 (LL). Subsequently, tilapia were fed basal diets (CT) and basal diets supplemented with 108 CFU/g of BT, BA1, BM, BV, BL, BA2 and LL, respectively. After 56 days of continuous feeding, the growth parameters (weight gain, final weight, and specific growth rate) showed significant improvement (p < 0.05) in both BM and BA2 groups. The total cholesterol and triglycerides of serum were significantly decreased in BV and LL groups (p < 0.05). The superoxide dismutase, glutathione reductase, and lysozyme of BV, BA2 and LL groups were increased, and the malondialdehyde of BV group was significantly decreased. The villous height and amylase of midgut were increased in BV, BA2 and LL groups. In addition, the expression levels of ZO-1 and occludin genes in the midgut of tilapia were enhanced in BM, BV, BA2 and LL groups. The supplementation of probiotics reduced the abundance of Cyanobacteria and increased the abundance of Actinobacteria at the phylum level. At the genus level, the addition of probiotics increased the abundance of Romboutsia. Furthermore, improvement in the expression of immune-related genes were observed, including interleukin 1ß, interleukin 10, tumor necrosis factor alpha, and transforming growth factor beta (p < 0.05). After challenging with S. agalactiae, the survival rates of BV, BA2 and LL groups were significantly higher than CT group (p < 0.05). Above results indicated that BM, BA2, BV and LL improved growth performance, gut health or immunity of tilapia, which can be applied in tilapia aquaculture.

Novel microbial fermentation for the preparation of iron-chelating scallop skirts peptides-its profile, identification, and possible binding mode.

Iron chelating peptides have been widely utilized as iron supplements due to their excellent absorption capacity, However, the high cost and cumbersome manufacturing process of these peptides significantly limit their industrial application. In this study, fermentation was used for the first time to prepare iron chelating peptides. Bacillus altitudinis 3*1-3 was selected as the most suitable strain from 50 strains. The hydrolysates of fermented scallop skirts showed excellent iron-chelating capacity (9.39 mg/g). Aspartic acid, glutamic acid, and histidine are crucial for the binding of peptides to ferrous ions. The heptapeptide (FEDPEFE) forms six binding bonds with ferrous irons. Compared with ferrous sulfate, peptide-ferrous chelate showed more stability in salt solution and simulated gastrointestinal juice (p < 0.05). Furthermore, the fermentation method could save >50% of the cost compared with the enzymatic method. The results can provide a theoretical basis for the preparation of ferrous-chelated peptides using the fermentation method.

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.

Assessment of antimicrobial activity and GC-MS using culture filtrate of local marine Bacillus strains.

Secondary metabolites produced by Bacillus species from marine sources encompass a variety of compounds such as lipopeptides, isocoumarins, polyketides, macrolactones, polypeptides and fatty acids. These bioactive substances exhibit various biological activities, including antibiotic, antifungal, antiviral, and antitumor properties. This study aimed to isolate and identify a particular species of Bacillus from marine water and organisms that can produce bioactive secondary metabolites. Among the 73 Bacillus isolates collected, only 5 exhibited antagonistic activity against various viral and bacterial pathogens. The active isolates were subjected to 16S rRNA sequencing to determine their taxonomical affiliation. Among them, Bacillus tequilensis CCASU-2024-66 strain no. 42, with the accession number ON 054302 in GenBank, exhibited the highest inhibitory potential. It displayed an inhibition zone of 21 mm against Bacillus cereus while showing a minimum zone of inhibition of 9 mm against Escherichia coli and gave different inhibition against pathogenic fungi, the highest inhibition zone 15 mm against Candida albicans but the lowest inhibition zone 10 mm was against Botrytis cinerea, Fusarium oxysporum. Furthermore, it demonstrated the highest percentage of virucidal effect against the Newcastle virus and influenza virus, with rates of 98.6% and 98.1%, respectively. Furthermore, GC-MS analysis was employed to examine the bioactive substance components, specifically focusing on volatile and polysaccharide compounds. Based on these results, Bacillus tequilensis strain 42 may have the potential to be employed as an antiviral agent in poultry cultures to combat Newcastle and influenza, two extremely destructive viruses, thus reducing economic losses in the poultry production sector. Bacteria can be harnessed for the purpose of preserving food and controlling pathogenic fungi in both human and plant environments. Molecular docking for the three highly active derivatives 2,3-Butanediol, 2TMS, D-Xylopyranose, 4TMS, and Glucofuranoside, methyl 2,3,5,6-tetrakis-O-(trimethylsilyl) was carried out against the active sites of Bacillus cereus, Listeria monocytogenes, Candida albicans, Newcastle virus and influenza virus. The data obtained from molecular docking is highly correlated with that obtained from biology. Moreover, these highly active compounds exhibited excellent proposed ADMET profile.

Effect of a phthalate derivative purified from Bacillus zhangzhouensis SK4 on quorum sensing regulated virulence factors of Pseudomonas aeruginosa.

Pseudomonas aeruginosa causes life-threatening diseases and is resistant to almost all conventional antibiotics. The quorum sensing (QS) system of P. aeruginosa contributes to many pathogenic factors some of which are pigment production, motility, and biofilm. The disruption of quorum sensing system may be an impactful strategy to deal with infections. The present study investigates the anti-quorum sensing property of a bioactive molecule extracted from marine epibiotic bacteria present on the surface of seaweeds. Among all the isolates tested against monitor strain Chromobacterium violaceum (MTCC 2656), the one with the highest activity was identified as Bacillus zhangzhouensis SK4. The culture supernatant was extracted with chloroform which was then partially purified by TLC and column chromatography. The probable anti-QS compound was identified as 1,2-benzenedicarboxylic acid, bis (2-methylpropyl ester) by GC-MS and NMR analysis. The treatment of P. aeruginosa MCC 3457 with the lead compound resulted in the reduced production of pyocyanin, rhamnolipids, exopolysaccharide, biofilm, and motility. The observations of light and scanning electron microscopy also supported the biofilm inhibition. The lead compound showed synergism with the meropenem antibiotic and significantly reduced MIC. The molecular docking and pharmacokinetics study predicted 1, 2-benzenedicarboxylic acid, bis (2-methylpropyl ester), a phthalate derivative as a good drug candidate. The molecular dynamics study was also performed to check the stability of the lead compound and LasR complex. Further, lead compounds did not exhibit any cytotoxicity when tested on human embryonic kidney cells. As per our knowledge, this is the first report on the anti-QS activity of B. zhangzhouensis SK4, indicating that epibiotic bacteria can be a possible source of novel compounds to deal with the multidrug resistance phenomenon.

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.

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.

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.

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.

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.