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Iturin, a structurally cyclic heptapeptides with a number of homologous derivatives has broad-spectrum antimicrobial and antibiofilm properties. The present study elucidates the structure and antimicrobial efficacy of iturin C 3 biosurfactant (BS) produced by the endophytic bacterium Bacillus sp. LLB-04. Bacillus sp. LLB-04 was isolated from the leaves of hemiparasite Scurrula parasitica L. during the winter season. The biosurfactant was produced in a static batch culture of 120 h using Nutrient Broth (NB) medium and was extracted by a series of solvent systems. The BS was purified by solvent fractionation and preparative High-Performance Liquid Chromatography (HPLC) method, and then it was structurally characterized through various analytical methods. Its antimicrobial and antibiofilm activities were tested against 0, to 18 h old topical and food-borne pathogens. Furthermore, the cellular aggregation and bacterial cell adhesion on polystyrene and urethral catheters were checked at the Biofilm inhibitory concentration (BIC). The cell line toxicity of BS (0-1.568 mg/ml) was tested against the human embryonic lung tissue L-132 and human alveolar epithelial cancer cell line, and the in silico mode of action was studied using standard methods. From the spectroscopic studies of 96 h culture harvested BS revealed that Bacillus sp. LLB-04 (GenBank Accession No.: MF037706) produced the BS as iturin C 3 . The BS had broad-spectrum antimicrobial with minimum inhibitory concentration (MIC) values ranging from 0.1 to 1.6 mg/ml and an average biofilm inhibition concentration (BIC) of 0.8-1.6 mg/ml in 18 h old cells where biofilm formation was inhibited up to 46.4 times at 1.6 mg/ml concentration. It could also destabilize 40-48 h old preformed biofilm and had a synergistic response with streptomycin (Bacillus subtilis MTCC 411, Escherichia coli MTCC 730), ciprofloxacin (B. subtilis MTCC 411, E. coli MTCC 730), and vancomycin (Staphylococcus epidermidis MTCC 3086, B. subtilis MTCC 411). It had antiproliferative activity (0.1-0.8 mg/ml) on cancer cell lines. In-silico protein-ligand interactions predicted that it could interact with different membrane proteins of topical and food-borne pathogens. Thus, the study revealed for the first time that the endophytic Bacillus sp. could be exploited for large-scale production of iturin C 3 that could be used in combating biofilm formation and cellular adhesion of topical and food-borne pathogens., Competing Interests: Declarations. Conflict of interest: The authors declare that they have no conflict of interest. NCBI database ID: MF037706., (© 2025. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The growing demand for efficient biosurfactants in various industrial sectors has driven the search for sustainable alternatives, enhanced production methods, and low-cost substrates. This study aimed to optimize the production, characterize, and assess the bioactivities of biosurfactants produced by an oligotrophic PET plastic-associated Bacillus sp. EIKU23. The bacterium yielded the highest amount of biosurfactant after 6 days of incubation in Luria broth medium (pH 7.0) at 30 °C without any additives. FTIR and NMR analyses confirmed the lipopeptide nature of the biosurfactant, which exhibited a negative charge. The biosurfactant remained stable at 4 °C-80 °C and pH 7.0-8.0 for at least 7 days. It exhibited antioxidant properties comparable to the ascorbic acid standard, with efficacy ranging from 23.61% to 89.96% in different antioxidant assays. It showed antibacterial activity against both Gram-positive and Gram-negative potential pathogens. The biosurfactant induced substantial DNA leakage at a concentration of 10 mg/mL and eradicated approximately 48.4% of pre-formed Staphylococcus aureus biofilm and showed anti-attachment behaviour to a polystyrene surface. Additionally, the biosurfactant precipitated up to 98.7% uranium from an aqueous solution, demonstrating its potential for bioremediation. These findings suggest that the biosurfactant produced by Bacillus sp. EIKU23 is multifunctional with promising applications in bioremediation, antibacterial activity, antibiofilm formation, and antioxidant defense, offering a novel solution for sustainable industrial practices and plastic waste management., Competing Interests: Declarations. Conflict of interest: The authors have no relevant financial or non-financial interests to disclose. Ethical Approval: This article did not require any ethical approval. Consent to Participate: Not applicable. Consent for Publication: Not applicable., (© 2025. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Post-weaning diarrhea (PWD) is a major concern for pig producers, as stress and early weaning increase susceptibility to enteropathogens like enterotoxigenic Escherichia coli (ETEC) and Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium). This study explores the immunomodulatory and antimicrobial properties of lipopeptide extracts (LPEs) from Bacillus strains which contain surfactin (SF) - an immunomodulatory lipopeptide - and their potential in mitigating PWD. Pre-incubation with LPEs from B. subtilis TC12, containing 1, 40 and 60 µg/ml SF, stimulated TNF-α production significantly (p < 0.05) in LPS-challenged RAW 264.7 cells. In contrast, B. velezensis MFF 2.2 LPE, containing 0.1 and 1 µg/ml SF, reduced TNF-α production by macrophages after LPS-challenge. The production of IFN-γ by these cells was not affected by pre-incubation with any of the extracts. On the other hand, all treatments significantly decreased (p < 0.05) IL-10 production. LPEs significantly reduced (p < 0.05) NO production; however, neither TC12 nor MFF 2.2 LPEs affected macrophage phagocytic activity or bactericidal capacity and, in addition, LPEs showed bacteriostatic activity towards S. Typhimurium and ETEC. Also, LPEs did not affect RAW 264.7 viability (> 90%) in concentrations up to 60 µg/ml SF for TC 12 and 1 µg/ml for MFF 2.2. These findings suggest that LPEs from native Bacillus possess immunomodulatory and antimicrobial properties, which could help control inflammation and inhibit pathogen growth during piglet weaning. According to preliminary in vivo studies, LPEs might offer a promising alternative to reduce antibiotic usage in pig husbandry., Competing Interests: Declarations. Animal ethics: No approval of research ethics committees was required to accomplish the goals of this study because experimental work was conducted in vitro using RAW 264.7 cell line. Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature B.V.)

Cyclic lipopeptides (CLPs) are potent secondary metabolites with diverse biological functions. Bacillus strains primarily produce CLPs of three key families, namely, iturins, fengycins, and surfactins, each comprising structural variants characterized by a cyclic peptide linked to a fatty acid chain. Despite extensive research on CLPs, the individual roles of these analogues and their proportion in driving biological activity have remained largely overlooked. In this study, we purified and chemically characterized CLP variants from Bacillus velezensis UMAF6639 and tested them individually for their antifungal and plant growth-promoting effects. We isolated 5 fractions containing iturin A analogues (from C 13 to C 17 ), 5 fengycin fractions (containing C 16 , C 17 , and C 18 fengycin A and C 14 , C 15 , C 16 , and C 17 fengycin B), and 5 surfactin fractions (from C 12 to C 16 ). We show how antifungal activity and seed radicle growth promotion relied on the lipopeptide structural variant and concentration based on the physiological ratio calculated for each lipopeptide variant. Notably, we found that the most toxic variants were the least abundant, which likely minimized autotoxicity while preserving bioactivity. This balance is achieved through synergistic interactions with more abundant, less aggressive analogues. Furthermore, certain fengycin and surfactin variants were shown to increase bacterial population density and exopolysaccharide production, crucial strategies for microbial competition with significant ecological impacts. In addition to advancing basic knowledge, our findings will support the development of precision biotechnological innovations, offering targeted solutions to drive sustainable food production and preservation strategies.

Sauerkraut contains various fermentative microorganisms that produce active metabolites, enhancing immunity and resistance to infections. However, its effects on methicillin-resistant Staphylococcus aureus (MRSA)-induced acute lung injury (ALI) remain unclear. Using RAW264.7 cells and a mouse model, we demonstrated that Bacillus safensis SK14 (BS SK14), an understudied fermentative bacterium, has an immunomodulatory effect on MRSA-induced ALI. BS SK14 significantly reduced the inflammatory responses. Supplementation with live BS SK14 or its culture supernatant increased survival rates, reduced lung damage, and attenuated inflammation in ALI model mice. LC-MS/MS analysis identified esculin as the key metabolite responsible for these effects. BS SK14 produces esculin via the gut-lung axis, inhibiting the TLR2-MyD88-NF-κB pathway, reducing Keap1 levels, and activating the Nrf2-ARE pathway. This decreased MRSA-induced M1 polarization and increased M2 polarization, enhancing antioxidant and anti-inflammatory activities in mice. Collectively, these results reveal that BS SK14 and its metabolite esculin exert therapeutic effects on MRSA-induced ALI through a multifactorial strategy.

Listeria monocytogenes, a pathogenic bacterium causing zoonotic diseases, necessitates the urgent search for novel anti-Listeria monocytogenes drugs due to the continuous emergence of drug-resistant bacteria. In this study, we isolated and identified a bacteriocin-producing strain CM7-4 from seawater as Bacillus velezensis through 16S rRNA sequence analysis. Moreover, we successfully purified a novel bacteriocin named PCM7-4 from Bacillus velezensis CM7-4. The molecular weight of PCM7-4 was determined to be 40,228.99 Da. Notably, PCM7-4 exhibited broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria with a minimum inhibitory concentration (MIC) of 5.625 μg/mL against Listeria monocytogenes specifically. It demonstrated heat resistance and high stability within the pH range of 2-12 while being sensitive to proteinase K degradation without any observed hemolytic activity. Furthermore, SEM analysis revealed that PCM7-4 effectively inhibited biofilm formation and disrupted cell membranes in Listeria monocytogenes cells. Transcriptome analysis revealed that PCM7-4 exerts an impact on genes associated with crucial metabolic pathways, encompassing the biosynthesis of secondary metabolites, phosphotransferase systems (PTS), and starch/sucrose metabolism. These findings highlight the significant potential of bacteriocin PCM7-4 for the development of effective antimicrobial interventions targeting food-borne pathogenic bacteria., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Application of melatonin and lipopeptides (LPs) derived from Bacillus strains is considered an efficient strategy to control plant diseases at both pre and postharvest stages. However, the combined application of melatonin and LPs has not been studied yet. Therefore, the present study presents the synergistic effect of melatonin and LPs produced by Bacillus atrophaeus strain MCM61 against gray mold disease and its impact on quality parameters and vase life of cut roses. The stems of cut roses along with flowers were dipped in the melatonin solution at concentration (0, 0.2, 0.4, 0.6, and 0.8 mM) and the results indicated that the vase life of flowers treated with the melatonin was enhanced compared to control. Melatonin at 0.6 mM concentration showed the highest vase life of cut roses at day 8. Synergistic treatment with melatonin and LPs of B. atrophaeus MCM61 revealed that MDA and H 2 O 2 content showed the highest decrease in cut roses. Similarly, relative water content, total phenol content, GSH content, and defense enzymes i.e., APX, SOD, PPO, POD, and CAT activities were increased in cut roses treated with co-application of B. atrophaeus MCM61 LPs and melatonin compared to single treatments and control treatment. Furthermore, the longevity of cut roses was also improved in flowers treated with a combined application of MCM61 and melatonin compared to other treatments., Competing Interests: Declarations. Conflict of Interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Bacillus lipopeptides have been reported to display anti-obesity effects. In the present study, Lipopeptides from Bacillus velezensis FJAT-45028 that consisted of iturin, fengycin and surfactin were reported. The lipopeptides exhibited a strong lipase inhibition activity in a concentration-dependent manner with a half maximal inhibitory concentration of 0.012 mg/mL, and the inhibition mechanism and type were reversible and competitive, respectively. Results of CCK8 assay showed that 3T3-L1 preadipocyte cells were completely viable under treatment of 0.050-0.2 mg/mL lipopeptides for 24 or 48 h. It was found that the lipopeptides could increase lipid droplets in the differentiated 3T3-L1 adipocytes in tested concentration and suppress the expression of peroxisome proliferator-activated receptor gamma (PPARγ). These results indicated the potential anti-obesity mechanism of the tested lipopeptides might be to inhibit lipase activity but not to suppress lipid accumulation in the adipocytes. Moreover, the lipopeptides could elevate glucose utilisation by 14.43%-33.81% in the differentiated 3T3-L1 adipocytes.

Recently, "Bacillus atrophaeus", which has a cell wall structure consisting of peptidoglycan layers, has attracted the attention of researchers due to its different usage areas. In particular, research focuses on the technology of obtaining bio‑hydrogen with various techniques. This research involves, for the first time, the use of the Bacillus atrophaeus bacteria as a bio-supporting material for monodisperse copper nanoparticles (CuNPs@Bacillus atrophaeus) and the manufacture of hydrogen through catalytic NaBH 4 -methanolysis (SB-methanolysis) in the presence of the resulting nanoparticles. Here, detailed kinetic studies were carried out during the SB-methanolysis by taking CuNPs and bacteria in varying amounts and at varying temperatures, and the activation energy and lifetime of monodisperse CuNPs@Bacillus atrophaeus was found to be 31.76 kJ mol -1 and 30,903 mol H 2 (mol Cu) -1 , respectively. The chemical and physical structure of the CuNPs@Bacillus atrophaeus was observed during the SB-methanolysis, so only detailed characterization of bacteria and monodisperse CuNPs@Bacillus atrophaeus was performed, and the particle size of the catalyst was calculated as 3.29 nm. The results showed that the monodisperse CuNPs@Bacillus atrophaeus, which has superior features and high catalytic activity, is a "clean", very well methanol-soluble, and quite surprising catalyst in terms of hydrogen production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Mechanical pressing in Daqu production has introduced quality-affecting variations. Up to now, clear elucidation has not yet been applied to the mechanisms behind this phenomenon, and the determinants of Daqu quality are not yet completely excavated. For this reason, the physicochemical factors, enzyme activity, metabolites, and microbial communities were compared between the mechanical Daqu (MDQ) and traditional Daqu (TDQ) in this paper. The results showed significant differences in amino acids between MDQ and TDQ, with arginine being the key differentiator. High temperature, high moisture, and low acidity were crucial to Daqu's amino acid richness. Additionally, mechanical pressing affected core community stability. Bacillus was the primary biological factor for the discrepancy in amino acids. Further investigation indicated that arginine contributed to the reduction of lactic acid and higher alcohols in Jiupei, confirming that amino acids were potential novel markers affecting the quality of Daqu., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Probiotics have excellent antioxidant and antibacterial effects and are also considered to be promising natural biological preservatives. This study examined the freeze-thaw stability of the antioxidant and antibacterial activity of probiotic Bacillus velezensis PJP10, and evaluated the effect of different concentrations of PJP10 (10 6 , 10 7 , and 10 8  CFU mL -1 ) on microbial growth, textural characteristics, physicochemical properties, and lipid oxidation of grass carp fillets during repeated freeze-thaw cycles., Results: Freeze-thaw cycles had little effect on the antioxidant and antibacterial stability of PJP10. Treatment with the PJP10 strain slowed effectively the increase in Aeromonas spp. counts (ASC), pH, total volatile basal nitrogen (TVB-N), and thiobarbituric acid (TBA) values of the freeze-thaw fish fillets. It maintained the textural quality of fillets, inhibiting the reduction of hardness, springiness, gumminess, cohesiveness, chewiness, and resilience values, and reduced lipid oxidation, as evidenced by decreased carbonyl values and conjugated diene values after multiple freeze-thaw cycles., Conclusion: Probiotic B. velezensis PJP10 had protective effects on microbiological, textural, and physicochemical properties, and lipid oxidation of grass carp fillets during repeated freeze-thaw storage, suggesting a potential application of this strain in the freeze-thaw preservation of freshwater fish. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Antimicrobial resistance is a growing global concern exacerbated by the scarcity of new medications and resistance to current antibiotics. Microbes from unexplored habitats are promising sources of natural products to combat this challenge. This study aimed to isolate bacteria producing secondary metabolites and assess their antimicrobial efficacy against human pathogens. Soil and liquid samples were collected from Afar region, Ethiopia. Bacterial isolates were obtained using standard serial dilution techniques. Antimicrobial activity was evaluated using agar plug and well diffusion methods. matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF MS) and whole-genome sequencing (WGS) were conducted for the isolate exhibiting the highest antimicrobial activity. Secondary metabolites were extracted and analyzed using gas chromatography-mass spectra (GC-MS). In this study, 301 bacteria isolates were identified, of which 68 (22.6%) demonstrated antagonistic activity against at least one reference pathogen. Whole-genome sequencing revealed that Sl00103 belongs to the genus Bacillus, designated as Bacillus sp. Sl00103. The extract of Sl00103 showed zones of inhibition ranging between 17.17 ± 0.43 and 26.2 ± 0.4 mm against bacterial pathogens and 19.5 ± 0.44 to 21.0 ± 1.01 mm against Candida albicans . GC-MS analysis of ethyl acetate and n -hexane extracts identified major compounds including (R,R)-butane-2,3-diol; 3-isobutylhexahydropyrrolo[1,2a] pyrazine-1,4-dione; cyclo(L-prolyl-L-valine); and tetradecanoic acid, 12-methyl-, methyl ester; hexadecanoic acid, methyl ester among other. In conclusion, this study isolated several promising bacterial strains from the Afar region in Ethiopia, with strain Sl00103 (Bacillus sp. Sl00103) demonstrating notable antimicrobial and antioxidant activities and warranting further studies., Importance: Antimicrobial resistance (AMR) is an escalating global health threat affecting humans, animals, and the environment, underscoring the urgent need for alternative pathogen control methods. Natural products, particularly secondary metabolites from bacteria, continue to be a vital source of antibiotics. However, microbial habitats and metabolites in Africa remain largely unexplored. In this study, we isolated and screened bacteria from Ethiopia's Afar region, characterized by extreme conditions like high temperatures, volcanic activity, high salinity, and hot springs to identify potential bioactive compounds. We discovered diverse bacterial isolates with antimicrobial activity against various pathogens, including strain Sl00103 (Bacillus sp. Sl00103), which demonstrated significant antimicrobial and antioxidant activities. GC-MS analysis identified several antimicrobial compounds, highlighting strain Sl00103 as a promising source of secondary metabolites with potential pharmaceutical applications and warranting further investigation., Competing Interests: The authors declare no conflict of interest.

Microbial polysaccharides derived from bacterial sources possess unique properties because of their structural complexity contributing to exceptional characteristics, including potent immunostimulatory effects. In this study, we extracted crude polysaccharide from Bacillus velezensis GV1 (BPS) which was isolated from Korean ginseng vinegar, and subsequently characterized for sugar composition and functional groups using FT-IR and methylation method. Structural analysis indicated that BPS was composed of mannan and glucan in a ratio of 7.5:2.5. The immunostimulatory effect of BPS was investigated in RAW264.7 macrophages. The results revealed that BPS significantly increased NO production, as well as the secretion and expression of key cytokines, such as IL-6, TNF-α, and IL-1β. These effects were confirmed using a TLR4 antagonist (TAK-242). Moreover, BPS exhibited immunostimulatory potential by promoting the NF-κB signaling pathway. In conclusion, this study establishes a foundation for the potential application of BPS as an immunostimulatory adjuvant or alternative component in functional foods, particularly for enhancing innate immune responses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Bacillus spp. produce numerous antimicrobial metabolites. Among these metabolites, cyclic lipopeptides (CLP) including fengycins, iturins, and surfactins are known to have varying antifungal activity against phytopathogenic fungi. The differential activities of CLP have been attributed to diverse mechanisms of action on fungal membranes. However, the precise biochemical determinants driving their antifungal modes of action have not been conclusively identified. In this study, three plant pathogenic fungi of varying lipopeptide sensitivities, Alternaria solani, Cladosporium cucumerinum, and Fusarium sambucinum, were studied to determine how their cell membrane lipid compositions may confer sensitivity and/or tolerance to fengycin, iturin, and surfactin. Results indicated that sensitivity to all three lipopeptides correlated with lower ergosterol content and elevated phospholipid fatty acid unsaturation. Fungal sensitivity to surfactin was also notably different than fengycin and iturin, as surfactin was influenced more by lower phosphatidylethanolamine amounts, higher levels of phosphatidylinositol, and less by phospholipid fatty acyl chain length. Results from this study provide insight into the fungal membrane composition of A. solani, F. sambucinum, and C. cucumerinum and the specific membrane characteristics influencing the antifungal effectiveness of fengycin, iturin, and surfactin. Understanding of these determinants should enable more accurate prediction of sensitivity-tolerance outcomes for other fungal species exposed to these important CLP., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Prolonged exposure of human dermal fibroblasts (HDF) to ultraviolet (UV) radiation triggers the production of reactive oxygen species by upregulating the expression of matrix metalloproteinases (MMPs), causing type-I collagen degradation and photoaging. A sulfated (1 → 3)/(1 → 4) mannogalactan exopolysaccharide (BVP-2) characterized as [→3)-α-Galp-{(1 → 4)-α-6-O-SO 3 -Manp}-(1 → 3)-α-6-O-SO 3 -Galp-(1→] was isolated from seaweed-associated heterotrophic bacterium Bacillus velezensis MTCC13097. Whole genome analysis of B. velezensis MTCC13097 (Accession number JAKYLL000000000) revealed saccharine biosynthetic gene clusters for exopolysaccharide production. BVP-2 administered cells showed noteworthy reduction in mitochondrial superoxide (∼85 %, p < 0.05) and ROS production (62 %) than those exhibited by UV-A irradiated HDF cells. Oxidative imbalance in HDF cells (after UV-A exposure) was recovered with BVP-2 treatment by significantly downregulating nitric oxide (NO) production (98.6 μM/mL, 1.9-fold) and DNA damage (⁓67 %) in comparison with UV-A induced cells (191.8 μM/mL and 98.7 %, respectively). UV-irradiated HDF cells showed a ∼30-50 % downregulation in the expression of MMPs (1, 2, and 9) following treatment with BVP-2. Considerable amount of sulfation (18 %) along with (1 → 3)/(1 → 4) glycosidic linkages in BVP-2 could be pivotal factors for down-regulation of the intracellular MMP-1, which was further supported by molecular docking and structure-activity studies. The (1 → 3)/(1 → 4)-linked bacterial exopolysaccharide (BVP-2) might be used as prospective natural lead to attenuate and mitigate UV-A-induced photoaging., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This study aims to develop an efficient chitin-based purification system, leveraging a novel design where the target proteins, superfolding green fluorescent protein (sfGFP) and Thermus antranikianii trehalose synthase (TaTS), fused with a chitin-binding domain (ChBD) from Bacillus circulans WL-12 chitinase A1 and a tobacco etch virus protease (TEVp) cleavage site. This configuration allows for the effective immobilization of the target proteins on chitin beads, facilitating the removal of endogenous proteins. A mutant TEVp, H-TEVS219V-ChBD, fused with the His-tag and ChBD, is employed to cleave the target proteins from the chitin beads specifically. Subsequently, fresh chitin beads are added for adsorption to remove H-TEVS219V-ChBD in the solution, thereby significantly improving the purity of the target protein. Our results confirm that this system can efficiently and specifically purify and recover sfGFP and TaTS, achieving electrophoretic-grade purity exceeding 90%. This system holds significant potential for industrial production and other applications.

Xanthomonas oryzae causes tremendous damage in rice plants (Oryza sativa L). Therefore, this study is focused on siderophore-producing Bacillus albus (CWTS 10) for managing BLB disease caused by X. oryzae. Both B. albus and its crude siderophore (methanolic and diethyl ether) extracts inhibited X. oryzae (10-12 mm). Fourier transform infrared spectroscopy (FTIR) analysis of the extracts indicated the presence of catecholate siderophore functional groups. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed the presence of antimicrobial compounds such as 2-deoxystreptamine, miserotoxin, fumitremorgin C, pipercide, pipernonaline, gingerone A, and deoxyvasicinone. Complete genome sequencing revealed the gene clusters for antibiotic, siderophore, antibacterial, antifungal, and secondary metabolite production. An in vivo study revealed that bacteria (CWTS 10) and their siderophore extracts effectively inhibited X. oryzae. The mode of application of bacterial or siderophore extracts in terms of DI and DSI percentage was as follows: soak method > inoculation method > spray method. In addition to providing enhanced antagonistic activity, there was a significant increase in root and shoot length and weight (wet and dry) of treated plants compared to control plants challenged with X. oryzae. Thus, the results clearly indicate that siderophore-producing B. albus and its siderophore extracts strongly inhibited X. oryzae. However, further field experiments are required before being formulated to protect rice crops from X. oryzae., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Intensive broiler production systems face challenges like enteric diseases, impacting global food security. Strategies to enhance broiler immunity and gut health, particularly amidst antibiotic growth promoter restrictions, are crucial. The present study investigated the combined effects of fenugreek seeds (FS) and Bacillus-based direct-fed microbials (DFM) on immune-related gene expression in the ileum and alteration of microbial population in the cecum of broiler. The study involved 160 Ross 308 broiler chicks, which were divided into four groups consisting of 5 replicates, each containing eight birds. The chicks were grown for a period of 42 d, during which they had ad libitum access to feed and water. Dietary treatments were: Control (basal diet), FS5 (basal + 5g/kg fenugreek seeds), FS5DFM (basal + 5g/kg fenugreek seeds + 0.1g/kg Bacillus-based DFM), and DFM (basal + 0.1g/kg Bacillus-based DFM). Ileum tissue and cecal contents were collected on d 42 for gene expression and gut microbiome analysis. Ileal gene expression analysis revealed the downregulation of IL-6, IL-8L2, CASP6, PTGS2, and IRF7 in both FSs and DFMs groups compared to the control, suggesting individual immunomodulatory effects. However, avian β-defensin genes exhibited complex regulation, highlighting the need for further investigation. Cecal microbiome diversity remained stable, with subtle shifts in specific taxa influenced by FSs and DFMs. Interestingly, the combination of the FSs and DFMs uniquely impacted specific taxa, including Clostridiales vadin BB60. These findings suggest that both FSs and DFMs demonstrated potential for improving broiler immunity through inflammation reduction. The combination of FSs and DFMs offers a synergistic effect in immune modulation and specific microbial modulation, warranting further investigation with pathogen challenge models for comprehensive understanding., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Published by Elsevier Inc.)

The soil hosts a wide array of bacterial species capable of producing diverse bioactive compounds. This research aimed to screen bacterial isolates for their bioactive potential from extreme environments in Pakistan. Out of the 69 isolates examined, only 7 exhibited antagonistic activity against Bacillus sp. and Escherichia coli test strains. Notably, the B. cereus DS-2 strain demonstrated the highest antibacterial potential (31 mm and 15 mm) against the Bacillus and E. coli test strains, respectively. Mode-of-action studies suggested that the crude extract might have induced morphological abnormalities in the Bacillus sp. (test strain), causing cell contraction, chain breakage, and deformation. Furthermore, the B. cereus DS-2 strain displayed significant antioxidant potential (64.8%) as revealed by the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Thin-layer chromatography (TLC) of the DS-2 crude extract led to the separation of six components, with only spots 3 and 4 exhibiting the antibacterial potential (3 mm and 5 mm, respectively). Subsequently, gas chromatography-mass spectrometry (GC-MS) analysis of the bioactive fraction extracted from TLC revealed the presence of diisooctyl phthalate, dibutyl phthalate, hexadecanoic acid methyl ester, and octadecanoic acid methyl ester. Molecular docking analysis of diisooctyl phthalate and dibutyl phthalate revealed their binding affinity against E. coli and Bacillus sp. targets. ADMET analysis confirmed the solubility, toxicity, and drug-like properties of the ligands based on Lipinski's rule of five. Current findings suggest that these compounds hold promise as antibacterial agents in drug development. This study underscores the diverse microbial community present in extreme environments and highlights the versatile applications of natural products derived from these strains., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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-1β (IL-1β), 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-1β 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-1β 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., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

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-1β, 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., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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 O 2 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 β-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 MnxE 3 F 3 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.

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 IC 50 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 Δ Bcpsd 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.

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 10 8  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 1β, 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., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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 β-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 β-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., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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.

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 × 10 6 mL -1 to 3 × 10 6 and 4 × 10 6 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., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., Competing Interests: Declaration of competing interest There is no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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.

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., (© 2024. The Author(s).)

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-β-ionone, β-ionone, dihydroactinidiolide, linalool oxide, and β-terpineol. The results of this study indicate that B. velezensis P1 presents desirable properties to be used as a biocontrol agent., Competing Interests: Declaration of competing interest The authors declare no competing interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 × 10 6 , and 1.2 × 10 7 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 × 10 7 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.

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.

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., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Amphipathic compounds known as biosurfactants are able to reduce surface and interfacial tensions. These substances produced by microbial organisms perform the same functions as chemical surfactants with several enhancements, the most significant of which is biocontrol activity. Lipopeptide is one of the five biosurfactants from natural resources and is identified as the best alternative for chemical surfactants and the major topic of interest for both scientific and industrial communities due to their increasingly growing potential applications in biological and commercial fields. These are the biological compounds with very less toxicity level that increase their importance in the pesticide industry. In this article we summarize the structural diversity of the microbial lipopeptide biosurfactants and focus on their applications as biocontrol agents in plants, covering (1) an intensive study of the structural diversity of lipopeptide biosurfactants originated primarily by the Bacillus, Pseudomonas, Cyanobacteria, and Actinomycetes species is presented, (2) the comparative study of advantages and disadvantages of characterization techniques and physicochemical properties which have a major role in biocontrol activity of microbial lipopeptides, and (3) their wide range biocontrol applications as systemic resistance inducers against different plant diseases, resistance against phytopathogens by alteration of wettability of plant surfaces and antimicrobial activities of important bioactive lipopeptides produced from Bacillus strains., Competing Interests: Conflict of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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 (LD 50 ), 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 LD 50 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 (C 14 -C 17 ), fengycin B (C 16 -C 17 ), surfactin (C 15 -C 17 ), and lichenysin (C 12 , C 13 , C 14 , and C 16 ), 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 (C 13 -C 17 ) (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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 β (Aβ) aggregation, whereas succinyl macrolactin O ( 2 ) inhibited Aβ aggregation, indicating their therapeutic potential for disassembling and removing Aβ aggregation.

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 10 7 ─10 8 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.

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 (IC 50 , half maximal inhibitory concentration ~ 29.5 µg/ml) on tetrazolium bromide analysis with less significant cytotoxicity on common fibroblast (HDF) cells (IC 50  ~ 77 µg/ml). The potential antioxidant ability of the organic extract of B. altitudinis MTCC13046 (IC 90 133 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 (IC 50 76.9 µg/ml). According to the findings, B. altitudinis MTCC13046 could be used to develop prospective anticancer agents., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (© 2022 John Wiley & Sons Ltd.)

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 Ca 2+ , which affected the growth process of CaCO 3 and inhibited the formation of CaCO 3 . This study provided deeper insight into scale inhibition performance related to the scale inhibition mechanism., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)