Effects of Dietary Gracilaria lichenoides and Bacillus amyloliquefaciens on Growth Performance, Antioxidant Capacity, and Intestinal Health of Penaeus monodon.

This research sought to assess the effects of dietary supplements with Gracilaria lichenoides and Bacillus amyloliquefaciens, either individually or combined, on the growth performance, antioxidant capacity, and intestinal function of Penaeus monodon. A total of 840 shrimps were randomly assigned to 28 tanks with an average initial weight of (1.04 ± 0.03) g (30 shrimp per tank) with 7 different treatment groups and 4 replicates per treatment. The control treatment (C) consisted of a basal diet; in contrast, the experimental groups were complement with varying levels of G. lichenoides (3% or 8%), either alone (S3 and S8) or in combination with B.amyloliquefaciens at different concentrations (3% G. lichenoides and 109 CFU/g-S3B9; 8% G. lichenoides and 1011 CFU/g B. amyloliquefaciens-S8B11; 109 CFU/g B. amyloliquefaciens-S9; 1011 CFU/g B. amyloliquefaciens-B11). The results indicated that the maximum values of final body weight (FBW) (10.49 ± 0.90) g, weight gain rate (WGR) (908.94 ± 33.58) g, and specific growth rate (SGR) (4.20 ± 0.06) g were perceived in the 3% G. lichenoide diet treatment, and compared with the control group, the difference was significant (p < 0.05). The whole-body lipid content of shrimp in the B9 group was significantly higher than that in the B11 group (p < 0.05), but no significant difference was observed when compared with shrimp fed other diets (p > 0.05). The ash content of shrimp in the B9 group was found to be significantly higher than that in the S3B9 group (p < 0.05). Furthermore, the lipase activity in the stomach and intestines of the experimental groups exhibited a statistically significantly increase compared to the control (p < 0.05). In comparison to the control group, the hepatopancreas of the S3 group exhibited a significant increase in the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and antioxidant genes [SOD, catalase (CAT), GSH-Px, thioredoxin (Trx), Hippo, and NF-E2-related factor 2 (Nrf2)] expression levels (p < 0.05). Additionally, the activities of total antioxidant capacity (T-AOC), SOD, peroxidase (POD), and antioxidant genes (CAT, GSH-Px, Trx, and Hippo) in the S3B9 treatment of hepatopancreas showed significant improvement (p < 0.05). The inclusion of dietary G. lichenoides and B. amyloliquefaciens resulted in enhanced relative expression of intestinal lipid metabolism genes (fatty acid synthetase (FAS), lipophorin receptor (LR), fatty acid transport protein 1 (FATP1)) and suppressed the expression of the long-chain fatty acid-CoA ligase 4 (LCL4) gene. Analysis of microbiota sequencing indicated improvements in composition and structure, with notable increases in Firmicutes at the phylum level and Vibrio at the genus level in the S3 group, as well as an increase in Tenericutes at the genus level in the S8B11 group. Overall, the inclusion of dietary G. lichenoides and B. amyloliquefaciens positively impacted the growth, antioxidant capacity, and microbial composition of shrimp, with particular enhancement observed in shrimp fed a supplementary 3% G. lichenoides diet.

Antimicrobial Activity of Bacillus amyloliquefaciens BS4 against Gram-Negative Pathogenic Bacteria.

Worldwide, bacterial resistance is one of the most severe public health problems. Currently, the failure of antibiotics to counteract superbugs highlights the need to search for new molecules with antimicrobial potential to combat them. The objective of this research was to evaluate the antimicrobial activity of Bacillus amyloliquefaciens BS4 against Gram-negative bacteria. Thirty yeasts and thirty-two Bacillus isolates were tested following the agar well-diffusion method. Four Bacillus sp. strains (BS3, BS4, BS17, and BS21) showed antagonistic activity against E. coli ATCC 25922 using bacterial culture (BC) and the cell-free supernatant (CFS), where the BS4 strain stood out, showing inhibitory values of 20.50 ± 0.70 mm and 19.67 ± 0.58 mm for BC and CFS, respectively. The Bacillus sp. BS4 strain can produce antioxidant, non-hemolytic, and antimicrobial metabolites that exhibit activity against several microorganisms such as Salmonella enterica, Klebsiella pneumoniae, Shigella flexneri, Enterobacter aerogenes, Proteus vulgaris, Yersinia enterocolitica, Serratia marcescens, Aeromonas sp., Pseudomonas aeruginosa, Candida albicans, and Candida tropicalis. According to the characterization of the supernatant, the metabolites could be proteinaceous. The production of these metabolites is influenced by carbon and nitrogen sources. The most suitable medium to produce antimicrobial metabolites was TSB broth. The one-factor-at-a-time method was used to standardize parameters such as pH, agitation, temperature, carbon source, nitrogen source, and salts, resulting in the best conditions of pH 7, 150 rpm, 28 °C, starch (2.5 g/L), tryptone (20 g/L), and magnesium sulfate (0.2 g/L), respectively. Moreover, the co-culture was an excellent strategy to improve antimicrobial activity, achieving maximum antimicrobial activity with an inhibition zone of 21.85 ± 1.03 mm. These findings position the Bacillus amyloliquefaciens BS4 strain as a promising candidate for producing bioactive molecules with potential applications in human health.

Characterization of Bacillus amyloliquefaciens PM415 as a potential bio-preserving probiotic.

Animal feed is vulnerable to fungal infections, and the use of bio-preserving probiotics has received increasing attention. In contrast to Lactobacillus and Bifidobacteria spp., fewer Bacillus spp. have been recognized as antifungal probiotics. Therefore, our objective was to screen antifungal strains and provide more Bacillus candidates to bridge this gap. Here, we screened 56 bacterial strains for cyclic lipopeptide genes and conducted an antifungal assay with Aspergillus niger as a representative fungus. We found that a Bacillus strain Bacillus amyloliquefaciens PM415, isolated from pigeon manure, exhibited the highest fungal inhibition activity as demonstrated by the confrontation assay and morphological observation under scanning electron microscope (SEM). Preliminary safety assessment and probiotic characterization revealed its non-pathogenic feature and stress tolerance capability. Whole genome sequencing of Bacillus amyloliquefaciens PM415 revealed a genome size of 4.16 Mbp and 84 housekeeping genes thereof were used for phylogenetic analysis showing that it is most closely related to Bacillus amyloliquefaciens LFB112. The in silico analysis further supported its non-pathogenic feature at the genomic level and revealed potential biosynthetic gene clusters responsible for its antifungal property. RNA-seq analysis revealed genome-wide changes in transportation, amino acid metabolism, non-ribosomal peptides (NRPs) biosynthesis and glycan degradation during fungal antagonism. Our results suggest that Bacillus amyloliquefaciens PM415 is a safe and effective probiotic strain that can prevent fungal growth in animal feeds.

Oral bacterial insights from a comparative study between healthy and comorbid diseased human individuals.

The human oral cavity is colonized by a diverse microbial community, which includes both native and transient colonizers. The microbial composition is crucial for maintaining oral homeostasis, but due to overgrowth or imbalances of these microbial communities, dysbiosis can occur. There is a lack of understanding of the research of native and transient colonizers in the oral cavity of the Indian subpopulation Therefore, in our present study, we explored the role and prevalence of transient and native colonizers between healthy and comorbid oral diseased human individuals. Culture-dependent techniques and culture-independent 16S r DNA metagenomic analyses were employed to isolate and study the interactions of native and transient colonizers from human oral samples. Among the 66 human individuals of both healthy and comorbid individuals, the most abundant isolate was found to be Bacillus amyloliquefaciens MCC 4424. In addition, the more prevalent culturable isolate from the healthy samples was Streptococcus salivarius MTCC 13009, whereas in comorbid samples Staphylococcus pasteuri MTCC 13076, Rothia dentocariosa MTCC 13010 and Pseudomonas aeruginosa MTCC 13077 were prevalent to a greater extent. 16S rDNA metagenomic analyses revealed the prevalence and abundance of genera such as Bacteroidetes and Proteobacteria in healthy individuals; consequently, Fusobacteria and Firmicutes were observed mostly in comorbid individuals. The significant differences in bacterial population density were observed in terms of the Shannon index (p = 0.5145) and Simpson index (p = 0.9061) between the healthy and comorbid groups. B. amyloliquefaciens MCC 4424 exhibits antagonistic behavior when grown as a dual-species with native and transient colonizers. This result is very consistent with the findings of antibiofilm studies using confocal laser scanning microscopy, which revealed a significant reduction in biofilm biovolume (73 %) and maximum thickness (80 %) and an increase in the rough coefficient of biofilms (30 %). Our data suggested that B. amyloliquefaciens MCC 4424 can be a native colonizer of Indian sub-populations. It may act as a novel candidate for oral healthcare applications and greatly aids in the regulation of transient species in the oral cavity.

Integrated Biofilm Modification and Transcriptional Analysis for Improving Fengycin Production in Bacillus amyloliquefaciens.

Although fengycin exhibits broad-spectrum antifungal properties, its application is hindered due to its low biosynthesis level and the co-existence of iturin A and surfactin in Bacillus amyloliquefaciens HM618, a probiotic strain. In this study, transcriptome analysis and gene editing were used to explore the potential mechanisms regulating fengycin production in B. amyloliquefaciens. The fengycin level of B. amyloliquefacien HM-3 (∆itu-ΔsrfAA) was 88.41 mg/L after simultaneously inhibiting the biosyntheses of iturin A and surfactin. The knockout of gene eps associated with biofilm formation significantly increased the fengycin level of the strain HM618, whereas the fengycin level decreased 32.05% after knocking out sinI, a regulator of biofilm formation. Transcriptome analysis revealed that the differentially expressed genes, involved in pathways of amino acid and fatty acid syntheses, were significantly down-regulated in the recombinant strains, which is likely associated with a decrease of fengycin production. The knockout of gene comQXPA and subsequent transcriptome analysis revealed that the ComQXPA quorum sensing system played a positive regulatory role in fengycin production. Through targeted genetic modifications and fermentation optimization, the fengycin production of the engineered strain HM-12 (∆itu-ΔsrfAA-ΔyvbJ) in a 5-L fermenter reached 1.172 g/L, a 12.26-fold increase compared to the fengycin level in the strain HM-3 (∆itu-ΔsrfAA) in the Erlenmeyer flask. Taken together, these results reveal the underlying metabolic mechanisms associated with fengycin synthesis and provide a potential strategy for improving fengycin production in B. amyloliquefaciens.

Transcriptomic analysis of the response of Avena sativa to Bacillus amyloliquefaciens DGL1.

Introduction: Bacillus amyloliquefaciens DGL1, isolated from the arid sandy areas in Dagler, Qinghai Province, China, promotes the growth of Avena sativa variety "Qing Yan 1". Methods: To elucidate the transcriptomic changes in the oat root system following interaction with DGL1 and to reveal the molecular mechanism by which DGL1 promotes oat growth, treatment and control groups of oat roots at 2, 4, 8, and 12 h after inoculation with a suspension of strain DGL1 were analyzed using Illumina high-throughput transcriptome sequencing technology. The differentially expressed genes were determined through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, and the metabolic pathways and key genes were analyzed. Results: The results showed that 7874, 13,392, 13,169, and 19,026 differentially expressed genes were significantly enriched in the glycolysis/gluconeogenesis pathway, amino acid metabolism, nitrogen metabolism, plant hormone signal transduction, and other related metabolic pathways in the oat roots at 2, 4, 8, and 12 h after inoculation with a DGL1 suspension. The GO and KEGG enrichment analyses revealed that the genes encoding plasma membrane ATPase, phosphoglycerate kinase gene PGK, ammonium transporter protein gene AMT, cellulose synthase gene CSLF6, and growth hormone response family gene IAA18 were significantly upregulated. Discussion: It is hypothesized that the pro-growth mechanism of strain DGL1 in oats is the result of the coordination of multiple pathways through the promotion of oat energy metabolism, phytohormone signaling, secondary metabolite synthesis, and amino acid metabolism.

Improving salt-tolerant artificial consortium of Bacillus amyloliquefaciens for bioconverting food waste to lipopeptides.

High-salt content in food waste (FW) affects its resource utilization during biotransformation. In this study, adaptive laboratory evolution (ALE), gene editing, and artificial consortia were performed out to improve the salt-tolerance of Bacillus amyloliquefaciens for producing lipopeptide under FW and seawater. High-salt stress significantly decreased lipopeptide production in the B. amyloliquefaciens HM618 and ALE strains. The total lipopeptide production in the recombinant B. amyloliquefaciens HM-4KSMSO after overexpressing the ion transportor gene ktrA and proline transporter gene opuE and replacing the promoter of gene mrp was 1.34 times higher than that in the strain HM618 in medium containing 30 g/L NaCl. Lipopeptide production under salt-tolerant consortia containing two strains (HM-4KSMSO and Corynebacterium glutamicum) and three-strains (HM-4KSMSO, salt-tolerant C. glutamicum, and Yarrowia lipolytica) was 1.81- and 2.28-fold higher than that under pure culture in a medium containing FW or both FW and seawater, respectively. These findings provide a new strategy for using high-salt FW and seawater to produce value-added chemicals.

Safety evaluation of the food enzyme bacillolysin from the non-genetically modified Bacillus amyloliquefaciens strain AE-NP.

The food enzyme bacillolysin (EC 3.4.24.28) is produced with the non-genetically modified Bacillus amyloliquefaciens strain AE-NP by Amano Enzyme Inc. The production strain meets the requirements for the qualified presumption of safety (QPS) approach to safety assessment. The food enzyme is intended to be used in 14 food manufacturing processes. Since residual amounts of total organic solids (TOS) are removed in three manufacturing processes, dietary exposure was calculated only for the remaining 11 food manufacturing processes in which the food enzyme-TOS is retained. It was estimated to be up to 35.251 mg TOS/kg body weight (bw) per day in European populations. As the production strain qualifies for the QPS approach and no issue of concern arising from the production process of the food enzyme were identified, the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Improvement of branched-chain amino acid production by isolated high-producing protease from Bacillus amyloliquefaciens NY130 on isolated soy/whey proteins and their muscle cell protection.

Branched-chain amino acids (BCAAs) are vital components of human and animal nutrition that contribute to the building blocks of proteins. In this study, 170 protease-producing strains were isolated and screened from soy-fermented foods. Bacillus amyloliquefaciens NY130 was obtained from Cheonggukjang with high production of BCAAs. Optimal production of protease from B. amyloliquefaciens NY130 (protease NY130) was achieved at 42 °C and pH 6.0 for 21 h. It was purified and determined as 27- and 40 kDa. Protease NY130 showed maximum activity at pH 9.0 and 45 °C with Km value of 10.95 mg for ISP and 1.69 mg for WPI. Protease-treated ISP and WPI showed increased sweetness and saltiness via electronic tongue analysis and enhanced the protective effect against oxidative stress in C2C12 myocytes by increasing p-mTOR/mTOR protein expression to 160%. This work possesses potential in producing BCAAs by using protease for utilization in food.

Genomic insights into combating anthracnose with an endophytic Bacillus amyloliquefaciens strain.

Anthracnose, caused by Colletotrichum spp., is a common disease of Camellia oleifera. In this study, a Bacillus amyloliquefaciens strain, GZY63, was isolated from fruit of the anthracnose-resistant cultivar of Ca. oleifera "Ganzhouyou7". Plate confrontation assays and field experiments demonstrated the strong inhibitory effect of GZY63 on anthracnose, and this strain exhibited broad-spectrum resistance to nine pathogenic Colletotrichum spp. This strain shows the potential as a fungicide alternative, but genetic information on this strain is critical for its optimal use. Combining Illumina and Nanopore sequencing, we assembled a high-quality circular genome of GZY63 that contained no plasmids. The GZY63 complete genome was approximately 3.93 Mb and had an average guanine-cytosine content of 46.5%. The genome comprised 4024 predicted coding sequences and 12 types of gene clusters involved in secondary metabolite production. This genome information provides insights into the mechanism underlying the antagonistic impact of the GZY63 strain on anthracnose and its symbiotic relationship with Ca. oleifera.

Metagenomic analysis of the intestinal microbiome reveals the potential mechanism involved in Bacillus amyloliquefaciens in treating schistosomiasis japonica in mice.

Schistosomiasis japonica is one of the neglected tropical diseases characterized by chronic hepatic, intestinal granulomatous inflammation and fibrosis, as well as dysbiosis of intestinal microbiome. Previously, the probiotic Bacillus amyloliquefaciens has been shown to alleviate the pathological injuries in mice infected with Schistosoma japonicum by improving the disturbance of the intestinal microbiota. However, the underlying mechanisms involved in this process remain unclear. In this study, metagenomics sequencing and functional analysis were employed to investigate the differential changes in taxonomic composition and functional genes of the intestinal microbiome in S. japonicum-infected mice treated with B. amyloliquefaciens. The results revealed that intervention with B. amyloliquefaciens altered the taxonomic composition of the intestinal microbiota at the species level in infected mice and significantly increased the abundance of beneficial bacteria. Moreover, the abundance of predicted genes in the intestinal microbiome was also significantly changed, and the abundance of xfp/xpk and genes translated to urease was significantly restored. Further analysis showed that Limosilactobacillus reuteri was positively correlated with several KEGG Orthology (KO) genes and metabolic reactions, which might play important roles in alleviating the pathological symptoms caused by S. japonicum infection, indicating that it has the potential to function as another effective therapeutic agent for schistosomiasis. These data suggested that treatment of murine schistosomiasis japonica by B. amyloliquefaciens might be induced by alterations in the taxonomic composition and functional gene of the intestinal microbiome in mice. We hope this study will provide adjuvant strategies and methods for the early prevention and treatment of schistosomiasis japonica. IMPORTANCE: Targeted interventions of probiotics on gut microbiome were used to explore the mechanism of alleviating schistosomiasis japonica. Through metagenomic analysis, there were significant changes in the composition of gut microbiota in mice infected with Schistosoma japonicum and significant increase in the abundance of beneficial bacteria after the intervention of Bacillus amyloliquefaciens. At the same time, the abundance of functional genes was found to change significantly. The abundance of genes related to urease metabolism and xfp/xpk related to D-erythrose 4-phosphate production was significantly restored, highlighting the importance of Limosilactobacillus reuteri in the recovery and abundance of predicted genes of the gut microbiome. These results indicated potential regulatory mechanism between the gene function of gut microbiome and host immune response. Our research lays the foundation for elucidating the regulatory mechanism of probiotic intervention in alleviating schistosomiasis japonica, and provides potential adjuvant treatment strategies for early prevention and treatment of schistosomiasis japonica.

Bacillus amyloliquefaciens SC06 Relieving Intestinal Inflammation by Modulating Intestinal Stem Cells Proliferation and Differentiation via AhR/STAT3 Pathway in LPS-Challenged Piglets.

Bacillus amyloliquefaciens is a well-accepted probiotic, with many benefits for both humans and animals. The ability of intestinal stem cells (ISCs) to develop into several intestinal epithelial cell types helps accelerate intestinal epithelial regeneration. Limited knowledge exists on how bacteria regulated ISCs proliferation and regeneration. Our study investigated the effects of Bacillus amyloliquefaciens supplementation on ISC proliferation and regeneration and intestinal mucosal barrier functions in piglets exposed to lipopolysaccharide (LPS). Eighteen piglets (male, 21 days old) were randomly split into 3 clusters: CON cluster, LPS cluster, and SC06+LPS cluster. On day 21, 100 µg/kg body weight of LPS was intraperitoneally administered to the SC06+LPS and LPS groups. We found SC06 supplementation maintained the intestinal barrier integrity, enhanced intestinal antioxidant capacity, reduced generation of inflammatory response, and suppressed enterocyte apoptosis against the deleterious effects triggered by LPS. In addition, our research indicated that the SC06 supplementation not only improved the ISC regeneration, but also resulted in upregulation of aryl hydrocarbon receptor (AhR) in LPS-challenge piglets. Further studies showed that SC06 also induced ISC differentiation toward goblet cells and inhibited their differentiation to intestinal absorptive cells and enterocytes. The coculture system of SC06 and ileum organoids revealed that SC06 increased the growth of ISCs and repaired LPS-induced organoid damage through activating the AhR/STAT3 signaling pathway. These findings showed that SC06, possibly through the AhR/STAT3 pathway, accelerated ISC proliferation and promoted epithelial barrier healing, providing a potential clinical treatment for IBD. Our research demonstrated that SC06 is effective in preventing intestinal epithelial damage after pathological injury, restoring intestinal homeostasis, and maintaining intestinal epithelial regeneration.

Bacillus amyloliquefaciens SC06 alleviated intestinal damage induced by inflammatory via modulating intestinal microbiota and intestinal stem cell proliferation and differentiation.

The aim of our research was to investigate the effects of Bacillus amyloliquefaciens SC06 on growth performance, immune status, intestinal stem cells (ISC) proliferation and differentiation, and gut microbiota in weaned piglets. Twelve piglets (male, 21 days old, 6.11 ± 0.12 kg) were randomly allocated to CON and SC06 (1 × 108 cfu/kg to diet) groups. This experiment lasted three weeks. Our results showed that SC06 increased (P < 0.05) growth performance and reduced the diarrhea rate in weaned piglets. In addition, SC06 increased intestinal morphology and interleukin (IL)-10 levels, and decreased (P < 0.01) necrosis factor (TNF-α) levels in jejunum and serum. Moreover, weaning piglets fed SC06 had a better balance of colonic microbiota, with an increase in the abundance of Lactobacillus. Furthermore, SC06 enhanced ISCs proliferation and induced its differentiation to goblet cells via activating wnt/ß-catenin pathway in weaned piglets and intestinal organoid. Taken together, SC06 supplementation improved the growth performance and decreased inflammatory response of piglets by modulating intestinal microbiota, thereby accelerating ISC proliferation and differentiation and promoting epithelial barrier healing.

Safety evaluation of the food enzyme bacillolysin from the non-genetically modified Bacillus amyloliquefaciens strain NZYM-NB.

The food enzyme bacillolysin (EC 3.4.24.28) is produced with the non-genetically modified Bacillus amyloliquefaciens strain NZYM-NB by Novozymes A/S. The production strain meets the requirements for qualified presumption of safety (QPS) approach to safety assessment. The food enzyme is intended to be used in eleven food manufacturing processes. Since residual amounts of total organic solids (TOS) are removed during two processes, dietary exposure was estimated only for the remaining nine food manufacturing processes. Exposure was estimated to be up to 1.327 mg TOS/kg body weight per day in European populations. As the production strain qualifies for the QPS approach and no issue of concern arising from the production process of the food enzyme was identified, the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded (except for distilled alcohol production), but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Transcriptomic and metabolomic analysis reveals the influence of carbohydrates on lignin degradation mediated by Bacillus amyloliquefaciens.

Introduction: Ligninolytic bacteria can secrete extracellular enzymes to depolymerize lignin into small-molecular aromatics that are subsequently metabolized and funneled into the TCA cycle. Carbohydrates, which are the preferred carbon sources of bacteria, influence the metabolism of lignin-derived aromatics through bacteria. Methods: In this study, untargeted metabolomics and transcriptomics analyses were performed to investigate the effect of carbohydrates on lignin degradation mediated by Bacillus amyloliquefaciens MN-13, a strain with lignin-degrading activity that was isolated in our previous work. Results: The results demonstrated that the cell growth of the MN-13 strain and lignin removal were promoted when carbohydrates such as glucose and sodium carboxymethyl cellulose were added to an alkaline lignin-minimal salt medium (AL-MSM) culture. Metabolomics analysis showed that lignin depolymerization took place outside the cells, and the addition of glucose regulated the uptake and metabolism of lignin-derived monomers and activated the downstream metabolism process in cells. In the transcriptomics analysis, 299 DEGs were screened after 24 h of inoculation in AL-MSM with free glucose and 2 g/L glucose, respectively, accounting for 8.3% of the total amount of annotated genes. These DEGs were primarily assigned to 30 subcategories, including flagellar assembly, the PTS system, RNA degradation, glycolysis/gluconeogenesis, the TCA cycle, pyruvate metabolism, and tryptophan metabolism. These subcategories were closely associated with the cell structure, generation of cellular energy, and precursors for biosynthetic pathways, based on a - log 10 (P adjust) value in the KEGG pathway analysis. Conclusion: In summary, the addition of glucose increased lignin degradation mediated by the MN-13 strain through regulating glycolysis, TCA cycle, and central carbon metabolism.

Safety evaluation of the food enzyme glutaminase from the non-genetically modified Bacillus amyloliquefaciens strain AE-GT.

The food enzyme glutaminase (l-glutamine amidohydrolase; EC 3.5.1.2) is produced with the non-genetically modified Bacillus amyloliquefaciens strain AE-GT by Amano Enzyme Inc. The production strain met the requirements for the qualified presumption of safety (QPS) approach. The food enzyme is intended to be used in five food manufacturing processes. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.462 mg TOS/kg body weight per day in European populations. Given the QPS status of the production strain and the absence of concerns resulting from the food enzyme's manufacturing process, toxicity tests were considered unnecessary by the Panel. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Characterization of soybean chitinase genes induced by rhizobacteria involved in the defense against Fusarium oxysporum.

Rhizobacteria are capable of inducing defense responses via the expression of pathogenesis-related proteins (PR-proteins) such as chitinases, and many studies have validated the functions of plant chitinases in defense responses. Soybean (Glycine max) is an economically important crop worldwide, but the functional validation of soybean chitinase in defense responses remains limited. In this study, genome-wide characterization of soybean chitinases was conducted, and the defense contribution of three chitinases (GmChi01, GmChi02, or GmChi16) was validated in Arabidopsis transgenic lines against the soil-borne pathogen Fusarium oxysporum. Compared to the Arabidopsis Col-0 and empty vector controls, the transgenic lines with GmChi02 or GmChi16 exhibited fewer chlorosis symptoms and wilting. While GmChi02 and GmChi16 enhanced defense to F. oxysporum, GmChi02 was the only one significantly induced by Burkholderia ambifaria. The observation indicated that plant chitinases may be induced by different rhizobacteria for defense responses. The survey of 37 soybean chitinase gene expressions in response to six rhizobacteria observed diverse inducibility, where only 10 genes were significantly upregulated by at least one rhizobacterium and 9 genes did not respond to any of the rhizobacteria. Motif analysis on soybean promoters further identified not only consensus but also rhizobacterium-specific transcription factor-binding sites for the inducible chitinase genes. Collectively, these results confirmed the involvement of GmChi02 and GmChi16 in defense enhancement and highlighted the diverse inducibility of 37 soybean chitinases encountering F. oxysporum and six rhizobacteria.

Safety evaluation of the food enzyme bacillolysin from the non-genetically modified Bacillus amyloliquefaciens strain DP-Cyb74.

The food enzyme bacillolysin (EC 3.4.24.28) is produced with the non-genetically modified Bacillus amyloliquefaciens strain DP-Cyb74 by Genencor International B.V. The production strain met all requirements for the qualified presumption of safety (QPS) approach to safety assessment. The food enzyme is intended to be used in six food manufacturing processes. Dietary exposure to the food enzyme total organic solids (TOS) was estimated to be up to 1.536 mg TOS/kg body weight per day in European populations. As the production strain qualifies for the QPS approach and no issue of concern arose from the production process of the food enzyme, the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Crude Lipopeptides Produced by Bacillus amyloliquefaciens Could Control the Growth of Alternaria alternata and Production of Alternaria Toxins in Processing Tomato.

Alternaria spp. and its toxins are the main contaminants in processing tomato. Based on our earlier research, the current study looked into the anti-fungal capacity of crude lipopeptides from B. amyloliquefaciens XJ-BV2007 against A. alternata. We found that the crude lipopeptides significantly inhibited A. alternata growth and reduced tomato black spot disease incidence. SEM analysis found that the crude lipopeptides could change the morphology of mycelium and spores of A. alternata. Four main Alternaria toxins were detected using UPLC-MS/MS, and the findings demonstrated that the crude lipopeptides could lessen the accumulation of Alternaria toxins in vivo and in vitro. Meanwhile, under the stress of crude lipopeptides, the expression of critical biosynthetic genes responsible for TeA, AOH, and AME was substantially down-regulated. The inhibitory mechanism of the crude lipopeptides was demonstrated to be the disruption of the mycelial structure of A. alternata, as well as the integrity and permeability of the membrane of A. alternata sporocytes. Taken together, crude lipopeptides extracted from B. amyloliquefaciens XJ-BV2007 are an effective biological agent for controlling tomato black spot disease and Alternaria toxins contamination.

Potentiation effect of the AI-2 signaling molecule on postharvest disease control of pear and loquat by Bacillus amyloliquefaciens and its mechanism.

An autoinducer-2 (AI-2) signaling molecule from Bacillus was synthesized, and its mechanism on the biofilm formation and biocontrol ability of B. amyloliquefaciens was verified in vitro and in vivo. The 16S/ITS amplicon sequencing was used to analyze the effect of B. amyloliquefaciens B4 with or without AI-2 on the microflora of pears during storage. The results showed that B. amyloliquefaciens B4 secreted AI-2, which promoted biofilm formation. Additionally, AI-2 at a concentration of 40 µmol/L enhanced the biocontrol ability of B. amyloliquefaciens B4 on postharvest pear and loquat fruits. Finally, amplicon sequencing demonstrated that the addition of AI-2 increased the abundance of B. amyloliquefaciens B4 in fruit by stimulating the growth and biofilm formation of this bacterium.