| Penicillum expansum and its water-soluble secondary metabolite patulin (PAT) were the main reason which caused the postharvest apple decay and apple product pollution. In order to reduce decay rate and enhance fruit quality during storage, a strain of BA-16-8 was isolated and screened through low-energy N+ implantation technology in this study. The anti-fungi activity and toxin-removing ability of the strain were studied. After the identification of the anti-fungal mechanism of the antibiotic substance produced by the strain BA-16-8, the research focus shifted to the biological safety and fresh-keeping application of bio-film activity composed by strain BA-16-8. The following results were obtained.An antagonistic strain, BA-16, that can effectively inhibit the growth of P. expansum was isolated from the surface of apples and was identified as Bacillus amyloliquefaciens after phenotypic analysis, API50CH identification, and phylogenetic (16S rDNA) analysis. The mutation of B. amyloliquefaciens BA-16 was induced through low-energy N+ implantation to obtain the mutant BA-16-8, which exhibited the strongest antagonistic capability and highest hereditary stability. Antifungal and patulin (PAT) removal experiments revealed that the cell-containing and cell-free fermentation broths of B. amyloliquefaciens BA-16-8 could significantly inhibit the growth and PAT production of P. expansum.To identify the key substances against P. expansum produced by B. amyloliquefaciens BA-16-8, the genes related to lipopeptide antibiotic synthesis were detected through polymerase chain reaction (PCR). The lipopeptideantibiotics were isolated and purified through high-performance liquid chromatography (HPLC). Then, the structure of lipopeptide was identified through matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The results showed that the fermentation broth of the strain contained surfactin and fengycin. After fenC gene knockout and antifungal experiment, the B. amyloliquefaciens BA-16-8-?fen mutant lost its fengycin-synthesizing capability and showed a significantly reduced inhibitory effect against P. expansum. These results demonstrate that fengycin is the key component of B. amyloliquefaciens BA-16-8 against P. expansum.The anti-fungal mechanism of fengycin in P. expansum was explored at the cell level through scanning electron microscopy (SEM), transmission electron microscopy (TEM), nucleic acid fluorescent dye (NCFD), fluorescence microscopy (FM), and electrophoretic mobility shift assays (EMSA). The anti-fungal effect of fengycin on P. expansum was discussed at physiological and biochemical levels through mycelia respiration detection (MRD) and mitochondria complexes activity assay (MCCA). The results show that fengycin could damage the cell membrane integrity and cell structure, such as mitochondrion. Moreover, fengycin inhibited the enzymatic activity of mitochondrial complexes ? and ? by combining with mitochondrial enzyme genes. The respiration and metabolism of P. expansum were also inhibited.To investigate the effect of fengycin on PAT production of P. expansum, SYBR Green I real-time PCR was performed to detect the expression levels of 6-MSAS and IDH, which are the key genes involved in the production of PAT by P. expansum mycelium under fengycin-treated and untreated conditions. Under the fengycin-treated conditions, the 6-MSAS expression level decreased significantly. Fengycin reduced PAT synthesis in P. expansum by decreasing the key synthetic PAT gene 6-MSAS.To verify the safety of B. amyloliquefaciens BA-16-8 (108 CFU/mL) in fresh-keeping application of postharvest apples, acute oral toxicity tests, a genetic toxicity experiment, and 28-day feeding trials were performed on mice. B. amyloliquefaciens BA-16-8 (108 CFU/mL) did not exert a mutagenic effect nor induce harmful effects on mice. Therefore, B. amyloliquefaciens BA-16-8 (108 CFU/mL) is safe to animals.The ideal concentration of chitosan and fermentation broth of B. amyloliquefaciens BA-16-8 was determined with decay incidence as the preservative adjustment index. The ideal concentration of chitosan is 2%, and the suitable concentration of fermentation broth of B. amyloliquefaciens BA-16-8 is 108 CFU/mL. Compared with the control treatment, the biofilm made up of 2% chitosan and fermentation broth of B. amyloliquefaciens BA-16-8 (108 CFU/mL) at an equal proportion showed a more significant preservative effect.With the contents of apple polyphenols as the fresh-keeping adjustment index, the ideal concentration of salicylic acid (SA), calcium chloride (CC), and methyl jasmonic acid (MeJA) was determined. The solution composed of 200 ?g/mL SA,2% CC, and 0.05 ?mol/mL MeJA at an equal proportion exhibited a significant fresh-keeping effect.Fuji apple was utilized as an experimental material. The fermentation broths of B. amyloliquefaciens BA-16-8 were mixed with chitosan, SA, CC, and MeJA to form a biological active film. After the apples were coated with this film, the preservation and fresh-keeping effects were evaluated. Compared with the control group, the biological active film formed by B. amyloliquefaciens BA-16-8, chitosan, SA, CC, and MeJA could effectively reduce the weightless and decay rates of apples. Meanwhile, MDA and nutrient contents (VC, TA, and SSC) were maintained by the bio-film. This result indicates that the freshness of the apples was enhanced.In summary, fengycin produced by B. amyloliquefaciens BA-16-8 can effectively inhibit the growth and toxin production of P. expansum by changing cell membrane permeability, destroying the cell structure, combining DNA to inhibit mitochondrial complex enzyme activity, and affecting the transcriptional level of toxin-producing genes. After coating on postharvest apples, the experiment group exhibited a better fresh-keeping effect than the control group. |
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