Investigation of Bactronophorus thoracites protein breakdown products as antimicrobials against Pantoea spp.

In addressing the challenge posed by Pantoea spp. pathogens in paddy cultivation, this study explores the potential of Bactronophorus thoracites protein breakdown products as innovative antimicrobial peptides. The research aimed to investigate these products’ efficacy against Pantoea spp. The initial phase involved optimizing enzymatic hydrolysis and lactic acid fermentation of B. thoracites crude protein (BTCP) using Response Surface Methodology (RSM). The resulting B. thoracites fermented protein (BTFP) exhibited superior antimicrobial activity and was further analyzed for its chemical and amino acid composition. Advanced techniques such as 1HNuclear Magnetic Resonance (NMR) spectroscopy and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) were employed to assess the fermentation’s impact and to profile the peptides. Optimal fermentation conditions were determined to be 4 days of fermentation with a 3% (w/v) glucose concentration and a 0.92% (w/v) substrate-to-water ratio. Under these conditions, the antimicrobial efficacy against Pantoea ananatis increased to 63.95% and against Pantoea stewartii to 53.53%, compared to the results from enzymatic hydrolysis (41.16% and 37.55%, respectively). Further, the most effective BTFP concentrations were identified as 125 μg/mL Minimum Inhibitory Concentration (MIC) and 500 μg/mL Minimum Bactericidal Concentration (MBC) against P. ananatis and P. stewartii. Nutritional composition analysis indicated a significant increase (p < 0.05) in protein and ash content to 12-15%, and a reduction in fat and carbohydrates to 7-19%. Lactic acid fermentation significantly enhanced (p < 0.05) the amino acid profile of BTFP, with leucine registering the highest increase (12.54 g/100g) and lysine the lowest (4.93 g/100g). Using 1H-NMR spectroscopy, 27 metabolites with notable differences (p < 0.05) were identified between BTCP and BTFP. Notably, indole-3-lactate was the most concentrated metabolite in BTFP (351.131 mmol/L), while arabinose was predominant in BTCP (69.117 mmol/L). The principal component analysis (PCA) highlighted metabolites such as 1,3-diaminopropane, acetoacetate, alanine, and others as key discriminators. LC-MS/MS analysis revealed 18 cationic peptides with molecular weights ranging from 393 to 14,500 Da, originating from various protein sources including mollusc, fish, and invertebrate proteins. In summary, the lactic acid fermentation of BTCP emerges as a promising source of bioactive compounds, exhibiting potent antimicrobial properties that could be harnessed to address plant pathogen challenges in agriculture.

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