Evaluation of growth stage and storage time on fermentation characteristics, microbial community structure, co-occurrence networks, and their functional shifts and pathogenic risk of fermented Italian ryegrass

Selecting an appropriate growth stage of herbage is critical for the promotion of cleaner production in a food company. This study aimed to evaluate the effect of the growth stages at harvest on fermentation characteristics, bacterial and fungal community diversity, co-occurrence networks, and their functionality and pathogenic contamination risk of fermented Italian ryegrass (Lolium multiflorum Lam.; IR). The IR harvested at two growth stages (flowering and filling stages) was spontaneously fermented (IR1 and IR2) for 1, 3, 7, 15, 30 and 60 days. Results showed that IR1 and IR2 both exhibited moderate fermentation quality after 60 days. The fermentation process effectively restricts the amino acid metabolism of harmful bacteria, while simultaneously enhancing some carbohydrate metabolism. The fermentation process facilitated the key Glycolysis pathway and associated enzymes related to ethanol and lactic acid synthesis. In fungal community function analysis, greater proportions of plant and animal pathogens were observed in IR2 than IR1. The findings indicated that both growth stages presented moderate fermentation quality, but the potential for microbial contamination and pathogens at filling stage remained after 60 days. Overall, a relatively early growth stage, e.g. flowering stage, is recommended to ensure the safety, hygiene, and clean in fermented IR production.