China Animal Husbandry & Veterinary Medicine ›› 2025, Vol. 52 ›› Issue (8): 3987-3998.doi: 10.16431/j.cnki.1671-7236.2025.08.046

• Clinical Veterinary Medicine • Previous Articles    

In vitro Safety Evaluation of Bacillus subtilis ZJ20 Based on Whole Genome Sequencing

GUO Jing1,2, HUANG Meixue1,2, GUO Linfeng1,2, FENG Wanli1,2, JIA Yanyan1,2, LIAO Chengshui1,2, YU Zuhua1,2   

  1. 1. Laboratory of Functional Microbiology and Animal Health, The Key Laboratory of Animal Disease and Public Health, College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471003, China;
    2. Luoyang Key Laboratory of Live Carrier Biomaterial and Animal Disease Prevention and Control, Luoyang 471003, China
  • Received:2024-12-06 Published:2025-08-02

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Abstract: 【Objective】 Bacillus subtilis (B.subtilis) ZJ20 is a strain with the ability to degrade aflatoxin B1(AFB1).The aim of this experiment was to evaluate the in vitro safety of B.subtilis ZJ20 with a view to provide a basis for the application of this strain in the degradation of AFB1 in feed and food. 【Method】 The genome-wide sequence of B.subtilis ZJ20 was used to predict virulence genes,drug resistance genes,and genes related to biofilm formation,and drug sensitivity and biofilm formation ability were experimentally verified based on the genome prediction results.In addition,the surface hydrophobicity,self-aggregation,and copolymerization of B.subtilis ZJ20 were examined.Using leghorn male hepatoma (LMH) cells as a model,B.subtilis ZJ20 cell-free supernatant (CFS) was administered to LMH cells at different concentrations.The effects of B.subtilis ZJ20 on LMH cell viability,apoptosis-related factors (Caspase-3,Caspase-9,BAX,and Bcl-2),and inflammatory cytokines (IL-1β,IL-6,TNF-α,and IFN-γ) were assessed to determine the potential cytotoxicity of B.subtilis ZJ20. 【Result】 There were 26 genes coding for virulence factors annotated in the genome of B.subtilis ZJ20,most of which were related to immune evasion and defense,and there were no genes coding for enterotoxins,and 11 antibiotic-resistant genes were identified,the results of the drug sensitivity test showed that B.subtilis ZJ20 was sensitive to a variety of antibiotics.Fourteen genes for the regulation of the bioepidermis were predicted,and the bioepidermal formation capacity of the strain reached its highest value at 24 h.The surface hydrophobicity was 47.842%,and the auto-aggregation rate was 59.334%.The co-aggregation rate with Salmonella Typhimurium ATCC 14028 was 60.905%,and with Escherichia coli CVCC 1527 was 47.309%.The results of cell viability assay showed that B.subtilis ZJ20 CFS added at 1% increased the cell viability to 107.34% at 36 h of interaction with LMH cells.The results of apoptosis and inflammation-related cytokine assays showed that compared with control group,the expression of Caspase-3 was significantly up-regulated with increasing dose (P＜0.05),but there was no significant change of Caspase-9 and BAX/Bcl-2 value (P＞0.05),and the expression of IL-6,IL-1β,TNF-α,and IFN-γ were all significantly down-regulated (P＜0.05). 【Conclusion】 The whole genome sequence analysis and its detection of the effects on the expression of LMH cell viability,apoptotic factors,and inflammation-related cytokines indicated that B.subtilis ZJ20 had a certain degree of safety,which provided a reference for the safety of its application in the development as a functional probiotic.

Key words: whole genome sequencing; Bacillus subtilis; security; drug resistance; virulence

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