基于全基因组测序的枯草芽孢杆菌ZJ20体外安全性评价

doi:10.16431/j.cnki.1671-7236.2025.08.046

摘要/Abstract

摘要： 【目的】枯草芽孢杆菌(Bacillus subtilis，B.subtilis) ZJ20是一株具有降解黄曲霉毒素 B₁ (aflatoxin B₁,AFB₁)的菌株。试验旨在评价枯草芽孢杆菌枯草芽孢杆菌ZJ20的体外安全性，以期为该菌株在降解饲料和食品中AFB₁的应用提供依据。【方法】对枯草芽孢杆菌ZJ20的全基因组序列中的毒力基因、耐药基因以及生物被膜形成相关基因等进行预测，根据基因分析结果对药物敏感性及生物被膜形成能力进行验证，并对枯草芽孢杆菌ZJ20的表面疏水性、自聚力、共聚力进行检测。以鸡肝癌细胞(leghorn male hepatoma，LMH)为模型，将枯草芽孢杆菌ZJ20无细胞上清液(CFS)按照不同剂量作用于LMH细胞，检测其对LMH细胞活力、凋亡因子(Caspase-3、Caspase-9、BAX、Bcl-2)和炎症相关细胞因子(IL-1β、IL-6、TNF-α、IFN-γ)表达水平的影响，以评价枯草芽孢杆菌ZJ20的潜在细胞毒性。【结果】枯草芽孢杆菌ZJ20基因组中被注释的毒力因子编码基因有26个，多与免疫逃避、防御作用相关，无肠毒素编码基因；具有11个抗生素耐药基因，药敏试验结果显示，枯草芽孢杆菌ZJ20对多种抗生素均敏感；预测到14个生物被膜调控基因，菌株在24 h时生物被膜形成能力达到最高值；表面疏水率为47.842%，自聚率为59.334%；与鼠伤寒沙门菌ATCC 14028的共聚率为60.905%，与大肠杆菌CVCC 1527的共聚率为47.309%。细胞活力检测结果表明，枯草芽孢杆菌ZJ20 CFS添加量为1%且与LMH细胞作用36 h时，细胞活力上升至107.34%。凋亡和炎症相关的细胞因子测定结果显示，随着剂量的增加，与对照组相比，Caspase-3表达显著上调(P＜0.05)，Caspase-9、BAX/Bcl-2比值无显著变化(P＞0.05)；IL-6、IL-1β、TNF-α和IFN-γ表达均显著下调(P＜0.05)。【结论】通过全基因组序列分析及其对LMH细胞活力、凋亡因子和炎症相关细胞因子表达水平影响的检测表明枯草芽孢杆菌ZJ20具有一定的安全性，为其开发为功能性益生菌的应用安全性提供了参考。

关键词: 全基因组测序; 枯草芽孢杆菌; 安全性; 耐药; 毒力

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

中图分类号:

S852.61⁺6

郭晶, 黄美雪, 郭霖丰, 冯婉丽, 贾艳艳, 廖成水, 余祖华. 基于全基因组测序的枯草芽孢杆菌ZJ20体外安全性评价[J]. 中国畜牧兽医, 2025, 52(8): 3987-3998.

GUO Jing, HUANG Meixue, GUO Linfeng, FENG Wanli, JIA Yanyan, LIAO Chengshui, YU Zuhua. In vitro Safety Evaluation of Bacillus subtilis ZJ20 Based on Whole Genome Sequencing[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3987-3998.

参考文献

[1] PATNAIK S,DAVILA C D,CHENNUPATI A,et al.Endocarditis of the native aortic valve caused by Lactobacillus jensenii[J].BMJ Case Reports,2015,2015:bcr2014206288.
[2] CAPURSO L.Thirty years of Lactobacillus rhamnosus GG:A review[J].Journal of Clinical Gastroenterology,2019,53(1):S1-S41.
[3] MARWA Y A M.Probiotics benefits,potential limitations and risks[J].Egyptian Academic Journal of Biological Sciences C,Physiology & Molecular Biology,2024,16(1):253-276.
[4] HUMPHRIES R,BOBENCHIK A M,HINDLER J A,et al.Overview of changes to the clinical and laboratory standards institute performance standards for antimicrobial susceptibility testing,M100,31st edition[J].Journal of Clinical Microbiology,2021,59(12):e0021321.
[5] 农业农村部.中华人民共和国农业农村部公告第226号[J].中华人民共和国农业农村部公报,2019,11:127. MINISTRY OF AGRICULTURE AND RURAL AFFAIRS. Announcement No.226 of the Ministry of Agriculture and Rural Affairs of the People’s Republic of China[J].Bulletin of the Ministry of Agriculture and Rural Affairs of the People’s Republic of China,2019,11:127.(in Chinese)
[6] 班博,戴维,张海涛,等.枯草芽孢杆菌对断奶竹鼠生长性能、血清生化指标和器官发育的影响[J].动物营养学报,2021,33(12):7192-7200. BAN B,DAI W,ZHANG H T,et al.Effects of Bacillus subtilis on growth performance,serum biochemical parameters and organ development of weaned Bamboo rats[J].Chinese Journal of Animal Nutrition,2021,33(12):7192-7200.(in Chinese)
[7] 罗燕儿,赵慧,郭道远,等.枯草芽孢杆菌对草鱼肝脏脂质代谢的调节作用[J].水生生物学报,2020,44(3):485-493. LUO Y E,ZHAO H,GUO D Y,et al.Regulation of lipid metabolism in liver of grass carp by Bacillus subtilis[J].Journal of Hydrobiology,2020,44(3):485-493.(in Chinese)
[8] 王红涛,冯颖,明美玉,等.枯草芽孢杆菌和蒲公英提取物对鲤鱼生长性能、血清生化指标、免疫指标及抗氧化功能的影响[J].中国畜牧杂志,2021,57(12):234-239. WANG H T,FENG Y,MING M Y,et al.Effects of Bacillus subtilis and dandelion extracts on growth performance,serum biochemical indices,immune indices and antioxidant function of common carp[J].Chinese Journal of Animal Science,2021,57(12):234-239.(in Chinese)
[9] 张灿,魏宇超,孙东岳,等.枯草芽孢杆菌对獭兔生长性能、营养物质表观消化率及毛皮品质的影响[J].饲料工业,2022,43(3):45-49. ZHANG C,WEI Y C,SUN D Y,et al.Effects of Bacillus subtilis on growth performance,apparent digestibility of nutrients and fur quality of Rex rabbits[J].Feed Industry,2022,43(3):45-49.(in Chinese)
[10] 李奥运.牦牛源枯草芽孢杆菌的筛选及对犊牦牛肠道菌群和代谢的影响[D].武汉:华中农业大学,2023. LI A Y.Screening of yak-derived Bacillus subtilis and its effects on intestinal microbiota and metabolism of calving yaks[D].Wuhan:Huazhong Agricultural University,2023.(in Chinese)
[11] 都振玉,田慧丽,张琦,等.枯草芽孢杆菌对肉用种母鸭生产性能、体尺指标和繁殖性能的影响[J].饲料工业,2024,45(3):30-35. DU Z Y,TIAN H L,ZHANG Q,et al.Effects of Bacillus subtilis on growth performance,body size index and reproductive performance of meat-type breeder female ducks[J].Feed Industry,2024,45(3):30-35.(in Chinese)
[12] 郑凯天,许英蕾,熊约飞,等.1株产纤维素酶猪源性枯草芽孢杆菌的筛选鉴定及其对仔猪生长性能、体液免疫的影响[J].中国畜牧杂志,2024,60(4):214-219. ZHENG K T,XU Y L,XIONG Y F,et al.Screening and identification of a cellulase-producing strain of porcine Bacillus subtilis and its effects on growth performance and humoral immunity of piglets[J].Chinese Journal of Animal Science,2024,60(4):214-219.(in Chinese)
[13] 高娟娟,贾丽艳,畅盼盼,等.枯草芽孢杆菌细菌素A32的抑菌机理研究[J].中国食品学报,2021,21(10):56-64. GAO J J,JIA L Y,CHANG P P,et al.Studies on the inhibition mechanism of bacteriocin A32 producing by Bacillus subtilis[J].Chinese Journal of Food Science,2021,21(10):56-64.(in Chinese)
[14] 钱荣,续晓琪,许宗奇,等.枯草芽孢杆菌KC-WQ发酵液中抗菌脂肽的分离鉴定及发酵条件优化[J].食品工业科技,2022,43(15):123-131. QIAN R,XU X Q,XU Z Q,et al.Isolation and identification of antibacterial lpopeptides from Bacillus subtilis KC-WQ fermentation broth and optimization offermentation conditions[J].Science and Technology of Food Industry,2022,43(15):123-131.(in Chinese)
[15] 方世纯,郝瑞霞,鲁志强.枯草芽孢杆菌(Bacillus subtilis)降解萘的动力学研究[J].高校地质学报,2007,4:682-687. FANG S C,HAO R X,LU Z Q.Dynamics of degrading naphthalene by Bacillus subtilis[J].Geological Journal of Universities,2007,4:682-687.(in Chinese)
[16] 雷元培,赵丽红,马秋刚,等.降解黄曲霉毒素枯草芽孢杆菌的解毒性、抗菌性及抗逆性研究[J].饲料工业,2011,32(24):23-27. LEI Y P,ZHAO L H,MA Q G,et al.Research on detoxification,antimicrobial activity and resistance to the simulated gut environments of Bacillus subtilis possesing aflatoxin degradation activity[J].Feed Industry,2011,32(24):23-27.(in Chinese)
[17] 周亮成,李运,李卓苗,等.枯草芽孢杆菌BSF01菌剂制备及对高效氯氰菊酯的降解效果[J].华南农业大学学报,2018,39(3):54-59. ZHOU L C,LI Y,LI Z M,et al.Preparation of bacterial agent based on Bacillus subtilis BSF01 and its biodegradation effect on beta-cypermethrin[J].Journal of South China Agricultural University,2018,39(3):54-59.(in Chinese)
[18] 陈宇婷,盛光遥,谢康颖,等.枯草芽孢杆菌耐镉能力驯化及镉吸附特性研究[J].工业水处理,2021,41(2):97-102. CHEN Y T,SHENG G Y,XIE K Y,et al.Acclimation of cadmium tolerance and cadmium adsorption characteristics of Bacillus subtilis[J].Industrial Water Treatment,2021,41(2):97-102.(in Chinese)
[19] 谭荣,龚杰,龚百川,等.用枯草芽孢杆菌吸附去除电镀废水中的铜[J].湿法冶金,2022,41(1):40-46. TAN R,GONG J,GONG B C,et al.Removal of copper from electroplating wastewater by microbia adsorption of Bacillus subtilis[J].Journal of Hydrometallurgy,2022,41(1):40-46.(in Chinese)
[20] JIN P,KANG Z,YUAN P,et al.Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168[J].Metabolic Engineering,2016,35:21-30.
[21] 张瑾,余祖华,丁轲,等.黄曲霉毒素B₁降解菌的筛选及生物学特性分析[J].食品与发酵工业,2022,48(14):175-180. ZHANG J,YU Z H,DING K,et al.Screening and biological characteristics analysis of biodegrading bacteria of aflatoxin B₁[J].Food and Fermentation Industry,2022,48(14):175-180.(in Chinese)
[22] GAUR P,HADA V,RATH R S,et al.Interpretation of antimicrobial susceptibility testing using European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) breakpoints:Analysis of agreement[J].Cureus,2023,15(3):e36977.
[23] BARAKATULLAH M,NATALIA G,MARTHA EVELIA P R,et al.Profiling toxin genes and antibiotic resistance in Bacillus cereus isolated from pre-launch spacecraft[J].Frontiers in Microbiology,2023,14:1231726.
[24] 刘伟杰,刘聪,蒋继宏.枯草芽孢杆菌形成生物被膜的研究进展[J].微生物学报,2014,54(9):977-983. LIU W J,LIU C,JIANG J H.Research progress on biofilm formation of Bacillus subtilis—A review[J].Acta Microbiologica Sinica,2014,54(9):977-983.(in Chinese)
[25] 廖才江,李会,王士源,等.生物被膜：益生菌肠道定植的新策略[J].生物工程学报,2022,38(8):2821-2839. LIAO C J,LI H,WANG S Y,et al.Bacterial biofilms:Novel strategies for intestinal colonization by probiotics[J].Chinese Journal of Bioengineering,2022,38(8):2821-2839.(in Chinese)
[26] PLOTNIKOV P V,MOVSESYAN N A,LEPTEEVA T N,et al.Immunity and bacterial biofilms:Current status of the matter (literature review)[J].Vestnik Vitebskogo Gosudarstvennogo Medicinskogo Universiteta,2021,20(3):7-15.
[27] 赵浩航,汪志文,温依铭,等.乌鳢肠道芽孢杆菌的分离鉴定及益生特性评估[J].水生生物学报,2024.Doi:10.7541/2025.2024.0270. ZHAO H H,WANG Z W,WEN Y M,et al.Isolation,identification,and evaluation of Bacillus properties of intestinal Bacillus from Channa argus[J].Chinese Journal of Hydrobiology,2024.Doi:10.7541/2025.2024.0270.(in Chinese)
[28] KIRAZ Y,ADAN A,KARTAL Y M,et al.Major apoptotic mechanisms and genes involved in apoptosis[J]. Tumor Biology,2016,37(7):8471-8486.
[29] ONANONG J,PORNPATTRA M,KUN K,et al.Corosolic acid induced apoptosis via upregulation of Bax/Bcl-2 ratio and Caspase-3 activation in cholangiocarcinoma cells[J].ACS Omega,2024,9(1):1278-1286.
[30] 冯骁,田聆,黄倩. Caspase-3基因表达调控研究进展[J].生命科学,2014,26(9):936-942. FENG X,TIAN L,HUANG Q.Advances in Caspase-3 gene expression regulation[J].Life Science,2014,26(9):936-942.(in Chinese)
[31] 王健,赵会君,史以超,等.枯草芽孢杆菌胶囊对溃疡性结肠炎患者炎症因子、氧化应激和肠黏膜功能的影响[J].转化医学杂志,2024,13(4):564-567. WANG J,ZHAO H J,SHI Y C,et al.Clinical efficacy of intestinal microecological changes induced by B.subtilis against ulcerative colitis[J].Translational Medicine,2024,13(4):564-567.(in Chinese)
[32] GUO C G,LIU S R,DI L J,et al.The impact of Bacillus pumilus TS2 isolated from yaks on growth performance,gut microbial community,antioxidant activity,and cytokines related to immunity and inflammation in broilers[J].Frontiers in Veterinary Science,2024,59(12):e00213-21.