牛源抗氧化芽孢杆菌的分离鉴定及生物特性研究

doi:10.16431/j.cnki.1671-7236.2023.01.036

摘要/Abstract

摘要： 【目的】从奶牛瘤胃中分离具有抗氧化能力且耐受性好的芽孢杆菌，为牛源益生菌作为抗氧化添加剂使用提供理论依据及参考。【方法】采集3头成年荷斯坦奶牛瘤胃内容物，通过细菌分离纯化、形态观察、生化检测及16S rDNA分子检测进行鉴定，同时检测筛选菌株的超氧自由基和羟自由基清除率、耐酸、耐胆盐能力及对抗生素的耐药性。【结果】从奶牛瘤胃液中共获得了12株菌，通过特异性培养基与革兰氏染色共筛选出5株芽孢杆菌（X-1~X-5）。对5株芽孢杆菌进行分子鉴定，确定X-1为热噬淀粉芽孢杆菌，X-2为短小芽孢杆菌，X-3为解蛋白芽孢杆菌，X-4为枯草芽孢杆菌，X-5为沙福芽孢杆菌。抗氧化能力检测结果显示，5株菌均具有一定的抗氧化能力，X-5菌株抗氧化能力最高，超氧自由基和羟自由基清除率分别达到73.81%和73.54%。当pH为2.5时，5株芽孢杆菌存活率均在25%以上；胆盐浓度为0.6%时，5株芽孢杆菌存活率均在60%以上，说明菌株具有良好的耐受性。药敏试验结果显示，试验分离出来的大多数菌株对氧氟沙星、呋喃妥因、红霉素等高度敏感；对头孢呋辛、头孢噻肟、链霉素等耐受；对卡那霉素、环丙沙星、氨苄西林等中度敏感。【结论】本研究分离的牛源芽孢杆菌具有抗氧化能力，部分菌株具有很好的耐酸、耐胆盐特性，为牛源芽孢杆菌作为抗氧化添加剂使用提供理论依据及参考。

关键词: 奶牛; 芽孢杆菌; 分离鉴定; 抗氧化; 生化特性

Abstract: 【Objective】 This study was aimed to isolate Bacillus with antioxidant capacity and good tolerance from the rumen of dairy cows, so as to provide theoretical basis and reference for the use of bovine probiotics as antioxidant additives.【Method】 The rumen contents of 3 adult Holstein cows were collected and identified by bacterial isolation and purification, morphological observation, physiological and biochemical detection and 16S rDNA molecular detection.The scavenging rates of superoxide free radicals and hydroxyl free radicals, acid resistance, bile salt resistance and antibiotic resistance of the screened strains were also detected.【Result】 A total of 12 strains of Bacillus were isolated from rumen fluid of dairy cows, and 5 strains of Bacillus (X-1 to X-5) were screened by specific medium and Gram staining.Five strains of Bacillus were identified by molecular identification, and X-1 was identified as Bacillus thermoamylovorans, X-2 as Bacillus pumilus, X-3 as Bacillus proteolyticus, X-4 as Bacillus subtilis, and X-5 as Bacillus safensis.The results of antioxidant capacity test showed that all strains had certain antioxidant capacity, and strain X-5 had the highest antioxidant capacity, with the scavenging rates of superoxide free radicals and hydroxyl free radicals being 73.81% and 73.54%, respectively.When pH was 2.5, the survival rates of 5 Bacillus strains were all more than 25%.When the bile salt concentration was 0.6%, the survival rates of five Bacillus strains were all more than 60%, indicating that the strains had good tolerance.The results of drug sensitivity test showed that most of the isolates were highly sensitive to ofloxacin, nitrofurantoin, erythromycin and so on.It was resistant to cefuroxime, cefotaxime, streptomycin and so on.It was moderately sensitive to several drugs such as kanamycin, ciprofloxacin and ampicillin.【Conclusion】 The isolated Bacillus from cattle in this study had antioxidant capacity, and some of the strains had good acid resistance and bile salt resistance, which provided theoretical basis and reference for the use of Bacillus from cattle as antioxidant additives.

Key words: dairy cows; Bacillus; isolation and identification; oxidation resistance; biochemical characteristics

中图分类号:

S816.7

孙愉, 张昊, 李畅洋, 陶燕子, 王秋菊. 牛源抗氧化芽孢杆菌的分离鉴定及生物特性研究[J]. 中国畜牧兽医, 2023, 50(1): 359-367.

SUN Yu, ZHANG Hao, LI Changyang, TAO Yanzi, WANG Qiuju. Isolation,Identification and Biological Characteristics of Antioxidant Bacillus from Cattle[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 359-367.

参考文献

[1] VERSCHUERE L, ROMBAUT G, SORGELOOS P, et al.Probiotic bacteria as biological control agents in aquaculture[J].Microbiology & Molecular Biology Reviews, 2000, 64(4):655-671.
[2] 高晓艺, 马贵达, 徐帆, 等.太行鸡肠道益生菌的分离与鉴定[J].黑龙江畜牧兽医, 2019, 11:1-5. GAO X Y, MA G D, XU F, et al.Isolation and identification of probiotics from intestinal of Taihang chickens[J].Heilongjiang Animal Science and Veterinary Medicine, 2019, 11:1-5.(in Chinese)
[3] 石璐璐, 徐元庆, 王哲奇, 等.热应激诱发的氧化应激对羊的影响及其作用机制[J].动物营养学报, 2019, 31(7):3016-3022. SHI L L, XU Y Q, WANG Z Q, et al.Effects of oxidative stress induced by heat stress and its mechanism in sheep and goats[J].Chinese Journal of Animal Nutrition, 2019, 31(7):3016-3022.(in Chinese)
[4] 黄帆, 吕秋凤.热应激诱导的氧化应激对动物肠道组织的损伤[J].动物营养学报, 2017, 29(6):1856-1860. HUANG F, LYU Q F.Oxidative stress induced by heat stress:Injury on intestinal tissue of animals[J].Chinese Journal of Animal Nutrition, 2017, 29(6):1856-1860.(in Chinese)
[5] 陈小慧, 张玲玲, 谭胜男, 等.奶牛氧化应激及抗氧化措施研究进展[J].黑龙江畜牧兽医, 2018, 21:53-56. CHEN X H, ZHANG L L, TAN S N, et al.Research progress of oxidative stress and antioxidant measures in dairy cows[J].Heilongjiang Animal Science and Veterinary Medicine, 2018, 21:53-56.(in Chinese)
[6] NAWAB A, IBTISHAM F, LI G, et al.Heat stress in poultry production:Mitigation strategies to overcome the future challenges facing the global poultry industry[J].Journal of Thermal Biology, 2018, 78:131-139.
[7] 占今舜, 魏明吉, 苏效双, 等.苜蓿黄酮对热应激下体外培养奶牛乳腺上皮细胞凋亡的影响[J].草业学报, 2016, 25(4):159-165. ZHAN J S, WEI M J, SU X S, et al.Effect of alfalfa flavonoids on bovine mammary epithelial cell cultures in vitro under heat stress[J].Acta Prataculturae Sinica, 2016, 25(4):159-165.(in Chinese)
[8] 张新宇, 邓文, 李霞, 等.硫辛酸对热应激肉鸡生产性能、肠道形态和通透性及抗氧化性能的影响[J].中国饲料, 2021, 19:34-38. ZHANG X Y, DENG W, LI X, et al.Effects of lipoic acid on performance, intestinal morphology, permeability and antioxidant performance of heat stressed broilers[J].China Feed, 2021, 19:34-38.(in Chinese)
[9] 张成, 王驰, 王孝成, 等.白藜芦醇对热应激肉鸡肝脏抗氧化功能和Nrf2通路相关基因表达的影响[J].南京农业大学学报, 2021, 44(6):1154-1161. ZHANG C, WANG C, WANG X C, et al.Effects of resveratrol on antioxidant function and the expression of Nrf2 signaling pathway-related genes in liver of heat-stressed broilers[J].Journal of Nanjing Agricultural University, 2021, 44(6):1154-1161.(in Chinese)
[10] 徐春兰, 钦传光, 牛卫宁, 等.Enterobacter cloacae Z0206细菌胞外多糖的体外抗氧化活性研究[J].天然产物研究与开发, 2010, 22(6):1098-1102. XU C L, QIN C G, NIU W N, et al.Antioxidative activity of exopolysaccharide produced by Enterobacter cloacae Z0206[J].Natural Product Research and Development, 2010, 22(6):1098-1102.(in Chinese)
[11] 赵云花.芽孢杆菌CL2在枸杞采后病害防控中的应用及机制研究[D].兰州:西北师范大学, 2021. ZHAO Y H.Study on the application and mechanism of Bacillus CL2 in the prevention and control of postharvest wolfberry diseases[D].Lanzhou:Northwest Normal University, 2021.(in Chinese)
[12] 田照辉, 徐绍刚, 董颖, 等.6株芽孢杆菌的分离鉴定和生物学特性[J].江苏农业科学, 2021, 49(13):157-161. TIAN Z H, XU S G, DONG Y, et al.Isolation and identification of six Bacillus strains and preliminary analysis of their biological characteristics[J].Jiangsu Agricultural Sciences, 2021, 49(13):157-161.(in Chinese)
[13] 王喜峰, 魏霖, 贺绍君, 等.枯草芽孢杆菌和乳酸菌对热应激肉鸡生长性能和肝肾功能的影响[J].安徽科技学院学报, 2019, 33(6):23-28. WANG X F, WEI L, HE S J, et al.Effects of Bacillus subtilis and Lactobacillus on growth performance and liver and kidney function of broilers under heat stress[J].Journal of Anhui Science and Technology University, 2019, 33(6):23-28.(in Chinese)
[14] 王乙茹, 柳成东, 白华毅, 等.一株凝结芽孢杆菌YNAU 5517对热应激肉鸡生长性能影响的初探[J].饲料工业, 2019, 40(5):16-22. WANG Y R, LIU C D, BAI H Y, et al.Effects of the diet supplement of Bacillus coagulans YNAU 5517 on broiler growth performance under heat stress conditions[J].Feed Industry, 2019, 40(5):16-22.(in Chinese)
[15] 韩昱, 张立永, 张富梅.地衣芽孢杆菌对热应激公兔肾上腺结构及血浆皮质醇含量的影响[J].云南农业大学学报(自然科学), 2021, 36(6):962-968. HAN Y, ZHANG L Y, ZHANG F M.Effect of Bacillus licheniformis on the heat stress to adrenal gland structure and plasma CORT content in male rabbits[J].Journal of Yunnan Agricultural University (Natural Science), 2021, 36(6):962-968.(in Chinese)
[16] 魏笑, 王秋菊, 孙愉, 等.鹅粪便中特基拉芽孢杆菌和类肠膜魏斯氏菌的分离鉴定[J].中国畜牧兽医, 2022, 49(7):2778-2787. WEI X, WANG Q J, SUN Y, et al.Isolation and identification of Bacillus tequilensis and Weissella paramesenteroides from goose feces[J].China Animal Husbandry & Veterinary Medicine, 2022, 49(7):2778-2787.(in Chinese)
[17] DENG W D, XI D M, MAO H M, et al.The use of molecular techniques based on ribosomal RNA and DNA for rumen microbial ecosystem studies:A review[J]. Molecular Biology Reports, 2008, 35(2):265-274.
[18] 张洁.奶牛瘤胃地衣芽孢杆菌的分离鉴定及其对两种不同碳源的响应[D].银川:宁夏大学, 2021. ZHANG J.Isolation and identification of Bacillus licheniformis from rumen of dairy cattle and its response to two different carbon sources[D].Yinchuan:Ningxia University, 2021.(in Chinese)
[19] 李靖.枯草芽孢杆菌对荷斯坦育成牛生长性能和瘤胃微生物区系的影响[D].泰安:山东农业大学, 2019. LI J.Effects of Bacillus subtilis on growth performance and rumen microflora in Holstein cows[D].Tai'an:Shandong Agricultural University, 2019.(in Chinese)
[20] 谈诗谣.瘤胃源11株芽孢杆菌的生理学特性研究及发酵条件优化[D].大连:大连工业大学, 2018. TAN S Y.Study on the physiological characteristics of 11 strains of bacteria in the rumen and optimization of fermentation conditions[D].Dalian:Dalian Polytechnic University, 2018.(in Chinese)
[21] 郭艺伟, 吕兰, 刘依山, 等.贝莱斯芽孢杆菌发酵蚯蚓浆对猪抗氧化功能和免疫功能的影响[J].中兽医医药杂志, 2022, 41(3):13-16. GUO Y W, LYU L, LIU Y S, et al.Effects of earthworm pulp fermented by Bacillus velezensis on antioxidant function and immune function in pigs[J].Journal of Traditional Chinese Veterinary Medicine, 2022, 41(3):13-16.(in Chinese)
[22] 李玉琴, 李义, 黄新, 等.解淀粉芽孢杆菌对奶牛泌乳性能、乳成分及血清生化、免疫和抗氧化指标的影响[J].动物营养学报, 2022, 34(2):979-988. LI Y Q, LI Y, HUANG X, et al.Effects of Bacillus amyloliquefaciens on lactation performance, milk composition and serum biochemical, immune and antioxidant indexes of dairy cows[J].Chinese Journal of Animal Nutrition, 2022, 34(2):979-988.(in Chinese)
[23] 王蕾, 孙娅静.枯草芽孢杆菌对AA肉仔鸡血清抗氧化功能和肉品质的影响[J].饲料研究, 2020, 43(9):36-39. WANG L, SUN Y J.Effect of Bacillus subtilis on serum antioxidant function and meat quality of AA broilers[J].Feed Research, 2020, 43(9):36-39.(in Chinese)
[24] 谢晓佩, 王琦, 孔新平, 等.西藏牦牛源枯草芽孢杆菌的分离培养及鉴定[J].家畜生态学报, 2021, 42(3):26-31. XIE X P, WANG Q, KONG X P, et al.Isolation, culture and identification of Bacillus subtilis from yak in Tibet[J].Journal of Domestic Animal Ecology, 2021, 42(3):26-31.(in Chinese)
[25] 冀婉玥, 林康, 孟林, 等.犬源芽孢杆菌分离鉴定及其耐受性的研究[J].天津农业科学, 2018, 24(8):11-15. JI W Y, LIN K, MENG L, et al.Study on isolation, identification and tolerance of Bacillus of canine[J].Tianjin Agricultural Sciences, 2018, 24(8):11-15.(in Chinese)
[26] 郭政宏, 周彪, 严亨秀.一株藏绵羊源短小芽孢杆菌的分离鉴定及生物学特性研究[J].中国畜牧兽医, 2016, 43(6):1610-1617. GUO Z H, ZHOU B, YAN H X.Study on the isolation, identification and biological characteristics of a strain of Bacillus pumilus isolated from Tibetan sheep[J].China Animal Husbandry & Veterinary Medicine, 2016, 43(6):1610-1617.(in Chinese)
[27] 王国艳, 乔国峰, 范建强, 等.肉牛场环境中枯草芽孢杆菌的16S rDNA分离鉴定[J].现代畜牧兽医, 2021, 12:9-11. WANG G Y, QIAO G F, FAN J Q, et al.Isolation and identification of 16S rDNA of Bacillus subtilis in beef cattle farm[J].Modern Journal of Animal Husbandry and Veterinary Medicine, 2021, 12:9-11.(in Chinese)