一株解淀粉芽孢杆菌的特性及抑菌活性研究
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摘要
试验旨在对一株牛瘤胃源解淀粉芽孢杆菌的生长曲线、耐酸性和耐胆酸盐性等特性进行分析,同时对该菌发酵产物的抑菌活性和抑菌活性稳定性进行测定。采用生长速率法测定该菌生长曲线,活菌计数法测定耐酸性和耐胆酸盐性等特性,牛津杯法测定抑菌活性和抑菌活性稳定性。结果显示,该菌生长4h时进入对数生长期,20h后生长趋于稳定,进入生长稳定期;同时该菌表现出较好的耐酸性,在pH 3.0~7.0时存活率为23.8%~90.6%;有较好的耐胆酸盐性,在0.1%~0.4%的胆酸盐中存活率为64.2%~83.6%。以大肠杆菌(Escherichia coli)、副溶血弧菌(Vibrio parahemolyticus)、藤黄八叠球菌(Sarcina lutea)和产气杆菌(Aerogenes)等细菌和黑曲霉(Aspergillus niger)、黑根霉(Rhizopus nigricans)等霉菌作指示菌进行抑菌试验发现,该菌的发酵产物具有抑菌活性,同时其抑菌活性物质的耐热性和耐酸性较好。本试验通过对该解淀粉芽孢杆菌的特性进行研究发现,该菌具有耐酸、耐胆酸盐等特性;其抑菌活性及抑菌稳定性较好,具有耐酸碱及耐高温特性。
The aim of the experiment was to analyze the growth curve,acid resistance and bile acid salt tolerance of a strain of Bacillus amyloliquefaciens from rumen,and to determine the bacteriostasis activity and antibacterial activity stability of the fermented product of the strain.The growth curve of the strain was determined by growth rate method,the viable count method was used to determine the acid resistance and bile acid salt tolerance,and the antibacterial activity and antibacterial activity stability were determined by the Oxford cup method.The results showed that the bacteria entered the logarithmic growth phase after 4 hof growth and tended to be stable after 20 hof growth and entered the stable growth phase.At the same time,the bacteria showed good acid resistance,and the survival rates were 23.8%to 90.6% at pH 3.0 to 7.0;And the bile acid salt tolerance was better,and the survival rates were 64.2%to 83.6%in bile acid concentration of 0.1% to 0.4%.Bacteria such as Escherichia coli,Vibrio parahemolyticus,Sarcina lutea and Aerogenes,and fungi such as Aspergillus niger and Rhizopus nigricans,as bacteriostatic indicator bacteria for antibacterial test,and the results showed that the fermentation products of the Bacillus amyloliquefaciens showed antibacterial activity,and its antibacterial active substance had good heat resistance and acid resistance.In this experiment,the properties of Bacillus amyloliquefaciens were studied and it had the properties of acid resistance and ile acid salt toler-ance;The bacteriostatic activity and bacteriostasis stability were good,and the bacterium had acid and alkali resistance and high temperature resistance.
The aim of the experiment was to analyze the growth curve,acid resistance and bile acid salt tolerance of a strain of Bacillus amyloliquefaciens from rumen,and to determine the bacteriostasis activity and antibacterial activity stability of the fermented product of the strain.The growth curve of the strain was determined by growth rate method,the viable count method was used to determine the acid resistance and bile acid salt tolerance,and the antibacterial activity and antibacterial activity stability were determined by the Oxford cup method.The results showed that the bacteria entered the logarithmic growth phase after 4 hof growth and tended to be stable after 20 hof growth and entered the stable growth phase.At the same time,the bacteria showed good acid resistance,and the survival rates were 23.8%to 90.6% at pH 3.0 to 7.0;And the bile acid salt tolerance was better,and the survival rates were 64.2%to 83.6%in bile acid concentration of 0.1% to 0.4%.Bacteria such as Escherichia coli,Vibrio parahemolyticus,Sarcina lutea and Aerogenes,and fungi such as Aspergillus niger and Rhizopus nigricans,as bacteriostatic indicator bacteria for antibacterial test,and the results showed that the fermentation products of the Bacillus amyloliquefaciens showed antibacterial activity,and its antibacterial active substance had good heat resistance and acid resistance.In this experiment,the properties of Bacillus amyloliquefaciens were studied and it had the properties of acid resistance and ile acid salt toler-ance;The bacteriostatic activity and bacteriostasis stability were good,and the bacterium had acid and alkali resistance and high temperature resistance.
引文
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[15]张晓云.枯草芽孢杆菌菌株CAB-1抑菌物质的分离鉴定及活性分析[D].保定:河北农业大学,2011.ZHANG X Y.Isolation and analysis of antifungal compounds produced by Bacillus subtilis strain CAB-1[D].Baoding:Hebei Agricultural University,2011.(in Chinese)
[16]刘秀侠,徐海燕,辛国芹,等.11株枯草芽孢杆菌益生特性研究[J].中国畜牧兽医,2017,8(44):2333-2341.LIU X X,XU H Y,XIN G Q,et al.Study on probiotic characteristics of eleven Bacillus subtilis strains[J].China Animal Husbandry&Veterinary Medicine,2017,8(44):2333-2341.(in Chinese)
[17]侯宝宏,徐秉良,郇町,等.抗苹果树腐烂病解淀粉芽孢杆菌TS-1203的抑菌活性物质稳定性及抑菌谱测定[J].甘肃农业大学学报,2017,52(1):80-86.HOU B H,XU B L,HUAN T,et al.Determination of the stability and antibacterial spectrum of antibacterial substances of the Bacillus amyloliquefaciens TS-1203against Valsa mali[J].Journal of Gansu Agricultural University,2017,52(1):80-86.(in Chinese)
[18]孙笑非,孙冬岩,王文娟,等.猪源性益生芽孢杆菌的筛分与鉴定[J].饲料研究,2013,1:39-40.SUN X F,SUN D Y,WANG W J,et al.Screening and identification of pig derived probiotic Bacillus[J].Feed Research,2013,1:39-40.(in Chinese)
[19]范会兰,姜军坡,王世英,等.抗犊牛腹泻益生菌株BN-9产芽孢条件的优化[J].湖北农业科学,2009,48(2):405-409.FAN H L,JIANG J P,WANG S Y,et al.Optimization of the spore production conditions of strain BN-9as probiotics to prevent calf diarrhea[J].Hubei Agricultural Sciences,2009,48(2):405-409.(in Chinese)
[20]张以侠,谭长营.牛消化系统疾病的诊治[J].山东畜牧兽医,2015,36(6):28-30.ZHANG Y X,TAN C Y.Diagnosis and treatment of digestive system diseases in rumen[J].Shandong Journal of Animal Science and Veterinary Medicine,2015,36(6):28-30.(in Chinese)
[21]CHANG X J,WU Z D,WU S L,et al.Degradation of ochratoxin A by Bacillus amyloliquefaciens ASAG1[J].Food Additives&Contaminants Part A Chemistry Analysis Control Exposure&Risk Assessment,2014,32(4):1-8.
[22]陈成,崔堂兵,于平儒,等.一株抗真菌的解淀粉芽孢杆菌的鉴定及其抗菌性研究[J].现代食品科技,2011,27(1):36-39.CHEN C,CUI T B,YU P R,et al.Identification of an anti-fungal strain of Amyloliquefaciens Bacillus and the properties of the antifungal substance[J].Modern Food Science&Technology,2011,27(1):36-39.(in Chinese)
[23]钟啸萍,熊智强,陈小龙.枯草芽孢杆菌a1所产抗真菌活性物质的稳定性研究[J].浙江农业科学,2011,1(4):878-881.ZHONG X P,XIONG Z Q,CHEN X L.Study on the stability of antifungal substances produced by Bacillus subtilis a1[J].Journal of Zhejiang Agricultural Sciences,2011,1(4):878-881.(in Chinese)
[24]张雪花,李琳玲,程华,等.解淀粉芽孢杆菌CMN1308抗真菌特性研究[J].北方园艺,2016,2:117-121.ZHANG X H,LI L L,CHENG H,et al.Characterizing a novel strain of Bacillus amyloliquefaciens CMN1308for potential biological control in pathogenic bacterium[J].Northern Horticulture,2016,2:117-121.(in Chinese)
[25]郝生宏,董晓芳,佟建明,等.不同饲料制粒条件和保存时间对益生菌活力的影响[J].中国畜牧杂志,2009,45(1):51-53.HAO S H,DONG X F,TONG J M,et al.Effects of different granulating condition and preservation time on the livability of probiotics[J].Chinese Journal of Animal Science,2009,45(1):51-53.(in Chinese)
[2]周淑萍.畜禽养殖中滥用抗生素的危害与对策[J].中国畜禽种业,2012,8(11):39-40.ZHOU S P.Harm and countermeasures of abuse of antibiotics in livestock and poultry breeding[J].The Chinese Livestock Breeding,2012,8(11):39-40.(in Chinese)
[3]刁其玉.微生物制剂在幼龄反刍动物营养与饲料中的应用[J].动物营养学报,2014,26(10):3159-3167.DIAO Q Y.Probiotics:Application in nutrition and feed of young ruminants[J].Chinese Journal of Animal Nutrition,2014,26(10):3159-3167.(in Chinese)
[4]MARTINEZ J L.Environmental pollution by antibiotics and by antibiotic resistance determinants[J].Environmental Pollution,2009,157(11):2893-2902.
[5]洪鹏,安国栋,胡美英,等.解淀粉芽孢杆菌防治果蔬采后病害研究进展[J].中国农学通报,2013,29(12):168-173.HONG P,AN G D,HU M Y,et al.Advance in research on biological control of postharvest diseases of fruits and vegetables by Bacillus[J].Chinese Agricultural Science Bulletin,2013,29(12):168-173.(in Chinese)
[6]栾素军,邢焕,孙永波,等.解淀粉芽孢杆菌在畜禽养殖中的应用研究进展[J].中国畜牧兽医,2016,43(10):2615-2620.LUAN S J,XING H,SUN Y B,et al.Research progress on Bacillus amyloliquefaciens in animal breeding industry[J].China Animal Husbandry&Veterinary Medicine,2016,43(10):2615-2620.(in Chinese)
[7]ROMY S,JOACHIM V,ANTO B,et al.Amylocyclicin,a novel circular bacteriocin produce by Bacillus amyloliquefaciens FZB42[J].Journal of Bacteriology,2014,196(10):1842-1852
[8]张彤,何剑斌,李鹏,等.两株鹿源芽孢杆菌的分离鉴定及生物学特性研究[J].沈阳农业大学学报,2017,48(2):166-173.ZHANG T,HE J B,LI P,et al.Isolation,identification and biological characteristics of two strains of deer-derived Bacillus[J].Journal of Shenyang Agricultural University,2017,48(2):166-173.(in Chinese)
[9]朱艳蕾.细菌生长曲线测定实验方法的研究[J].微生物学杂志,2016,36(5):108-112.ZHU Y L.Experimental method of bacteria growth curve determination[J].Journal of Microbiology,2016,36(5):108-112.(in Chinese)
[10]宁豫昌,赵绪永,郑鸣.猪源芽孢杆菌的分离鉴定及生物学特性研究[J].中国畜牧兽医,2012,39(9):65-68.NING Y C,ZHAO X Y,ZHENG M.Isolation,identification of Bacillaceae from swine and study on its biological characteristics[J].China Animal Husbandry&Veterinary Medicine,2012,39(9):65-68.(in Chinese)
[11]李善仁,陈济琛,蔡海松,等.三株芽孢杆菌作为益生菌的生物特性[J].营养学报,2010,32(1):75-78.LI S R,CHEN J C,CAI H S,et al.The properties of three Bacillus strains as potential probiotics[J].Acta Nutrimenta Sinica,2010,32(1):75-78.(in Chinese)
[12]杨冬静,张成玲,徐振,等.一株解淀粉芽孢杆菌的分离鉴定及其生物学特性研究[J].江西农业学报,2017,29(9):69-74.YANG D J,ZHANG C L,XU Z,et al.Isolation and identification of a strain of Bacillus amyloliquefaciens and its biological characteristics[J].Acta Agriculturae Jiangxi,2017,29(9):69-74.(in Chinese)
[13]刘冬梅,李理,杨晓泉,等.用牛津杯法测定益生菌的抑菌活力[J].食品研究与开发,2006,27(3):110-111.LIU D M,LI L,YANG X Q,et al.Determination of the antimicrobial activity of probiotic by oxford plate assay system[J].Food Research and Development,2006,27(3):110-111.(in Chinese)
[14]张华,赵辉,王鹏涛,等.解淀粉芽孢杆菌B10-26抑菌物质的稳定性研究与初步分离纯化[J].河南农业科学,2014,43(5):93-96.ZHANG H,ZHAO H,WANG P T,et al.Stability determination and prefractionation of antifungal substance produced by Bacillus amyloliquefaciens B10-26[J].Journal of Henan Agricultural Sciences,2014,43(5):93-96.(in Chinese)
[15]张晓云.枯草芽孢杆菌菌株CAB-1抑菌物质的分离鉴定及活性分析[D].保定:河北农业大学,2011.ZHANG X Y.Isolation and analysis of antifungal compounds produced by Bacillus subtilis strain CAB-1[D].Baoding:Hebei Agricultural University,2011.(in Chinese)
[16]刘秀侠,徐海燕,辛国芹,等.11株枯草芽孢杆菌益生特性研究[J].中国畜牧兽医,2017,8(44):2333-2341.LIU X X,XU H Y,XIN G Q,et al.Study on probiotic characteristics of eleven Bacillus subtilis strains[J].China Animal Husbandry&Veterinary Medicine,2017,8(44):2333-2341.(in Chinese)
[17]侯宝宏,徐秉良,郇町,等.抗苹果树腐烂病解淀粉芽孢杆菌TS-1203的抑菌活性物质稳定性及抑菌谱测定[J].甘肃农业大学学报,2017,52(1):80-86.HOU B H,XU B L,HUAN T,et al.Determination of the stability and antibacterial spectrum of antibacterial substances of the Bacillus amyloliquefaciens TS-1203against Valsa mali[J].Journal of Gansu Agricultural University,2017,52(1):80-86.(in Chinese)
[18]孙笑非,孙冬岩,王文娟,等.猪源性益生芽孢杆菌的筛分与鉴定[J].饲料研究,2013,1:39-40.SUN X F,SUN D Y,WANG W J,et al.Screening and identification of pig derived probiotic Bacillus[J].Feed Research,2013,1:39-40.(in Chinese)
[19]范会兰,姜军坡,王世英,等.抗犊牛腹泻益生菌株BN-9产芽孢条件的优化[J].湖北农业科学,2009,48(2):405-409.FAN H L,JIANG J P,WANG S Y,et al.Optimization of the spore production conditions of strain BN-9as probiotics to prevent calf diarrhea[J].Hubei Agricultural Sciences,2009,48(2):405-409.(in Chinese)
[20]张以侠,谭长营.牛消化系统疾病的诊治[J].山东畜牧兽医,2015,36(6):28-30.ZHANG Y X,TAN C Y.Diagnosis and treatment of digestive system diseases in rumen[J].Shandong Journal of Animal Science and Veterinary Medicine,2015,36(6):28-30.(in Chinese)
[21]CHANG X J,WU Z D,WU S L,et al.Degradation of ochratoxin A by Bacillus amyloliquefaciens ASAG1[J].Food Additives&Contaminants Part A Chemistry Analysis Control Exposure&Risk Assessment,2014,32(4):1-8.
[22]陈成,崔堂兵,于平儒,等.一株抗真菌的解淀粉芽孢杆菌的鉴定及其抗菌性研究[J].现代食品科技,2011,27(1):36-39.CHEN C,CUI T B,YU P R,et al.Identification of an anti-fungal strain of Amyloliquefaciens Bacillus and the properties of the antifungal substance[J].Modern Food Science&Technology,2011,27(1):36-39.(in Chinese)
[23]钟啸萍,熊智强,陈小龙.枯草芽孢杆菌a1所产抗真菌活性物质的稳定性研究[J].浙江农业科学,2011,1(4):878-881.ZHONG X P,XIONG Z Q,CHEN X L.Study on the stability of antifungal substances produced by Bacillus subtilis a1[J].Journal of Zhejiang Agricultural Sciences,2011,1(4):878-881.(in Chinese)
[24]张雪花,李琳玲,程华,等.解淀粉芽孢杆菌CMN1308抗真菌特性研究[J].北方园艺,2016,2:117-121.ZHANG X H,LI L L,CHENG H,et al.Characterizing a novel strain of Bacillus amyloliquefaciens CMN1308for potential biological control in pathogenic bacterium[J].Northern Horticulture,2016,2:117-121.(in Chinese)
[25]郝生宏,董晓芳,佟建明,等.不同饲料制粒条件和保存时间对益生菌活力的影响[J].中国畜牧杂志,2009,45(1):51-53.HAO S H,DONG X F,TONG J M,et al.Effects of different granulating condition and preservation time on the livability of probiotics[J].Chinese Journal of Animal Science,2009,45(1):51-53.(in Chinese)