大菱鲆肠道中广谱拮抗活性乳酸菌的筛选及其细菌素鉴定
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摘要
应用牛津杯法从大菱鲆肠道分离的乳酸菌中筛选出对革兰氏阳性和阴性细菌均具有抑制作用的菌株LP1-4,经生理生化反应和16SrRNA鉴定为植物乳杆菌(Lactobacillusplantarum)。通过温度、pH值和蛋白酶等因素分析,结果表明菌株LP1-4无细胞上清液(cell-free supernatant,CFS)中抑菌物质具有良好的热稳定性,在pH2.5～5.0具有抑菌活性,且对蛋白酶敏感,初步判断为细菌素类抑菌物质。菌株LP1-4在最佳培养时间24 h时抑菌活性达到峰值,应用乙酸乙酯萃取后抑菌圈直径达25.42 mm。采用Labscale TFF System超滤分离和Sephadex G-25凝胶层析获得小于1.0 ku分子质量的细菌素。经高效液相色谱纯化分析,抑菌物质在液相分离条件下目标峰的保留时间为5.0 min。采用基质辅助激光解吸电离串联飞行时间质谱确定细菌素分子质量为0.854 ku,其氨基酸排列顺序为谷氨酸-天冬酰胺-赖氨酸-脯氨酸-丙氨酸-丙氨酸-脯氨酸-赖氨酸(ENKPAAPK)。本研究从大菱鲆肠道筛选具有广谱抗菌活性的菌株LP1-4,对研发控制水产品腐败菌和致病菌的乳酸菌生物防腐保鲜制剂具有潜在的应用价值。
Strain LP1-4, with strong antagonistic activity against both Gram-negative bacteria and Gram-positive bacteria,was screened out of lactic acid bacteria(LAB) isolated from the turbot intestine using the Oxford cup method. LP1-4 was identi?ed as Lactobacillus plantarum according to physiological and biochemical characteristics and 16 S rRNA sequence analysis. The antimicrobial substances in the cell-free supernatant(CFS) of LP1-4 were heat stable and effective in the pH range of 2.5–5.0, but were sensitive to protease. These substances were identi?ed as bacteriocins. The antibacterial activity of LP1-4 reached the peak after 24 h of culture, and the diameter of inhibition zone of the ethyl acetate extract was 25.42 mm.A bacteriocin with molecular mass less than 1.0 ku was purified from the extract by ultrafiltration using Labscale TFF system and Sephadex G-25 column chromatography. The retention time of the target peak in high performance liquid chromatography(HPLC) was 5.0 min. The molecular mass of the bacteriocin was estimated to be 0.854 ku by matrix-assisted laser desorption ionisation/time-of-flight/time-of-flight mass spectrometry(MALDI-TOF/TOF MS).The amino acid sequence of this bacteriocin was Glu-Asn-Lys-Pro-Ala-Ala-Pro-Lys. In conclusion, LP1-4 has potential applications in controlling spoilage and pathogenic bacteria in aquatic products as a biological preservative.
Strain LP1-4, with strong antagonistic activity against both Gram-negative bacteria and Gram-positive bacteria,was screened out of lactic acid bacteria(LAB) isolated from the turbot intestine using the Oxford cup method. LP1-4 was identi?ed as Lactobacillus plantarum according to physiological and biochemical characteristics and 16 S rRNA sequence analysis. The antimicrobial substances in the cell-free supernatant(CFS) of LP1-4 were heat stable and effective in the pH range of 2.5–5.0, but were sensitive to protease. These substances were identi?ed as bacteriocins. The antibacterial activity of LP1-4 reached the peak after 24 h of culture, and the diameter of inhibition zone of the ethyl acetate extract was 25.42 mm.A bacteriocin with molecular mass less than 1.0 ku was purified from the extract by ultrafiltration using Labscale TFF system and Sephadex G-25 column chromatography. The retention time of the target peak in high performance liquid chromatography(HPLC) was 5.0 min. The molecular mass of the bacteriocin was estimated to be 0.854 ku by matrix-assisted laser desorption ionisation/time-of-flight/time-of-flight mass spectrometry(MALDI-TOF/TOF MS).The amino acid sequence of this bacteriocin was Glu-Asn-Lys-Pro-Ala-Ala-Pro-Lys. In conclusion, LP1-4 has potential applications in controlling spoilage and pathogenic bacteria in aquatic products as a biological preservative.
引文
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[2]SCATAMBURLO T M,YAMAZI A K,CAVICCHIOLI V Q,et al.Spoilage potential of Pseudomonas,species isolated from goat milk[J].Journal of Dairy Science,2015,98(2):759-764.DOI:10.3168/jds.2014-8747.
[3]王振杰,孙晔,顾欣哲,等.计算机视觉技术对食品腐败检测的研究进展[J].食品安全质量检测学报,2015(8):2969-2975.
[4]SHARMA P,SIHAG R C,BHRADWAJ A.Isolation and identification of pathogenic bacteria and fungi isolated from skin ulcers of Cirrhinus mrigala[J].Indian Journal of Animal Research,2013,47(4):283-291.
[5]HAMMOND S T,BROWN J H,BURGER J R,et al.food spoilage,storage,and transport:implications for a sustainable future[J].Bioscience,2015,1(8):1-11.DOI:10.1093/biosci/biv081.
[6]刘晓雪,葛菁萍,宋刚,等.Nisin的防腐特性及其在食品工业中的应用[J].黑龙江医学,2016,29(2):195-199.DOI:10.14035/j.cnki.hljyy.2016.02.002.
[7]RAUTA P R,DHUPAL M,NAYAK B.Screening and characterization of potential probiotic lactic acid bacteria isolated from vegetable waste and fish intestine[J].International Journal of Current Microbiology and Applied Sciences,2013,2(8):234-244.
[8]GHOMRASSI H,BEN B O,CHOISET Y,et al.Evaluation of marine bacteriocinogenic Enterococci strains with inhibitory activity against fish-pathogenic Gram-negative bacteria[J].Diseases of Aquatic Organisms,2016,118(1):31-43.DOI:10.3354/dao02953.
[9]GEREZ C L,TORRES M J,VALDEZ G F D,et al.Control of spoilage fungi by lactic acid bacteria[J].Biological Control,2013,64(3):231-237.DOI:10.1016/j.biocontrol.2012.10.009.
[10]ULLAH N,WANG X,WU J,et al.Purification and primary characterization of a novel bacteriocin,LiN333,from Lactobacillus casei,an isolate from a Chinese fermented food[J].LWT-Food Science and Technology,2017,84:867-875.DOI:10.1016/j.lwt.2017.04.056.
[11]?ALOMSKIEN?J,ABRAITIEN?A,JONKUVIEN?D,et al.Selection of enhanced antimicrobial activity posing lactic acid bacteria characterised by(GTG)5-PCR fingerprinting[J].Journal of Food Science and Technology,2015,52(7):1-11.
[12]凌代文,东秀珠.乳酸细菌分类鉴定及实验方法[M].北京:中国轻工业出版社,1999.
[13]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001.
[14]BUDANAN R E,GIBBONR N E.伯杰细菌鉴定手册[M].8版.北京:科学出版社,1984:477-478.
[15]邢德明,李新华,袁慎亮,等.SOD乳酸菌高产菌株筛选鉴定及SOD分离纯化[J].食品与生物技术学报,2016,35(2):156-161.DOI:10.3969/j.issn.1673-1689.2016.02.007.
[16]MUHIALDIN B J,HASSAN Z,BAKAR F A,et al.Identification of antifungal peptides produced by Lactobacillus plantarum,IS10 grown in the MRS broth[J].Food Control,2016,59:27-30.DOI:10.1016/j.foodcont.2015.05.022.
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[18]TATIANA M,ALZBETA M,L’UBOMIR V.Production of antimicrobial substances by lactic acid bacteria[J].Chemicke Listy,2016,110:179-184.
[19]任士菊,王俊钢,周红,等.冷水鱼肠道抑菌活性乳酸菌的筛选及其生理特性分析[J].食品工业科技,2014,35(14):178-181.DOI:10.13386/j.issn1002-0306.2014.14.031.
[20]刘国荣,宋振芹,任丽,等.一株产广谱细菌素乳酸菌菌株的分离鉴定及其细菌素特性研究[J].食品工业科技,2015,36(4):180-184.DOI:10.13386/j.issn1002-0306.2015.04.031.
[21]葛菁萍,辛星,王,等.三步法分离纯化副干酪乳杆菌细菌素的初步研究[J].中国农学通报,2014(24):316-320.
[22]BORRERO J,KELLY E,O’CONNOR PM,et al.Plantaricyclin A,a novel circular bacteriocin produced by Lactobacillus plantarum NI326:purification,characterization,and heterologous production[J].Applied and Environmental Microbiology,2017,84(1):1-10.DOI:10.1128/AEM.01801-17.
[23]WANG Y,SHANG N,QIN Y,et al.The complete genome sequence of Lactobacillus plantarum,LPL-1,a novel antibacterial probiotic producing class IIa bacteriocin[J].Journal of Biotechnology,2018,266:84-88.DOI:10.1016/j.jbiotec.2017.12.006.
[24]AN Y,WANG Y,LIANG X,et al.Purification and partial characterization of M1-UVs300,a novel bacteriocin produced by Lactobacillus plantarum,isolated from fermented sausage[J].Food Control,2017,81:211-217.DOI:10.1016/j.foodcont.2017.05.030.
[25]TODOROV S D,STOJANOVSKI S,ILIEV I,et al.Technology and safety assessment for lactic acid bacteria isolated from traditional Bulgarian fermented meat product“lukanka”[J].Brazilian Journal of Microbiology,2017,48(3):576-586.DOI:10.1016/j.bjm.2017.02.005.
[26]胡盼.酸驼奶中产细菌素乳酸菌的分离及细菌素特性[D].杨凌:西北农林科技大学,2014.
[27]杨瑾.乳酸菌细菌素分离纯化及生物学特性研究[D].杭州:浙江工商大学,2007.
[28]FIELD D,ROSS R P,HILL C.Developing bacteriocins of lactic acid bacteria into next generation biopreservatives[J].Current Opinion in Food Science,2018,20:1-6.DOI:10.1016/j.cofs.2018.02.004.
[29]CASABURI A,MARTINO V D,FERRANTI P,et al.Technological properties and bacteriocins production by Lactobacillus curvatus 54M16and its use as starter culture for fermented sausage manufacture[J].Food Control,2016,59:31-45.DOI:10.1016/j.foodcont.2015.05.016.
[30]DU L,LIU F,ZHAO P,et al.Characterization of Enterococcus durans 152 bacteriocins and their in hibition of Lister ia monocytogenes in ham[J].Food Microbiology,2017,68:97-103.DOI:10.1016/j.fm.2017.07.002.