复合诱变选育新型高产细菌素植物乳杆菌
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摘要
目的从东北传统发酵肉制品中分离和再诱变选育广谱高产细菌素的植物乳酸菌,并对其进行鉴定。方法四轮复合诱变[微波诱变、亚硝基胍(nitroso-guanidin,NTG)诱变、常压室温等离子体(atmospheric room temperatureplasma,ARTP)诱变、紫外诱变突]具有降胆固醇能力的的植物乳杆菌M1株,获得还可产生广谱细菌素的植物乳杆菌。对其进行稳定性、微生物形态特征、生理生化特征及PCR鉴定。结果原始菌株经四轮复合诱变后获得1株高产细菌素菌株,该菌株兼具遗传稳定性、优良发酵特性和抑菌功效。鉴定该菌株为植物乳杆菌,命名为M1-UVs300,其分泌的细菌素命名为M1-UVs301;菌株保藏号为CGMCC No. 7972。结论通过四轮复合诱变获得的植物乳杆菌M1-UVs300抑菌谱较宽,且兼具降胆固醇和防腐的优良加工特性,工业化推广前景广泛。
Objective To isolate high bacteriocin-producing Lactobacillus platarum from traditional fermented meat products in Northeast China,breed by complex mutagenesis and identify the obtained strain. Methods L. plantarum M1 strain with ability of decreasing cholesterol was subjected to four rounds of complex mutagenesis,consisting of microwave mutagenesis,nitroso-guanidin(NTG) mutagenesis,atomospheric room temperature plasma(ARTP) mutagenesis and ultraviolet mutagenesis,to obtain the L. plantarum strain producing broad-spectrum bacteriocin. The obtained strain was analyzed for stability,microbial morphological characteristics as well as physiological and biochemical characteristics,and identified by PCR. Results A high bacteriocin-producing L. plantarum was obtained by four rounds of complex mutagenesis,which showed genetic stability,excellent fermentation characteristics and antibacterial effect,was identified as L. plantarum and named as M1-UVs300,with an accession number of CGMCC No.7972. The bacteriocin secreted by the strain was named as M1-UVs301. Conclusion The L. plantarum M1-UVs300 with broad bacteriostatic spectrum as well as cholesterol lowering and antiseptic effects were obtained by four rounds of complex mutagenesis,which was worthy of popularization in industrial production.
Objective To isolate high bacteriocin-producing Lactobacillus platarum from traditional fermented meat products in Northeast China,breed by complex mutagenesis and identify the obtained strain. Methods L. plantarum M1 strain with ability of decreasing cholesterol was subjected to four rounds of complex mutagenesis,consisting of microwave mutagenesis,nitroso-guanidin(NTG) mutagenesis,atomospheric room temperature plasma(ARTP) mutagenesis and ultraviolet mutagenesis,to obtain the L. plantarum strain producing broad-spectrum bacteriocin. The obtained strain was analyzed for stability,microbial morphological characteristics as well as physiological and biochemical characteristics,and identified by PCR. Results A high bacteriocin-producing L. plantarum was obtained by four rounds of complex mutagenesis,which showed genetic stability,excellent fermentation characteristics and antibacterial effect,was identified as L. plantarum and named as M1-UVs300,with an accession number of CGMCC No.7972. The bacteriocin secreted by the strain was named as M1-UVs301. Conclusion The L. plantarum M1-UVs300 with broad bacteriostatic spectrum as well as cholesterol lowering and antiseptic effects were obtained by four rounds of complex mutagenesis,which was worthy of popularization in industrial production.
引文
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[13]REN L,LIU G R,WANG C T,et al.Separation and identification of bacteriocin-producing Lactobacillus curvatus and characterization of its bacteriocin[J].Sci Technol of Food Indust,2013,34(6):218-223.(in Chinese)任丽,刘国荣,王成涛,等.产细菌素弯曲乳杆菌的分离鉴定及细菌素特性初步研究[J].食品工业科技,2013,34(6):218-223.
[14]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,1999:117-129.
[15]KUWAHARA T,NORIMATSU I,NAKAYAMA H,et al.Genetic varialion in 16S-23S rDNA infernal transcribed spacer regions and the possible use of this genentic variation for molecular diagnosis of Bateriodes species[J].Microbiol Munol,2001,45(3):191-199.
[16]MACWANA S J,MURIANA P M.A'bacteriocin PCR array'for identification of bacteriocin-related structural genes in lactic acid bacteria[J].J Microbiol Methods,2012,88(2):197-204.
[17]XIN L,YI L,DANG J,et al.Purification of novel bacteriocin produced by Lactobacillus coryniformis,MXJ 32 for inhibiting bacterial foodborne pathogens including antibiotic-resistant microorganisms[J].Food Control,2014,46(11):264-271.
[18]LI S C,ZHU C Y,WANG M.Screening of high-yeild L(+)-lactic acid strains by microwave irradiation mutagen[J].Acta Laser Biol Sin,2009,18(5):647-651.(in Chinese)李市场,朱朝阳,王敏.微波诱变L(+)-乳酸高产菌株的选育[J].激光生物学报,2009,18(5):647-651.
[19]ZHENG H Y,ZHAO W T,CHANG Y X,et al.Screening and identification and mutagenesis of high-yielding strains for producing GABA[J].Food Res Dev,2016,37(2):177-182.(in Chinese)郑鸿雁,赵炜彤,昌妍希,等.GABA高产菌株的筛选、鉴定及诱变选育[J].食品研究与开发,2016,37(2):177-182.
[20]WANG X Q,LOU X,ZHANG X X,et al.Screening of high daptomycin-producing strains by complex mutagenesis[J].Chem Bioengi,2015,32(8):60-62,66.(in Chinese)王秀琴,娄忻,张雪霞,等.复合诱变法筛选达托霉素高产菌株[J].化学与生物工程,2015,32(8):60-62,66.
[21]GE J P,SUN Y Y,LI X L,et al.Induced mutation breeding to improve Nisin production ability by Lactococcus lactis[J].Chin Agri Sci Bull,2015,31(24):173-178.(in Chinese)葛菁萍,孙艳阳,李兴霖,等.诱变育种提高乳酸乳球菌Lactococcus lactis产Nisin的能力[J].中国农学通报,2015,31(24):173-178.
[22]CHEN X L,SHAO Q B,WANG C,et al.Isolation and identification of Lactobacillus salivarius from chicken intestine and its biological characteristics[J].Food Sci,2016,37(13):157-161.(in Chinese)陈新亮,邵启兵,王超,等.唾液乳杆菌的分离鉴定及生物特性研究[J].食品科学,2016,37(13):157-161.
[23]TIAN X J,ZHANG L P.Screening of high-yield phenyllactic acid strains by NTG mutation[J].J Chin Inst of Food Sci Technol,2014,14(4):104-110.(in Chinese)田雪娇,张丽萍.亚硝基胍法诱变选育高产苯乳酸菌株[J].中国食品学报,2014,14(4):104-110.
[24]WANG F,LU W J,YANG J,et al.Study on screening of high-yield bacteriocin producing Lactobacillus plantarum strains induced by mutations[J].Sci Technol of Food Indust,2017,38(2):191-195.(in Chinese)王芳,陆文俊,杨静,等.高产细菌素植物乳杆菌的诱变选育研究[J].食品工业科技,2017,38(2):191-195.
[25]QIU W W,REN Y L,CHEN C S,et al.Research on mutation of high lactase activity Kluyveromyces lactis by ARTP[J].JChin Inst of Food Sci Technol,2014,14(2):132-137.(in Chinese)邱雯雯,任雅琳,陈存社,等.常压室温等离子体诱变筛选高乳糖酶活力酵母的研究[J].中国食品学报,2014,14(2):132-137.
[2]DEEGAN L H,COTTER P D,HILL C,et al.Bacteriocins:biological tools for bio-preservation and shelf-life extension[J].Int Dairy J,2006,16(9):1058-1071.
[3]李伟栋.产细菌素乳酸菌的筛选、培养条件优化及生物学特性的研究[D].黑龙江大庆:黑龙江八一农垦大学,2016:4.
[4]CINTAS L M,CASAUS M P,HERRANZ C,et al.Review:bacteriocins of lactic acid bacteria[J].Food Sci&Technol Int,2001,7(4):281-305.
[5]REA M C,ROSS R P,COTTER P D,et al.Classification of bacteriocins from Gram-positive bacteria//Prokaryotic antimicrobial peptides[M].Springer New York,2011:29-53.
[6]WANG Y W.Screening of bacteriocin-producing lactic acid bacteria and studies on its bacteriocin,leucocin B-K7[D].Wuxi:Jiangnan University,2016:2.(in Chinese)王彦伟.产细菌素乳酸菌的筛选及其所产细菌素leucocin B-K7的研究[D].江苏无锡:江南大学,2016:2.
[7]WANG T.Screening of enterocins and expression of its structure gene[D].Wuhan:Huazhong Agricutural University,2013:7.(in Chinese)王涛.肠球菌属细菌素的筛选及其结构基因的表达[D].湖北武汉:华中农业大学,2013:7.
[8]ZHANG X,ZHAO B,ZHANG X M,et al.Screening of bacteriocin producing lactic acid bacteria and the analysis of bacteriocin related genes[J].J China Agri Univ,2013,18(4):168-177.(in Chinese)张旭,赵斌,张香美,等.产细菌素乳酸菌的筛选及细菌素相关基因的分析[J].中国农业大学学报,2013,18(4):168-177.
[9]ZHANG J M,REN L Y,YI H X,et al.Strategies to increase bacteriocin production by lactic acid bacteria[J].China Brew,2014,33(7):29-33.(in Chinese)章检明,任璐雅,易华西,等.乳酸菌细菌素的高效表达方法研究[J].中国酿造,2014,33(7):29-33.
[10]GAO Y Q,DANG L J,BIE X M,et al.Different mutation methods of Lactobacillus acidophilus NX2-6 for improving antifungal activity[J].Sci Technol of Food Indust,2015,36(9):184-187.(in Chinese)高钰淇,党丽娟,别小妹,等.不同诱变方法选育嗜酸乳杆菌NX2-6类细菌素高产菌株[J].食品工业科技,2015,36(9):184-187.
[11]TAN C D,ZHU M J,DU S X,et al.Study on the inhibition zone method in antimicrobial test[J].Food Indust,2016,37(11):122-125.(in Chinese)谭才邓,朱美娟,杜淑霞,等.抑菌试验中抑菌圈法的比较研究[J].食品工业,2016,37(11):122-125.
[12]XUE G,CHEN L J,WU B,et al.Selection of high-yield thermostanle protease producing strain by ARTP and the study on its-enzymological properties[J].Sci Technol of Food Indust,2015,36(1):177-180.(in Chinese)薛刚,陈利娟,吴斌,等.ARTP诱变选育高温蛋白酶高产菌株及其酶学性质研究[J].食品工业科技,2015,36(1):177-180.
[13]REN L,LIU G R,WANG C T,et al.Separation and identification of bacteriocin-producing Lactobacillus curvatus and characterization of its bacteriocin[J].Sci Technol of Food Indust,2013,34(6):218-223.(in Chinese)任丽,刘国荣,王成涛,等.产细菌素弯曲乳杆菌的分离鉴定及细菌素特性初步研究[J].食品工业科技,2013,34(6):218-223.
[14]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,1999:117-129.
[15]KUWAHARA T,NORIMATSU I,NAKAYAMA H,et al.Genetic varialion in 16S-23S rDNA infernal transcribed spacer regions and the possible use of this genentic variation for molecular diagnosis of Bateriodes species[J].Microbiol Munol,2001,45(3):191-199.
[16]MACWANA S J,MURIANA P M.A'bacteriocin PCR array'for identification of bacteriocin-related structural genes in lactic acid bacteria[J].J Microbiol Methods,2012,88(2):197-204.
[17]XIN L,YI L,DANG J,et al.Purification of novel bacteriocin produced by Lactobacillus coryniformis,MXJ 32 for inhibiting bacterial foodborne pathogens including antibiotic-resistant microorganisms[J].Food Control,2014,46(11):264-271.
[18]LI S C,ZHU C Y,WANG M.Screening of high-yeild L(+)-lactic acid strains by microwave irradiation mutagen[J].Acta Laser Biol Sin,2009,18(5):647-651.(in Chinese)李市场,朱朝阳,王敏.微波诱变L(+)-乳酸高产菌株的选育[J].激光生物学报,2009,18(5):647-651.
[19]ZHENG H Y,ZHAO W T,CHANG Y X,et al.Screening and identification and mutagenesis of high-yielding strains for producing GABA[J].Food Res Dev,2016,37(2):177-182.(in Chinese)郑鸿雁,赵炜彤,昌妍希,等.GABA高产菌株的筛选、鉴定及诱变选育[J].食品研究与开发,2016,37(2):177-182.
[20]WANG X Q,LOU X,ZHANG X X,et al.Screening of high daptomycin-producing strains by complex mutagenesis[J].Chem Bioengi,2015,32(8):60-62,66.(in Chinese)王秀琴,娄忻,张雪霞,等.复合诱变法筛选达托霉素高产菌株[J].化学与生物工程,2015,32(8):60-62,66.
[21]GE J P,SUN Y Y,LI X L,et al.Induced mutation breeding to improve Nisin production ability by Lactococcus lactis[J].Chin Agri Sci Bull,2015,31(24):173-178.(in Chinese)葛菁萍,孙艳阳,李兴霖,等.诱变育种提高乳酸乳球菌Lactococcus lactis产Nisin的能力[J].中国农学通报,2015,31(24):173-178.
[22]CHEN X L,SHAO Q B,WANG C,et al.Isolation and identification of Lactobacillus salivarius from chicken intestine and its biological characteristics[J].Food Sci,2016,37(13):157-161.(in Chinese)陈新亮,邵启兵,王超,等.唾液乳杆菌的分离鉴定及生物特性研究[J].食品科学,2016,37(13):157-161.
[23]TIAN X J,ZHANG L P.Screening of high-yield phenyllactic acid strains by NTG mutation[J].J Chin Inst of Food Sci Technol,2014,14(4):104-110.(in Chinese)田雪娇,张丽萍.亚硝基胍法诱变选育高产苯乳酸菌株[J].中国食品学报,2014,14(4):104-110.
[24]WANG F,LU W J,YANG J,et al.Study on screening of high-yield bacteriocin producing Lactobacillus plantarum strains induced by mutations[J].Sci Technol of Food Indust,2017,38(2):191-195.(in Chinese)王芳,陆文俊,杨静,等.高产细菌素植物乳杆菌的诱变选育研究[J].食品工业科技,2017,38(2):191-195.
[25]QIU W W,REN Y L,CHEN C S,et al.Research on mutation of high lactase activity Kluyveromyces lactis by ARTP[J].JChin Inst of Food Sci Technol,2014,14(2):132-137.(in Chinese)邱雯雯,任雅琳,陈存社,等.常压室温等离子体诱变筛选高乳糖酶活力酵母的研究[J].中国食品学报,2014,14(2):132-137.