甘蔗尾中乳酸菌的分离筛选和鉴定
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摘要
该研究利用传统培养法从0–90 d打包青贮发酵的甘蔗(Saccharum sinense)尾中分离得到20株乳酸菌。通过形态学观察、API 50 CH生化试剂盒鉴定及16S rDNA基因序列分析法对分离纯化的菌株进行鉴定,并测定其产酸能力。结果显示,菌株Y42是干酪乳杆菌(Lactobacillus casei),菌株Y32、Y59是副干酪乳杆菌(L. paracasei),其余的17株是植物乳酸杆菌(L. plantarum);分离的20株菌株在培养18 h后,菌株发酵液的pH可降低至3.70以下,Y67菌株的pH最低,为3.57。从甘蔗尾中分离的20株乳酸菌产酸能力均较强,具备良好的青贮潜力,可作为制备青贮饲料发酵剂的菌种。
Twenty strains of lactic acid bacteria were isolated from sugarcane sheaths packed in silage for 0 –90 d and fermented by traditional culture. The isolated and purified strains were identified by morphological observation, API 50 CH biochemical kit identification, and 16 S rDNA sequence analysis, and their acid production capacity was determined. The results showed that strain Y42 was Lactobacillus casei, strains Y32 and Y59 were L. paracasei, and all other strains were L.plantarum. After fermentation for 18 h, the strains' fermented liquid pH values can be reduced to less than 3.70. The pH of strain Y67 was the lowest at 3.57. The 20 strains of lactic acid bacteria isolated from sugarcane sheaths showed a strong capacity for producing acid and good silage potential. These strains could be used for preparing fermentation agents for silage.
Twenty strains of lactic acid bacteria were isolated from sugarcane sheaths packed in silage for 0 –90 d and fermented by traditional culture. The isolated and purified strains were identified by morphological observation, API 50 CH biochemical kit identification, and 16 S rDNA sequence analysis, and their acid production capacity was determined. The results showed that strain Y42 was Lactobacillus casei, strains Y32 and Y59 were L. paracasei, and all other strains were L.plantarum. After fermentation for 18 h, the strains' fermented liquid pH values can be reduced to less than 3.70. The pH of strain Y67 was the lowest at 3.57. The 20 strains of lactic acid bacteria isolated from sugarcane sheaths showed a strong capacity for producing acid and good silage potential. These strains could be used for preparing fermentation agents for silage.
引文
[1]中国报告网.(2017-05-12)[2018-02-22].http://market.Chinabaogao.Com/nonglinmuyu/05122RB32017.Html.Insight&Info Consulting Ltd.(2017-05-12)[2018-02-22].http://market.Chinabaogao.Com/nonglinmuyu/05122RB32017.Html.
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[8]MASANORI T,HISAMI K,MASARU N.Genotypic characterization of lactic acid bacteria isolated from Itatian ryegrass silage.Animal Science Journal,2012,83(2):110-112.
[9]朱军莉,赵进,朱雅霏.自然发酵蔬菜制品中降解亚硝酸盐乳酸菌的分离与鉴定.中国酿造,2015,34(6):39-42.ZHU J L,ZHAO J,ZHU Y F.Isolation and identification of nitrite-degrading lactic acid bacteria from natural fermented vegetable.China Brewing,2015,34(6):39-42.
[10]TAN Z F,PANG H L,Duan Y H,QIN G Y,CAI Y M.16S ribosomal DNA analysis and characterization of lactic acid bacteria associated with traditional Tibetan Qula cheese made form yak milk.Animal Science Journal,2010,81(6):706-713.
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[12]KOBAYASHI H,CAI Y,UEGAKI R.Microorganism composition,chemical composition and fermentation characteristics of the mixed sowing silage of timothy and orchard grass at different cutting number.Japanese Journal of Zoo Technical Science,2010,81(3):345-352.
[13]邵冰洁,陶永清,王素英,毕连克,朱晓昊,陈瑾.传统法糯米发酵炸糕中酵母菌的分离鉴定及优势菌株筛选.现代食品科技,2016,32(7):92-93.SHAO B J,TAO Y Q,WANG S Y,BI L K,ZHU X H,CHEN J.Isolation,identification,and screening of dominant yeast strains in fried cake made from natural fermented glutinous rice.Modern Food Science and Technology,2016,32(7):92-93.
[14]魏超,代晓航,郭灵安,刘炜,赵欢.MALDI-TOF MS与16S rDNA方法对肠杆菌科微生物鉴定比较分析.质谱学报,2018,39(2):209-215.WEI C,DAI X H,GUO L A,LIU W,ZHAO H.Analysis and comparison of MALDI-TOF MS and 16S rDNA for the identification of Enterobacteriaceae.Journal of Chinese Mass Spectrometry Society,2018,39(2):209-215.
[15]ELNAZ M,FAKHRI S,SEYED A M.16S ribosomal DNA analysis and characterization of lactic acid bacteria associated.Journal of Food Processing and Preservation,2017,41(4):1-8.
[16]SUNTHORNTHUMMAS S,DOII K,RANGSIRUJ A,SARAWANEEYARUK S,PRINGSULAKA O.Isolation and characterization of Lactobacillus paracasei LPC and phageΦT25 from fermented milk.Food Control,2017,73:1353-1361.
[17]杨承剑,谢芳,梁辛,梁贤威,李孟伟,李丽莉,彭开屏.木薯渣中乳酸菌的分离鉴定及其产酸能力分析.中国酿造,2017,36(2):89-92.YANG C J,XIE F,LIANG X,LIANG X W,LI M W,LI L L,PENG K P.Isolation,identification and acid-producing ability of lactic acid bacteria from cassava residues.China Brewing,2017,36(2):89-92.
[18]崔棹茗,郭刚,原现军,李君风,杨晓丹,丁良,余成群,邵涛.青稞秸秆青贮饲料中优良乳酸菌的筛选及鉴定.草地学报,2015,23(3):607-615.CUI Z M,GUO G,YUAN X J,LI J F,YANG X D,DING L,YU C Q,SHAO T.Characterization and identification of high quality lactic acid bacteria from hulless barley straw silage.Acta Agrestia Sinica,2015,23(3):607-615.
[19]杨子燕,赵世伟,马万平,赵旭燕,尹柯茹,陈敏,杨子彪.干酪乳杆菌的筛选及其在活性乳酸菌饮料中的应用.食品工业科技,2018,36(3):106-111.YANG Z Y,ZHAO S W,MA W P,ZHAO X Y,YIN K R,CHEN M,YANG Z B.Screening of Lactobacillus casei and application in active lactic bacteria beverage.Science and Technology of Food Industry,2018,36(3):106-111.
[20]MEDONALD P,HENDERSON A R,HERON S J E.The Biochemistry of Silage.Marlow,UK:Chalcombe Publications,1991:399.
[21]WOOLFORD M K.The detrimental effects of air on silage.Journal of Applied Bacteriology,1990,68:101-112.
[2]FAO.Food and Agriculture Organization of the United Nations.http://www.fao.org/statistics/databases/zh/,2016.
[3]郑勇,王金丽,李明,邓怡国,王刚,焦静.热带农业废弃物资源利用现状与分析:甘蔗废弃物综合利用.广东农业科学,2011(1):15-18.ZHENG Y,WANG J L,LI M,DENG Y G,WANG G,JIAO J.Status quo and analysis of utilization of agricultural waste resources in the tropics:Comprehensive utilization of sugarcane waste.Guangdong Agricultural Sciences,2011(1):15-18.
[4]梁碧军.甘蔗尾叶青贮技术及利用.广西畜牧兽医,2008,24(6):358-359.LIANG B J.Technology and utilization of sugarcane tail leaf silage.Guangxi Journal of Animal Husbandry&Veterinary Medicine,2008,24(6):358-359.
[5]刘建勇,余梅,王安奎,黄必志,李瑞生,缪世明,孙维庭.EM菌剂调制甘蔗梢及饲喂德宏水牛的效果.中国牛业科学,2010,36(6):40-43.LIU J Y,YU M,WANG A K,HUANG B Z,LI R S,MIU S M,SUN W T.Feeding effects of sugarcane tip silage treated by EMmicrobial agent on dehong buffalo.China Cattle Science,2010,36(6):40-43.
[6]穆胜龙,周波,杨冉冉,刘敏燕,靳艺,李媛媛,邹彩霞,何仁春.枯草芽孢杆菌和布氏乳杆菌对甘蔗尾青贮品质的影响.饲料研究,2017(15):24-30.MU S L,ZHOU B,YANG R R,LIU M Y,JIN Y,LI Y Y,ZOU C X,HE R C.Effects of Bacillus Subtilis and Lactobacillus Brucella on the quality of sugarcane tail silage.Feed Research,2017(15):24-30.
[7]DANIEL L P,CHECOLLI M,ZWIELEHNER J,JUNGES D,FERNANDES J,NUSSIO L G.The effects of Lactobacillus kefiri and L.brevis on the fermentation and aerobic stability of sugarcane silage.Animal Feed Science and Technology,2015,205(2015):69-74.
[8]MASANORI T,HISAMI K,MASARU N.Genotypic characterization of lactic acid bacteria isolated from Itatian ryegrass silage.Animal Science Journal,2012,83(2):110-112.
[9]朱军莉,赵进,朱雅霏.自然发酵蔬菜制品中降解亚硝酸盐乳酸菌的分离与鉴定.中国酿造,2015,34(6):39-42.ZHU J L,ZHAO J,ZHU Y F.Isolation and identification of nitrite-degrading lactic acid bacteria from natural fermented vegetable.China Brewing,2015,34(6):39-42.
[10]TAN Z F,PANG H L,Duan Y H,QIN G Y,CAI Y M.16S ribosomal DNA analysis and characterization of lactic acid bacteria associated with traditional Tibetan Qula cheese made form yak milk.Animal Science Journal,2010,81(6):706-713.
[11]PANG H L,TAN Z F,QIN G Y,WANG Y P,LI Z M,JIN Q S,CAI Y M.Phenotypic and phylogenetic analysis of lactic acid bacteria isolated from forage crops and grasses in the Tibetan plateau.The Journal of Microbiology,2012,50(1):63-71.
[12]KOBAYASHI H,CAI Y,UEGAKI R.Microorganism composition,chemical composition and fermentation characteristics of the mixed sowing silage of timothy and orchard grass at different cutting number.Japanese Journal of Zoo Technical Science,2010,81(3):345-352.
[13]邵冰洁,陶永清,王素英,毕连克,朱晓昊,陈瑾.传统法糯米发酵炸糕中酵母菌的分离鉴定及优势菌株筛选.现代食品科技,2016,32(7):92-93.SHAO B J,TAO Y Q,WANG S Y,BI L K,ZHU X H,CHEN J.Isolation,identification,and screening of dominant yeast strains in fried cake made from natural fermented glutinous rice.Modern Food Science and Technology,2016,32(7):92-93.
[14]魏超,代晓航,郭灵安,刘炜,赵欢.MALDI-TOF MS与16S rDNA方法对肠杆菌科微生物鉴定比较分析.质谱学报,2018,39(2):209-215.WEI C,DAI X H,GUO L A,LIU W,ZHAO H.Analysis and comparison of MALDI-TOF MS and 16S rDNA for the identification of Enterobacteriaceae.Journal of Chinese Mass Spectrometry Society,2018,39(2):209-215.
[15]ELNAZ M,FAKHRI S,SEYED A M.16S ribosomal DNA analysis and characterization of lactic acid bacteria associated.Journal of Food Processing and Preservation,2017,41(4):1-8.
[16]SUNTHORNTHUMMAS S,DOII K,RANGSIRUJ A,SARAWANEEYARUK S,PRINGSULAKA O.Isolation and characterization of Lactobacillus paracasei LPC and phageΦT25 from fermented milk.Food Control,2017,73:1353-1361.
[17]杨承剑,谢芳,梁辛,梁贤威,李孟伟,李丽莉,彭开屏.木薯渣中乳酸菌的分离鉴定及其产酸能力分析.中国酿造,2017,36(2):89-92.YANG C J,XIE F,LIANG X,LIANG X W,LI M W,LI L L,PENG K P.Isolation,identification and acid-producing ability of lactic acid bacteria from cassava residues.China Brewing,2017,36(2):89-92.
[18]崔棹茗,郭刚,原现军,李君风,杨晓丹,丁良,余成群,邵涛.青稞秸秆青贮饲料中优良乳酸菌的筛选及鉴定.草地学报,2015,23(3):607-615.CUI Z M,GUO G,YUAN X J,LI J F,YANG X D,DING L,YU C Q,SHAO T.Characterization and identification of high quality lactic acid bacteria from hulless barley straw silage.Acta Agrestia Sinica,2015,23(3):607-615.
[19]杨子燕,赵世伟,马万平,赵旭燕,尹柯茹,陈敏,杨子彪.干酪乳杆菌的筛选及其在活性乳酸菌饮料中的应用.食品工业科技,2018,36(3):106-111.YANG Z Y,ZHAO S W,MA W P,ZHAO X Y,YIN K R,CHEN M,YANG Z B.Screening of Lactobacillus casei and application in active lactic bacteria beverage.Science and Technology of Food Industry,2018,36(3):106-111.
[20]MEDONALD P,HENDERSON A R,HERON S J E.The Biochemistry of Silage.Marlow,UK:Chalcombe Publications,1991:399.
[21]WOOLFORD M K.The detrimental effects of air on silage.Journal of Applied Bacteriology,1990,68:101-112.