乳酸菌噬菌体的分离及生物学特性鉴定

doi:10.16431/j.cnki.1671-7236.2021.06.027

Abstract

Abstract: In order to understand the biological characteristics of Lactobacillus phage,and provide experimental data and reference for the prevention and control of phage pollution during fermentation production,in this study,Lactobacillus phages were isolated from dairy products and kimchi samples by spot test and double-layer agar plate method,L.casei ATCC 393,L.pentosus KLDS 1.0413 and L.brevis ATCC 367 were used as indicator bacteria.The morphology of the phage was observed by transmission electron microscopy,and the packaging mechanism of the phage was analyzed by the restriction enzyme activity of the phage genome,the host range and one-step growth curve were measured,the structural proteins of the phage were analyzed by SDS-PAGE to analyse the biological characteristics of the obtained phage.The results showed that three virulent Lactobacillus phages were isolated and named as Lc,Lpen and Lbre,respectively.The results of electron microscopy showed that phage Lc and Lpen were consisted of polyhedral heads and non-contractive tails,while phage Lbre was consisted of polyhedral heads and contractile tails.According to morphology analysis,Lc and Lpen belonged to the Siphoviridae family,B1 category,and Lbre belonged to the Myoviridae family,A1 category.The genomes of phages were extracted and subjected to restriction endonuclease.The results of nucleic acid electrophoresis showed that the genomes of phage Lc and Lpen both had heterogeneous blunt ends,and the packaging mechanism was full-head packaging,namely pac-type,while Lbre contained stickiness end,the packaging mechanism belonged to cos-type.The phage Lc and Lbre were host-specific,and the host spectrum of phage Lpen was broader.The one-step growth curve showed that the latent period of Lc,Lpen and Lbre were 60,45 and 150 min respectively,the burst period were 45,90 and 105 min respectively,and the burst size were 47,24 and 30 PFU/cell respectively.SDS-PAGE testing indicated that phage Lc had 7 structural proteins,while phage Lpen,Lbre had 5 structural proteins,their protein molecular weight were about 50,55 and 50 ku,respectively.In conclusion,three virulent Lactobacillus phages were isolated and their biological characteristics were indentified,which provided important experimental data and theoretical basis for understanding the prevention and control of virulent Lactobacillus phages contamination.

Key words: Lactobacillus; phage; biological characteristics

CLC Number:

TS252

QIN Songyue, ZHAO Li, DI Yaxin, YU Xiaoli, WANG Li, TANG Lijie, XU Yigang, ZHOU Han, JIANG Yanping, CUI Wen, LI Yijing, QIAO Xinyuan. Isolation and Biological Characterization of Lactobacillus Phage[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(6): 2140-2149.

References

[1] 尹胜利,杜鉴,徐晨.乳酸菌的研究现状及其应用[J].食品科技,2012,37(9):25-29. YIN S L,DU J,XU C.Advances in the research and application of Lactobacillus[J].Food Science and Technology,2012,37(9):25-29.(in Chinese)
[2] 刘晶,杨桂连,王春凤,等.乳酸菌作为DNA疫苗载体的研究进展[J].中国兽医杂志,2016,52(7):67-69. LIU J,YANG G L,WANG C F,et al.Research progress of lactic acid bacteria as DNA vaccine carriers[J].Chinese Journal of Veterinary Medicine,2016,52(7):67-69.(in Chinese)
[3] CHATEL J M,POTHELUNE L,AH-LEUNG S,et al.In vivo transfer of plasmid from food-grade transiting lactococci to murine epithelial cells[J].Gene Therapy,2008,15:1184-1190.
[4] 刘乃芝,亓秀晔,郭杨丽,等.基因工程重组乳酸菌防控动物疫病的研究进展[J].中国畜牧兽医,2020,47(4):1199-1208. LIU N Z,QI X Y,GUO Y L,et al.Research progress of genetic engineering recombinant lactic acid bacteria on epidemic prevention and control of animal diseases[J].China Animal Husbandry & Veterinary Medicine,2020,47(4):1199-1208.(in Chinese)
[5] 王芳芳,刁华杰,夏九龙,等.乳酸菌及其发酵饲料在动物生产中的应用[J].饲料研究,2016,1:15-19. WANG F F,DIAO H J,XIA J L,et al.The application of lactic acid bacteria and its fermented feed in animal production[J].Feed Research,2016,1:15-19.(in Chingese)
[6] MARCÓ M B,MOINEAU S,QUIBERONI A.Bacteriophages and dairy fermentations[J].Bacterio-phage,2012,2(3):149-158.
[7] BRIGGILER M M,REINHEIMER J,QUIBERONI A.Phage adsorption and lytic propagation in Lactobacillus plantarum:Could host cell starvation affect them?[J].BMC Microbiology,2015,15:273.
[8] CHEN X,GUO J,LIU Y,et al.Characterization and adsorption of a Lactobacillus plantarum virulent phage[J].Journal of Dairy Science,2019,102:3879-3886.
[9] WHITEHEAD H R,HUNTER G J E.Observations on the activity of bacteriophage in the group of lactic streptococci[J].Journal of Pathology & Bacteriology,2005,44(2):337-347.
[10] HERRERO M,DE L,CSOA J L,et al.Characterization of φ393-A2,a bacteriophage that infects Lactobacillus casei[J].Microbiology,1994,140(10):2585-2590.
[11] ZAGO M,DE L A,CARMINATI D,et al.Detection and identification of Lactobacillus delbrueckii subsp.lactis bacteriophages by PCR[J].Journal of Dairy Research,2006,73(2):146-153.
[12] ZHANG X,KONG J,QU Y.Isolation and characterization of a Lactobacillus fermentum temperate bacteriophage from Chinese yogurt[J].Journal of Applied Microbiology,2006,101(4):857-863.
[13] PEI Z M,SADIQ F A,HAN X,et al.Identification,characterization,and phylogenetic analysis of eight new inducible prophages in Lactobacillus[J].Virus Research,2020,286:198003.
[14] 于美玲.两株乳杆菌噬菌体的分离鉴定及抗噬菌体菌株的筛选[D].哈尔滨:东北农业大学,2015. YU M L.Isolation and identification of two Lactobacillus phages and screening of phage-resisted strains[D].Harbin:Northeast Agricultural University,2015.(in Chinese)
[15] EBRECHT A C,GUGLIELMOTTI D M,TREMMEL G,et al.Temperate and virulent Lactobacillus delbrueckii bacteriophages:Comparison of their thermal and chemical resistance[J].Food Microbiology,2010,27(4):515-520.
[16] WANG S H,JIAN K,CHEN G,et al.Isolation and characterization of a novel virulent phage (phiLdb) of Lactobacillus delbrueckii[J].International Journal of Food Microbiology,2010,137(1):22-27.
[17] 赵影,刘秀侠,李金敏,等.一株鼠伤寒沙门氏菌噬菌体的分离鉴定及对小鼠的防制效果研究[J].中国畜牧兽医,2018,45(1):234-241. ZHAO Y,LIU X X,LI J M,et al.Isolation and identification of a phage against Salmonella Typhimurium and its control effect on mice[J].China Animal Husbandry & Veterinary Medicine,2018,45(1):234-241.(in Chinese)
[18] 汪祥燕,辛国芹,董佩佩,等.1株宽噬菌谱鸡白痢沙门氏菌噬菌体的分离及生物学特性分析[J].中国畜牧兽医,2018,45(9):2575-2581. WANG X Y,XIN G Q,DONG P P,et al.Isolation and biological properties analysis of a Salmonella Pullorum lytic phage with wide host range[J].China Animal Husbandry & Veterinary Medicine,2018,45(9):2575-2581.(in Chinese)
[19] QUIBERONI A,GUGLIELMOTTI D,BINETTI A,et al.Characterization of three Lactobacillus delbrueckii subsp.bulgaricus phages and the physicochemical analysis of phage adsorption[J].Journal of Applied Microbiology,2004,96(2):340-351.
[20] 张丽娜,于长青.乳酸菌的降胆固醇作用及其在发酵制品中的应用[J].食品工业科技,2007,7:228-231. ZHANG L N,YU C Q.The cholesterol-lowering effect of lactic acid bacteria and its application in fermented products[J].Food Industry Science and Technology,2007,7:228-231.(in Chinese)
[21] MATTHEWS R.Classification and nomenclature of viruses.Fourth report of the international committee on taxonomy of viruses[J].Intervirology,1982,12(3-5):129.
[22] MULLERMERBACH M,RAUSCHER T,HINRICHS J.Inactivation of bacteriophages by thermal and high-pressure treatment[J].International Dairy Journal,2005,15(6-9):777-784.
[23] SOZZI T,POULIN J M,MARET R.Effect of incubation temperature on the development of lactic acid bacteria and their phages[J].Journal of Dairy Research,1978,45(2):259-265.
[24] DEASY T,MAHONY J,NEVE H,et al.Isolation of a virulent Lactobacillus brevis phage and its application in the control of beer spoilage[J].Journal of Food Protection,2011,74:2157-2161.
[25] WATANABE K,TAKESUE S,ISHIBASHI K,et al.Simulation of the process of pl-1 phage adsorption to its host bacterium,ATCC 27092[J].Agricultural & Biological Chemistry,1980,44(4):869-875.
[26] SHIMIZU-KADOTA M,SAKURAI T,TSUCHIDA N.Prophage origin of a virulent phage appearing on fermentations of Lactobacillus casei S-1[J].Applied & Environmental Microbiology,1983,45(2):669-674.
[27] CHIBANI-CHENNOUFI S,DILLMANN M L,MARVIN-GUY L,et al.Lactobacillus plantarum bacteriophage LP65:A new member of the spo1-like genus of the family Myoviridae[J].Journal of Bacteriology,2004,186(21):7069-7083.
[28] WATANABE K,TAKESUE S,JIN-NAI K,et al.Bacteriophage active against the lactic acid beverage-producing bacterium Lactobacillus casei[J].Applied Microbiology,1970,20(3):409-415.
[29] CAPRA M L,PATRIGNANI F,QUIBERONI A D L,et al.Effect of high pressure homogenization on lactic acid bacteria phages and probiotic bacteria phage[J].International Dairy Journal,2009,19(5):336-341.

Isolation and Biological Characterization of Lactobacillus Phage

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	WANG Yangyang, QIN Shizhen, TANG Defu, SHAO Yuxin, SHI Zhaoguo, WANG Zheng, LI Xing, ZHAO Dongdong. Effects of Zinc, Lactobacillus on Immune and Antioxidant Function of Squabs Infected with Salmonella [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2276-2285.
[2]	WU Liting, LU Rui, LIU Banhong, BAO Hongduo, WANG Yongjuan, ZHOU Yan, WANG Ran, ZHANG Hui. Combination of Clostridium perfringens Phage and Lactobacillus Improves Growth Performance and Regulates Intestinal Flora Function of Broilers [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2321-2332.
[3]	LI Xiaoyu, LI Ruoyu, WANG Xiao, WANG Lili, LI Jibin, LI Shuying, XU Yongping. Research Progress on the Influence of Filamentous Phages on Host Bacterial Adaptability [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2496-2506.
[4]	CHEN Jianshi, ZHANG Ning, PU Fangxiong, MIAO Jianjia, ZENG Nan, LI Bingxue. Prediction of Bacillus anthracis Carrying Prophages and Analysis of Drug Resistance and Virulence [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2573-2584.
[5]	ZHANG Jingyi, CHEN Li, HUANG Siyang, CHEN Xiang, LIU Zongping, TONG Xishuai. Research Progress on Pathogenic Characteristics and Pathogenicity of Ehrlichia canis [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2023-2031.
[6]	MA Jifu, Adil·Ketan, XIE Jingang, Bakhet·Bodai, LI Zhenwei, Oralhazi·Hazikhan. Screening of Nanobody Specific to Inhibin α Subunit Based on Phage Display Technology [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2032-2043.
[7]	WANG Leping, PAN Yan, TAN Yizhou, LI Lei, HAN Kaiou, CAO Yajie, LI Jun, ZHOU Yuqing, LIAO Yuying, MA Dongxin, GONG Yu, PENG Hao, WANG Xiaoye. Isolation,Identification and Genome Analysis of Escherichia coli Phage vB_EcoP_GN07 [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1522-1531.
[8]	TIAN Changqing, ZHANG Kunzhong, QI Yumei, SHI Wenjing, DONG Zhijie, ZHANG Haohao, HE Zengwen, ZHI Ji, ZHAO Xuehui, CHONG Qian, XUE Huiwen, GOU Huitian. Isolation,Identification and Biological Characteristics Analysis of a Strain of Listeria monocytogenes from Environment [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1543-1555.
[9]	GUO Bingyu, LI Li, LI Chen, KONG Xianghui. Role and Molecular Mechanism of Mpeg1 in Pathogen Clearance [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1584-1592.
[10]	HUANG Qiaochen, LIAO Binru, YU Chunyan, MO Yupeng, HUANG Weiqian, LI Lei, HAN Kaiou, WANG Leping, LI Xun, WANG Xiaoye. Isolation,Identification and Biological Characteristics Analysis of a Bacteriophage EP_H11 from Escherichia coli [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1684-1694.
[11]	CHENG Longfei, LIAO Weilian, CHEN Hongmei, JIANG Nansong, FU Qiuling, WAN Chunhe, LIU Rongchang, SHI Shaohua, HUANG Yu, FU Guanghua. Screening and Biological Characteristics of Broad-spectrum Riemerella anatipestifer Phage [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1169-1176.
[12]	GAN Lu, ZHENG Huizhen, NUO Mingdalai, LIU Yan, JIN Min, HE Wenwen, WEN Licui, LI Yongchang, GAILIKE·Bayinchahan. Propagation of Trypanosoma evansi Yili Strain and Cloning, Expression and Bioinformatics Analysis of Its PFR Gene [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 469-478.
[13]	LIU Bin, LI Meng, XI Qianyun, SUN Jiajie, LUO Junyi, ZHANG Yongliang, CHEN Ting. Research Progress on Biological Characteristics of Hermetia illucens L. and Its Application in Poultry Production [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 586-597.
[14]	YIN Yangyan, PAN Yan, LI Xiaoning, LI Jun, LIAO Yuying, LI Changting, WEI Tianchao, PENG Hao. Study on Biological Characteristics and Antibacterial Activity in vitro of a Lactobacillus plantarum Strain [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 666-673.
[15]	GAO Yefan, SONG Hanan, WANG Yunan, WU Yue, NIU Ruili, ZONG Xianchun, GUAN Weijun. Study on the Isolation, Culture and Differentiation Potential of Limbal Stem Cells from Beijing You Chickens [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3372-3381.