该研究通过牛津杯双层平板法,从分离自四川泡菜的114株植物乳杆菌中筛选对大肠杆菌O157:H7具有较强拮抗活性的菌株,并对其所产抑菌物质的特性进行了初步的研究。结果表明:114株植物乳杆菌的上清液均可抑制大肠杆菌,其中16株菌的上清液经排酸后仍有抑菌作用,并且抑菌作用不受过氧化氢酶影响,经蛋白酶处理后大幅降低甚至消失;在进一步筛选中发现植物乳杆菌IMAU80122所产抑菌物质具有耐酸(pH 3)、耐弱碱(pH 8)的特性,在80 ℃具有一定的稳定性,对不同的表面活性剂、有机溶剂、金属离子、盐浓度、紫外照射时间都具有良好的耐受性;对G-菌株的抑菌活性强于G+菌株,但对供试的乳酸菌没有作用。综上所述,植物乳杆菌IMAU80122所产抑菌物质具有蛋白性质,不仅抑菌活性强,而且具有良好的稳定性和广谱抑菌效果,在食品加工和医药行业中具有一定的开发应用潜能。
In this study, 114 strains of Lactiplantibacillus plantarum isolated from Sichuan pickles were screened by Oxford cup double-layer plate method, which had strong antagonistic activity against Escherichia coli O157∶H7, and the characteristics of protein antibacterial substances produced by L. plantarum were studied. The results showed that the supernatant of 114 strains of L. plantarum could inhibit E. coli, and 16 strains of L. plantarum could still inhibit E. coli after acid drainage, and the inhibitory effect was not affected by catalase, but was significantly reduced or even disappeared after protease treatment. Further screening showed that the antibacterial substances produced by L. plantarum IMAU80122 had the characteristics of acid resistance (pH 3) and weak alkali resistance (pH 8), had certain stability at 80 ℃, and had good resistance to different surfactants, organic solvents, metal ions, salt concentration and UV irradiation time. The antibacterial activity against G- strain was stronger than G+ strain, but it had no antibacterial activity against tested lactic acid bacteria. In conclusion, the bacteriostatic substances produced by L. plantarum IMAU80122 have protein properties, which not only have strong bacteriostatic activity, but also have good stability and broad-spectrum bacteriostatic effect, and have certain development and application potential in food processing and pharmaceutical industry.
[1] LIM J Y, YOON J, HOVDE C J.A brief overview of Escherichia coli O157:H7 and its plasmid O157[J].Journal of Microbiology and Biotechnology, 2010, 20(1):5-14.
[2] WANG R F, FANG M M, HU X Y, et al.Kojic acid and tea polyphenols inactivate Escherichia coli O157:H7 in vitro and on salmon fillets by inflicting damage on cell membrane and binding to genomic DNA[J].International Journal of Food Science & Technology, 2021, 56(11):6 039-6 051.
[3] MURDOCK C A, CLEVELAND J, MATTHEWS K R, et al.The synergistic effect of nisin and lactoferrin on the inhibition of Listeria monocytogenes and Escherichia coli O157:H7[J].Letters in Applied Microbiology, 2007, 44(3):255-261.
[4] PREVENTION E, CONTROL (ECDC).Antimicrobial resistance surveillance in Europe 2016.Annual report of the European Antimicrobial Resistance Surveillance Network (EARS-Net)[J].Stockholm:ECDC, 2017:1-86.
[5] YI L H, LUO L L, LYU X.Efficient exploitation of multiple novel bacteriocins by combination of complete genome and peptidome[J].Frontiers in Microbiology, 2018, 9:1 567.
[6] DENKOVA R, YANAKIEVA V, DENKOVA Z, et al.In vitro inhibitory activity of Bifidobacterium and Lactobacillus strains against Candida albicans[J].Bulgarian Journal of Veterinary Medicine, 2013, 16(3):186-197.
[7] TENEVA D, DENKOVA R, GORANOV B, et al.Antimicrobial activity of Lactobacillus plantarum strains against Salmonella pathogens[J].Ukrainian Food Journal, 2017, 6(1):125-133.
[8] KAILASAPATHY K, CHIN J.Survival and therapeutic potential of probiotic organisms with reference to Lactobacillus acidophilus and Bifidobacterium spp.[J].Immunology and Cell Biology, 2000,78(1):80-88.
[9] DA SILVA SABO S, VITOLO M, GONZÁLEZ J M D, et al.Overview of Lactobacillus plantarum as a promising bacteriocin producer among lactic acid bacteria[J].Food Research International, 2014, 64:527-536.
[10] SOBRINO-LÓPEZ A, MARTÍN-BELLOSO O.Use of nisin and other bacteriocins for preservation of dairy products[J].International Dairy Journal, 2008, 18(4):329-343.
[11] DEY B C, RAI N, DAS S, et al.Partial purification, characterization and mode of action of bacteriocins produced by three strains of Pediococcus sp.[J].Journal of Food Science and Technology, 2019, 56(5):2 594-2 604.
[12] WIEDEMANN I, BÖTTIGER T, BONELLI R R, et al.Lipid II-based antimicrobial activity of the lantibiotic plantaricin C[J].Applied and Environmental Microbiology, 2006, 72(4):2 809-2 814.
[13] LIU Y X, LI Z F, LV Y J, et al.Chlamydomonas reinhardtii-expressed multimer of Bacteriocin LS2 potently inhibits the growth of bacteria[J].Process Biochemistry, 2020, 95:139-147.
[14] ZHANG L, PORNPATTANANANGKU D, HU C M J, et al.Development of nanoparticles for antimicrobial drug delivery[J].Current Medicinal Chemistry, 2010, 17(6):585-594.
[15] ANSARI A, ZOHRA R R, TARAR O M, et al.Screening, purification and characterization of thermostable, protease resistant Bacteriocin active against methicillin resistant Staphylococcus aureus (MRSA)[J].BMC Microbiology, 2018, 18(1):192.
[16] YAO Z, KIM J A, KIM J H.Characterization of a fibrinolytic enzyme secreted by Bacillus velezensis BS2 isolated from sea squirt jeotgal[J].Journal of Microbiology and Biotechnology, 2019, 29(3):347-356.
[17] 郭行. 鼠李糖乳杆菌LS-8中新型细菌素的挖掘及抑菌机制的研究[D].杨凌:西北农林科技大学, 2020.
GUO X.Mining novel bacteriocins from Lactobacillus rhamnosus LS-8 and study of their antibacterial mechanism[D].Yangling:Northwest A & F University, 2020.
[18] BARMAN S, GHOSH R, MANDAL N C.Production optimization of broad spectrum bacteriocin of three strains of Lactococcus lactis isolated from homemade buttermilk[J].Annals of Agrarian Science, 2018, 16(3):286-296.
[19] BAGDE P, VIGNESHWARAN N.Improving the stability of bacteriocin extracted from Enterococcus faecium by immobilization onto cellulose nanocrystals[J].Carbohydrate Polymers, 2019, 209:172-180.
[20] ZHANG J M, YANG Y Y, YANG H, et al.Purification and partial characterization of bacteriocin lac-B23, a novel bacteriocin production by Lactobacillus plantarum J23, isolated from Chinese traditional fermented milk[J].Frontiers in Microbiology, 2018, 9:2165.
[21] 冉军舰, 焦凌霞, 梁新红, 等.嗜酸乳杆菌产细菌素的提取及其生物特性研究[J].食品工业科技, 2017, 38(13):112-118.
RAN J J, JIAO L X, LIANG X H, et al.Extraction of bacteriocin produced by Lactobacillus acidophilus and biology characteristic analysis[J].Science and Technology of Food Industry, 2017, 38(13):112-118.
[22] 舒梨, 何义国, 赵兴秀, 等.浓香型窖泥中高产细菌素乳酸菌的鉴定及特性[J].食品工业科技, 2019, 40(4):119-124.
SHU L, HE Y G, ZHAO X X, et al.Identification and characteristic of lactic acid bacteria with high production of bacteriocin from pit mud of strong-flavor[J].Science and Technology of Food Industry, 2019, 40(4):119-124.
[23] CHEN L, GU Q, LI P, et al.Purification and characterization of plantaricin ZJ316, a novel bacteriocin against Listeria monocytogenes from Lactobacillus plantarum ZJ316[J].Journal of Food Protection, 2018, 81(12):1 929-1 935.
