Screening of Probiotics for Inhibiting Pathogens and Preliminary Determination of Antimicrobial Substances

doi:10.13560/j.cnki.biotech.bull.1985.2017-0411

Abstract

Abstract: In order to obtain probiotics that inhibit pathogens and to determine their antimicrobial substances,strains that can produce inhibitory zones were isolated from the soil,feed additives and fertilizer additives. Strains isolated and preserved in the laboratory were re-screened with the Oxford Cup method. Then the unknown strains were identified with morphological observation,16S rRNA -based phylogeny and physiological and biochemical reaction. The antimicrobial activity of fermentation supernatant was determined after treated by acid-base reaction,high temperature,protease decomposition,salting-out and dialysis. Results showed that 8 strains presented the strong inhibitory effect on Escherichia coli,Staphylococcus aureus and Micrococcus luteus,and 4 strains were known as Lactobacillus bulgaricus,Lactobacillus casei,Lactobacillus rhamnosus and Lactobacillus plantarum. Three of the 4 unknown strains were identified as Bacillus licheniformis and one was identified as Pediococcus lactis. Bacillus licheniformis can produce a variety of antimicrobial substances with different molecular weights and high temperature resistance,which can or cannot pass through 8-14 kD dialysis bags,and the antimicrobial substances produced by Bacillus licheniformis are preliminarily determined as polypeptide,and antimicrobial substances from 5 acid-producing strains are mainly acidic.

Key words: probiotics, Oxford Cup method, 16S rRNA, antimicrobial substances

WANG Li-jie, YU Xiao-bin, FANG Yin-bing, GU Qiu-ya. Screening of Probiotics for Inhibiting Pathogens and Preliminary Determination of Antimicrobial Substances[J]. Biotechnology Bulletin, 2017, 33(11): 123-129.

References

[1] 张群. 饲用微生态制剂的开发与应用[J]. 食品与生物技术学报, 2016, 35（8）：896.
[2] Rasić JL, Kurmann JA. Bifidobacteria and their role. Microbiological, nutritional-physiological, medical and technological aspects and bibliography[J]. Experientia Suppl 1983, 39：1-295.
[3] Alexander Y, Le BG, Boisrame-Gastrin S, et al. Probiotics：a new way to fight bacterial pulmonary infections?[J]. Med Mal Infect, 2014, 44（1）：9-17.
[4] Simova ED, Beshkova DM, Angelov MP, et al. Bacteriocin production by strain Lactobacillus delbrueckii ssp. bulgaricus BB18 during continuous prefermentation of yogurt starter culture and subsequent batch coagulation of milk[J]. Journal of industrial microbiology & biotechnology, 2008, 35（6）：559-567.
[5] Milioni C, Martnez B, Degl’Innocenti S, et al. A novel bacteriocin produced by Lactobacillus plantarum LpU4 as a valuable candidate for biopreservation in artisanal raw milk cheese[J]. Dairy Science & Technology, 2015, 95（4）：479-494.
[6] Lv X, Hu P, Dang Y, et al. Purification and partial characterization of a novel bacteriocin produced by Lactobacillus casei TN-2 isolated from fermented camel milk（Shubat）of Xinjiang Uygur Autonomous region, China[J]. Food Control, 2014, 43：276-283.
[7] Srinivasan R, Kumawat DK, Kumar S, et al. Purification and characterization of a bacteriocin from Lactobacillus rhamnosus L34[J]. Annals of Microbiology, 2012, 63（1）：387-392.
[8] Lv HX, Wang W, Tan Z, et al. Isolation and Preliminary Identifica-tion of Broad-spectrum Bacteriocin-producing Lactobacillus Strain CW3[J]. Agricultural Biotechnology, 2014, 3（1）：38-39.
[9] Milner JL, Raffel SJ, Lethbridge BJ, et al. Culture conditions that influence accumulation of zwittermicin A by Bacillus cereus UW85[J]. Appl Microbiol Biotechnol, 1995, 43（4）：685-691.
[10] 马俊美, 宁亚维, 王志新, 等. 侧孢短芽孢杆菌抗菌肽的结构与性质[J]. 食品与生物技术学报, 2016, 06）：629-634.
[11] 石举然, 别小妹, 吕凤霞, 等. 广谱抗菌芽孢杆菌的筛选及其抗酵母物质的纯化和鉴定[J]. 食品科学, 2017, 38（6）：6-12.
[12] 剧建格, 于宏伟, 韩军, 等. 广谱高效抑菌物质产生菌的筛选及鉴定[J]. 微生物学通报, 2009, 36（5）：689-693.
[13] Manhar AK, Bashir Y, et al. Cellulolytic potential of probiotic Bacillus Subtilis AMS6 isolated from traditional fermented soybean（Churpi）：An in-vitro study with regards to application as an animal feed additive[J]. Microbiol Res, 2016, 186-187：62-70.
[14] Larsen N, Thorsen L, Kpikpi EN, et al. Characterization of Bacillus spp. strains for use as probiotic additives in pig feed[J]. Appl Microbiol Biotechnol, 2014, 98（3）：1105-1118.
[15] 王际辉, 刘诗文, 肖珊, 等. 现代生物技术与饲用微生态制剂[J]. 微生物学杂志, 2015, 35（2）：1-8.
[16] 高林, 白子金, 冯波, 等. 微生物饲料添加剂研究与应用进展[J]. 微生物学杂志, 2014, 34（2）：1-6.
[17] 诸葛健, 李华钟. 微生物学[M]. 第2版. 北京：科学出版社, 2009.
[18] 李清, 王英, 刘小莉, 等. 一株广谱抑菌活性乳酸菌的筛选及特性研究[J]. 微生物学通报, 2015, （2）：332-339.
[19] 刘冬梅, 李理, 杨晓泉, 等. 用牛津杯法测定益生菌的抑菌活力[J]. 食品研究与开发, 2006, 27（3）：110-111.
[20] 蔡妙英, 东秀珠. 常见细菌系统鉴定手册[M]. 北京：科学出版社, 2001：52-66.
[21] R. E. 布坎南, N. E. 吉本斯, 等. 伯杰细菌鉴定手册[M]. 第8版. 北京：科学出版社, 1984.
[22] 王春晓, 胡永红, 杨文革, 等. 地衣芽孢杆菌NJWGYH 833051的抑菌作用[J]. 湖北农业科学, 2016, 55（4）：904-907.
[23] Wang Z, He Z, et al. Comparison of two live Bacillus species as feed additives for improving in vitro fermentation of cereal straws[J]. Anim Sci J, 2016, 87（1）：27-36.
[24] Wang G, Ning J, Zhao J, et al. Partial characterisation of an anti-listeria substance produced by Pediococcus acidilactici P9[J]. International Dairy Journal, 2014, 34（2）：275-279.
[25] 饲料添加剂品种目录（2013）[J]. 中国饲料, 2014, 44（1）：66-67.
[26] Kim Y, Cho JY, Kuk JH, et al. Identification and Antimicrobial Activity of Phenylacetic Acid Produced by Bacillus licheniformis Isolated from Fermented Soybean, Chungkook-Jang[J]. Current Microbiology, 2004, 48（4）：312-317.
[27] 黄浦, 肖瑜, 覃妍, 等. 地衣芽孢杆菌降解茶皂素的研究[J]. 安徽大学学报自科版, 2016, 40（1）：91-97.
[28] Wang J, Li L, Zhao X, et al. Partial characteristics and antimicrobial mode of pediocin produced by Pediococcus acidilactici PA003[J]. Annals of Microbiology, 2015, 65（3）：1753-1762.