Identification and Antimicrobial Activity Detection of Lactic Acid Bacteria Isolated from Corn Stover Silage

  • Li, Dongxia (Henan Provincial Key Laboratory of Ion Beam Bio-engineering, Zhengzhou University) ;
  • Ni, Kuikui (Henan Provincial Key Laboratory of Ion Beam Bio-engineering, Zhengzhou University) ;
  • Pang, Huili (Henan Provincial Key Laboratory of Ion Beam Bio-engineering, Zhengzhou University) ;
  • Wang, Yanping (Henan Provincial Key Laboratory of Ion Beam Bio-engineering, Zhengzhou University) ;
  • Cai, Yimin (Animal Physiology and Nutrition Division, National Institute of Livestock and Grassland Science) ;
  • Jin, Qingsheng (Institute of Crops and Utilization of Nuclear Technology, Zhejiang Academy of Agricultural Sciences)
  • Received : 2014.06.12
  • Accepted : 2014.11.04
  • Published : 2015.05.01


A total of 59 lactic acid bacteria (LAB) strains were isolated from corn stover silage. According to phenotypic and chemotaxonomic characteristics, 16S ribosomal DNA (rDNA) sequences and recA gene polymerase chain reaction amplification, these LAB isolates were identified as five species: Lactobacillus (L.) plantarum subsp. plantarum, Pediococcus pentosaceus, Enterococcus mundtii, Weissella cibaria and Leuconostoc pseudomesenteroides, respectively. Those strains were also screened for antimicrobial activity using a dual-culture agar plate assay. Based on excluding the effects of organic acids and hydrogen peroxide, two L. plantarum subsp. plantarum strains ZZU 203 and 204, which strongly inhibited Salmonella enterica ATCC $43971^T$, Micrococcus luteus ATCC $4698^T$ and Escherichia coli ATCC $11775^T$ were selected for further research on sensitivity of the antimicrobial substance to heat, pH and protease. Cell-free culture supernatants of the two strains exhibited strong heat stability (60 min at $100^{\circ}C$), but the antimicrobial activity was eliminated after treatment at $121^{\circ}C$ for 15 min. The antimicrobial substance remained active under acidic condition (pH 2.0 to 6.0), but became inactive under neutral and alkaline condition (pH 7.0 to 9.0). In addition, the antimicrobial activities of these two strains decreased remarkably after digestion by protease K. These results preliminarily suggest that the desirable antimicrobial activity of strains ZZU 203 and 204 is the result of the production of a bacteriocin-like substance, and these two strains with antimicrobial activity could be used as silage additives to inhibit proliferation of unwanted microorganism during ensiling and preserve nutrients of silage. The nature of the antimicrobial substances is being investigated in our laboratory.


Antimicrobial Activity;Corn Stover Silage;Identification;Lactic Acid Bacteria


Supported by : Henan Science and Technology Committee


  1. AOAC. 1990. Official Methods of Analysis. 15th edn. Association of Official Analytical Chemists, Arlington, VA, USA.
  2. Benitez, L. B., K. Caumo, A. Brandelli, and M. B. Rott. 2011. Bacteriocin-like substance from Bacillus amyloliquefaciens shows remarkable inhibition of Acanthamoeba polyphaga. Parasitol. Res. 108:687-691.
  3. Cai, Y. and S. Kumai. 1994. The proportion of lactate isomers in farm silage and the influence of inoculation with lactic acid bacteria on the proportion of L-lactate in silage. Jpn. J. Zootech. Sci. 65:788-795.
  4. Cai, Y., S. Ohmomo, M. Ogawa, and S. Kumai. 1997. Effect of NaCl-tolerant lactic acid bacteria and NaCl on the fermentation characteristics and aerobic stability of silage. J. Appl. Microbiol. 83:307-313.
  5. Cai, Y., Y. Benno, M. Ogawa, S. Ohmomo, S. Kumai, and K. Nakase. 1998. Influence of Lactobacillus spp. from an inoculant and of Weissella and Leuconostoc spp. from forage crops on silage. Appl. Environ. Microbiol. 64:2982-2987.
  6. Cai, Y. 1999. Identification and characterization of Enterococcus species isolated from forage crops and their influence on silage fermentation. J. Dairy Sci. 82:2466-2471.
  7. Chaudhari, A. A. and S. Kariyawasam. 2014. An experimental infection model for Escherichia coli egg peritonitis in layer chickens. Avian Dis. 58:25-33.
  8. Cleveland, J., T. J. Montville, I. F. Nes, and M. L. Chikindas. 2001. Bacteriocins: Safe, natural antimicrobials for food preservation. Int. J. Food Microbiol. 71:1-20.
  9. Eitan, B. D., O. H. Shapiro, N. Siboni, and A. Kushmaro. 2006. Advantage of using inosine at the 3' Termini of 16S rRNA gene universal primers for the study of microbial diversity. Appl. Environ. Microbiol. 72:6902-6906.
  10. Ennahar, S., T. Sashihara, K. Sonomoto, and A. Ishizaki. 2000. Class IIa bacteriocins: Biosynthesis, structure and activity. FEMS Microbiol. Rev. 24:85-106.
  11. Ennahar, S., Y. Cai, and Y. Fujita. 2003. Phylogenetic diversity of Lactic acid bacteria associated with paddy rice silage as determined by 16S ribosomal DNA analysis. Appl. Environ. Microbiol. 69:444-451.
  12. Hata, T., R. Tanaka, and S. Ohmomo. 2010. Isolation and characterization of plantaricin ASM1: A new bacteriocin produced by Lactobacillus plantarum A-1. Int. J. Food Microbiol. 137:94-99.
  13. Hellal, A., L. Amrouche., Z. Ferhat, and F. Laraba. 2012. Characterization of bacteriocin from Lactococcus isolated from traditional Algerian dairy products. Ann. Microbiol. 62: 177-185.
  14. Karska-Wysocki, B., M. Bazo, and W. Smoragiewicz. 2010. Antibacterial activity of Lactobacillus acidophilus and Lactobacillus casei against methicillin-resistant Staphylococcus aureus (MRSA). Microbiol. Res. 165:674-686.
  15. Kozaki, M., T. Uchimura, and S. Okada. 1992. Experimental Manual for Lactic Acid Bacteria. Asakurasyoten, Tokyo, Japan.
  16. Kraus, E., H. H. Kiltz, and U. F. Femfert. 1976. The specificity of proteinase K against oxidized insulin B chain. Hoppe. Seylers. Z. Physiol. Chem. 357:233-237.
  17. Lin, C., K. K. Bolsen, B. E. Brent, and D. Y. C. Fung. 1992. Epiphytic lactic acid bacteria succession during the preensiling and ensiling periods of alfalfa and maize. J. Appl. Bacteriol. 73:375-387.
  18. Magnusson, J. and J. Schnurer. 2001. Lactobacillus coryniformis subsp. coryniformis strain Si3 produces a broad-spectrum proteinaceous antifungal compound. Appl. Environ. Microbiol. 67:1-5.
  19. Muck, R. E. 1989. Initial bacterial numbers on lucerne prior to ensiling. Grass Forage Sci. 44:19-25.
  20. Pang, H., M. Zhang, G. Qin, Z. Tan, Z. Li, Y. Wang, and Y. Cai. 2011. Identification of lactic acid bacteria isolated from corn stovers. Anim. Sci. J. 82:642-653.
  21. Pang, H., Z. Tan, G. Qin, Y. Wang, Z. Li, Q. Jin, and Y. Cai. 2012. Phenotypic and phylogenetic analysis of lactic acid bacteria isolated from forage crops and grasses in the Tibetan Plateau. J. Microbiol. 50:63-71.
  22. Rawlings, N. D. and A. J. Barrett. 1994. Families of serine peptidases. Method Enzymol. 244:19-61.
  23. Riley, M. A. 2009. Bacteriocins, biology, ecology, and evolution. Encyclopedia of Microbiology, 3rd Ed. (Ed S. Moselio). Academic Press, Massachusetts. USA. pp. 32-44.
  24. Saitou, H. and K. I. Miura. 1963. Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta. 72:619-629.
  25. Saitou, N. and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425.
  26. Sharma, P. and R. C. Sihag. 2013. Pathogenicity test of bacterial and fungal fish pathogens in Cirrihinus mrigala infected with EUS disease. Pak. J. Biol. Sci.16:1204-1207.
  27. Simova, E. D., D. M. Beshkova, M. P. Angelov, Zh. P. Dimitrov. 2008. Bacteriocin production by strain Lactobacillus delbrueckii ssp. bulgaricus BB18 during continuous prefermentation of yogurt starter culture and subsequent batch coagulation of milk. J. Ind. Microbiol. Biotechnol. 35:559-567.
  28. Smaoui, S., L. Elleuch, W. Bejar, I. Karray-Rebai, I. Ayadi, B. Jaouadi, F. Mathieu, H. Chouayekh, S. Bejar, and L. Mellouli. 2010. Inhibition of Fungi and Gram-Negative Bacteria by Bacteriocin BacTN635 Produced by Lactobacillus plantarum sp. TN635. Appl. Biochem. Biotechnol. 162:1132-1146.
  29. Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24:1596-1599.
  30. Tohno, M., H. Kobayashi, M. Nomura, M. Kitahara, M. Ohkuma, R. Uegaki, and Y. Cai. 2012. Genotypic and phenotypic characterization of lactic acid bacteria isolated from Italian ryegrass silage. Anim. Sci. J. 83:111-120.
  31. Todorov, S. D., H. Prevost, M. Lebois, X. Dousset, J. G. LeBlanc, and B. D. G. M. Franco. 2011. Bacteriocinogenic Lactobacillus plantarum ST16Pa isolated from papaya (Carica papaya) - From isolation to application: Characterization of a bacteriocin. Food Res. Int. 44:1351-1363.
  32. Torriani, S., G. E. Felis, and F. Dellaglio. 2001. Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers. Appl. Environ. Microbiol. 67:3450-3454.
  33. Weinberg, Z. G., R. E. Muck, P. J. Weimer, Y. Chen, and M. Gamburg. 2004. Lactic acid bacteria used in inoculants for silage as probiotics for ruminants. Appl. Biochem. Biotechnol. 118:1-9.
  34. Yang, E., L. Fan, Y. Jiang, C. Doucette, and S. Fillmore. 2012. Antimicrobial activity of bacteriocin-producing lactic acid bacteria isolated from cheeses and yogurts. AMB Express. 2:48.

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