A Bacillus spp. Strain against Penicillium expansum: Screening, Identification, Phylogenetic Analysis and Activity of Extracellular Antibacterial Substances from It

Abstract

Abstract: In order to obtain a Bacillus spp. that can protect post-harvest fruits from blue mould and patulin, plate confrontation test and double layer agar diffusion method were used for strain screening in this study. The Bacillus strain LPL-T12 isolated from oolong tea exhibited the best antibacterial effect, whose fermentation supernatant had an inhibitory zone diameter of (22.74 ± 0.44) mm against Penicillium expansum 3.3703. In addition, LPL-T12 was identified as Bacillus amyloliquefaciens through morphological observation, physiological and biochemical experiments, 16S rDNA sequence analysis and specific PCR amplification. After the fermentation supernatant of the strain was subjected to ammonium sulfate precipitation and dialysis against several different pore-size membranes, a thermosensitive protein with molecular weight of more than 8000 D was obtained based on antibacterial activity analysis, protease hydrolysis and heating treatment.

Key words: Penicillium expansum, Bacillus spp., screening, identification, antibacterial substance

CLC Number:

References

[1]Morales H, Marín S, Ramos A J. Influence of post-harvest technologies applied during cold storage of apples in Penicillium expansum growth and patulin accumulation: A review [J]. Food Control 2010, 21 (7):953-962.

[2]Berdy J. Recent development of antibiotic research and classification of antibiotics according to chemical structure [J]. Advances in applied microbiology, 1974, 18:309-406.

[3]Pusey P L, Wilson C L. Post-harvest biological control of stone fruit brown rot by Bacillus subtilis[J]. Plant Disease, 1984, 68:753-756.

[4]Leelasuphakul W, Hemmanee P, Chuenchitt S. Growth inhibitory properties of Bacillus subtilis strains and their metabolites against the green mold pathogen (Penicillium digitatum Sacc.) of citrus fruit[J]. Postharvest Biology and Technology, 2008, 48:113-121.

[5]Hao Weining, Li Hui, Hu Meiying, et al. Integrated control of citrus green and blue mold and sour rot by Bacillus amyloliquefaciens in combination with tea saponin [J]. Postharvest Biology and Technology, 2011, 59:316-323.

[6]黄继翔.抑扩展青霉芽孢杆菌的筛选鉴定及抑菌物质研究[D]. 北京, 中国农业大学,2006.

[7]东秀珠.常见细菌系统鉴定手册[M]. 北京:科学出版社, 2001, 62-64.

[8]别小妹, 陆兆新, 房耀维,等.利用16S r DNA序列分析鉴定一株产抗菌物质的微生物菌株[J]. 食品科学, 2006, 27 (11):466-470.

[9]Oleg N R, Christina D, Johan M, et a1.Taxononfic characterization and plant colonizing abilities of some bacteria related to Bacillus amyloliquefaciens and Bacillus subtilis[J]. FEMS Microbiology Ecology, 2004, 48:249-259.

[10]贾洁.枯草芽孢杆菌R21-4抗菌蛋白的分离提取与应用[D]. 北京, 中国农业大学, 2005.

[11]Zhou W W, Huang J X, Niu T G. Isolation of an antifungal Paenibacillus strain HT16 fromlocusts and purification of its medium-dependent antagonistic component[J]. Journal of Applied Microbiology, 2008, 105:912-919.

[12]车晓曦, 李校堃.解淀粉芽孢杆菌（Bacillus amyloliquefaciens）的研究进展[J]. 北京农业, 2010, 1:7-10.

[13]Wang San-liang, Shih Ing-lung, Liang Tze-wun W, et al. Purification and Characterization of Two Antifungal Chitinases Extracellularly Produced by Bacillus amyloliquefaciens V656 in a Shrimp and Crab Shell Powder Medium[J]. Journal of Agriculture and Food Chem., 2002, 50 (8):2241-2248.

[14]Jeong H Y, Lim J H, Kim B K, et al.Selection and mechanisms of indigenous antagonistic microorganisms against sheath rot and dry rot disease of garlic [J]. Korean Journal of Microbiology and Biotechnology, 2010, 38(3):295-301.

[15]Kim P I, Chung K C. Production of an antifungal protein for control of Colletotrichum lagenarium by Bacillus amyloliquefaciens MET0908 [J]. FEMS Microbiology Letters, 2004, 234:177-183.

[16]Wong J H, Hao J, Cao Z, et al. An antifungal protein from Bacillus amyloliquefaciens [J]. Journal of Applied Microbiology, 2008, 105:1888-1898.