大黄鱼病原副溶血弧菌拮抗菌的筛选

Screening of antagonistic bacteria against pathogenic Vibrio parahaemolyticus of Pseudosciaena crocea

用点种法从大黄鱼肠道优势菌群中筛选出104株病原性副溶血弧菌的拮抗菌,进一步研究拮抗效果最好的11株拮抗菌的抗菌谱和生长曲线,其中NAC平板上筛选的4株拮抗菌的抗菌范围较广,锰营养琼脂培养基上筛选的3株拮抗菌具有生长优势。用BIOLOG板测定了抗菌谱较广、有一定生长优势3株拮抗菌(R22,Y58和J312)的碳源利用情况,结果表明,3株拮抗菌的碳源谱都比较广,都能利用糖类、有机酸和氨基酸等,而且与鱼类营养的竞争较小。16S rRNA基因同源性分析的结果显示与R22,Y58,J312同源性达99%以上的菌株分别来自弧菌属、芽孢杆菌属和假单胞菌属。进一步对R22,J312和Y58三株拮抗菌进行生化鉴定,结果显示R22为溶藻弧菌,J312为荧光假单胞菌,但Y58的种属未能得到确认。所得到的3株拮抗菌R22,Y58,J312在拮抗病原菌、抗菌谱、生长特性和营养利用等方面都初步满足益生菌的筛选条件,有待进一步检测其安全性和实用效果以便能够成为能实用的益生菌。

104 strains of antagonistic bacteria against pathogenic Vibrio parahaernolyticus were isolated from dominant flora of Pseudosciaena crocea intestine by dot-inoculating method. The results of antibacterial spectra and growth curves of eleven strains with better antagonistic effect showed that： 4 strains isolated from NAC plate had wider range of antibacterial spectra, 3 strains isolated from manganic nutrient agar plate had advantage on growth. The utilization of carbon source of three strains（R22,Y58 and J312） which had a wide antibacterial spectra and growth advantages were mensurated by BIOLOG. The results showed that： all of the 3 strains had wider carbon-source spectra; they could utilize saccharide, organic acid and amino acid; there were no severity competition on nutriment between antagonistic bacteria and marine fish. All results above indicate that these three strains of antagonistic bacteria accord with the properties of probiotics, which should be further studied. 16S rRNA gene homology of the three strains were analyzed in Genebank, and the results showed that strains had homology more than 99% with R22, Y58 and J312 were Vibrio, Bacillus and Pseudornonas respectively. Results of further biochemical identification showed that： R22 was V. alginolyticus, J312 was P. fluorescens, while Y58 could not be identified by bioMerieux. Although antagonistic bacteria isolated （strain R22, Y58, J312） measure up to probiotics for their antagonistic effect, fast growth and carbon source utlity, their security to fish and application effect should be confirmed before they can be use as probiotics.