Preliminary screening, identification and biological characteristic analysis of Bacillus probiotics isolated from Cynoglossus semilaevis

Abstract

To screen local probiotic strains to promote antibiotic-free farming, two potential probiotic strains (S3, S5) were recognized among 89 cultivable bacterial strains isolated from the intestine of healthy Cynoglossus semilaevis. The two potential probiotic isolates were analyzed in terms of their morphology, physiology, biochemistry, the similarity of 16S rDNA sequences, growth characteristics, enzyme production capacity, bacterial antagonism, and safety in C. semilaevis. The results revealed that the bacterial morphology and physiological and biochemical characteristics of S3 and S5 were similar to those of Bacillus subtilis. The 16S rDNA sequences had 99.9 % similarity to that of Bacillus subtilis MH 145363.1. Therefore, S3 and S5 were identified as B. subtilis. In addition, we found that S3 and S5 had a strong ability to secrete amylase, protease, and lipase. During the safety tests of S3 and S5 in C. semilaevis with high concentrations, C. semilaevis in immersion, injection, and feeding groups remained in good condition without falling ill or dying. Moreover, we found that S3 and S5 exhibited superior growth at 25~50℃, salinities of 10 to 40, and pH values of 5 to 9. Furthermore, S3 and S5 had significant bacteriostatic activity against Vibrio anguillarum, Aeromonas salmonicida, and Shewanella algae, which are the main pathogenic bacteria of mariculture fish. In summary, S3 and S5 showed superb inhibition of the pathogenic bacteria of marine fish, rapid growth, eurythermal and euryhaline features, and suitability for the intestinal environment of C. semilaevis. Thus, strains S3 and S5 have excellent commercial development potential. These results provide a basis for ecological disease prevention strategies and are also valuable for developing and utilizing probiotics.