Screening Of Lactic Acid Bacteria In Hainan And Its Effect On Litopenaeus Vannamei Growth

Lactic acid bacteria (LAB) is a group of gram-positive bacteria which has similar morphology, metabolism, physiological characteristics. Its main fermentation production of sugar is lactic acid. LAB can produce massive extracellular secretions such as lactic acid,protease, lipase and antibacterial peptides, which can stimulate animal's immune system and improve immune ability. Meanwhile LAB can be colonized in gut, digestion,absorption of the organism and improve the metabolism ability. Therefore, LAB is one of the most important probiotics in the world and has been used widely.In this study, we adopt the MRS Soluble calcium circle method. We screened 40 strains with activity of lactic acid bacteria from 113 tropical marine environmental strains preserved in laboratory and 110 isolated strains from marine environment samples as well as shrimp digestive. These bacteria are identified as gram-positive bacteria by gram staining and 16S rDNA sequence analysis, including 13 Lactococus lactis, 12 Enterococcus faecalis,5 Enterococcus hirae,2 Lactobacillus fermentum, 2 Enterococcus faecium,2 eptococcus lutetiensistrs, 1 Enterococcus durans, 1 Streptococcus infantarius, 1 Streptococcus equinus and 1 Streptococcus agalactiae strains. By measuring the hemolytic activity of all strains, the result showed that 32 isolates including Lactococus lactis , Enterococcus faecalis, Enterococcus hirae and Lactobacillus fermentum were gamma hemolysis . We selected 2 Lactococus lactis (S1,S2) and 2 Enterococcus faecalis(F3, F7) strains to test the strongest capacity of producing acid, resistance of gastric juice,surface hydrophobic rate, self-aggregation rate antagonism effect on pathogenic antagonism and safety evaluation. The result showed that all above strains can adapt to the shrimp digestive tract environment and were non-toxic or harmless for shrimp . They also have strong antagonism effect of Vibrio harveyi, Vibrio alginolyticus Sakazaki,Streptococcus agalactiae, Edwardsiella and other pathogens.By feeding test in which 108 cfu/g S1, S2, F3 and F7 were concentrated as experiment group, commercial compound lactic acid bacteria spices as positive control and 0.9%physiological saline as negative control (0.63±0.15 g initial weight of shrimp),we measured the effect of S1, S2, F3 and F7 on the bait coefficient, specific growth rate,weight gain rate, survival rate and mortality of Vibrio sinensis. The result showed that there was significant difference between experiment group and control group (P <0.05). The bait coefficient of the test group was between 0.868 and 0.989, and the positive control group was 0.922, which was lower than that of the negative control group (P <0.05). The survival rate of S2 group was significantly higher than that of negative control group (P <0.05).The weight gain rate of all groups ranged from 614.28% to 768.67%, all of which were higher than control group, the highest group is S1 and lowest group is negative control respectively. The specific growth rate of the test group was 6.55% ?7.20%,which was significantly different from the negative control group (6.55%). According to the principle of "survival rate priority and prominent weight gain rate", S2 and F7 may be the most suitable strain for Litopenaeus vannamei spices.When the initial weight of 0.73 ± 0.20 g of shrimp were fed with three concentrations of S2 and F7 (concentration of 106, 107, 108 cfu/g), the result showed that the bait coefficient, survival rate, weight gain rate, specific growth rate and Vibrio stress mortality were significantly different from those of negative control (P <0.05). The bait coefficient of the test group was between 0.845 and 0.937 , in which the lowest was concentrated by 108 cfu/g S2 (0.845) and the highest was negative control group (1.010). The survival rate of the test group was between 80.5% and 85.00%, in which the highest was concentrated by 106 cfu/g S2 (85.00%) and the lowest was negative control group (76.79%). The weight gain rate of the test group was between 1914.25% and 2306.37%, in which the highest was concentrated by 106 cfu/g F7 (2306.37%) and the lowest was negative control group(1665.11%). The specific growth rate in the test group was between 4.91% and 5.28%, in which the highest was concentrated by 106 cfu/g F7 (5.28%) and the lowest was negative control group (4.77%) . Therefore, the indigenous lactic acid bacteria isolated from the tropical marine are more suitable for the feeding of Litopenaeus vannamei in Hainan.In this study, we adopted the method of single factor test and Response surface methodology optimized the culture conditions and culture medium of S2 and F7. The single factor experiment result showed that the optimum culture conditions of S2 and F7 were pH 6.0 ?7.0, temperature 30 ?37 ?,speed 160 ?180 r/min,volume 45%?50%and inoculation quantity 8% ?10%. The result of response surface test showed that the optimal medium of S2 was consisted of molasses 87.40 g/L and NaCl 23.90 g/L, in condition of 30?, ventilation 1L/min, 24 h, the viable count was 1.06 × 10~9 cfu/mL; while the optimal medium of F7 was consisted of yeast protein 91.95 g/L and tryptone 83.91 g/L,in condition of 30?, ventilation 1 L/min, 24 h, the viable count was 1.12 ×1010 cfu/mL.In conclusion, in this study we obtained 2 lactic acid bacteria in 223 aborigines strains from Hainan tropical marine environment suitable for feeding of Litopenaeus vannamei,then we determined the optimal medium and optimum culture medium of the two strains.This will lay an important foundation for the future development of tropical indigenous lactic acid bacteria preparations suitable for feeding Litopenaeus vannamei.