牙鲆肠道益生菌的分离筛选及益生效果评价

Isolation,Screening and Evaluation of Intestinal Probiotics from Paralichthys olivaceus

为获得安全有效的牙鲆益生菌,从牙鲆(Paralichthys olivaceus)肠道分离到94株菌,经溶血性、消化酶活力测试筛选出3株候选菌:Y1、Y5、I66,进行16S rDNA分子鉴定,分别为枯草芽孢杆菌(Bacillus subtilis)、解淀粉芽孢杆菌(Bacillus amyloidophilus)和蜡样芽孢杆菌(Bacillus cereus)。经安全性检验后,开展为期4周的投喂试验,分别将3株菌以1×10^(8) CFU·g^(-1)添加到饲料中投喂牙鲆,为3个试验组,同时设对照组(C),定期检测各组鱼血液先天免疫指标。结果发现,与对照组相比,试验组鱼淋巴细胞吞噬百分比显著升高(P<0.05),血清碱性磷酸酶(AKP)和溶菌酶(LYZ)活性显著升高(P<0.05),血清超氧化物歧化酶(SOD)活性极显著升高(P<0.01)。投喂相应饲料4周,腹腔注射溶藻弧菌(Vibrio alginolyticus)攻毒,试验组鱼死亡延迟,累积死亡率显著低于对照组(P<0.05)。综上,Y1、Y5和I66是牙鲆安全有效的益生菌,能促进先天免疫,提高抗病力,具有推广应用优势和潜力。

To obtain safe and effective probiotics derived from flounder(Paralichthys olivaceus),94 strains were collected from the intestine of flounder.3 candidate probiotics were screened out by hemolysis and digestive enzyme activity assays.Bacillus subtilis(Y1),Bacillus amyloidophilus(Y5)and Bacillus cereus(I66)were identified by 16S rDNA molecular approach,respectively.After the safety inspection,a four-week feeding test was carried out.The 3 strains were added to the diet at 1×10^(8)CFU·g^(-1),which were used as three experimental groups,and a control group(C)was set up.The blood innate immune indexes in each group were tested regularly.The results indicated that the phagocytosis percentage of lymphocytes,the activity of alkaline phosphatase(AKP)and lysozyme(LYZ)of probiotics groups were significantly higher(P<0.05)than those of the control group(C),and the serum superoxide dismutase(SOD)activity of probiotics groups was extremely significantly increased(P<0.01).After feeding corresponding diet for four weeks,each group subjects were injected to seroperitoneum with pathogenic Vibrio alginolyticus.The death of the probiotics group was delayed,and the cumulative mortality was significantly lower than that of the control group(P<0.05).In conclusion,Y1,Y5 and I66 are safe and effective probiotics for flounder,which can promote innate immunity and improve disease resistance,and have advantages and potential for popularization and application.