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长牡蛎苗种培育用益生菌的筛选鉴定
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Abstract:
为筛选出长牡蛎Crassostrea gigas苗种培育用益生菌，采用培养方法从牡蛎成体组织共分离出345株细菌，经过产淀粉酶、脂酶、蛋白酶和纤维素酶能力测定从中筛选出8株菌产四种酶。对8株菌进行溶血试验，7株为γ-溶血，即无潜在致病性。选出DX10和QX14菌株以终浓度为1 × 106 cells/mL浸浴攻毒健康长牡蛎幼体7 d，结果显示两株菌对长牡蛎幼体无毒性；16S rRNA基因序列同源性分析表明，DX10菌株与Shewanella aquimarina SW-120菌株的序列相似性99.31%，QX14菌株与Pseudoalteromonas sp. P113-L017a菌株的序列相似性为100%；将两菌株以终浓度1 × 105 cells/mL添加在长牡蛎幼体培育水体中，以不加菌为对照组，分别在选育后的第3、6、12和15 d测定幼体壳高，结果显示，DX10 (第6天至第15天)和QX14菌株(第3天至第15天)组幼体的壳高显著高于对照组(P < 0.05)；幼体培育15 d后开始投放附着基以便苗种附着，投放附着基10 d后(即选育后25 d)出库至海上养成，并统计稚贝数量，结果显示，DX10和QX14菌株组稚贝数量显著高于对照组(P < 0.05)。研究表明，益生菌希瓦氏菌DX10和假交替单胞菌QX14促进长牡蛎幼体生长和/或存活。
In order to select the probiotics for larviculture of the oyster Crassostrea gigas, 345 bacterial strains were isolated from tissues of adult C. gigas using culture method. Eight strains were screened based on the abilities of the isolates producing amylase, lipase, protease and cellulase. Hemolysis was conducted on the 8 strains, and 7 strains showed γ-hemolysis, that is, they did not have potential pathogenicity. Healthy larvae were both challenged by strains DX10 and QX14 selected at 1 × 106 cells/mL for 7 d, confirming that both strains were non-toxic for oyster larvae. Similarity analysis of 16S rRNA gene sequences indicated that the strain DX10 had 99.31% similarity to Shewanella aquimarina SW-120 and the strain QX14 had 100% similarity to Pseudoalteromonas sp. P113-L017a. Two strains were added to seawater in tanks for larviculture of the oyster at final concentration of 1 × 105 cells/mL and no potential probiont was added to control tanks. The larvae were cultured and the shell height of the larvae were measured on the 3rd, 6th, 12th, and 15th days after breeding. The results showed that the shell height of the larvae in the strains DX10 (6th to 15th day) and QX14 (3rd to 15th day) groups was significantly higher than that of the control group (P < 0.05). After 15 days of larvae rearing, substrates were introduced for seedling attachment. Ten days later (that is, 25 days after breeding), the juveniles were transferred to sea area for farming, and the quantity of juveniles of each group were counted. The results showed that the quantity of juveniles of strains DX10 and QX14 groups were significantly higher than that of the control group (P < 0.05). These findings suggest
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