工厂化循环水养殖条件下云纹石斑鱼消化道产酶菌的分离鉴定

采用16S r DNA-PCR菌群分离鉴定的方法,对循环水养殖条件下云纹石斑鱼(Epinehelus moara)幼鱼的胃、幽门盲囊、前肠、中肠和后肠的菌群结构进行了鉴定,用产酶菌筛选培养法对产消化酶的菌株进行了分离鉴定,并测试了各菌株消化酶的活力。研究发现,云纹石斑鱼幼鱼消化道内可培养的主要菌群为假单胞菌属(Pseudomonas)、微小杆菌属(Exiguobacterium)、不动杆菌属(Acinetobacter)、寡养单胞菌属(Stenotrophomonas)和葡萄球菌属(Staphylococcus),其中产消化酶的菌株占可培养菌的55.6%。在产酶菌中,同一株菌产3种酶的有5株;产2种酶的有9株;中肠和后肠的菌株数最为丰富,胃次之,幽门盲囊和前肠菌群种类较少;产脂肪酶的菌株都集中在中肠。产消化酶的菌株主要以产蛋白酶和淀粉酶为主,且产酶量丰富,产蛋白酶活力最高达(87.732±1.134)U/m L;淀粉酶活力为(77.176±0.599)~(73.458±0.574)U/m L;产纤维素酶的菌仅一株,且酶活力较低。分析得知,消化道的菌群结构直接影响了外源性消化酶的种类与活性。本研究为工厂化循环水养殖条件下产酶有益菌的筛选提供了理论依据。

Isolation and identification of enzyme-producing bacteria from thedigestive tract of Epinehelus moara in re-circulating aquaculture systems

The purpose of this research was to study the bacterial community structure in digestive tract and enzymeproduction capacity of enzyme-producing bacteria, and provide reference for selection and application ofprobiotics for carnivorous fish culture. In this experiment, samples of juvenile saladfish (Epinehelus moara)stomach, pyloric caeca, foregut, midgut, and hindgut were obtained in recirculating aquaculture systems. Bacterialcommunity structure was analyzed using 16S rDNA-PCR. The enzyme-producing bacteria were isolated and identifiedby isolating and screening enzyme-producing bacteria. Moreover, the enzyme activities were tested.Twenty-seven strains were isolated and cultured under experimental conditions, including 13 strains of Pseudomonas,5 strains of Exiguobacterium, 7 strains of Acinetobacter, 1 strain of Stenotrophomonas, and 1 strain ofStaphylococcus, which accounted for 48.2%, 18.5%, 25.9%, 3.7%, and 3.7%, respectively, of the isolated bacteria.The sequence homology of corresponding genes was greater than 98%. Fifteen strains produced enzymes and accountedfor 55.6% of all bacteria; these bacteria included 7 strains of Pseudomonas, 5 strains of Exiguobacterium,2 strains of Acinetobacter and 1 strain of Stenotrophomonas. Among these bacteria, 13 strains can produce bothprotease and amylase, whereas 4 strains can produce protease, amylase, and lipase. Among the enzyme-producingbacteria, 5 strains can produce 3 enzymes and 9 strains can produce 2 enzymes. Moreover, the bacteria in themidgut and hindgut were most abundant, and those in the stomach, diverticulum pyloricum and foregut were lessabundant; the bacteria that produce lipase were concentrated in the midgut. Protease and amylase were the mainenzymes produced by these bacteria; these two enzymes were highly productive, with protease activity up to(87.732±1.134) U/mL and amylase activity between (77.176±0.599) U/mL and (73.458±0.574) U/mL. Only onestrain produced cellulase, and the activity was low. Under the experimental conditions, the isolated bacteria wereall culturable. However, non-culturable bacteria cannot be isolated. Moreover, some culturable bacteria in the digestivetract could not be isolated because of limited testing conditions such as temperature, pH, culture medium,and other factors that may affect normal bacteria growth. In addition, isolation and identification took place underaerobic conditions, which is not similar to real gut conditions; thus, a large number of anaerobic bacteria were notisolated. Therefore, further investigation is needed to determine the actual bacterial community structure of the E.moara digestive tract. Our data showed that the bacterial community structure of the digestive tract directly affectedthe activity and diversity of exogenous digestive enzymes. This research provides a theoretical basis forselection of enzyme-producing bacteria in recirculating aquaculture conditions.