| Lactic acid bacteria(LAB)is recognized as an important probiotic in the world.In the process of aquaculture,people may feed LAB mixture to improve the survival rate,weight gain rate and immunity of aquatic animals.In addition,directly spray LAB into the water body to improve the water quality of the culture system.At present,these LAB are mainly derived from fermented foods and livestock,and the scientific problems.Up to date,whether the LAB can colonize in the intestines of shrimps and whether the water quality can be improved by LAB are not clear.This paper intends to isolate LAB from the intestines of Litopenaeus vannamei,cultured water,natural seawater,feed,LAB preparations,disinfectant reservoir water and the intestines of wild shrimps in Changjiang,Hainan.Then,by 16S rDNA sequencing and RAPD analysis,etc,we also try to clarify the species,source,colonization ability,genetic diversity and acid production capacity of the isolated LAB.It would not only provide excellent strains for the development of LAB preparations suitable for tropical marine aquaculture in Chanjiang,but also for precise application for LAB preparations provide strong scientific guidance.Details as follows:472 strains of LAB were screened out from 37 samples,including the shrimp intestines,aquaculture water,natural seawater,feed,LAB preparations,water quality improvers from the two main L.vannamei breeding bases in Changjiang,Hainan,(Haiwei Base and Changhua Base,referred to as HW and CH,respectively;HW in the shrimp culture process,used a different type of LAB preparations for the whole pool splashing and mixing Feeding,CH does not use LAB preparation,but·the entire pond is sprinkled with a photosynthetic bacteria as the main water quality improver)and the local wild Penaeus scmisulcatus.Interestingly,only 2 strains were selected from the shrimp culture water.This might be that the lactic acid bacteria were anaerobic bacteria and could not survive in a highly dissolved oxygen aquaculture environment.The results also indicated that spraying LAB into the culture water was with little effect.The 16S rDNA sequences of all LAB were aligned with the Blastn sequence alignment in the EzBioCloud database(www.ezbiocloud.net).The results showed that Lactobacillus penlosus,Lactococcus lactis,Leuconostoc suionicum,sere Weissella cibaria,and other LAB were 216 st.rains,67 strains,35 strains,27 strains and 119 strains,respectively.A total of 293 strains of LAB were obtained from the intestines of L.vannamei in HW.Among them,L.pentosus was the dominant bacteria,up to 156 strains,accounting for 53.24%of the total LAB in the intestinal tract of L.vannamei in HW;30 strains of L lactis accounted for only 10.24%;27 strains were L.suionicum,and 76 strains were other LAB.131 strains of LAB were obtained from the intestines of L.vannamei in CH,including 52 strains of L.pentosus and 37 strains of L.lactis,which accounted for 39,69%and 28.24%of the total amount LAB from the intestine of L.vannamei in CH,respectively.This indicated that L.pentosus and L.latis are the dominant bacteria in the intestinal tract of L.vannamei.The artificial feeding of L.pentosus can cause this strain to become the only dominant species in the intestinal tract of the shrimp.Despite this operation increased the proportion of LAB in the intestine(53.24%),can temporarily ensure the health of the cultured shrimp,but at the same time,the structure of the microbial flora in the intestinal tract of the shrimp tends to be singular,and there is a risk of structural imbalance of the intestinal microflora of the shrimp.From the intestinal tract of wild shrimp,23 strains of Lactococcus garvieae,four strains of and three strains of were obtained.Four strains of L.pentosus,one strain of L.suionicum,two strains of Lactobacillus paracasei,one strain of L.parcicasei were obtained from the LAB preparation in HW.But Lactobacillus brevis was not found in the intestines of cultured shrimps.This indicates that L.brevis are not suitable for colonization in the intestine of L.vannamei.One strain of L.suionicum and 1 strain of Aerococcus Williams were obtained from the culture water in HW;Six strains of V.carniphilus,one strain of Enterococcus lactis and 1 strain of Enterococcus hirae were obtained from natural seawater in HW.Among them,L.suionicum might be derived from a spilled LAB preparation,which may be due to that the strain is a facultative anaerobic bacterium.Therefore,the strain can be simultaneously present in the LAB preparation,the culture water body and the intestine of the shrimp,but neither can become a dominant bacteria.The results also showed that LAB in natural seawater do not appear in aquaculture water bodies due to the strict disinfection of water storage.The RAPD analysis of the dominant LAB(L.pentosus)showed that 28 bands were produced in 212 strains,the polymorphic ratio was 96.4%,and the similarity coefficient was 0.056～1.When the similarity coefficient is 0.5,the strains are divided into 13 categories,of which ⅩⅢ strains have the largest number of 172 strains,of which 171 strains are the same strain,which are derived from the LAB preparation(4 strains)and intestines of shrimp in HW(142 strains,mainly isolated from small shrimp intestines)and intestines of shrimp in CH(25 strains).It indicated that L.pentosus in LAB preparations can colonize well in the intestines of shrimps(especially juvenile prawn).The Ⅸ class consists of 25 strains,which can be finely divided into 2 small branches.One is derived from the intestine of the shrimp of CH,and the other is derived from the intestine of the shrimp in HW.The branches indicated that there are different geographical populations of the bacteria in the two bases.Through the research on the growth rate and acid production capacity of LAB,it is shown that the growth rate and acid-producing of the dominant LAB(Z.pentosus and L.lactis)in the intestine of cultured shrimp are significantly higher than those in the intestinal tract of wild shrimp(L.garvieae,V.carniphilust and E.faecalis).The pH values of L.pentosus and L.laclis were the lowest when cultured for 22h,and they were 3.67 and 3.79,respectively.When cultured for 24h,the pH values of V.carniphilus,L.garvieae and E.faecalis were the lowest,and they were 5.19,4.70 and 4.56,respectively.This is closer to the optimum pH(7.5,7.0 and 6.5)for proteases,lipases and amylases in the intestines of L.vannamei.This indicated that LAB in the intestines of wild shrimp may be important as potential bacterial resources,laying the foundation for the development of LAB preparations in the future. |
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