Screening Of Marine Fish-derived Probiotics And Effects Of The Probiotics On Homologous Host Growth, Enzyme Activity And Gut Microbiota

In recent years,with the intensification and high density of marine fish aquaculture,the incidence of marine fish has been increasing,which has become one of the factors that restrict the development of marine fish industry,therefore,how to prevent and control fish diseases at the same time as high-speed and efficient culture is a problem that researchers need to solve.Probiotics are a kind of active microorganisms that can bring health benefits to the host when added in sufficient amount,and can play a good effect in promoting animal growth,enhancing the body’s resistance to disease and improving the host’s immunity.Probiotics such as Bacillus and Lactic acid bacteria have been developed and used in marine-fish culture,but the non-fish origin of some strains makes it difficult for them to play an effective role in marine-fish culture.Ideal probiotic bacteria from animal gastrointestinal tract,therefore,for screening of both enzyme activities and the characteristic of disease-causing bacteria sea source probiotics,this study aquaculture economic species in north China the Sebastes schlegelii and Hexagrammos otakii as the research object,through the separation of intestinal bacteria identification,character analysis and breeding application test,The effects of probiotics on growth performance,intestinal digestive enzymes,liver antioxidant enzymes and intestinal microflora structure of homologous hosts were studied.The results provided a new probiotics strain for marine-fish culture industry and provided scientific basis for further application and promotion in production.1.Screening,Identification and Characteristic Analysis of Probiotics from Marine Fish80 strains of culturable bacteria were obtained by separating bacteria from the mucosa samples of digestive tract of wild S.schlegelii and H.otakii.The enzyme-producing ability of the strains was determined by using selective culture medium,and the common pathogenic bacteria of marine fish were selected as indicator bacteria to determine the antibacterial activity of the isolated strains.Two potential probiotics,screening out two potential probiotics TS2 and TH8,and carrying out physiological and biochemical test,16S rDNA sequential analysis,growth characteristics and host safety were studied.The results showed that TS2 has the strongest ability to hydrolyze starch,protein and fat,and its sterile culture products can significantly inhibit the growth of Pseudoalteromonas nigrifaciens,Vibrio anguillarum,V.parahaemolyticus and V.harvey.TH8 has the strongest ability to hydrolyze protein and fat,and its sterile culture products can significantly inhibit the growth of Staphyloccocus aureus,Escherichia coli,Aeromonas hydrophila,P.nigrifaciens,V.anguillarum,V.alginolyticus and V.parahaemolyticus.Based on physiological and biochemical detection of bacteria and16S rDNA sequence alignment and identification,TS2 was identified as Bacillus subtilis and TH8 as Vagococcus fluvialis.D72-2 showed significant growth at 15～45℃,Na Cl content of 0～8%and p H of 4～10,got into the logarithmic growth period after 6hours and the stable growth period after 26 hours;D39 grew rapidly at 20～40℃,Na Cl content of 0～8%and p H of 5～12,got into the logarithmic growth period after 6 hours and the stable growth period after 26 hours.The safety test of strain on homologous host showed that TS2 and TH8 were relatively safe to homologous host at the concentration of 10~8CFU/m L.The research indicated that B.subtilis TS2 and V.fluvialis TH8,which have strong enzyme production ability and inhibit the growth of various pathogens by its products,with the advantages of wide temperature and salt tolerance,acid and alkali resistance,fast growth speed,could be considered as potential probiotic candidates for the development of microbial pharmaceutics,and shall be developed more applications in marine aquaculture.2.Effects of TS2 on growth performance,intestinal digestive enzymes,liver antioxidant enzymes and intestinal microflora structure of S.schlegeliiIn this experiment,different concentrations of TS2 were applied to the S.schlegelii for 56d,and the growth performance,intestinal digestive enzymes,liver antioxidant enzymes and intestinal microflora structure of S.schlegelii were observed on 28d and56d,respectively.The results showed that the final average body weight of each experimental group was significantly higher than that of the control group,TS2supplementation with 1×10~6CFU/g could significantly improve the weight gain rate of S.schlegelii,and supplementation at 1×10~5CFU/g and 1×10~6CFU/g could significantly improve the specific growth rate of S.schlegelii.In terms of intestinal digestive enzymes,in the middle of the experiment,the addition of TS2 at 1×10~7CFU/g significantly increased trypsin activity,the addition of TS2 at 1×10~5CFU/g and 1×10~7CFU/g significantly increased amylase activity,and the addition of TS2 at 1×10~6CFU/g and1×10~7CFU/g significantly increased lipase activity;At the end of the experiment,the activities of trypsin,amylase and lipase were significantly increased in each experimental group.In terms of liver antioxidant enzymes,in the middle of the experiment TS2 supplementation at 1×10~5CFU/g and 1×10~7CFU/g could significantly increase the activity of SOD,and CAT activity in all experimental groups could be significantly increased;at the end of the experiment,TS2 supplementation at 1×10~6CFU/g and 1×10~7CFU/g could significantly increase the activity of SOD,and CAT activity could be significantly increased in all experimental groups.In terms of intestinal microflora structure,the addition of TS2 can significantly affect the diversity of intestinal microflora of Scorpene Schrispene,in the middle of the experiment TS2supplementation at 1×10~6CFU/g could significantly increase the number of Sediminibacterium,and the proportion of Methylovirgula decreased first and then increased with the increase of TS2 concentration;When TS2 supplemental level was1×10~5CFU/g at the end of the experiment,the number of beneficial bacteria Sediminibacterium was significantly increased,and the number of harmful bacteria Bradyrhizobium was significantly decreased;The proportion of Methylovirgula increased first and then decreased with the increase of TS2 concentration,and the proportion of Rhodanobacter decreased first and then increased throughout the experiment.Therefore,B.subtilis TS2 can promote the growth of S.schlegelii,significantly improve the intestinal digestive enzyme activity and liver antioxidant enzyme activity,enrich the diversity of intestinal flora,increase the number of beneficial bacteria,reduce the number of harmful bacteria.3.Effects of TH8 on growth performance,intestinal digestive enzymes,liver antioxidant enzymes and intestinal microflora structure of H.otakii.In this experiment,different concentrations of TH8 were also applied to H.otakii,for 56d,and the growth performance,intestinal digestive enzymes,liver antioxidant enzymes and intestinal microflora of H.otakii were observed on 28d and 56d,respectively.The results showed that TH8 supplemental level of 1×10~7CFU/g could significantly increase the average body weight,weight gain rate and specific growth rate of H.otakii.In terms of intestinal digestive enzymes,in the middle of the experiment the trypsin activity could be significantly increased when TH8 supplemental level was1×10~6CFU/g and 1×10~7CFU/g,and the activities of amylase and lipase were significantly increased in all experimental groups;at the end of the experiment,the trypsin activity and amylase activity were significantly increased in each experimental group.In terms of antioxidant enzymes in the liver,the SOD activity and CAT activity could be significantly increased when the TH8 supplemental level was 1×10~6CFU/g and 1×10~7CFU/g in the middle of the experiment;the SOD activity could be significantly increased when the TH8 supplemental level was 1×10~6CFU/g and 1×10~7CFU/g and CAT activity was significantly increased in each experimental group at the end of the experiment.In the intestinal flora structure,TH8 could significantly improve the diversity of intestinal microflora of H.otakii.In addition,in the middle of the experiment,the addition level of V.fluvialis TH8 at 1×10~6CFU/g could significantly reduce the number of Rodinella.With the increase of YH8 concentration,The proportion of Bradyrhizobium,Sediminibacterium,Rhodanobacter and Methylovirgula remained unchanged.However,at the end of the experiment,the number of Rodanobacter could be significantly increased when the TH8 supplemental level was1×10~7CFU/g,with the increase of YH8 concentration,Sediminibacterium gradually decreased,Rodanobacter gradually increased,Methylovirgula increased first and then decreased.It can be seen that V.fluvialis TH8 can not only significantly improve the intestinal digestive enzyme activity and liver antioxidant enzyme activity,but also significantly improve the diversity of intestinal flora,which may be the main factor that TH8 promotes the growth of H.otakii.In conclusion,this study from the wild S.schlegelii and H.otakii digestive tract two strains of probiotics TS2 and TH8 separation filter,the enzyme production ability,bacteriostatic effect is good,with the advantages of wide temperature and salt tolerance,acid and alkali resistance and fast growth speed,applied in the homologous host breeding,can promote the growth of the body,and improve the digestive enzymes activity and liver antioxidant enzymes,diversity structure of intestinal flora.The results provide data reference for the research and development of probiotics and their application in Marine fish.