| Plectropomus leopardus,also known as Leopard Coral Grouper,is a valuable seafood product containing astaxanthin,which has high nutritional value;But as the breeding density increases,the problem of diseases and medication become more prominent.Therefore,there is an urgent need for scientific and effective green health and safe breeding technology.Lactic acid bacteria are“generally recognized as safe”,which are considered to improve the gut microbiota and enhance the digestibility of the host.In this study,Lactobacillus salivarius GZPH2 was used in the production of Plectropomus leopardus,and two experimental groups with different concentrations were set up:group B(1.3×10~7 CFU/g)and group C(7.3×10~6CFU/g),together with control group A(0 CFU/g),interval feeding for 8 days(1 d is the background value,2-4 d with GZPH2 in feed,5-8 d to stop),sampling every day for microbiome and metabolomics research.(1)Firstly,the effect of GZPH2 on the total culturable bacteria counts(TCBC)and total culturable vibrios counts(TCVC)in the body surface and intestine of Plectropomus leopardus was studied by traditional bacteriological methods.The results showed that compared with the control group,GZPH2 significantly reduced the concentration of TCBC and TCVC in the body and intestine of the experimental group(p<0.05),and the low concentration of GZPH2had a better control effect on TCBC and TCVC.Pearson correlation analysis showed that the concentration of GZPH2 was only significantly negatively correlated with body surface TCBC(p<0.05),and no statistical correlation was found with intestinal TCBC,body surface and intestinal TCVC(p>0.05).In addition,compared with the control group,both groups of GZPH2 significantly increased the body length and weight gain rate of Plectropomus leopardus(p<0.05).(2)Secondly,the 16S rDNA gene V3-V4 region High-throughput sequencing technology of genomics was used to carry out microbiome research on intestinal flora of Plectropomus leopardus on 1 d,4 d and 8 d samples.The results showed that 466977effective sequences were obtained from 9 samples.After OTU clustering analysis,the intrinsic bacteria in the intestine of Plectropomus leopardus were obtained,which were Firmicutes,Proteobacteria,Actinobacteria,Patescibacteria,Bacteroidetes.Bacteroidetes/Firmicutes is an important indicator which indicates the growing of fish,the lower the ratio is,the faster the fish grows.In the early feeding period(1-4 d),the ratio of Bacteroidetes/Firmicutes in groups B and C both decreased(0.250 and 0.003 respectively).In the late feeding period(5-8 d),this ratio decreased in group B and increased a little in group C(0.001 and 0.004,respectively),while in group A,it has been maintained at a relatively high level(4 d;0.503,8 d:0.454).the result was proved by length gain and weight gain formerly described.PCoA(principal coordinate analysis)found that the samples in group A were separated from group B and C,that is,there was a large difference in the composition of intestinal flora.Through the analysis of species composition,this difference was found:on 4 d,the abundance of Bacilli?Actinobacteria?Gammaproteobacteria in A group were 6.58%?13.54%?29.56%,in B group were 4.84%?14.18%?49.26%,in C group were 91.57%?0.52%?6.06%;on 8 d,these data were 4.91%?12.55%?47.54%in A group,20.21%?0.36%?6.50%in B group and44.70%?1.21%?23.87%in C group.Bacilli and Actinobacteria is beneficial to enhance the resistance to diseases,while Gammaproteobacteria has the possibility of retarded fish growth and even cause disease.the result showed the positive effect of high concentration of GZPH2on Gammaproteobacteria.Among the top 20 abundant genera in the samples,during 1-4 d,high concentration of GZPH2 can control 30%(6/20)of which,4 were Gram-negative(G-)bacteria,2 were Gram-positive(G+)bacteria,low concentration GZPH2 can control 35%(7/20)of which,4were G-bacteria,3 were G+bacteria;during 4-8 d,high concentration GZPH2 can control 50%(10/20),G-bacteria and G+bacteria were both half,low concentration GZPH2 can control 25%(5/20),2 were G-bacteria and 3 were G+bacteria.In addition,it was also found that the high concentration of GZPH2 on 8 d increased the total relative abundance of probiotics/functional flora(+76.89%)while reducing the total relative abundance of(opportunistic)pathogenic bacteria(-11.77%).Low concentration GZPH2 has no such effect.Differences in the composition of intestinal flora will inevitably bring about functional changes.On 4 d and 8 d,the functional abundances of the three groups A,B,and C were significantly different(p<0.01).There was little change in group A(p>0.05),and most of the functional abundance in groups B and C increased,which helped to improve the utilization of protein,carbohydrates and other nutrients,as well as biosynthesis,transportation and catabolism of secondary metabolites.In terms of the mechanism by which GZPH2improves the function of the intestinal flora,the synergistic effect(positive correlation)within the functional flora and its inhibitory effect with the pathogenic bacteria(negative correlation)play a role at the same time,thereby achieving the inhibition of pathogenicity bacteria and increase the relative abundance of functional flora.Such as the negative correlation between Lactobacillus and Burkholderia(p<0.05),and the positive correlation between Clostridium?sensu?stricto?1 and Lactobacillus(p>0.05).(3)Thirdly,LC-MS/MS technology was used to carry out metabolomic studies on intestinal flora of Plectropomus leopardus.Compared with the control,GZPH2 promoted the intestinal metabolism of Plectropomus leopardus to a better mode,the content of metabolites such as isoflavone and organic acids increased,and pentose and glucuronate interconversions,glycerophospholipid metabolism,arginine biosynthesis,Linoleic acid metabolism were significantly accumulated.The disturbance of the metabolic activity of the control group affects the synthesis of functional intermediate products,and then poses a potential threat to the immunity of Plectropomus leopardus.The high concentration of GZPH2 helps to regulate the intestinal metabolic level,remove the body's oxidative metabolites,and promote the synthesis of arginine and functional fatty acids,as well as functional substances such as glucuronic acid and phenyllactic acid,performs a positive effect on gut health of Plectropomus leopardus.Low concentration GZPH2 also has the same effect,but it is not as good in the concentration of glucuronic acid and isoflavone.(4)Finally,using statistical analysis,the correlation between microbiome and metabolomics of the intestinal microflora of Plectropomus leopardus was studied.The results found that,on the one hand,GZPH2 changed the intestinal flora structure of Plectropomus leopardus,affecting the electron transfer in the biochemical reactions of the body's metabolic pathways,and the correlation with pentose and glucuronate interconversions,glycerophospholipid metabolism,drug metabolism-cytochrome P450 and other pathways'differential metabolites(p<0.05);on the other hand,GZPH2 caused changes in metabolites,which in turn reacted on the intestinal flora,such as the significant negative correlation(p<0.05)between botryodiplodin and Burkholderia-Caballeronia-Paraburkholderia,Pseudomonas.To sum up,it's obvious that GZPH2 has a regulating effect on the intestinal micro-ecology of Plectropomus leopardus during the application period and within a few days after it was stopped.It achieves the function of maintaining the health of fishes by increasing the amount of beneficial bacteria,reducing the accumulation of harmful bacteria,and promotes the production of functional metabolites,enhances the body's antioxidant capacity.so we ca say our study is of practical significance in the promotion of Plectropomus leopardus healthy green farming and guarantee food security. |
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