Study On Preparation,Pharmacological Effect,and Toxicology Of The Gan Liao Compound

In recent years,various bacterial diseases have had a major impact on the economic benefits of aquaculture industry in China.The drawbacks of using antibiotics and other chemical drugs have gradually arised,which has seriously threatened the health of animals and food safety.It was hot problem to search for green and effective antibiotic substitutes.With the deepening of research,it had been found that Chinese herbal medicines were used in combination with microecological preparations,which have both the functions of Chinese herbal medicines and the advantages of probiotics,and they can complement each other and enhance their advantages.Its have good development potential and application prospects in the animal production industry.The purpose of this study was to prepare the Gan Liao compound preparation,a Chinese herbal compound microecological preparation which was combined with Polygonum hydropiper L.,Glycyrrhiza uralensis Fisch and MOS.Then,the theoretical basis for application of probiotics in healthy culture of Penaeus vannamei is provided,based on the results of pharmacological toxicological tests and the culture experiment of P.vannamei.Method:（1）The contents of each component added（Chinese herbal,probiotics,prebiotics）was determined via the Oxford cup test,a single factor test,and an orthogonal design test.（2）The acute toxicity test and the rat 30-day feeding test were carried out to evaluate the safety of Gan Liao compound preparation.（3）Feeding shrimp diet with differ dosage（1/2part,1part,2part）Gan Liao compound preparation for 21 days,its effects on the growth performance,immunity index at 14 d and 21 d were determined and compared,and the llumina miaeq high-throughput sequencing technology was used to sequence 16Sr DNA macrogenome and analyze biological information of intestinal flora samples for observing the effect of applicationResult:（1）The 31.25 g/L decoction of Glycyrrhiza and 250 g/L decoction of P.hydropiper can affect the growth of three common aquatic pathogens（Vibrio alginolyticus,Vibrio parahaemolyticus,Aeromonas hydrophila）,and it does not inhibit the growth of B.licheniformis.The 1%～3%concentration of MOS,the 0.5%concentration of Glycyrrhiza,and the 0.5%～1%concentration of P.hydropiper promoted the growth of B.licheniformis.The orthogonal test results showed that when B.licheniformis was cultured at 2%in broth,P.hydropiper,Glycyrrhiza and MOS were added in a mass ratio of 1:0.1:2（%）,the maximum number of viable bacteria（6.25×10⁷）can be obtained.So we can make Ganliao compound preparation with 1 g P.hydropiper,0.1 g Glycyrrhiza,2 g MOS and B.licheniformis（10⁶）mixed.（2）Kunming mice were given the drug by intragastric administration,and the LD₅₀ was not detected.The maximum tolerance test showed that Gan Liao compound preparation could not cause death in mice at the dose of 30 g/kg BW.The results of 30-day feeding test in rats showed that the Gan Liao compound preparation had no significant,effect on body weight,organ index and hematological index of rats at 30 g/kg BW/d、15 g/kg BW/d、7.5 g/kg BW/d,respectively.（3）Gan Liao Compound preparation could significantly improve the lipase activity of P.vannamei（p<0.05）,and high-dose Gan Liao Compound preparation significantly increased amylase activity（p<0.05）;The probiotics had no significant effect on the amylase activity of P.vannamei（p>0.05）,but could increase the lipase activity in the intestine（p<0.05）;The antibiotic had no significant positive effect on the amylase and lipase activity of P.vannamei（p>0.05）.The low-dose Gan Liao Compound preparation obviously improved the level of ACP of P.vannamei（p<0.05）;The medium-dose Gan Liao Compound preparation significantly increased T-AOC and the activity of PO of P.vannamei（p<0.05）;The high-dose Gan Liao Compound preparation impose on the activity of i NOS of P.vannamei（p<0.05）;The ACP activity was significantly inhibited by antibiotics（p<0.05）;There was no significant difference in GSH content among the 6 experimental groups（p>0.05）.The results of Illumina Hi Seq Sequencing and Analysis showed that the ACE and Chao1 index of the Gan Liao compound preparation group appeared an upward trend,and higher than that of control group.Compared with control group,the Simpson index of probiotic group and antibiotic group was significantly higher（p<0.05）.There was no significant difference in other alpha diversity indices.At the phylum level,the distribution and abundance of Proteobacteria,Firmicutes and Bacteroidetes in the middle and high dosage group of the intestine of P.vannamei were close to those in the control group,while the relative abundance of Proteobacteria in probiotics and antibiotics group was higher.At the level of class,Alphaproteobacteria,Flavobacteriia and Gamma Proteobacteria were dominant,but their relative abundance was different.The Spongiimonas,Vibrio,Photobacterium and Shewanella dominated at the genus level.The intestinal abundance of Spongiimonas in antibiotic group was less than that in other groups;Photobacterium were more abundant in antibiotic group and probiotic group than other groups.The abundance of Shewanella is reduced in this order:Probiotics group,low dose group,high dose group,ENR group,middle dose group and control group.Cluster tree analysis showed that the similarity of intestinal flora distribution between Gan Liao compound preparation group and control group was high,while that between probiotic group and antibiotic group was low.The results showed that the Gan Liao compound preparation can affect intestinal flora composition of P.vannamei,and its effect on microbial community is more mitigated than that of antibiotics.Conclusion:The the Gan Liao compound preparation prepared in this study is safe,which can improve the digestive performance and immunity of P.vannamei,and improve the intestinal flora structure of P.vannamei.It has practical value for healthy culture of P.vannamei.