Screening And Functional Study Of Potential Probiotics In Crucian Carp(Carassius Auratus)intestine

The current aquaculture industry is facing a serious threat of bacterial diseases.Antibiotics are still the first choice for the prevention and treatment of bacterialophilic diseases.The abuse of antibiotics will lead to the development of resistance to pathogenic bacteria and cause huge pollution to the environment.Therefore,the use of some new methods to replace the use of antibiotics has become a hot research topic in the prevention and treatment of bacterial diseases.Studies have shown that probiotics have a potential role in promoting the growth and improving immunity of aquatic animals.At the same time,endogenous probiotics have strong colonization ability in the body,are safe for the host and the environment,and can indirectly regulate the body's immunity by improving the structure of the intestinal flora.Therefore,the development and research of new probiotics play an important role in promoting the development of aquaculture.This study preliminarily analyzed the structural characteristics of crucian carp(Carassius auratus)intestinal flora,screened and isolated potential probiotics that are non-hemolytic and have inhibitory effect on Aeromonas hydrophila in vitro,and further evaluated their safety through in-body safety test.Potential probiotics were fed to the crucian carp in the later period to study its effects on the growth,physiology,immunity and intestinal flora of the crucian carp.The results obtained are as follows:1.FWA medium was usd to isolate the strains from the intestine of the crucian carp.According to the morphological characteristics of the colonies,36 strains were randomly selected according to the ratio and identified by 16S rDNA sequencing.Among them,the dominant bacteria were Aeromonas,Bacillus,Plesiomonas,Geobacillus and Enterobacter,which accounted for 46.88%,34.38%,6.25%,6.25%and 6.25%,respectively.A total of 16 strains of 36 strains isolated from crucian carp were non-hemolytic,of which three strains were Aeromonas veronii C19,Enterobacter asburiae C28,and Bacillus tequilensis C35,which had antagonistic effects on Aeromonas hydrophila KS02.Healthy crucian carp were injected at three concentrations of C19,C28,and C35 at 2×10~6,2×10~7,and 2×10~8 CFU/mL,0.5 m L of bacterial solution was injected intraperitoneally per 100 g of fish body weight,and an equal amount of normal saline was injected in the control group.After 10consecutive days of observation,no disease or death occurred in all the strain injection groups and the control group,indicating that all three strains were not pathogenic to the crucian carp.2.Effects of potential probiotics on growth,physiology,immunity and disease resistance of crucian carp:three bacteria were added to the conventional crucian carp feed and fed to crucian carp for 35 days;During this period,blood was collected every7 days and serum biochemical indicators were determined.After feeding,growth status were determined,collect blood to determine hematological indicators,liver was collected to determine biochemical indicators;In each group,10 fishes were randomly selected and intraperitoneally injected with Aeromonas hydrophila to determine the immune protection effect of feeding strain on crucian carp.The results showed that the body weight,body length and full length of crucian carp fed with Aeromonas veronii C19 were higher than that of the control group.Serum lipase,amylase,SOD and CAT activities of crucian carp fed with Aeromonas veronii C19 were significantly increased,and serum MDA content was significantly reduced.C19 could play a better protective effect on crucian carp when infected by pathogenic bacteria Aeromonas hydrophila;Bacillus tequilensis C35 showed the effect of improving the serum lysozyme,lipase and liver alanine aminotransferase in crucian carp,played the role on promoting digestive capacity and immunity of crucian carp.3.Effects of three potential probiotics on intestinal health of crucian carp:on the35th day of the experiment,intestinal samples were collected for the determination of intestinal digestive enzyme activity and the preparation of midgut tissue sections.The intestinal contents of crucian carp midgut were taken out by aseptic operation,the total DNA of the microbiome was extracted,and the variable region of 16S rDNA(V3+V4)was amplified by PCR.The intestinal microbial community structure of crucian carp was analyzed by high-throughput sequencing technology.The results showed that feeding potential probiotics C19 and C28 had positive effects on the digestion and absorption of crucian carp.All three strains of potential probiotics could increase the abundance of beneficial bacteria such as Cetobacterium,Romboutsia and Akkermansia in the intestinal tract of crucian carp,and decrease the abundance of harmful bacteria such as Plesiomonas and Pseudoxanthomonas.In conclusion,Aeromonas veronii C19 and Bacillus tequilensis C35 can improve the digestive capacity,antioxidant capacity and immunity of crucian carp,which can be further developed and utilized.