The Study Of Isolation, Biological Characteristics And Mechanism Of Probiotics From Cyprinus Carpio

In this study,probiotics with the ability of heat tolerant and to inhibit pathogenic bacterium were screened from intestinal tract of Cyprinus carpio and breeding water. Followed identification,some probiotics strains were tested for ability to adhere to the foregut mucus of Cyprinus carpio and against binding of pathogenic bacteria.Mechanism of three isolates,which are Bacillus subtilis,Enterococcus and Photosynthetic Bacteria effect on immune function,digestive enzymes,and flora diversity of Cyprinus carpio intestinal tract, muscle quality,and growth performance of Cyprinus carpio were discussed.The results of study are as follows:In the first and second experiments,twenty strains isolated from breeding pools, aquariums and intestinal tracts of healthy Cyprinus carpios,and eight strains provided by Animal Micro-ecology Research Center of Sichuan Agricultural University were tested by heat-treatment and inhibitory experiment.There were five strains were screened according to the results,and then identified as two strains of Bacillus,two strains of Enterococci,and one strain of Citrobacter by 16S rDNA sequencing.Further,the method of immobilizing Cyprinus carpio foregut mucus albumen in vitro,combined with labeling bacteria with isotope ³²P was conducted to study the adhesion activity of Citrobacter and Enterococci strains from Cyprinus carpio intestinal tract,and Bacillus strains from breeding water.The method of screening probiotics from Cyprinus carpio was established in this study.The results indicated that all five strains can adhere to the mucus of in vitro model,and while the adhesion ratio of Enterococci strains was extremely higher than Citrobacter and Bacillus strains(P＜0.01),the difference between Citrobacter and Bacillus strains was not significant (P＞0.05),and the adhesion ratio of Enterococci strain L2 was extremely higher than Enterococci strain E2(P＜0.01).The results suggested that the Enterococci strains isolated from fish are easier to adhere to Cyprinus carpio foregut mucus than the heterogenous Bacillus strains,and the adhesion activity depended on different strain genus.In the third and forth experiments,in order to investigate the effect of probiotics on immune function and intestinal digestive enzymes of Cyprinus carpio,four treatment groups fed with different probiotics preparations were conducted,including Group A(control group), Group B(fed with Bacillus subtilis strain Z2),Group C(fed with mixing preparation of Bacillus subtilis and Photosynthetic bacteria),Group D(fed with mixing preparation of Bacillus subtilis and Enterococci strain L2),and each preparation was compared with Group A within the same level.The results showed that phagocytosis and bactericidal activity of leukocyte and serum enzymatic activity of Group B and C was significantly different from Group A(P＜0.05),and the performance of Group D was not as well as Group B and C. There was no significant difference in amount of lymphocyte among Group A,B and C(P＞0.05).The results indicated that Bacillus subtilis rather than compound of Bacillus subtilis and Photosynthetic bacteria,Bacillus subtilis and Enterococci efficiently improved the phagocytosis and bactericida activity of leukocyte,and serum lysozyme activity.Further, amylase specific activity of Group B was enhanced significantly in the later stage of experiment(P＜0.05),while lipase and trypsin specific activity were extremely improved(P＜0.01).Trypsin specific activity of Group C and D was extremely enhanced,but the change of amylase and lipase were not as well as trypsin,especially the difference of amylase was not significant in every stage of Group D and A(P＞0.05).In this study,Group B showed the best performance to improve the activity of intestinal digestive enzymes.In the fifth experiment,Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis(PCR-DGGE) method was used to investigate the effect of different preparation of probiotics on intestinal tract flora construction and diversity of Cyprinus carpio. The results indicated that Group B,C and D can improve the intestinal tract flora diversity. Flora diversity and belts of dominance strains of Group B were significantly increased compared with Group A,and the belts added with nine at 42 days,meanwhile,Group C showed less efficiency than Group B,and the belts increased with four at 42 days.The effect of Group D was not significant.In the sixth experiment,the effect of different probiotics preparations on amino acid content,muscle quality and growth performance was studied.The results showed that growth performance index of Group B,C and D were higher than control group.The fullness, absolute growth rate,relative growth rate,transient growth rate and growth specific speed of Group B reached the peak at every stage.Absolute growth rate and transient growth rate of Group B and C were significantly improved(P＜0.05).Compared with the control group, muscle crude fat content and dehydration rate of treatment groups fed with probiotics were decreased significantly(P＜0.01),and muscle crude protein was increased significantly(P＜0.05).The results indicated that three strains of probiotics in this study can promote the growth performance,and improve the muscle quality,and Bacillus subtilis has the best effect.In conclusion,probiotics are distributed widely in intestinal tracts of Cyprinus carpio and breeding water.Bacillus subtilis strain Z2 and Enterococci strain L2 screened by common and selecting cultivation method,heat-treatment,and inhibitory experiment in vitro showed strong inhibition activity to enteropathogenic E.coli and Aeromonas.For the first time,the method of immobilizing Cyprinus carpio foregut mucus albumen in vitro,combined with labeling bacteria with isotope ³²P was used to study chemical composition of the lectin receptors on bacterial surface,and ability of pathogenic bacteria to adhere to mucus.Further, the mechanism of bacteriostasis of prebiotics was discussed.Three different prebiotics combination groups enhanced immune function and digestive enzymes activity,increased intestinal tract flora diversity,and improved muscle quality and growth performance of Cyprinus carpio.Bacillus subtilis strain Z2 has the potential to utilize as prebiotics in breeding Cyprinus carpio and other fish.