Research On The Probiotic Effects Of Adding Rhodotorula Glutinis To Feed Litopenaeus Vannamei

As a kind of safe and efficient probiotics,Rhodotorula glutinis has been widely used in shrimp culture,but its probiotic mechanism remains to be improved and further studied.In this paper,Lito Litopenaeus vannamei,an important shrimp breed in China,was taken as the research object.Different doses and different forms of R.glutinis were fed to analyze its growth performance,digestive enzyme activity,intestinal flora composition,non-specific immunity and related gene expression,in order to explore the probiotic mechanism of R.glutinis on L.vannamei.Through the subculture of the selected R.glutinis,the effects of serial re-pitching on the physiological and biochemical ability and probiotics of R.glutinis were explored,and the industrial application potential of R.glutinis was explored.In order to study the effects of R.glutinis on the growth performance,anti-infection ability and intestinal microbial composition of L.vannamei,the freeze-dried powder of R.glutinis was added to the commercial feed at the concentrations of 0(CG),0.025%(LA),0.05%(MA)and 0.1%(HA),and fed L.vannamei for 40 days.The results showed that the specific growth rates of LA,MA and HA experimental groups were 4.20、3.80 and 3.63 respectively,which were significantly higher than those of the control group 3.17.There was no significant difference in culture survival rate between the experimental group and the control group.The activities of CAT and ACP in the experimental group increased with the increasing of feeding time,and in LA and HA groups,the activities of SOD decreased at first and then increased,but in the control group,it increased continuously.At the end of the experiment,the activities of the four immunoenzyme in LA group were significantly higher than those in the control group.The cumulative mortality(96h)of Vibrio harveyi immersion infection in the control group was 81.67%,which was significantly higher than that in the experimental group23.67%(LA)、25.00%(MA)and 23.67%(HA).The addition of R.glutinis changed the composition of intestinal microbiota of shrimp at the family and genus level,reduced the intestinal microbial diversity,inhibited the abundance of Pseudomonas and Flavobacterium in the intestine,and increased the abundance of Rhodobacillaceae and Mycoplasma.With the increasing of concentration,the abundance of Myxobacteria and Ruegeria decreased.The study shows that under the experimental conditions,feeding freeze-dried powder of R.glutinis can effectively improve the growth performance,immune and disease resistance of L.vannamei,and effectively inhibit the growth of some conditional pathogenic bacteria in the intestinal tract.It is suggested that 0.025% should be added to the feed.In order to study the probiotic mechanism of R.glutinis on L.vannamei,live bacteria group(LB),dead bacteria group(DB),cell-free extract group(CE)and control group(CG)were added to the commercial feed of L.vannamei for 40 days.The results showed that the specific growth rate of dead bacteria group and cell-free extract group was slightly higher than that of other groups,but there was no significant difference among the three groups.The addition of R.glutinis changed the composition of intestinal microorganisms and reduced the intestinal microbial diversity of L.vannamei at the family and genus level.Vibrio was the dominant species in the control group and living bacteria group,and Rhodobacteraceae in the dead bacteria group and cell-free extract group became the dominant species.During the whole experiment,there was no significant difference in the relative expression of IMD among the three groups.The relative expression of crustin in dead bacteria group and cellfree extract group was significantly higher than that in live bacteria group and control group.The relative expression of penaeidin-3a in dead bacteria group was significantly higher than that in live bacteria group and control group.The relative expression of LZM in dead bacteria group,live bacteria group and cell-free extract group was significantly down-regulated.The results show that under the experimental conditions,the feeding method of dead bacteria group and cell-free extract group can exert the probiotic ability of R.glutinis better,and the probiotic ability of R.glutinis may be through some active substances in the cell,not the activity of the yeast itself.In order to explore the effect of serial re-pitching on the physiological and biochemical ability and probiotics of R.glutinis,to explore the industrial application potential,and to analyze the changes of physiological and biochemical properties of R.glutinis.The fragments of R.glutinis from different serial re-pitchings were added to the commercial feed of L.vannamei for 40 days.The results showed that the best culture time of R.glutinis was 40 h.The cell activity of the 11 th and 21 st generations decreased by 26% and 47% respectively after 48 h culture,and the flocculation ability increased by 38.90% and 89.49% respectively after 18 h culture.serial re-pitching had no significant effect on the contents of amino acids and fatty acids in R.glutinis.The morphological changes of the 5th and 10 th generations of R.glutinis were small,but there were slight wrinkles on the surface,and then with the increase of serial re-pitching,the proPOrtion of folds increased,the folds deepened step by step,and the phenomenon of yeast cell aggregation was obvious.in the experimental group fed with yeast,the specific growth rate of L.vannamei increased significantly,the total OTU number of intestinal microorganisms decreased,and the abundance of Vibrio,Ruegeria,Flavobacterium and Candidudatus Bacilloplasma decreased.Compared with the control group,the expression of IMD in the two feeding groups was up-regulated,the relative expression of penaeidin-3a and crustin was significantly up-regulated,the relative expression of proPO was down-regulated,and the relative expression of LZM was significantly downregulated.The results showed that under the experimental conditions,the serial re-pitching had no significant effect on the intracellular amino acid and fatty acid content of R.glutinis,but had a great effect on the cell morphology,which could reduce the cell activity and enhance the flocculation ability of R.glutinis.Serial re-pitching will affect the probiotic effect of active substances in R.glutinis.The comprehensive probiotic effect of the first generation of R.glutinis is better than that of the experimental group without R.glutinis.