| Monascus spp.are filamentous fungi,which have been used as fermentation strains of traditional food red mould rice(red yeast rice)for thousands of years in Asia.Monascus can produce various secondary metabolites,including pigments,antibacterial substances,monacolin K,ergosterol and a variety of digestive enzymes,and show a wide range of biological functions to mammals and poultry.However,there is no research report on Monascus spp.as probiotics of aquatic animals.In this study,Monascus purpureus M-32 was isolated and identified from Fujian red yeast rice,and its functionl genes were predicted by genome sequencing analysis,and then the prebiotic function was predicted.Then Monascus purpureus M-32 powder and fermented functional soybean meal were prepared and added to the feed in different proportions to study their effects on the growth and health of L.vannamei.The main research results are as follows:(1)Four filamentous fungi producing red pigment were isolated from Fujian red mould rice.Among them,strain M-32 had the highest ergosterol and pigment contents,reaching 805.09 μ/g and 2684.98 μ/g,respectively.According to ITS r DNA sequence alignment,the strain was identified as Monascus purpureus and deposited in the China General Microbiological Culture Collection Cente(Strain No.CGMCC No.19377).The draft genome of M.purpureus M-32 was completed by genome sequencing technology.Its genome size was estimated to be about 26 Mb and it is predicted that there were 8035 coding genes and 144 t RNA genes.The results of amino acid sequence annotation revealed 93 glycoside hydrolases,51 glycosyl transferases,45 auxiliary activities and 44 carbohydrate esterases,which indicated that strain M-32 has strong carbohydrate metabolism.A total of 19 secondary metabolite gene clusters were predicted in M-32 genome,mainly including type I polyketide synthase(T1PKS)and non ribosomal polypeptide synthetase(NRPS),with a small amount of terpenoids and betalactone biosynthetic gene cluster.These gene clusters can make M.purpureus M-32 have antibacterial and antioxidant abilities.(2)M.purpurea M-32 powder was prepared and fed to L.vannamei with different dosage(T0: 0 g/kg,T1: 5 g/kg,T2: 10 g/kg,T3: 20 g/kg)added to the basic diet.The results showed that after 8 weeks,compared with the control group(T0),the feed conversion rate(FCR)of T1 and T2 groups decreased significantly(P<0.05),and the survival rate(SR)of T3 group increased significantly(P<0.05);The activities of trypsin,lipase and amylase in hepatopancreas of experimental group had no significant changes(P>0.05);The addition of M.purpureus M-32 powder to the feed significantly increased the activity of glutathione peroxidase(GSH-Px)and decreased the content of malondialdehyde(MDA)in hepatopancreas(P<0.05);Feeding M.purpureus M-32 powder significantly increased the activities of alkaline phosphatase(AKP)and lysozyme(LSZ)in serum(P<0.05),and significantly improved the intestinal morphology including villus height,villus width and muscle thickness;The intestinal microflora composition of T1,T2 and T3 groups was significantly different from that of the control group.The addition of M.purpureus M-32 powder to the diet could significantly affect the intestinal microflora composition of L.vannamei,and significantly increase the relative abundance of Lactobacillus;Addition of M.purpureus M-32 powder could enhance the resistance of shrimp to V.parahaemolyticus infection.(3)M.purpurea M-32 was used to prepare fermented soybean meal.The results showed that after the soybean meal was fermented by M.purpureus M-32,the crude protein content increased from 46.03% to 51.60%,the acid soluble protein content increased from 5.36% to 19.16%,and the proportion of macromolecular protein decreased;The content of ergosterol in fermented soybean meal is 149.15 μg/g.The soybean meal fermented by M.purpureus M-32 was used to replace 20%(S20),40%(S40)and 60%(S60)of the soybean meal in the basic feed,respectively,and was fed to L.vannamei for 42 days.The results showed that the weight gain rate(WGR)and specific growth rate(SGR)of L.vannamei in the three Monascus fermented soybean meal groups were significantly higher than those in the control group(P<0.05),the feed conversion rate(FCR)was significantly lower than that in the control group(P<0.05),and the survival rate(SR)of L.vannamei in the S60 group was significantly higher than that in the control group(P<0.05);the content of malondialdehyde(MDA)in hepatopancreas of the three Monascus fermented soybean meal groups was significantly lower than that of the control group(P<0.05),the activity of superoxide dismutase(SOD)was significantly higher than that of the control group(P<0.05),and the activity of lysozyme(LZM)in serum and the phosphorus content in muscle of L.vannamei in S60 group were significantly higher than that of the control group,S20 and S40 groups(P<0.05);the content of calcium in muscle of L.vannamei in S40 and S60 groups was significantly higher than that in control group and S20 group(P<0.05);feeding Monascus fermented soybean meal could significantly reduce the cumulative mortality of L.vannamei after challenge by V.parahaemolyticus(P<0.05)and significantly improved the intestinal morphology;feeding Monascus fermented soybean meal could affect the intestinal microflora composition of L.vannamei.The Sob,Chao and ACE indexes of S60 group were significantly lower than those of the control group(P<0.05),and the abundance of Actinobacteria and Verrucomicrobia were significantly increased,while Proteobacteria and Firmicutes were decreased;in the group of Monascus fermented soybean meal,Demequina,Vibrio,Pseudoruegeria,Hoppeia and Haloferula increased,while Ruegeria,Shewanella,Spongiimonas,Lactobacillus and Rhodococcus decreased significantly.The results of intestinal metabolomic analysis showed that,compared with the control group,there were 54 metabolites up-regulated and 85 metabolites down-regulated in S60 group,the contents of leukotriene C5 and prostaglandin A1(PGA1)increased significantly,while the contents of taurochenodeoxycholic acid(TCDCA),carnosine and itaconic acid decreased significantly.(4)The soybean meal fermented by M.purpureus M-32 was used to replace 20%(F20),40%(F40)and 60%(F60)fish meal in the basic feed respectively,and then fed to L.vannamei for 42 days.The results showed that the WGR,FCR,SGR and SR of L.vannamei had no significant difference compared with the control group when the fish meal replacement ratio was 20% and40%(P>0.05).When the substitution ratio was 60%,FCR increased significantly(P<0.05);The MDA content of hepatopancreas of L.vannamei in the Monascus fermented soybean meal group was significantly lower than that in the control group(P<0.05).The LZM and PO activities in serum,calcium content in muscle and phosphorus content in blood of L.vannamei in F60 group were significantly higher than those in the control group(P<0.05);the fish meal replaced by Monascus fermented soybean meal could significantly reduce the cumulative mortality after challenge by V.parahaemolyticus(P<0.05);replacing fish meal with Monascus fermented soybean meal can affect the intestinal structure,when the replacement ratio was 20%,the intestinal morphology and structure were improved,and when the replacement ratio were 40% and 60%,the intestinal structure was damaged;replacing fish meal with Monascus fermented soybean meal could affect the composition of intestinal flora.The Sob,Chao and ACE indexes of the Monascus fermented soybean meal group were significantly lower than those of the control group(P<0.05).At the level of phylum,the abundance of Proteobacteria and Firmicutes decreased,and the abundance of Actinobacteria and Bacteroides increased significantly.At the genus level,the abundance of Demequina,Vibrio,Pseudouegeria and Shimia in F60 group increased,while the abundance of Ruegeria,Shewanella,Spongiimonas,Lactobacillus and Rhodococcus decreased.The results of intestinal metabolomic analysis showed that there were 49 metabolites up-regulated and 140 metabolites down-regulated in F60 group.The contents of leukotriene C5,prostaglandin A1,and daidzein in F60 group were significantly higher than those in the control group,while the contents of taurinechenodeoxycholic acid,carnosine,and itaconic acid in F60 group were significantly lower than those in the control groupTo sum up,a strain of M.purpureus M-32 with high ergosterol and pigments production was isolated from Fujian red yeast rice in this study.Genomic analysis showed that the strain contained a large number of carbohydrates metabolizing enzyme genes and secondary metabolite gene clusters,which had the potential to develop into aquatic probiotic.Animal experiments showed that the addition of M.purpureus M-32 powder to the basic diet could improve the growth performance,intestinal health,intestinal flora and anti-infection ability of L.vannamei.Fermentation of soybean meal by M.purpureus M-32 can increase the content of crude protein and acid soluble protein,and increase the functional components of ergosterol and monascus pigments.The functional feed prepared by adding M.purpureus M-32 fermented soybean meal to replace soybean meal or fish meal in the basic feed could improve the growth performance,intestinal flora and intestinal metabolism of L.vannamei. |
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