Supplementation Of Enzyme-treated Soy Protein To Save Dietary Protein Of Juvenile Jian Carp(Cyprinus Carpio Var. Jian) And The Mechanism Studies

Through animal trail,firstly,we studied the effect of adding enzyme-treated soy protein(ETSP)on growth performance,nutrient deposition,nitrogen and phosphorus excretion of juvenile Jian carp,to investigate whether the ETSP has the effect of saving dietary protein and the optimal level.Further,we studied the effect of ETSP on the digestion and absorption ability,intestinal antioxidant ability and immunity of juvenile Jian carp to investigate the mechanism.Finally,we studied the effective substances in ETSP through separation,purification,structural identification and cell verification test.The main contents and results are as follows:1 Effects of ETSP on saving dietary protein of juvenile Jian carp and the possible mechanisms1.1 Effects of ETSP on growth performance,nutrients deposition,N and P excretion of juvenile Jian carpA total of 1400 juvenile Jian carp(11.96±0.05 g)were randomly distributed into seven groups,feeding diet 1(34%CP),diet 2(32%CP),and diet 3 to 7 with graded levels of ETSP(1.0,1.5,2.0,2.5 and 3.0%)for 56 d.Based on the growth trail,we also conducted the digestibility test at the same time.The results indicated that after reducing2%of dietary protein(from 34%to 32%),the FBW,PWG,SGR and FI significantly decreased(P<0.05).The deposition rate of fat significantly reduced,while the deposition rate of ash,calcium and phosphorus significantly increased(P<0.05).The nitrogen excretion significantly reduced(P<0.05).After adding 1%of ETSP to the 32%CP diet,the FBW,PWG,SGR and FI significantly increased(P<0.05),having no significant difference with 34%CP diet(P>0.05).The deposition rate of protein,ash,calcium and phosphorus were much higher than 34%CP diet(P<0.05).The nitrogen excretion significantly reduced and was lower than 34%CP diet(P<0.05).These results suggested that adding 1%of ETSP obtained similar growth performance as the 34%CP diet,which only provided 0.46%crude protein to save 2%of dietary crude protein.1.2 Effects of ETSP on the digestive and absorptive ability of juvenile Jian carp and the mechanismsThis study had the same test design as the first trail.We studied effects of adding ETSP on intestinal folds height,the growth and development of hepatopancreas and intestine,activities of digestive and absorptive enzymes and correspond gene expression,to further reveal the mechanism of ETSP to save fish dietary protein.The results indicated that after reducing 2%of dietary protein(from 34%to 32%),IL,ISI and IW significantly decreased(P<0.05).The activities of intestinal lipase and amylase,chymotrypsin in hepatopancreas significantly decreased(P<0.05).The activities of CK in PI,MI DI,and Na⁺-K⁺-ATPase in DI significantly decreased(P<0.05).The relative expression of Na⁺-K⁺-ATPase(?1a)in MI significantly increased(P<0.05),and relative expression of trypsin 2 in hepatopancreas significantly reduced(P<0.05).After adding 1.0-1.5%of ETSP to the 32%CP diet,HW,HSI,HPC,IL,IW,ISI and IPC significantly increased(P<0.05).Moreover,HW,HSI,HPC and IL were significantly higher than 34%CP diet(P<0.05).Folds height of MI and DI significantly increased(P<0.05).The activities of digestive enzymes in hepatopancreas and intestine significantly increased(P<0.05).Further,the activities of trypsin,chymotrypsin and lipase in hepatopancreas and intestine were much higher than 34%CP diet(P<0.05).Except for the activities of Na⁺-K⁺-ATPase,?-GT and AKP in DI,the activities of other absorptive enzymes were significantly higher than 34%CP diet(P<0.05).The relative expressions of chymotrypsin,trypsin 2,lipase and amylase in hepatopancreas significantly increased(P<0.05).Further,the relative expressions of lipase and amylase were much higher than 34%CP diet(P<0.05).The relative expressions of Na⁺-K⁺-ATPase(?1a)in MI and DI significantly increased(P<0.05),which were also higher than 34%CP diet(P<0.05).The relative expressions of?-GT and AKP in MI,?-GT in DI significantly increased(P<0.05),while the relative expressions of AKP in DI significantly decreased(P<0.05).The relative expressions of CK1 in MI and DI,CK2 in MI significantly increased(P<0.05),which were also higher than 34%CP diet(P<0.05).The relative expressions of TOR and 4E-BP2 in PI,MI and DI significantly decreased(P<0.05).1.3 Effects of ETSP on immunity and antioxidant ability in intestine of juvenile Jian carp and the mechanismsThis study had the same test design as the first trail.We studied effects of adding ETSP on intestinal antioxidant ability and immunity to explore the effect of ETSP on intestinal health of juvenile Jian carp and the potential pathway,which can further reveal the mechanism of ETSP to save fish dietary protein.The results indicated that after reducing 2%of dietary protein(from 34%to 32%),all the immune indicators in intestine had no significant change(P>0.05).The relative expressions of IL-1?2-1 and TNF-?significantly increased(P<0.05),while the relative expressions of anti-inflammatory factors such as TGF-?2 and IL-10 had no significant change(P>0.05).PC content significantly increased(P<0.05).The SOD activity significantly decreased and GSH content significantly increased(P<0.05).The relative expressions of MnSOD,CuSOD,CAT,GR,GPx1a and Nrf2 had a downward trend,while the relative expression of Keap1a had an upward trend(P>0.05).After adding 1.5%of ETSP to the 32%CP diet,lysozyme activity and IgM content significantly increased which were also higher than 34%CP diet(P<0.05).The relative expression of TGF-?2 significantly increased(P<0.05).The relative expressions of IL-1?2-1,IL-1?2-2 and TNF-?significantly decreased(P<0.05).MDA and PC content were lower than 34%CP group(P<0.05).T-AOC and AHR were higher than 34%CP group(P<0.05).The SOD,CAT and GST activities were higher than 34%CP group(P<0.05).The relative expressions of SOD,CAT,GR,GPx1a and Nrf2 significantly increased,while the relative expression of Keap1a significantly decreased(P<0.05).2 Structural identification of antioxidant peptide in ETSPThis study determined the amino acid structure of bioactive peptide with antioxidant property in ETSP by means of separation,purification and structural identification.The results indicated that the molecular weight of water-soluble portion in ETSP mainly concentrated in 100¹000 Da,which was accounted for 80%.And then,we analyzed antioxidant activities of five molecular weight segments(<1 kDa,1-3 kDa,3-5 kDa,5-10kDa and>10 kDa)from the water-soluble portion of ETSP.The results indicated that ABTS⁺·clearance ability and FRAP reduction ability increased with the reduction of molecular weight.These two indicators were most active when the molecular weight was less than 1000 Da.Iron chelation ability had no correlation with molecular weight,but it was most active when the molecular weight was less than 1000 Da.Further,we obtained13 components from MW<1000 Da components.Among them,the ABTS⁺·clearance ability of six components were strong,which almost reached 50%or more of GSH.Finally,we determined that the amino acid sequences of two peptides,which could be found in soy protein.There were two possibilities for the component 5,which was 82.4% for Glu-Tyr and 17.6%for Tyr-Glu.The component 9 was Ala-Phe.3 Test effects of Glu-Tyr and Ala-Phe on antioxidant ability in intestine cells of Jian carp and the possible mechanisms3.1 Effects of Glu-Tyr and Ala-Phe on oxidation damage in intestine cells of Jian carpThrough cell tests,we investigated different levels of Glu-Tyr and Ala-Phe on the MDA and PC content in cell and LDH activity in culture liquid with 4 duplications per group,to study whether these two antioxidant peptides affected the oxidation damage in intestine cells of Jian carp and the optimum level.The results indicated that adding Glu-Tyr significantly decreased MDA and PC content(P<0.05),and the data was minimum when the level reached 320 mg/L.LDH activity in culture liquid also significantly decreased with graded levels of Glu-Tyr(P<0.05),and the data remained stable when the level reached 320 mg/L.Adding Ala-Phe also significantly decreased MDA and PC content(P<0.05),and the data were minimum when the level reached 240 mg/L.LDH activity in culture liquid had no significant change with the graded levels of Ala-Phe(P>0.05).3.2 Protect effects of Glu-Tyr and Ala-Phe on the oxidation damage induced by H2O2in intestine cell of Jian carp and possible mechanismsThrough two cell experiments to investigate the protect effect and the mechanism of Glu-Tyr and Ala-Phe on the oxidation damage induced by H₂O₂ in intestine cell of Jian carp.The results showed that MDA and PC content significantly increased,while CAT and GPx activities significantly decreased with H₂O₂ stress(P<0.05).When adding Glu-Tyr before H₂O₂ stress,MDA and PC content decreased,and CAT and GPx activities increased(P<0.05).Data of relative gene expression indicated that when adding Glu-Tyr before H₂O₂ stress,Nrf2 significantly increased(P<0.05),Keap1a significantly decreased(P<0.05),and MnSOD,GPx1a/4a significantly increased(P<0.05).When H₂O₂ stress after adding Glu-Tyr,compared to Glu-Tyr group,Nrf2 had tendency to decrease(P>0.05),Keap1a significantly increased(P<0.05),and MnSOD,GPx1a,GPx4a had a downward trend(P>0.05).When treated with ML385 and then adding Glu-Tyr before H₂O₂ stress,compared to control group,Nrf2 had tendency to increase(P>0.05),Keap1a significantly decreased(P<0.05),and MnSOD,GPx1a/4a had tendency to increased(P>0.05).When adding Ala-Phe before H₂O₂ stress,MDA content significantly decreased(P<0.05),and CAT,GPx,T-SOD activities significantly increased(P<0.05).Data of relative gene expression indicated that when adding Ala-Phe before H₂O₂ stress,Nrf2 significantly increased(P<0.05),Keap1a had a downward trend(P>0.05),GPx1a and GPx4a significantly increased(P<0.05).When H₂O₂ stress after adding Ala-Phe,compared to Ala-Phe group,Nrf2 had a downward trend(P>0.05),Keap1a had an upward trend(P>0.05),and MnSOD significantly decreased(P<0.05).When treated with ML385 and then adding Ala-Phe before H₂O₂ stress,compared to control group,Nrf2 significantly increased(P<0.05),GPx1a and GPx4a had an upward trend(P>0.05).3.3 Repair effects of Glu-Tyr and Ala-Phe on the oxidation damage induced by H2O2in intestine cell of Jian carp and possible mechanismsThe results showed that compared to H₂O₂ group,MDA and PC content significantly decreased when adding Glu-Tyr after H₂O₂ stress,while CAT and GPx activities significantly increased(P<0.05).Data of relative gene expression indicated that compared to H₂O₂ group,Nrf2 significantly increased by adding Glu-Tyr after H₂O₂ stress(P<0.05),Keap1b significantly decreased(P<0.05),MnSOD and GPx1a significantly increased(P<0.05).When treated with ML385 and then adding Glu-Tyr after H₂O₂ stress,compared to control group,Nrf2 had a downward trend(P>0.05),Keap1b and MnSOD had an upward trend(P>0.05),GPx1a significantly decreased(P<0.05).When adding Ala-Phe after H₂O₂ stress,compared to H₂O₂ group,MDA and PC content significantly decreased,while CAT,GPx,T-SOD activities significantly increased(P<0.05).Data of relative gene expression indicated that when adding Ala-Phe after H₂O₂ stress,Nrf2 had an upward trend(P>0.05),Keap1a had a downward trend(P>0.05),Keap1b significantly decreased(P<0.05),MnSOD and GPx1a significantly increased(P<0.05).When treated with ML385,H₂O₂ stress,and then adding Ala-Phe,compared to the control group,Keap1a and Keap1b had an upward trend(P>0.05),Nrf2 significantly decreased(P<0.05),MnSOD significantly decreased(P<0.05),GPx1a had a downward trend(P>0.05).3.4 Intercept effects of Glu-Tyr and Ala-Phe on the oxidation damage induced by H2O2 in intestine cell of Jian carp and possible mechanismsThe results showed that compared to H₂O₂ group,MDA and PC content significantly decreased(P<0.05),and CAT,GPx,T-SOD activity had an upward trend when adding Glu-Tyr and H₂O₂ at the same time(P>0.05).Data of relative gene expression indicated that when adding Glu-Tyr and H₂O₂ at the same time,compared to H₂O₂ group,Nrf2significantly increased(P<0.05),Keap1b significantly decreased(P<0.05),MnSOD and GPx1a significantly increased(P<0.05).When treated with ML385 and then adding Glu-Tyr and H₂O₂ at the same time,compared to control group,Nrf2 had a downward trend(P>0.05),Keap1a and Keap1b had an upward trend(P>0.05),MnSOD and GPx1a had a downward trend(P>0.05).When adding Ala-Phe and H₂O₂ at the same time,compared to the H₂O₂ group,MDA and PC content,CAT,GPx,T-SOD activity had no significant change(P>0.05).Data of relative gene expression indicated that when adding Ala-Phe and H₂O₂ at the same time,compared to H₂O₂ group,Nrf2,Keap1a/1b,CuZn/MnSOD and GPx1a/4a had no significant change(P>0.05).When treated with ML385 and then adding Ala-Phe and H₂O₂ at the same time,compared to the control group,Keap1a significantly increased(P<0.05),Nrf2 significantly decreased(P<0.05),MnSOD and GPx1a significantly decreased(P<0.05).Collectively:(1)Reduction 2%of dietary protein(from 34%CP to 32%CP)reduced the growth performance of Jian carp,which may be related to the reduction of digestion and absorption capacity,intestinal immunity and antioxidant capacity.(2)Adding 1.0%ETSP(only providing 0.46%CP)to the diet with 32%CP obtained similar performance as the 34%CP group.The saving effect of fish dietary protein by ETSP may be related to the improvement of fish digestive and absorptive ability and intestinal health through enhancing immunity and antioxidant capacity.(3)The ability of scavenging oxygen radical was strongest in section of MW<1000 Da.From this section,we obtained two peptides with strong antioxidant activity and the structure were Glu-Tyr and Ala-Phe.(4)Glu-Tyr reduced oxidation damage in intestine cell of Jian carp through protect,repair and intercept effect,while Ala-Phe only had protect and repair effect.Glu-Tyr and Ala-Phe could protect and repair the oxidation damage of intestine cell by activating Keap1/Nrf2 signaling pathway to increase activities of antioxidant enzymes.