Effect Of Enzyme-treated Soy Protein On Growth Performance And Flesh Quality As Well As The Related Mechanisms In On-growing Grass Carp (Ctenopharyngodon Idella)

This growth experiment firstly investigated the effects of enzyme-treated soy protein on growth performance and flesh quality as well as the related mechanisms in on-growing grass carp(Ctenopharyngodon idella).Then,the growth experiment secondly investigated the influence of enzyme-treated soy protein on muscle antioxidant enzyme activities and gene expressions and Nrf2 signaling pathway-related genes and protein expressions;meanwhile,in vitro experiments,we studied the effects of enzyme-treated soy protein effectors on antioxidant ability of grass carp myotubes and the related mechanism;Thirdly,the growth experiment also investigated the influence of enzyme-treated soy protein on myofiber histological characteristics,development-related genes and protein expressions,ERK1/2 signaling pathway-related genes and protein expressions,TOR and Fox O1 signaling pathway-related genes and protein expressions;meanwhile,in vitro experiments,we investigated the effects of enzyme-treated soy protein effectors on the proliferation,differentiation and protein synthesis of grass carp myoblasts as well as the related mechanism,which explored the effect of enzyme-treated soy protein on myofiber development and the mechanism.The main contents and results are presented as fellow:1.The effect of enzyme-treated soy protein(ETSP)on growth performance and flesh quality in on-growing grass carpA total of 540 grass carp(325.72 ± 0.60 g)were randomly distributed into six groups of each three replicates.The fish were fed six diets,diet 1 was formulated to contain 28% CP,and set as the positive control group.Low protein diets were formulated to contain 26% CP.ETSP was added to the low protein diets to provide graded concentrations of 0.0%(diet2),0.8%(diet 3),1.2%(diet 4),1.6%(diet 5)and 2.0 %(diet 6)ETSP diets,respectively.This study was conducted to evaluate the effect of ETSP on growth performance and flesh quality in on-growing grass carp.Results indicated that:(1)reducing dietary protein level by 2%(dietary protein level from 28 to 26%)significantly decreased FBW,PWG,SGR and FI(P < 0.05).Meanwhile,we observed that the reduction of 2% dietary protein significantly increased muscle moisture content as well as saturated fatty acid palmitic acid(C16: 0)and stearic acid(C18: 0)contents(P < 0.05),and significantly decreased muscle protein,linolenic acid(C18:3n-3)and docosahexaenoic acid(DHA,c22:6 n-3)contents as well as the ratio of total unsaturated fatty acids and saturated fatty acids contents,umami amino acids(aspartic acid and glutamic acid)contents,5'-inosinic acid(IMP)content and aromatic amino acid histidine content(P < 0.05).There was no significant difference in flesh cooking loss,shear force,p H and aroma precursor amino acid methionine content(P > 0.05).These above data suggested that compared with 28% CP group,low protein diet(26% CP,un-supplemented ETSP)decreased growth performance,and deteriorated flesh quality(nutritive value,healthiness and flavor)in on-growing grass carp.(2)When ETSP supplementation levels are 0.8-1.2%,FBW,PWG,SGR,FI and FE significantly increased(P < 0.05);muscle moisture,C16: 0 and C18: 0 contents,cooking loss and shear force significantly reduced(P < 0.05);muscle protein,oleic acid(C18: 1),C18: 3n-3,DHA,aspartic acid,glutamic acid,histidine,methionine and IMP contents and p H as well as the ratio of total unsaturated fatty acids and saturated fatty acids increased.These above data suggested that compared with the positive control group,0.8-1.2% ETSP supplementation in low protein diets improved growth performance and flesh quality in on-growing grass carp.2.The effect of ETSP on muscle antioxidant capacity and the mechanism in on-growing grass carp2.1 Effect of ETSP on muscle antioxidant capacity and possible mechanism in on-growing grass carpThis study investigated the effect of ETSP on muscle antioxidant capacity and possible mechanism in on-growing grass carp.Results showed that:(1)reducing dietary protein levels from 28% to 26% significantly decreased muscle malondialdehyde(MDA)and protein carbonyl(PC)contents(P < 0.05);significantly reduced muscle GSH content and antioxidant enzymes(Cu/Zn-SOD,Mn SOD,CAT,GPx,GST,and GR)activities(P < 0.05).Those data suggested that the reduction of dietary protein levels by 2% decreased muscle antioxidant capacity in on-growing grass carp.(2)when the low protein diets were supplemented 0.8-1.2% ETSP,muscle MDA and PC contents significantly reduced(P < 0.05),when the low protein diets were supplemented 0.8-1.6% ETSP,GSH content and all the antioxidant enzymes activities increased.Those above results indicate that supplemented 0.8-1.6% ETSP diets may increase muscle antioxidant ability relating to increasing GSH content and antioxidant enzymes activities in growing grass carp.(3)when the low protein diets were supplemented 0.8-1.6% ETSP,the mRNA levels of antioxidant enzymes and GCLC and GCLM as well as the mRNA and protein expression levels of Nrf2 in muscle significantly up-regulated(P <0.05),and the mRNA level of Keap1 b in the muscle significantly down-regulated(P < 0.05).Those above data indicated that ETSP may promote the nuclear translocation of Nrf2 relating to down-regulate Keap1 b mRNA level,thereby up-regulating the mRNA levels of antioxidant enzymes,and finally increasing the antioxidant enzyme activities in the muscle of on-growing grass carp.2.2 Effects of ETSP effectors(Glu-Tyr and Ala-Phe dipeptide)on antioxidant ability and related mechanism in the myotubes of grass carpIn this study,we used the trial in vitro to further investigate the effect of ETSP effectors(Glu-Ty and Ala-Phe dipeptides)on antioxidant capacity of myotubes and related mechanism in grass carp.Four treatments were designed in this experiment,each with six replicates: the control group(0 mg/L dipeptide),dipeptide group,Nrf2 inhibitor group,and dipeptide+Nrf2 inhibitor group.Results showed that:(1)Compared with the control group,Glu-Tyr and Ala-Phe significantly decreased MDA and PC contents(P < 0.05),and significantly increased most of antioxidant enzymes activities in the myotubes of grass carp(P < 0.05).Those above results indicated that Glu-Tyr and Ala-Phe could decrease myotubes oxidative damage and increase antioxidant enzyme activities,thereby improving myotubes antioxidant capacity in grass carp.(2)Compared with the control group,Glu-Tyr and Ala-Phe significantly increased myotubes Nrf2 protein expression(P < 0.05),indicating that Glu-Tyr and Ala-Phe may enhance myotubes antioxidant capacity relating to activate Nrf2 signaling pathway in grass carp.Compared with the Glu-Tyr and Ala-Phe treatment groups,Nrf2 inhibitor(ML385)+Glu-Tyr and Nrf2 inhibitor(ML385)+Ala-Phe groups significantly down-regulated myotubes Nrf2 protein expression(P < 0.05),significantly increased myotubes MDA and PC contents(P < 0.05),significantly down-regulated myotubes antioxidant enzymes activities(P < 0.05).Those above results indicated that Glu-Tyr and Ala-Phe could enhance myotubes antioxidant capacity through activating Nrf2 signaling pathway in grass carp.3.Effect of ETSP on myofiber development and the mechanism in on-growing grass carp3.1 Effect of ETSP on myofiber development and possible mechanism in on-growing grass carp3.1.1 Effect of ETSP on myofiber histological characteristics in on-growing grass carpThis study had the same test design as the first trail.We studied the effect of ETSP on myofiber histological characteristics in on-growing grass carp.Results showed that reducing dietary protein levels from 28% to 26% significantly decreased muscle fiber diameter and significantly increased muscle fiber density(P < 0.05),indicating that low protein diet inhibited myofiber development.Moreover,muscle fiber diameter in 0.8-1.2% ETSP supplementation diets significantly increased(P < 0.05);muscle fiber density in 0.8-1.2% ETSP supplementation diets significantly decreased,indicating that supplemented 0.8-1.2% ETSP diets promoted myofiber development in on-growing grass carp.3.1.2 Effect of ETSP on myoblast proliferation and differentiation as well as possible mechanism in on-growing grass carpThis study investigated the effect of ETSP on myoblast proliferation and differentiation as well as possible mechanism in on-growing grass carp.Results showed that:(1)when the low protein diets were supplemented with 0.8-1.2% ETSP,the mRNA levels of myoblast proliferation-related genes cyclin B,cyclin D,cyclin E,E2F4 and PCNA significantly increased,suggesting that appropriate ETSP supplementation in low protein diet could promote myoblast proliferation in on-growing grass carp.(2)when the low protein diets were supplemented with 0.8-1.2% ETSP,the mRNA levels of myoblast differentiation-related genes My OD,Mrf5,My OG,MRF4 and My HC mRNA levels and the protein levels of My OD,My OG and My HC significantly up-regulated(P < 0.05),indicating that appropriate ETSP supplementation in low protein diet could promote myoblast differentiation in on-growing grass carp.(3)when the low protein diets were supplemented 0.8-1.2% ETSP,the gene expressions of ERK1 and ERK2 in the muscle significantly increased(P < 0.05),indicating that supplemented 0.8-1.2% ETSP diets may activate the ERK1/2 signaling pathway,thereby promoting myoblast proliferation and differentiation in on-growing grass carp.3.1.3 Effect of ETSP on muscle protein synthesis as well as possible mechanism in on-growing grass carpThis study investigated the effect of ETSP on muscle protein synthesis in on-growing grass carp.Results showed that when the low protein diets were supplemented 0.8-1.2% ETSP,the mRNA levels of TOR and S6K1 and the protein levels of p-TOR and T-TOR significantly increased(P < 0.05);the mRNA levels of 4E-BP1,Fox O1 a,MURF1 and MAFbx significantly reduced(P < 0.05).Those above results indicated that supplemented 0.8-1.2% ETSP diets may activate TOR signaling pathway and inhibit the Fox O1 signaling pathway,thereby promoting muscle protein synthesis and inhibit protein degradation,and ultimately increasing muscle protein content in on-growing grass carp.3.2 Effect of ETSP effectors(Glu-Tyr and Ala-Phe dipeptides)on myoblast proliferation,differentiation and protein synthesis as well as related mechanisms in grass carp3.2.1 Effect of ETSP effectors(Glu-Ty and Ala-Phe dipeptides)on myoblast proliferation and the mechanism in grass carpIn this study,we used the trial in vitro to further investigate the effect of ETSP effectors(Glu-Ty and Ala-Phe dipeptides)on myoblast proliferation and related mechanism grass carp.Four treatments were designed in this experiment,each with six replicates: the control group(0 mg/L dipeptide),dipeptide group,ERK1/2 inhibitor group,and dipeptide+ERK1/2 inhibitor group.Results showed that:(1)Glu-Tyr and Ala-Phe treatment could promote myoblast proliferation of grass carp,and myoblast in the groups of Glu-Tyr and Ala-Phe treatment for 48 h had the optimal proliferative potential.(2)Compared with the control group,Glu-Tyr or Ala-Phe treatment for 48 h significantly increased cyclin B?cyclin D?cyclin E and E2F4 as well as ERK1 and ERK2 mRNA levels(P < 0.05),indicating that Glu-Tyr or Ala-Phe may up-regulate the gene expressions of cyclins and transcription factor E2F4 through activating ERK1/2 signaling pathway,thereby promoting myoblast proliferation in grass carp.Compared with the Glu-Tyr treatment group,OD value and the mRNA levels of ERK1 and cell cycle regulatory proteins significantly decreased in the ERK1/2 inhibitor(U0126)+Glu-Tyr treatment group;the mRNA level of ERK2 significantly decreased(P < 0.05),and had no significant change compared with the U0126 group(P > 0.05).Those above data indicated that Glu-Tyr up-regulated gene expressions of cyclins and transcription factor E2F4 through activating ERK1 signaling pathway(but not ERK2 signaling pathway),thereby promoting myoblasts proliferation in grass carp.Meanwhie,compared with the Ala-Phe treatment group,OD value and the mRNA levels of ERK1,ERK2,cyclin B,cyclin E and E2F4 significantly decreased in the ERK1/2 inhibitor(U0126)+Ala-Phe treatment group,which were significantly higher than those in the U0126 group(P < 0.05).Those results suggested that Ala-Phe up-regulated the gene expressions of cyclins and transcription factor E2F4 through activating ERK1 and ERK2 signaling pathway,thereby promoting myoblasts proliferation in grass carp.3.2.2 Effect of ETSP effectors(Glu-Ty and Ala-Phe dipeptides)on myoblast differentiation and the mechanism in grass carpIn this study,we used the trial in vitro to further investigate the effect of ETSP effectors(Glu-Ty and Ala-Phe dipeptides)on myoblast differentiation and related mechanism in grass carp.We used the same test design as the trail 3.2.1.The results show that:(1)Compared with the control group,Glu-Tyr significantly increased the expressions of myogenic regulatory factors(My OD,Myf5,My OG and MRF4 gene expressions as well as My OD and My OG protein expressions)and the gene expressions of My HC and ERK1(P < 0.05),suggesting that Glu-Tyr could promote myoblasts differentiation relating to activate ERK1 signaling pathway in grass carp.Meanwhile,Ala-Phe significantly increased the gene expressions of myogenic regulatory factors,My HC,ERK1 and ERK2(P < 0.05),indicating that Ala-Phe could promote myoblasts differentiation relating to activate ERK1 and ERK2 signaling pathway in grass carp.(2)Compared with the Glu-Tyr treatment group,the expressions of myogenic regulatory factors and the gene expression of ERK1 significantly decreased in the ERK1/2 inhibitor(U0126)+Glu-Tyr treatment group,which were significantly higher than those in the U0126 group(P < 0.05).Those results suggested that Glu-Tyr could promote myoblasts differentiation through activating ERK1 signaling pathway in grass carp.Compared with the Ala-Phe treatment group,the expressions of myogenic regulatory factors and the gene expression of ERK1 and ERK2 significantly decreased in the ERK1/2 inhibitor(U0126)+Glu-Tyr treatment group,which were significantly higher than those in the U0126 group(P < 0.05).Those results indicated that Ala-Phe could promote myoblasts differentiation through activating ERK1 and ERK2 signaling pathway in grass carp.3.2.3 Effect of ETSP effectors(Glu-Tyr and Ala-Phe dipeptides)on myoblast protein synthesis and the mechanism in grass carpIn this study,we used the trial in vitro to further investigate the effect of ETSP effectors(Glu-Ty and Ala-Phe dipeptides)on myoblast protein synthesis and related mechanism in grass carp.Four treatments were designed in this experiment,each with six replicates: the control group(0 mg/L dipeptide),dipeptide group,TOR inhibitor group(or Fox O1 activator group),and dipeptide+TOR inhibitor group(or Fox O1 activator group).Results showed that:(1)Compared with the control group,Glu-Tyr and Ala-Phe significantly increased myoblasts protein synthesis rate in grass carp(P < 0.05),suggesting that Glu-Tyr and Ala-Phe could promote myoblasts protein synthesis in grass carp.(2)Compared with the control group,Glu-Tyr and Ala-Phe significantly increased the mRNA level of TOR and the ratio of p-TOR and T-TOR protein levels(P < 0.05).Those data suggested that Glu-Tyr and Ala-Phe could promote myoblast protein synthesis relating to activate TOR signaling pathway.Compared with the Glu-Tyr and Ala-Phe treatment groups,protein synthesis rate,the mRNA levels of TOR and S6K1 and the ratio of p-TOR and T-TOR protein levels significantly decreased in TOR inhibitor(Rapamycin)+Glu-Tyr and TOR inhibitor(Rapamycin)+Ala-Phe groups(P < 0.05).Those above results indicated that Glu-Tyr and Ala-Phe could promote myoblasts protein synthesis through activating TOR signaling pathway in grass carp.(3)Compared with the control group,Glu-Tyr and Ala-Phe significantly reduced 3-methylhistidine content in the culture medium of myoblasts(P < 0.05),indicating that both Glu-Tyr and Ala-Phe could decrease protein degradation rate in the myoblast of grass carp.(4)Compared with the control group,Glu-Tyr significantly down-regulated the gene expression of Fox O1b(P < 0.05),and Ala-Phe significantly down-regulated the gene expressions of Fox O1 a and Fox O1b(P < 0.05),indicating that Glu-Tyr may reduce myoblast protein degradation relating to inhibit Fox O1 b signaling pathway,and Ala-Phe may reduce myoblast protein degradation relating to inhibit Fox O1 a and Fox O1 b signaling pathway.Compared with the Glu-Tyr and Ala-Phe treatment groups,3-methylhistidine content in the culture medium of myoblasts significantly decreased in the Glu-Tyr+Fox O1 activator(Wortmannin)and Ala-Phe+Fox O1 activator(Wortmannin)treatment groups(P < 0.05),Glu-Tyr+Fox O1 activator(Wortmannin)significantly up-regulated the gene expression of Fox O1 b in the myoblasts(P < 0.05),and Ala-Phe+Fox O1activator(Wortmannin)significantly up-regulated the gene expressions of Fox O1 a and Fox O1 b in the myoblasts(P < 0.05).Those above results indicated that Glu-Tyr could decrease myoblast protein degradation through inhibiting Fox O1 b signaling pathway,and Ala-Phe could decrease myoblast protein degradation through inhibiting Fox O1 a and Fox O1 b signaling pathway in grass carp.Collectively:(1)Reducing dietary protein levels by 2% on the base of 28%CP diet decreased growth performance and flesh quality in on-growing grass carp;0.8-1.2% ETSP supplementation in low protein(26%)diets improve growth performance and flesh quality in on-growing grass carp.(2)ETSP-improved flesh quality was related to enhanced muscle antioxidant ability in on-growing grass carp.Glu-Tyr and Ala-Phe could enhance myotubes antioxidant ability through activating Nrf2 signaling pathway in grass carp.(3)ETSP-improved flesh quality was also related to promoted myofiber development.Ala-Phe activated ERK1 and ERK2 signaling pathway,thereby promoting the proliferation and differentiation of myoblasts in grass carp;while Glu-Tyr only activated ERK1(but not ERK2)signaling pathway,thereby promoting the proliferation and differentiation of myoblasts in grass carp.Glu-Tyr and Ala-Phe promoted myoblast protein synthesis through activating TOR signaling pathway in grass carp.Ala-Phe inhibited myoblast protein degradation through inhibiting Fox O1 a and Fox O1 b signaling pathway,while Glu-Tyr inhibited myoblast protein degradation through inhibiting Fox O1 b signaling pathway in grass carp.