Study On Immunomodulatory Activity Of Golden Threadfin Bream Peptides And The Relating Regulation Mechanism On Inflammation Stress

Golden threadfin bream(Nemipterus virgatus)belongs to Perciformes,Nemipterus family.The muscle of golden threadfin bream is rich in protein,where myosin accounts for more than 50%.Golden threadfin bream is an important endemic fish in China,in which northern Fujian is the main producing area.Generally,the majority of golden threadfin bream are sold fresh or other rough processed products,and the deep processing technology is relatively backward.Meanwhile,poor gel characteristics and thermal properties further limit its application in traditional fishery processing.Development of bioactive peptides by hydrolyzing golden threadfin bream myosin(GTM)is one of the potential ways to improve deep-processing of the related products.In present study,combined ultrasound-microwave(UM)was applied to modify structural properties and aggregation behavior of GTM,which would transform to a more suitable conformation after treatment.The immunomodulating peptides were prepared from GTM by controlled enzymatic hydrolysis,and then the resulting hydrolysate(GTMP)were fractionated by a series of ultrafiltration separation technology.After comparing the structural and bioactive characteristics of different components,we selected the optimal component with the strongest immunomodulatory activity based on inhibition rate of pro-inflammatory factors in LPS-treated RAW264.7 macrophage.Moreover,the effects of the resulting component on expression of various cellar cytokines and stability of during the immune response of RAW264.7 macrophages were investigated,after that,its absorption rate and immunomodulatory activity in the Caco-2 cell layer were further studied.Furthermore,the immunological activity of the selected component was explored in vivo by DSS-induced colitis model,and its immunoregulatory mechanism could be revealed at the molecular level.At the same time,the physicochemical properties of GTMP and the stability of its immunoregulatory in various environments were also studied.The aim of this study is to provide the theoretical and technical basis for the further development of deep-processing of golden threadfin bream products.The origin GTM was highly-ordered with low solubility and molecular flexibility,which is not easy to hydrolyze during peptide preparation.Thus,different microwave,ultrasound and combined UM methods were used to modify GTM structure before it was hydrolyzed.100 W microwave and 100-200 W ultrasound treatment could dissociate complex myosin aggregates into low-molecular-weight fragments,where the ordered secondary structure(α-helix and β-turn)was expanded into β-turn and random coil,and compact tertiary structure gradually became loose.Meanwhile,the thermal denaturation degradation temperature were also increased after mild microwave and ultrasound treatment.Combined UM treatment could further enhance these modification effects.100 W microwave coupled with 300 W ultrasound treatment(M100-U300)promoted the dissociation of myosin fragments into oligomers and monomers with regular and stable morphology,and accelerated changes in molecular conformation and thermal properties.This meant more potential hydrolysis sites were exposed after combined UM pre-treatment.Enzymatic hydrolysis also confirmed that GTM treated by M100-U300 exhibited the optimal degree of hydrolysis and yield of peptide.In addition,compared with neutral protease and pepsin,alcalase showed better efficiency of hydrolysis.According to the amino acid composition of myosin of GTM,it revealed that the essential amino acids contained 32.24% and the umami amino acid content was35.32%,which was obviously lower than that of other marine economic fish,and thus GTM was a typical low value protein.However,the ration of hydrophobic amino acid reached 38.02%,and aromatic amino acids accounted for 16.91%,which can be considered as a high-quality raw material for development of bioactive peptide.Alcalase was chosen to hydrolyze GTM,and the degree of hydrolysis and inhibition effect of TNF-α in the inflammatory cell model were used as evaluation indicators.The related hydrolysis parameters optimized by single factor experiment and response surface experimental design.The best process conditions of alcalase was determined as follows: hydrolysis p H was 9,substrate concentration was 15%,hydrolysis temperature was 58.5 ℃,enzyme storage was 5.6% and hydrolysis time was 150 minutes.Under this condition,the degree of hydrolysis reached 27.29%,the yield of the peptide was 66.67%,and the inhibition rate of TNF-α in LPS-treated RAW264.7macrophage was 41.89%.Subsequently,the GTMP was isolated by a series of ultrafiltration membrane with molecular weight cut-off 10 k Da,5 k Da and 3 k Da,and four different molecular weight ranges of peptides were obtained as followed: GTMP-I(> 10 k Da),GTMP-II(5-10 k Da),GTMP-III(3-5 k Da)and GTMP-IV(< 3 k Da),in which GTMP-IV had the highest proportion(76.94%).With the help of Raman spectroscopy and fluorescence spectroscopy,it was found that the conformation became more loose with higher high molecular flexibility as the molecular weight of GTMP decreased.The random coil of GTMP-IV reached 49.79%,where nearly all aromatic amino acids are exposed to a polar environment.According to invitro chemical test,GTMP-IV showed stronger antioxidant and anti-hypertensive activities than other components.When peptide concentration reached 15 mg/m L,DPPH scavenging ability and ACE inhibitory activity accounted for 83.28% and 70.61%,respectively.Comparing with the regulatory effects of different components on the RAW264.7 macrophage inflammation model,GTMP-IV could further enhanced the inhibitory effects on the pro-inflammatory factors(TNF-α,IL-1β).The physicochemical properties of GTMP-IV under various conditions were examined.The results showed that the solubility of GTMP-IV was more than 80% at the p H range from p H 2 to 10,and GTMP-IV has good water absorption and water holding capacity,which were further improved as the peptide concentration increased.Moreover,GTMP-IV showed desirable surface hydrophobicity with abundant reactive sulfydryl groups(R-SH)at room temperature,which suggested that GTMP-IV molecule have a good hydrophilic/hydrophobic ratio.However,as temperature increased,hydrophobic aggregation and disulfide cross-linking occurred,resulting in a decrease in surface hydrophobicity and R-SH.Emulsification tests revealed that 15mg/m L GTMP-IV could develop a smaller and more stable emulsions with more homogeneous distribution,and the resulting emulsions exhibited strong flocculation stability against 3000 × g gravity field for 4 h.Meanwhile,GTMP-IV exhibited the optimal foaming ability and stability at p H 6(PI),which was further improved with increasing in peptide concentration.Furthermore,the study on immunoregulatory stability showed the immunoregulatory ability of GTMP-IV did not significantly decrease until p H is less than 4 or more than 10;when the temperature exceeded 70℃ or the amount of Na Cl was above 6%,GTMP-IV would also gradually loses immunoregulatory ability;GTMP-IV had strong stability under ultrasound and ultraviolet radiation fields,and the immunomodulatory ability did not change significantly in a short time,while microwave treatment can rapidly reduce the functionality of GTMP-IV;There was no obvious effect on GTMP-IV with glucose at room temperature,while glucose could react with GTMP-IV and form Maillard reaction product(MRP)as the temperature was above 70 ℃,which contributed to enhancing its immunomodulatory ability.In addition,immunomodulatory ability was increased when GTMP-IV was exposed to pepsin within 10 min,but it would be declined with extended gastric digestion or further intestinal digestion.Further study focused on the completely evaluation the regulatory effect of GTMP-IV on immune stress in RAW264.7 macrophage.The results showed that GTMP-IV with different dose groups could inhibit the production of various pro-inflammatory factors(TNF-α,IL-1β,IL-6 and IL-8)and increase the expression of anti-inflammatory factors(IL-4,IL-10).At the meantime,GTMP-IV with different dose groups could also reduce the damage of intracellular antioxidant enzyme activity caused by inflammation and enhance the stability of the antioxidant defense system.The main mechanism of effect was as follow: 1.GTMP-IV treatment would activate m RNAs expression of antioxidant enzymes(SOD,CAT,Gr);2.GTMP-IV treatment contributed to suppressing the unstability of the antioxidant defense system by inhibiting the inflammatory response;3.Part of GTMP-IV had antioxidant activity to a certain degree,which could reduce the pressure of the antioxidant defense system by scavenging excess ROS.These effects further enhanced immunomodulatory ability,and its activity had a good dose-effect relationship with peptide concentration.Moreover,we constructed a compact cellular monolayer based on Caco-2 cell,where GTMP-IV could be well absorbed.GTMP-IV could effectively reduce LPS-induced overexpression of TNF-α,IFN-γ and IL-8 due to by inhibition of the TLR4 signaling pathway,which also contributed to repairing the dense damage of cellular monolayer caused by inflammation.Furthermore,Dextran Sodium Sulfate(DSS)was applied to construct colitis model in mice.It could be found that GTMP-IV effectively inhibited the secretion of intestinal pro-inflammatory factors and the content of diamine oxidase in the blood,and the immunomodulatory effect of high-dose GTMP-IV group was significantly stronger than the positive control group.However,GTMP-IV has no significant effect on colonic connexin,which suggested that GTMP-IV might not reduce inflammation damage by enhancing the stability of colonic tissue structure.In addition,the present study reflected that GTMP-IV could effectively reduce the phosphorylation of TLR4 and NF-κB,therefore we speculated that GTMP-IV played an immunomodulatory role by blocking TLR4,NF-κB and related inflammation signals.