Study On The Formation Of Adducts Between Rosmarinic Acid And Myofibrillar Protein And Its Effects On Meat Quality

With the consumer’s demand for healthy diets,natural polyphenol are increasingly widely used as antioxidants in meat products;Adducts between polyphenol and proteins were formed during meat processing,which had influence on the protein secondary structure,gel properties,nutrition value,safety and other aspects of the meat qualities;The formation of adduct mechanism and its impact on the quality of meat mechanism is not clear.Therefore,the purpose of this research is to determine the mechanism of polyphenol-protein adduct formation in meat products,and to investigate the influence of polyphenol-protein adduct on the function and safety(protein structure,gel properties,antioxidant activity and cytotoxicity),and ultimately make the effective application of polyphenols antioxidants in the meat industry and provide a strong theoretical support for improving the quality of meat products.In the first part,adducts between Rosmarinic Acid and Cys,GSH,Peptides,myosin were firstly identified by UPLC-LTQ-Orbitrap and MALDI-TOF/TOF mass spectrometry in the presence of oxidative stress(0.1 mM FeSO4,30mM H2O2),then the influence of processing parameters(reaction time,temperature and oxidation stress)on the regulation of adducts formation was investigated.As the results shown:(1)Two adducts of RosA-Cys and Cys-RosA-Cys were identified by extracted ion and MS2 fragment ion chromatograms,the addition site of the former occurred on the right phenyl ring of RosA,and the addition sites of the latter were located on the phenyl ring on both sides of RosA.Adducts between RosA and thiol groups on the cysteine side chain of the peptides was P1-RosA,P2-RosA and P3-RosA.(2)The adduction between myosin and RosA was confirmed,and the modified peptides by RosA was LEDEC*SELK,m/z = 1425.73,and the binding site between myosin and RosA was proved to be Cys949.For the first time the precise addition site between RosA and myosin was identified,which strongly proved the cocurrance of adduction between polyphenols and myofirillar proteins during meat storage and processing.(3)The addition of Cys-RosA showed a significant increase with the increase of reaction time(1-7h),and the growth of GSH-RosA was not significant,which indicated that Cys was more reactive than GSH.With the reaction temperature(30-50℃),the addition of Cys-RosA and GSH-RosA increased significantly.GSH-RosA showed a significant increase with the increase of oxidation intensity(10-70mM H2O2),while Cys-RosA increased first and then decreased significantly.In the second part,RosA and meat proteins were reacted at 4℃ for 12h in Fenton oxidation systerm(10μm FeCl3,100pm ascorbic acid and ImM H2O2),and RosA modified protein and its modified position were analyzed by Triple TOF MS/MS,and the quantitative analysis of different amino acid modification sites and the number of modification sites in proteins were carried out to reveal the regularity of polyphenols on the addition of meat proteins.As the results shown:(1)75 RosA-modified peptides responded to 67 proteins made adduction of RosA.These proteins were widely distributed in myofibrillar protein,sarcoplasmic protein and transmembrane protein,not only affect the meat gel performance,but also involved in a variety of biological metabolic pathways,such as glycolysis,purine metabolism.(2)75 RosA-modified polypeptides were analyzed,8 Cys-containing peptides,14 His-containing peptides,48 Arg-containing peptides,64 Lys-containing peptides,and 5 N-terminal peptides.77 addition sites were analyzed,including 2 N-terminal adduction sites,3 Cys adduct sites,4 His adduct sites,29 Arg adduct sites and 39 Lys sites.(3)Protein modification site analysis showed that about 80.597%of the protein carries a single RosA modification site,14.925%carried two modification sites,1.492%contains three modification sites,and the remaining 2.985%had four or more modifications Site.In the third part,the effects of different concentrations(0,0.05,0.25,and 1.25mM)of rosmarinic acid(RosA)on the gelling characterization of porcine myofibrillar protein(MP)were evaluated under Fenton oxidation system((10 μmol/L FeCl3,100 μmol/L ascorbic acid,1 mmol/L H2O2,pH 6.25)with high salt concentration(0.6 mol/L NaCl).The protein gel properties were investigated by rheological properties,protein secondary structure,water holding capacity and gel strength,and adducts were quantified by UPLC-Q-TOF MS/MS.As the results shown:(1)Supplementation of RosA significantly decreased the contents of a-helix and β-sheet,while increased the contents of β-turn and random coil.The rheological and gelation properties with0.05 and 0.25mM RosA-treated samples were improved,while 1.25mM RosA was detrimental to MP gelation.(2)According to the Michael Addition reaction for adducts prediction,three hydrolytic adducts were identified by UPLC-Q-TOF MS/MS including 3,4-dihydroxy,5-N6-argininocinnamic acid,3,4-dihydroxy,5-N4-histidinocinnamic acid and 3-(3,4-dihydroxyphenyl,5-cystein-S-yl.(3)In the presence of low concentrations of RosA(0.05,0.25mM),both RosA-Cys and Arg/His-RosA-Cys adducts formed,which exerted positive effects on the protein gelation properties.With high concentration of RosA(1.25mM),RosA-Cys became the dominant adduct and blocked the thiol groups on MP from forming disulfide cross-link,which reduced water holding capacity and weakened gel strength by undermining protein gelation.It was the first time to elucidate formation mechanism of polyphenol-protein adducts and its effects on gel properties with the molecular explanation.(4)It was also observed from gel microstructure that the samples treated with low concentration of RosA(0.05 mM and 0.25 mM)had more fiber filaments,and good crosslinking was formed between the proteins.In the high concentration RosA(1.25 mM)Most of the protein denaturation is a less spherical,coarser network,forming a larger gap,and the gel structure of the protein is destroyed.In the fourth part,GSH-RosA adduct was prepared by RosA and GSH for 48 h under oxidation stress.LO-2 cell lines samples with Control,oxidation,RosA and GSH-RosA treatment were determined with antioxidant enzymes(SOD),the expression levels of antioxidant enzymes(SOD1,CAT),cell cycle and apoptosis to evaluate the properties of antioxidant and safety of adducts.As the results shown:(1)GSH-RosA synthesized and purified by HPLC,the antioxidant capacity of which increased according to the concentration,and up to 90.04%of RosA at 150μM.(2)In cell antioxidant analysis,RosA-GSH inhibited MDA formation significantly,and the concentration of MDA had;no difference between RosA-GSH and RosA treated samples at 72h incubation;the SOD activity of RosA-GSH was up to 166.30nmol/mg Pro.,which was significant higher than oxidation model sample but lower than RosA treated sample.(3)Western blot analysis revealed that the expression of CAT and SOD1 in GSH-RosA treated sample was higher than in oxidation model sample.Comparing to RosA treated sample,the expressions of CAT in GSH-RosA treated sample was lower,but SOD1 expression had no significant difference at 72h incubation.(4)In the cytotoxicity analysis by MTT,RosA had significant inhibitons on normal cells the concentration increased,while RosA-GSH had fewer negative effects to normal cells,which indicated that RosA took cytotoxic effect at a certain concentration and RosA-GSH was highly safe even in high-dose additions.(5)In the cell cycle analysis,the percentage of G1 phase cells increased from 36.81%to 62.41%and G2+M phase cells decreased from 23.90%to 13.41%in RosA treated sample.While there were fewer changes in GSH-RosA treated sample compare to the control sample.(6)In the apoptotic experiment,the percentage of viable cells decreased significantly(p<0.05)compared with the control group,and the percentage of apoptotic cells increased significantly(p<0.05)to 4.96%in the LO-2 cells treated with 500 μM RosA for 24h.While the inhibitory effect of LO-2 on GSH-RosA treatment group was significantly lower than that of RosA treatment group and the apoptosis process was alleviated.According to this research,we can conclude that the Michael addition reaction between RosA and thiol containing compounds occurred under oxidative stress,and the formation of adducts were influenced by the processing parameters(reaction time,temperature and oxidative intensity).The regulation of RosA modification for meat proteins was investigated,and the formation of adducts could make effects on the gel properties of meat products;Compared to RosA,the GSH-RosA adduct also has efficient antioxidant capacity,but had less influence on the cytotoxicity,cell cycle and cell apoptosis.The understanding on phenol-protein adductions obtained from this research provides fundamental theory and guides to make safety and efficient applications of polyphenols in meat industry.Furthermore,the theory of phenol-protein influence on gelling properties light on the novel way to improve meat qualities in molecular level.