| High glycolytic potential and fast acidification rate,which are induced by preslaughter stress,co-lead to PSE-like chicken breast.As a kind of dysfunctional chicken fillets,PSE-like chicken obtained pale appearance,soft texture and high expressible moisture.As a raw material for meat product processing,it would lead to unsatisfied quality and low yield,which both resulted in economic loss.Isoelectric solubilization/precipitation(ISP)process has been applied in meat processing field.For now,ISP process was generally studied in marine-derived protein modification.It could change the native structural properties of protein by adjusting unfolding and refolding behavior,and affect the gelation and emulsification properties of protein,thus enhance the protein gel quality.Therefore,pursuing a proper ISP strategy for improving gelation properties of PSE-like chicken protein and elucidating the mechanism were meaningful.In this paper,the effects of different solubilization pH on the quality of PSE chicken protein gel and emulsion gel were studied.Based on this,the optimal recovery pHs were studied to confirm the favorable ISP treatment strategy for improving the properties of PSE-like meat protein gel.Secondly,the conformational changes and aggregation properties of PSE-like meat proteins before and after ISP treatment were studied,and their physicochemical properties during heating process were compared.By analyzing the contribution of ISP-modified sarcoplasmic protein on the gelation properties of myofibrillar protein,the mechanism of ISP treatment on improving the properties of PSE gel was comprehensively elucidated.Finally,the oxidation properties and in vitro digestibility of ISP-extracted protein were explored,providing a theoretical basis for the application of ISP isolated protein in meat product,and providing new ideas for improving the value of PSE-like chicken.Detailed contents and results are as follows:1.Effects of solubilization pH on gelation properties of ISP-treated PSE-like chicken breast meat protein.In order to establish an effective ISP strategy to improve the gel properties of PSE-like chicken protein,the solubility curves of proteins under different pH conditions were determined.Based on the results,the ISP-isolated protein was extracted,and the salt solubility,total protein and salt soluble protein profile of the ISP-isolated protein and the control PSE-like chicken meat paste were compared,while the gel texture,water retention and color properties of the heat-induced gel were also compared.The results showed that the solubility curve in the range of pH 2.0-12.0 was a classic U-shaped curve.In order to ensure the protein recovery,pH 2.5-3.5 and pH 11.0-12.0 were selected for subsequent study.There was no obvious difference in total protein profile among various treatment groups,but salt-solubilized protein profile of the ISP group was changed notably compared with the control group,and the alkali-extracted group was more similar than the acid group.The hardness of protein isolate treated by alkaline pH 11.0 and acidic pH 3.5 was significantly increased from 2158.0 N to 2967.0 N and 2441.0 N,respectively.In addition,cooking loss was significantly decreased(P<0.05)from 8.9%to 4.8%and 6.5%.respectively.There was no significant(P<0.05)change in gel springiness,while all the alkaline solubilization pH had a positive effect on the hardness of the gel,but pH 2.5 and 3.0 treatments were negative to the texture properties of PSE like meat protein gel.In addition,pH 2.5 and alkaline pH treatments significantly increased the whiteness of protein gel(P>0.05),and the whiteness of pH 3.5 group showed the highest W*value,and there was no significant difference between the 11 groups(P<0.05).These results indicated that the solubilization of pH 3.5 and pH 11 were beneficial to improving the texture,water retention and color characteristics of PSE chicken protein gel.2.Effects of ISP processing on the O/W protein emulsions stabilized by isolated PSE-like chicken protein.In order to clarify the effects of ISP treatment on PSE chicken protein emulsified gel.ISP-isolate protein and PSE-like chicken meat/soybean oil O/W protein emulsion were prepared under various protein concentrations(50,100.150 mg/g).Physicochemical and rheological properties of the protein emulsions before heating were studied.The textural properties,total expressible fluid retention ability and color characteristics of the thermal induced emulsion gels were compared.The results were as follows:compared with the control group,the stability of the emulsion increased in the ISP-treated group during the refrigeration period of 72 h,and the droplet distribution in the ISP group was more finer and more dispersed.The apparent viscosity of all protein emulsions fitted well with the Ostwald-de-Waele model distribution(R2>0.9),and the K value of the consistency coefficient of protein emulsion viscosity is significantly increased after ISP treatment(P<0.05).According to frequency sweep tests,all emulsion systems,especially 100 and 150 mg/g protein concentration groups,showed 'strong gel' feature.In addition,the rheological behavior of ISP group was different from that of control group.Excessive protein concentration showed negative effects on the formation of ISP-isolated protein emulsion gel.When the protein concentration was 50 and 100 mg/g,the gel hardness of ISP group was significantly higher than that ofthe control group(P<0.05),but when protein concentration was 150 mg/g,the trend was opposite.In addition,when the protein concentration of the control group increased from 50 to 150 mg/g,the total expressible fluid(TEF)of the control group decreased from 30.3%to 12.7%.However,in ISP group,when protein concentration increased from 50 to 100 mg/g,TEF decreased significantly from 21.9%to 13.4%(P<0.05),while when protein concentration further increase to 150 mg/g,TEF increased slightly to 16.1%(P<0.05).Overall,ISP treatment could increase the viscosity of PSE-like chicken protein emulsion system,make the emulsion more unifonmly dispersing,improve the stability of emulsion,and enhance the emulsion gel texture and fluid retention ability.However,the high protein concentration(150 mg/g)will adversely affect the quality of ISP-treated emulsion gel.3.Effects of recovery pH on gelation properties of ISP-treated PSE-like chicken breast meat protein.To optimize the ISP process strategy and further improve the gel properties of ISPisolate protein,pH 5.5(isoelectric point)and pH 6.2(relatively far from isoelectric point)were chosen as different recovery conditions,with acidic pH 3.5 and alkaline pH 11.0 for solubilization conditions.The effects of four ISP strategies on recovery yield,protein solubility,protein profile,viscosity,aggregation properties,surface hydrophobicity and gelation properties were compared.The results showed that the recovery yield decreased slightly for 6.2 groups(P<0.05).Although alkali-treatment resulted in more water-soluble protein loss,the protein profile proportion of functional salt-soluble protein increased.Compared with the solubilization pH,the recovery pH has little effect on the salt-soluble and water-soluble protein profile in ISP groups.According to the SDS results,the loss of myosin light chain may be caused during ISP treatment.After ISP treatment.the surface hydrophobicity of PSE-like chicken protein increased significantly.Compared with pH 5.5 treatment group,pH 6.2 groups showed reduced zeta potential(P<0.05),resulting in a higher degree of protein aggregation and a significant increase in particle size of protein aggregates(P<0.05).In addition,compared with pH 5.5 groups,the heat-induced gel network formed by pH 6.2 recovered protein isolate was more homogeneous,and gel hardness increased significantly(P<0.05).Cooking loss in acid treatment group increased significantly(P<0.05).Based on the above results,alkaline pH 11 solubilization combined with pH 6.2 recovery can effectively promote the formation of PSE-like chicken gel network,increase the gel textural properties and water retention ability.1.The mechanism of ISP process on modifying the functional properties of PSElike chicken meat protein:a molecular perspective.To study the effects of different ISP processing strategies on PSE like meat protein,the molecular changes of PSE-like protein induced by ISP treatment were tested.The structural changes and enthalpy changes of protein before and after ISP treatment were studied,including the fluorescence intensity of tryptophan(TFI)?the content of total sulfhydryl group,intermolecular forces,water distribution characteristics,rheological characteristics,and the secondary structural contents of ISP-isolated protein and control group were measured by fourier infrared spectroscopy.The results showed that after ISP treatment,the enthalpy transition peak of myosin and actin migrated to a lower temperature(P<0.05).which indicated that ISP treatment reduced the heat stability of protein;the TFI in ISP treatment group decreased compared with the control group,which indicated that the exposure degree of tryptophan increased,and the content of total sulfhydryl group decreased significantly(P<0.05),indicating a certain amount of disulfide bonds formation.However,after heating,there was no significant difference in total sulfhydryl content between the acid treatment group and the control group(P>0.05).For acid treatment group and alkali treatment group,pH 6.2-recovery could increase the elasticity of heat-induced gel.After ISP treatment,the content of ?-helix of PSE-like chicken protein decreased significantly(P<0.05).For acid group,the ?-helix content of pH 5.5 and 6.2 recovered protein decreased from 40.8%to 34.9%and 32.7%,respectively;alkali-treated sample with pH 5.5 and 6.2 recovery decreased to 28.9%and 29.4%,respectively,however,there was no difference(P>0.05)in ?-helix content among groups after heating;according to the changes of intermolecular force,the hydrophobic interaction in control group continued to increase with the temperature rising,but the amplification of ISP-treated group decreased,among which for the alkali-treated pH 6.2 recovered group,the hydrophobic interaction decreased slightly before 45?.According to the above results,ISP treatment leads to the change of molecular structure of PSE-like chicken protein,more orderly secondary structure transforms to disorder stuctures,hydrophobic interaction increases,sulfhydryl was oxidized to disulfide bond,protein denaturation caused by ISP treatment may antagonize the heat-induced denaturation to some extent,and the heat-induced denaturation retards,thus changing the gel formation behavior.5.The mechanism of ISP process on modifying the functional properties of PSElike chicken meat protein:contribution of sarcoplasmic protein.In order to further elucidate the mechanism of ISP treatment improving the gelation properties of PSE-like chicken protein,the micromorphological changes,aggregation characteristics and intermolecular forces of sarcoplasmic proteins under different ISP treatment strategies were compared,and then the contribution of ISP-modified sarcoplasmic protein(ISP-SP)to the gel quality of myofibrillar protein(MP)were studied,including thermal denaturation,rheological behavior,textural characteristics and water retention capacity.The results showed that SP in the shape of native spherical particles was driven to form loose floating aggregates under intermolecular force after ISP treatment,and different recovery pH had significant effect on the size of aggregates(P<0.05),but there was no significant difference in the flow properties of SP suspension between ISP treatment group and control group.The main molecular forces maintaining these aggregates are hydrophobic interaction and a certain amount of disulfide bonds.The changes of intermolecular force during heating were affected by ISP treatment.Different from SP in control group,the hydrophobic interaction in ISP-SP group changed little during heating.Even after ISP treatment,SP alone cannot form orderly elastic network,which is determined by the protein structure and amino acid distribution of SP itself.ISP-SP addition did not change the rheological behavior curve of MP.After mixing the SP and MP system with or without ISP treatment,it was found that native SP had almost no positive effect on the gel formation of MP system due to its strong mobility and small molecular weight.While adding ISP-SP to MP,the gel breaking force increased from 91.7 g to 293.7 g.adding ISP-SP in ISP-MP,the gel breaking force increased from 275.7 g to 304.5 g.In addition,adding ISP-SP to MP could reduce cooking loss from 23.0%to 6.8%hence effectively reducing water loss(P<0.05).The results showed that the addition of ISPdenatured sarcoplasmic protein did not affect the thermal behavior of MP gelation,but enhanced the gel quality of PSE-like MP.6.Oxidative stability of ISP-isolated PSE-like chicken protein under hydroxyl radical generating system.To study the oxidative stability of ISP-isolated PSE-like chicken protein,the effects of a hydroxyl radical generating system(Fe3+/H2O2)at different H2O2 concentrations(0,1 and 10 mM)on the chemical and structural properties of isoelectric solubilization/precipitation(ISP)-isolated PSE(pale,soft,exudative)-like chicken protein were investigated,including the content of pro-oxidant,the formation of carbonyl group,the types of hydrolyzed amino acids,the changes of content of sulfhydryl group,Schiff base and free amino acids,and the rheological properties of protein after oxidation.The results showed that acid treatment and alkali treatment could reduce the content of pro-oxidants in PSE-like chicken system,including lipids,pigments and myoglobin.The removal efficiency of acid treatment was higher.The content of lipid decreased significantly from 6.21 in the control group(P<0.05)to 5.58/100 g dry basis,and the content of myoglobin decreased significantly from 3.08 to 0.37 mg/g dry samples.ISP treatment had no significant effect on carbonyl formation and free amino loss in PSE-like paste(P>0.05).However,in the simulated oxidation system,ISP-isolate protein generated fewer carbonyl derivatives(P<0.05),but resulted in higher level(P<0.05)of sulfhydryl and free amino loss.Accordingly,under mild oxidation conditions(1 mM H2O2),the sulfur-containing amino acids in ISP protein isolate were more easily transformed into disulfide bonds than those in the control group.In addition,compared with the control group PSE-like meat,the G' value of ISP isolate was higher after 10 mM oxidation.Based on the above results,after ISP treatment,the sensitivity of sulfur amino acids increased,and it was more easily oxidized to form disulfide bonds.However,oxidation sensitive amino acids increased their resistance,and better maintained their gel formation ability under the oxidation stress.7.In vitro stimulated digestion properties of ISP-isolated PSE-like chicken protein.In order to further explore the effect of ISP treatment on the digestibility of PSE-like chicken protein,the impacts of different heating strategies(72?,20 min,100?,60 min)under in vitro simulated gastrointestinal digestion model were studied.The differences of digestibility between ISP protein isolates and PSE like paste in control group,including the changes of physicochemical properties related to digestibility,in vitro digestibility kinetics in gastrointestinal stage,changes of protein profile,etc.,and the differences of digested products were analyzed by proteomics and polypeptide peptideomics.The results showed that the advantage of ISP treatment in increasing water retention ability and textural properties was more significant under the condition of high temperature and long time heating;although the structure of protein changed greatly before heating,especially the exposure of hydrophobic groups,the difference of structure after heating was diminished,and the size of thermal aggregation particles formed was similar to that of the control group(P>0.05);the results of SDS-page showed that 100? 60 min heating could cause oxidative denaturation of protein and form non-disulfide covalent bond;heating can significantly increase the digestion properties of PSE-like meat protein;analysis of digestion kinetic parameters shows that heating can improve the digestion potential and rate of gastric digestion stage,in which the digestion ability of ISP protein isolate after heating at 72? is higher than that of the control group(P<0.05).but there was no significant difference in the 100? heating group(P>0.05);in the simulated intestinal stage,except for the 72? treatment group,the digestion kinetics of different groups were similar;for the final digestion products,the control group and ISP treatment group showed similar characteristics under 100? treatment,but for the 72? and unheated group,the digestion products were more affected by the protein source;100? 60 min heating may lead to the crosslinking and aggregation between sarcoplasmic protein and myofibrillar protein,so its digestibility will be reduced.Compared with the control group,ISP group showed higher peptide abundance before and after heating.In summary,ISP treatment can increase digestibility of pepsin of 72? 20 min heated PSE-meat protein and increase the eptide abundance of digestion products,but there is no significant difference between the ISP group and the control group under intestinal digestion stage.For ISP protein isolates,72?20 min heating is beneficial to increase its digestion properties,but after this heating strategy,the gel properties are decreaed than that of 100? 60 min treatment group.In summary,ISP treatment can effectively improve the gelation properties of PSE-like chicken protein,and the solubilization pH of 11.0 combined with recovery pH of 6.2 was proved as optimal strategies.ISP treatment can modify the conformation of native protein and regulate the formation of protein aggregates to modify gel formation behavior,thereby affecting the final gelation properties.The ISP-modified sarcoplasmic protein can be distributed in the myofibrillar domained gel network,and plays an important role in improving the gel properties of PSE chicken protein.PSE-like meat protein with or without ISP treatment exhibited different oxidaition properties under hydroxyl radical generating system.ISP-treated protein retained higher level of gel forming ability under high oxidation stress compared to the control.ISP treatment combined with low-temperature short-term heating could increase the digestibility of PSE-like meat protein and increase the abundance of final digestion products. |
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