【作者】 李春强；

【导师】 熊幼翎；

【作者基本信息】 江南大学 ， 食品科学， 2013， 博士

【摘要】 微生物谷氨酰胺转移酶（MTG）可催化蛋白中谷氨酰胺和赖氨酸残基发生酰基转移反应，使蛋白之间发生交联，进而改善蛋白的功能性质，现已被应用于肉制品加工中以提高产品的品质。然而，MTG在实际应用中存在产品质量不稳定的问题。鉴于氧化引起的蛋白结构变化可能改变MTG可利用底物的含量（例如氧化引起的结构展开会暴露更多的谷氨酰胺和赖氨酸底物），本研究假设羟自由基氧化能够改变MTG催化交联反应，从而改变肌原纤维蛋白（MFP）的功能性质。以期通过探索蛋白氧化与MTG交联反应之间的关系，为肉制品加工品质改善提供理论依据。本研究以猪肌原纤维蛋白（MFP）或肌球蛋白为研究对象，经羟自由基产生体系（FeCl3/抗坏血酸/H₂O₂，pH6.25）氧化后进行MTG交联（E:S=1:20），观察MFP或肌球蛋白的氧化对MTG交联反应的影响。主要研究内容包括：（1）氧化引起的MFP结构变化对MTG催化交联程度的影响；（2）MFP氧化对MTG催化交联后二级结构及交联部位（肌球蛋白亚片段，如S1、S2、Rod、HMM和LMM）的影响；（3）肌球蛋白氧化对MTG交联后异肽键（-CONH-）形成以及特定交联位点的影响；（4）在影响肉制品加工的常见因素条件（不同盐浓度和温度）下，MFP的氧化对MTG交联反应程度和功能性质的影响。通过研究在高盐条件（0.6mol/L NaCl）下氧化引起的MFP结构变化与MTG交联程度之间的关系，发现蛋白氧化过程中结构展开、蛋白聚集和氨基酸修饰是同时存在的，且MTG的交联程度受优势方的影响。相对于未氧化MFP的交联程度11.6%，轻度氧化MFP（1mmol/L H₂O₂）中结构展开占优势，暴露更多可利用的谷氨酰胺和赖氨酸底物，导致交联程度提高至27.6%；而深度氧化MFP（5和10mmol/LH₂O₂）中蛋白聚集和氨基酸修饰占优势，使得部分可利用的谷氨酰胺和赖氨酸残基被遮蔽或被氧化，导致交联程度低至9.6%。通过CD和FTIR光谱进一步测定了在高盐条件（0.6mol/L NaCl）下MFP氧化对MTG催化交联后二级结构的影响，发现氧化和MTG交联均导致MFP解螺旋，MTG交联引起的ɑ-螺旋变化与氧化程度相关：轻度氧化（0.1–1mmol/LH₂O₂）>未氧化>深度氧化（5–20mmol/L H₂O₂）。同时借助胰凝乳蛋白酶将MFP中的肌球蛋白水解为不同片段（S1/Rod和HMM/LMM），观察氧化对MTG交联部位的影响。结果表明，对于未氧化肌球蛋白，MTG主要交联部位为S1；对于因轻度氧化而结构展开的肌球蛋白（0.1–1mmol/L H₂O₂），所有片段的交联概率均增加，但S1仍是重点交联部位；对于因过度氧化（5–20mmol/LH₂O₂）而聚集的肌球蛋白，S2是重点交联部位。将MFP中的主要蛋白—肌球蛋白提纯，观察高盐条件下肌球蛋白氧化对MTG交联程度、交联部位和交联位点的影响，并与MFP进行比较。ε（γ-G）L测定结果表明轻度氧化肌球蛋白（1mmol/L H₂O₂）的反应程度为87.6%，显著高于（P<0.05）未氧化蛋白的64.7%和过度氧化蛋白（200mmol/L H₂O₂）的33.8%，交联程度的趋势与MFP相似。氧化肌球蛋白的交联部位与MFP也大体相似：对于未氧化肌球蛋白，交联部位主要为S1；对于轻度氧化肌球蛋白，Rod和S1交联概率均增加，S1仍是重点交联部位；过度氧化肌球蛋白因无法严格水解为特定片段（S1/Rod或HMM/LMM）而无法判断其交联部位。此外，与过度氧化的肌球蛋白不同的是，未氧化和轻度氧化的肌球蛋白经MTG交联后会生成不易水解为S1和Rod的聚集物。通过MALDI-TOF/TOF MS找到了该聚集物中两个可能的交联位点：由肌球蛋白头部789–794（IQAQAR）肽段中的790谷氨酰胺残基与800–804（IEFKK）肽段中的803赖氨酸残基交联所形成的IQ（IEFKK）AQAR，以及由肌球蛋白头部560–567（SNNFQKPR）肽段中的565赖氨酸残基与肌球蛋白尾部1872–1875（LQLK）肽段中的1872谷氨酰胺残基交联所形成的SNNFQK（LQLK）PR。将MFP在三种盐浓度（0.15、0.45和0.6mol/L NaCl）下进行氧化，随后在三种温度（4、15和30°C）下进行交联，观察在常见的肉制品加工条件下氧化对MTG交联反应以及后续凝胶性能的影响。发现在上述所有条件下，氧化MFP的交联程度几乎均高于未氧化MFP，且随着盐浓度和温度的增加而升高。例如，15°C高盐2h氧化样品的交联程度为46.8%，高于相同条件下未氧化样品的31.6%，远远高于15°C低盐2h氧化样品的27.2%以及4°C高盐2h氧化样品的18.2%。MFP的氧化对MTG交联后凝胶性能的影响主要通过凝胶强度和动态流变学进行测定，结果表明，高盐氧化样品经4°CMTG处理后凝胶强度的增加量（92.6%–120.3%）大于未氧化样品（79.0%），然而提高温度等过度交联条件反而不利于凝胶网络结构的形成。在两种交联温度（4和15°C）下，低盐氧化样品经MTG处理后凝胶强度的增加量均小于未氧化样品。动态流变学测定结果表明两种盐浓度的氧化MFP经MTG处理后最终储能模量的变化幅度均大于未氧化MFP的。可见，高盐MFP的氧化是一个辅助MTG提高蛋白功能性质的有效手段。总之，本课题研究了MFP氧化对MTG催化交联反应（交联程度、交联部位和交联位点）的影响，并通过MFP的凝胶性能进行了验证，阐明了MFP氧化对MTG交联的影响机理，为解决MTG实际应用中出现的问题提供了理论依据，以利于制定应对策略。更多还原

【Abstract】 Microbial transglutaminase （MTG） has been applied cmmerically to improve the texturalproperties of meat products for its catalysis of protein cross-linking through acyl transferreaction between glutamine and lysine residues. However, the quality of meat productsprocessed with MTG is often found variable. Based on the structural changes of MFP induedby oxidation may alter the content of avaliable MTG substrates （e.g. protein unfoldinginduced by oxidation can cause the exposure of more glutamine and lysine residues）, ahopothesis that radical stress affects the cross-linking catalysis of MTG was tested. Theultimate goal of the study is to produce scientific evidence that supports the understanding ofthe relationship between protein structural modification and MTG-mediated cross-linking foroptimized quality of processed meats.Myofibrillar protein （MFP） and myosin were oxidatively stressed in a hydroxylradical-generating system （FeCl3/ascorbic acid/H₂O₂, pH6.25） with different H₂O₂concentrations and then subjected to MTG （E:S=1:20）. With this general oxidizing system,four individual experiments were performed to investigate:（1） effects of MFP oxidation onthe cross-linking degree of MTG catalysis;（2） effects of MFP oxidation on secondarystructure and cross-linking location （myosin subfragments as S1, S2, rod, LMM, HMM） inMFP after MTG catalysis;（3） effects of myosin oxidation on isopeptide bond （-CONH-）formation and the specific mass spectrometry-verified cross-linking sites after MTG catalysis;（4） effects of MFP oxidation on MTG cross-linking degree and functionality of MFP undercommon factors influencing the manufacture of meat products （different salt concentrationsand temperatures）.The relationship between structure changes of oxidized MFP and MTG cross-linkingdegree at high salt concentration （0.6mol/L NaCl） was studied. The results showed thatprotein unfolding, polymerization and the modification of amino acid residues coexistedduring oxidation. The degree of MTG cross-linking was dependent on dominant factors. Fornonoxidized MFP, the cross-linking degree was11.6%. For mildly oxidized MFP （1mmol/LH₂O₂）, protein unfolding was the main facor, which promoted MTG cross-linking reactionbecause of the exposure of more available glutamine and lysine substrates.The cross-linkingdegree of mildly oxidized MFP was improved to27.6%. For deeply oxidized MFP （5and10mmol/L H₂O₂）, polymerization and the modification of amino acid residues dominated, whichblocked MTG cross-linking reaction because of reburying or modifying of many availableglutamine and lysine residues. The cross-linking degree of deeply oxidized MFP was as lowas9.6%.The effect of protein oxidation at high salt concentration （0.6mol/L NaCl） on thesecondary structure of MFP when treated with MTG was determined by CD and FTIR. Theuncoiling of MFP occurred when subjected to either oxidation or MTG cross-linking. Thechange of ɑ-helix induced by MTG was related to oxidation degree: mild oxidation （0.1–1mmol/L H₂O₂）>nonoxidation>advanced oxidation （5–20mmol/L H₂O₂）. The influence ofprotein oxidation on the cross-linking location of MTG was studied using chymotrypsinthrough digesting myosin into different fragments （S1/Rod and HMM/LMM）. The results indicated that the main cross-linking location of nonoxidized myosin was S1. For unfoldedmyosin caused by mild oxitaion （0.1–1mmol/L H₂O₂）, every fragment was the target of MTG,especially S1. For polymerized myosin caused by excessive oxidation （5–20mmol/L H₂O₂）,S2played an important role in MTG cross-linking.Myosin was purified to study the cross-linking degree, cross-linking location andcross-linking sites of differently oxidized myosin and to compare with those of MFP. Theε（γ-G）L result showed that the cross-linking degree of mildly oxidized myosin （1mmol/LH₂O₂） was87.6%, which was significantly higher （P<0.05） than nonoxidized （64.7%） andexcessively oxidized myosin （200mmol/L H₂O₂,33.8%）. The trend of cross-linking degreewas similar with that of MFP. The cross-linking location of oxidized myosin was also similarwith that of MFP: for nonoxidized myosin, the main cross-linking position was S1; for mildlyoxidized myosin, both Rod and S1were easily oxidized, in which S1was the main one; thecross-linking location of excessively oxidized myosin can not be obtained since it can not bedigested strictly into specific fragments （S1/Rod or HMM/LMM）. Unlike excessivelyoxidized myosin, an aggregate which can not be digested into S1and Rod was founed innonoxidized and mildly oxidized myosin. Two possible cross-linked peptides were founed inthis aggregate by MALDI-TOF/TOF MS: IQ（IEFKK）AQAR was formed throughcross-linking between the790glutamine of peptide789–794（IQAQAR） and the803lysine ofpeptide800–804（IEFKK） in myosin head; SNNFQK（LQLK）PR was formed throughcross-linking between the565lysine of peptide560–567（SNNFQKPR） in myosin head andthe1872glutamine of peptide1872–1875（LQLK） in myosin tail.MFP was oxidized at three salt concentrations （0.15,0.45and0.6mol/L NaCl） andsubsequently cross-linked at three temperatures （4,15and30°C）, and then the effect ofprotein oxidation on the MTG cross-linking and gelation was studied. At all saltconcentrations and temperatures, the cross-linking degree of almost all oxidized MFP washigher than the nonoxidized, and increased with elevated salt concentration and temperature.For example, the cross-linking degree of2h-oxidized sample at0.6mol/L NaCl was46.8%when treated with MTG at15°C, higher than the31.6%of nonoxidized sample. It was alsohigher than the27.2%of the corresponding sample at0.15mol/L NaCl and15°C as well asthe18.2%of the corresponding sample at0.6mol/L NaCl and4°C. The effect of proteinoxidation on the gelation of MFP when treated with MTG was determined by gel strength anddynamic rheology. Gels formed by oxidized MFP at0.6mol/L NaCl （92.6%–120.3%） werenotably stronger （P<0.05） than that of nonoxidized protein gels （79.0%） when treated withMTG at4°C. However, excessive cross-linking treatment （e.g. improving temperature） washarmful to the network structure of gels. The improvement of gel strength caused by MTGcross-linking at4and15°C in MFP oxidized at0.15M NaCl was lower than the nonoxidized.The final storage modulus change of oxidized MFP at both salt concentrations when treatedwith MTG was higher than the nonoxidized. Therefore, the oxidation of MFP at high saltconcentration was a good method to improve the quality of meat products when treated withMTG.In summary, the effect of oxidation on the MTG-catalyzed of MFP and myosincross-linking （degree, location, and site） was studied, and then verified by protein gelation. The mechanism of MTG catalysis influenced by protein oxidation was clarified, whichprovided a theoretical basis to explain the problems occurring in situ and to develop strategiesto over the problem when MTG is used as a meat processing aid.更多还原