【作者】 孙群；

【导师】 杨志荣；

【作者基本信息】 四川大学 ， 遗传学， 2007， 博士

【摘要】 食品的氧化品质是影响消费者感官接受程度的重要因素，包括水活度在内的很多因素对食物的氧化变质都具有影响。对含水量中等的肉品（半干肉）（intermediate moisture meat，IMM）来说，可通过对水活度（water activity，a_w）的调节以获得最佳感官指标和最长货架期。长期以来，很多研究认为在高水活度和低水活度下脂质氧化更易发生，而美拉德非酶促褐变反应（Maillardreaction）的强度则随着水活度的升高而增加。脂质氧化、蛋白质氧化和非酶促褐变反应相互联系，并且三者可协同作用引起半干肉品的氧化变质。我们的前期研究表明，在450 nm下测定的硫代巴比妥酸反应物质(TBARS₄₅₀)而非经典的在532 nm下测定的TBARS值(TBARS₅₃₂)可在肉品加速贮藏条件下（49℃）随时间延长而增加，由于TBARS₄₅₀可由脂质氧化和美拉德非酶促褐变共同引起，因而该反应更能反映在加速存储过程中由脂质氧化和美拉德反应引起的氧化变质情况。该研究用含15％脂肪的牛肉为材料，通过添加甘油醇（0％、20％、40％、60％）获得水活度范围为0.76-0.98的牛肉香肠样品，探讨了水活度对牛肉在低温（4℃）和加速贮藏条件（49℃）包括脂质氧化、蛋白质氧化、美拉德非酶促褐变的各氧化品质及肉品表面和内部颜色（L、a、b值）的影响。结果表明，在4℃和49℃两个贮藏温度下，经典的TBARS₅₃₂均值随着贮藏时间的增加而降低。TBARS₄₅₀在4℃贮藏样品中不随贮藏时间和水活度而有显著改变。在49℃时，不同水活度样品的TBARS₄₅₀均随贮藏时间延长而有显著性增加（P＜0.05），且对照（0％甘油醇）与各处理样品间无显著差异（P＞0.05），因此TBARS₄₅₀受加速贮藏温度（49℃）和贮藏时间的影响比受水活度的影响更大。美拉德反应（MR@370）的变化趋势与TBARS450非常一致。蛋白质氧化在4℃贮藏样品中随贮藏时间的延长而降低直至第4周基本达到稳定，而对照和样品之间无显著差异（P＜0.05）。在49℃贮藏4周后，蛋白质氧化随贮藏时间的延长有明显提高（P＜0.05）。在所有取样检测的贮藏时间点，对照和处理样品间均无显著性差异（P＞0.05）。因此，与脂质氧化和美拉德非酶促褐变相似，蛋白质氧化受贮藏时间和温度的影响比受水活度的影响更大。采用完全冷冻干燥的牛肉分别经饱和的NaCl、NaBr、K₂CO₃、MgCl₂、LiCl溶液复水获得了具不同水活度（0.008-0.754）的牛肉样品，然后在49℃下进行加速贮藏。在用1H固态核磁共振（nuclear magnetic resonance，NMR）研究该系列样品中水分子迁移率与脂质氧化相关性的机理及核磁共振成像（nuclearresonance image，NRI）观察样品精细结构时发现，水和脂质的质子分别产生两个吸收峰，其质子扫描光谱表明吸收峰与样品中水分含量有很好的相关性。1H强度和水自旋-自旋弛缓时间（T₂）均随着水活度、存储温度和存储时间的增加而增加。谱线宽度分散现象随含水量增加而减少，它在短期存储（0-30天）的水活度为0.3的样品中出现，经更长存储时间后消失，这表明即使样品中含水量达到平衡后，在贮藏过程中仍然存在着动态变化。用核磁共振成像对贮藏12周的对照（a_w=0.008）和复水样品（a_w=0.753）检测时显示，对照中的水分子排布有位置局限性，因而迁移率低，而复水后的样品中水分子呈弥散状分布，相应地其迁移率也会更高。因此，以TBARS₄₅₀检测的脂质氧化以及美拉德非酶促反应产物随水活度的降低而增加的现象有可能与观测到的水分子迁移率和肉品细微结构的改变有关。对IMM牛肉中水活度对包括脂质氧化、蛋白质氧化和美拉德非酶促反应等氧化品质的影响及水分子迁移率与脂质氧化相关性机理的研究，可以帮助我们更好地理解肉品中水合作用动力学的机制及其对肉品脂质氧化以及其他氧化品质的影响，这将为改善半干肉品品质和延长其贮藏期提供理论依据。由于作为普通畜产品的鲜肉中的脂质氧化与蛋白质氧化，特别是与肉品中最重要的呈色蛋白质肌红蛋白氧化间的相关性已经研究得非常彻底，因此该论文的第二部分就着重在对高原特有物种牦牛的肌红蛋白基因序列、功能及其氧化特性与脂质氧化的相关性进行研究。牦牛（Bos grunniens）是牛属动物中唯一能在海拔3，500米以上高寒地区繁衍生存的种群，对高寒、缺氧、日温差大等严酷的自然环境具有很强的抗逆能力。肌红蛋白有运输和暂时储存氧气的功能，并可携带氧在肌肉中运动，当动物急剧运动时肌红蛋白就把氧释放出来，以保障肌肉代谢对氧的强烈需求。在同样的氧分压下，肌红蛋白结合的氧量是血红蛋白结合氧量的6倍，因此，在高海拔地区氧分压低的环境下，肌红蛋白对动物体内氧气的运输和贮藏起着至关重要的作用。本论文对四川若而盖高原（平均海拔3，500米）的麦洼牦牛的肌红蛋白进行了cDNA序列和含量的测定及心肌肌红蛋白氧化和脂质氧化相关性的研究，并和成都平原上普通黄牛的肌红蛋白进行了比较，初步探讨了牦牛适应高原低氧环境的机理。对牦牛与黄牛肌红蛋白的cDNA序列进行测序并比较，发现二者只有2个碱基的差异（89位氨基酸由cac变为cat，91位氨基酸由gcc变为gct），但由于氨基酸密码子的简并性，两者的氨基酸序列没有差异，因而两者的肌红蛋白在蛋白质序列和结构上没有差异。牦牛的骨骼肌和心肌的肌红蛋白含量分别为488.3nmol／g和823.4nmol／g，而黄牛则分别为410.96±71.9nmol／g和510.7±29.5nmol／g，牦牛的骨骼肌和心肌的肌红蛋白含量比黄牛的分别高18.8％（P＞0.05）和61.2％（P＜0.05）。在4℃贮藏70小时过程中，不管是心肌还是骨胳肌，黄牛肌红蛋白氧化形成高铁肌红蛋白的量都大于牦牛，贮藏70小时后，黄牛心肌中和骨骼肌中高铁肌红蛋白的累积量比牦牛分别高出6.9％（P＞0.05）和25.8％（P＜0.05），即牦牛心肌和骨骼肌中肌红蛋白的氧化速率比黄牛均要慢。在4℃贮藏的6天中，牦牛和黄牛的硫代巴比妥酸反应物质（TBARS）值都随着贮藏时间的延长而缓慢升高，牦牛的TBARS值从第1天的0.021上升到第7天的0.266，平均每天上升0.04；而黄牛从0.017上升到0.435，平均每天上升0.07，氧化的速度是牦牛的近两倍。第7天时，黄牛的TBARS值比牦牛高出了63.5％（P＜0.05），即牦牛肌肉中脂质氧化的速度显著低于黄牛（P＜0.05）。对牦牛肌红蛋白的cDNA序列分析及其氧化性与脂质氧化相关性的研究表明，牦牛能够适应高原低氧分压和高氧化胁迫的低氧环境，并非通过改变其肌红蛋白氨基酸序列从而引起蛋白质结构的变化来实现的，而可能是通过提高肌红蛋白在体内的含量，以及减缓肌红蛋白氧化和脂质氧化的速率，达到降低肌红蛋白转变为高铁肌红蛋白的量的目的，从而使更多的肌红蛋白保持与氧结合的能力，满足牦牛在低氧条件下对氧气的需求。本论文开创性地研究了肉品中水活度对肉品中多个氧化品质的影响，并用核磁共振技术研究了水迁移率与脂质氧化的关系，这对于我们更好地理解肉品中水—蛋白质—脂质的相互作用提供了理论基础；同时，该论文首次将牦牛肌红蛋白对氧化的易感性与脂质氧化联系起来，发现了二者之间的相关性，以及肌红蛋白在牦牛适应高原低氧环境中所受到的保护作用。更多还原

【Abstract】 The oxidative quality of foods is a critical factor influencing sensory acceptance. A variety of factors, including water activity, impact the potential for foods to undergo oxidative deterioration. Maximizing sensory and microbiological shelf life of intermediate moisture meat（IMM） may be approached by manipulating water activity（aw）. The interactions between lipid oxidation, protein oxidation and Maillard reaction may play together to impair the oxidative quality of IMM. Understanding the molecular mobility of water and its relationship to lipid oxidation would provide critical information related to quality of dehydrated foods. Nuclear Magnetic Resonance（NMR） is a powerful technique that can measure the molecular dynamics of water and can be utilized to address the heterogeneity, be specifically, to measure moisture mobility and migration in rehydrated freeze-dried meat products of different water activity values.The objective of this part of study was to understand the effects of an important meat processing variable, water activity here, on oxidative deterioration of intermediate-moisture meats, and reveal water molecular mobility observed by Nuclear Magnetic Resonance（NMR） in rehydrated freeze-dried meat patties.To evaluate the effect of water activity on oxidative quality in beef, intermediate moisture mega sausage was prepared with glycerol as humectant to reach different water activity, and then subjected to storage at cold storage at 4℃and 49℃, an accelerated condition typical of storage studies for military ration components. Water activity（a_w）, total moisture content（％）, lipid oxidation（TBA test）, protein oxidation, and non-enzymatic browning reaction at week 0, 4, and 8 were monitored. The values of water activity for fresh prepared samples treated with glycerol 0％, 25％, 40％, 60％were about 0.98, 0.92, 0.88, and 0.84 at week 0, with 0％as control. At both storage temperatures, TBARS532 consistently decreased with storage length and there was no significant difference（P＞0.05） between control and treatments as well as among treatment. Interestingly, TBARS532 from control（higher aw） was even higher than those of treatments（lower a_w） at any time points, although there was no differences between treatments（P＞0.05）. At 4℃of storage, TBARS450 did not change with storage time or a_w（P＞0.05）, while significantly increased with storage time at 49℃（P＜0.05）, but no difference between control and treatments was found（P＞0.05）. The changes of Maillard reaction（MR@370） showed the same pattern as TBARS450. Protein oxidation decreased with storage time from week 0 to week 4 in samples stored at 4℃, but not further decreased after week 4. No difference between any control and treatments was found（P＞0.05）. At 49℃, protein oxidation significantly increased with storage length after week 4. There was no difference between any control and treatments at any time point. Accordingly, in IMF beef system with aw range from 0.98 to 0.84, water activity had no significant effect on lipid oxidation measured by TBARS450, Maillard reaction and protein oxidation. At the conditions described above, lipid-oxidation measured by TBARS450, Maillard reaction and protein oxidation were significantly influenced by accelerated storage condition（49℃） and storage time at this temperature.To study water mobility related to lipid oxidation with using NMR, freeze-dried beef patties were adjusted from 0 to 0.75 a_w and stored at 4℃and 49℃. During 16 weeks of storage, lipid oxidation was monitored by TBARS at 450 and 532 nm, and water mobility by wide-line, solid-state proton NMR. The results showed that water and lipid protons contributed to two peaks; both water and lipid protons spectra showed a line narrowing effect at increasing moisture. ¹H intensity and water T₂ increased with increasing a_w, temperature and storage time. The LW dispersion （decrease with moisture content） occurred at around 0.3 a_w at shorter storage time （0-30 days） and disappeared after extended storage, indicating a complex dynamic behavior even when equilibrium moisture content has been reached. The TBARS increase with decreasing a_w was probably related to the observed molecular and structural changes. The results above indicated the importance of hydration dynamics and its effect on lipid oxidation in muscle foods. A better understanding of water-protein-lipid interaction and the roles of water and lipid mobility in affecting lipid oxidation could also provide the basis for improving product quality and shelf life.Due to the comprehensive study of the effect of lipid oxidation on protein oxidation, especially the specific oxygen-transportative myoglobin oxidation, in fresh meats, this study on the oxidative quality in fresh meat would be focused on myoglobin in yak, including its cDNA sequence, its content and oxidation with related to lipid oxidation, in order to discover the mechanism by which yak adapts to hypoxia condition on high attitude.The mechanism by which yak（Bos grunniens） adapts to the hypoxia environment, especially myoglobin（Mb） kept its role for oxygen transportation and storage in this extreme condition, have not been fully understood. The cDNA sequence of yak Mb, as well as Mb content and oxidation related to lipid oxidation in yak skeletal and cardiac muscle, were investigated to examine the relationship of myoglobin to yak’s adaptation to hypoxia environment. The cDNA sequence of yak myoglobin showed that, in the open reading frame coding 153 amino acids of Mb, there was a two-base-difference （cac→cat and gcc→gct, at the 89th and 91th amino acids） between cattle（Bos taurus） and yak, although their protein sequence after translation was the same due to code degeneracy. Mb contents in yak skeletal and cardiac muscles were 488.3 nmol/g and 823.4 nmol/g, respectively, which were 18.8％and 61.2％higher than those of cattle. During the storage at 4℃, oxidation of yak Mb was slower by 10％in skeletal and 30％in cardiac muscle compared with cattle, while thiobarbituric acid-reactive substance（TBARS） in yak skeletal muscle was 63.5％lower than that of cattle after 6 days of storage. Our data indicated that yak adapted to hypoxia environment not by the change of cDNA sequence of myoglobin thus the modification of its structure during evolution, but at least partially by the increased content of myoglobin and its improved resistance to oxidation stress, as well as the potential protection effect of lipids in yak muscle.更多还原