【作者】 韩剑众；

【导师】 邓少平；

【作者基本信息】 浙江工商大学 ， 食品科学， 2008， 博士

【摘要】 猪肉的生鲜品质具有复杂而多元的特性,对其形成机理、控制和评价一直是肉品科学研究的焦点。迄今为止,对猪肉生鲜品质的形成机理及其评价研究大量地集中在正常肉和劣质肉（如PSE肉）之间的差异及胴体瘦肉率等方面,而对不同品种、饲养方式特别是规模化快速养殖生产下猪肉生鲜品质的形成、控制和评价研究尚缺乏令人满意的解释和有效方法。本研究以浙江优质地方品种金华两头乌和目前国内普遍养殖的纯外来的三元杂交品种“杜长大”为研究对象,利用差异蛋白质组学、低场核磁共振和电子舌等技术对现代猪肉生产条件下,不同品种和饲养时间的猪肉生鲜品质进行了深入的研究,通过宰前处理对宰后生鲜猪肉品质影响的机理和实际作用效果研究,探索并提出了提高生鲜猪肉品质可操作和实际应用的方法和途径;通过对构成肌肉的主要物质蛋白质表达的差异、水分存在状态的变化及其与抗氧化能力等之间的关系,探索了猪肉生鲜品质的形成机理,建立了用于品质评价的相应方法;利用本实验室前期研制的多频脉冲电子舌对肉质进行辩识和评价的研究,探索了利用电子舌对宰后各猪肉生鲜品质的评价监测和货架寿命（新鲜度）的控制方法。实验结果为不同品种,养殖时间,饲料和营养浓度,饲喂模式,动物福利,屠宰加工等变化导致的肉质差异进行快速监测、评定和等级标注提供了理论和技术依据。主要研究内容和结果如下:1、一水肌酸和丁氨丙磷宰前处理对生鲜猪肉品质的影响在杜长大肥育猪饲料中添加一水肌酸（CMH）（500g/t）,可以有效改善宰后猪肉的品质。本研究结果表明,使用CMH后,肥育猪的眼肌面积提高了9.04%（P＜0.01）,背最长肌比率提高了15.73%（P＜0.01）,脂肪比率及第十肋骨处的背膘厚则有所下降;与对照组比较,背最长肌粗脂肪含量提高了34.75%（P＜0.01）,而水分则下降了4.06%（P＜0.05）,肌酸含量上升了14.27%（P＜0.01）,糖原含量上升了8.81%（P＜0.05）,肌苷酸含量提高29.21%（P＜0.01）,肌苷含量提高32.14%（P＜0.01）,而胶原蛋白下降了13.79%（P＜0.01）;背最长肌24小时的滴水损失降低了30.43%（P＜0.01）,肌酸激酶活率提高了16.4%（P＜0.01）;血清中的游离脂肪酸和葡萄糖的含量,分别提高了13.33%（P＜0.05）和5.35%（P＜0.05）,肌酸激酶的活率提高了18.84%（P＜0.05）。宰前40-48小时开始用丁氨丙磷饮水处理,剂量为150mg/kg体重和300mg/kg体重,间隔8小时一次,可有效改善由于应激等对宰后肉质的影响。和对照组比较,试验组背最长肌24h的滴水损失分别下降25.1%和26.05%（p＜0.01）;肌酸含量分别升高了12.85%和19.34%（p＜0.01）;糖原含量分别上升了10.29%（p＜0.05）和16.87%（p＜0.01）;而肌酸激酶的活率则提高了19.56%和24.18%（p＜0.05）。2、基于双向电泳的猪肉生鲜品质的差异蛋白质组学研究通过猪肉样品的制备和双向凝胶电泳实验条件方法的优化,建立了猪肌肉组织双向凝胶电泳（2-DE）方法,获得了具有统计学意义的图谱,并从图中筛选到了差异蛋白质点共345个,其中具有统计学意义（P＜0.05）又具有量分析意义的点共有33个,根据软件的进一步比对和分析,结合质谱的要求,选择了12个有意义的点作为后续质谱分析的待切点。利用MALDI-TOF-MS检测技术,对差异表达的蛋白质进行检出、分析和鉴定,通过利用肽质量指纹图谱（PMF）和MASCOT软件搜索对比分析,结果表明,地方品种猪金华“两头乌”和杜长大三元杂交猪宰后0h的样品得到了6个差异蛋白质和一个未知蛋白（斑点编号为1101）,差异蛋白质分别为Predicted Protein、Actinchain A Hypothetical Protein、EST1和Myoglobin（chain B）;同样,宰后24h的样品也得到了7个差异蛋白质,分别为β-2 globin、Lymphocyte antigen、SOD、β-globin、Myosin regulatory、Actin-cappingprotein和Dj-1 with sulfinic acid,提示宰后不同品种的猪背最长肌品质的差异可能是由于这些蛋白质量的变化即斑点的上调或者下调引起。由于地方品种金华“两头乌”和杜长大三元杂交猪的蛋白质数据库还没有建立,而且已建数据库中与猪肉品质相关的蛋白质也非常少,因此通过MALDI-TOF-TOF-MS只能鉴定出接近70%的差异蛋白质。3、基于低场核磁共振的生鲜猪肉水分存在状态和分布规律研究利用磁场强度为0.5T、共振频率为21-23MHz的核磁共振仪对宰后不同时间生鲜猪肉的横向弛豫时间T₂进行研究。结果表明,代表猪肉结合水的T₂₁,各猪肉样品几乎没有差异且十分恒定一致;但代表猪肉不易流动水的T₂₂,金华两头乌和杜长大存在较明显的差异,金华两头乌猪无论屠宰体重如何,在宰后的0h、12h、24h和36h的T₂₂明显大于杜长大所有屠宰体重的猪肉;从同一品种和屠宰体重来比较,金华两头乌猪肉的变化规律十分明显,而杜长大仅在宰后6h和30h稍有增加,其它时间始终维持在较低水平;反映自由水的T₂₃,金华两头乌亦高于杜长大。宰后生鲜猪肉核磁共振的横向弛豫时间能很好地反映各时间点的滴水损失,通过对T₂的进一步区分,获得了宰后猪肉水分分布和变化的详细情形,这对于认识和理解不同品质猪肉之间不易流动水的分布具有重要意义。4、宰后肌肉组织抗氧化性、酶学变化与生鲜猪肉持水性的关系研究宰后生鲜猪肉的抗氧化能力金华猪明显大于杜长大,而且与肉的滴水损失高度相关。表现明显的主要有猪肉超氧化物歧化酶活力、谷胱甘肽过氧化物酶（GSH-PX）活力、猪肉抑制羟自由基能力、宰后肌肉的总抗氧化能力、猪肉游离脂肪酸的含量等。谷胱甘肽过氧化物酶（GSH-PX）活力在金华猪,不管其上市体重如何,均明显高于杜长大猪,平均高出6-8倍（P＜0.05）;宰后肌肉的总抗氧化能力大大高于杜长大猪,以宰后0h的数据看,60Kg的金华猪分别比各上市体重的杜长大高588%、439%和327%;75Kg的则分别高533%、395%和293%;90Kg的则分别高616%、452%和337%。对于杜长大猪,体重越小,总抗氧化能力越差,其中90Kg的比75Kg高27.7%,105Kg的则高61.1%;宰后生鲜猪肉游离脂肪酸的含量和品种关系较大,而与上市体重影响较小,金华猪明显高于杜长大（平均在3倍以上）。宰后生鲜猪肉的pH值金华猪均明显高于杜长大猪,但变化规律两个品种及各上市体重之间十分一致,即在0-12h内逐渐下降并在12h时至最低,后基本不变;杜长大猪在各个时间点的滴水损失均大于金华猪,而且随着屠宰体重的增大,滴水损失也随之增大,105Kg的杜长大猪肉在24h的滴水损失比75Kg的大87.29%,比90Kg的大64.98%;48h的滴水损失,75Kg和105Kg的杜长大均明显升高,但90Kg的滴水损失则与24h时的基本一致,72h的滴水损失75Kg明显下降,但105Kg的则仍继续增大,达5.48%,比75Kg高232%。金华猪的滴水损失的也有随屠宰体重增大而升高的趋势,但没有杜长大明显,而且在随宰后时间变化的影响较小。5、不同品种生鲜猪肉质构变化的比较研究对不同品种的生鲜猪肉质构研究表明,宰后生鲜猪肉的硬度、咀嚼性、胶着性变化不仅与上市体重相关,而且与品种也有很大的关系,但弹性的变化与品种无关。金华猪不论大小,其总的硬度比杜长大要高（三个上市体重的硬度平均大1倍以上）;而咀嚼性整体高于杜长大猪（高1.5-2倍）。6、生鲜猪肉品质的电子舌评价研究利用本实验室前期研制的多频大幅脉冲电子舌,对其在宰后肉质整体特性品质辩识及评价中应用的关键技术进行了研究,通过铂金电极、金电极、钛电极、钨电极和钯电极等传感器阵列及相关检测参数的比较研究,建立了电子舌用于生鲜猪肉品质评价和新鲜度监测的方法。适合生鲜猪肉整体特性品质差异区分辨识的传感器阵列为铂金电极、金电极、钛电极、钨电极和钯电极;猪肉样品采用1∶5（g/ml）的匀浆配比检测结果较稳定;盲样检测结果表明,电子舌的辨识准确率可达85%以上（p＜0.05）;基于比较计算类别之间判别距离（马氏距离）与区分效果之间的关系,发现当不同肉样之间的判别距离d＞2时,即可实现有效区分;结合主成分分析与余弦夹角度量方式,设计了评价生鲜猪肉品质的相似度,结果表明,在预先给定标准样品的情况下,利用本研究设计的相似度,可以对不同品种、贮藏时间下的各肉样进行了有效评价,且方便有效地解决了肉品新鲜度和货架寿命的监控指标和方法。以上结果表明,宰后猪肉生鲜品质虽涉及很多因素,然后利用差异蛋白质组学、核磁共振技术来研究蛋白质表达变化和水分分布及存在状态,不仅为宰后猪肉生鲜品质的形成机理探索,而且为其评价提供了创新的思路和方法;肉品品质的电子舌研究更为现代集约化养殖下肉品质量的分级、标注以及新鲜度（货架寿命）的监测控制提供了全新的技术和方法。更多还原

【Abstract】 The quality of fresh pork meat is a complicated and multipleunit character. Studies on its intrinsic mechanism, evaluation and management are always the hottest research fields of the meat science. Such kinds of researches have been generally focused on lean rates and differences in the quality between normal and poor meat （such as PSE）. However, studies on why or how different species or feed modes especially in intensive and fast breeding conditions influence the quality of the fresh pork have not been investigated yet. Moreover, methods for effectively evaluating the quality of the fresh pork are still not well established as far as we know.Therefore, this study was conducted to evaluate the quality of fresh pork of two different species （Jinhua pigs in the original area of Zhejiang, China （termed as JH）, and hybrid pigs of Duroc×Landrace×Yorkshire, termed as DLY） with different feeding periods by using technologies of differential proteomics, low field nuclear magnetic resonance （NMR） and electronic tongue. Intrinsic mechanism underlying the determination of the quality was investigated by analyzing differences in protein expression profiles, moisture existence and antioxidative abilities among different fresh porks. In addition, feasible methods and approaches for improving the meat quality were also determined according to effects of preslaughter treatment on the quality of fresh pork. Results obtained in the present study not only provided evidence for understanding the intrinsic mechanism of the meat quality, but also valuable methods for quick monitor, evaluation and grade mark of meat quality under different conditions such as species, period, feed, breeding mode, animal welfare, slaughter and processing. The main results of the present study were as follows. 1. Effects of pre-slaughter treatment of creatine monohydrate and butafosfan on the quality of fresh porkCreatine monohydrate （CMH） supplemented into diet of DLY at the level of 500 g/t could effectively improve the quality of fresh pork in the present study. The results showed that eye muscle area and rate of longissimus dorsi muscle after CMH-treatment increased （p < 0.01） by 9.04% and 15.73%, respectively. The contents of crude fat, moisture, creatine, glycogen, inosinic acid, and inosine in treatment groups （12.02%, 71.02%, 41.63umol/g, 92.73mg/100g, 2.30mg/g and 0.39mg/g, respectively） were significantly increased or decreased （p < 0.01） as compared with respective ones of the control group （8.92%, 74.21%, 36.43umol/g, 85.21mg/100g, 1.78mg/g and 0.28 mg/g, respectively）. Moreover, CMH-treatment caused a remarkable decrease in drip loss （p < 0.01） and a significant increase in creatine kinase activity （p < 0.01） in longissimus dorsi after 24h postmortem. As for the free fat acid and glucose concentrations and creatine kinase activity in blood serum treated with CMH, they were increased （p < 0.05） by 13.33%, 5.35% and 18.84%, respectively, as compared with that of the control.Butafosfan （T1 and T2, 150 mg/kg and 300 mg/kg by body weight, respectively） supplemented into drinking water beginning at 40-48 h of pre-slaughter （once at 8h-interval） could improve availably the influence of environment such as stress on the pork quality. Drop losses of longissimus dorsi treated with T1 and T2 were decreased （p < 0.01） by 25.10% and 26.05%, respectively, as compared with that of the control. In contrast, the contents of creatine, glycogen and creatine kinase in longissimus dorsi of both treatment groups （42.13umol/g, 93.52mg/100g and 188.36U/mg in the T1-treated group; 44.53umol/g, 99.72mg/100g, 195.63U/mg in the T2-treated group, respectively） were significantly higher than the respective one of the control （37.33umol/g, 84.79mg/100g, 157.54U/mg）. 2. Differential proteomics of fresh porkThe procedure and method of differential proteomics of fresh pork were established with the pork sample preparation and experimental optimization of 2-DE. Differences in the expression profile of 345 pretein were observed between the fresh pork of JH and DLY. Among them, the profile of 33 proteins showed a significant difference as compared with their corresponding ones. Twelve points were chosen from the 33 protein points before the next measurement according to the statistical and quantity analysis and the demand of mass spectrum （MS）. Then different expressed proteins were identified using the MALDI-TOF-MS and contrastively analyzed with the peptide mass fingerprinting （PMF） and MASCOT software. The results obtained from the samples prepared at 0 h indicated that there were six different proteins （predicted protein; actin chain A; hypothetical protein; EST1 and myoglobin, chain B） and an unknown protein （No. 1101） between two species of pig. As for the samples at 24 h, seven differential proteins （β-2 globin; lymphocyte antigen; SOD;β-globin; myosin regulatory; Actin-capping protein and Dj-1 with sulfinic acid） were observed. These results suggested that meat quality of porcine longissimus dorsi might be associated with these differential proteins. However, only 70% of differential proteins were identified using MALDI-TOF-TOF-MS because of the scarcity of the protein database responding to pork meat quality.3. Effect of water-holding capacity, water status and muscle antioxidation on meat qualityTransverse relaxation time （T₂） of fresh pork sampled at different postmortern time was studied using NMR under magnetic field intensity 0.5T and resonance frequency 21-23 MHz. T₂₁ representing bound water indicated that there was no significant difference in each pork meat. However, T₂₂ representing uneasy fluid of water was higher （P < 0.05） in JH meat at all postmortem time （0h, 12 h, 24 h and 36 h） than that of DLY and showed significant trend from the same species or slaughter weight. The T₂₂ of DLY was relatively low in the present study except the samples prepared at 6 h and 30 h. As for T₂₃ representing free water, similar results were observed. The data suggested that T2 could well reflect the drip loss of each period and the detailed water distribution and variety could be obtained with the T₂ subdivision. Thus, results obtained here would provide important cues for the recognition of uneasy flow water distribution in pork meat taken from different pig species.The antioxidation and specific enzyme activities of different meat samples were also determined. As a result, the antioxidative activity of JH fresh meat represented by some factors such as SOD, GSH-Px, inhibition ability of hydroxyl radical, total antioxidant capacity and free fat acid concentration was higher than that of DLY and exhibited a positive correlation with drip loss and texture. The activity of GSH-Px in JH was 6-8 times higher （p<0.05） than that of DLY. Total antioxidant capacity （TOAC） of JH was also greatly stronger than that of DLY. For example, TOAC in the meat of 60kg-JH at 0h-postmortern point showed about 6-, 4- and 3-fold higher than that of all three weight of DLY, respectively. And TOAC in the meat of 75kg- or 90kg-JH was about 5-, 4- and 3-fold higher, or 6-, 5- and 3-fold higher than that of all three weight of DLY, respectively. With regard to TOAC in the meat of DLY, it increased with the increase in the body weight. Free fat acid concentrations in the postmortern meat were greatly associated with the pig species, while they were almost not affected by the market weight. The free fat acid concentrations in JH meat were significantly higher （p < 0.05） than those of DLY meat, being more than 3-fold higher at average.The results in meat texture, pH and drip loss showed that pork hardness, chewiness and gumminess were not only associated with the market weight, but also greatly depended on pig species. However, the meat elasticity was not associated with the pig species. Higher meat hardness, chewiness and pH were observed in JH pork as compared with those in DLY pork, respectively. The variation trends of pH in JH pork were consistent with that of DLY pork based on the same market weight. The pH values in pork of both species decreased continiously from 0h to 12h postmortem and then kept unchanged almostly. Drip loss of DLY pork at each postmortem period was higher than that of JH pork and increased obviously with the slaughter weight. As for JH pork, the drip loss also increased with the slaughter weight. However, no significant difference （p > 0.05） was observed.4. Evaluation of electronic tongue on fresh pork qualityThe core technique in whole characters for identificating and evaluating postmortem pork quality was studied using the pulse electronic tongue with multi-frequency and broad width prepared previously in our laboratory. And the corresponding methods and procedures were established. The sensors array and correlated test parameters were analyzed comparatively with the different electrodes and the adaptive electrodes were manufactured by platinum, aurum, titanium, tungsten and palladium. The results indicated that the homogenate proportion of pork samples was 1:5 （g/ml） and the identification accuracy of electronic tongue was up to 85% （p < 0.05） using the blind sample analysis. Different pork samples were identified effectively when the Mahalanobis distance （d） was more than 2 based on the correlation between distance arithmetic and identification result. Moreover, the similarity to assess the fresh pork quality was designed and determined by combinations with the principal component analysis and cosine angle metric mode. Thus, the meat samples from different species and storage time were evaluated effectively using the above similarity under the standard samples and the control indexes. Moreover, these methods were also confirmed to solve the corresponding problems of meat freshness and shelf life. Based on the findings of the presnt study, the innovative approaches and methods including differential proteomics, water-holding capacity and water status were provided to discuss the intrinsic mechanism and quality evaluation of the postmortern pork characters. Furthermore, electronic tongue developed in the present study would provide one of the new methods for meat classification, label and freshness （shelf life） control under modern intensive swine production.更多还原