【作者】 孙为正；

【导师】 赵谋明；

【作者基本信息】 华南理工大学 ， 食品科学， 2011， 博士

【摘要】 广式腊肠是我国大宗传统特色肉制品之一,以其独特的风味深受世界各地消费者的喜爱。风味是消费者选择食品的主要依据之一。肉制品的风味形成是一个复杂生化反应过程,至今还未完全明晰。脂质水解和蛋白质降解是广式腊肠加工过程中两个最重要的生化变化,对最终产品的风味起着重要贡献。本文通过分离和鉴定广式腊肠加工过程中的风味物质,阐述了广式腊肠风味物质形成规律,分析了脂类物质及其降解产物、蛋白质及其降解产物等风味前体物质对广式腊肠风味物质形成的影响,分析了风味物质的形成途径,研究了蛋白质氧化及其结构的变化,分析其对风味物质释放的影响。为科学认识广式腊肠风味物质的形成机理、传统工艺改进和参数优化提供理论依据。1、按照传统工艺制备广式腊肠,分别在烘烤0h、6h、18h、36h、54h和72h取样,将肌肉部分和肥丁部分分离,测定其脂质组成及各组分的脂肪酸组成。研究结果显示,两部分的中性脂肪的甘油酯均主要以POO+SLO+OOO(P:棕榈酸;O:油酸;L:亚油酸;S:硬脂酸)形式存在,脂肪酸组成以油酸为主;磷脂均主要以卵磷脂(PC)的形式存在,但两部分磷脂的其他组分含量差异较大。肌肉部分磷脂脂肪酸组成以多不饱和脂肪酸为主,而肥丁部分则以单不饱和脂肪酸为主。肌肉部分主要降解部分是磷脂,尤其是PC,肥丁部分中性脂肪和磷脂的降解程度相当。无论是肌肉部分还是肥丁部分,游离脂肪酸均显著升高,不饱和脂肪酸,尤其是多不饱和脂肪酸的释放速率大于饱和脂肪酸。2、研究了加工过程中脂肪酶和磷脂酶的活力变化及其与加工条件的相关性。结果显示:中性脂肪酶、酸性脂肪酶和磷脂酶活力在烘烤过程中均呈现下降趋势。但在整个过程保持一定的活力,对脂质水解具有重要影响。其活力与加工过程中的盐含量、水分活度、总糖含量、pH显著相关。3、通过对加工过程中脂质氧化情况分析,结果表明:酸价和羰基值逐渐升高,过氧化值先升高后降低,表明脂质氧化逐渐加强,脂肪氧合酶起到了一定作用。过氧化氢酶和超氧化物歧化酶在加工过程中均保持一定的活力,表明其在抑制脂质氧化中起到一定的作用。分离并测定了加工过程中小分子肽的抗氧化性能,蛋白质降解产生的小分子肽具有较强的抗氧化性,分子量>5kDa肽段的抗氧化能力强于<5kDa肽段,其抗氧化性能与脂质氧化具有较好的相关性。4、研究了加工过程中的蛋白质降解特性,结果表明:肌浆蛋白和肌原纤维蛋白降解显著,同时伴随着非蛋白态氮和碱溶蛋白的增加。广式腊肠的可溶性氮主要以>10kDa和3–5kDa的组分为主,其在加工过程中处于生成和降解的动态变化中,烘烤结束后,其含量分别占可溶性总氮的40.66%和42.05%。在加工过程中,游离氨基酸显著升高,尤其是疏水性氨基酸,游离氨基酸在加工过程中具有不同变化规律,主要与内源性蛋白酶的优先酶切位点以及氨基酸参与风味形成的活性有关。5、分析了加工过程中蛋白质的变化,结果表明:稳定广式腊肠蛋白质结构的作用力主要是氢键、疏水性相互作用和二硫键。热作用、蛋白质氧化和降解显著影响了肌浆蛋白和肌原纤维蛋白的表面疏水性,进而影响了蛋白质在加工过程中粒径的变化。其相关性表明肌原纤维蛋白的粒径变化除了上述因素外,巯基和二硫键的相互转化也是导致其粒径变化的重要原因。圆二色谱结果显示肌浆蛋白的二级结构在加工过程中由于氢键的作用增强,α-螺旋含量降低,而β-折叠含量上升。利用傅里叶变换红外光谱和拉曼光谱研究了肌原纤维蛋白在加工过程中结构变化,结果表明其结构变化显著,在加工后期发生结构重组,且蛋白质的降解起到关键作用。6、采用SPME-GC-MS分离鉴定出广式腊肠挥发性风味物质104种,主要以乙醇和乙酯类化合物为主,在不同阶段分别占到16.94%–50.12%和30.01%–65.54%。广式腊肠风味形成的主导因素是配方中加入较大量的曲酒及脂质降解和氧化所致;此外,氨基酸降解、微生物作用和美拉德反应对广式腊肠风味形成有着积极作用。聚类分析结果显示,18-36h和储存阶段是广式腊肠风味形成的重要阶段。加工过程中蛋白质结构、分子间相互作用力的变化以及脂质含量变化等因素对风味物质的释放具有重要影响。7、分别在90℃(M1)、100℃(M2)、110℃(M3)、120℃(M4)制备了美拉德反应物,研究了添加美拉德反应物及混合菌种发酵对广式腊肠品质及风味的影响,结果表明:美拉德反应物影响广式腊肠的脂质降解和氧化,其中M1和M3可抑制游离脂肪酸的释放和延长脂质氧化的诱导期,提高广式腊肠的抗氧化性能;M1和M2对广式腊肠的风味有积极作用,但M3和M4对其有负面影响。混合菌种发酵(S.condimenti和M.caseolyticus)可显著降低广式腊肠的亚硝酸盐残留量,促进加工初期乳酸菌和加工后期酵母菌的生长,对广式腊肠的发色和风味形成具有很好的作用。更多还原

【Abstract】 Cantonese sausage is a traditional Chinese meat product that is well accepted by consumers over the world due to its unique qualities. Flavor is considered as one of the most important attributes determining the acceptance of food by the consumer. The development of flavour in meat products is a very complex process, not yet fully understood due to the high number of reactions involved. Lipolysis and proteolysis constitute two of the most important biochemical mechanisms during the processing of Cantonese sausage with relevant consequences for the final sensory quality. The objectives of this work were to identify the predominant volatile components in Cantonese sausage and to track its changes during the ripening and storage. The mechanisms for the formation of these volatile compounds were also discussed. Changes in lipolysis and proteolysis, and substrates released during lipolysis and proteolysis were investigated to evaluate the impact of these changes on the formation of Cantonese sausage volatile compounds. The results will help to understand which substances are responsible for the flavor of Cantonese sausage and the relevant mechanism. This information will be useful to select the technological strategies to enhance flavor characteristics of Cantonese sausage.1. Cantonese sausage was prepared according to traditional practices. Samples were periodically taken at 0th, 6th, 18th, 36th, 54th and 72nd h for analyses. Lean pork and back fat were separated. Total lipids were fractionated, and fatty acids of each fraction were determined. The results indicated that the main triacylglycerols of neutral lipid were POO+SLO+OOO (P: palmitic, S: stearic, L: linoleic, O: oleic) for both lean pork and back fat, the main fatty acid was oleic acid. PC was the most abundant in phospholipids for both lean pork and back fat, and the other fractions were different. The most abundant fatty acid in phospholipids fraction was polyunsaturated fatty acids (PUFA) in lean pork, while monounsaturated (MUFA) in back fat. The lipolysis of phospholipids in lean pork, especially that of PC was the main contributor to the increase of free fatty acids, while in back fat neutral lipid and phospholipids were equivalent to release free fatty acids. Total free fatty acids, whether in lean pork or in back fat, increased (P < 0.05) greatly through the processing of Cantonese sausage. The rate of release of unsaturated fatty acids, especially PUFA was higher than saturated fatty acids.2. Lipase and phospholipases were extracted from Cantonese sausages at different processing periods and to assay their activities. Changes in the main indices during processing were also investigated to evaluate their relationships with lipase and phospholipases activities. The activities of these enzymes decreased throughout processing. Lipolysis influenced by lipase and phospholipasesdue, which remain active all along the process. A highly significant correlation was measured between their activities and salt content, water activity, total carbohydrate content, pH.3. Lipid oxidation was estimated. Results showed that lipid oxidation occurred during processing. The acidity value and TBARS value of Cantonese sausage increased during the process (P < 0.05), and the peroxide value increased at the first stage (P < 0.05), and decreased at the last stage (P < 0.05). The activity of antioxidant enzymes was determined. The activity of catalase and SOD remain active all along the process, indicating that lipid oxidation was retarded. Two peptide fractions (P1 and P2) were isolated from Cantonese sausages at different processing periods and their antioxidant activities were assayed. A good antioxidant activity was observed for each peptide fraction. Peptides with molecular weights more than 5 kDa possess a higher antioxidant activity than peptides with molecular weights less than 5 kDa. A significant (P < 0.05) correlationship between lipid oxidation and antioxidant activity was observed.4. Proteolysis was investigated during Cantonese sausage processing. Changes in the protein composition of Cantonese sausage were illustrated by the progressive decrease in sarcoplasmic and myofibrillar protein fractions, accompanied by an increase in the alkali-soluble and non-protein fractions. Peptides of > 10 and 3–5 kDa were the main WSN fractions, and their contents at final products were 40.66% and 42.05% of total WSN, respectively. A stable balance between the formation and degradation of peptides was observed. The total free amino acid concentration increased during Cantonese sausage processing, especially for hydrophobic amino acids. The discrepancies in changes of individual amino acids were due to restriction Enzyme cutting site of endogenous enzyme and active activities of individual amino acids. 5. Dynamic evolvement of Cantonese sausage protein was estimated. The protein solubility determinations suggested that hydrogen bonds, hydrophobic interactions and disulfide bonds gave rise to a three-dimensional network structure of Cantonese sausage proteins. Heat treatment, protein oxidation and proteolysis were implicated at least in changes of protein surface hydrophobicity of sarcoplasmic proteins and myofibrillar proteins, and further resulting in protein particle size changes. Not only above reasons but also the SH/S-S exchange reaction could lead to changes of myofibrillar proteins particle size. Results from CD suggested thatα-helical content markedly decreased, whileβ-strand increased. Structural changes of myofibrillar proteins from raw muscle and Cantonese sausage at different processing periods were elucidated using Fourier transform infrared (FTIR) spectroscopy and Raman spectroscopy. Structural changes of myofibrillar proteins were significant. The rebuilding process of myofibrillar proteins secondary structures was observed at the rest stage due to proteolysis.6. The volatile compounds of Cantonese sausage at different stages of processing and storage were analyzed by gas chromatography/mass spectrometry (GC/MS) with solid-phase microextraction (SPME). One hundred and four volatile compounds were identified. Esters and alcohols were the most important chemicals in all the samples, accounting for 16.94–50.12% and 30.01–65.54% of the total area, respectively. Alcohol and lipid oxidation played a key role on Cantonese sausage flavor formation. Amino acid catabolism and microbial action also plays an active role in the formation of volatile compounds. The hierarchical cluster analysis results show that the period of 18–36 h and storage were important to volatile compounds formation. Releases of volatile compounds of Cantonese sausage were influenced by structural changes of Cantonese sausage proteins, the distribution and extents of protein inter- and intra-molecular interactions and lipid content changes.7. The Maillard reaction products (MRPs) were prepared at 90 (M1), 100 (M2), 110 (M3) and 120°C (M4), respectively. The effects of MRPs and mixed starter cultures on Cantonese sausage quality characteristics and flavour were investigated. Addition of MRPs affected lipolysis and lipid oxidation during Cantonese sausage storage. M1 and M3 had an inhibitory action on total free fatty acids levels and extended the induction period. Antioxidant quality of Cantonese sausage treated with M1 and M3 was improved. Cantonese sausages treated with M1 and M2 showed good sensory properties. However, M3 and M4 had a negative (P < 0.05) effect on the flavour of Cantonese sausages compared to control. The residual nitrite content of Cantonese sausage decreased by inoculation with mixed starter cultures (S.condimenti and M.caseolyticus). Mixed starter cultures could accelerate growth of Lactic acid bacteria during initial processing stage and yeast during last processing stage. Inoculation with mixed starter cultures had effective on colour formation and flavour of Cantonese sausages.更多还原