【作者】 邱春江；

【导师】 夏文水； 薛长湖；

【作者基本信息】 江南大学 ， 水产品加工及贮藏工程， 2014， 博士

【摘要】 我国是淡水鱼养殖大国，鲢鱼（Hypophthalmichthys molitrix）因其生长周期短、抗病能力强、产量高等特点，养殖产量位居第二。土腥味严重一直是制约其深加工的技术“瓶颈”，脂肪氧合酶在鱼肉脂肪氧化和风味形成中有重要作用，研究发现鲢鱼中所含的脂肪氧合酶主要存在形式是12-isozyme，并且与鱼腥味紧密相关。鲢鱼质优价廉，凝胶形成能力较强，较适合加工成鱼糜，是目前加工前景最被看好的淡水经济鱼种。加工成鱼糜是提高其附加值的有效途径，也是解决鱼糜原料短缺的重要措施，但是，鲢鱼鱼糜制品在加工过程中容易发生凝胶劣化，从而导致鱼糜制品的品质下降。目前，越来越多的研究表明肌原纤维结合型丝氨酸蛋白酶（Myofibril-bound Serine Proteinases, MBSP）对肌原纤维蛋白，尤其是对肌球蛋白重链（MHC）的分解，是引起鱼糜制品弹性下降的主要原因。鲢鱼可为我国提供丰富的优质肌肉蛋白质来源，肌原纤维蛋白是肌肉蛋白中最重要的结构蛋白，肌肉蛋白的很多功能特性直接由它决定。近年来超高压技术在食品的杀菌、灭酶及改善食品品质等应用方面是最有潜力和发展前途的重点开发技术之一，超高压处理改变蛋白质构象并引起蛋白质变性、聚集或胶凝，进而影响其质构品质。本研究的目的是采用超高压技术对鲢鱼鱼腥味产生的关键酶—脂肪氧合酶的钝化作用抑制加工过程中腥味产生，同时不仅通过直接影响肌原纤维蛋白的超微结构来影响质构，还可以通过对热稳定组织蛋白酶--肌原纤维结合型丝氨酸蛋白酶的作用以防止凝胶劣化，探讨超高压钝酶与改善品质等方面的机制和在淡水鱼加工中应用的可能性。首先研究了超高压协同温度处理鱼腥味产生关键酶--脂肪氧合酶（LOX）的失活变化趋势，总体上，压力对鲢鱼脂肪氧合酶有显著影响，同时钝化酶的效果随温度升高而增加明显，在实验设定的所有温度范围内，随着处理压力的增大残存酶活均不断下降，而且处理压力越高，随着时间延长，LOX酶活残存率就越低。研究表明鲢鱼脂肪氧合酶的超高压失活是不可逆的并且符合一阶反应动力学规律。采用响应面分析建立了鲢鱼脂肪氧合酶粗提液的超高压LOX失活速率常数（k）失活动力学模型。经由响应面回归分析，采用CCRD设计方法，对压力和温度分别以X1及X2表示，得到超高压LOX失活速率常数（k）标准回归方程为：k0.2780.3991E004X0.01X6.167E006X X3.608E007X22121219.525E005X2采用超高压技术研究了不同压力不同保压时间下对肌原纤维结合型丝氨酸蛋白酶（MBSP）活力影响趋势，发现压力对MBSP酶活力有显著影响，随着时间延长，酶活的降低就越明显。结果表明MBSP的超高压失活同样符合一阶反应动力学规律。相对于粗酶液，肌原纤维蛋白对肌原纤维结合型丝氨酸蛋白酶的酶活具有一定的保护作用。通过SDS-PAGE电泳图谱分析表明，肌球蛋白重链（MHC）降解程度与高压处理导致肌原纤维结合型丝氨酸蛋白酶的酶活降低高度吻合。动态流变特性研究表明，高压处理对肌原纤维结合型丝氨酸蛋白酶活性以及肌原纤维蛋白影响显著，首次明确了300MPa（≥10分钟）的工艺条件就可以对肌原纤维结合型丝氨酸蛋白酶显著失活从而有效防止凝胶劣化。以鲢鱼肌原纤维蛋白作为研究对象，利用示差扫描量热法（DSC）、表面疏水性、紫外吸收光谱及其二价导图谱、内源荧光光谱、圆二色谱、拉曼光谱等方法研究超高压加工对其构象影响。DSC分析表明，当处理压力超过200MPa后，肌原纤维蛋白构象稳定性随压力不断增加而不断变差。内源荧光光谱、紫外吸收光谱及其二价导图谱分析表明，当处理压力超过300兆帕时，更多的疏水性氨基酸残基所处微环境极性增强，这种变化对表面疏水性影响明显。圆二色光谱分析表明，随着处理压力的升高，鲢鱼肌原纤维蛋白二级结构的α-螺旋含量逐渐降低，发现400MPa处理10分钟，α-螺旋破坏比较严重，同时这种变化进一步验证了蛋白质的表面疏水性和α-螺旋结构呈现负相关。拉曼光谱分析表明鲢鱼肌原纤维蛋白二级结构、三级结构均有改变。超高压影响蛋白构象变化并引起蛋白变性与胶凝等特性的改变，进而影响其质构品质，从而为开发鲢鱼新型调理食品奠定基础。通过对超高压与热处理对白鲢鱼糜凝胶性能的比较研究证实超高压加工的白鲢鱼糜制品鱼腥味较淡，在凝胶强度、弹性等方面都优于传统热加工，电镜扫描发现300MPa处理20min的鱼糜凝胶细腻，网状结构更趋紧密，所有这些与理论研究基本吻合。但在硬度方面超高压处理较热处理其硬度变小，显示了与理论研究相矛盾的地方。更多还原

【Abstract】 China is the largest producer of freshwater fish in the world, and silver carp(Hypophthalmichthys molitrix) is one of the main freshwater fish species. However, it issusceptible to lipid oxidation and the subsequent development of rancid and fishy odourduring processing and storage. It has been reported that Lipoxygenase (LOX) primarilyaccounting for peroxidation participated in enzymatic reaction and resulted in lipid oxidationand the rapid formation of fishy odour in silver carp processing. High hydrostatic pressure(HHP) processing is an alternative method for enzyme inactivation. Inactivation kinetics ofendogenous lipoxygenase in crude silver carpextract was studied for thermal (50–80°C,ambient pressure) and high hydrostatic pressure combined with heat treatment (300–700MPaat50–80°C) process conditions. LOX inactivation followed first-order kinetics at allpressure–temperature combinations used. Moreover, LOX inactivation rates became lesspressure dependent with increasing pressure at all temperatures studied and also showed lesstemperature dependence of the enzyme inactivation rate at various high pressure levels tested.It is evident that inactivation kinetics is complex affected by both pressure and temperature.Therefore, response surface methodology (RSM) was attempted to investigate combinedtemperature–pressure processing conditions on LOX inactivation kinetics in crude silver carpextract samples. In conclusion, the LOX inactivation rate constant was modelled as a functionof both temperature and pressure conditions by a quadratic polynomial equation as follows:k0.2780.3991E004X0.01X6.167E006X X3.608E007X29.525E005X2121212Silver carp is a popular cultured freshwater fish in China, and becomes a potential rawmaterial for surimi product. However, silver carp surimi exhibits considerable gel softening(modori) and myofibril-bound serine proteinases (MBSP) involved in the degradation ofmyofibrillar proteins have been assumed to be crucial to the modori phenomenon.High-pressure technology has demonstrated its potential application to control enzyme-relatedseafood texture deterioration. Herefrom, it could be further applied to the freshwater fishprocessing and the effect of high pressure treatments on the activity of MBSP implicated intexture deterioration in myofibrils and crude enzyme extracts was evaluated. The inactivationkinetics of MBSP pressured from200MPa to500MPa at room temperature (20°C) werefitted first-order kinetics, The extent of enzyme inactivation was lower in silver carpmyofibrils in comparison with the crude enzyme extracts from myofibrils showing aprotective effect against the high pressure treatment. The effect of HHP on MBSP myofibrillardegradation shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE) was associated with a corresponding decrease coincided with the elevated highpressure levels in intensity of myosin heavy chain (MHC). Dynamic rheological measurements indicated that high-pressure treatment exerted considerable influence on MBSPactivities as well as proteins (mainly myofibrils), resulting in structural modifications andtexture changes. In this study,300MPa (≥10min) was the process condition (pressure andtime) that caused apparent MBSP inactivation derived from the first order kinetic model andhad the efficacy in controlling silver carp texture deterioration both verified by SDS-PAGEand dynamic rheological measurements.Silver carp is an abundant protein resource and is widely used as a material for processedfoods. Myofibrillar proteins play an important role in the quality of fish products because ofits responsibility for imparting textural attributes to fish muscle and endowing functionalproperties of fish meat such as gel-forming ability, emulsifying property, and water-holdingability. Conformational changes of myofibrillar proteins have an enormous potential tomanufacture desirably processed fish meats with lower cost and more desirable nutritionalproperties. Modification of myofibrillar proteins induced by high pressure processing (HPP)has been investigated at pressures ranging from200to500MPa for10min at20°C. Influenceof high pressure on conformational changes of myofibrils was studied by means of differentialscanning calorimetry (DSC), surface hydrophobicity measurement, ultraviolet absorptionspectra and second-derivative spectroscopy, intrinsic spectrofluorimetry, Raman spectroscopicanalysis and circular dichroism (CD). DSC analyses revealed that the conformational stabilityof myofibrils is reduced to different extents when they are exposed to a pressure over200MPa. More sulfydryl groups as well as hydrophobic regions and amino acid residues whichhad ultraviolet absorbance had been found significantly after HPP (≥300MPa). Changes inthe Raman spectra were interpreted as the occurrence of secondary structural changes inpressure-induced myofibrillar proteins. CD analysis results demonstrated that α-helix wasdestroyed after processed at400MPa for10min. The results implicated that HPP can be usedas a possible means of improving the functional properties for growing demand for healthierfish products.The comparative study between HHP and thermal treatment on silver carp surimi gelproperties of silver carp surimi confirmed that pressured surimi products with smallerunpleasant fishy taste are better in gel strength, elasticity and so on than the traditionalthermal processed, and SEM showed that surimi pressured at300MPa for20min is morecompacted in reticular structure and refined in gel, which are consistent with theoreticalresearch. But in terms of hardness the pressured surimi gel is the hardness smaller than theheat treated, which is contradictory to our study.更多还原