摘要
超高压技术是21世纪生物制品和食品领域最有潜力和发展前途的技术之一。超高压处理对肉及肉制品产生重要影响,如抑制微生物生长、改变肉的颜色、对肉品组织结构产生影响、对风味及蛋白质凝胶品质有重要影响。
肌球蛋白是肉品中含量最高最重要的蛋白质,肌球蛋白的凝胶特性决定产品的质量,对产品的质构、赋形、保水性和保留其他食品成分有重要意义。本研究以兔腰大肌肌球蛋白为对象,研究超高压与热结合处理对肌球蛋白凝胶特性的影响,初步探讨超高压凝胶形成机理。研究内容包括：(1)研究不同加工条件及加入功能性食品胶对超高压-热凝胶特性的影响；(2)运用傅立叶红外光谱(FT-IR)技术研究超高压条件下蛋白质二级结构的变化,探讨蛋白质结构与功能特性之间的关系；(3)利用非变性电泳、扫描电镜等技术初步研究超高压肌球蛋白凝胶形成机理。为超高压技术在肉品中的应用提供技术支持和理论指导。具体研究内容和结果如下：
1不同加工条件对超高压-热肌球蛋白凝胶特性的影响
研究不同pH、NaCl浓度、保压时间、超高压温度对超高压-热肌球蛋白凝胶质构特性、保水性及微观结构的影响。结果显示,pH5.0和5.5时未见形成网络凝胶；pH6.0～7.0时,形成有序的丝状、多孔且孔径均一的细致网络凝胶。凝胶的硬度随pH的增加逐渐下降,pH6.0时,硬度最大。超高压凝胶保水性在pH5-7范围内,先降低后升高,在pH5.5时保水性最差。氯化钠浓度在0.1～0.5mol·L-1范围内,凝胶硬度和保水性逐渐增加,0.6mol·L-1时两者都显著下降。低离子强度(0.1～0.2mol·L-1)时,不能形成硬度较好的凝胶,0.5mol·L-1时凝胶硬度达最大,凝胶网络结构最好。保压时间对凝胶质构和保水性影响不显著。超高压处理温度在20-60℃范围内,随温度升高硬度显著增加,20℃时凝胶为颗粒状连接,40℃时凝胶孔洞均一、有序,结构光滑、细腻,保水性最高。通过主成分分析研究不同加工条件下凝胶7个质构指标和保水性的关系,结果表明,质构特性与保水性存在很强的相关性。
2添加魔芋胶、酪蛋白酸钠对超高压-热肌球蛋白凝胶特性的影响
模拟经典乳化肠配方,设四个处理组：肌球蛋白、肌球蛋白：酪蛋白酸钠质量比为15：4、肌球蛋白：酪蛋白酸钠：魔芋胶为15：4：2、肌球蛋白：酪蛋白酸钠：魔芋胶为15：4：4。分别进行两种处理：低压结合热处理(200MPaa,5min+80℃,40min)与超高压处理(500MPa,60℃,30min),比较两种凝胶特性的差异。结果表明,魔芋胶和酪蛋白酸钠结合使用,改善凝胶质构,提高保水性。肌球蛋白：酪蛋白酸钠：魔芋胶比例为15：4：2时凝胶质构特性最好。扫描电镜结果显示,加入酪蛋白酸钠,凝胶微结构发生显著改变,呈小孔状,结构有序、均一、光滑、结实的网络结构。加入魔芋胶的凝胶结构粗糙、孔径变大。比较低压-热结合凝胶与超高压凝胶差异结果显示,低压-热结合凝胶质构特性更好,保水性更高。
3不同压力对肌球蛋白凝胶特性及蛋白质二级结构的影响
肌球蛋白溶液先经100～600MPa压力预处理10min,处理温度20℃,后加热制备凝胶,以未超高压处理组为对照组,测定凝胶质构和保水性,采用傅立叶红外光谱法测定肌球蛋白二级结构变化,然后通过相关系数矩阵法研究肌球蛋白7种质构特性、保水性与各二级结构含量的关系。结果表明,不同超高压处理对肌球蛋白凝胶硬度的影响不显著,对凝胶保水性影响显著,随着压力升高,凝胶保水性逐渐下降。与对照组相比,100、200MPa处理的凝胶保水性无显著差异；与对照组相比,300MPa以上处理的凝胶保水性显著减少。超高压致使肌球蛋白分子结构展开,使蛋白质p-折叠等有序结构减少而p-转角等无序结构增加。
从相关性分析结果得出,凝胶的粘附性、弹性、保水性与各二级结构之间存在显著的相关性。
4超高压肌球蛋白凝胶形成机理的初步研究
肌球蛋白溶液(20mg·mL-1,0.6mol·L-1NaCl.pH6.5)在各种压力(100～400MPa)下处理10min,温度20℃,以未超高压处理组为对照。通过测定蛋白质表面疏水性、自由巯基含量、分子变化(native-page电泳法)和超微结构(扫描电镜法)揭示凝胶形成机理。结果表明,表面疏水性在100MPa变化不显著,200MPa以上,疏水性显著增加；自由巯基含量在300MPa以上增加显著。超微结构显示,200MPa以下的肌球蛋白凝胶呈丝状结构,孔洞小而多,300MPa以上的凝胶呈球状聚集。Native-page电泳结果显示,肌球蛋白重链参与了凝胶化过程。
初步研究超高压肌球蛋白凝胶形成机理为：超高压改变肌球蛋白二、三级、四级结构,暴露出巯基和疏水基团,减少与水分子的接触,同时增加蛋白质和蛋白质之间的相互作用,然后蛋白质分子相互聚集形成凝胶。
The ultra-high pressure technology is considered as one of the most potential and promising technologies in the field of biological products and food filed in the21st century. Ultra-high pressure processing has important effects on meat and meat products such as the inhibition of microbial growth, changing the color of the meat, impacting on the organizational structure,flavor of meat and changing protein gel quality.
Myosin which has highest content is the most important protein in meat. The quality of meat is determined by property of myosin gel in that it contributes to textural properties, shaping the product, retaining water, and holding other food components in the product. The material was rabbit psoas myosin. This research studied the effects of ultra-high pressure combining with heating on myosin gel properties and the mechanism of ultra-high pressure induced gels formation. The contents of this research included:(1) studying the effects of ultra-high pressure combining with heating on myosin gel properties with different processing conditions and adding functional foods gum;(2) studying changes of secondary structure of myosin by Fourier transform infrared spectroscopy (FT-IR) after ultra-high pressure pretreatment10min at20℃from100to600MPa then heating and studying the relationship of the protein structure and functional properties of myosin gel;(3) studying the preliminary mechanism of ultra-high pressure induced myosin gels formation with various pressure processing by determining changes of chemical force, native-page electrophoresis, scanning electron microscopy.
The aim was to provide technical support and theoretical guidance for ultra-high pressure processing used in the meat industrial production.The detailed contents and results are shown as follows:
1Effects of different processing conditions on the characteristics of ultra-high pressure-heating myosin gel properties
The effect of various pH, concentration of NaCl,keeping time of ultra-high pressure and ultra-high pressure temperature on textural properties (TPA), water holding capacity (WHC) and micro-structure of myosin gels was investigated. The results showed that at pH5.0and5.5there are not well formed network gel. At pH6.0-7.0, the gels have ordered filaments, porous and uniform pore size network structure. Hardness of gels increased with pH decrease. The hardness was highest in pH6.0. WHC of ultra-high pressure gel in the pH5-7, was first decreased then increased. The worst WHC myosin gels were at isoelectric points of pH5.5. Hardness and WHC of gels gradually increased when concentration of sodium chloride at0.1～0.5mol·L-1range. At sodium chloride concentration0.6mol·L-1, WHC and hardness were both decreased significantly. Low ionic strength (0.1-0.2mol·L-1), good gel was cannot form while at0.5mol·L-1, the gel hardness and WHC was highest. Keeping time of high pressure did not significantly affect gel textures and WHC. With the increase of ultra-high pressure processing temperature from20to60℃, hardness increased significantly.20℃gels had filament link while40℃gels had good network, uniform, orderly holes with smooth and delicate structure, accompanied maximum water holding capacity. The relationship of the7kinds of TPA variables and WHC were determined by principal component analysis.Significant correlations were found between TPA and WHC.
2Effects of adding konjac glucomannan and sodium caseinate on ultra-high pressure-heating gels properties of myosin
This experiment with simulated the classic sausage formulations, was divided into four treatment groups:myosin;myosin:sodium caseinate (SC) mass ratio of15:4; myosin: sodium caseinate (SC):konjac glucomannan(KGM),15:4:2;myosin:sodium caseinate (SC):konjac glucomannan (KGM) is15:4:4.Two kinds of processing respectively:low pressure-heating gel(200MPa,5min+80℃,40min)and ultra-high pressure (500MPa,60℃,30min),in order to compare the differences of two gel properties.The results showed that SC and KGM can greatly improve TPA and WHC of the myosin gel.When the ratio of gel of myosin:sodium caseinate (SC):Konjac glucomannan(KGM) was15:4:2,the properties of gels was best. Results of scanning electron microscopy showed SC significantly improved the structure of gels, showing a small,porous,and orderly, uniform, smooth,and strong network structure. Adding KGM, gel structure was coarse with big pore size. Comparing with low pressure-heating and the ultra-high pressure gels, different gel properties was found:low pressure-heating of gel had good TPA properties and higher WHC.
3Effects of various pressures on gel properties and the secondary structure of myosin
TPA, WHC and secondary structure of the gel was determined after ultra-high pressure pretreatment10min at20℃,from100to600MPa then heated, the not ultra-high pressure treated sample as the control. Secondary structure, TPA and WHC of the gel were determined with the Fourier transform infrared spectroscopy methods. The relationship of the protein structure and functional properties of myosin gel was determined by correlation matrix. The results showed that the hardness of gel did not change significantly with various ultra-high pressure treatments. WHC decreased with the pressure increased. Compared with the control, the WHC of100and200MPa have no significant difference. Compared with the control, the WHC of above300MPa have a significant decrease.
The structure of myosin was unfolded by ultra-high pressure. The order structure such β-sheet decreased while disordered structure such as β-turn increased.
Significant correlations were found among in springiness, adhesiveness, WHC, the secondary structure of gels.
4The preliminary mechanism of high pressure-induced myosin gels
Myosin (20mg·mL-1of0.6mol·L-1NaCl, pH6.5) was subjected to various pressures (0.1～400MPa) at20℃for10min to investigate pressure-induced gels mechanism by measurements of protein surface hydrophobicity, free sulfhydryl contents, molecular size by native polyacrylamide gel electrophoresis and ultra-structure using SEM. Surface hydrophobicity had no significant increase at100MPa but had significant increase upon200MPa. Free sulfhydryl groups had little increase below200MPa,but had significant increase at300and400MPa. Ultra-structure revealed that gels below200MPa were filament structure with many small cavities,while gels upon300MPa were globular aggregates with big cavities.Native-page electrophoresis showed that myosin heavy chain was involved in the process of gelation.
Preliminary mechanism of myosin gels formation in ultra-high pressure treatment was that the secondary, tertiary, quaternary of myosin were changed by ultra-high pressure, and the sulfhydryl and hydrophobic groups were exposed. The contact between myosin and water were reduced, while the interaction of protein molecules with each other was increased. Then the protein molecules were aggregated to form a gel.
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