超高压对大豆脂肪氧合酶、营养抑制因子和蛋白性质的影响

摘要近二十年来超高压食品加工技术飞速发展并逐渐步入产业化。

但是，和其他的新技术一样，超高压技术的产业化突破必须通过建立一个评价其对食品安全、质量方面影响的科学基础来实现，这样的定量评价无论是对满足立法安全需要还是对满足目前消费者的食品质量需求都是必不可少的。

大豆富含丰富的蛋白质和合理的氨基酸组成，是国际上公认的一种全营养食品。

大豆蛋白具有重要的营养价值和理化及功能特性（如凝胶性、乳化性、起泡性等），所以被作为一种具有加工功能性的食品添加用中间原料而广泛应用于食品行业。

但大豆中含有多种酶类和一些抗营养因子，传统的热处理技术虽然能有效杀死致病微生物和钝化酶类，但是同样会导致一些不良的化学变化从而影响产品的品质。

本研究的目的是利用新型超高压加工技术处理豆浆及大豆分离蛋白溶液，初步探讨超高压处理对豆浆品质、大豆脂肪氧合酶失活、营养抑制因子失活、大豆分离蛋白理化及功能性质的影响，为超高压加工技术在大豆制品加工中的应用、大豆蛋白的改性以及食品安全提供理论参考。

以豆浆和脂肪氧合酶粗提液为对象，研究了大豆脂肪氧合酶的超高压失活动力学。

结果表明，大豆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；在某一恒定的温度下，脂肪氧合酶的失活速率常数k 随着超高压处理压力的增加而增大，表明增加压力可以加快脂肪氧合酶失活；在某一恒定的压力下，脂肪氧合酶的失活速率常数在10-20℃出现最小值，表明Arrhenius 方程不能适用于整个温度区间；在中温区域（20℃≤T≤60℃），温度对脂肪氧合酶失活速率常数的影响随着压力的增加而降低；而脂肪氧合酶失活速率常数对压力的敏感性大约在30℃最大。

豆浆体系中脂肪氧合酶的失活速率常数要比粗酶提取液中小，但是从动力学角度来看，体系的不同并没有影响到脂肪氧合酶超高压失活的反应级数以及失活速率常数的温度敏感性和压力敏感性。

在此基础上，采用两种完全不同的数学模型来描述压力与温度对脂肪氧合酶超高压失活速率常数的影响。

结果表明，不管以Eyring 方程为起点建立的经验数学模型还是以Hawley 提出的热力学方程为基础建立的热动力学数学模型，都能够成功地模拟两个体系中压力与温度对大豆脂肪氧合酶超高压失活速率常数的影响，但热动力学模型要比经验数学模型更加精确。

以豆浆作为研究对象，研究并优化了大豆营养抑制因子的超高压失活条件。

同样的超高压处理条件下，尿素酶发生失活的温度（室温）低于胰蛋白酶抑制剂（≥40℃），温度升高、压力增大和时间延长有利于营养抑制因子的失活。

中心组合旋转设计优化显示，在所考察的因素中，对尿素酶和胰蛋白酶抑制剂超高压失活的影响程度从大到小的排序为压力、时间、温度；理想的大豆营养抑制因子的超高压失活条件为压力750MPa、温度60℃、时间5min。

两种不同pH 缓冲溶液体系中超高压处理对大豆分离蛋白理化及功能性质的研究发现，pH3.0 的Gly-HCl 缓冲溶液中超高压处理提高大豆分离蛋白溶解度的程度显著大于pH8.0 的Tris-HCl 缓冲溶液。

游离巯基含量和蛋白质表面疏水性的测定结果表明，压力I处理很可能导致了蛋白质结构的展开、内部疏水基团的暴露以及新的二硫键的形成。

超高压处理前后大豆分离蛋白的体积排阻高效液相、动态光散射技术和凝胶电泳分析发现，在pH3.0 的Gly-HCl 缓冲溶液中超高压处理使大豆分离蛋白（包括不溶部分）发生结构重组，形成可溶性大分子聚集体；在pH8.0 的Tris-HCl 缓冲溶液中超高压处理可能造成了可溶性大分子聚集体的解聚。

这些现象说明，由于在不同pH 缓冲溶液中大豆分离蛋白的存在形式不同，可能造成了其在超高压处理过程中产生不同的结构变化。

超高压处理可以改善大豆分离蛋白的乳化性能和起泡性能，这种改善作用在pH3.0Gly-HCl 缓冲溶液中得到显著体现，这是因为超高压处理不但显著提高了酸性条件下大豆分离蛋白的溶解度，而且还使其表面疏水性得到显著的提高。

超高压能够诱导一定浓度的大豆分离蛋白溶液形成凝胶。

与处理时间以及温度相比，压力的变化对凝胶质构性质的影响最大。

凝胶的硬度随着压力的增加、温度的升高以及处理时间的延长而增大。

12%大豆分离蛋白溶液在20℃、700MPa 条件下处理15min 后所形成的凝胶，其硬度已经超过了常压下85℃热处理20min 后形成的热凝胶的硬度值。

未处理、热处理和超高压处理豆浆的理化、风味、色泽和流变等性质比较表明，超高压处理和热处理不影响豆浆的pH 值和电导率；热处理提高了豆浆的表观粘度，这是豆浆中蛋白质的热聚集效应造成的；热处理和超高压处理对豆浆色泽影响较小；超高压处理降低豆浆中已生成挥发性风味成分的效果甚微，打浆前的超高压处理可有效降低豆腥味；超高压处理和热处理不影响豆浆中蛋白质的氨基酸组成；热处理和超高压处理减小豆浆的流态特性指数，流变特性趋向假塑性流体；热处理后豆浆样品的稠度系数显著提高，说明热处理显著增加豆浆的表观粘度。

总之，热处理对豆浆样品流变特性的影响远大于超高压处理，这可能是由于热处理导致豆浆中蛋白质展开、变性和聚集的程度远大于超高压处理。

关键词：超高压；脂肪氧合酶；营养抑制因子；失活；动力学；模型；大豆蛋白ⅡAbstractOver the last twenty years, high pressure technology has developed rapidly and its applications in food processing are gradually stepping into industrialization. But, the industrial breakthrough of high pressure technology in food processing, like that of other novel technologies, can only be forced by the establishment of a scientific basis to assess its impact on food safety and quality aspects. Such quantitative assessment is indispensable to fulfill legislative food safety requirements as well as to respond to the consumers’ increasing demand for high quality food. Soybean is generally acknowledged as full-nutrient food because of its abounding proteins and rational amino acids composition. And soybean protein is widely used as functional food ingredient or food additive because of its good nutritional values and excellent functional properties, such as gelatin, emulsification, foamability etc. Besides its favorable features, soybean is known to contain some adverse enzymes and nutritional inhibitors. Traditional heat treatment could effectively inactivate these enzymes and destroy microorganisms. However, it would simultaneously cause some adverse chemical changes which could affect the final quality of products. The purpose of this study is to investigate the effects of high pressure treatment on the quality of soymilk, the lipoxygenase and nutritional inhibitors in soybean, and also the physicochemical properties of soybean protein isolates, thus provide some theoretical references for the application and safety assessment of high pressure treatment in soybean products processing, and possible modification of soybean proteins by high pressure treatment.The high pressure inactivation of lipoxygenase in soy milk and crude soybean extract was studied in the pressure range 200-650 MPa with temperature varying from 5 to 60 ℃. And the results suggest that, for both systems, the isobaric-isothermal inactivation of lipoxygenase was irreversible and followed a first-order reaction at all pressure-temperature combinations tested. In the entire pressure-temperature area studied, the lipoxygenase inactivation rate constants increased with increasing pressure at constant temperature for both systems, indicating an acceleration of the lipoxygenase inactivation by increasing pressure. At constant elevated pressure, lipoxygenase exhibited the greatest stability around 10-20 ℃ in both systems, indicating the Arrhenius equation not to be valid over the entire temperature range. For both systems, the temperature dependence of the lipoxygenase inactivation rate constants in mild temperature area (20℃≤T≤60℃) decreased with increasing pressure, while the highest sensitivity of the lipoxygenase inactivation rate constants to pressure was observed at about 30℃. The lipoxygenase inactivation rate constants in soy milk system were somewhat smaller than those in crude soybean extract, but on a kinetic basis, neither the reaction order of inactivation nor the pressure and temperature sensitivities of the inactivation rate constants were influenced by the different levels of food complexity between the twosystems.Based on thoroughly studies of the kinetics for high pressure inactivation of lipoxygenase, two absolutely different mathematical models were used to describe the combined pressure-temperature dependence of the high pressure inactivation rate constants for lipoxygenase in both systems. Results showed that the pressure-temperature dependence of the high pressure inactivation rate constants for lipoxygenase in both systems could be described by either the thermodynamic kinetic model which built on the basis of the thermodynamic equation proposed by Hawley or the empirical mathematical model which used the Eyring equation as a starting point. By comparison, the former could do more accurately than the latter.The high pressure inactivation of nutritional inhibitors in soy milk was studied and the inactivation conditions were optimized. Results showed that the high pressure inactivation of urease in soy milk could occur at room temperature, while high pressure inactivation of trypsin inhibitors was possible only trough combination with elevated temperature (T≥40℃). The inactivation could be speeded up by increasing any one of the three influencing factors (pressure, temperature and processing time). For both urease and trypsin inhibitors inactivation, the three process parameters were optimized by using central composite rotatable design and response surface methodology. Results showed that pressure is the uppermost influencing factor, while temperature is the most minor factor. The ideal high pressure inactivation conditions of nutritional inhibitors in soy milk were as follows: pressure, 750 MPa; temperature, 60 ℃ and processing time, 5 min.Effects of high pressure treatment on physicochemical and functional properties of soybean protein isolates (SPI) in two different buffers were studied. Results showed that the increase of SPI’s solubility in pH3.0 Gly-HCl buffer induced by high pressure treatment was more remarkable than that in pH8.0 Tris-HCl buffer. Analysis of free sulfhydryl group and surface hydrophobicity of SPI showed that high pressure treatment produced a molecular unfolding of the protein with the exposure of the hydrophobic groups to the medium and thus formed new S-S bonds through SH/S-S interchange reactions. Aggregation of SPI after high pressure treatment was investigated by size exclusion chromatography, laser light scattering analysis and SDS-PAGE. High pressure treatment could induce structural reorganization of SPI in pH3.0 Gly-HCl buffer with the formation of soluble high molecular aggregates, whereas in pH8.0 Tris-HCl buffer high pressure might produce disaggregation of the protein aggregates. These phenomena demonstrated that the different existing forms of SPI in different buffers might result in the different structural changes of SPI under high pressure treatment. High pressure treatment could improve the emulsifying and foaming properties of SPI and this improvement was much remarkable in pH3.0 Gly-HCl buffer because of the significant increase of SPI’s solubility and surface hydrophobicity. High pressure couldinduce the dispersions of SPI with a certain concentration to form a gel. Compared with temperature and processing time, pressure is the uppermost influencing factor of high pressure treatment on textural properties of the gel. The hardness of the gel is increasing when elevate the pressure, temperature and prolong the processing time. The hardness of pressure-induced gel of SPI (12% protein concentration), treated under 700MPa and 20 ℃ for 15 minutes, was greater than that of heat-induced gel of SPI with the same protein concentration, treated under ambient pressure and 85 ℃ for 20 minutes.Comparing the physicochemical properties, color, flavor and rheological properties of the untreated, heat treated and high pressure treated soy milk, it was shown that high pressure treatment and heat treatment did not affect the pH and electric conductivity of the soy milk; the apparent viscosity of the soy milk was increased by the heat treatment, which might because of the thermo-aggregation effect of the soybean protein; heat treatment and high pressure treatment had little effect on the color of the soy milk; high pressure treatment had little effect on reducing the existed volatile flavor compounds; however, the beany flavor of the soy milk were much decreased when high pressure treatment were done before the refining process; high pressure treatment and heat treatment had no effect on the protein based amino acid composition of the soy milk; high pressure treatment and heat treatment reduced the flow behavior index of soy milk, and the flow behavior tended to be the pseudo-plastic fluid; the consistency factor of the soy milk was dramatically increased after heat treatment, which indicated that heat treatment could elevate the apparent viscosity of the soy milk. In summary, heat treatment had greater impact on the rheological property of the soy milk than high pressure treatment, which might because of the soybean protein folded, denatured and aggregated to a far greater extent under heat treatment than high pressure treatment.Keywords: High pressure, Lipoxygenase, Nutritional inhibitors, Inactivation, Kinetics, Model, Soybean proteins。