【作者】 李迎秋；

【导师】 陈正行；

【作者基本信息】 江南大学 ， 粮食、油脂及植物蛋白工程， 2007， 博士

【摘要】 脉冲电场(Pulsed Electric Fields, PEF)处理是一种新型的非热食品杀菌技术。它是以较高的电场强度(10-50kV/cm)、较短的脉冲宽度(0-100μs)和较高的脉冲频率(0-2000Hz)对液体、半固体食品进行处理,并且可以组成连续杀菌和无菌灌装的生产线。脉冲电场技术要在食品加工中大规模推广使用,还需要解决很多问题(如杀菌钝酶机理及动力学模型的建立、脉冲电场对食品组分的影响和食品安全性评价等等)。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(如粘弹性、凝胶性、乳化性)等,在食品配方中作为一种重要的功能成分和食品添加剂广泛应用于食品工业。但大豆中含有多种酶类和一些抗营养因子,使得大豆及其制品有一定的毒副作用和产生不愉快的豆腥味。传统的加热杀菌易使大豆蛋白变性,大豆蛋白饮料易于沉淀,从而影响产品的质量;蛋白质天然结构的破坏会使理化及其功能性质发生变化,温度太低对抗营养因子和豆腥味祛除不彻底。本研究的目的就是利用脉冲电场这一新型的杀菌技术处理豆奶及大豆分离蛋白溶液,初步探讨脉冲电场对豆奶品质、大豆脂肪氧化酶和大豆分离蛋白功能性质及结构的影响,为脉冲电场技术在大豆制品加工中的应用、大豆蛋白的改性及食品安全提供理论参考。以豆奶为真实的食品体系,测定了脉冲电场处理后豆奶理化性质、色泽、风味、酶类及微生物等指标,结果表明,由于较高的脉冲电场能使带电粒子趋向一致,致使黏度下降;脉冲电场可能导致轻微褐变的发生,豆奶L值略有上升;pH值、电导率没有发生改变。通过GC-MS测定结果表明,脉冲处理后的豆奶的风味物质几乎没有变化;处理后豆奶中总游离氨基酸含量降低,半胱氨酸、酪氨酸、苯丙氨酸和丝氨酸含量降低的原因可能是电场作用使半胱氨酸之间通过巯基氧化形成了二硫键、对疏水相互作用和氢键有一定的影响。脉冲电场对大豆脂肪氧化酶(SLOX)、大豆胰蛋白酶抑制剂(STI)及脲酶有显著的钝化效果(P<0.05),脲酶对脉冲电场较为敏感,SLOX次之,STI对脉冲电场具有一定的抵抗力。脉冲强度和处理时间对大肠杆菌、沙门氏菌及金黄色葡萄球菌的钝化都有显著的影响(P<0.05)。大肠杆菌对脉冲电场比较敏感,沙门氏菌次之,金黄色葡萄球菌对脉冲电场的抵抗力较强。以大豆分离蛋白溶液为研究对象,研究了脉冲电场对其理化及功能性质的影响,脉冲电场处理后大豆分离蛋白的pH值和表观黏度略微降低,溶解度、乳化性及起泡性都有所提高,较强脉冲条件则使其功能性质呈现下降趋势。原因可能是脉冲电场对其疏水相互作用有一定的影响,使大豆分离蛋白分子部分伸展,疏水基团暴露,因此溶解度、乳化性及起泡性都提高;较强的脉冲条件则使极化的蛋白分子之间相互吸引重新形成分子聚集体,其功能性质下降。脉冲处理后的大豆分离蛋白的7S和11S的变性温度都有所下降,7S的变性温度下降的较大,说明7S对脉冲处理较为敏感。热变性温度的下降说明蛋白的构象发生了变化,其结构发生了解折叠,变为松散的结构。应用Ellman试剂法、ANS荧光探针法、分子排阻法及激光光散射法研究了脉冲处理前后大豆分离蛋白聚集情况。结果发现,随着脉冲强度的增大或时间的延长,大豆分离蛋白的表面游离巯基含量和疏水性提高,较强的脉冲则使其巯基含量和疏水性下降。短的脉冲处理时间对大豆分离蛋白分子量和粒径分布几乎没有影响;但脉冲处理时间长于432μs,高于1000kDa和1000nm大分子蛋白聚集体含量增加。这说明脉冲电场诱导了大豆蛋白分子的极化;破坏了维持蛋白四级结构的作用力如疏水相互作用、二硫键、静电相互作用及氢键等;导致了蛋白亚基解离和疏水基团的暴露;极化的亚基相互吸引通过非共价键重新形成更大的分子聚集体。应用SDS-PAGE聚丙烯酰胺凝胶电泳、圆二色谱法(CD)、拉曼光谱法(Raman)和FT-IR光谱法等方法研究了PEF处理后大豆分离蛋白结构的变化。脉冲处理后11S球蛋白和7S球蛋白的各亚基的电泳谱带都没有变化,说明脉冲电场对大豆分离蛋白的亚基没有影响。CD色谱法分析结果表明,随着脉冲处理时间的延长,α-螺旋结构的含量逐渐减小,β-折叠逐渐增大,无规则卷曲稍微增大;但当脉冲处理时间为547μs时β-折叠含量变小,而无规则卷曲含量增大。这说明,脉冲电场诱导了维持蛋白二级结构的作用力如氢键等改变了二级结构,从而使四种结构的含量发生了变化,Raman和FT-IR光谱分析结果进一步证明了这一点。Raman光谱中通过S-S和C-C伸缩振动特征谱带的变化,说明脉冲电场对巯基和二硫键有一定的影响;通过酪氨酸特征振动频率的变化情况,说明脉冲电场对疏水相互作用有一定的影响。在两极方波脉冲条件下,随着PEF处理时间延长、强度增强、频率及宽度增大,大豆脂肪氧化酶的钝化程度增强。一级分数转换模型、韦布分布函数和Fermi’s模型都可用来很好地拟合大豆脂肪氧化酶活性和PEF参数之间的关系,韦布分布函数最适于预测PEF处理参数对大豆脂肪氧化酶的钝化,它可以解释在低PEF强度时的时间延迟,通过高的形状系数来反应;而一级分数转换模型可以提供PEF的最大钝化酶活;Fermi’s模型可以提供给酶活性为50%时所需的临界电场强度。这些数据为PEF技术在豆奶加工中的应用提供了很重要的信息。更多还原

【Abstract】 Pulsed electric fields (PEF) are a new non-thermal food preservation method, which treats liquid or semi-solid food with high electric field intensities (10-50 kV/cm), very short electric pulse (0-100μs) and very high pulse frequency (0-2000 Hz). PEF can is convenient to continuously sterilize and aseptically can. There are many problems to need solve for the industrial application of PEF, for example, mechanism and modeling of inactivation microorganisms and enzymes; effects of PEF on food constituents and structures; product safety assurance after PEF processing etc. Soybean is a full-nutrient food due to have abounding proteins and rational amino acids. So soybean proteins are commonly used as a functional ingredient and additive for food formulation because of its excellent functional properties (stickiness, elasticity, gelling, and emulsification) and good nutritional values. However, soybean and soy products have a rancid off-flavor and negative aspects to human health due to the presence of some enzymes and antinutritional factors. Thermal preservation results in denaturation of soybean proteins and precipitation of beverage containing soybean proteins, thus affects the quality of products. Disruption of nature proteins structure can affect their physicochemical and functional properties. Low thermal does completely remove the rancid off-flavor and antinutritional factors. The objective of this study is to investigate the effect of PEF on the quality of soymilk, inactivation of soybean lipoxygenase (SLOX), and physicochemical properties and structure of soybean protein isolates (SPI) in order to provide the reference for the application of soybean products, modification of soybean proteins, and food safety.Physicochemical properties, color, flavor, enzymes, and microorganisms of soymilk have detected. The viscosity of soymilk decreased possibly due to identical tendency of electric particles in PEF inducement. PEF resulted in mild browning and L increased a little. Electric conductivity, flavor and pH did not change. The contents decrease of cysteine, hydrophobic amino acid (tyrosine and phenylalanine) and serine suggested that PEF had affected on disulfide bond, hydrophobicity and hydrogen bond. PEF inactivated markedly SLOX, soybean trypsin inhibitors (STI) and urease (p<0.05), which urease is more sensitive, SLOX second, STI resistent to PEF. PEF strength and time had remarkable effects on inactivation of E.coil, salmonella and S. aureus (p<0.05), which E.coil is more sensitive, salmonella second, S. aureus resistent to PEF.Effects of PEF treatment (0 to 547μs and 0 to 40 kV/cm) on physicochemical and functional properties of SPI were studied. The viscosity and pH of SPI PEF-treated decreased slightly. Solubility, emulsibility, foaming capacity increased with the increment of the pulsed electric fields strength and treatment time. When the PEF strength and treatment time were above 30 kV/cm and 288μs, solubility, emulsibility, foaming capacity of SPI decreased due to denaturation and aggregation of SPI by hydrophobic interactions and disulfide bonds. The denaturation temperatures of 7S reduced more than 11S after PEF treatment, which indicated 7S was more sensitive to PEF processing. The decrease of denaturation temperatures of SPI showed that PEF changed the structure of SPI and make protein unfold and relax.Aggregation of SPI after PEF was investigated by Ellman reagent, ANS fluorescence probe, size exclusion chromatography (SEC) and laser light scattering analyses (LLS). Surface free sulfhydryls and hydrophobicity of SPI increased with the increment of the PEF strength and treatment time. The stronger PEF conditions caused surface free sulfhydryls and hydrophobicity to decrease due to dissociation, denaturation and aggregation of SPI detected by SEC and LLS. Over 432μs the content of molecule of weight higher than 1000 kDa and larger than 1000 nm increased. These results showed PEF treatment induced polarization of SPI and dissociation of sub-units and molecular unfolding; changed molecular conformation and caused hydrophobic groups and free sulfhydryls buried inside the molecules to expose. Too strong PEF conditions produced stronger molecular polarization and polarized molecules attracted each other and converged again to form larger aggregations by non-covalent bonds such as hydrophobic interactions, electrostatic interactions, hydrogen bonds and S-S bonds.The structure of SPI after PEF was investigated by SDS-PAGE, CD, Raman and FT-IR spectra. Subunits of 11S and 7S fractions did not change after PEF by SDS-PAGE spectra. CD spectra showed thatα-helix content decreased, andβ-sheet content and random coil content increased with the increment of treatment time. But theβ-sheet content reduced and the random coil content increased when PEF treatment time was 547μs. These results showed PEF may destrory the interactional force (for example, hydrogen bond) maintaining the second structure of protein and change the second structure of protein. This fact was confirmed further by Raman and FT-IR spectra. Changes in S-S and C-C stretching vibration frequency by Raman spectra indicated that PEF have certain effects on sulfhydryl and disulphide bond. PEF influenced hydrophobicity by changes in tyrosine vibration frequency of Raman spectra.Residual activity of SLOX decreased with the increase of PEF treatment time, strength, frequency, and width in square wave pulse of bipolar mode. The first-order fractional conversion model, Weibull distribution function, and Fermi’s model described successfully the relationship between residual activity of SLOX and PEF paremeters, which Weibull distribution function was the best model. The model showed greater capability to make predictions than the other tested models and was reflected by a higher shape parameter if the curves of inactivation of enzyme exhibited some lag time at low PEF strength. The first-order fractional conversion model provided the residual enzyme activity after a prolonged time of treatment (stabilization). Fermi’s model provided the strength when residual enzyme activity is 50%. The data provide the important reference for the application of PEF in soymilk and soybean products.更多还原