【作者】 尹寿伟；

【导师】 温其标； 唐传核；

【作者基本信息】 华南理工大学 ， 食品科学， 2009， 博士

【摘要】 本论文旨在探寻适于芸豆蛋白资源利用的途径。研究了芸豆球蛋白(Phaseolin)和分离蛋白(KPI)的物化和功能特性及其相关机制,探讨了KPI的物化改性及其相关构效机理。主要研究结果如下:对芸豆球蛋白和KPI的物化和功能特性进行了分析和比较,从蛋白结构与功能性关系角度揭示了两者功能性差异的机制。结果表明,芸豆球蛋白具有良好的功能特性,其溶解度(PS)、乳化活性指数(EAI)、乳化稳定性指数(ESI)和凝胶能力均显著高于KPI (P < 0.05);芸豆球蛋白的变性焓变(ΔH)为20.3 J g-1,而KPI的ΔH仅为11.2 J g-1;芸豆球蛋白的暴露巯基(SHE)、总离巯基(SHT)和二硫键(SS)含量显著低于KPI(P < 0.05)。圆二色光谱(CD)、荧光光谱和表面疏水性(H0)分析表明,KPI是部分变性的,其制备过程中的酸碱处理导致蛋白分子伸展、H0增加及三级结构的改变。蛋白变性是导致KPI功能性降低的原因。运用溶解度(PS)、比浊法、热分析(DSC)、荧光光谱和CD光谱法探讨了不同压力微射流处理(HM)对KPI构象和功能特性的影响。HM诱导不溶性蛋白聚合物解聚,增加了KPI的PS和EAI;CD和荧光光谱分析表明,HM对KPI的二级、三级结构没有明显的影响;DSC表明,蛋白的Td和ΔH均未受到显著的影响(P > 0.05)。研究了高静压处理(HP)对KPI物化和功能特性的影响,通过Try荧光光谱、ANS荧光光谱和SHE分析以揭示HP导致KPI构象改变的规律。结果表明,200 MPa处理诱导KPI中的可溶性高聚物(空体积处)解离,而400和600 MPa处理导致的不溶性聚集物解离,改善KPI的PS;200和400 MPa处理改善KPI的EAI和ESI,而600 MPa处理造成EAI和ESI下降;400和600 MPa处理降低KPI消化性能。HP导致KPI分子伸展、SHE和H0的增加,但仅600 MPa处理导致KPI三级结构的改变;HP不影响KPI的Td,降低KPI的ΔH,但600 MPa处理KPI的ΔH高达11.2 J g-1(约占对照KPI的60%)。在酸酐-蛋白比为0~0.1(乙酰化)和0~0.2(琥珀酰化) g g-1时,N-酰化度从0迅速增至93~94%;再增加酸酐与蛋白比,N-酰化度仅增加2~3%,羟基(Thr, Ser)开始参与酰化反应。酰化诱导KPI的PS-pH曲线向酸性偏移;S-KPI(琥珀酰化)的PS随酸酐-蛋白比的增加而增加,而A-KPI(乙酰化)的PS却是先增加后降低;酰化(尤其琥珀酰化)显著改善KPI的EAI(P<0.05);琥珀酰化弱化KPI的凝胶能力,而乙酰化改善KPI的凝胶能力;酰化改善KPI的体外消化性能。酰化蛋白的Iep随N-酰化度增加而线性降低,羟基酰化不影响KPI的Iep;在相同的N-酰化度,S-KPI具有与A-KPI相似或略低的Iep;酰化导致KPI的Zeta电势-pH曲线整体向下平移,回归分析表明,酰化蛋白的Zeta电势随N-酰化度的增加而线性增加(pH 7.0)。酰化影响KPI的亲水/疏水平衡。ε-氨基(lys)酰化阶段,KPI的H0逐渐下降;在羟基酰化阶段,琥珀酰化导致H0下降,乙酰化却导致H0增加。荧光光谱分析表明,ε-氨基酰化不影响KPI的三级构象,羟基酰化诱导KPI分子伸展和三级构象的改变,CD研究印证了这一结果。DSC也表明ε-氨基酰化不影响KPI的Td;羟基酰化导致KPI的Td和ΔH逐渐下降,表明蛋白变性(或分子伸展)。总之,HM、HP、乙酰化和琥珀酰化都可改善KPI的PS、EAI和ESI,琥珀酰化的改性效果最明显,其PS、EAI和ESI是接近于芸豆球蛋白的。四种技术手段均可诱导不溶性聚集物解离,HM不影响KPI的构象;HP导致KPI分子伸展,600 MPa处理导致KPI三级结构的改变;在羟基酰化阶段,乙酰化和琥珀酰化导致KPI分子伸展及三级结构的改变。更多还原

【Abstract】 The aim of this study was to explore utilization means for proteins from red kidney bean (Phaseolus vulgaris L). Physiochemical and functional properties of phaseolin (the main storage globulin) and red kidney bean protein isolate (KPI) was studied and compared, the related mechanism was elucidated. Physiochemical modifications were used to modify KPI for improving its functional properties. The possible relationship between structure and functional properties of KPI was also discussed. Main results are as follows:Phaseolin showed excellent functional properties, its protein solubility (PS), emulsifying activity index (EAI), and gel-forming ability were much higher than those of KPI (P < 0.05). Differential scanning calorimetry analyses suggested that phaseolin was less denatured than KPI, the enthalpy change (ΔH) of phaseolin was about 20 J g?1, while enthalpy change (ΔH) of KPI was only 11.2 J g?1. The exposed SH (SHE), total SH (SHT) and SS content of phaseolin was significantly lower than those of KPI. Near-UV CD spectra and intrinsic fluorescence spectrum analyses confirmed much loss of tertiary conformation of KPI, relative to phaseolin, which may be attributed to acid and alkaline treatment during KPI preparation resulted in protein denaturation, exposure of hydrophobic groups.The effects of microfluidization on functional properties as well as conformational properties of KPI were investigated by solubility and turbidimetric measurements, DSC, fluorescence spectrum and Far-UV CD. The microfluidization led to dissociation of insoluble aggregates, thus improved PS and EAI of KPI, in a pressure dependent manner. Fluorescence emission spectra and far- UV CD spectrum analyses showed that both the tertiary conformation and the secondary structure of the proteins in KPI were nearly unaffected by the microfluidization treatment. DSC analysis indicated the microfluidization-treated KPI samples presented similar Td andΔH, relative to that of untreated KPI.The effects of high-pressure (HP) treatment at 200–600 MPa on functional properties and in vitro trypsin digestibility of vicilin-rich red kidney bean (Phaseolus vulgaris L.) protein isolate (KPI) were investigated. HP-induced conformation changes were also evaluated by Try fluorescence spectrum, surface hydrophobicity (H0) and free sulfhydryl (SH) contents analyses. HP treatment at 200 MPa led to dissociation of soluble aggregates (at void volume), while HP treatment at 400 and 600 MPa led to dissociation of insoluble aggregates in KPI, thus improve PS. HP treatment at 200 and 400 MPa significantly increased emulsifying activity index (EAI) and emulsion stability index (ESI); however, EAI and ESI were significantly decreased at 600 MPa (relative to untreated KPI). The in vitro trypsin digestibility of KPI was decreased only at a pressure above 200 MPa and for long incubation time (e.g., 120 min). HP treatment resulted in gradual unfolding of protein structure, as evidenced by gradual increases in SHE and H0 as well as decrease inΔH. However, only 600 MPa HP treatment resulted in loss of tertiary conformation of KPI, as evidenced by Try fluorescence spectrum analyses. Interestingly, theΔH of HP treated KPI (at 600 MPa) was 11.2 J g-1, account for 60% of that of untreated KPI.The degree of N-acylation sharply increased to about 93-94 % with the anhydride levels increasing from 0 to 0.1 (acetylation) or 0.2 g g-1 (succinylation). Further increase in the ratio just resulted in a slight increase (about 2% to 3%) in extent of acylation. The O-acylation (Thr, Ser) distinctly occurred only when degree of N-acylation was higher than 93-94 %.The acylation treatment (at a ratio of anhydride to protein of 0.05 g/g) resulted in a shift of the minimal of PS profile of KPI toward a more acidic pH. In the succinylation case, the PS progressively increased with the increase in ratio of anhydride to protein. Whereas in the acetylation case, the PS gradually increased to a maximum (from 70% to about 85%) at an anhydrideto-protein ratio of 0.2 g/g, and then on the contrary decreased upon further increase in ratio of anhydride to protein. Acylation, especially succinylation remarkably improved EAI at neutral pH. Succinylation resulted in a marked decrease in mechanical moduli of heat-induced gels of KPI, while the mechanical moduli were, on the contrary, increased by acetylation. Additionally, in vitro trypsin digestibility was improved by the acylation in an anhydride-type and level-dependentmanner.The succinylation led to progressive and significant decrease in H0, from 879 (control) to 118-119 (at degree of N-acylation of 97 %). Whereas in the acetylation case, the H0 decreased first, and reached a minimum (at degree of N-acylation of about 93 %) and then increased to a value (957) even higher than control. There was a close and negative relationship between Ip and degree of N-acylation, the Ip of acetylated KPI was similar or slightly higher than that of succinylated KPI. On the other hand, zeta potential at neutral pH of acylated KPI samples also linearly decreased with the increase in degree of N-acylation.In short, microfluidization, high-pressure, acetylation and succinylation resulted in increase in PS, EAI and ESI of KPI. Succinylation is the most effective for improving functional properties of KPI, the PS, EAI and ESI of succinylated KPI are close to those of phaseolin. These modification technologies led to dissociation of insoluble aggregate. Microfluidization treatment had little effect on the conformation of KPI. HP led to protein unfolding of KPI, however, only HP treatment at 600 MPa resulted in change in tertary conformation of KPI. Upon acylation marked structural unfolding (or change in tertiary conformation) occurred when the degree of O-acylation began to increase.更多还原