大米蛋白水解机制研究——Ⅲ酶水解对大米蛋白-糖结合特性的影响

凝胶色谱洗脱组分分析和β-消去反应结果表明,酶水解所溶出的大米蛋白与糖分子仍以N-糖肽键形式连接。红外光谱分析显示酶解后可溶性蛋白和残余物蛋白中均含有糖组分。SDS-PAGE分离和Schiff试剂染色结果表明,13ku亚基是能抵抗碱性蛋白酶水解的糖蛋白。     

高压处理对热变性米蛋白酶解特性的影响

研究高压处理热变性米蛋白在碱性蛋白酶酶解时其溶解性、水解度、-SH含量、分子特征和微观形态的变化特征。结果显示：米蛋白经100～700MPa处理后酶解所得可溶性蛋白的含量均高于未高压处理者,其中500MPa时增加幅度最大,-SH含量也最高。当酶解120min时,高压处理米蛋白可溶性米蛋白的比例达73.0%,而未高压处理米蛋白的可溶性米蛋白只有53.6%,但二者水解度(分别为7.40%和7.05%)的差异并不明显;凝胶过滤色谱分析表明,高压处理后酶解时可溶物的相对分子质量小于未高压处理的酶解物;SDS-PAGE分析表明,高压处理米蛋白不溶性酶解物中大分子组分的含量明显减少,小分子组分明显增加;扫描电镜观察显示,经高压处理的米蛋白形态相对蓬松。因此,适当高压处理有利于米蛋白的酶解,且酶解特性与米蛋白高压处理后的结构变化有直接关系。     

高压处理大米蛋白酶解过程中糖-蛋白结合特性的变化

研究了热变性大米蛋白经100MPa高压处理的酶解过程中蛋白质与糖结合特性的变化,并与未高压处理的酶解物进行了比较.凝胶色谱分析显示,经高压处理的大米蛋白可溶性酶解物中蛋白/糖含量比值低于常压处理者.β-消去反应证明了蛋白和糖的连接方式为N-糖肽键结合.FT-IR分析表明,高压和常压处理后大米蛋白的酶解残余物中均含有糖组分,且高压处理者的糖相对含量较低.SDS-PAGE和PAS-Schiff试剂染色显示,高压和未高压处理者的酶解残余物中14ku均是糖蛋白.上述结果表明,高压处理有利于糖蛋白的酶解,但部分糖蛋白仍呈不溶解状态.     

4种花生粕蛋白的理化性质及功能特性研究

以花生粕为原料,采用分级提取工艺提取花生清蛋白、球蛋白、醇溶蛋白和谷蛋白,研究4种花生粕蛋白的理化性质和功能特性。扫描电镜观察,4种花生粕蛋白的形态结构各不相同。SDS-PAGE法测定分子质量表明,清蛋白含有4种亚基,分子质量为70、40、30、25和15 ku;醇溶蛋白含有2种亚基,分子质量分别为25和1 5 ku;球蛋白含有5种亚基,相对分子质量分别为40、38、30、25和15 ku;谷蛋白含有4种亚基,相对分子质量分别为40、30、25和15 ku。花生清蛋白、醇溶蛋白、球蛋白、谷蛋白的等电点分别为pH 3.6、pH 5.2、pH 4.6、pH 5.0。功能性质研究表明,球蛋白的持水性最好,为1.52 mL/g,其次为谷蛋白1.10 mL/g,清蛋白和醇溶蛋白的持水性较低分别为0.49、0.14 mL/g;清蛋白的持油量相对较高为8.21mL/g,其次为球蛋白为7.16 mL/g,谷蛋白和醇溶蛋白的持油量相对较低,分别为3.82 mL/g和5.49 mL/g;清蛋白的乳化性和乳化稳定性相对较高,乳化能力(EC)值和乳化稳定性(ES)值分别为7 1.4%和83.33%,谷蛋白次之,EC和ES值分别为66.7%和82.86%,醇溶蛋白和球蛋白相对较低,EC值分别为64.0%和62.2%,ES值分别为82.35%和76.67%。综上,花生粕清蛋白的持油性、乳化性和乳化稳定性相对较好。     

小米蛋白提取、测定以及SDS-PAGE电泳

依照Osborne法对小米清蛋白、球蛋白、醇溶蛋白和谷蛋白进行分离提取,通过单因素试验确定小米蛋白提取时的最佳条件,并通过SDS-PAGE对小米蛋白组分进行了亚基分析。结果表明,小米清蛋白提取的最佳温度为40℃,球蛋白提取的最佳氯化钠质量分数为2%,醇溶蛋白提取的最佳醇体积分数为80%,谷蛋白提取的最佳氢氧化钠浓度为0.05 mol/L。小米蛋白各组分SDS-PAGE凝胶电泳图谱显示,小米清蛋白的亚基主要分布在(97.4~22)ku范围内且含有二硫键;小米球蛋白的亚基主要分布在(66.2~10)ku范围内;小米醇溶蛋白的亚基条带分布广泛且含有二硫键;小米谷蛋白的亚基条带分布在(66.2~10)ku范围内。不同提取条件不仅会影响提取率,同时也会影响蛋白组分的亚基组成。     

大米蛋白的酶水解机制研究——Ⅱ酶水解过程中蛋白质的组分变化

凝胶色谱和高效液相色谱分析表明,大米蛋白在碱性蛋白酶Alcalase水解过程中,其可溶性蛋白组分的相对分子质量(Mr.)分布范围相对稳定,但Mr.1350u以下小分子组分的相对含量不断增加;十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析显示,随着酶水解反应的进行,Mr.为57、39、26、22ku的亚基逐渐消失,而29ku和13ku两个亚基的含量相应增加,且这两个亚基表现出可抵抗酶水解的特性。氨基酸分析表明,酶解后残余物蛋白中胱氨酸、蛋氨酸等含硫氨基酸的含量显著高于可溶性蛋白中的含量。圆二色光谱(CD)分析显示,酶水解前后大米蛋白的二级结构发生显著变化,在残余物蛋白质组分中自由回转的比例高达71.3%,α-螺旋结构完全消失。     

储藏时间对大米蛋白质组分的影响

主要研究储藏时间对大米4种蛋白质亚基组分变化的影响。实验以市售普通大米为初始原料,在高温高湿的环境下进行储藏,以5个不同储藏时间段(0、24、48、72、96h)的大米为实验对象,对不同储藏时间段大米的4种蛋白质(清蛋白、球蛋白、醇溶性蛋白和谷蛋白)进行SDS-PAGE电泳实验,分析大米储藏过程中4种蛋白质各自亚基组成的变化。研究发现,储藏过程中水溶性清蛋白的亚基组成变化很大,有低分子质量亚基发生了类似于酶的作用,即先分解后合成,平均分子质量增加;盐溶性球蛋白减少较明显;醇溶性蛋白质组分基本无变化,十分稳定;碱溶性蛋白质高、中分子质量的亚基都存在分解和合成现象,并且在储藏96h后出现一个分子质量约为22ku的亚基。     

酸法脱酰胺改善大米蛋白的溶解性及其功能性质分析

大米蛋白是一种优质的植物蛋白，但较低的溶解特性限制了大米蛋白的应用范围，为提高大米蛋白的溶解性，该研究采用酸法脱酰胺对其进行改性处理，处理条件为料液比1∶25（g/mL）加入0.4 mol/L HCl 溶液，95 ℃水浴加热搅拌4 h，分析改性前后蛋白的溶解性、起泡及起泡稳定性、乳化及乳化稳定性、表面疏水性、持水性、持油性等功能性质，以及亚基分子量、表面微观结构的变化。结果表明，酸法脱酰胺处理改善大米蛋白溶解性效果显著，中性环境（pH7）中由0.58%提高至56.60%，碱性环境（pH 值为12、13）溶解性达到90.00%以上。pH 值为3、7 时起泡性、乳化性改善效果较好，起泡性分别由22.50%、80.40%提高至78.50%、151.70%，乳化性分别由4.4、6.6 m2/g 提高至14.3、16.5 m2/g，过度的水解也会导致表面疏水性的降低，进一步影响界面性质的改善效果。酸法脱酰胺改性对蛋白质的持水性、持油性改善效果不明显。改性后蛋白亚基分子量大幅度降低，主要分布在6.5～20 kDa 之间，有序的β-折叠结构转变成无序的无规则卷曲结构，蛋白微观结构由聚集变得松散，致密的球状结构变为不规则块状结构，表面由粗糙变得光滑。     

超高压处理对大米蛋白功能特性及结构的影响

以不同压力(200、400、600 MPa)对大米进行超高压处理。研究了超高压处理对大米中谷蛋白功能特性以及清蛋白、球蛋白和谷蛋白结构的影响。结果表明:超高压处理后大米蛋白的功能特性和二级结构均发生变化,不同压力影响效果不同。200 MPa时蛋白质的溶解性、持水性和乳化性提高,持油性降低;400 MPa时持水性和乳化性降低,持油性提高,溶解性升高不明显;600 MPa时溶解性、持水性、持油性和乳化性均降低。超高压处理后清蛋白、球蛋白和谷蛋白的二级结构发生改变,β-折叠结构含量降低,无序结构增多。相关性分析结果表明压力、功能特性和二级结构三者之间存在相关性。     

不同pH及离子强度对葡萄籽分离蛋白功能特性的影响

以葡萄酒酿造完成后的废弃料赤霞珠葡萄籽为原料,对葡萄籽蛋白质在不同p H及离子强度下的功能特性和亚基组成进行了研究。结果表明:在p H为12,离子强度为0.01时溶解性较好。在等电点附近,葡萄籽分离蛋白的乳化性、溶解性较差。葡萄籽分离蛋白在低盐浓度下有较好的溶解性和乳化性、起泡性、泡沫稳定性;由SDS-PAGE分析可知,在碱性条件下,随pH的升高,葡萄籽分离蛋白亚基聚集,形成了大分子质量的可溶性聚集体,正是这些聚集体的形成造成了溶解度的增加。在低离子强度下,葡萄籽分离蛋白只有部分介于43～66 ku的亚基可溶,随盐离子的不断增加,随后也进入沉淀。     

酸性条件下花生分离蛋白亚基结构的变化规律

本文通过SDS-PAGE凝胶电泳、Zeta电位、溶解性、内源荧光光谱以及粒径的测定分析,探讨了酸性条件下花生分离蛋白亚基结构的变化规律。电泳分析表明,当p H3.5时,部分亚基酸解产生约33 ku的新条带,18 ku条带亮度增加。亚基的酸解受时间和离子强度的影响。花生分离蛋白亚基在常温p H 2.5的条件下处理10 min后开始酸解,且随着时间的延长高分子量的亚基逐渐减少;在离子强度0~0.1 mmol/L时酸解程度较大,当离子强度大于0.2 mmol/L时,酸解作用被抑制。进一步研究表明,在p H 2.0~2.5间,花生分离蛋白结构较为伸展,内部基团暴露,溶解性较高,荧光光谱最大吸收波长比中性条件时红移约13 nm;当p H进一步降低时,Zeta电位较低,花生分离蛋白形成大分子量的可溶性聚集体,荧光光谱最大吸收波长蓝移至335.27 nm。     

FUNCTIONAL PROPERTIES OF HIGH HYDROSTATIC PRESSURE-TREATED WHEY PROTEIN

Surface hydrophobicity, solubility, gelation and emulsifying properties of high hydrostatic pressure (HHP)‐treated whey protein were evaluated. HHP treatment of whey protein buffer or salt solutions were performed at 690 MPa and initial ambient temperature for 5, 10, 20 or 30 min. Untreated whey protein was used as a control. The surface hydrophobicity of whey protein in 0.1 M phosphate buffers treated at pH 7.0 increased with an increase in HHP treatment time from 10 to 30 min. HHP treatments of whey protein in salt solutions at pH 7.0 for 5, 10, 20 or 30 min decreased the solubility of whey proteins. A significant correlation was observed between the surface hydrophobicity and solubility of untreated and HHP‐treated whey protein with r = ?0.946. Hardness of HHP‐induced 20, 25 or 30% whey protein gels increased with an increase in HHP treatment time from 5 to 30 min. An increase in the hardness of whey protein gels was observed as whey protein concentration increased. Whey proteins treated in phosphate buffer at pH 5.8 and 690 MPa for 5 min exhibited increased emulsifying activity. Whey proteins treated in phosphate buffer at pH 7.0 and 690 MPa for 10, 20 or 30 min exhibited decreased emulsifying activity. HHP‐treated whey proteins in phosphate buffer at pH 5.8 or 7.0 contributed to an increase in emulsion stability of model oil‐in‐water emulsions. This study demonstrates that HHP treatment of whey protein in phosphate buffer or salt solutions leads to whey protein unfolding observed as increased surface hydrophobicity. Whey proteins treated in phosphate buffers at pH 5.8 and 690 MPa for 5 min may potentially be used to enhance emulsion stability in foods such as salad dressings, sausage and processed cheese.     

High hydrostatic pressure modification of whey protein concentrate for improved functional properties

Whey protein concentrate (WPC) has many applications in the food industry. Previous research demonstrated that treatment of whey proteins with high hydrostatic pressure (HHP) can enhance solubility and foaming properties of whey proteins. The objective of this study was to use HHP to improve functional properties of fresh WPC, compared with functional properties of reconstituted commercial whey protein concentrate 35 (WPC 35) powder. Fluid whey was ultrafiltered to concentrate proteins and reconstituted to equivalent total solids (8.23%) as reconstituted commercial WPC 35 powder. Solutions of WPC were treated with 300 and 400 MPa (0- and 15-min holding time) and 600 MPa (0-min holding time) pressure. After HHP, the solubility of the WPC was determined at both pH 4.6 and 7.0 using UDY and BioRad protein assay methods. Overrun and foam stability were determined after protein dispersions were whipped for 15 min. The protein solubility was greater at pH 7.0 than at pH 4.6, but there were no significant differences at different HHP treatment conditions. The maintenance of protein solubility after HHP indicates that HHP-treated WPC might be appropriate for applications to food systems. Untreated WPC exhibited the smallest overrun percentage, whereas the largest percentage for overrun and foam stability was obtained for WPC treated at 300 MPa for 15 min. Additionally, HHP-WPC treated at 300 MPa for 15 min acquired larger overrun than commercial WPC 35. The HHP treatment of 300 MPa for 0 min did not improve foam stability of WPC. However, WPC treated at 300 or 400 MPa for 15 min and 600 MPa for 0 min exhibited significantly greater foam stability than commercial WPC 35. The HHP treatment was beneficial to enhance overrun and foam stability of WPC, showing promise for ice cream and whipping cream applications.     

Characterization of Physicochemical Properties and IgE‐Binding of Soybean Proteins Derived from the HHP‐Treated Seeds

The aim of this work was to evaluate the characterization of physicochemical properties and IgE‐binding of soybean proteins derived from the high hydrostatic pressure (HHP) treated seeds. Soybean seeds were treated by HHP at different pressures, and changes in the physicochemical properties of soybean proteins were characterized by proteins solubility, free sulfhydryl (SH) content, surface hydrophobicity, and secondary structures. Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and enzyme‐linked immunoabsorbent assay (ELISA) were used to define the proteins patterns and IgE‐binding ability. The results showed that HHP treatment in the ranges of 0 to 500 MPa led to a slight but gradual decline in free SH content. The solubility and hydrophobicity of soybean proteins increased sharply from 100 to 200 MPa, and gradually decreased upon the further increase of pressure. The α‐helix and β‐sheets contents of soybean proteins decreased, while the random coil content increased. The SDS‐PAGE showed that HHP treatment of 100 to 200 MPa could dissociate the proteins, breaking the aggregates into smaller units, while the treatment ranging from 300 to 500 MPa could induce the proteins aggregation into larger units. Moreover, the ELISA revealed that the IgE‐binding of soybean proteins after HHP treatment at 200 MPa decreased 61.7% compared to the untreated group. Our findings suggested that HHP processing could not only modify the physicochemical properties of soybean proteins, but also significantly reduce its IgE‐binding at an appropriate pressure level.     

高静水压和热处理对荞麦蛋白功能性质的影响

高静水压（high hydrostatic pressure,HHP）处理技术作为一种新型的非热食品加工技术,在食品加工中具有对食物营养和感官品质破坏较小等多种优势。本实验通过HHP处理与传统热处理法（heat treatment,HT）处理荞麦蛋白（buckwheat protein,BWP）,并测定BWP溶解性、起泡性和乳化性等性质,此外通过BWP羰基含量、巯基含量和表面疏水性测定以及傅里叶变换红外光谱分析,研究不同加工处理方式对BWP功能性质的影响。结果表明：随着HHP处理压力的增加,BWP的羰基含量、表面疏水性显著升高（P＜0.05）,而巯基含量显著降低（P＜0.05）;在500 MPa、30 min时,BWP乳化性、起泡性均达到最大值（67.3 m2/g和91.5%）,较空白对照组（未经HHP处理）分别提高33.0%、16.1%,较HT处理组分别提高61.5%、52.8%。傅里叶变换红外光谱结果表明,HHP处理后BWP的α-螺旋和β-折叠相对含量减少,β-转角和无规卷曲相对含量增加。经相关性分析发现,BWP的溶解性、乳化性、起泡性、羰基含量与巯基含量之间均存在相关性。其中,BWP的乳化性与起泡性、表面疏水性呈极显著正相关（P＜0.01）,并且与巯基含量呈极显著负相关（P＜0.01）。综上,HHP处理可以改变BWP的功能性质。可为进一步研究BWP在功能食品的应用提供理论和实验依据。     

高静压协同酶法处理对白果蛋白抗原性的影响

本文研究了高静压结合酶解处理对白果蛋白抗原性的影响,分别采用4种蛋白酶水解白果蛋白,水解前分别采用不同压力的高静压对白果蛋白进行预处理,酶解产物水解率和分子量采用OPA法和SDS-PAGE测定,致敏性采用western-blotting和ELISA法测定。结果表明,木瓜蛋白酶,碱性蛋白酶或胃蛋白酶为水解酶时,高静压能显著提高白果蛋白的水解率和降低其致敏性;而中性蛋白酶为水解酶时,白果蛋白的水解和脱敏效果很差,即使高压处理也未见明显提高。木瓜蛋白酶或碱性蛋白酶在处理压力为300 MPa时,而胃蛋白酶在400 MPa时,其水解和脱敏效果最好,在此条件下白果蛋白能被水解为分子量小于15 ku的多肽,95%以上的白果蛋白致敏性能被消除,酶解产物中致敏蛋白条带全部消失。因此,高静压处理能明显提高蛋白酶对白果蛋白的水解效率和脱敏效果,但是取决于选择的蛋白酶种类和处理压力的大小。     

Selective-Targeted Effect of High-Pressure Processing on Proteins Related to Quality: a Proteomics Evidence in Atlantic Mackerel (Scomber scombrus)

The effect of high hydrostatic pressure (HHP) treatment (150, 300, and 450 MPa for 0, 2.5, and 5 min) on total sodium dodecyl sulfate (SDS)-soluble and sarcoplasmic proteins in frozen (?10 °C for 3 months) Atlantic mackerel (Scomber scombrus) was evaluated. Proteomics tools based on image analysis of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) protein gels and protein identification by tandem mass spectrometry (MS/MS) were applied. Total SDS-soluble proteins, composed in high proportion of myofibrillar proteins, were stable under pressurization treatment in terms of solubility and electrophoretic gel profiles. However, pressurization reduced sarcoplasmic proteins’ solubility, modified their one-dimensional (1-D)/two-dimensional (2-D) SDS-PAGE patterns in a direct-dependent manner, and exerted a selective effect on particular sarcoplasmic proteins depending on processing conditions. Thus, protein bands assigned to creatine kinase, fructose-bisphosphate aldolase A, glycogen phosphorylase, and β-enolase were degraded at 300–450 MPa. Additionally, the stability of triosephosphate isomerase B, phosphoglucomutase, and phosphoglycerate kinase-1 was found to be HHP-reduced when submitted at 450 MPa. HHP processing (300–450 MPa) also induced a cross-linking product formation of pyruvate kinase and two compounds derived from tropomyosin at 450 MPa. Frozen storage time of pressurized samples induced an additional lessening in protein solubility, but electrophoretic patterns were not modified. The present investigation emphasizes the higher lability of sarcoplasmic proteins under HHP treatment and the important role of these proteins in the sensory quality enhancement provided by milder HHP conditions on frozen mackerel. HHP technology is expected to boost the development of novel tailored processing approaches to tackle food quality challenges.     

Low temperature dry extrusion and high-pressure processing prior to enzyme-assisted aqueous extraction of full fat soybean flakes

Oil, protein and solid extraction yields obtained during aqueous extraction processing (AEP) of full fat soybean flakes (FFSF), FFSF extruded at a die temperature of 100 °C and FFSF pressurised at 200 and 500 MPa for 15 min at 25 °C, were compared to those obtained during enzyme-assisted aqueous extraction processing (EAEP) using 0.5% of protease Protex 7L. Without enzyme addition, pretreatment of the FFSF with extrusion and 500 MPa increased and decreased, respectively, the oil extraction yield while protein extraction yield was significantly decreased after both treatments. The best treatment in terms of oil and protein recovery was EAEP of extruded flakes with 90% and 82% of oil and protein extraction yield, respectively, and 17% of free oil. Addition of protease during extraction significantly decreased the yield of isolated soy protein (ISP) due to an increased solubility of the proteins at pH 4.5. ISP from extruded EAEP had higher solubility at pH 7.0 and better functionality. The DSC results, combined with the protein extraction yields, showed that a proportion of the proteins became insoluble after extrusion and 500 MPa treatment, while only those extracted from 500 MPa FFSF had a reduced native state.     

Effects of high hydrostatic pressure on the structure and retrogradation inhibition of oat starch

As a non-thermal processing technology, high hydrostatic pressure (HHP) can be used for starch modification without affecting the quality and flavour constituents. The effect of HHP on starch is closely related to the treatment pressure of HHP. In this paper, we investigated the impacts of HHP treatment pressure (0, 100, 200, 300, 400, 500, 600 MPa) on the microstructure and retrogradation characteristics of oat starch, established the retrogradation kinetic model and elaborated the mechanism of HHP treatment inhibiting the retrogradation of oat starch. Results show that HHP treatment caused the microstructure of oat starch experienced crystallisation perfection (100–300 MPa), crystallisation destruction (400 MPa), crystallisation disintegration and gelatinisation (500–600 MPa). Results of oat starch retrogradation showed that, after treated at 500 MPa for 15 min, the recrystallisation rate of oat starch was reduced, the formation of nuclei at the early stage of oat starch retrogradation suppressed and its nucleation mode was changed from instantaneous to spontaneous, otherwise, the mobility of water in oat starch gel system reduced. Therefore, 500 MPa treated for 15 min can inhibits the retrogradation of oat starch. This study provides theoretical guidance for the application of HHP technology in starch modification and food processing.