美拉德反应制备等电点澄清透明的乳清分离蛋白研究

本文旨在以乳清分离蛋白(WPI)为原料,研究其与糖类物质的美拉德反应制备得到产物在等电点附近的溶解性、乳化性(EAI)、乳化稳定性(ESI)和热稳定性等功能特性,为其在透明饮料中的应用奠定基础。在相对湿度79%,温度70℃的反应条件下制备得到乳清分离蛋白与L-乳糖的美拉德反应产物,聚丙烯酰胺凝胶电泳(SDS-PAGE)实验结果证实了美拉德反应使WPI中α-乳清蛋白和β-乳球蛋白分子量分别增加2~3 ku左右;邻苯二甲醛(OPA)结果表明在美拉德反应时间为12 h时,WPI中游离氨基酸含量减少35%左右;美拉德反应改性使其溶解性、乳化性(EAI)、乳化稳定性(ESI)和热稳定性都有显著提高(在10 min时EAI和ESI分别为0.35 m~2/g和20 min,为WPI的1.5倍和1.7倍);电位和粒径结果显示,乳清分离蛋白糖基化产物的溶解性和热稳定性增强是因为蛋白质表面引入糖分子的位阻效应所致,而非静电作用。     

湿热条件下低聚木糖-乳清分离蛋白的美拉德反应及产物乳化性与流变性研究

本文研究了湿热条件下不同混合质量比(1:1、1:2、1:3、1:4,W/W)对低聚木糖与乳清分离蛋白(WPI)美拉德反应及其产物的乳化性与流变性的影响。UV-Vis吸光度值,pH和粒径大小显著变化表明,该条件下成功制备了WPI和低聚木糖的美拉德反应产物 (MRPs)。数据显示,质量比为1:2的溶液体系美拉德反应程度最高;与未经过美拉德反应的体系相比,MRPs的平均粒径均减小而质量比为1:2的体系平均粒径最大;同时,美拉德反应提高了WPI的乳化活性和乳化稳定性,比例为1:2时MRPs具有最佳的乳化活性(38.63 m2/g)和乳化稳定性(65.23%);流变学测试表明糖基化修饰增强了WPI的凝胶性,比例为1:3时MRPs的储存模量提高最大,G''值高达约97,000 Pa (约WPI的7倍);在相同剪切速率下,MRPs溶液的表观粘度增加,而WPI的添加比例对体系粘度的影响占主导作用。上述研究表明,美拉德反应可改善WPI的乳化特性和凝胶特性。     

美拉德反应中乳清分离蛋白特性的变化

乳清分离蛋白(WPI)与两种还原糖(D-葡萄糖和D-半乳糖)按质量比1:1配制成混合液,分别在95℃条件下加热0～6h,得到不同反应时间的美拉德反应产物(MRPs)。测定MRPs的pH值、吸光度(A294nm和A420nm)、荧光强度、游离氨基含量以及蛋白聚合物变化趋势。结果表明:随着加热时间的延长,各反应体系的pH值显著降低(P<0.05);非荧光中间产物(A294nm)、褐变产物(A420nm)以及荧光强度显著增加(P<0.05);游离氨基的含量逐渐降低(P<0.05);SDS-PAGE测定表明,在美拉德反应过程中,α-乳白蛋白和β-乳球蛋白都发生了不同程度的聚合,生成更大分子的物质;WPI与D-半乳糖反应体系比D-葡萄糖体系反应产生的MRPs具有更多的中间产物和发生更大程度的褐变。     

乳清分离蛋白-葡聚糖接枝物Pickering乳液的稳定性研究

为提高蛋白基Pickering乳液稳定性,采用美拉德反应制备乳清分离蛋白(WPI)-葡聚糖(Dex)接枝物,然后利用该接枝物制得蛋白基固体颗粒,再与中链甘油三酯制备Pickering乳液,考察WPI-Dex接枝物对蛋白基固体颗粒乳化活性、乳化稳定性和蛋白基Pickering乳液乳析指数的影响,以及Pickering乳液在不同pH、加热温度、贮藏时间下粒径的变化。结果表明：扫描电镜观察到共价接枝Dex将WPI形貌结构由球状转变为片状,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳证实干法美拉德反应成功制备了WPI-Dex接枝物;与WPI相比,WPI-Dex接枝物的乳化活性和乳化稳定性分别增加了57.8%和138.5%;WPI和WPI-Dex接枝物Pickering乳液贮藏30 d时的乳析指数分别为52.3%和36.0%,WPI-Dex接枝物使Pickering乳液的乳析稳定性提高了31.2%;WPI-Dex接枝物Pickering乳液具有良好的pH稳定性、热稳定性和贮藏稳定性。综上,蛋白质糖基化接枝修饰是提高天然蛋白质Pickering乳液稳定性的有效方法。     

乳清蛋白Maillard反应产物对Caco-2细胞抗增殖抑制的影响

利用Caco-2细胞培养技术,结合MTT检测法,研究乳清分离蛋白(WPI)和还原糖(核糖、半乳糖以及乳糖)的美拉德(Maillard)反应产物对Caco-2细胞生长抑制的影响,以判断乳清蛋白Maillard反应产物的毒理学特性.结果表明,加热处理时间为0,2和4h的乳清蛋白Maillard反应产物,在作用Caco-2细胞质量浓度在0.01～2 g/L范围内,随着Maillard反应产物浓度的增加,Caco-2细胞存活率降低.在相同的作用质量浓度条件下,不同加热处理时间的乳清蛋白Maillard反应产物对Caco-2细胞存活率影响差异显著,即加热处理时间越长的乳清蛋白Maillard反应产物,对Caco-2细胞抑制作用越大.加热处理时间为4h的WPI、WPI-核糖、WPI-半乳糖和WPI-乳糖的Maillard反应产物对Caco-2细胞存活率分别为68.7％,87.5％,86.8％和70.9％,这说明乳清蛋白Maillard反应产物对Caco-2细胞生长具有微弱抑制作用,可作为功能性基料,在食品工业中广泛应用.     

大豆分离蛋白超声处理对其与木糖美拉德反应及产物乳化能力的影响

将大豆分离蛋白（soybean protein isolate,SPI）经过超声波预处理不同时间,然后与木糖（xylose,XYL）以质量比4∶1在湿热条件（90?℃、6?h）下发生美拉德反应制备美拉德产物（Maillard reaction products,MRPs）,并以此产物为乳化剂、大豆油为分散相制备乳状液,研究SPI超声处理对其与XYL美拉德反应及所得MRPs乳化能力的影响。结果表明：对SPI进行超声处理可以显著促进其与XYL之间的美拉德反应,并提高相应MRPs的Zeta电位、荧光强度及乳化能力。粒径分析和分层指数分析表明,与天然SPI相比,其经超声处理后再与XYL发生美拉德反应降低了相应MRPs稳定乳液在环境离子强度、加热及pH值发生变化时的聚集程度,但是对乳液的分层情况没有明显的改善作用。     

米糠多糖-乳清分离蛋白美拉德反应及其产物表征

目的：通过米糠多糖(rice bran polysaccharide,RBP)与乳清分离蛋白(whey protein isolate,WPI)非酶褐变反应,探究反应时间对美拉德反应的影响,开发一种新型乳化剂制备纳米乳液包埋脂溶性物质。方法：RBP与WPI在干热条件下反应不同的时间,以A_{294 nm}、褐变程度、色泽变化、游离氨基含量等的变化监控美拉德反应进程,对美拉德反应产物的分子质量、粒径、乳化活性、乳化稳定性、微观结构进行表征,并用美拉德反应产物制备大豆油纳米乳液。结果：A_{294 nm}、褐变程度、色泽变化及游离氨基含量的变化,表明混合体系发生了美拉德反应,且随反应时间的延长产物颜色变暗;美拉德反应产物分子质量为17 kDa;反应12 h时的产物粒径分布较为均匀;RBP接枝到WPI分子上显著提高了WPI的乳化活性和乳化稳定性(P<0.05);WPI-RBP(12 h)@纳米乳液比WPI@纳米乳液更稳定、粒径均匀,能抵抗酸性条件下和盐离子存在条件下对乳液的不利影响。结论：WPI与RBP间的美拉德反应有效改善了WPI的特性,可作为一种新型...     

乳清分离蛋白与壳聚糖美拉德反应初级阶段产物乳化性研究

采用干热美拉德反应制备乳清分离蛋白(WPI)-壳聚糖复合物,通过对复合物的乳液粒径及稳定性分析,研究反应温度、WPI与壳聚糖质量比及反应时间对复合物乳化性的影响。荧光光谱分析表明：WPI和壳聚糖在干热反应后发生共价交联并且结构发生了变化。WPI与壳聚糖在50℃、相对湿度79%干热条件下,反应1～4d的产物为Maillard反应初级阶段产物,该产物的乳化性质得到改善。其中,WPI与壳聚糖以质量比1:4混合,50℃反应1d的产物乳化稳定性是反应前的10倍。     

超声对乳清蛋白及其水解物的美拉德产物抗氧化性及结构的影响

为了提高乳清蛋白及其水解物的抗氧化性,以乳清分离蛋白及其水解物为原料,在超声辅助水浴条件下分别与半乳糖发生美拉德反应,研究超声时间对美拉德反应进程、产物结构及抗氧化性的影响。结果表明,乳清分离蛋白美拉德反应中间产物、褐变程度和接枝度分别在超声60 min、未超声及超声30 min时达到最大值,分别为1.37,0.24,20.89%;而乳清蛋白水解物美拉德反应均在未超声时达到最大值,分别为1.08,0.29,21.80%。乳清分离蛋白及其水解物美拉德反应产物还原力均在超声作用60 min时达到最大值,分别为0.481和0.699,显著高于未超声处理(P0.05);两者反应产物DPPH自由基清除能力均在超声30 min时达到最大值,分别为54.89%和83.83%,显著高于未超声处理组(P0.05)。同时,内源荧光光谱及紫外吸收光谱表明,超声作用可以改变美拉德反应产物的结构,使其结构更加松散。以上结果表明,超声作用可改变美拉德反应进程及产物的三级结构,提高产物抗氧化性。     

乳清分离蛋白美拉德反应产物的体外抗氧化特性

以乳清分离蛋白和木糖、葡萄糖、果糖、蔗糖、麦芽糖、乳糖为美拉德反应原料,研究蛋白与糖的种类及混合比例对褐变程度的影响,并对反应产物进行抗氧化活性检测。结果表明:木糖与乳清分离蛋白按质量比2:1混合,湿热糖基化反应后,褐变程度最强,且在乳清分离蛋白与木糖按质量比2:1条件下反应后,乳清分离蛋白-木糖体系的美拉德反应产物(MRPs)具有较高还原能力和抗脂质过氧化能力及对DPPH自由基和O-2.具有较高的清除能力。     

乳清分离蛋白-甜菜果胶共价复合物理化特性分析

蛋白质-多糖通过Maillard反应生成共价复合物,其乳化稳定性等功能特性得到提高,为研究与开发新型食品添加剂提供了一条新的途径,但共价复合物的乳化稳定性提高机理尚不明确。本研究通过界面张力仪测定界面张力、红外光谱分析二级结构,并结合GPC-动态光散射法考察其分子量分布,初步探讨乳清分离蛋白(WPI)-甜菜果胶(BP)共价复合物乳化稳定提高原因。研究结果表明:WPI-BP共价复合物与混合物相比,界面张力降低;WPI-BP共价复合作用使得蛋白质分子中的羟基含量增加,亲水性增强;共价复合物的分子量增大,dn/dc降低,分子量分布更集中,提高其在乳状液界面上的空间位阻作用,从而提高其乳化稳定性。因此,界面张力降低、亲水性增强与空间位阻增大是WPI-BP共价复合物乳化稳定性提高的主要原因。     

Gel mechanical properties of milk whey protein–dextran conjugates obtained by Maillard reaction

The effect of Maillard reaction on the mechanical properties of whey protein isolate (WPI) heat-induced gels was evaluated. WPI and dextran (15–25 kDa) conjugates were obtained by controlled dry heating during storage at 60 °C and 63% relative humidity for 2, 5 and 9 days. Changes in browning intensity and content of free amino groups were used to estimate the Maillard reaction. A decrease in free amino groups of WPI was observed when increasing polysaccharide concentration and reaction time. An increase in both a* and b* CIE Lab colour parameters indicated the development of a reddish-brown colour, typical of the Maillard reaction. Uniaxial compression and stress relaxation tests were performed to measure the mechanical properties of mixed and conjugate gels. Maillard reaction significantly modified the mechanical properties of WPI/DX gels, and even prevented fracture when conjugate gels were subjected to 80% deformation in uniaxial compression test.     

Ultrasound assisted vis-à-vis classical heating for the conjugation of whey protein isolate-gellan gum: Process optimization,structural characterization and physico-functional evaluation

In the present work, ultrasonic assisted synthesis and characterization of Maillard conjugates of whey protein isolate (WPI) and gellan gum (GG) was carried out and compared with the classical heating method. Conjugation was confirmed by FTIR, secondary structure, intrinsic fluorescence analysis, and degree of glycation (DG). The structural analysis revealed high protein unfolding by sonication confirming loss of α-helix content and exposing more amino groups to the conjugation reaction. Color change analysis confirmed that ultrasonication accelerated the initial stage of Maillard reaction but did not form the advanced Maillard reaction products. Ultrasonication gave higher DG in lesser time (17.22% in 60 min) than classical heating method (8.41% in 4 h). Physico-functional properties such as pH and thermal stability, emulsifying activity index, emulsion stability index, foaming capability and antioxidant activity of WPI-GG conjugates prepared by ultrasonic treatment were superior to classical heating method suggesting ultrasonication as a potentially alternative method for Maillard conjugation.Industrial relevanceImprovising supplementary protein functionality by conjugation with food grade polysaccharides like gellan gum through ultrasonication could make the process more controllable and economical. The undesired browning can be inhibited in ultrasonic assisted conjugate synthesis and hence the overall acceptance of the final product can be increased.     

Pilot-scale formation of whey protein aggregates determine the stability of heat-treated whey protein solutions—Effect of pH and protein concentration

Denaturation and consequent aggregation in whey protein solutions is critical to product functionality during processing. Solutions of whey protein isolate (WPI) prepared at 1, 4, 8, and 12% (wt/wt) and pH 6.2, 6.7, or 7.2 were subjected to heat treatment (85°C × 30 s) using a pilot-scale heat exchanger. The effects of heat treatment on whey protein denaturation and aggregation were determined by chromatography, particle size, turbidity, and rheological analyses. The influence of pH and WPI concentration during heat treatment on the thermal stability of the resulting dispersions was also investigated. Whey protein isolate solutions heated at pH 6.2 were more extensively denatured, had a greater proportion of insoluble aggregates, higher particle size and turbidity, and were significantly less heat-stable than equivalent samples prepared at pH 6.7 and 7.2. The effects of WPI concentration on denaturation/aggregation behavior were more apparent at higher pH where the stabilizing effects of charge repulsion became increasingly influential. Solutions containing 12% (wt/wt) WPI had significantly higher apparent viscosities, at each pH, compared with lower protein concentrations, with solutions prepared at pH 6.2 forming a gel. Smaller average particle size and a higher proportion of soluble aggregates in WPI solutions, pre-heated at pH 6.7 and 7.2, resulted in improved thermal stability on subsequent heating. Higher pH during secondary heating also increased thermal stability. This study offers insight into the interactive effects of pH and whey protein concentration during pilot-scale processing and demonstrates how protein functionality can be controlled through manipulation of these factors.     

Heat-induced whey protein isolate fibrils: Conversion,hydrolysis, and disulphide bond formation

Fibril formation of individual pure whey proteins and whey protein isolate (WPI) was studied. The heat-induced conversion of WPI monomers into fibrils at pH 2 and low ionic strength increased with heating time and protein concentration. Previous studies, using a precipitation method, size-exclusion method, or proton NMR spectroscopy, reported a wide range of values for the conversion. An alternative method was developed, namely centrifugal filtration, giving a consistent picture of the conversion. The present results help to explain the disparities reported in literature. No fibrils formed upon heating pure α-lactalbumin or pure BSA at pH 2, whereas fibrils formed in pure β-lactoglobulin (β-lg) and WPI solutions. Experiments indicate that β-lg was the only whey protein involved in fibril formation. In all whey protein samples, hydrolysis occurred during heating at pH 2, as determined by HPLC and SDS-PAGE. When WPI fibrils formed at pH 2 were stored at pH 7 or 10, disulphide bonds were formed in the samples.     

Fibrillation of whey protein isolate by radio frequency heating for process efficiency: Assembly behavior,structural characteristics,and in-vitro digestion

In this study, a novel technology involving radio frequency heating (RF) was applied to promote the production of whey protein isolate (WPI) nanofibrils. As the prolonged treatment time, gradual increases were observed in the conversion rate (>16 h with the conventional processes), fluorescence intensity, and free amino acid content of WPI. During the fibrillation process, WPI was hydrolyzed into small molecular weight peptides that self-assembled into fibrils, resulting in a significant increase in the ζ-potential and surface hydrophobicity. Conformational and structural changes that favored an increased content of β-sheets were confirmed by Fourier transform infrared and circular dichroism spectroscopy. A linear fibril structure with a proper aspect ratio (length/diameter, 35.50 ± 0.39) formed at 7 h. Moreover, the good resistance to proteolytic digestion (especially gastric stage) of certain fibrils makes them suitable for application as nutraceutical delivery vehicles. Our findings move a step forward for WPI fibrillation by RF heating.     

乳清分离蛋白酶解制备抗氧化和ACE抑制活性产物

以乳清分离蛋白为原料,通过对肽浓度、游离氨基酸含量及水解度的研究,探讨了碱性蛋白酶Alkaline、中性蛋白酶NPU、肽酶AX和风味蛋白酶Flavourzyme对乳清蛋白酶解的影响。在优化碱性蛋白酶Alkaline水解条件的基础上,考察了中性蛋白酶NPU、风味酶Flavourzyme添加时间,获得了复合酶分步酶解工艺:酶解温度55℃,加入1.5% Alkaline([E/S]),维持pH8.5酶解10 min,待pH7.2时加入1% NPU([E/S]),酶解240 min,加入1% Flavourzyme([E/S])继续酶解20 min。在此条件下,肽浓度达到60 g/L以上,游离氨基酸含量为4348 mg/kg。获得的酶解产物基本无苦味。酶解物分子量主要分布在<1 kDa(69.62%)。酶解产物具有ABTS自由基清除和ACE抑制活性,IC₅₀值分别为1.02(0.377 μmol Trolox当量/mg)和1.38 mg/mL。综上,采用复合酶酶解乳清分离蛋白可获得具有抗氧化活性和ACE抑制活性产物,为乳清酶解物在功能食品或保健品领域的运用提供科学参考。