Effect of ascorbic acid and protein concentration on the molecular weight profile of bovine serum albumin and β-lactoglobulin by γ-irradiation

The effects of ascorbic acid and protein concentration on the molecular weight size distribution of BSA and β-lactoglobulin were examined after irradiation of proteins at various doses. Gamma-irradiation of protein solutions caused disruption of the ordered structure of protein molecules resulting in degradation, cross-linking, and aggregation of the polypeptide chains. SDS–PAGE and gel permeation chromatography study showed that ascorbic acid protected the aggregation and degradation of proteins by scavenging oxygen radicals produced by irradiation and the effect of irradiation on protein conformation was more significant at lower concentrations of proteins.     

Effect of gamma-irradiation on the physicochemical properties of porcine and bovine blood plasma proteins

To elucidate the effect of γ-irradiation on the physicochemical properties of blood plasma proteins, bovine and porcine blood, released from a slaughterhouse, was collected and plasma proteins were prepared. Physicochemical properties of blood plasma protein powders and solutions, such as molecular weight distribution, secondary structure, solubility, and viscosity, were examined after γ-irradiation at 1, 5, 7, and 10 kGy. Oxygen radicals, such as hydroxyl and superoxide anion radicals, generated by γ-irradiation, affected the physicochemical properties of the blood plasma protein solutions, but not the plasma protein powders. Circular dichroism and sodium dodecyl sulphate–polyacrylamide gel electrophoresis studies showed that an increase of γ-irradiation decreased the ordered structure of plasma protein solutions and caused initial fragmentation of the polypeptide chains and subsequent aggregation. However, solubility and viscosity of the irradiated plasma protein powders, as well as secondary structure and molecular weight profile, were not changed significantly with radiation dose.     

蛋白质氧化对核桃蛋白质结构的影响

为了深入明晰氧化对核桃蛋白质结构性的影响,本文以核桃分离蛋白为研究对象,采用不同浓度的2,2'-盐酸脒基丙烷(AAPH)热降解形成烷过氧自由基(ROO·)代表脂质过氧化反应过程中产生的脂质自由基,对核桃分离蛋白进行氧化修饰得到不同氧化程度的核桃氧化蛋白。对不同氧化程度蛋白的羰基、巯基和总巯基,表面疏水性(H₀)、内源荧光、粒径分布、相对分子质量和二级结构进行研究和分析。结果表明,随着AAPH浓度的增加,核桃氧化蛋白羰基含量显著增加(p<0.05),巯基和H₀显著降低(p<0.05);圆二色谱结果表明,随着AAPH浓度的增加,核桃蛋白α-螺旋结构和β-折叠含量下降,而无规卷曲结构含量增加,说明氧化破坏了蛋白质的二级结构,使有序结构变为无序结构。最大内源荧光值降低,且出现蓝移现象,说明烷过氧自由基氧化导致分子聚集,使得色氨酸残基包埋;粒径分布结果表明,核桃氧化蛋白可溶性聚集体可被共价交联和非共价聚集转变成不可溶性部分;体积排阻色谱结果表明,核桃氧化蛋白相对分子质量随着氧化浓度的增大聚集程度逐渐增大。研究结果表明,核桃蛋白在烷过氧自由基(ROO·)氧化体系中产生了显著氧化作用并导致其结构发生一定的改变,为进一步阐明核桃蛋白氧化机理提供理论依据。     

Electrophoretic patterns of microwaved and γ‐irradiated beef liver proteins

The effects of γ‐irradiation treatments (2.5, 5 and 10 kGy) and microwaves generated from an oven at low and defrost power settings for 0.5, 1 and 2 min on the total proteins and protein patterns of beef liver immediately after treatment and during frozen storage (?18 °C) for different periods were studied. Chemical analyses indicated that the protein content of beef liver was reduced after exposure to γ‐radiation or microwaves and also during frozen storage. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) was used to illustrate the changes in protein bands of different molecular weights and their percentages before and after exposure to gamma and microwave radiation. The main effect of γ‐radiation on the protein patterns of beef liver was the disappearance of some high‐molecular‐weight protein bands and the development of other bands characterised by moderate and low molecular weights. This finding indicates the degradation of beef liver proteins by γ‐irradiation. In contrast, microwave treatment caused an increase in the levels of high‐molecular‐weight protein bands with a concomitant decrease in low‐molecular‐weight protein bands. This phenomenon demonstrates the polymerisation of low‐molecular‐weight proteins under the influence of microwaves. © 2001 Society of Chemical Industry     

Effects of microwave heating,steaming, boiling and baking on the structure and functional properties of quinoa (Chenopodium quinoa Willd.) protein isolates

The effects of microwave heating (MW), steaming (SM), boiling (BL) and baking (BK) on the structure and functional properties of quinoa protein isolates (QPI) were investigated. SDS-PAGE showed the 20–30 kDa band strength of QPI-BL was enhanced, which indicated the disulphide bond was broken and protein molecules dissociated. Due to the recombination of subunits, some large molecular weight or insoluble polymer components of QPI-SM and QPI-BK did not pass through the gel. Microwave heating and boiling showed negative effects for α-helix and positive effects for β-sheet, which implied the molecular structure was transformed from ordered to disordered, the secondary structure became loose. The reduction of free SH (sulfhydryl) and surface hydrophobicity implied that aggregation and cross-linking of protein molecules for QPI-SM and QPI-BK. QPI, QPI-MW and QPI-BL exhibited better solubility, which was related to the water holding capacity (WHC) and emulsification. For QPI-SM and QPI-BK, functional properties (including solubility, WHC and gel-forming ability) decreased due to molecular aggregation. Heat treatments significantly affect the structure and functional properties of QPI, the current research showed that microwave heating and boiling might be better heat treatment methods for QPI and would help the development of quinoa protein products.     

FTIR spectroscopic characterization of irradiated hazelnut (Corylus avellana L.)

Radiation induced molecular changes in macromolecular components of hazelnut tissues were investigated by mid-Fourier transform infrared (FTIR) spectroscopy. Irradiation dose of 1.5 kGy (low) and 10 kGy (high) were applied. The changes in frequency, signal intensity and intensity ratio of IR bands revealed that the unsaturated lipid concentration increased for low dose treated samples whereas it decreased and peroxidation appeared at high dose treatment. The low dose irradiation treatment, slightly increased the total lipid content whereas it dramatically decreased for high dose treatment. A slight increase in the lipid to protein ratio was observed for low dose treatment, whilst this ratio significantly decreased for high dose treatment. In addition, the high dose γ-irradiation caused alterations in the structure of hazelnut proteins, as cross-linking and aggregation occured in protein molecules. These results indicate that FTIR spectroscopy can be successfully used to monitor food irradiation.     

Changes in protein-protein and protein-polysaccharide interactions induced by high pressure

β-Lactoglobulin and bovine serum albumin (BSA) protein solutions (0.1%, 0.2% and 2.5%), when subjected to high pressure treatment (800 MPa for 20 min) at neutral pH, were denatured and some aggregates formed. The total calorimetric enthalpy of 2.5% solutions of the pressure-treated proteins decreased to virtually zero for both β-lactoglobulin and BSA following pressure treatment. Isoelectric focussing patterns (IEF) indicated that aggregation occurred in both proteins and there was a concomitant loss of sulphydryl groups (42% for β-lactoglobulin and 55% for BSA), suggesting that protein aggregation after high pressure processing was caused, at least in part, by the formation of -S-S- bridges. The surface hydrophobicity of the two proteins was modified, increasing (40%) with β-lactoglobulin and decreasing (41%) with BSA. Pressure treatment of 1:1 mixtures of BSA and dextran sulphate (DS) yielded structures with a significant enthalpy. However, addition of DS to β-lactoglobulin had little effect on the thermograms, suggesting that the DS either protects the protein against pressure induced unfolding or enables the pressure-denatured protein to regain some secondary structure.     

超声波辅助泡沫分离回收溶液中牛血清白蛋白的性质研究

本文提出了一种超声波辅助泡沫分离富集水溶液中牛血清白蛋白的新方法。首先利用傅立叶红外光谱研究泡沫分离前后牛血清白蛋白分子结构的变化,利用高效分子排阻色谱测定超声波对牛血清白蛋白聚集效果的影响,随后,研究了装液高度、气体体积流量、分布器孔径对泡沫分离回收牛血清白蛋白效率的影响,并对回收的牛血清白蛋白的理化性质进行评价。结果表明,泡沫分离过程中牛血清白蛋白在气液界面上的吸附会诱导分子间二硫键的产生,超声波辅助泡沫分离能有效抑制牛血清白蛋白分子聚集。在温度25℃,pH6.0,牛血清白蛋白浓度0.20 g/L,气体分布器的孔径180 μm,液体高度300 mm,进料速率2 mL/min,气体体积流量150 mL/min,超声波功率600 W和超声波处理时间2 min的条件下,牛血清白蛋白的富集比和回收率分别达到6.3%和85.3%。超声波处理能够明显抑制泡沫分离过程中牛血清白蛋白分子聚集体的产生,进而提高消泡液的表面疏水性、泡沫性能和乳化性。     

Effect of ultraviolet irradiation on molecular properties and immunoglobulin production-regulating activity of β-lactoglobulin

To utilize ultraviolet (UV) irradiation as a means to prepare hypo-allergenic food, we investigated the molecular weight profile, secondary structure content, fluorescence spectrum, and immunoglobulin (Ig) production-regulating activity of β-lactoglobulin after UV-irradiation. UV-irradiation caused a change on the molecular size distribution, disruption of the ordered structure, and decrease of emission intensity of β-lactoglobulin. The alteration on Igs production regulating activity of β-lactoglobulin by UV-irradiation was observed in the mouse spleen lymphocytes system. These results suggest that UV-irradiation is effective for alteration of molecular properties and antigenicities of β-lactoglobulin and such treatment should be useful for the preparation of low allergenic foods.     

Probing the heat‐induced structural changes in bovine serum albumin by fluorescence spectroscopy and molecular modelling

The fluorescence spectroscopy and in silico methods were used to evaluate the structural changes in bovine serum albumin (BSA) over a temperature range of 25–70 °C for 15 min. Experimental results indicated that the polypeptide chain rearrangements at temperatures higher than 40 °C lead to a lower exposure of hydrophobic residues causing the decrease in fluorescence intensity. The nonlinearity of the phase diagram indicated a sequential denaturation process involving several molecular species. The molecular dynamics simulations at the single‐molecule level showed the high stability of the BSA structure, compensating for the entropic costs associated with the prevalent helical folding.     

生物解离对豆奶粉致敏性及其结构的影响

本文研究了不同生物解离时间下豆奶粉的致敏性、蛋白质分子量、水解度及氨基酸组成,并利用原子力显微镜与拉曼光谱分析豆奶粉蛋白的结构变化。研究结果表明,生物解离可有效降低豆奶粉的致敏性,过敏原含量最低为1.80 mg/g。随着生物解离时间的增加,水解度随之增加,蛋白质分子量不断减少,在生物解离时间为30 min时,水解度最大为13.2%。原子力显微镜观察可知在生物解离20 min时,蛋白质以单个分子状态存在,未出现分子的聚集。拉曼光谱分析显示蛋白质中的化学键及基团受到了一定程度的破坏。     

Effects of malondialdehyde-induced protein oxidation on the structural characteristics of rice protein

Malondialdehyde (MDA) was selected to represent a secondary by-product of lipid peroxidation during rice ageing. This study aimed to investigate the effects of MDA modification on the structural characteristics of rice protein. The results showed that as MDA concentration increased, rice protein carbonyl and disulphide groups increased, but sulphydryl content decreased. The blue shift of maximum fluorescence peak, the decrease of rice protein intrinsic fluorescence intensity and the reduction of surface hydrophobicity indicated the formation of protein aggregates caused by MDA oxidative modification. The results of molecular weight distribution and particle size distribution showed that MDA modification resulted in the formation of soluble protein aggregates, and the decrease of rice protein solubility indicated that insoluble protein aggregates were formed. Results of protein electrophoresis showed that MDA modification contributed to rice protein aggregation via non-disulphide covalent bonds. The results showed that rice protein gradually aggregated with increasing MDA concentration.     

酯化淀粉薄膜中增塑剂与淀粉分子间相互作用的研究

采用酯化淀粉以邻苯二甲酸二乙酯（DEP）为增塑剂,经流延法制备了酯化淀粉薄膜。通过扫描电子显微（SEM）、傅立叶变换红外光谱分析仪（FT-IR）、小角/广角X射线散射（SAXS/WAXS）等现代分析技术对酯化淀粉薄膜的结构及增塑剂DEP与淀粉分子间的相互作用变化进行了系统分析。结果表明,随着增塑剂DEP含量的增加,增塑剂DEP分子减弱了淀粉分子内/间相互作用力,且导致了无定形区域中淀粉分子形成部分排列更规整的微晶结构,促使淀粉分子形成有序微区,但有序微区结构尺寸Rg在37 nm~38 nm之间变化不大。通过调节酯化淀粉薄膜中增塑剂DEP含量,可控制增塑剂分子与淀粉分子间的相互作用、淀粉分子链段在薄膜中微区的有序排列程度及其规整程度,为设计抑制增塑剂迁移的酯化淀粉薄膜材料提供了基础数据。     

Aggregation and disaggregation of humic supramolecular assemblies by NMR diffusion ordered spectroscopy (DOSY-NMR)

Diffusion ordered nuclear magnetic resonance spectroscopy (DOSY-NMR) was applied to a number of fulvic (FA) and humic (HA) acids of different origin. Spectral separation achieved by DOSY based on diffusion coefficients (D), and correlated to molecular sizes by calibration standards, showed that carbohydrates had the largest molecular size in FA, whereas alkyl or aromatic components were the most slowly diffusing moieties in HA. At increasing concentrations, these components had invariably lower D values in DOSY spectra for all humic samples,thereby indicating an aggregation into apparently larger associations, whose increased hydrodynamic radius was confirmed by viscosity measurements. When humic solutions were broughtfrom alkaline to acidic pH (3.6), components diffusivity detected by DOSY increased significantly, suggesting a decrease of aggregation and molecular size. A general comparison of HA and FA molecular sizes was achieved by multivariate statistical analysis. While a larger extent of aggregation and disaggregation was observed for HA than for FA, no aggregation was detected, under similar conditions, for a true macropolymeric standard. Such difference in diffusion between a polymeric molecule and humic samples, is in line with the supramolecular nature of humic matter. The possible formation of humic micelles was also investigated by both changes of diffusivity in DOSY spectra and shift of 1H NMR signals. Except for HA of peat and soil origin, revealing a self-assembling in micelle-like structures at the 4 mg mL(-1) concentration, no other humic sample showed evidence of critical micelle concentration (cmc) up to 20 mg mL(-1). These results indicated that DOSY-NMR spectroscopy is a useful technique to evaluate components of different molecular size in natural humic superstructures.     

热处理羊乳酪蛋白结构与抗原性的变化规律

热加工处理羊乳α-酪蛋白(α-casein,α-CN)和β-酪蛋白(β-casein,β-CN),通过圆二色谱、荧光光谱等方法探索不同热加工条件下羊乳的蛋白结构变化与抗原性的关系。结果表明:随着对蛋白热处理温度的升高,会破坏羊α-CN和β-CN的天然结构,使得分子内部发生交联或聚集,导致分子量发生改变,分子内游离羰基含量升高,在134℃时,羰基含量分别增长了134.72%、110.98%;自由巯基含量则不断下降;疏水性探针检测最大荧光吸收强度分别增加了50.38%和9.61%,升温引起疏水基团或二硫键等被暴露出来,导致蛋白疏水性随着温度的升高而增大。圆二色谱显示,二级结构中卷曲程度或弹性结构发生复杂的转化,使蛋白质空间结构发生改变,α-螺旋呈现增加趋势,β-转角和无规则卷曲则在减少。两种蛋白的抗原性随着温度的升高而降低,经模拟胃肠消化,134℃处理后抗原性分别减少了80.63%和84.12%。故热处理可降低羊乳酪蛋白的抗原性,且抗原性变化与蛋白的游离羰基含量变化呈反比,还与二级结构中的无规则卷曲及β-转角含量呈正相关关系。     

Study of the denaturation/aggregation behaviour of whole porcine plasma and its protein fractions during heating under acidic pH by variable-temperature FTIR spectroscopy

Variable-temperature (VT) Fourier transform infrared (FTIR) spectroscopy was employed to examine changes in secondary structure of whole plasma proteins as well as of plasma protein fractions (serum, serum albumin and globulins) upon heating at pH 4.5 and to establish their kinetics of thermally induced protein aggregation through formation of non-native intermolecular beta-sheets. A detailed analysis of the amide I′ band in the VT-FTIR spectra indicated that plasma proteins were more thermally sensitive at pH 4.5 than at pH 7.5 both when found as mixtures and in monomolecular systems, with the thermal aggregation being strongly enhanced under acidic conditions, particularly in the case of serum albumin. Comparison of the spectral changes of plasma and serum (fibrinogen-depleted plasma) during heating indicated that fibrinogen has no role in protein aggregation under acidic conditions, in contrast to findings at pH 7.5. Considering the particular characteristics of the different plasma proteins, the strong predominance of positive charges in the plasma as a whole at pH 4.5 along with the effects of these pH conditions on the conformation of fibrinogen could be suggested as the main factors responsible for the lack of a contribution by fibrinogen to protein aggregation. Moreover, 2D correlation spectroscopy indicated that the sequence of structural changes occurring during heating was practically identical among the different protein fractions examined and completely different from that established at pH 7.5, with the native beta-sheets being now more heat-sensitive than the alpha-helical structures and with protein aggregation through the formation of intermolecular beta-sheets beginning after native beta-sheets started to unfold.     

毛葱水溶性多糖的超声波微波协同法提取工艺优化及结构分析

以毛葱为原料,利用超声波微波协同法提取毛葱水溶性多糖。以多糖得率为考察指标,在单因素试验的基础上,通过响应面分析方法确定毛葱水溶性多糖提取的最佳工艺为液料比17∶1(mL/g)、超声波功率360 W、微波功率200 W、协同时间16 min,此条件下毛葱水溶性多糖得率为(11.92±0.13)%。溶解性实验结果表明:该毛葱水溶性多糖可溶于水,不溶于有机溶剂;显色反应实验结果表明:该毛葱水溶性多糖含有糖醛酸,但不含淀粉及酚类物质。紫外光谱分析显示该毛葱水溶性多糖无明显的核酸和蛋白质的特征吸收峰,傅里叶变换红外光谱分析显示该毛葱水溶性多糖具有多糖类的特征吸收峰。高效凝胶渗透色谱法分析该毛葱水溶性多糖的重均分子质量,可初步得出该毛葱水溶性多糖不是由均一组分构成的。     

Structural feature and antioxidant activity of low molecular weight laminarin degraded by gamma irradiation

Recently, it has been reported that low molecular weight laminarin had enhanced biological activities. In this study, laminarin was degraded by gamma irradiation, and the changes in its structure and antioxidant property were investigated. Gel permeation chromatography data showed that the average molecular weight of the irradiated laminarin decreased significantly as the irradiation dose increased. The absorbance at 290 nm, from UV spectra, was increased, depending on the irradiation dose and caused by the formation of carbonyl groups. From the NMR analysis, the ratio of β-1,3- to β-1,6-linkages was not changed, indicating the random scission of chains by gamma irradiation. The antioxidative activities, measured by Rancimat test, were all increased in the gamma-irradiated laminarin, depending on the absorbed dose, presumably due to the carbonyl groups formed by gamma irradiation. Therefore, gamma irradiation could be a promising method for preparing low molecular weight laminarin with enhanced biological activities.     

Aggregation of hydrophobic soybean protein hydrolysates: Changes in molecular weight distribution during storage

The stability and molecular weight of peptides play important roles in their functional and biological properties. In this study, soybean protein hydrolysates (SPHs) with different hydrophobic characteristics were used to evaluate the aggregation of peptides during storage at −20 °C. SPHs were isolated from immobilized metal affinity chromatography, and size expulsion HPLC showed the molecular weight distribution of them at different storage times. As time elapsed, some high molecular weight peptides formed from the original SPHs, and the content of the newly formed high molecular weight peptides produced from the highly hydrophobic SPHs was considerably large. The aggregation could be broken by 6 mol/l urea and 20 g/l SDS. The damage caused by 6 mol/l urea was stronger than that of 20 g/l SDS. The results suggested that hydrophobic interaction may promote the aggregation of SPHs during storage.     

荧光光谱法探究pH值对叶黄素与牛血清白蛋白相互作用的影响

采用荧光光谱法研究不同pH值（3.0、5.2、7.4）条件下叶黄素与牛血清白蛋白（bovine serum albumin，BSA）的相互作用，利用数学模型计算得到二者的猝灭常数和热力学参数等，分析叶黄素与BSA的荧光猝灭现象及蛋白构象的变化，最后通过位点竞争实验和分子模拟技术确定了二者的结合位点。荧光光谱表明，不同pH值条件下叶黄素对BSA均产生荧光猝灭效应，在298 K时，pH 7.4的猝灭常数最大，结合常数较大；热力学参数和分子对接结果表明，叶黄素与BSA间的作用力主要是疏水相互作用；同步荧光光谱表明BSA的构象在叶黄素与pH值的共同作用下发生改变；位点竞争性实验和分子对接实验表明，pH 7.4和pH 5.2时，二者的结合位点在亚结构域IIA和IIIA之间，但更接近于Sudlow’s site II；pH 3.0时，结合位点不在亚结构域IIA及IIIA附近。结果表明，pH值对叶黄素和BSA的相互作用有影响，这为蛋白质与生物活性成分的相互作用提供一定的理论依据。