过氧自由基氧化对大米蛋白结构的影响

采用不同浓度2,2’-盐酸脒基丙烷(AAPH)在有氧条件下热分解产生的过氧自由基代表脂质氧化过程中的脂质自由基,研究过氧自由基氧化对大米蛋白结构的影响。结果表明,当AAPH浓度从0增加至25 mmol/L,大米蛋白羰基和二硫键含量分别由3.77和13.88 nmol/mg增加至7.26和15.73 nmol/mg,游离巯基由7.32 nmol/mg下降至1.97 nmol/mg,表明过氧自由基诱使大米蛋白发生了氧化;傅里叶红外分析表明蛋白质氧化导致大米蛋白α-螺旋和β-折叠含量下降,β-转角和无规卷曲含量上升。随着大米蛋白氧化程度的增加,大米蛋白粒径从126 nm增加到216 nm,表面疏水性从1053下降到568,内源荧光最大荧光峰位蓝移,内源荧光强度下降,并且在分子量分布图中高分子量聚集体含量逐渐增加。表明过氧自由基氧化修饰导致大米蛋白结构改变和形成氧化聚集体。     

脂质过氧自由基诱导兔肉肌浆蛋白聚集机制

为全面理解兔肉品质的形成过程,利用偶氮二异丁脒盐酸盐（2,2’-azobis(2-amidinopropane)dihydrochloride,AAPH）的热分解作用生成过氧自由基（ROO·）诱导兔肉肌浆蛋白氧化,通过AAPH处理后兔肉肌浆蛋白中高铁肌红蛋白相对含量、超铁肌红蛋白浓度、羰基含量、总巯基含量以及肌浆蛋白表面疏水性、粒度、浊度和溶解性测定,肌浆蛋白十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE）和内源荧光光谱分析,研究ROO·胁迫肌浆蛋白氧化聚集机制。结果表明,随着AAPH浓度的升高,高铁肌红蛋白和超铁肌红蛋白相对含量显著增加（P＜0.05）,蛋白质羰基含量显著增加（P＜0.05）,总巯基含量显著下降（P＜0.05）。内源荧光光谱和表面疏水性分析结果表明ROO·可以引起肌浆蛋白的去折叠和疏水基团暴露。SDS-PAGE分析结果表明ROO·可以导致肌浆蛋白二硫键交联。此外,随着AAPH浓度的增大,肌浆蛋白粒度和浊度呈现出逐渐增大的趋势,表明ROO·可以引发肌浆蛋白的聚集。疏水相互作用和二硫键交联是ROO·胁迫肌浆蛋白聚集的主要作用力。ROO·不仅可以直接引发肌浆蛋白聚集,还可以通过影响肌红蛋白诱导氧化体系促进肌浆蛋白交联聚集。     

3种多酚干预过氧自由基诱导核桃蛋白氧化的研究

为研究没食子酸、儿茶素、杨梅素对核桃蛋白氧化稳定性的影响,以2,2-偶氮二异丁基脒二盐酸盐(2,2’-Azobis[2-Methylpropionamidine]dihydrochloride,AAPH)为引发剂,建立过氧自由基氧化核桃蛋白的模拟体系,分析经3种多酚干预后其羰基、巯基、内源荧光、表面疏水性、溶解度等指标的变化规律。结果表明,经过氧自由基氧化后,核桃蛋白的羰基、巯基含量和表面疏水性显著增大,荧光强度和溶解度显著下降(P<0.05)。而经3种多酚干预后,核桃蛋白的羰基含量和表面疏水性相比氧化组显著降低,最大降幅分别为72.2%和58.0%;巯基含量和溶解度相比氧化模型组显著增加,最大增幅分别为2.1倍和53.0%;此外,核桃蛋白的荧光强度显著上升(P<0.05)。相关性热图和主成分分析进一步说明多酚干预可在一定程度降低核桃蛋白的氧化程度,且3种多酚干预对核桃蛋白的氧化指标存在显著区别。总体上,儿茶素对过氧自由基氧化核桃蛋白的抑制效果最好,可为核桃制品的合理储藏和加工提供一定参考。     

过氧自由基对兔肉肌原纤维蛋白理化性质及结构的影响

为探讨脂质氧化对蛋白质氧化的影响,用不同浓度2,2-偶氮二(2-甲基丙基咪)二盐酸盐[2,2′-azobis(2-amidinopropane)dihydrochloride,AAPH]热解产生的脂质初级代谢产物过氧自由基处理兔肉肌原纤维蛋白(myofibril protein,MP)。结果表明,随着AAPH浓度的增加,兔肉MP羰基含量、表面疏水性、亮度值和黄度值显著增加(P<0.05),而MP总巯基、游离氨、溶解度、内源性荧光强度和Zeta电位绝对值都呈显著降低趋势(P<0.05)。拉曼光谱结果显示,MP的α-螺旋含量下降,无规则卷曲含量上升,进一步证实蛋白质二级结构发生改变。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gelelectrophoresis,SDS-PAGE)结果表明,AAPH会导致MP发生交联和聚集,形成分子质量较大的聚集物。因此,过氧自由基会通过促进兔肉肌原纤维蛋白氧化,影响MP的理化性质,研究可为通过调控脂质氧化来控制兔肉加工过程中的蛋白氧化提供一定的理论依据。     

氧化对兔肉肌原纤维蛋白结构、乳化性和凝胶性的影响研究

为探讨氧化对兔肉肌原纤维蛋白结构的氧化修饰及对肌原纤维蛋白乳化性质和凝胶质构特性的影响,本文利用0~10.0 mmol/L 2,2'-盐酸脒基丙烷(2,2'-azobis(2-amidinopropane)dihydrochloride,AAPH)热解产生的烷过氧自由基对兔肉肌原纤维蛋白进行氧化处理。结果表明,随着AAPH浓度的增加,兔肉肌原纤维蛋白游离巯基含量、内源性荧光强度和蛋白乳化稳定性不断减小,而羰基含量、二聚酪氨酸含量、蛋白粒径、乳化活性显著增加(P<0.05),表面疏水性则呈现先增后减再增的趋势。聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis,SDS-PAGE)结果显示,低浓度AAPH(0~1.0 mmol/L)和高浓度(10.0 mmol/L)分别诱导了蛋白的聚集和裂解,这些结构的改变引起了肌原纤维蛋白凝胶性质的改变。适度的氧化改性(AAPH 0~1.0 mmol/L)能提高兔肉肌原纤维蛋白凝胶的质构特性,而进一步氧化(AAPH 1.0~10.0 mmol/L)则显著降低了凝胶的性能(P<0.05)。因此,烷过氧自由基的氧化修饰能显著影响兔肉肌原纤维蛋白的结构和乳化凝胶性质(P<0.05)。     

氢过氧化物氧化对核桃蛋白结构和乳化特性的影响

采用脂肪氧合酶催化亚油酸氧化的反应体系进行氧化修饰核桃分离蛋白,代表脂质氢过氧化物氧化核桃分离蛋白。通过对不同亚油酸含量下核桃分离蛋白的结构指标进行表征,探讨氢过氧化物氧化对其结构的影响。采用氧化修饰后的核桃分离蛋白制备乳状液,对其乳化特性进行综合评价,结果表明,随着亚油酸添加量的增加,羰基和二硫键含量增加,巯基含量减少,表面疏水性下降,溶液可溶性聚集体转变成不可溶性聚集体,粒径变大。乳液内源荧光光谱分析检测到核桃分离蛋白乳液的构象发生改变。不同的氧化程度,乳液油滴粒径分布不同。适度的氧化,使蛋白质结构展开,暴露出更多的疏水基团,形成可溶性聚合物,乳液油滴粒径降低,乳化活性有所提高,进一步氧化,促进液滴的聚合,致乳化活性下降。结构和乳化功能指标之间的皮尔逊相关系数表明,由HPODE氧化引起的蛋白质结构的改变对核桃蛋白乳化性的影响并不显著。     

过氧自由基氧化对米糠蛋白结构和功能性质的影响

采用不同浓度的2,2’-盐酸脒基丙烷（2,2’-azobis(2-amidinopropane),AAPH）有氧热分解产生的过氧自由基氧化米糠蛋白,研究过氧自由基氧化对米糠蛋白结构和功能性质的影响。结果显示：随着AAPH浓度的增加,米糠蛋白羰基、二硫键和二酪氨酸含量分别从2.0?nmol/mg、12.04?nmol/mg和84.54增加到7.09?nmol/mg、14.69?nmol/mg和127.1,游离巯基含量从23.97?nmol/mg下降到15.29?nmol/mg。过氧自由基氧化导致米糠蛋白α-螺旋和无规卷曲相对含量下降,β-折叠相对含量增加,氨基酸残基侧链含量先增加后略微下降;同时使得米糠蛋白表面疏水性和内源荧光强度下降,最大荧光峰位蓝移。过氧自由基氧化对蛋白质亚基结构影响较小,可同时使得米糠蛋白多肽链聚集和主肽链部分断裂形成多肽。随着AAPH浓度的增加,米糠蛋白溶解性逐渐降低,持水性、持油性、起泡能力、泡沫稳定性、乳化性和乳化稳定性先上升后下降。     

脂质自由基AAPH体外氧化对富硒大豆分离蛋白性质和结构的影响

本文采用2,2'-偶氮二(2-脒基丙烷)二盐酸(AAPH)在有氧条件下热分解制备过氧自由基,代表脂肪氧合酶诱导多不饱和脂肪酸脂质过氧化反应产生脂质自由基,研究AAPH体外氧化对富硒大豆分离蛋白性质和结构的影响。随着AAPH添加浓度的增加,FT-IR结果表明大豆分离蛋白(SPI)的α-螺旋、β-转角和无规卷曲结构增加,β-折叠结构相对减少;SDS-PAGE结果表明SPI的亚基在浓缩胶顶端逐步聚集;蛋白溶解性、游离巯基含量下降;表面疏水性、乳化性、乳化稳定性呈现先上升后下降趋势;內源荧光峰位(λmax)表现为逐渐蓝移,且荧光强度先增加后减少。富硒大豆分离蛋白(SSPI)在结构、亚基分布、性质等多项测试指标方面均优于普通大豆分离蛋白(OSPI),可能与SSPI中含有较高的硒含量,因而表现出更强的抗氧化性有关。     

蛋白质氧化对核桃蛋白界面性质的影响

采用不同浓度(0～10 mmol/L)的丙二醛替代脂质过氧化反应中产生的活性醛氧化核桃蛋白,研究蛋白质氧化对核桃蛋白界面性质的影响。试验结果表明:随着丙二醛浓度的增加,核桃蛋白发生显著氧化,羰基含量从3.12 nmol/mg增至20.61 nmol/mg。随着核桃蛋白氧化程度的增加,核桃蛋白粒径分布发生偏移,蛋白质相对分子质量先减小后增加,表面张力呈先下降后上升的趋势,Zeta电位绝对值、表面疏水性、起泡性和泡沫稳定性呈先增加后减小的趋势。当蛋白质氧化体系中的丙二醛浓度为0.1 mmol/L时,表面张力达到最小值,即55.64 mN/m;核桃蛋白的Zeta电位绝对值、表面疏水性、起泡性和起泡稳定性达到最大值,分别为10.38 mV,476,21.43%,17.80%。研究结果表明:丙二醛氧化诱导使得核桃蛋白的分子结构和界面性质发生改变,较低程度的氧化有利于改善核桃蛋白的界面性质。     

过氧自由基氧化对花生球蛋白结构及聚集行为的影响

采用不同浓度的2,2′-盐酸脒基丙烷[2,2′-azobis（2-amidinopropane）,AAPH]有氧热分解产生的过氧自由基氧化花生球蛋白,研究AAPH 氧化对花生球蛋白分子结构及聚集行为的影响。结果显示：随着AAPH 浓度的增加,花生球蛋白表示浊度的吸光度由0.20±0.01 增加至0.44±0.01,平均粒径从（153.94±3.31）nm 增加到（196.80±3.06）nm,而游离巯基含量从（14.62±0.08）μmol/g 下降到（7.77±0.25）μmol/g,Zeta 电位绝对值从（24.17±0.06）mV 下降到（15.93±0.05）mV。对荧光最大发射波长的研究表明,在AAPH 浓度为0～1 mmol/L 时,内源荧光最大荧光峰位红移,在AAPH 浓度为5～15 mmol/L 时,内源荧光最大荧光峰位蓝移。过氧自由基氧化导致花生球蛋白α-螺旋结构含量下降,β-折叠和无规则卷曲含量上升,呈现出α-螺旋向β-折叠和无规则卷曲转化的趋势。扫描电镜观察结果显示,花生球蛋白经过氧自由基氧化后,蛋白由聚集紧实逐渐变得发散,浓度从10 mmol/L 升高到15 mmol/L 时,花生球蛋白表面形态变疏松,孔隙显著增大。因此,AAPH 适度氧化可以通过改变花生球蛋白的结构影响其聚集行为,特别是浓度为5～10 mmol/L 时有利于花生球蛋白的聚集。     

Oxidative modification of soy protein by peroxyl radicals

Oxidative modification of soy protein by peroxyl radicals generated in a solution containing 2,2’-azobis (2-amidinopropane) dihydrochloride (AAPH) under aerobic condition was investigated. Incubation of soy protein with increasing concentration of AAPH resulted in gradual generation of protein carbonyl derivatives and loss of protein sulphydryl groups. Circular dichroism spectra indicated that exposure of soy protein to AAPH led to loss of α-helix structure. Effect of oxidation on tertiary structure was demonstrated by surface hydrophobicity and tryptophan fluorescence. Surface hydrophobicity steadily decreased, accompanied by loss and burial of some tryptophan residues, indicating that soy protein gradually aggregated. The results of the size exclusion chromatogram (SEC) implied that incubation caused an AAPH-dose-dependent increase of fragmentation and aggregation of oxidised soy protein. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) indicated that non-disulphide linkages were involved in aggregate formation, and β-conglycinin was more vulnerable to peroxyl radicals than glycinin.     

Structural modification of soy protein by 13-hydroperoxyoctadecadienoic acid

To reveal the role of primary products of lipid peroxidation during soy protein oxidation process, oxidative modification of soy protein by 13-hydroperoxyoctadecadienoic acid (13-HPODE) generated by lipoxygenase-catalyzed oxidation of linoleic acid was investigated in this article. Incubation of soy protein with increasing concentration of 13-HPODE resulted in generation of protein carbonyl derivatives and loss of protein sulfhydryl groups. Circular dichroism spectra indicated that exposure of soy protein to 13-HPODE led to loss of α-helix structure. Effect of oxidation on tertiary structure was demonstrated by surface hydrophobicity and tryptophan fluorescence. Surface hydrophobicity gradually decreased, accompanied by loss and burial of some tryptophan residues. The results of surface hydrophobicity and tryptophan fluorescence implied that aggregation was induced by oxidation. Size exclusion chromatogram indicated that the extent of aggregation was increased in a 13-HPODE dose-dependent manner. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that non-disulfide linkages were involved in aggregate formation, and β-conglycinin was more vulnerable to 13-HPODE than glycinin.     

Effect of protein oxidation on kinetics of droplets stability probed by microrheology in O/W and W/O emulsions of whey protein concentrate

Whey protein concentrate (WPC) was oxidized by peroxyl radicals derived from 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) and the kinetics of droplet stability in O/W and W/O emulsions stabilized by oxidized WPC were evaluated by studying the micro-rheology. Degrees of protein oxidation were indicated by carbonyl concentration and emulsion types were distinguished by fluorescence microscopy. Oxidation resulted in free sulfhydryl groups degradation and surface hydrophobicity decrease. Moderate protein oxidation promoted to form diminutive droplets, which aggregated quickly to gel-network structure and decreased the motion rate of droplets, leading to the increased elasticity and viscosity, which led to better stability. Over-oxidation underwent severe droplet aggregation and sediment with increased motion rate, which resulted in instability of emulsions. The W/O emulsions of oxidized WPC were more inclined to block the motion of droplets and form a stable structure with higher viscosity, compared with the O/W emulsions.     

Structural modification of soy protein by the lipid peroxidation product malondialdehyde

BACKGROUND: Protein oxidation results in covalent modification of structure and deterioration of functional properties of target protein. Oxidation extent of soy protein was affected by the content and type of lipid peroxidation (LPO) products in defatted soybean flours during storage and processing. Malondialdehyde (MDA) was selected as a secondary byproduct of LPO to investigate the effects of oxidative modification of LPO‐derived reactive aldehyde on soy protein structure. RESULTS: MDA reacted with ε‐amino and sulfhydryl groups of soy protein, and resulted in an increase in protein carbonyl groups but a decrease in sulfhydryl/disulfide, free amines and lysine. The decrease in solubility, surface hydrophobicity and intrinsic fluorescence indirectly indicated that MDA induced soy protein aggregation, and results of high‐performance size‐exclusion chromatography directly showed that gradual aggregation of soy protein was induced by increasing concentration of MDA. Results of electrophoresis indicated that MDA caused soy protein aggregation, and non‐disulfide covalent bonds were involved in aggregate formation. CONCLUSION: The results showed that sensitivity of soy protein was related to MDA concentration. Soy protein gradually aggregated with increase of MDA concentration; β‐conglycinin was more sensitive to MDA modification than glycinin. Copyright © 2009 Society of Chemical Industry     

Effects of oxidative modification on thermal aggregation and gel properties of soy protein by peroxyl radicals

Effects of protein oxidation on thermal aggregation and gel properties of soy protein by 2,2′‐azobis (2‐amidinopropane) dihydrochloride (AAPH)‐derived peroxyl radicals were investigated in this article. Incubation of soy protein to increase concentration of AAPH resulted in a decrease in particle size and content of thermal aggregates during thermal‐induced denaturation. Protein oxidation resulted in a decrease in water‐holding capacity (WHC), gel hardness and gel strength of soy protein gel. An increase in coarseness and interstice of the gel network was accompanied by uneven distribution of interstice as extent of oxidation of soy protein increased. A decrease in disulphide content and formation of oxidation aggregates in the process of oxidative modification were contributed to the decline of particle size and content of thermal aggregates during thermal‐induced denaturation, leading to a decrease in WHC, gel hardness and gel strength of soy protein gel.     

贮藏条件对核桃仁蛋白质品质的影响

以核桃仁为研究对象,探讨了温度和真空度对其贮藏过程中蛋白质结构和功能的影响。结果表明:核桃仁蛋白质品质受温度和真空度影响显著,且温度>真空度,恒温(35℃)>室温(25℃)>冷藏(4℃);随着贮藏时间的延长,核桃仁蛋白质物质组成中游离巯基含量下降,二硫键、羰基化合物含量增加;由电泳和红外分析可知,核桃仁蛋白质发生了聚集现象,二级结构中β-折叠和无规卷曲度比例上升,α-螺旋和β-转角比例减少;在功能特性方面其表面疏水性上升,溶解度下降,消化特性发生了显著变化。