脂肪氧合酶催化亚油酸氧化对酸沉大豆蛋白理化性质的影响(Ⅱ)——聚集体的相对分子质量分布

模拟大豆蛋白质制备过程,应用毛细管电泳和凝胶体积排阻色谱探讨了脂肪氧合酶催化亚油酸氧化诱导的大豆蛋白质聚集体的相对分子质量分布.毛细管电泳分析表明,随着反应的进行,酸沉大豆蛋白各亚基的迁移时间延长、峰形展宽,说明反应后大豆蛋白各亚基的构象发生了改变,且随着模拟体系反应时间的延长,形成C-C或C-N共价交联形式聚集体的相对分子质量增大、数量增加.凝胶体积排阻色谱分析表明,酸沉大豆蛋白的相对分子质量随反应时间延长而增加,且分布具有多态性.这些数据说明模拟反应体系中的蛋白质聚集行为和聚集程度随反应时间的不同而不同.     

重水环境下的氧化大豆蛋白Raman光谱分析

应用Raman光谱分析了不同亚油酸浓度下脂肪氧合酶催化诱导产生的大豆蛋白聚集体的结构变化。结果表明,当模拟反应体系中亚油酸浓度在3.14~6.09 mmol/L范围之间时,拉曼光谱中归属于CH和CH2弯曲振动的谱带强度增强,而归属于位于非极性环境中的色氨酸和酪氨酸残基的谱带强度减弱;当亚油酸浓度达到7.51 mmol/L时,则出现相反的结果,这表明大豆蛋白质的聚集涉及到疏水相互作用。此外,在蛋白质拉曼光谱中还观察到胱氨酸残基二硫键的伸缩振动变化。二级结构分析表明反应后大豆蛋白α-螺旋减少,β-折叠增加。     

脂质氢过氧化物氧化对核桃分离蛋白结构的影响

采用脂肪氧合酶催化亚油酸构建脂质氢过氧化物——核桃分离蛋白氧化体系,研究脂质氧化反应中产生的氢过氧化物对核桃分离蛋白结构特性的影响。结果显示,向100 m L 0. 05 mg/m L蛋白溶液中添加9 m L亚油酸,核桃分离蛋白溶解度下降至6. 89%,游离巯基含量由2. 82μmol SH/g下降至1. 92μmol SH/g,羰基含量由2. 26 nmol/mg增加至4. 23 nmol/mg,表明氧化后核桃蛋白理化特性产生变化。圆二色谱分析表明,蛋白质氧化导致核桃分离蛋白α-螺旋和β-折叠含量下降,β-转角和无规则卷曲含量上升。随着亚油酸添加量的增加,蛋白质氧化程度加大,核桃分离蛋白表面疏水性从429. 66下降到405. 24。内源荧光最大荧光峰位红移,内源荧光强度下降,此结果表明,脂肪氧合酶(lipoxygenase,LOX)催化亚油酸氧化诱导核桃分离蛋白聚集,并进一步使蛋白质二级和三级结构发生变化。体积凝胶色谱法结果显示,核桃分离蛋白氧化后,小分子多肽含量下降,高分子量聚集体含量增加,说明脂质氢过氧化物氧化修饰导致核桃蛋白结构改变,形成氧化聚集体。     

亚油酸氧化诱导大豆分离蛋白氧化对其结构的影响

利用脂肪氧合酶催化不同底物浓度亚油酸氧化大豆分离蛋白,通过测定氧化后大豆分离蛋白的羰基值、巯基、表面疏水性、内源荧光以及凝胶电泳等指标,对氧化修饰后大豆分离蛋白的结构进行分析,并通过分析氧化后大豆分离蛋白在不同溶剂中溶解性能表征其分子间相互作用力。结果表明,随体系中亚油酸浓度的增加,大豆分离蛋白羰基含量增加56.3%,总巯基和游离巯基含量降低,表面疏水性呈先增大后减小趋势,内源荧光强度降低,发生先红移后蓝移现象,大豆分离蛋白粒径先减小后增大,主要分布于5~40 nm之间,蛋白质氧化过程发生去折叠和重聚集,蛋白质交联中存在非二硫键共价键生成。     

亚油酸氢过氧化物氧化修饰对大豆蛋白热变性和聚集的影响

以亚油酸氢过氧化物代表脂质氢过氧化物氧化修饰大豆分离蛋白,采用圆二色光谱、内源荧光光谱、粒径分析以及相对分子质量分布研究氢过氧化物氧化修饰对大豆蛋白热变性和聚集的影响.结果发现亚油酸氢过氧化物氧化修饰使得大豆蛋白热稳定性下降,热变性过程中形成聚集体粒径随着蛋白质氧化程度的升高呈现先增加后减小的趋势,热变性大豆蛋白冷却后的粒径和聚集体含量则随着蛋白质氧化程度的增加而下降.     

脂肪氧合酶催化亚油酸氧化对花生蛋白结构的影响

利用脂肪氧合酶催化亚油酸氧化的反应体系对花生分离蛋白进行不同程度的氧化修饰,通过研究不同亚油酸含量下花生分离蛋白的羰基值、游离巯基含量、粒径分布、表面疏水性、溶解度以及荧光光谱的变化规律,从而探讨氧化对花生分离蛋白结构的影响。结果表明:随着反应体系中底物亚油酸含量的增加,花生分离蛋白的羰基值先增加后略微下降,游离巯基含量下降,表面疏水性先增加后减小,说明氧化后花生分离蛋白的结构已经发生了改变。其粒径分布和溶解度的变化规律可以表征花生分离蛋白氧化聚集体的状态,同时其荧光峰位λmax的变化规律可反映花生分离蛋白三级结构的具体变化,表明脂肪氧合酶催化亚油酸氧化可以诱导花生分离蛋白分子发生聚集,使其结构发生显著变化。     

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。     

氧化对大豆蛋白结构、乳液稳定性及消化特性的影响

以大豆分离蛋白为原料,以脂质过氧化产物丙二醛（malondialdehyde,MDA）为氧化引发剂,逐级研究氧化对大豆蛋白结构、乳液稳定性及乳液消化特性的影响。结果发现：随着MDA浓度的升高,蛋白羰基及席夫碱含量明显升高而巯基含量显著降低。同时,MDA可促进蛋白聚集并诱导β-伴大豆球蛋白（7S）组分形成二硫键和非二硫键诱导的共价交联。进一步制备O/W型乳液,发现不同浓度MDA处理蛋白对乳液的形成影响较小,但可以显著改变界面蛋白组成。其中经中高浓度（2.5～10 mmol/L）MDA氧化后,更多7S组分以聚集状态参与界面组成。体外模拟胃肠道消化实验进一步表明,乳液消化主要在肠道进行,氧化诱导的蛋白交联/聚集可延缓或降低胆盐在界面的替代,进而减缓乳液消化并降低脂质释放率。     

脂肪氧合酶催化亚油酸氧化对大豆蛋白氨基酸组成的影响

利用低脂质含量大豆蛋白、脂肪氧合酶和亚油酸组成的三元体系,模拟大豆蛋白质制备过程,结合TBA值与羰基含量测定.研究反应后大豆蛋白的氨基酸组成变化情况.结果证明,反应后大豆蛋白的TBA值和蛋白质氧化值增加,氨基酸组成中天冬氨酸和脯氨酸含量增加,而组氨酸、精氨酸、赖氨酸、酪氨酸和半胱氨酸(胱氨酸)含量减少;进一步分析表明,反应后大豆蛋白的天冬氨酸含量增加,组氨酸、赖氨酸、精氨酸、酪氨酸和半胱氨酸(胱氨酸)含量减少可归属于大豆蛋白氨基酸残基与脂肪氧合酶催化亚油酸氧化产生的氢过氧化物及其降解产物的反应有关.     

脂肪氧合酶催化亚油酸氧化与大豆蛋白相互作用的荧光光谱分析

利用低脂质含量大豆蛋白、脂肪氧合酶和亚油酸组成的三元体系,模拟大豆蛋白质制备过程,通过荧光光谱结合可溶性蛋白表面疏水性分析,研究了反应后大豆蛋白芳族氨基酸的氧化情况,结果证明,在LOX的催化作用下,LA过氧化所产生的氢过氧化物及其降解产物可与大豆蛋白作用,反应后大豆蛋白的荧光光谱最大激发波长在350nm,最大发射波长在440nm.LOX催化LA氧化与大豆蛋白的相互作用,在LRSP LA LOX模拟体系中产生荧光物质.该荧光物质部分可溶于氯仿-甲醇溶液的有机相中,其荧光光谱最大激发波长在350nm,最大发射波长在430nm,且荧光强度随着反应程度的增加而增强.伴随着荧光物质的形成,反应后大豆蛋白的可溶性蛋白的表面疏水性降低.使用抗氧化剂(BHT)可抑制荧光物质的产生和蛋白质疏水性的丧失.     

Soybean protein aggregation induced by lipoxygenase catalyzed linoleic acid oxidation

Aggregation of lipid-reduced soybean proteins (LRSP) was investigated by chemical analysis, spectroscopy, electrophoresis, SEC-HPLC and light scattering. Soybean proteins obtained from the model systems consisting of LRSP and different levels of linoleic acid and lipoxygenase (RSP 4 and 5) showed increased turbidity, protein oxidation, surface hydrophobicity but decreased sulfhydryl and disulfide contents. SDS-PAGE of RSP 4 and 5 revealed remarkable difference of electrophoretic bands for 7S subunits, comparing with those samples without linoleic acid and lipoxygenase. Fluorescence spectroscopy suggested other covalent linkages than disulfide bonds formed during the formation of aggregates. SEC-HPLC and laser light scattering indicated that aggregates with high molecular weight and large particle size existed in samples of RSP 4 and 5. The experimental evidences suggest that the aggregates were formed via non-covalent interactions, but covalent bonds were also involved.     

Effect of lipoxygenase activity in defatted soybean flour on the gelling properties of soybean protein isolate

Soybean lipoxygenase was inactivated to different degrees by dry heating of defatted soybean flour for 0, 5, 10, 15, 20 and 25 min and soy protein isolates were prepared thereof by isoelectric precipitation of the water extract of the defatted soybean flour. The fluorescence emission intensity at 420 nm of the chloroform–methanol extract of soy protein isolates, which was indicator of the existence of peroxidized lipid, varied in parallel with the lipoxygenase residual activity in defatted soybean flours. The dispersion of soy protein isolate showed an increasing turbidity with the increase of lipoxygenase residual activity in the starting defatted soybean flour, suggesting an elevated tendency to form insoluble aggregates during the preparation of soy protein isolate. Small deformation rheological test revealed that the gelling times were shorter for those soy protein isolates derived from low lipoxygenase activity defatted soybean flours than that of high lipoxygenase activity. Frequency sweep showed that G′ of soy protein isolate derived from low lipoxygenase defatted soybean flour was independent of oscillation frequency in contrast to that of derived from non dry-heated defatted soybean flour, the latter showed a marked frequency dependence. Large deformation test revealed that the gel hardness increased about 10 times after dry heating of defatted soybean flour for 20 min. As the increase of the lipoxygenase residual activity, the gel permeability increased markedly, suggesting that soy protein isolate from high lipoxygenase defatted soybean flour produced coarser textured gel, which corresponded well with the results of scanning electron microscopy.     

Mechanisms of Oxidative Processes in Meat and Toxicity Induced by Postprandial Degradation Products: A Review

Antioxidant system loss after slaughtering, reactive species production, cell disruption, contact with oxygen and light, heme and nonheme iron, and irradiation starts up mainly by 2 related oxidative processes: lipid peroxidation and protein oxidation. Products generated in these processes are responsible for meat quality loss, and some of them are suspected to be toxic to humans. This review article is focused on reactive species implicated in oxidative processes in meat, on lipid peroxidation mechanisms, heme protein, and nonheme protein oxidation, and on some toxic oxidation and digestion products. Nonenzymatic fatty acid peroxidation is exemplified by an arachidonic acyl group, and the initiation of chain reaction can be described by 3 pathways: singlet oxygen, hydroxyl radical from the Fenton reaction, and perferrylmyoglobin. Enzymatic oxidation of fatty acids is exemplified using linoleic acid, and the main characteristics of lipoxygenase are also presented. Heme protein oxidation is described in an interrelation with lipid peroxidation and the significance for food quality is shown. For protein oxidation, 3 different mechanism types are described: oxidation of amino acid residues, oxidation of protein backbone, and reactions of proteins with carbonyl compounds from lipid peroxidation. The effects of oxidative damage on protein properties and bioavailability are also shown. At the end of each oxidative process, the postprandial toxicity induced by oxidation products and the dietary degradation products are presented. Also discussed are reports by some researchers who suggest that dietary lipid and protein oxidation products and heme iron from red meat are in part cytotoxic and/or genotoxic.     

Composition and structure of carob (Ceratonia siliqua L.) germ proteins

This study was focused on the analysis of the chemical composition of defatted carob germ flour and the protein isolate. The amino acid composition and the nature of the subunits that compose carob germ proteins were also studied. Isolate was obtained by alkaline extraction followed by isoelectric precipitation of proteins. Results showed that an isolate of 96.5% of protein content was obtained. A high amount of amino acids like glutamic acid, aspartic acid and arginine was detected. Carob proteins were found to be composed by aggregates formed by a 131 and 70 kDa subunits linked by non-covalent bonds, and other peptides strongly bounded by disulfide interactions. Both, aggregates and subunits were formed mainly by 100 and 48 kDa monomers linked by disulfide bonds. A considerable content of high molecular mass peptides (HMWP) strongly bounded were also found. Proteins became partially denatured and thermally stabilized at acid pH (pH 2). These results could be useful in the study of different functional properties of carob germ proteins, and the application of these proteins as nutritional ingredients in formulated food.     

大豆脂肪氧化酶与籽粒营养品质关系的研究

利用大豆近等基因研究了脂肪氧化酶缺失对大豆种子中蛋白南、氨基酸、脂肪及脂肪酸含量的影响。结果表明,脂肪氧化酶及同功酶缺失,对蛋白质、脂肪及脂肪酸含量无影响。脂肪氧化酶及同功酶缺失,可能引起种子中脯氨酸含量降低,对其它氨基酸无影响。脯氨不在人必须氨基本我,脂肪氧化酶缺失不会引起大豆营养品种降低。     

豆浆模拟体系中挥发性风味物质的比较研究

选择大豆蛋白、大豆甘油三酯、大豆脂肪氧合酶和胰脂肪酶4种主要组分,按照实际豆浆的组分含量和酶活大小进行混合制备豆浆模拟体系。分别利用二甲基酚橙法和顶空固相微萃取-气质联用法测定体系中脂质氢过氧化物和挥发性风味物质。结果表明:脂肪氧合酶催化甘油三酯氧化生成的脂质氢过氧化物相当于3. 958 mmol/L H2O2,生成的挥发性风味物质总量为43. 80 mg/kg;而脂肪氧合酶催化甘油三酯水解产物生成的脂质氢过氧化物相当于52. 243 mmol/L H2O2,生成的挥发性风味物质总量为668. 42 mg/kg。同时在该反应条件下对模拟体系和豆浆产生的挥发性风味物质进行主成分分析,模拟体系和3个豆浆样品集中在得分图相近区域,结果判定豆浆模拟体系产生的风味与真实豆浆的特征风味具有较好的相似性。     

Effect of microbial transglutaminase on the protein fractions of rice,pea and their blends

BACKGROUND: Transglutaminase (TG) is a transferase that has been used for crosslinking proteins. In general, those interactions are promoted within proteins of the same nature, and very few studies have been conducted for creating new bonds between proteins from different sources catalysed by TG. The effect of TG on the protein fractions of rice flour, pea protein isolate and their blends was studied by using different electrophoretic analyses (simple sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) and multistaking SDS‐PAGE under reducing and non‐reducing conditions). RESULTS: TG induced the disappearance of numerous protein bands as a consequence of the formation of large protein polymers, linked by isopeptidic and disulfide bonds, with reduced solubility. The main protein fractions involved in those interactions were the albumins and globulins, from the pea protein isolate, and the rice flour; and the glutelins were also crosslinked. CONCLUSION: Composite flours containing the rice flour and the pea protein isolate are proposed for obtaining a protein‐enriched dough with better amino acid balance. Also a protein network formed of protein aggregates of high molecular weight can be created in the presence of transglutaminase. Copyright © 2007 Society of Chemical Industry     

脂肪氧化对蛋白质结构的影响

为了探索脂肪氧化对蛋白质结构的影响,利用不同条件下制备的氧化脂肪,加入蛋白后在同样条件下作用一定时间,测定蛋白质的各项指标。结果表明：加入蛋白后硫代巴比妥酸值(TBA)值下降,蛋白氧化值升高(羰基含量),表面疏水性增加且最大发射波长(λmax)发生红移,二级结构变化明显。说明脂肪氧化对蛋白质结构产生了影响。     

猪肉12-脂肪氧合酶催化结构域的表达、纯化及其酶学性质分析

研究脂肪氧合酶（lipoxygenase,LOX）在猪肉贮藏、加工过程中对脂肪氧化及风味形成的作用机制。通过序列分析和聚合酶链式反应扩增获得了猪肉12-脂肪氧合酶催化结构域（12-lipoxygenase catalytic domain,12-LOXcd）的编码基因,采用大肠杆菌表达系统,经镍柱亲和层析和Superdex G200凝胶过滤层析纯化得到12-LOXcd蛋白,并研究其酶学性质。结果表明,构建的原核表达载体pMBP-12-LOXcd在大肠杆菌中成功可溶性表达了猪肉12-LOXcd,该重组蛋白经纯化可达电泳纯。12-LOXcd以亚油酸为底物的比活力为2 826.7 U/mg,最适pH值为6.0,最适作用温度为30 ℃。亚油酸Km为0.40 mmol/L,亚麻酸Km为0.55 mmol/L,花生四烯酸Km为4.15 mmol/L,表明最适底物为亚油酸。与大豆LOX相比,该酶在较高NaCl质量分数（9%）时仍保持活性稳定;对热较不稳定,在60 ℃条件下失活,但优于大豆LOX的热稳定性;此外,12-LOXcd的pH值稳定性也优于大豆LOX,在碱性条件下仍能保留部分活力。     

Gelation of soybean proteins induced by sequential high-pressure and thermal treatments

The effect of high-pressure treatment on structural and rheological properties of soybean protein dispersions was studied. A sequential high-pressure/thermal treatment was also analyzed. Dissimilar effects on soy protein isolate (SPI) and the enriched soybean protein fractions: β-conglycinin (βCEF) and glycinin (GEF) were observed. High pressure (600 MPa) promoted βCEF gelation, but did not modify the rheological properties of GEF in spite of its complete denaturation. Pressure treatment also induced the establishment of hydrophobic interactions and disulfide bonds that allowed the formation of soluble high molecular mass aggregates from the different polypeptides of both β-conglycinin and glycinin. Protein strands formation was detected in matrix microstructure of HP-treated SPI and βCEF dispersions in accordance with their rheological behavior of weak gels. In the case of GEF modifications induced by HP in the microstructure (apparition of large granules) were not accompanied by rheological changes.