酶法与非酶法磷酸化改性食品蛋白质的研究进展

磷酸化是一种重要的蛋白质修饰改性手段,可以有效改善食物蛋白质的功能性质,如乳化性、溶解性、凝胶性、热稳定性等。蛋白质磷酸化改性方法主要可分为酶法与非酶法两种,其中酶法磷酸化常使用蛋白激酶作为磷酸基团的供体,对蛋白质进行修饰。主要的蛋白激酶包括环磷酸腺苷依赖蛋白激酶(CAMPdPK)和酪蛋白激酶Ⅱ(CK-Ⅱ)。非酶法磷酸化常见的改性试剂主要包括三氯氧磷、焦磷酸钠、三聚磷酸钠和葡萄糖-6-磷酸等。本文对近年来蛋白质磷酸化的有关报道进行了总结归纳,并通过介绍磷酸化蛋白的磷酸键性质、磷酸化肽段及位点的鉴定,阐述了不同磷酸化试剂的反应机制及其对蛋白质构效关系的影响,为通过定向的磷酸化修饰而改善蛋白质特定的功能性质提供了理论依据与参考。     

蛋白质磷酸化对肉品质影响的研究进展

蛋白质磷酸化反应是生物界最普遍、最重要的蛋白质翻译后修饰方式。本文综述了磷酸化蛋白质的富集、位点分析、定量分析方法的最新研究进展。从蛋白质磷酸化反应与糖酵解、肌肉收缩、蛋白质降解的关系探讨了蛋白质磷酸化反应对肉品质的影响,并对磷酸化蛋白质组学的发展方向以及蛋白质磷酸化反应在肉品中的研究进行了展望。     

牛初乳和牛常乳乳清N-糖蛋白质的差异分析

为阐明牛乳蛋白质N-糖基化,采用糖蛋白质组学技术,在牛初乳和牛常乳乳清中共鉴定到154 个N-糖蛋白和246 个糖基化位点,其中,牛初乳鉴定到117 个N-糖蛋白和183 个糖基化位点;牛常乳鉴定到109 个N-糖蛋白和145 个糖基化位点。初乳中丛生蛋白（P17697）糖基化位点N-283表达量最高,常乳中α-乳白蛋白糖基化位点N-93表达量最高。考虑定量差异及有无差异,在牛初乳和牛常乳乳清中共鉴定到129 个糖蛋白的190 个差异表达糖基化位点。基因本体论功能注释表明,差异表达糖蛋白参与的生物学过程是生物调节、刺激性反应、多细胞生物过程、定位、免疫系统过程等;主要分布为细胞外区域和细胞器;主要的分子功能是结合作用、催化活性和分子功能调节。差异表达糖蛋白参与的代谢通路主要是补体与凝血级联、金黄色葡萄球菌感染和溶酶体等。此外,通过蛋白互作分析,找到一些具有高连接度的重要糖蛋白。本研究丰富了牛乳N-糖蛋白质组成及其糖基化位点信息,阐明了牛乳乳清N-糖基化的功能,为评价和改善牛乳品质、研发婴幼儿配方乳中糖蛋白质的改良及功能食品的生产提供了理论依据。     

蛋白质磷酸化对宰后肉品质影响研究进展

畜禽肌肉在宰后变为可食肉的过程中,需要经过一系列的生化变化直到成熟,其中蛋白质磷酸化反应作为一种蛋白质的动态调控机制,几乎调节每一个主要的生物过程,是生物界常见的翻译后修饰形式之一。因此,研究蛋白质磷酸化与宰后肌肉的关系有助于肉类食品行业的发展。本文从介绍蛋白质磷酸化概念出发,讨论磷酸化蛋白质检测技术的发展,总结宰后肌肉中会发生磷酸化的蛋白质及影响因素,从蛋白质磷酸化反应与宰后肉的嫩度、肉的色泽和保水性3 个方面的研究入手综述蛋白质磷酸化反应对肉品质的影响,以及食盐与氨基酸在该过程中的应用,并对蛋白质磷酸化反应在肉品品质中的研究做出展望。     

牛乳与山羊乳中乳桥蛋白的比较研究

山羊乳是近年来备受瞩目的动物乳源，含有在婴儿早期发育中发挥多种生物活性的乳源性骨桥蛋白（osteopontin, OPN），也被称为乳桥蛋白（lactopontin, LPN），但目前关于山羊乳LPN的研究还较少，对于其结构特点及与牛乳LPN的差异还未见相关报告。通过对山羊乳和牛乳中LPN进行序列相似度分析，并进行富集和测定，比较两者在磷酸化修饰的差异，探讨山羊乳LPN与牛乳LPN结构的异同。结果显示，山羊乳和牛乳LPN完全相同的氨基酸比例高达90.94%，并且均存在同样的整合素蛋白结合序列。山羊乳LPN未鉴定到O-糖基化修饰，山羊乳LPN和牛乳LPN的磷酸化修饰位点基本一致，但同一位点的磷酸化修饰位点覆盖率在两者间略有差异。结果说明，山羊乳LPN可能具有与牛乳LPN相似的生物活性。     

定位酶解与蛋白质表面性质及结构特性关系研究进展

定位酶修饰对蛋白质表面性质(如乳化性、起泡性)有一定程度改善,这些表面性质变化产生原因是蛋白酶特异性断裂蛋白质肽键,从而导致蛋白质结构发生变化,结构变化主要包括蛋白质表面张力、疏水性、柔性等。该文从蛋白酶作用位点及其处理程度对底物蛋白结构改变的角度,综述酶修饰蛋白质水解后表面性质变化,并分析蛋白质结构对其表面性质影响。     

糖基化联合磷酸化降低鲢鱼小清蛋白致敏性的机制

以鲢鱼小清蛋白（parvalbumin,PV）为研究对象,采用糖基化联合磷酸化对其进行修饰,运用光谱、质谱和KU812细胞实验等方法研究修饰后鲢鱼PV抗原表位和致敏性的变化。结果表明：糖基化联合磷酸化修饰可增加鲢鱼PV分子质量,降低游离氨基含量,且改变其二级结构和构象结构;糖基化联合磷酸化修饰后的鲢鱼PV含有8个糖基化位点（K33、K46、K55、K65、K84、K88、K97和K108）和1个磷酸化位点（S56）。糖基化联合磷酸化修饰能显著降低鲢鱼PV与IgG/IgE的结合能力,也能降低KU812细胞中组胺和白介素-6的释放能力。因此,糖基化联合磷酸化修饰通过糖基化和磷酸化位点遮掩鲢鱼PV的线性表位和破坏其构象表位,降低其致敏性。研究结果为开发低致敏性鱼类制品提供重要的理论依据。     

低钾胁迫下高钾烤烟苗期根系差异表达蛋白分析

为了解高钾烤烟苗期根系在低钾胁迫下的差异表达蛋白及其作用机理,本研究分别提取低钾和正常钾营养液培育的高钾烤烟品系HKDN-5的苗期根系蛋白,酶解后采用液相色谱串联质谱和label-free蛋白定量技术对差异表达蛋白进行鉴定分析。结果表明,与正常钾相比,低钾组共发现681个差异表达蛋白,其中有359个发生下调表达,322个上调表达。差异表达蛋白主要参与脂质代谢、次生代谢、碳水化合物代谢和遗传信息加工等途径。其中,下调蛋白具有翻译后修饰、蛋白质转换、伴侣蛋白相关功能的数量最多,上调蛋白主要为一般功能相关蛋白。差异倍数较大的蛋白主要与代谢和应激反应过程相关。以上结果表明,低钾胁迫产生的差异蛋白主要参与物质代谢及应激反应等相关过程。     

糖基化反应对鸡蛋清蛋白与低聚麦芽糖交联物活性和功能性的影响

本文以鸡蛋清蛋白与低聚麦芽糖为原料,进行糖基化修饰反应,以接枝度和褐变程度等指标分析糖基化程度,并研究糖基化修饰反应对接枝产物生物活性和功能特性的影响。结果表明,湿热法加热2 h的反应产物接枝度最高,褐变程度较小,即反应产物大多是初级阶段末期或中间阶段产物。糖基化修饰反应提高了鸡蛋清蛋白的金属还原力和清除DPPH自由基的能力,同时还可增强其螯合亚铁离子的能力。功能特性分析表明,糖基化修饰反应可提高鸡蛋清蛋白的溶解度和热稳定性,而不影响其乳化稳定性和乳化活性。同时,体外模拟消化实验表明,糖基化修饰反应降低了鸡蛋清蛋白的消化性,这可能与其在大肠中被肠道微生物降解有一定的关联。上述分析表明,糖基化反应可作为一种有效的修饰方法提高蛋白质的功能特性和生物活性。     

GⅡ.4型诺如病毒VP2蛋白生物信息学分析

为了解诺如病毒VP2蛋白的生物学特征，该研究以当前诺如病毒GII.4型流行毒株VP2蛋白为研究对象，应用生物信息学软件对VP2蛋白的理化性质、磷酸化位点、糖基化位点、跨膜区、信号肽、二级结构、三级结构、抗原决定簇和B细胞抗原表位进行预测分析。结果表明，诺如病毒流行毒株VP2蛋白为稳定的亲水性蛋白，平均等电点为10.47、吸水系数为-0.47、不稳定系数为47.91，碱性氨基酸约占含量的11.60%；该蛋白含有约43个潜在的磷酸化修饰位点和2个潜在的糖基化修饰位点；大多数毒株不存在跨膜区和信号肽，然而GI.3型诺如病毒VP2蛋白存在跨膜区和信号肽；诺如病毒VP2蛋白二级结构中α-螺旋平均占比38.17%、延伸链平均占比7.45%、β-折叠平均占比6.25%、无规则卷曲平均占比48.13%；三级结构预测模型的蛋白覆盖率为56.70%；平均存在8个潜在的蛋白质抗原决定簇和25个B细胞抗原表位。该研究运用生物信息学分析方法预测当前GII.4型诺如病毒流行毒株VP2蛋白的理化性质和结构等方面，为进一步深入研究诺如病毒VP2蛋白功能奠定了理论基础。     

Coagulation of Egg White of Soft-Shell Turtle (Pelodiscus Sinensis) at High Pressure or High Temperature

The coagulations of soft-shell turtle egg white at high pressure or at high temperature were investigated and compared to chicken egg white. Unlike chicken egg white, no coagulation of soft-shell turtle egg white was observed after high-pressure processing (HPP; 200–600 MPa) or heating treatment (75°C). Basic amino acids, histidine, lysine, and arginine were present at higher percentages in soft-shell turtle egg white proteins than in chicken egg white proteins. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis showed that the intensities of soft-shell turtle egg white components were balanced, there were more components with high molecular mass in soft-shell turtle egg white, and that aggregates formed in both soft-shell turtle egg white and chicken egg white. This work revealed some differences in soft-shell turtle egg white compositions. The low protein content in egg white was responsible for the non-coagulation of soft-shell turtle egg white in response to high pressure or temperature.     

Separation of Native and Acylated Egg White Proteins with Gel Chromatography and DEAE-Cellulose Ion Exchange

Chemical modification of egg white has been shown to improve its functional properties. In order to fully understand the changes in functionality it is necessary to separate the proteins and study their corresponding structural changes. This paper describes two chromatographic techniques applied to the separation of native and acylated egg white. A molified DEAE—cellulose ion exchange methodology was developed which reduced separation time and expense. It was found that increases in net charge introduced by acylation with succinic or acetic anhydride changed the ionic state of the proteins, limiting their separation by ion-exchange chromatography. Using Sephacryl S-200, a gel chiomatographic technique was developed which separated over 80% of the total succinylated egg white proteins; viz., ovomucin, ovalbumin, conalbumin, ovomacroglobulin, and lysozyme. Most proteins of native and acetylated egg white were separated with S-200 chromatography, but required rechromatography of the ovalbumin-conalbumin fractions for complete resolution.     

不同孵化时期鸡蛋蛋清小分子多肽的鉴定及功能分析

通过聚丙烯酰胺凝胶电泳(SDS-PAGE)和基质辅助激光解吸电离飞行时间串联质谱(MALDI-TOF MS/MS)技术,比较不同孵化时期鸡胚蛋蛋清中产生的小分子多肽,研究肽段与鸡胚发育之间的联系。结果显示在孵化第0、6、14、16 d分别得到837、879、872和842条肽段(共3430条)。除丛生蛋白肽段外,大部分被鉴定到的肽段来源于蛋清中低丰度蛋白。其中926条肽段来源于与鸡胚先天免疫有关的9种蛋白质,如防御素、白细胞介素6等;256条肽段来源于与鸡胚呼吸系统发育相关的3种蛋白质,cx9C基序蛋白、sprouty蛋白和碳酸酐酶。结果表明由蛋清蛋白质内源性酶降解形成的小分子多肽可能对鸡胚发育时形成的呼吸和免疫系统等发挥重要作用。     

Production of dialysable iron by in vitro digestion of chicken muscle protein fractions: the size of the dialysable iron

Muscle foods enhance the absorption of non‐haem iron. We studied the size of the dialysable iron species produced following in vitro digestion of soluble and insoluble chicken muscle proteins and compared the results with those for egg white and whey protein. Digestion of chicken muscle proteins caused a four‐ to fivefold increase in dialysable iron, whereas egg white showed only a slight increase and whey protein caused a reduction, as compared with the control. All the dialysable iron species produced by egg white and whey were smaller than 1 kDa. In contrast, chicken muscle proteins produced dialysable iron species of all sizes up to 10 kDa, but the majority were in the range 2–3.5 kDa for both soluble and insoluble muscle proteins. Copyright © 2005 Society of Chemical Industry     

腌制剂及复配对无重金属盐皮蛋品质的影响

为解决无重金属盐皮蛋易出现的“碱伤”、“烂头”等问题,改善无重金属盐皮蛋品质,本研究以鸡蛋为原料,采用清料法腌制皮蛋,加入葡萄糖、单宁、葡萄糖酸-δ-内酯三种非金属腌制剂,通过单因素实验选择最佳添加量,之后进行复配,以蛋清硬度、游离碱度、色度以及蛋黄硬化率为指标,结合腌制20 d皮蛋的感官评价及蛋壳微观结构,考察各复配组对皮蛋品质的影响。结果表明:单独添加0.7%葡萄糖、0.5%单宁、0.7%葡萄糖酸-δ-内酯腌制组皮蛋碱伤程度均有所改善,其中0.7%葡萄糖酸-δ-内酯改腌制组改善效果最好。最优复配组为0.7%葡萄糖+0.7%葡萄糖酸-δ-内酯,腌制20 d,皮蛋蛋黄硬化率为86.69%,蛋清硬度值为183.584 g,蛋清游离碱度为240.371 mg/100 g,色泽为深红棕色,感官品质最好,未出现“碱伤”和“烂头”的现象。     

Cross-reactivity and Heat Lability of Antigenic Determinants of Duck and Goose Lysozymes

Two panels of monoclonal antibodies (Mabs) raised against duck (Barbary) egg white lysozyme or hen egg white lysozyme, were tested in antigen-coated plate (ACP) and double antibody sandwich (DAS) ELISA for cross-reaction with various avian lysozymes. The antibodies to hen lysozyme cross-reacted with goose lysozyme, but most antibodies to duck lysozyme reacted with it. One antibody to duck lysozyme reacted more strongly with goose lysozyme than with the homologous antigen, a heterospecific reaction confirmed by biosensor technology. Many lysozyme epitopes recognized by different antibodies showed considerable resistance to heat denaturation. Such Mabs may be useful for detecting chicken liver adulterants in “foies gras” of goose or duck origin.     

Effects of spray-drying and freezing on the proteins of liquid whole egg

Commercial pasteurised liquid whole egg was spray-dried, both alone and mixed with liquid milk, and some of it was frozen. Paper electrophoresis revealed that lipoprotein fractions from the pasteurised liquid egg were mainly immobile, in contrast to unpasteurised new-laid egg which contained both mobile and immobile lipoprotein fractions. The major difference between the soluble proteins of the commercial chilled pasteurised liquid egg and those of the same egg after freezing and thawing was the presence, in the former, of an inert lipoprotein fraction, amounting to 3.6% of the total egg solids and containing 88% lipid. The “soluble” proteins of spray-dried egg and egg-milk mixture also included large “inert” lipoprotein fractions eluted with the void volume. They contained 72–81% of lipid and accounted for 7–13% of the total egg solids. Larger fractions were eluted by the stronger salt buffer solutions and by hydrochloric acid from the dried egg-milk mixture than from the dried egg, and these were probably derived from the milk. All the spray-dried samples possessed the “inert insoluble” protein fraction characteristic of liquid egg, and the presence of a high molecular weight lipoprotein fraction was confirmed in each case. The “insoluble proteins” of the spray-dried samples had higher protein contents than those from the original chilled liquid egg and the thawed frozen egg.     

鸡豆花的工艺优化及品质分析

"鸡豆花"是四川的传统美食,因其成型困难及口感特殊,难以推广.以鸡胸肉为原料,采用单因素实验(鸡肉糜与水的比例、鸡蛋清的添加量、马铃薯淀粉的添加量)和正交实验确定最佳工艺参数,并通过感官评定、持水率和质构仪(texture profile analysis,TPA)指标,分析蒸、煮方式及加热时间对鸡豆花品质的影响.结果...     

Separation of the proteins of egg white and egg yolk and a study of their interactions in whole egg. 3. Amino-acid composition of protein fractions

A number of the fractions and sub-fractions isolated from the soluble proteins of white, yolk and whole egg have been identified by their amino-acid compositions, and others have compositions that differ from those of known proteins. It is concluded that the relative abundance of the different amino acids is similar for nearly all egg proteins. Part of the low density lipoprotein in whole egg is in a soluble form and can be partially fractionated by ion-exchange chromatography; the resulting fractions contain apoproteins with slightly differing amino-acid compositions.     

Tryptophan determination in proteins and feedstuffs by ion exchange chromatography

A chromatographic method was developed for the determination of tryptophan content in food and feed proteins. The method involves separation and quantitation of tryptophan (released from protein by alkaline hydrolysis with NaOH) by isocratic ion-exchange chromatography with O-phthalaldehyde derivatization followed by fluorescence detection. In this procedure, chromatographic separation of the tryptophan and α-methyl tryptophan, the internal standard, was complete in 15 min, without any interference from other compounds. The precision of the method was 1–4% relative standard deviation. Accuracy was validated by agreement with the value for chicken egg white lysozyme, a sequenced protein, and by quantitative recoveries after spiking with lysozyme. The method allows determination in a range of feed proteins, containing varied concentrations of tryptophan, and is applicable to systems used for routine amino acid analysis by ion-exchange chromatography.