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。     

牛乳中不同部分蛋白质的组成及功能分析

本研究通过SDS-PAGE电泳将牛乳中不同蛋白质组成部分进行分离鉴定发现,乳脂肪球膜中存在201种蛋白,乳清中存在96种蛋白,酪蛋白中存在21种蛋白,乳粒中存在43种蛋白,其中有27种相同表达的蛋白。通过GO功能注释分析发现,在生物过程中乳脂肪球膜蛋白发挥的作用大于乳清、乳粒蛋白,尤其是生物的调控作用;在分子功能上,牛乳蛋白的主要分子功能是结合作用,其中乳脂肪球膜蛋白的结合作用最强;而乳粒蛋白参与的转运活性分子功能大于乳脂肪球膜、乳清蛋白。在细胞组成上,与乳清、乳粒蛋白相比乳脂肪球膜蛋白参与的细胞组成均较多,而在细胞膜的组成上乳粒蛋白参与较多。通过京都基因与基因组百科全书(KEGG)代谢通路分析可知,乳脂肪球膜、乳清、乳粒中的蛋白均参与过氧化物酶体增殖物激活受体信号通路。对牛乳蛋白质组成进行研究,不仅能够增加牛乳的利用率,并且为日后以乳脂肪球膜、乳粒蛋白作为原料生产乳制品提供理论依据。     

基于iTRAQ技术分析牛初乳与常乳乳清差异蛋白质组

为阐明牛初乳、牛常乳乳清蛋白的差异,利用同位素标记相对和绝对定量（isobaric tags for relative and absolute quantitation,iTRAQ）蛋白质组学技术对二者进行蛋白质组差异分析,在得到的599 种具有定量信息的乳清蛋白中,鉴定出60 种差异蛋白。将牛初乳与牛常乳乳清丰度差异蛋白进行生物信息学分析发现,差异蛋白主要参与的生物过程为转运、定位、单一生物作用等;主要参与的分子功能为顶端质膜、细胞外区域、细胞外区域部分等;主要参与的细胞组成为蛋白结合和阴离子结合。丰度差异蛋白中有9 种是与信号传导相关,有6 种糖基化乳清蛋白。此外,利用蛋白质网络互作分析发现,差异蛋白中存在具有高连接度的关键乳清蛋白因子。本研究采用iTRAO技术对牛初乳与牛常乳乳清差异蛋白进行鉴定及生物信息学分析,为今后改善牛初、常乳品质,开发婴幼儿乳粉以及功能性乳制品提供了一定参考。     

羊乳乳清蛋白组成和功能及其与人乳、牛乳的对比分析

本研究将羊乳、牛乳、人乳中乳清蛋白进行分离并结合液质联用技术鉴定,在羊乳、牛乳、人乳乳清蛋白中分别鉴定出156、278、454种蛋白质。与牛乳与人乳乳清蛋白对比显示,羊乳含有99种特异性表达蛋白质,与牛乳和人乳分别有31种和15种相同表达蛋白质。通过分析基因本体(gene ontology,GO)功能注释发现,羊乳乳清蛋白在生物过程中主要发挥生物调节作用;在分子功能上,主要体现在结合作用方面;在细胞组成上,参与的细胞组成主要为细胞器区和胞外区。羊乳乳清蛋白在以上三种功能上与人乳有较大差距,但与牛乳相近。通过分析京都基因与基因组百科全书系统(Kyoto Encyclopedia of Genes and Genomes,KEGG)代谢通路可知,羊乳主要参与补体和凝血级联反应以及吞噬作用,对人体免疫能力有积极影响。对羊乳与人乳、牛乳乳清蛋白组成及功能区别的研究,为羊乳的进一步研究和开发提供一定的理论参考。     

人乳与牛乳天然小分子蛋白肽的差异对比及功能分析

研究利用超滤技术和Tricine-SDS-PAGE电泳将人乳与牛乳中天然存在的小分子蛋白肽进行分离。蛋白条带经质谱鉴定发现,人乳小分子蛋白肽含有28种,牛乳小分子蛋白肽含有24种,二者存在18种相同蛋白肽。Gene Ontology(GO)功能注释分析发现,在生物过程中,人乳小分子蛋白肽发挥的作用要高于牛乳小分子蛋白肽,尤其体现在免疫过程中的作用;在分子功能上,二者主要分子功能是结合作用,其中人乳小分子蛋白肽的结合作用强,而牛乳小分子蛋白肽参与的转运活性的分子功能大于人乳小分子蛋白肽;在细胞组成上,二者均参与了细胞与细胞膜的组成。通过Kyoto Encyclopedia of Genes and Genomes(KEGG)代谢通路分析可知,人乳小分子蛋白肽参与酪氨酸和抗原的加工和呈递2个代谢通路,而牛乳小分子蛋白肽仅参与系统性红斑狼疮代谢通路。对人乳和牛乳中的小分子蛋白肽组成进行研究,不仅为揭示人乳蛋白和牛乳蛋白的差异及其生理功能提供了理论依据,而且为婴幼儿食品、母乳化制品达到母乳水平提供有益探索。     

人乳与牛乳乳脂肪球膜蛋白质组的对比研究

人乳是婴幼儿生命初始阶段唯一能够摄入的食物,蛋白质是人乳中重要的营养成分,而牛乳作为代替人乳的常用原料已经被广泛的应用于婴幼儿食品中。本研究利用SDS-PAGE电泳和LC-MALDL-TOF蛋白组学方法将人乳与牛乳中乳脂肪球膜蛋白进行分离,能够发现人乳与牛乳脂肪球膜蛋白存在较大的差异。牛乳脂肪球膜中已鉴定出488种蛋白,人乳脂肪球膜中鉴定出的蛋白为1545种。牛乳脂肪球膜具有173种特异性蛋白,人乳脂肪球膜具有1230种特异性蛋白,在人乳与牛乳中存在315种同源蛋白。从蛋白质的GO(Gene Ontology)功能注释上来看,人乳脂肪球膜蛋白参与的生物过程有37%为代谢过程;具有的分子功能55%为结合作用;34%为参与细胞器构成。人乳脂肪球膜中有24种蛋白参与免疫相关的通路,主要为抗原加工和呈递。与牛乳相比,对人乳中乳脂肪球膜蛋白质在组成及功能上的研究,能够促进深入地了解人乳蛋白,并为以牛乳为原料的婴幼儿产品添加功能性蛋白提供参考。     

驴乳中蛋白质组分的分离纯化与鉴定

目的：分离纯化并鉴定驴乳乳清中蛋白质组分,为进一步研究驴乳用于辅助治疗疾病,以及为作为人乳替代品提供参考。方法：采用DEAE-52 离子交换层析和Sephadex G-100 凝胶层析分离纯化驴乳乳清中蛋白质组分,并通过十二烷基硫酸钠--聚丙烯酰胺凝胶电泳法和高效凝胶渗透色谱法对所纯化蛋白质进行鉴定。结果：与牛乳乳清中蛋白组分相对比,发现驴乳乳清中存在3 种未知蛋白质,分子质量分别为32、70.1、72.2kD。结论：驴乳中除了含有与牛乳相似的营养成分外,还具有其他生物活性成分,它们可能是一些保护性蛋白,在机体的抗病机制方面起着重要作用。     

牛初乳与牛常乳乳脂肪球膜中丰度差异蛋白的表征与分析

为阐明不同泌乳期间牛乳乳脂肪球膜蛋白的差异,选定牛初乳和牛常乳中的乳脂肪球膜蛋白作为研究对象,利用定量蛋白质组学技术对其中的蛋白质进行表征,并对两组间的丰度差异蛋白进行筛选及生物信息学分析。本研究共鉴定到763 种蛋白,其中197 种为两组共有蛋白,进一步从其中筛选出80 种丰度差异蛋白,包括41 种上调蛋白和39 种下调蛋白（牛初乳/牛常乳）。对上述丰度差异蛋白进行生物信息学分析发现,这些蛋白参与的主要细胞组分为细胞外泌体、细胞外空间、细胞外区域等,参与的主要代谢通路为代谢途径、嘌呤代谢、苯丙氨酸代谢、酪氨酸代谢、丙酮酸代谢、糖酵解/糖异生等,并进一步筛选出31 种与其他蛋白存在相互作用的关键蛋白,包括结合珠蛋白、2-磷酸-D-甘油酸水解酶等。研究结果将有助于了解泌乳期间乳蛋白成分的差异及其功能性,为牛乳制品的精深加工奠定基础。     

牛乳外泌体中miRNA的测序与分析

采用密度梯度离心法提取牛乳外泌体，利用Illumina测序技术对外泌体中非编码小RNA(sRNA)进行测序，探索牛乳microRNA(miRNA)的表达谱。对原始序列进行质量控制，共获得3 899 629条纯净sRNA序列，长度集中于28 nt，与数据库比对后鉴定到61种已知miRNA,346种新miRNA。基因本体论富集结果表明，牛乳外泌体miRNA在细胞过程、单一生物体过程、代谢过程等生物过程发挥作用；主要构成细胞、细胞器等组成；主要参与结合、催化活性、转运蛋白活性等分子功能。京都基因与基因组百科全书通路富集结果表明，已知miRNA与新miRNA靶基因均显著富集在百日咳（ko05133）、趋化因子信号通路（ko04062）、内吞作用（ko04144）、溶酶体（ko04142）等通路，牛乳外泌体miRNA在特定信号通路中发挥重要作用。     

不同泌乳期羊乳和牛乳的高通量定量乳清蛋白质组学

利用高分辨质谱技术获取牛初乳、牛常乳、羊初乳和羊常乳的乳清蛋白组轮廓,基于其非标记定量强度建立偏最小二乘判别分析模型。结果发现牛初乳和牛常乳在蛋白质组成方面较羊初乳和羊常乳更相似,同时筛选出羊乳中丰度较高的9 种蛋白用作标志物区分这4 组乳样。生物信息学分析发现羊乳中的高丰度蛋白大部分与免疫应答和代谢过程有关,说明羊乳更有助于新生儿建立抗微生物感染的免疫系统。该研究可加深对羊乳蛋白的认识,对牛乳及其制品的营养改良和母乳替代品的生产具有重要意义。     

Quantitative analysis of differentially expressed milk fat globule membrane proteins between donkey and bovine colostrum based on high-performance liquid chromatography with tandem mass spectrometry proteomics

This study was designed to provide novel insights into milk fat globule membrane (MFGM) proteins in donkey colostrum (DC) and bovine colostrum (BC) using quantitative proteomics. In total, 179 (DC) and 195 (BC) MFGM proteins were characterized, including 71 shared, 108 DC-specific, and 124 BC-specific proteins. Fifty-one shared proteins were selected as differentially expressed MFGM proteins, including 21 upregulated and 30 downregulated proteins in DC. Gene ontology analysis showed that these proteins were mainly enriched in cellular components, including the extracellular exosome, extracellular space, and plasma membrane. Additionally, they were further involved in metabolic pathways, including cholesterol metabolism, the peroxisome proliferator-activated receptor signaling pathway, and purine metabolism. Furthermore, several key protein factors with high connectivity were identified via protein–protein interaction analysis. These results provide more comprehensive knowledge of differences in the biological properties of MFGM proteins in DC and BC as well as pave the way for future studies of the nutritional and functional requirements of these important ingredients toward the development of dairy products based on multiple milk sources.     

毛细管电泳法对乳及乳制品中真蛋白的测定

利用毛细管电泳法对市售乳清蛋白粉、采自当地奶牛场的牛初乳及原料奶、同一品牌不同阶段或同一阶段内不同厂家、不同国别的婴儿配方奶粉、不同保质期的液态奶、不同国别的超高温灭菌(UHT)奶和酸奶、国产复原乳中α- 乳白蛋白(α-Lac)、β- 乳球蛋白A (β-LgA)及β- 乳球蛋白B (β-LgB)的质量分数进行测定。结果表明：α-Lac、β-LgA 及β-LgB 的质量分数在乳清蛋白粉、牛初乳及原料奶中依次降低。液态奶中上述3 种蛋白的质量分数与保质期有关,一般保质期越久的产品,上述3 种蛋白的质量分数越低。大多数酸奶中α-Lac 的质量分数均高于UHT 奶。婴儿配方奶粉中α-Lac 质量分数随着阶段数的增加而增加,而且同一阶段内不同厂家不同国别配方奶粉中α-Lac 质量分数差异也较大,并非进口奶粉中α-Lac 质量分数均比国产奶粉高。     

牛乳和双峰驼乳中乳脂球膜蛋白的差异分析

为阐明双峰驼乳中乳脂球膜蛋白（milk fat globule membrane proteins,MFGMP）的组成以及与牛乳MFGMP之间的差异,以牛乳和双峰驼乳中的MFGMP为研究对象,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、双向电泳和液相色谱-串联质谱（liquid chromatograph-tandem mass spectrometry,LC-MS/MS）技术对其中的高丰度蛋白质进行表征,并对双峰驼乳MFGMP提取方法进行选择。结果显示,双峰驼乳和牛乳MFGMP中含量最高的蛋白组分依次为嗜乳脂蛋白、黄嘌呤氧化还原酶/脱氢酶、乳凝集素（MFG-E8或PAS6/7）、脂肪分化相关蛋白（ADRP或PLIN2）,3 种方法分析双峰驼乳和牛乳中MFGMP中高丰度蛋白组分的结果具有一致性。基于LC-MS/MS技术对双峰驼乳的MFGMP进行鉴定,并将对鉴定出的蛋白质进行基因本体功能注释和京都基因与基因组百科全书路径分析,发现这些蛋白主要是细胞膜、线粒体、内质网膜等组分,参与的生物途径有翻译和翻译的起始、氧化还原过程、细胞内蛋白质转运、蛋白质折叠、细胞氧化还原稳态等,具有结合活性和分子催化等分子功能。     

Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions

We previously reported that microRNA (miRNA) is present in human breast milk. Recently, other groups have reported that bovine milk also contains miRNA; however, these reports are few. We therefore investigated bovine milk miRNA using microarray and quantitative PCR analyses to identify the differences between colostrum and mature milk. The RNA concentration in a colostrum whey fraction was higher than that in a mature milk whey fraction. In total, 102 miRNA were detected in bovine milk by microarray analysis (100 in colostrum and 53 in mature milk; 51 were common to both). Among these miRNA, we selected several immune- and development-related miRNA, including miR-15b, miR-27b, miR-34a, miR-106b, miR-130a, miR-155, and miR-223. These miRNA were detected in bovine milk by quantitative PCR, and each of these miRNA was significantly more highly expressed in colostrum than in mature milk. We also confirmed the presence of some mRNA in bovine milk. Nevertheless, synthesized miRNA spiked in the raw milk whey were degraded, and naturally existing miRNA and mRNA in raw milk were resistant to acidic conditions and RNase treatment. The RNA molecules in milk were stable. We also detected miRNA and mRNA in infant formulas purchased from Japanese markets. It is still unknown whether milk-derived RNA molecules play biological roles in infants; however, if milk-derived RNA do show functions in infants, our data will help guide future studies.     

Comparative Study on Heat Stability and Functionality of Camel and Bovine Milk Whey Proteins

Heat stability, emulsifying, and foaming properties of camel whey have been investigated and compared with that of bovine whey. Camel whey is similar to bovine whey in composition, but is deficient in β-lactoglubulin (β-LG), a major component of bovine whey. Whether the deficiency in β-LG will affect stability and functional properties is not yet known. Substantial information on the functional properties of bovine milk whey proteins is available; however, there is little research done on functional properties of camel whey proteins. Therefore, the objective of this study was to investigate the heat stability, emulsifying, and foaming characteristics of camel whey proteins. Calorimetric studies showed no significant difference in heat stability between bovine and camel whey proteins in liquid form. Upon drying, thermograms indicated that the 2 proteins are different in composition and thermal stability. The difference is represented in the absence of β-LG and the occurrence of protein denaturation peak at a lesser temperature in camel whey. The first marginal thermal transition in bovine whey appeared at 81°C, followed by 2 other transitions at 146 and 198°C. For camel whey, the transitions appeared at 139, 180, and 207°C respectively. The first marginal denaturation peak in bovine whey is due to β-LG, which is essentially absent in camel whey, while the second peak is due to the mixture of α-lactalbumin, serum albumin, and possibly part of the partially stabilized β-LG structure during the denaturation process. Because camel whey is deficient in β-LG, the denaturation peak at 139 must be due to the mixture of α-lactalbumin and camel serum albumin. In both proteins, the highest thermal transition is due to sugars such as lactose. The solubility study has shown that camel whey is more sensitive to pH than bovine milk whey and that heat stability is lowest near the isoelectric point of the proteins at pH 4.5. The sensitivity to pH resulted in partial denaturation and increased tendency to aggregate, which caused poor and unstable emulsion at pH 5. Both bovine and camel whey proteins have demonstrated good foaming properties; however, the magnitudes of these properties were considerably greater in bovine milk for all of the conditions studied.