蜂王浆主蛋白1低聚体的分离纯化及其圆二色谱分析

蜂王浆主蛋白1（major royal jelly protein 1,MRJP1）是蜂王浆主蛋白家族中最重要的成员之一,MRJP1存在单体和低聚体两种形式。为了研究MRJP1低聚体的二级结构信息,首先利用ToyoScreen GigaCap Q-650M离子交换柱从新鲜蜂王浆中纯化出MRJP1低聚体,经分析超速离心和高效液相色谱-串联质谱技术鉴定其为MRJP1低聚体后,再通过圆二色谱技术检测MRJP1低聚体在不同浓度Ca2+条件下（10、50、200 mmol/L）二级结构的变化情况,并测定其变性温度。结果表明：该分离方法可以一步纯化出MRJP1低聚体,MRJP1低聚体的二级结构中β-折叠占比最高（55%左右）,其次是无规卷曲（20%左右）和α-螺旋（15%左右）,β-转角（10%左右）含量最低;MRJP1低聚体的变性温度为55 ℃。实验提出了MRJP1低聚体新的纯化方法及其二级结构的基本信息。     

利用不同疏水色谱柱分离蜂王浆主蛋白1

目的:比较不同疏水色谱柱对蜂王浆主蛋白1(major royal jelly protein 1,MRJP 1)的分离效果,并筛选出适合分离MRJP 1寡聚体和单体的色谱柱。方法:首先分别采用12种疏水色谱柱对蜂王浆蛋白粗提液进行分离,然后使用非变性电泳验证所分离MRJP 1组分的寡聚体和单体形式。结果:Hitrap Butyl HP疏水色谱柱可以将所有的MRJP 1从蜂王浆蛋白粗提液中分离出来,并得到分别以MRJP 1寡聚体和MRJP 1单体为主的2个组分。结论:利用疏水色谱柱可以有效地分离MRJP 1。     

蜂王浆的主要活性成分及其保健作用

蜂王浆是工蜂上颚分泌的专供蜂王和蜜蜂幼虫食用的乳白色或淡黄色的浆状物质.文中重点介绍蜂王浆中的蛋白类、氨基酸、有机酸和低聚糖等主要活性成分,以及其对人体保健所发挥的作用.     

蜂王浆冷冻干燥工艺

蜂王浆为蜜蜂(工蜂)咽腺中分泌出来的一种物质,是蜜蜂用来喂饲蜂王的食料。它具有较高的营养价值及药理价值,受到国内外食品、化妆和医药等行业的青睐。蜂王浆具有一定的生物活性,并具有较高的热敏感性质。因此,新鲜蜂王浆的常温贮藏,会很快丧失其生物活性,以致腐败、变质。我国规定蜂王浆应在—18℃下低温保存。     

不同贮存条件的蜂王浆蛋白质组的分析

利用双向电泳(2-DE)及基质辅助激光解析/电离-飞行时间串联质谱(MAIDI-TOF-TOF MS)对新鲜蜂王浆及在-20,4℃和常温条件(23±2)℃下贮存的蜂王浆蛋白质组对比分析,结果发现蜂王浆主蛋白(MRJP)1~3相对较稳定,在3种条件下贮存1年后仍可检出;MRJP4、MRJP5和葡萄糖氧化酶(GOD)对贮存条件较敏感,在4℃或常温下贮存1年后均完全降解。结合对6种产地、蜜源蜂王浆中蛋白质组的分析,初步确定MRJP4、MRJP5和GOD为蜂王浆中对贮存温度较敏感的蛋白,3种蛋白均可能应用到蜂王浆新鲜度的评价及检测中。     

蜂王浆与医疗保健

蜂王浆又叫蜂乳,它是哺育工蜂的王浆腺分泌的一种高浓度的用来饲喂蜂王和早期幼虫的粘稠状物质。蜂王浆不仅对蜜蜂来说是十分完美的食品,而且也是人类理想的医疗保健食品。 蜂王和工蜂都是由受精卵发育而成的,以蜂王浆为食的蜂王,每昼夜要产1500～2000粒卵,其总重量是自身重量的二倍以     

RP-HPLC法测定蜂王浆水溶性蛋白及其相关产物对ACE的抑制作用

采用反相高效液相色谱(RP-HPLC),测定蜂王浆水溶性蛋白(WSPs)、WSPs的纯化蛋白1(MRJP1)和2(MRJP2)、WSPs的酶解产物(P-WSPs、T-WSPs和PT-WSPs)及从WSPs和酶解产物中分离到的不同肽段(>3 ku,1～3 ku和<1 ku)等对血管紧张素转换酶(ACE)的抑制作用。结果表明,WSPs、MRJP1和MRJP2等水溶性蛋白本身对ACE抑制作用很弱,而它们的酶解产物对ACE的抑制作用却大大增强了,其IC50值分别为,P-WSPs:0.262 mg/mL;T-WSPs:0.870 mg/mL;PT-WSPs:0.046 mg/mL。在WSPs和酶解产物的膜分离肽段中,分子质量小于1 ku的肽段对ACE具有非常强的抑制作用。     

不同蛋白水平的饲粮对蜂王浆腺苷含量的影响

采用不同蛋白含量的人工饲粮喂食蜜蜂,观察对蜂王浆中腺苷含量的影响。实验组喂食不同蛋白含量(15%、20%、25%、30%、35%、40%)的饲粮,以油菜花粉作对照,每3d取浆1次,测定第0、6、12、18、24天所取蜂王浆中腺苷的含量。结果表明:饲粮蛋白含量对蜂王浆中腺苷的影响存在最适剂量,蛋白含量35%水平的为蛋白饲粮组蜂王浆中腺苷的含量在第12天和第18天均为最高,油菜花粉组所产的蜂王浆中腺苷的含量几乎是最低的。     

蜂王浆蛋白和脂类降血脂功能的研究

为研究蜂王浆中的蛋白和脂类的降血脂功能,建立高血脂症大鼠模型,以大鼠血清中TC、TG和HDL-C水平,动脉粥样硬化指数(AI)以及体重,肝重、脾重与体重比作为评价指标,考察蜂王浆蛋白和脂类的降血脂作用。结果表明,蜂王浆蛋白能显著降低高血脂症大鼠血清中TC、TG水平,提高血清HDL-C水平,抑制AI值的上升,减少体重、肝脏和脾脏重量的增加,且与鲜蜂王浆效果接近;但是脂类没有降血脂功能。因此,蜂王浆的降血脂功效成分主要是蜂王浆蛋白。     

蜂王浆蛋白多肽及其生物活性研究进展

蜂王浆(RJ)是6 d~18 d龄青年工蜂发达的下咽头腺和上颚腺等腺体所分泌的特殊浆状物质。蜂王浆中的干物质以蛋白质含量最多,蜂王浆中的主要王浆蛋白(MRJPs)具有广泛的生物学活性。通过直接分离或者先用蛋白酶水解再分离蜂王浆中的活性蛋白质和肽类,发现了其具有抗氧化、抗菌、免疫调节、降血糖、促进细胞增殖、抗肿瘤、抗疲劳、降血压、保护神经细胞等众多功能。随着现代分离分析技术及基因组学、蛋白质组学的发展和蜜蜂基因组测序的完成,蜂王浆蛋白复杂的物质基础和作用机制都将逐一呈现,并且能够广泛应用于生物医药、科研、食品、化妆品等领域。     

A rapid method to isolate soluble royal jelly proteins

Soluble royal jelly (RJ) proteins (SRJPs) include the major RJ protein (MRJP) family, which contribute to the physiological actions of RJ. Although SRJPs are prepared using conventional methods involving dialysis and centrifugation, dialysis is a time-consuming process. We have therefore developed a simple method to isolate SRJPs from RJ. This new method produces 20-fold higher levels of SRJPs than that of the conventional procedure; hence, the levels obtained by the new and existing methods were compared. A 1-h ultracentrifugation separated SRJPs in the supernatant into upper, middle and lower layers. Each layer was analyzed by size-exclusion HPLC, SDS–PAGE and 2-DE. The upper and middle layers contained MRJP2 (52 kDa) and MRJP3 (60–70 kDa), while the lower layer contained MRJP1 (290 kDa). In nature, MRJP1 is a monomer and/or oligomer. When the lower layer was analyzed by Superose 12 HPLC, MRJP1 was predominantly an oligomer. Our MRJP isolation method reduces the procedure time by using ultracentrifugation without dialysis to obtain SRJPs and produces layers containing MRJP1 oligomers, MRJP2 and MRJP3.     

Fast determination of royal jelly freshness by a chromogenic reaction

Royal jelly is one of the most important products of honeybees. Given its role in development of bee brood into fertile individuals of the royal caste it is also used in health products for human consumption. Royal jelly spoils and loses its health-promoting properties depending on storage duration and conditions. To ensure product quality before selling, it is therefore necessary to assess royal jelly freshness. Many indexes of freshness have been suggested, but they all lack reliability or require complex and time-consuming analyses. Here we describe a method to detect royal jelly freshness based on a chromogenic reaction between royal jelly and HCl. We demonstrate that analyses based on color parameters allow for the discrimination of royal jelly samples based on the duration of their storage. Color parameters of royal jelly stored at -18 and 4 °C for 28 d remained comparable to that of fresh samples, which supports the reliability of the method. The method of freshness determination described is practical, cheap, and fast and can thus be used in real-time when trading royal jelly. PRACTICAL APPLICATION: The method developed can be used to assess royal jelly freshness. It is practical, cheap, and fast and can thus be used in real-time when trading royal jelly.     

蜂王浆在常温储存条件下品质变化的研究

本实验对常温条件下储存5个月的蜂王浆的品质进行了研究。通过SDS-PAGE凝胶电泳,发现王浆中两种水溶性蛋白85.2kD(葡萄糖氧化酶)和60.7kD(MRJP-4)发生降解,并且比57kD蛋白降解得明显,这表明85.2kD(葡萄糖氧化酶)和60.7kD(MRJP-4)可以作为王浆新鲜程度的标志性成分。研究还发现蜂王浆褐变的程度随着时间的增加而增加,而且蜂王浆的褐变与蛋白质有关,是蛋白质的糖基化结果,因此蜂王浆在储存过程中的褐变是美拉德反应的结果。     

Estimation and characterisation of major royal jelly proteins obtained from the honeybee Apis merifera

Royal jelly (RJ) contains many components, including proteins. We focused on major royal jelly proteins (MRJPs) under natural conditions, and attempted to determine the content ratios and molecular forms of MRJPs by size-exclusion HPLC, SDS–PAGE, 2-DE and MALDI TOF/TOF MS. Soluble RJ proteins were extracted by dialysis followed by several centrifugation techniques. Soluble RJ proteins were universally separated into five peaks (640 kDa, 280 kDa, 100 kDa, 72 kDa and 4.5 kDa) by size-exclusion HPLC on a Superose 12 column. Among these peaks, both the 280 kDa and 72 kDa peaks were major, but the intensity of the 280 kDa peak differed markedly among original RJ samples (n = 70). The main 280 kDa protein was separated into a 55 kDa band by reducing and non-reducing SDS–PAGE. This protein was also separated into multiple spots ranging from pH 4.2 to 6.5 by 2-DE. These spots were identified as MRJP 1 by MALDI TOF/TOF MS. From these results, MRJP 1 was thought to comprise an oligomer complex linked by non-covalent bonds under natural conditions. Another major protein, the 72 kDa peak on Superose 12 HPLC, was identified as MRJP 2.     

蜂王浆醇溶性成分分析及其抗氧化性研究

蜂王浆经体积分数95%乙醇提取、MCI-Gel、ODS等反相色谱柱分离,得到单磷酸腺苷(AMP)和7种脂肪酸。采用1,1-二苯基苦基苯肼自由基(DPPH.)清除率和总抗氧化能力试剂盒(T-AOC)等方法探索蜂王浆醇溶性物质的抗氧化能力,结果表明:蜂王浆醇提后抗氧化活性提高,分离得到的AMP和3,10-二羟基癸酸(3,10-DDA)的抗氧化性相对较强,且清除DPPH.的IC50值分别为6.274 mg/mL和9.153 mg/mL,是蜂王浆中的抗氧化活性物质。     

Using proteomics platform to develop a potential immunoassay method of royal jelly freshness

So far royal jelly (RJ) has been widely used as a kind of popular and traditional food for health promotion. However, the quality of RJ is vulnerable to improper storage conditions. In order to prohibit the low quality RJ products entering the market and consequently affect the health of humans, it is necessary to define the quality parameters and establish corresponding detection methods for the freshness of RJ. In this research, we applied two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight mass spectrometry to research major royal jelly proteins (MRJPs) changes under different storage conditions after 6-month storage, looking for a stable and reliable protein marker which was also feasible to detect the freshness of RJ. Further research with the help of Western blotting analysis (WB) confirmed that, under room temperature, MRJP5 began to hydrolyze within 30 days and would completely degrade within 75 days, indicating that MRJP5 can be adapted as a freshness marker for RJ products. Moreover, the assessment results of the freshness of 12 commercial RJ products with WB showed that MRJP5 was present in twelve RJ samples at different abundance levels which further confirmed the detection of MRJP5 could be a feasible method to assess the quality and freshness of commercial RJ products.     

蜂王浆中活性因子对衰老小鼠的保护作用

为了探索蜂王浆及其主要的蛋白质类、脂类功能活性因子对D-半乳糖（D-gal）致衰老小鼠的保护作用。实验通过透析、超滤、有机相萃取的方法分别提取分离了蜂王浆中粗蛋白、蛋白质类功能因子及脂类功能因子。通过连续7周每日腹腔注射100 mg/kg（以干质量计） 的D-gal建立衰老小鼠模型,并分别给予蜂王浆及不同剂量的粗蛋白组分、蛋白质类或脂类功能因子干预后,发现蜂王浆可以显著提高小鼠在T-迷宫中的准确率,并且降低肝脏器官指数,证实蜂王浆可能提高衰老小鼠的认知记忆水平,改善小鼠的衰老症状;脂类功能因子及高剂量的蛋白质类功能因子能够显著提高 T-迷宫准确率、缓解肝脏肿大。因此推测,蜂王浆中的蛋白质类功能因子和脂类功能因子都具有良好的抗衰老作用,其机制可能与提高大脑的记忆能力有关。