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。     

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。     

蜜蜂蜂王不同于工蜂的关键因素-蜂王浆主蛋白1

蜂王浆是决定蜜蜂幼虫发育中级型分化,即成为蜂王还是工蜂的关键环境因素,而蜂王浆主蛋白(main royal jelly proteins,MRJPs)是反映蜂王浆新鲜度的重要指标。日本镰仓昌树以蜜蜂和果蝇为模型的最新研究表明,MRJP1是蜂王浆中决定蜜蜂级型分化的关键因子,该蛋白可通过激活虫体脂肪体中的表皮生长因子信号通路,引发个体增大、发育时间缩短和卵巢发育等蜂王特征的出现。因此,今后很有必要进一步开展MRJP1对人体的营养功能和作用机理研究,为MRJP1应用于功能食品提供科学依据。     

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。     

植物源抗烟草花叶病毒活性物质的初步筛选

利用半叶枯斑法,测定了13科18种植物蛋白粗提液对烟草花叶病(TMV)的抑制效果。结果表明,不同植物蛋白粗提液对TMV侵染心叶烟的抑制效果存在差异,6种植物蛋白粗提液对TMV的抑制效果达到50%以上,其中菠菜和合欢最高,分别达到了97.44%和96.92%。进一步通过(NH4)2SO4盐析,对菠菜和合欢蛋白粗提液抗病毒活性成分进行了初步分离,并测试(NH4)2SO4盐析的不同蛋白组分对TMV的抑制效果。结果显示,菠菜蛋白粗提液盐析后的两个蛋白组分都表现出很高的抗病毒活性,而合欢蛋白粗提液只在40%~80%饱和度盐析的蛋白组分具有较强的抗病毒活性。     

蜂王浆中链霉素残留的提取、净化及液相色谱测定方法研究

本实验对蜂王浆中链霉素残留的提取净化及液相色谱检测方法进行研究。蜂王浆样品用含有蛋白沉淀剂的磷酸提取液(pH2.0)溶解,超声波辅助提取蜂王浆中的链霉素残留。样液经离心后,分别用阳离子交换柱和反相固相萃取柱净化。采用柱后衍生-高效液相色谱梯度洗脱与荧光检测分析蜂王浆中的链霉素残留。以Atlantis HILIC Silica(2.1mm×150mm,3.0μm)为色谱柱,以酸性水溶液和乙腈为流动相,链霉素在25min内实现较好的分离,方法快速准确,灵敏度高,是一种较好的定性和定量方法。所研究的提取净化方法及色谱分离条件能有效排除蜂王浆中的杂质干扰,添加回收率在71.7%～77.4%之间,方法检出限为10μg/kg。     

超滤膜法制备豌豆蛋白的工艺

研究了用超滤技术从蛋白粗提液中分离提纯豌豆分离蛋白的工艺参数.通过单因素试验,分析了不同温度、压力、pH值对膜通量的影响,并通过正交试验确定膜分离的最佳温度、压力、pH值分别 :45℃、0.8 MPa、pH8.0.     

胡麻分离蛋白提取工艺研究

为促进胡麻分离蛋白在食品工业中应用,采用碱溶酸沉原理研究胡麻分离蛋白提取工艺。分别讨论料液比、浸提液pH值、浸提温度和时间等条件对蛋白提取率影响,确定最佳提取条件为:料液比1:13、碱提液pH值9.5、时间60、温度60℃,在此条件下,胡麻分离蛋白提取率为42%,纯度达89.37%。     

瑞氏木霉疏水蛋白HFBI的分离纯化

通过对瑞氏木霉(Trichodermareesei)疏水蛋白HFBI的一系列抽提纯化,确定了瑞氏木霉疏水蛋白的抽提纯化工艺。比较几种疏水蛋白粗提方法,选用了1%的SDS进行抽提,KCl沉淀后,经过Berol532双水相萃取得到粗提品;通过SephadexG-25、Q-Sepharose的纯化后,样品中疏水蛋白HFBI占总蛋白含量的98%以上,总回收率可达62·42%。     

库拉索芦荟凝集素的分离与纯化

目的:对库拉索芦荟凝集素进行分离、纯化与初步鉴定。方法:库拉索芦荟经捣碎匀浆、盐析、透析后得到库拉索芦荟凝集素的粗提液;兔血凝集反应测试初步检测粗提液成分;将粗提液进行SDS-聚丙烯酰胺凝胶电泳和葡聚糖G-75凝胶柱层析,对分离组分进行分析;将洗脱液浓缩,再用SDS-聚丙烯酰胺凝胶电泳和葡聚糖G-200凝胶柱层析对组分进行分析。结果:发现库拉索芦荟粗提液可以凝集兔血,证明在粗提液中具有芦荟凝集素成分。对粗提液检测、分离,获得三种蛋白质组分;经凝血活性检测,只有第一个蛋白峰具有凝集活性且凝集效果较粗提液进一步提高。再次分离纯化洗脱液,可知其由两个组分组成。结论:库拉索芦荟凝集素由两种组分组成。     

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.     

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.     

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.     

Analysis of chloramphenicol in honey and royal jelly by LC/MS/MS

A sensitive and selective method using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was developed for the determination of chloramphenicol (CAP) in honey and royal jelly. Mass spectral acquisition was performed in the negative mode by applying multiple reaction monitoring. In LC separation, Mightyl RP-18GP and 10 mmol/L ammonium acetate-acetonitrile were used as the column and mobile phase, respectively. CAP in honey samples was diluted with water, while CAP in royal jelly was extracted with 1% metaphosphoric acid-methanol (4 : 6). The solutions were cleaned up with an Oasis HLB cartridge. The quantification limits of CAP in honey and royal jelly were 0.3 ng/g and 1.5 ng/g, respectively. The recoveries of CAP from both honey and royal jelly at the quantification limits were over 92%. Twenty honey products and seven royal jelly products were analyzed by the developed method. CAP was detected in one honey product at 0.6 ng/g and in six royal jelly products at the level of 1.5-17.8 ng/g. These results show that the developed method has satisfactory sensitivity selectivity and is useful for the determination of CAP residues in honey and royal jelly.     

蜂王浆蛋白肽的制备及其降血糖和抗氧化活性研究

目的:研究蜂王浆蛋白的最佳酶解工艺条件,并分析所制备酶解肽的氨基酸组成、分子量分布、降血糖及抗氧化活性。方法:以蜂王浆为原料,采用碱提酸沉法提取蜂王浆粗蛋白,以α-葡萄糖苷酶抑制率和ABTS自由基清除率为评价指标,筛选出酶解蜂王浆蛋白制备降血糖及抗氧化活性肽的最佳蛋白酶,并研究蜂王浆酶解肽的体外降血糖和抗氧化活性。结果:蜂王浆降血糖及抗氧化活性肽的最佳制备工艺为:以酸性蛋白酶为酶制剂,酶添加量为6000 U/g,酶解温度为43℃,酶解pH为4.0,酶解时间为4 h,料液比为1:10。在上述条件下,制备的蜂王浆蛋白肽的氨基酸总量为82.19%,相对分子质量集中在1000 Da以下。蜂王浆蛋白肽对α-葡萄糖苷酶的半抑制浓度(IC₅₀)为6.94 mg/mL,清除ABTS自由基、羟自由基、1,1-二苯基-2-三硝基苯肼(DPPH)自由基及超氧阴离子自由基的半抑制浓度(IC₅₀)分别为14.18、0.45、11.02和18.38 mg/mL。试验结果表明,蜂王浆蛋白肽具有一定的降血糖和抗氧化活性,可为蜂王浆高值化利用及活性肽产品开发提供理论依据。     

离子色谱法测定蜂花粉与蜂王浆中的葡萄糖、果糖、蔗糖和麦芽糖

研究了蜂花粉与蜂王浆中葡萄糖、果糖、蔗糖和麦芽糖的离子色谱分析方法。样品用热水溶解,乙腈沉降蛋白,使用2个固相萃取小柱去除色素与脂肪等干扰成分后,以CarboPac PA10分析柱为色谱柱,氢氧化钠和乙酸钠溶液为流动相进行离子色谱分离。葡萄糖、果糖、蔗糖和麦芽糖的检出限分别为0.12,0.14,0.21,0.33mg/L。蜂花粉的回收率93.5%～102.2%,精密度为4.4%～6.3%,蜂王浆的回收率为95.0%～103.5%,精密度为6.7%～10.2%。利用该方法对20个蜂花粉和20个蜂王浆样品进行了分析,花粉中葡萄糖含量为9.9%～20.6%,果糖14.2%～26.9%,蔗糖1.8%～8.3%,麦芽糖0.33%～1.4%;王浆中葡萄糖含量为6.2%～8.3%,果糖7.0%～8.7%,蔗糖0.38%～3.6%,麦芽糖0.27%～0.83%。该方法简单、灵敏度高,可作为标准方法对蜂花粉与蜂王浆中葡萄糖、果糖、蔗糖和麦芽糖进行分析。     

不同花期蜂王浆主要成分和抗氧化活性分析

为探究不同花期蜂王浆品质和抗氧化活性的差异,以同产地同蜂种同饲养条件下所生产的蜂王浆作为研究对象,分析不同花期蜂王浆的10-羟基-2-癸烯酸（10-HDA）、总蛋白、水分、总酚酸的含量变化,同时采用DPPH法和FRAP法分析比较其体外抗氧化能力。结果表明,不同花期之间蜂王浆的10-HDA含量存在显著差异（P<0.05）,葡萄蜂王浆10-HDA含量最高达到了2.20%。总蛋白含量在13.58%~15.26%之间。蜂王浆的水分含量均≤67.5%,达到国标（GB 9697-2008）关于优等品蜂王浆水分含量的要求。与其他花期蜂王浆相比,荆条花期的总酚酸含量、DPPH自由基清除能力和总抗氧化能力均为最优,且不同花期蜂王浆之间的DPPH自由基清除率存在显著差异（P<0.05）。相关性分析表明,DPPH自由基清除能力和总酚酸以及总抗氧化能力之间均存在极显著正相关性（P<0.01）。