Major royal jelly proteins accelerate onset of puberty and promote ovarian follicular development in immature female mice

The major royal jelly proteins(MRJPs)are the central constituents responsible for the specific activities of royal jelly.Here MRJPs via oral administration daily for 45 consecutive days were evaluated the effects on the reproductive parameters in immature female mice(FM).Neonatal FM were divided into four groups fed MRJPs with doses of 0,125,250 and 500 mg/kg/body weight(M125,M250 and M500).The results in M125,M250 and M500 showed that the times of estrus were accelerated by 10.7%,15.5%and 10.7%,the secondary follicles number were increased by 50.7%,78.8%and 38.6%,the Graafian follicles were increased by 600.0%and 774.0%and 150.0%,respectively.M500 induced multi-oocyte follicles.The serum estradiol levels of the three groups were increased by 47.1%,64.9%and 31.1%,the action of MRJPs raising hormone secretion level is mainly via upregulating expression of ERˇgene.Antioxidant parameters of ovarian tissue showed that the malondialdehyde levels in M125 and M250 were decreased,the superoxide dismutase activities and glutathione peroxidase activities in M125 and M250 were increased.In conclusion,MRJPs may accelerate onset of puberty and promote follicular development in FM.Our findings would facilitate better understanding of the benefit effect of MRJPs as the key ingredient in royal jelly on promoting fertility performance.

Structural and antioxidative properties of royal jelly protein by partial enzymatic hydrolysis

The objective of this study was to investigate the structural and antioxidative properties of royal jelly protein(RJP)at different degrees of hydrolysis(DH)by partial enzymatic hydrolysis. RJP was hydrolyzed by alcalase for 0 min, 15 min, 1 h, 5 h and 8 h to obtain hydrolysates at DH of 5.34%, 11.65%, 15.19%, 21.38% and 23.91%, respectively. With the increased DH, the RJP hydrolysates showed elevated antioxidative activities. The molecular weight of RJP hydrolysates was significantly decreased but their primary backbone kept unchanged. Analysis of circular dichroism spectra revealed that the enzymolysis reduced the content of α-helix but increased the contents of β-sheet, β-turn and random coil. Meanwhile, the surface hydrophobicity and fluorescence intensity of RJP hydrolysates were decreased and a red shift occurred. As the enzymolysis continued, the surface morphology of RJP was gradually changed from a sheet-like structure into microparticles. Changes in antioxidative activities and structures generally followed a DH-dependent manner, however these changes became insignificant for samples at DH beyond 20%. Taking into consideration of both effectiveness and productivity, the optimum enzymatic duration was determined at 5 h.

Anti-senescence effect and molecular mechanism of the major royal jelly proteins on human embryonic lung fibroblast(HFL-I) cell line

Royal jelly (R J)from honeybee has been widely used as a health promotion supplement.The major royal jelly proteins (MRJPs)have been identified as the functional component of RJ.However,the question of whether MRJPs have anti-senescence activity for human cells remains.Human embryonic lung fibroblast (HFL-I)cells were cultured in media containing no MRJPs (A),MRJPs at 0.1mg/ml (B),0.2mg/ml (C),or 0.3mg/ml (D),or bovine serum albumin (BSA)at 0.2mg/ml (E).The mean population doubling levels of cells in media B,C,D,and E were increased by 12.4%,31.2%,24.0%,and 10.4%,respectively,compared with that in medium A.The cells in medium C also exhibited the highest relative proliferation activity,the lowest senescence,and the longest telomeres.Moreover, MRJPs up-regulated the expression of superoxide dismutase-1(SOD1)and down-regulated the expression of mammalian target of rapamycin (MTOR),catenin beta like-1(CTNNB1),and tumor protein p53(TP53).Raman spectra analysis showed that there were two unique bands related to DNA synthesis materials,amide carbonyl group vibrations and aromatic hydrogens.These results suggest that MRJPs possess anti-senescence activity for the HFL-I cell line,and provide new knowledge illustrating the molecular mechanism of MRJPs as anti-senescence factors.

Evaluation of the major royal jelly proteins as an alternative to fetal bovine serum in culturing human cell lines

Royal jelly （R J） is a well-known bioactive substance. It contains large amounts of major royal jelly proteins （MRJPs）, which express growth-factor-like activity in several animal and human cell lines. However, the question on whether MRJPs possess growth-factor-like activity on all types of cell cultures remains. In order to determine whether MRJPs can be used as an alternative to fetal bovine serum （FBS） in different types of human cell culture, the prolif- eration of the complex serum with different ratios of MRJPs/FBS （M/F） was evaluated on five cell lines： 293T, HFL-I, 231, HCT116, and Changliver using MTT （3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2H-tetrazolium bromide） assay. The proliferation activity of the combination of the complex M/F serum with cytokines on the test cell lines was also measured. The results demonstrated that the complex serum with M/F 6/4 possessed the highest proliferation activity similar to or in excess of FBS. However, no activity of complex medium with M/F 6/4 was observed in 231 cells, indicating a selectivity of MRJPs on cell types. Compared with the complex medium with M/F 6/4, the complex medium with M/F 6/4 together with two cytokines, epidermal growth factor （EGF） and insulin-transferrin-selenium （ITS）, pro- moted proliferations of Changliver, 293T, HCT116, and H FL-I by 18.73%-56.19% （P〈0.01）. Our findings demonstrate that MRJPs could partially replace FBS in culturing many human cell lines.

王浆主蛋白1(MRJP-1)及其对蜜蜂学习记忆影响的研究进展

蜂王浆由蜜蜂幼年工蜂所产,是蜂王及工蜂小幼虫的唯一食物来源,同时也因蜂王浆的丰富营养价值而为人们所熟知。蜂王浆主蛋白(MRJPs)是蜂王浆中最为丰富的水溶性蛋白,其中的MRJP-1除提供营养外,对蜜蜂的个体发育、神经系统发育、行为调控、级型分化等方面均有很好的生理调节作用。文章介绍了MRJP-1的理化特性和在蜜蜂体内的表达情况,并对MRJP-1在蜜蜂学习记忆方面的影响研究作简要概括,以期为王浆主蛋白在蜜蜂认知行为方面的研究提供一些思路和理论支撑。

Determination of royal jelly freshness by ELISA with a highly specific anti-apalbumin 1,major royal jelly protein 1 antibody

Major royal jelly protein 1（MRJP1）, designated apalbumin 1, has been regarded as a freshness marker of royal jelly（RJ）. A MRJP1-specific peptide（IKEALPHVPIFD） identified by bioinformatics analysis of homologous members of the major royal protein family was synthesized and used to raise polyclonal anti-MRJP1 antibody（antiSP-MRJP1 antibody）. Western blot analysis showed that anti-SP-MRJP1 antibody only reacted with MRJP1 in RJ. In contrast, the previously reported antibody against recombinant MRJP1（anti-R-MRJP1 antibody） reacted with other members of MRJP family in RJ. Enzyme-linked immunosorbent assay（ELISA） using anti-SP-MRJP1 antibody demonstrated that MRJP1 content in RJ stored at 40 °C significantly degraded by 37.3%, 55.9%, 58.0%, 60.6%, 65.7%, 72.7%, and 73.1% at 7, 14, 21, 28, 35, 42, and 49 d, respectively, when compared with MRJP1 content in fresh RJ（0 d）. Optical density analysis of MRJP bands from sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） profiles demonstrated that the degradation of MRJP1, MRJP2, MRJP3, and MRJP5 in RJ was strongly and positively correlated with the period of storage（P〈0.0001）. Our results indicated anti-SP-MRJP1 antibody was highly specific for MRJP1, and ELISA using the antibody is a sensitive and easy-to-use method to determine the freshness and authenticity of RJ.

蜂王浆水溶性蛋白及其酶解产物的抗氧化活性

本研究分别采用清除二苯基-2-苦肼自由基(DPPH·),抑制亚油酸氧化和总抗氧化能力测定等方法,探讨蜂王浆水溶性蛋白及其酶解产物的抗氧化特性。结果表明,蜂王浆水溶性蛋白(WSPs)本身的抗氧化活性很弱,但酶解后抗氧化活性增强,其中胃蛋白酶和胰蛋白酶共同酶解产物(PT-WSPs)的抗氧化活性最强。对WSPs及其酶解产物进行膜分离后发现,分子量小于3kD,尤其是小于1kD的肽类物质其清除DPPH·、抑制亚油酸氧化及总抗氧化能力最强。

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。

不同蜂花粉对西方蜜蜂蜂王浆主蛋白基因表达及蜂王浆产量、质量和组成的影响

本试验旨在研究不同蜂花粉对西方蜜蜂(Apis mellifera)蜂王浆主蛋白基因(mrjps)表达及蜂王浆产量、质量和组成的影响。选取蜂王、群势基本一致的意大利蜜蜂9群,随机分为3个组,每组3群蜜蜂:油菜花粉组,饲喂油菜花粉;土连翘花粉组,饲喂土连翘花粉;对照组,不饲喂花粉,自然采粉(主要为鬼针草花粉)。试验周期为2个月,试验时间为2017年9月中旬至11月中旬。利用凯氏定氮法和水解法分别检测油菜花粉、土连翘花粉和鬼针草花粉粗蛋白质含量和氨基酸组成与含量;每组选3只9日龄工蜂进行mrjp1~mrjp9和黄体基因e3(yellow⁃e3)相对表达量的检测;检测各组蜂王浆产量、质量及水溶性蛋白和氨基酸组成与含量。结果显示:3种蜂花粉粗蛋白质含量、总氨基酸含量均为油菜花粉>土连翘花粉>鬼针草花粉,且组间差异显著(P<0.05),3种蜂花粉单一氨基酸(组氨酸除外)含量也表现为油菜花粉>土连翘花粉>鬼针草花粉,且组间差异显著(P<0.05)。在9日龄工蜂头部,mrjp1、mrjp2、mrjp3、mrjp4、mrjp5和mrjp7相对高表达,mrjp6、mrjp8和mrjp9相对低表达,油菜花粉组、土连翘花粉组和对照组mrjp1、mrjp2、mrjp3、mrjp4、mrjp5和mrjp7的相对表达量之和分别占mrjps总相对表达量的99.94%、99.94%和98.60%。与对照组相比,饲喂油菜花粉和土连翘花粉显著提高了mrjp1、mrjp2、mrjp3、mrjp4、mrjp5、mrjp7和yellow⁃e3的相对表达量(P<0.05),且油菜花粉组上述基因的相对表达量显著高于土连翘花粉组(P<0.05);饲喂油菜花粉和土连翘花粉显著降低了mrjp6、mrjp8、mrjp9的相对表达量(P<0.05)。油菜花粉组蜂王浆产量最高,其次为土连翘花粉组,对照组最低,各组之间差异显著(P<0.05)。油菜花粉组和土连翘花粉组蜂王浆的质量与对照组相比无显著差异(P>0.05)。3组蜜蜂所产蜂王浆水溶性蛋白和氨基酸组成基本一致。综上所述,不同粗蛋白质含量的蜂花粉对西方蜜蜂mrjps基因的表达及蜂王浆产量的影响不同,对蜂王浆质量和组成的影响不大。3组蜂群相比,采食油菜花粉组具有营养功能的mrjps基因的相对表达量和蜂王浆产量最高。由此可见,对于西方蜜蜂繁育或蜂王浆生产,油菜花粉可作为一种优质的营养饲料。

蜂王浆中水溶性蛋白质和活性肽的研究进展

蜂王浆中的蛋白质和肽对蜂王浆的生理活性有重要影响。综述了近年来蜂王浆水溶性蛋白和活性肽的分离纯化、蛋白质结构,以及功能特性等方面的研究状况。

蜂王浆冻干粉体外抗氧化作用

对青海、陕西、安徽和浙江产油菜蜂王浆的冻干粉中水溶性蛋白、总酚和10-羟基-2-葵烯酸(10-HDA)含量进行测定,并通过DPPH法和FRAP法评价蜂王浆冻干粉的抗氧化活性。结果表明:不同产地油菜蜂王浆的冻干粉中总酚含量无显著性差异,但青海产油菜蜂王浆的冻干粉中水溶性蛋白含量最高(190.32mg/g),10-HDA含量最高的为陕西产油菜蜂王浆的冻干粉(4.28%);不同产地油菜蜂王浆的冻干粉均具有良好的DPPH自由基清除能力和总抗氧化能力,且样品的总抗氧化能力与其所含有的水溶性蛋白(r=0.88)和总酚含量(r=0.95)间呈显著正相关。

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。

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。

贮藏温度和时间对蜂王浆水溶性蛋白质的影响

采用SDS-PAGE和Folin-酚试剂法,对贮存不同温度和时间下的蜂王浆水溶性蛋白进行了初步研究。结果表明:-20℃时,蜂王浆水溶性蛋白相对稳定,40 d后蛋白质中相对分子质量为88 000(P1)的谱带才开始降解,且降解较慢;4℃时,蜂王浆水溶性蛋白中相对分子质量为88 000(P1)的谱带在5 d内降解迅速,水溶性蛋白中相对分子质量80 000(P2)的谱带在40 d时候已经降解;在20℃时蜂王浆水溶性蛋白中P1谱带迅速降解,P2谱带在28 d至40 d降解较快。水溶性蛋白中P1谱带的变化在一定程度上能够代表蜂王浆的新鲜度的变化。

饲粮蛋白质水平对意大利蜜蜂咽下腺发育及产浆量的影响

本试验旨在研究饲粮蛋白质水平对产浆期蜜蜂咽下腺发育及产浆量的影响。选择35群群势、蜂王年龄和质量一致的意大利蜜蜂(Apis mellifera ligustica),随机分为7组,每组5群,每群为1个重复。对照组饲喂油菜花粉,试验组分别饲喂蛋白质水平为15%、20%、25%、30%、35%及40%的试验饲粮。试验测定各群采食量、工蜂咽下腺蛋白质含量、腺小囊面积、蜂王浆产量(产浆量)及王台接受率。结果表明,饲粮蛋白质水平对蜜蜂采食量、产浆量及工蜂咽下腺发育均具有显著影响(P<0.05),且有很强的规律性,即随着饲粮蛋白质水平的提高,各指标呈现先升高后降低的变化趋势,并以饲粮蛋白质水平为30%时,蜂群采食量、工蜂咽下腺小囊面积最大,咽下腺蛋白质含量、产浆量、王台接受率最高。由此得出,饲粮蛋白质水平达到30%时,工蜂咽下腺发育最好,且能够获得最好的生产性能。

三种蜂王浆蛋白质的营养评价

采用国际上通用的营养价值评价方法,全面评价了中蜂王浆、意蜂王浆和卡蜂王浆蛋白质的营养价值。三种王浆的必需氨基酸含量分别占其氨基酸总量的42.88%,40.98%和42.72%,含硫氨基酸为第一限制氨基酸。蛋白质的化学评分(CS)、氨基酸评分(AAS)、必需氨基酸指数(EAAI)、生物价(BV)、营养指数(NI)和氨基酸比值系数分(SRCAA)计算结果表明,三种蜂王浆都是良好的蛋白源,但中蜂王浆蛋白质的营养价值高于意蜂王浆和卡蜂王浆,明显与常规品质评价指标10-HDA的含量高低顺序不同。

蜜蜂属六蜂种间MRJP2基因C-端重复序列的比较分析

构建了中华蜜蜂(Apis cerana cerana,中蜂)8日龄工蜂头部cDNA文库,获得了中蜂王浆主蛋白2(major royaljelly protein2,MRJP2)的全长cDNA序列,该序列长1605bp,包含一个编码468个氨基酸的开放阅读框(open readingframe,ORF)。在中蜂MRJP2的cDNA序列的C-端,首次发现存在串联重复片段长度多态性(variable numbers oftandemrepeat,VNTR)。克隆并测定了蜜蜂属Apis内其他5个种的MRJP2基因的C-端重复序列,结果表明:在蜜蜂属的其他5个种中,C-端重复片段的核心序列是以碱基高度突变方式而表现出个体之间的多态性,而重复片段长度基本一致。中蜂与西方蜜蜂A.mellifera,大蜜蜂A.dorsata与黑大蜜蜂A.laboriosa,以及小蜜蜂A.florea与黑小蜜蜂A.andreniformis分别形成3个进化枝。中蜂和西方蜜蜂与大蜜蜂和黑大蜜蜂之间的亲缘关系较近,而与小蜜蜂和黑小蜜蜂的亲缘关系较远。

酶解与包合技术制备水溶性蜂王浆

为研究酶解与包合技术制备水溶性蜂王浆的效果,本研究选用4种蛋白酶及β-环糊精,参考不同反应条件,比较酶解及包合产物的水溶性蛋白质提取率。结果显示:木瓜蛋白酶、无花果蛋白酶、中性蛋白酶、菠萝蛋白酶、β-环糊精4∶1;β-环糊精1∶1以及β-环糊精1∶4的水溶性蛋白质提取率分别为14.10%,13.97%,24.91%,7.20%,89.38%,92.06%,91.21%,酶解及包合产物在热、酸、碱环境中具有较强的稳定性;β-环糊精1∶1、1∶4的包合产物在72 h后的水溶性蛋白质提取率为68.79%,89.45%。采用β-环糊精包合技术制备水溶性蜂王浆明显优于酶解技术,同时包合技术具有条件简易、成本低廉等优势,可用于水溶性蜂王浆的生产。研究显示,制备水溶性蜂王浆的最佳条件为添加β-环糊精1∶4为包合原料,在p H 8.737℃下搅拌反应4 h。

贮存温度、时间对蜂王浆水溶性蛋白的影响

通过福林酚试剂法和SDS PAGE 2种方法 ,分析了不同温度和时间处理蜂王浆水溶性蛋白的变化。结果表明 ,蜂王浆水溶性蛋白由P1( 18 4ku)、P2 ( 2 3 1ku)、P3( 2 5 0ku)、P4( 34 5ku)、P5 ( 4 1 1ku)和P6 ( 4 5 0ku) 6种常量蛋白和其他微量蛋白组成 ,这些蛋白在 4～35℃及不同时间下都会降解 ,其中P6的降解最明显 ;相同贮存时间内 ,蜂王浆水溶性蛋白含量随贮存温度升高而降低 ;相同温度条件下 ,随贮存时间的延长其含量亦逐渐降低。

超声波处理对蜂王浆蛋白功能和结构的影响

探讨不同功率的超声波处理(20 kHz,200、400、600 W)对蜂王浆蛋白溶解性、乳化性、起泡性、水解度以及表面疏水性的影响。同时,通过对王浆蛋白二级结构、三级结构和微观结构的分析,探讨超声波处理改善蜂王浆蛋白功能特性的机制。结果表明,与未处理组相比较,蜂王浆蛋白经过400 W处理后,其溶解度增加10.90%,乳化活性增加67.18%,乳化稳定性增加15.87%,起泡性增加60.00%,起泡稳定性增加118.75%,水解度增加83.33%,表面疏水性增加18.65%。蛋白质的二、三级结构分析显示,该处理条件可以诱导蛋白质发生去折叠化。微观结构分析显示,蛋白质经400 W处理后,其表面较为粗糙,碎片化程度增加。这些结果表明,超声波处理所诱导的蛋白质构象的变化,是其功能特性得以改善的主要原因。