两蜂种蜂王浆蛋白质组分析

采用蛋白质组双向电泳技术和已鉴定蜂王浆蛋白质组的比对,对王浆高产蜜蜂（浆蜂）和中华蜜蜂（中蜂）的蜂王浆蛋白质组进行了差异比较。结果表明,浆蜂蜂王浆的总蛋白质点数（93个）显著高于中蜂蜂王浆总蛋白质点数（70个）,它们的等电点主要集中在5-8之间,分子量在50-80kDa之间,其中共有蛋白质点为30个;通过与已鉴定的浆蜂蜂王浆蛋白质组比较,这些共有蛋白质点推断为蜂王浆主蛋白1、2、3和葡萄糖氧化酶。同时两蜂种蜂王浆的蛋白质丰度也存在较大差异,共有点中有4个蛋白质的丰度中蜂显著高于浆蜂,7个浆蜂显著高于中蜂中。结果表明浆蜂和中蜂蜂王浆蛋白质种类及丰度均有较大差异。     

Novel royal jelly proteins identified by gel-based and gel-free proteomics

Royal jelly (RJ) plays an important role in caste determination of the honeybee; the genetically same female egg develops into either a queen or worker bee depending on the time and amount of RJ fed to the larvae. RJ also has numerous health-promoting properties for humans. Gel-based and gel-free proteomics approaches and high-performance liquid chromatography-chip quadruple time-of-flight tandem mass spectrometry were applied to comprehensively investigate the protein components of RJ. Overall, 37 and 22 nonredundant proteins were identified by one-dimensional gel electrophoresis and gel-free analysis, respectively, and 19 new proteins were found by these two proteomics approaches. Major royal jelly proteins (MRJPs) were identified as the principal protein components of RJ, and proteins related to carbohydrate metabolism such as glucose oxidase, α-glucosidase precursor, and glucose dehydrogenase were also successfully identified. Importantly, the 19 newly identified proteins were mainly classified into three functional categories: oxidation-reduction (ergic53 CG6822-PA isoform A isoform 1, Sec61 CG9539-PA, and ADP/ATP translocase), protein binding (regucalcin and translationally controlled tumor protein CG4800-PA isoform 1), and lipid transport (apolipophorin-III-like protein). These new findings not only significantly increase the RJ proteome coverage but also help to provide new knowledge of RJ for honeybee biology and potential use for human health promotion.     

蜂王浆三针夹取式拣虫机的设计与试验

蜂王浆拣虫是挖浆前必须完成的工序。由于蜜蜂幼虫个体较小、数目庞大,因此人工拣虫过程劳动强度非常大;然而中国养蜂人员老龄化日益严重,且目前蜂王浆拣虫机械化尚未成熟。该文针对中国养蜂现状提出一种以三针夹取的方式将王台中蜜蜂幼虫夹出的方法。以三针夹取的拣虫方式构建蜂王浆拣虫机整机方案。三根针在王台孔内壁同时做向心运动或分离运动,将蜜蜂幼虫夹住或松开。对传动机构中球面槽轮和凸轮的参数进行计算。通过有限元分析得出动力输入端脚踏板受力情况。计算得出球面槽轮圆柱销弧长7.5π,槽深20.68 mm;得出凸轮的理论廓线;有限元分析结果得出动力输入方式可靠。该机器在保留蜂王浆幼虫完整的前提下,一次性将整条王台(64孔)中蜜蜂幼虫全部拣出,可实现连续作业,拣虫效率为10条/min时为人工的10倍,克服了蜂场手工作业时间长和劳动强度大问题。对扩大中国蜂业养蜂规模有着极大的促进作用。     

Storage stability assessment of freeze-dried royal jelly by furosine determination

The effect of freeze-drying and the assessment of the storage stability of freeze-dried royal jelly (RJ) were investigated by the determination of furosine and blocked lysine. The level of furosine in the RJ samples collected from cells at different times (1, 2, and 3 days after grafting) showed that the Maillard reaction had already occurred in the hive as indicated by the increase in furosine: from 9.6 to 20.8 mg/100 g of protein. Freeze-dried RJ was more prone to the early stage of the Maillard reaction than fresh RJ, as confirmed by the significantly higher furosine values found after 12 months, both at 4 degrees C (253.4 versus 54.9 mg/100 g of protein) and at room temperature (884.3 versus 332.5 mg/100 g of protein). After 18 months at room temperature, the lyophilized samples reached a furosine level of 1440.4 mg/100 g of protein, which corresponded to the blocked lysine levels, amounting to 24% of total lysine.     

Proteomic analysis of royal jelly from three strains of western honeybees (Apis mellifera)

To compare the protein complement of royal jelly (RJ) from high RJ producing honeybees ( Apis mellifera L.), a strain of A. mellifera artificially selected for increased RJ production from Italian honeybees in China for more than two decades was compared to those of native Italian honeybees ( A. mellifera L.) and Carnica honeybees ( A. mellifera C.); the protein in RJ from these three strains of honeybees was partially identified by using a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS), and a protein engine identification tool applied to the honeybee genome. The results showed that 152, 157, and 137 proteins were detected in the three species of RJ; among which 57, 57, 51 high abundant proteins ere identified, respectively. Most identifited spots, 45, 45, 41, were assigned to major royal jelly proteins (MRJPs). Remarkable differences were found in the heterogeneity of the MRJPs, in particular, MRJP3. Also, 3-glucose oxidase, 1-peroxiredoxin (PRDX), and 1-glutathione S-transferase (GST) S1 were identified in three RJ samples. Furthermore, during the determination of the peptides mass fingerprinting (PMF) of each spot, for the first time, PRDX and GST S1 proteins have been identified in RJ. Thus, the results suggest that the protein complement of high RJ producing honeybees is not different compared to native Italian honeybees, while a difference remains between Carnica honeybees.     

Specific hydroxy fatty acids in royal jelly activate TRPA1

This is the first report of TRPA1 activation by fatty acids. Activation of TRPA1 and TRPV1 induces thermogenesis and energy expenditure enhancement. In this study, we searched for novel agonists of TRPA1 and TRPV1 from a nonpungent food, royal jelly (RJ). We measured the activation of human TRPA1 and TRPV1 by RJ extracts and found that the hexane extract contains TRPA1 agonists. The main functional compounds in the hexane extract were trans-10-hydroxy-2-decenoic acid (HDEA) and 10-hydroxydecanoic acid (HDAA). These are characteristic fatty acids of RJ. Their EC50 values were about 1,000 times larger than that of AITC, and their maximal responses were equal. They activated TRPA1 more strongly than TRPV1. Their EC50 values for TRPV1 were 2 times larger, and the maximal response was less than half of that for TRPA1. Next, we studied the potencies of other lipid components for both receptors. Most of them have higher affinity to TRPA1 than TRPV1. Among them, dicarboxylic acids showed equal efficacy for both receptors, but those are present in only small amounts in RJ. We concluded that the main function of RJ is TRPA1 activation by HDEA and HDAA, the major components of the RJ lipid fraction.     

Furosine: a suitable marker for assessing the freshness of royal jelly

Fifteen commercial samples of royal jelly, consisting of 10 imported samples, and 5 samples of known origin obtained freshly harvested from beekeepers, were analyzed for protein, lysine, and furosine content. In addition, a commercial sample of royal jelly, at the beginning of its commercial shelf life, was stored for 10 months both at 4 degrees C and at room temperature in order to assess the development of the Maillard reaction (furosine) and relative nutritional damage (blocked lysine). The commercial royal jelly products contained different amounts of furosine, ranging from 37.1 to 113.3 mg/100 g protein, evidence of different storage times and conditions. The average furosine content of the royal jelly samples of known origin and harvesting was significantly lower than that of the imported samples (41.7 versus 73.6 mg/100 g protein, respectively). With regard to shelf life, furosine content increased significantly from 72.0 mg/100 g protein to 500.8 mg/100 g protein after 10 months of storage at room temperature, while it increased to a much lower level (100.5 mg/100 g protein) when the royal jelly was stored at 4 degrees C. However, nutritional damage, expressed as blocked lysine (calculated indirectly from the furosine content), was minor or negligible, 11.9 and 2.3% of total lysine, in samples stored at room temperature and at 4 degrees C, respectively. Lysine was determined by an innovative procedure based on high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The results showed that furosine is a suitable index for assessing the quality and freshness of royal jelly.     

Application of solid/liquid extraction for the gravimetric determination of lipids in royal jelly

Gravimetric lipid determination is a major parameter for the characterization and the authentication of royal jelly quality. A solid/liquid extraction was compared to the reference method, which is based on liquid/liquid extraction. The amount of royal jelly and the time of the extraction were optimized in comparison to the reference method. Boiling/rinsing ratio and spread of royal jelly onto the extraction thimble were identified as critical parameters, resulting in good accuracy and precision for the alternative method. Comparison of reproducibility and repeatability of both methods associated with gas chromatographic analysis of the composition of the extracted lipids showed no differences between the two methods. As the intra-laboratory validation tests were comparable to the reference method, while offering rapidity and a decrease in amount of solvent used, it was concluded that the proposed method should be used with no modification of quality criteria and norms established for royal jelly characterization.     

Fast determination of adenosine 5'-triphosphate (ATP) and its catabolites in royal jelly using ultraperformance liquid chromatography

To obtain insight into the metabolic regulation of adenosine 5'-triphosphate (ATP) in royal jelly and to determine whether ATP and its catabolites can be used as objective parameters to evaluate the freshness and quality of royal jelly (RJ), a rapid ultraperformance liquid chromatography (UPLC) method has been developed for feasible separation and quantitation of ATP and its catabolites in RJ, namely, adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), inosine monophosphate (IMP), inosine (HxR), and hypoxanthine (Hx). The analytes in the sample were extracted using 5% precooled perchloric acid. Chromatographic separation was performed on a Waters Acquity UPLC system with a Waters BEH Shield RP18 column and gradient elution based on a mixture of two solvents: solvent A, 50 mM phosphate buffer (pH 6.5); and solvent B, acetonitrile. The recoveries were in the range of 86.0-102.3% with RSD of no more than 3.6%. The correlation coefficients of six analytes were high (r(2) ≥ 0.9988) and within the test ranges. The limits of detection and quantification for the investigated compounds were lower, at 0.36-0.68 and 1.22-2.30 mg/kg, respectively. The overall intra- and interday RSDs were no more than 1.8%. The developed method was successfully applied to the analysis of the analytes in samples. The results showed that ATP in RJ sequentially degrades to ADP, AMP, IMP, HxR, and Hx during storage.     

Characterization of royal jelly proteins in both Africanized and European honeybees (Apis mellifera) by two-dimensional gel electrophoresis

In this study, the proteins contained in royal jelly (RJ) produced by Africanized honeybees and European honeybees (Apis mellifera) haven been analyzed in detail and compared using two-dimensional gel electrophoresis, and the N-terminal amino acid sequence of each spot has been determined. Most spots were assigned to major royal jelly proteins (MRJPs). Remarkable differences were found in the heterogeneity of the MRJPs, in particular MRJP3, in terms of molecular weights and isoelectric points between the two species of RJ. Furthermore, during the determination of the N-terminal amino acid sequence of each spot, for the first time, MRJP4 protein has been identified, the existence of which had been only implied by cloning of its cDNA sequence. The presence of heterogeneous bands of glucose oxidase was also identified. Thus, the results suggest that two-dimensional gel electrophoresis provides a suitable method for the qualitative analysis of the proteins contained in RJ derived from different honeybee species.     

Immunochemical approach to detection of adulteration in honey: physiologically active royal jelly protein stimulating TNF-alpha release is a regular component of honey

The presence of royal jelly (RJ) proteins in honey collected from nectars of different plants, origin, and regions and in honeybee's pollen was detected by Western-blot analysis using polyclonal antibodies raised against water-soluble RJ-proteins. The most abundant RJ-protein in honeybee products corresponded to a 55 kDa protein. The N-terminal amino acid sequence of 55 kDa protein was N-I-L-R-G-E. This sequence is identical to the apalbumin-1, the most abundant protein of RJ. Apalbumin-1 is a regular component of honeybee products and thus is a suitable marker tool for proving adulteration of honey by means of immunochemical detection. Its presence in all tested samples of honeys and honeybee pollen was confirmed also by Western-blot analysis using polyclonal antibodies raised against recombinant apalbumin-1. It has been found that major RJ-proteins, apalbumin-1, and apalbumin-2, stimulate mouse macrophages to release TNF-alpha, which demonstrates that physiologically active proteins of honey could be used for its biological valuation.     

羊胎盘提取剩余物发酵制备活性肽工艺及产物生物活性

为实现羊胎盘提取剩余物的高值化利用,该文以超速冻冻藏羊胎盘提取剩余物为原料,利用枯草芽孢杆菌对其进行生物降解制备生物活性肽。通过对发酵过程监控研究了发酵过程中蛋白酶活、产物的平均肽链长度、产物的抗氧化和免疫活性的变化,实现了可控制发酵羊胎盘提取剩余物制备免疫活性肽。研究结果表明:随着发酵时间的延长,蛋白酶活、水解度逐渐升高;平均肽链长度逐渐变小;产物的抗氧化活性和免疫活性均呈现先升高后降低的趋势。在发酵时间35 h下产物具有较好的抗氧化活性和免疫活性,产物的免疫活性与抗氧化活性均随浓度的增大而升高,发酵产物对淋巴细胞的刺激指数最高可达23.37%,发酵产物对1,1-二苯基-2-三硝基苯肼(DPPH:1,1-Diphenyl-2-picrylhydrazyl radical2,2-Diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl)自由基清除率为0.667 mg/m L。超滤结果表明:不同分子量组分的发酵液生物活性有较大差异,其中3~10 k Da组分的免疫活性最高,≤3 k Da组分的抗氧化活性最高。各组分之间的生物活性与原样品相比不具有加和性但是具有明显的协同作用。研究结果为实现可控制发酵制备羊胎盘生物活性肽提供了参考。     

Cloning and expression of an acidic pectin methylesterase from jelly fig (Ficus awkeotsang)

Pectin methylesterase (PME) is the key enzyme responsible for the gelation of jelly curd in the water extract of jelly fig (Ficus awkeotasang) achenes. The jelly fig PME extracted from achenes was isoelectrofocused at pH 2.5 and subjected to N-terminal amino acid sequencing. A cDNA fragment encoding the mature protein of this acidic PME was obtained by PCR cloning using a poly(T) primer and a degenerate primer designed according to the N-terminal sequence of the purified PME. The complete cDNA sequence of its precursor protein was further obtained by PCR using the same strategy. The PME clone was overexpressed in Escherichia coli, and its expressed protein was immunologically recognized as strongly as the original antigen using antibodies against purified PME. Fractionation analysis revealed that the overexpressed PME was predominantly present in the pellet and thus presumably formed insoluble inclusion bodies in E. coli cells.     

Phosphate sorption by red mediterranean soils from Greece

Abstract

In nineteen surface horizons of red Mediterranean soils from various locations of Greece, phosphorus (P) sorption experiments were conducted and the sorption characteristics were studied in relation to soil properties. Phosphate sorption data were fitted both to the Langmuir and Freundlich equations. From these equations, the following P sorption parameters were determined from the Freundlich equation, X = ACⁿ, the parameters A (the phosphate sorbed at C = 1 mg P/L), n (the P sorption intensity), the P sorption index (PS = X/log C) and maximum P sorption (Xmfr). From the Langmuir equation, C/X = 1/KXm + C/Xm, the parameters K (showing the bonding energy), maximum P sorption (Xmla), the quantity of P adsorbed at a standard concentration of 0.2 mg P/L (P0.2), and P maximum buffering capacity (PMBC). The Freundlich parameter A was strongly correlated to the clay and sesquioxides ("free”; iron and aluminum oxides and amorphous iron oxides) content. Seventy‐four percent of the variance of this parameter was explained by clay and “free”; iron (Fe) content. The Freundlich parameter n was significantly correlated with pH and amorphous iron oxides content, while 52% of its variance was explained by amorphous Fe and dithionite extrac‐table aluminum (Al). The P sorption maxima calculated from the Freundlich equation were in general lower than those calculated by the Langmuir equation. Both these parameters were strongly correlated with clay and more slightly with sesquioxides content. About 50% of their variance was explained by clay content of the soils. The P sorption index was strongly correlated with the clay content and less strongly with dithionite‐extractable Fe and Al. The P‐buffering capacity calculated from the data of Langmuir equation was also strongly correlated with these two parameters. In addition, clay content and dithionite‐extractable Fe and Al were well correlated to the amounts of P required to obtain an equilibrium concentration of 0.2 mg P/L while 61% of the variation of this parameter was explained by the clay and the dithionite‐extractable Fe content. From these findings, it seems that for the red Mediterranean soils from Greece, P sorption is affected by clay content and iron and aluminum oxide contents.     

Structure and bioactivity of thiosulfinates resulting from suppression of lachrymatory factor synthase in onion

In normal onion (Allium cepa), trans-S-1-propenyl-L-cysteine sulfoxide is transformed via 1-propenesulfenic acid into propanethial S-oxide, a lachrymatory factor, through successive reactions catalyzed by alliinase and lachrymatory factor synthase (LFS). A recent report showed that suppression of the LFS activity caused a dramatic increase in thiosulfinates previously reported as "zwiebelane isomers". After purification by recycle high-performance liquid chromatography and subsequent analyses, we established the planar structure of the putative "zwiebelane isomers" as S-3,4-dimethyl-5-hydroxythiolane-2-yl 1-propenethiosulfinate, in which two of the three molecules of 1-propenesulfenic acid involved in the formation gave the thiolane backbone, and the third molecule gave the thiosulfinate structure. Of at least three stereoisomers observed, one in the (2'R,3'R,4'R,5'R)-configuration was collected as an isolated fraction, and the other isomers were collected as a combined fraction because spontaneous tautomerization prevented further purification. Both fractions showed inhibitory activities against cyclooxygenase-1 and α-glucosidase in vitro.     

Nitrogen leaching from soils in the Kopais area of Greece

Abstract. The contribution of agriculture to the nitrogen pollution of surface and ground waters of calcareous lake soils in the Kopais area (190 km²) of Greece was studied over three cropping seasons. Sample fields were chosen from seven representative soil units under different crop rotations. The distribution of mineral N (NO₃-N + NH₄-N) throughout the soil profile and the concentration of NO₃-N in the ground water and drainage water were measured and allocated to 6-month winter or spring periods. For all fields N was leached to the deeper soil layers and to the saturated zones by both excess winter rainfall and spring irrigation of different crops The amount of nitrogen leached depended on the amount of nitrogen fertilizer applied; the depth of leaching varied with the physical properties of the soils. Losses in individual fields accounted for the equivalent of 17.6–80.8% of the nitrogen applied to maize and 70.5–94.1% of that applied to wheat. For the whole region estimated minimum N losses ranged from 175, 912 to 783, 564 kg for the 6-month period. Nitrate concentrations in the ground and surface waters were often more than the EC target level of 25 mg/1.     

The release of nonexchangeable potassium from certain soils of northern Greece

The release of nonexchangeable potassium from the surface layers of nine red soils, sampled in different districts of northern Greece, was studied by treatment of the samples with H⁺-resin at various soil: resin ratios and temperatures and in a pot-experiment with rye grass. It was thus found that, the phenomenon of K-release from the soil samples during their treatment with the H⁺-resin, follows typically the kinetics of the first order opposing reactions under all conditions tested. A close relationship was shown to exist between nonexchangeable potassium released by cropping (defined as K-supplying capacity of the soil) and that released by treatment with the resin. The K-supplying capacity of the soils studied seems to be more closely related to the total amount of lattice-K released by the resin than to the rate of the release. The latter is affected considerably not only by temperature changes but also by changes in the soil:resin ratio. A value for the activation energy of the release reaction was calculated which might be considered as lending additional support to the view, already widely accepted, that K-release in the soil is a diffusion-dependent phenomenon.     

Physicochemical properties and bioactivity of fungal chitin and chitosan

Chitinous material was extracted from mycelia of Aspergillus niger and Mucor rouxii grown in yeast peptone dextrose broth for 15 and 21 days, respectively. The extracted material was characterized for purity, degree of acetylation, and crystallinity and tested for antibacterial and eliciting properties. The maximum glucosamine level determined in the mycelium of A. niger was 11.10% dw and in the mycelium of M. rouxii was 20.13% dw. On the basis of the stepwise extraction of freeze-dried mycelia, it appeared that M. rouxii mycelia contained both chitin and chitosan, whereas A. niger contained only chitin. The yields of crude chitin from A. niger and M. rouxii were 24.01 and 13.25%, respectively, and the yield of chitosan from M. rouxii was 12.49%. Significant amounts (7.42-39.81%) of glucan were associated with chitinous compounds from both species and could not be eliminated by the extraction method used. The degrees of acetylation were determined to be 76.53 and 50.07% for chitin from A. niger and M. rouxii, respectively, and 19.5% for M. rouxii chitosan. The crystallinity of fungal chitin and chitosan was estimated to be less intense than in corresponding materials from shrimp shells. The extracted chitin and chitosan in a concentration of 0.1% reduced Salmonella Typhimurium DT104 2576 counts by 0.5-1.5 logs during a 4 day incubation in tryptic soy broth at 25 degrees C. Furthermore, all tested chitinous materials from fungal sources significantly reduced lesions caused by Botrytis cinerea and Penicillium expansum in harvested apples.     

A nutritional disorder in pistachio from near Athens,Greece

Abstract

A problem pistachio orchard near Athens, Greece had many trees with severely scorched leaves. Half or more of each leaflet v/as brown and dry but tips and edges were most affected. Affected leaves were also of lighter green color than normal leaves and slightly resembled Fe deficiency. Leaf analysis indicated low K, low Fe, and high Mg. Irrigation water contained moderate levels of Mg and Na chlorides and chloride toxicity was possibly another factor. Improvement was observed when Fe chelate was applied to the orchard but Mg‐induced K deficiency was possibly a major factor. Response to K applied to soil was not observed after four months, however.     

印楝素对植物病原菌抑菌作用初步研究

印楝(Azadirachta indica A.Juss)系楝科楝属乔木,原产于印度与缅甸,广泛种植于热带、亚热带地区,在亚洲南部、非洲和南美洲的70多个国家已有分布[J].印楝是一种多用途树种,并含有以印楝素为主的多种能防虫、杀虫、杀菌的活性物质[2],在我国云南、四川、海南、广西、广东等地均有种植,其中云南省的种植面积最大.