【作者】 吴黎明；

【导师】 胡福良； 孙素琴；

【作者基本信息】 浙江大学 ， 特种经济动物饲养， 2008， 博士

【摘要】 长期的科学研究和实践经验表明,蜂王浆的品质和保健功效易受其贮存条件的影响。若贮存温度过高或时间过长,蜂王浆的物理性状、化学组成会发生变化,保健功效降低甚至丧失。也就是说,蜂王浆的新鲜度与其品质密切相关,并具有一定的正相关性。但迄今为止,尚没有一个公认的蜂王浆新鲜度的控制指标,也没有一种适合的评价方法。缺少新鲜度指标及相应的评价方法是现行蜂王浆国家质量标准的一大缺陷,也使质量监管部门及加工、贸易企业无法在蜂王浆生产和流通过程中进行有效地监管和控制,难以对蜂王浆质量做出准确的评价。因此,探求并确立能如实反映蜂王浆新鲜度的指标及评价方法是蜂王浆研究的一项重要课题,也是完善现行蜂王浆质量标准,有效监控蜂王浆质量,进一步规范蜂王浆市场的关键所在。针对上述问题,本文利用液相色谱技术对不同贮存条件下的蜂王浆中6种糠醛和26种氨基酸进行了定量分析和比较,同时利用红外光谱技术对不同贮存条件下的蜂王浆进行了相关分析和蛋白质的二级结构鉴定,初步建立了蜂王浆新鲜度监控的方法。研究结果如下：1.选定有可能反映蜂王浆新鲜度的糠醛类物质为研究对象,建立了同时测定蜂王浆中5-羟甲基-2-糠醛(5-HMF)、5-甲基糠醛(5-MF)、2-糠醛(F)、2-糠酸(2-OIC)、3-糠酸(3-OIC)、5-羟甲基-2-糠酸(5-HMFacid)6种糠醛类物质含量的高效液相色谱分析方法。蜂王浆样品用去离子水提取,离心沉淀后,取上清液加入亚铁氰化钾溶液和乙酸锌溶液沉降蛋白,0.45μm滤膜过滤,用高效液相色谱仪在不同波长下测定。在测试范围内,6种糠醛类物质的浓度与相应的峰面积呈线性关系,相关系数(R2)不小于0.9993。方法定量和定性检出限分别在0.07-0.26μg/mL和0.02-0.08μg/mL之间,加标回收率为86.2%-99.7%,相对标准偏差小于2.40%。方法操作简单,灵敏度高,是一种适合蜂王浆中糠醛类化合物测定的方法。利用该方法定量测定了在不同温度下(-18℃,4℃,16℃和25℃)经过不同时间(1,3,6,9和12个月)贮存的蜂王浆中6种糠醛类物质的含量。结果表明,在新采收的蜂王浆中没有检出这6种化合物；而在经过不同时间贮存后,4个温度梯度的样品中均检出了5-HMF,其含量随着贮存时间增加和温度升高而增加；在12个月的试验期内,仅在25℃下贮存的蜂王浆中检出了F。根据5-HMF和F含量与贮存时间和温度的相关性,结合蜂王浆贮存的实践经验,本文提出以5-HMF和F作为评价蜂王浆新鲜度的指标,并初步确定5-HMF含量小于150μg/kg和F不得检出作为评判蜂王浆是否新鲜的标准。2.选定可能能代表蜂王浆新鲜度的26种氨基酸,探索了它们的提取过程以及利用超高效液相色谱(UPLC)分析的条件,建立了一种分析蜂王浆中26种氨基酸的UPLC分析方法。该方法采用6-氨基喹啉基-N-羟基琥珀酰亚氨基甲酸酯(AQC)柱前衍生,通过梯度洗脱,使用ACCQ·TagC18氨基酸分析柱在8 min以内同时分离并定量测定了蜂王浆中的26种氨基酸含量。在测试范围内,26种氨基酸的浓度与相应的峰面积呈线性关系,相关系数(R2)不小于0.9978。方法定量(LOQ)和定性检出限(LOD)分别在42.7-235.1 ng/mL和12.9-69.3 ng／mL之间,加标回收率为90.1%-100.9%,相对标准偏差小于2.8%。方法操作简单,耗时短,灵敏度高,是一种适合蜂王浆的氨基酸测定方法。利用该方法定量测定了在不同温度下(-18℃,4℃和25℃)经过不同时间(1,3,6和10个月)贮存的蜂王浆中游离氨基酸和总氨基酸含量。结果表明,新鲜蜂王浆的游离氨基酸和总氨基酸总量分别为9.21mg/g和111.27mmg/g；其中最主要的游离氨基酸为Pro、Gln、Lys和Glu,而含量最高的总氨基酸分别为Asp、Glu、Lys和Leu。尽管多数游离氨基酸和总氨基酸含量在蜂王浆的贮存过程中并没有发生明显的变化或变化无规律,但总Met和游离Gin在贮存过程中含量持续下降而且差异显著。因此,本文提出总Met和游离Gln有可能作为评价蜂王浆品质和新鲜度的指标。3.测定了在不同温度下经过不同时间贮存后蜂王浆的Fourier变换红外光谱,以新采收蜂王浆的红外谱图为参考标准谱,利用光谱比对软件进行了一系列的相关分析,同时比较了红外谱图中1647、1541、1409、1247和1054 cm-15个峰位的相对峰强。结果表明：不同贮存条件下的蜂王浆红外谱图之间存在着明显差异；红外光谱图中酰胺Ⅰ带(1700-1600cm-1)波段的相关系数随着蜂王浆贮存时间延长和温度升高而降低；相对峰强度I1647/I1541、I1647/I1409、I1647/I1247和I1647/I1054也随着蜂王浆贮存时间延长和温度升高而降低,且与贮存时间存在着良好的线性关系,变化幅度为28℃>16℃>4℃>-18℃。因此,初步选定红外光谱图中酰胺Ⅰ带特征波段的相关系数和4个相对峰强度(I1647/I1541、I1647/I1409、I1647／I1247和I1647/I1054)作为蜂王浆新鲜度的评价指标,相关系数的阈值设定为0.9100,4个相对峰强度的阈值分别设定为1.744、2.430、3.345和1.412。只要有1个或多个指标低于相应的阈值,可初步判定此蜂王浆是不新鲜的。该方法利用FTIR光谱法并结合计算机辅助解析技术能从宏观上和整体上快速地评价蜂王浆的新鲜度。4.测定了在不同温度下经过不同时间贮存后蜂王浆Fourier变换红外光谱图,应用去卷积和曲线拟合方法对蜂王浆的酰胺Ⅰ带进行了蛋白质的二级结构分析,半定量地分析了在不同储存条件下蛋白质二级结构的相对含量的变化规律。结果表明：经过不同条件贮存的蜂王浆蛋白质二级结构的相对含量存在显著差异。随着贮存时间增加和温度升高,蛋白质二级结构的a-螺旋相对含量显著减少,而p-折叠相对含量显著增加。其变化幅度为28℃>16℃>4℃>-18℃。因此,FTIR光谱法结合去卷积和曲线拟合技术可以对蜂王浆中蛋白质的二级结构进行宏观评价和进行半定量分析,也可以作为蜂王浆新鲜度评价的参考依据。综上所述,本文利用液相色谱法分析测定了不同条件下贮存的蜂王浆6种糠醛类物质和26种氨基酸的含量,选定了5-HMF、F、总Met和游离Gln作为蜂王浆新鲜度的评价指标,并设定了相应的阈值；同时利用红外光谱法并结合相关分析和曲线拟合技术分析了在不同贮存条件下蜂王浆红外谱图的相关性和蛋白质二级结构相对含量的变化规律。本文利用液相色谱和红外光谱技术,从微观到宏观,从定性、半定量到定量建立了多种评判蜂王浆新鲜度的简单易行方法,为进一步进行蜂王浆新鲜度和品质相关研究打下了基础,也为完善现行蜂王浆质量标准,如实评价和有效监控蜂王浆质量,进一步规范蜂王浆市场提供了理论依据。更多还原

【Abstract】 Long-term research and practical experiences has shown that the quality of royal jelly (RJ) is related to its storage conditions. Physical properties and chemical compositions of RJ will alter with conditions of high temperature or long time, and its health function will reduce or even loss. Namely, the quality of RJ is positive correlated with its freshness significantly.But so far, there is no accepted indicator or suitable method for evaluating freshness of RJ. It results in a limitation of the existing RJ national quality standards and also brings problems to monitor and control the production and circulation processes of RJ by quality control departments and processors effectively. Therefore, to explore and establish indicators and evaluation methods that can reflect RJ freshness and quality accurately is an important topic of RJ research, and also a key to improve the existing quality standards, monitor quality effectively and further regulate the RJ market.To address the above issues, a study was launched on freshness indicators and assessment methods of RJ. Results were as follows:1. A reversed-phase high performance liquid chromatographic method was described for determining 5-hydroxymethyl-2-furfural (5-HMF),5-methyl-2-furaldehyde (5-MF),2-furaldehyde (F), furan-2-carboxylic acid (2-OIC), furan-3-carboxylic acid (3-OIC) and 5-Hydroxymethyl-2-furancarboxylic acid (5-HMFacid) in RJ. Samples were extracted with purity water and proteins in them were removed by zinc acetate and potassium ferrocyanide solutions. Before injection, the solutions were passed through 0.45μm filters for determination by a liquid chromatography UV detection at different wavelengths. Within the test ranges,6 furfural compounds concentrations and their peak areas showed good linear correlations (R2>0.9993). The limits of quantitation and detection were 0.07-0.26μg/mL and 0.02-0.08μg/mL, respectively. The recoveries ranged from 86.2% to 99.7% and the overall relative standards were lower than 2.40%. The method was simple, sensitive and suitable for analyzing those furfural compounds in RJ. The method was applied to quantitatively determine 6 furfural compounds in RJ samples stored at different temperatures (-18℃,4℃,16℃and 25℃) for different time intervals (1,3,6,9 and 12 months). Results showed that no furfural compounds was detected in new collected RJ samples; 5-HMF was detected in most samples that stored for different periods, and its contents increased with the increase of storage time and temperature; F was only detected in the samples stored at 25℃in the 12-month trial period. On the basis of the correlations between the content of 5-HMF (or F) and storage time (or temperature), combined with storage experiences,5-HMF and F were selected as assessment indexes of RJ freshness, and their threshold values were set as 150μg/kg and not detected respectively.2. A rapid ultra-performance liquid chromatography (UPLC) method was developed for feasible separation and quantification of 26 amino acids in RJ. The analysis was performed on Acquity UPLC system with Acquity UPLC AccQ·Tag Ultra Column in 8 min. The correlation coefficient (R2>0.9978) values indicated good correlations between the investigated compounds concentrations and their peak areas within the test ranges. The limits of quantitation and detection of 26 amino acids were 42.7-235.1 ng/mL and 12.9-69.3 ng/mL respectively. The recoveries ranged from 90.1% to 100.9% and the overall relative standard deviations for intra-and inter-day were lower than 2.8%. The results showed that UPLC was a powerful tool for analysis of amino acids in RJ.The method was also applied to quantitatively determine free amino acid (FAA) and total amino acid (TAA) profiles in RJ samples stored at different temperatures (-18℃,4℃and 25℃) for different time intervals (1,3,6 and 10 months). Results showed that the average contents of FAA and TAA in fresh RJ were 9.21 mg/g and 111.27 mg/g, respectively; the major FAAs were Pro, Gin, Lys, Glu, and the most abundant TAAs were Asp, Glu, Lys and Leu. Although the concentrations of most FAAs and TAAs showed no significant difference during storage, contents of total Met and free Gln decreased constantly and obviously. So these might be a parameter to predict the quality of RJ.3. Fourier transformation infrared spectroscopy (FTIR) of RJ stored at different temperatures and after different storage periods were measured, a series of correlation analysis among the spectra was carried out by using the spectra of new-harvested RJ as a standard. Results showed that the correlation coefficient of amide band I and the relative intensity ratios of I1647/I1541, I1647/I1409, I1647/I1247, I1647/I1054 of RJ samples’spectra decreased with extension of storage time and temperature, and presented good linear correlations with the storage time, while the order of their change extent was 28℃>16℃>4℃>-18℃. According to the spectra change laws and practical experiences of RJ storage, the correlation coefficient of amide band I and four relative intensity ratios I1647/I1541, I1647/I1409, I1647/I1247 and I1647/I1054 were selected as assessment indexes of RJ freshness. The threshold value of correlation coefficient was set as 0.9100, and the threshold values of the four relative intensity ratios were set as 1.744,2.430,3.345 and 1.412 respectively. Once one or more indexes were lower than the corresponding threshold values, the RJ sample would be considered as a stale one. So, FT-IR spectroscopy combined with several data-processing methods would be an effective method for overall assessing the freshness of RJ.4. An effective method for analyzing the secondary structure of proteins in RJ using FT-IR spectroscopy combined with secondary derivative, deconvolution, and curve-fitting was established. FTIR spectroscopy of RJ samples were measured at different temperatures and storage periods, while compositions of the secondary structure of proteins were determined by curve-fitting analysis of the amide I bands in the FTIR spectra. Results showed that the compositions of the secondary structure of proteins appeared extremely different, and the rates of a-helix decreased and P-sheet increased dramatically with the increase of storage temperature and periods, the order of their change extent was 28℃>16℃>4℃>-18℃. These results have met the theory that RJ should be kept under lower temperature. So it is a new suitable way for evaluating the quality and freshness of RJ.In this paper, liquid chromatography methods were applied to quantitatively determine 6 furfural compounds and 26 amino acids in RJ samples stored under different conditions respectively.5-HMF, F, total Met and free Gln were selected as RJ freshness assessment indexes, and their corresponding threshold values were also set up. The correlation coefficients of amide band I among spectra and the secondary structure of proteins in RJ stored under different conditions were also analyzed using FT-IR spectroscopy combined with several data-processing methods, such as correlation analysis and curve-fitting.To sum up, several indexes as well as their relevant simple evaluation methods were established for evaluating RJ freshness and quality in this paper. The results have provided a theoretical basis for improving existing RJ quality standards, monitoring RJ’s quality effectively, and further regulating the RJ market.更多还原