| 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. |
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