| Maillard reaction is one of the most important reactions of food in processing and storage, which had an important influence of the flavor, color and nutrition values. Maillard reacton is composed of glucose-amino acids system and fructose-amino acids system. Amadori and Heyns compounds are the key intermediates of Maillard reaction derived from glucose-amino acids system and fructose-amino acids system respectively. We can monitor the extent of Maillard reaction in food by detecting the content of Amadori and Heyns compounds, which can prove theoretical basic for monitoring and controlling the extent of Maillard reaction in food scientific analysis. Many researches focused on the analysis of Amadori compounds, but few of studies on Heyns compounds were carried out, especially about the quality and quantity of them in complex matrix. It is because Amadori and Heyns compounds are isomerides and the structures of Amadori and Heyns compounds are very similar. They both have high molecular polarity. So, the aim of this research was to develop a method to distinguish and analyze the Amadori and Heyns compounds simultaneously by high performance liquid chromatography tandem mass spectrometry. We analyzed and quantified the intermediates of Maillard reaction in processing food and tobacco by using the characteristic fraction ions, which provide scientific basis the changes of Maillard reaction in processing food. The main results are as follows:1. The main 18 Heyns compounds were prepared by the reaction between the 18 common Amino acids. After purification by ion-exchange resin, the mass spectrometry (ESI-MS/MS) was applied to analyze these compounds to analyze the fragment pathways. Fragmentor, CE voltage, Chromatographic and MS Conditions were optimized. The systemized study of mass spectrometry fragment modes of the 18 Heyns compounds under electrospray ionization condition was carried out in the research. According to the experiment data, the fragment pathways of Heyns compounds are summarized, which clearly illuminates the mass spectral cleavage mechanism of the compounds. Furthermore, their main fragment pathways of the compounds are summarized, which are useful to know about the character of the compounds and identify them.2. The fragment patterns of the two compounds were compared, and the mechanism of characteristic fragment ion production of the two compounds was deduced. The characteristic fragment ion pairs of Amadori and Heyns are at [AA-H+CH2]+/[M+H]+ and [M+H-2H2O-CH3OH-CO]+/[M+H]+. We established an efficient method to distinguish and analyze Amadori compounds and Heyns compounds simultaneously according to the characteristic fragment ion production of the two compounds, which can provide analytical basic for determining the Amadori and Heyns compounds. The method was established to distinguish and analyze Amadori compounds and Heyns compounds simultaneously in dried fruits by using the characteristic fragment ion pairs of them. The preparation of samples was optimized. Compared to the past literatures, a column filled with ion-exchange resin was used for purifying samples to remove sugars and organic acids, which can decrease the effect of matrix effects and improve the sensitivity of the method. The method displayed good linearity with correlation coefficient (R2) no more than 0.9989. The limit of detection was no more man 0.267 ng/mL. The relative standard deviation (RSD) was less than 4.31% and the spiked recoveries ranged from 90.56% to 110.91%. The method is accurate and reliable to determine Amadori and Heyns compounds in dried fruits simultaneously3. The Maillard reaction between proline and fructose/glucose had an important influence on the color and flavor of tobacco, owing to the abundant content of proline. In this research, an analytical method of Amadori and Heyns compounds was used to measure the content of Fru-Pro (Amadori) and Glu-Pro (Heyns) compounds in tobacco. Since tobacco contained a lot of sugar, polyol and pigment, a cleansing method was made a modification on the basis of the purification of dried fruit samples. The ion exchange resins and C18 filler material were combined in order to remove sugar, polyol, pigments and other impurities in tobacco extract. The effect of matrix effect on the analysis results was reduced by this purification method, and it also can improve the accuracy and sensitivity of the method. HPLC-MS/MS was used to detect the purified tobacco extract, Fru-Pro and Glu-Pro qualitative ion pairs were both 116/278, and their quantitative ion pairs were 128/278 and 182/278 respectively. The results showed that the method displayed good linearity with correlation coefficient at 0.999, the spiked recoveries ranged from 93.19% to 101.46%, the relative standard deviation was between 3.53%-8.54% and the limit of detection was at 0.12 ng/mL. The contents of Fru-Pro and Glu-Pro measured among 10 kinds of flue-cured tobacco samples were 6.25-11.33mg/g and 1.15-1.96mg/g respectively. This method was accurate and reliable to determine fructose-proline and glucose-proline compounds in flue-cured tobacco simultaneously, which was helpful to evaluate and control the quality of tobacco.4. The analysis on the contents of sugar and key intermediates of Maillard reaction in raw garlic and black garlic were studied. The results showed that the contents of fructan in the black garlics were decreased in comparison with fresh raw garlics ranged from 84.63% to 99.24%. The water-soluble sugar (the sum of glucose, fructose and sucrose) content was drastically increased by values ranging from 187.79% to 790.96% compared with fresh raw garlic. The sucrose content in black garlic was almost equivalent to fresh garlic. By using the method established above, the contents of Amadori and Heyns compounds in raw garlic and black garlic were detected. The results showed that Heyns and Amadori compounds were drastically increased in black garlics, but the compounds were very few in fresh raw garlics. It was a significant proof that the Maillard reaction was occurred in processing black garlic and abundant Maillard reaction intermediate were generated, which had important influence of the color and flavor of products.5. To confirm the above reaction mechanism, black garlic were prepared by thermal processing. The fresh raw garlics were prepared at 55 ℃ and 80% H for 90 days, and the samples were taken for each 5 to 10 days. By determination the contents of fructan, fructose, Amadori and Heyns compounds, and the changes of color and pH of black garlic, we found the change rules of Maillard reaction in black garlic. The changes of the corresponding Marlliard reaction products during the thermal processing was showed that, the content of fructan was decreased by 84.79% and the content of fructose increased by 508.11% in black garlic. The content of Maillard reaction intermediates (Amadori and Heyns compounds) was increased at first and then declined; The absorbance under 420 nm was increased with the deepened color of black garlic, which represented that the Maillard reaction products was gradually increased in the processing. The pH value of black garlic was reduced from 6.13 to 4.00. The changes about these substances showed that the change mechanisms of colar and flavor (color turned black and taste sweet and sour) generated from Maillard reaction, which occurred between the fructose and glucose derived from the degradation of fructan and amino acids in the thermal processing of black garlic. |
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