| Vaccinium bracteatum Thunb.?VBT?is a wildly distributed plant resource with a long medicinal history.Vaccinium bracteatum Thunb.leaves?VBTL?are rich in active phytochemicals which show many nutritional and pharmacological functions.The dyed rice produced by VBTL juice?named as‘Wu mi'in China?is widely consumed with bright indigo and the fragrance of the fresh leaves as a traditional healthy cereal food in the Jiangsu,Zhejiang,Fujian,and other provences in China.The natural plant pigment in VBTL has showed the promising prospects which can be used for foods,cosmetics,textiles and other fields.However,the problems of VBTL pigments,such as the seasonal difference,poor stability and backward production technology,have limited the further development of the VBT industry.The key problems of the development VBTL pigment were focusing on the lack of unified conclusion of the compositions,the lack of research on the processing technology,and the unclear reason of seasonal difference,etc.In this thesis,we are focusing on the VBTL dark blue pigments based on the the complex phytochemicals as the pigment precursor.The co-factors of pigment formation were discussed for the first time,and the mechanism of pigment formation was preliminarily proposed.Then,metabonomics at different growth stages of VBTL was used to investigate the dynamic changes of pigment precursors to explain the seasonal differences of pigment.Furthermore,based on the fundamental substrates,the reaction process of pigment was simulated and the application potential as a new pigment source was evaluated.Finally,the digestion behavior and transport property of pigment in vitro was studied in order to verify the potential beneficial effect of pigment.The main results of this thesis are shown as follows:Firstly,the dynamic changes of‘Wu mi'prepared from the whole year VBTL samples were analyzed.The principal component analysis?PCA?and partial least squares discrimination analysis?PLS-DA?were used to conduct multivariate statistical analysis on UPLC-QTOF-MS datasets of spring VBTL and dyeing solution samples in order to screen out the differential secondary metabolites between three groups of spring VBTL and three groups of dyeing solution samples.The results showed that the blue color on the surface of‘Wu mi'gradually became lighter with the ageing leaves,and the spring leaves owned the best dyeing effect.The multivariate statistical analysis revealed that the differential metabolites screened by PLS-DA model were identified as iridoid compounds,which were p-coumaroyl-monotropein,p-coumaroyl-monotropein dimer,p-coumaroyl-hydroxy-dihydromonotropein,and p-coumaroyl-dihydromonotropein repectively.Meanwhile,the precursors,co-factors and the reaction process of pigment were speculated.The main secondary metabolites in spring VBTL were detected by UPLC-QTOF-MS.By comparing the changes of secondary metabolites in dyeing solution before and after dyeing,the pigment precursors were speculated.Through the analysis of co-factors of pigment in VBTL and the detection of intermediate products in the reaction process,the mechanism of pigment formation was confirmed.The results showed that the pigment precursors were iridoid glycosides.The acidic conditions and?-glucosidase are key co-factors of pigment formation,and amino is an essential substrate for pigment formation.The reaction mechanism of pigment formation in VBTL was proposed for the first time by systematic verification of pigment formation conditions.Then,the dynamic changes on the substrates of pigment in VBTL at different growth stages were investigated to analyze the reason of seasonal difference of VBTL pigment.Based on PLS-DA model analysis of untargeted UPLC-QTOF-MS datasets,the differential secondary metabolites in VBTL at different growth stages were investigated.Through PLS-DA model of free amino acid datastes,the differencial free amino acids between VBTL samples were also screened.The O2PLS model was used to analyze the correlation between secondary metabolites and free amino acids.The results showed that the differential secondary metabolites were mainly three types of compounds:the first type was iridoid glycosides and their derivatives,the second type was chlorogenic acid and its dimer,and the third type was flavonoid glycosides.With the prolonging of growth time,most of the differential secondary metabolites showed a trend of increasing first and then decreasing.Nine free amino acids which contributed greatly to the differences between VBTLsamples were screened.The O2PLS analysis showed that both secondary metabolites and free amino acids were closely related to the seasonality of VBTL,and iridoid glycosides and most amino acids all showed high correlation with VBTL samples from April to July.Next,based on the results of key pigment precursors,exogenous glycine was selected to react with VBTL juice to prepare dark blue pigment.Fourier transform infrared spectroscopy and the full-band scanning spectrum were used to characterize the specific absorption peak of pigment.The color retention of the pigment solution was evaluated under irradiation and dark conditions.In the thermal accelerated storage experiment,the thermal stability of pigment was also evaluated.FTIR showed that C-N bond was successfully introduced into the end-pigment during the pigment reaction.The full-band scanning spectrum shows that the pigment sample has a characteristic absorption peak at 585 nm in the visible region.Under normal sunlight and UV irradiation conditions,the color of the pigment solution gradually decreases with the prolonging of storage time,and the color decay in pigment solution was significantly accelerated under UV irradiation conditions.Under the dark condition,the pigment has better color retention rate in weak acid system?pH=4.0,5.0 and 6.0?than in high pH system?pH=7.0,8.0 and 9.0?.In the thermal accelerated storage experiment,the pigment showed good thermal stability in weak acid system,while in alkaline and strong acid systems,the blue hue was stable under moderate heating conditions.Furthermore,the simulated digestion behavior of pigment in vitro was investigated.The stability of pigment in the simulated gastrointestinal tract?GIT?in vitro was studied.The inhibition of pigment on pancreas?-amylase activity was studied via the fluorescence spectrum analysis and circular dichroism analysis.The results showed that the color retention rate of pigment decreased gradually with the digestion time in GIT.The retention rate of pigment was more than 65%in the simulated intestinal digestion stage.The results of inhibition of porcine pancreas?-amylase showed that both unheated and heated pigment all had good inhibitory effects on?-amylase with IC50 values of 2.915 and 3.692 mg/m L,respectively.The inhibition kinetics analysis showed that pigment was a non-competitive inhibitor.The fluorescence spectrum analysis showed that the structure of?-amylase unfolded after binding with pigment.Circular dichroism analysis showed that the pigment inhibited the?-amylase activity by changing the secondary structure of?-amylase.The relative content of?-sheet in?-amylase increased after interacting with pigment,while the relative content of?-helix and?-turn decreased.Finally,Caco-2 cell was used to test the toxicity of VBTL pigment and Caco-2 monolayer was established to study the transport properties of pigment to evaluate the absorption in intestinal tract.The results showed that there was no significant difference between the concentration of 0.25?1.5 mg/m L and control.The absorption of pigment in Caco-2 cell monolayer transport model was found as the concentration-dependent,and the absorption type was a passive absorption process dominated by diffusion.Orthogonal Partial Least Squares Discriminant?OPLS-DA?was used to conduct multivariate statistical analysis on intracellular metabolites of Caco-2 cells affected by pigment,and 28 different metabolites were screened out.Among them,13 metabolites were up-regulated and 15 metabolites were down-regulated between experimental and control groups.The pathway of arginine and proline metabolism was significantly affected. |
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