Study On The Effect Of Enzymatic Browning On The Quality Of Chrysanthemum

1 ObjectiveThe enzymatic Browning is the main reason for effecting the quality of medicinal flowers.Chrysanthemum is rich in phenolic compounds,and if flowers can not be dried in time or in an improper method,enzymatic browning is easy to occur,resulting in a decrease in the content of phenolic compounds and their appearance and quality are also affected.How to improve the processing quality of chrysanthemum?One of the key factors is how to control the enzymatic browning in time to minimize the loss of phenolic acids in the process of processing and prevent the change of chrysanthemum appearance.So it is necessary to strengthen the basic research of the chrysanthemum enzymatic Browning,which can standardize the processing technology and improve the appearance and quality of chrysanthemum on the basis of realizing the enzymatic Browning mechanism,and it can also provides some ideas for the enzymatic Browning control of medicinal materials which is rich in phenolic compounds.2 MethodsThe enzymatic characteristics of the PPO and POD enzymes which were the key enzyme of the chrysanthemum enzymatic browning in the drying in shade were studied in this paper.The effect of enzymatic Browning on the main chemical constituents of chrysanthemum was also studied.The reaction of PPO enzyme or POD enzyme and substrate chlorogenic acid was simulated to identify the reaction products and reveal the mechanism of the enzymatic browning.Finally,the effect of enzymatic Browning on the quality of chrysanthemum was discussed by comparing the antioxidant activities before and after the browning reaction of chlorogenic acid.2.1 The dynamic changes of the activities of chrysanthemum PPO and POD enzymes in the drying in shade.The fresh chrysanthemum petals was picked and observed the appearance changes in the drying in shade.Then,the moisture of chrysanthemums and the dynamic changes activitiies of the chrysanthemum PPO and POD enzyme were monitored daily to systematically evaluate the role of PPO and POD enzymes in the enzymatic Browning of chrysanthemum.2.2 Changes in the chemical composition of chrysanthemum in the process of drying in shade.To investigate the effects of enzymatic Browning and drought stress on the chemical composition and quality of chrysanthemum,HPLC was used to detect changes in the chemical composition of chrysanthemum and monitor the chemical composition changes during the 24h water conservation and the remove water conservation experiment.2.3 Research on the enzymatic characteristics of PPO and POD enzymes.Ultraviolet spectrophotometer was used to study the effects of different reaction substrates and their concentrations,PH value of buffer,reaction temperatures and heat stability on the activities of PPO and POD enzymes.The enzymatic characteristics of enzymatic browning key enzymes were masterd to provide a basis for the study of enzymatic reaction mechanism and how to effectively inhibit the enzymatic Browning.2.4 Study on the reaction mechanism of PPO and POD enzymes in chrysant hemum.Chlorogenic acid which is one of the main active ingredients was used as the substrate of the enzymatic browning reactions.First,PPO and POD crude enzymes were extracted.Then,to stimulate enzymatic browning reactions,an enzymatic Browning reaction system in vitro was constructed on the basis of the optimum condition from research on the enzymatic characteristics.Qualitative and quantitative analysis was used by HPLC to monitor the substrates and products.After that,the structure and the molecular weight of the product were speculated by UPLC-MSⁿ.The target product was isolated,purified and the structure was identified by spectral data analysis.And the reaction mechanism of enzymatic browning was elucidated in chrysanthemum,finally.2.5 Comparison of substrate and product antioxidant activitiesTo study the effect of enzymatic Browning on the quality of chrysanthemum,the changes in antioxidant activity of the reaction fluid before and after the simulated reaction in vitro was used to evaluate the antioxidant activities by DPPH method.3 Results3.1 The results of dynamic activitiy changes of the chrysanthemum PPO and POD enzymes in the drying in shade.In this chapter,the results showed that the activities of PPO and POD enzymes in chrysanthemum was increased at first and then decreased gradually in the drying in shade.It was speculated that in the process of drying chrysanthemum,the tissue integrity of the chrysanthemum was damaged and polyphenols distributed in cell vacuoles were in contact with PPO in cytoplasm,then the PPO and POD enzymes were activated.The appearance of the chrysanthemum was turning black.All these indicated that the key enzymes of enzymatic browning occurred in the process of chrysanthemum were PPO and POD.3.2 The results of changes in the chemical composition of chrysanthemum in the process of drying in shade.The results of this chapter showed that enzymatic browning and drought stress were two main factors that affected the effective composition of chrysanthemum.The chemical composition of chrysanthemum in the process of drying decreased firstly and then rised.And the reason was because at the beginning of the drying,Chrysanthemum has a high miosture and the enzymatic Browning was the dominant factor resulted in a decrease in the constituents of phenolic acids.After 24 hours,the enzymatic browning phenomenon occurred,phenolic acids were completely degraded,and flavonoids content was basically unchanged.It was speculated that phenolic acid was the main substrate of enzymatic Browning.As the water content decreased,PPO and POD enzymatic activities decreased,so the drying process of chrysanthemum entered the second stage that plants activated drought-resistant mechanisms and promoted the increase of effective components to resist drought stress.3.3 The results of the enzymatic characteristics of PPO and POD enzymesThe results showd that the optimal substrates of PPO and POD were pyrocatechol and guaiacol,and the optimal concentration of POD were 30 mmol·L^-1 and 50mmol·L^-1.The optimal pH value were 7.0 and 4.4,the optimal reaction temperature were25?and 30-35?.The PPO and POD enzyme activities were inactived when the temperature at 80?for 6 min and 4 min.The above results provided a basis for construct an enzymatic browning reaction system in vitro and do research on the mechanism of enzymatic browning reaction further.3.4 The results of the reaction mechanism study of PPO and POD enzymes in chrysanthemum.In this chapter,it was found that the color of the solution was deepened after the reaction of PPO or POD crude enzyme and chlorogenic acid.In addition,it was detected that the degradation of chlorogenic acid and the growth of product content by HPLC.All of these showed that the enzymatic browning occurred in chrysanthemum.Then,the product was analyzed by UPLC-MSⁿ after it was separated and purified.At last,the structure of the compound was identified by spectrum analysis that it was a dimer of chlorogenic acids.The possible reaction mechanism of the enzymatic browning in chrysanthemum was under the action of PPO or POD enzyme,chlorogenic acid was polymerized to produce dimer of chlorogenic acid.3.5 The results of comparison of substrate and product antioxidant activitiesAfter the simulated enzymatic browning reaction in vitro,it was found that the ability of reaction liquid scavenging DPPH radical activity was reduced.It was shown that the oxidation resistance of the reaction product was significantly lower than that of the reaction substrate.Therefore,It was considered that enzymatic browning reduced the antioxidant properties of chrysanthemum,and it also affected the efficacy and quality of chrysanthemum.4 Conclusion4.1 Enzymatic browning occurred in the process of chrysanthemum drying in shade and the key enzymes of enzymatic browning were PPO and POD.The content of chrysanthemum phenolic acid was significantly decreased by enzymatic browning.Because of the enzymatic browning and plants resist drought stress,the effective composition of chrysanthemum in the drying process of chrysanthemum was increased first and then decreased.4.2 The possible reaction mechanism of the enzymatic browning in chrysanthemum is under the action of PPO or POD enzyme,chlorogenic acid is polymerized to produce dimer of chlorogenic acid.4.3 The enzymatic browning reduces the antioxidant properties of chrysanthemum,and it also affects the efficacy and quality of chrysanthemum.