Purification,Preliminary Structure Identificantion And Antioxidant Activity Of Products Derived From Non-enzymatic Browning In Lotus Root Filled With Sticky Rice And Control Of Non-enzymatic Browning

Lotus root（Nelumbo nucifern G.）is a kind of perennial and aquatic macrophytes belonging to Nymphaeaceae speice.As an aquatic vegetable with high economic value,the cultivated area of Lotus root in China is the largest.The main edible part of lotus root is the rhizome.Lotus root filled with sticky rice is a traditional Chinese snack,which is characterized by sweet tastes,sticky mouth feel,high nutrition level and easy consumption.Therefore,it is very popular.However,the knowledge about the mechanism,control of browning during heating processing of lotus root filled with sticky rice,as well as the properties of browning products,is limited.In the study,the problem of browning excessively in the heat processing of lotus root filled with sticky rice was studied.To be exact,the main factors affecting colour of lotus root during poaching were identified.After that,melanoidins,which was the non-enzymatic browning product were purified from lotus root with sticky rice and identified preliminarily.Furthermore,the antioxidant activity of melanoidins was assayed.In addition,the methodology about how to control excessive browning during processing of lotus root with sticky rice was explored.All these studies can provide guidance for producing lotus root filled with sticky rice.The main results are shown as follows:Fresh lotus root named Dahongzi variety was used as experimental material.The non-enzymatic browning reactions during poaching were studied,so as to find the factors affecting the colour of lotus root filled with sticky rice.It was found that water content in lotus root was 88.06%and the dry matter was mainly composed of starch,carbohydrates and free amino acids.Specifically,97.87%of starch was amylopectin.There were three types of sugars in lotus root,including glucose,fructose and sucrose.75.93%of total sugar was fructose.There were 16 types of free amino acids,the content of threonine being the highest（50.75%）followed by arginine（12.44%）.After processing,lotus root became darker and the contents of all components inside decreased.The contents of fructose of glucose were 29.77 and 136.20 mg/g DW,while their corresponding consumption rates were 33.90%and 81.56%.The consumption rates of threonine and arginine reached 65.62%and 74.27%,which were significantly higher than that of other components（P<0.05）.Therefore,it was deduced that Maillard reaction was the main non-enzymatic browning reaction in the process of lotus root.Glucose was first involved and fructose was the main carbohydrate taking part in Maillard reaction.Threonine and arginine were the main free amino acids involved.The product from non-enzymatic browning of lotus root filled with sticky rice,namely melanoidin by aqueous ethanol extraction was used as solvent.The extraction process was optimized by single-factor experiments and L9（34）orthogonal experimental design.It was found that temperature was the main factor affecting the extraction yield,followed by ethanol concentration,extraction time,and solid-liquid ratio.The optimal extraction conditions were 10%for ethanol concentration,1:40（w/v）for solid to solvent ratio,80 ℃for extraction temperature and 1.5 h for extraction time.Under these conditions,the extraction yield of melanoidin was 8.235.Furthermore,the antioxidant activities of melanoidin were estimated by means of scavenging capacities of a 2,2-diphenyl-picrylhydrazyl radical（DPPH·）,hydroxyl radical（·OH）and reducing power under different concentrations.It was found that the antioxidant activities of the aforementioned indexes significantly increased（P<0.05）with the increase of melanoidin concentration.When the relative concentration was 1.0,the three indexes of antioxidant activities reached 53.39%,10.16%and 15.95%,respectively.Under the same concentration,the value of DPPH· scavenging capacity was the highest,followed by the reducing power and ·OH scavenging capacity.Therefore,it was concluded thatmelanoidin in Chinese traditional lotus roots filled with sticky rice had antioxidant activity,which was affected by the dose applied.Based on optimization of extraction of melanoidins in lotus root filled with sticky rice,macroporous resin was used to purify melanoidins in the extract.AB-8 macroporous resin was found to be the best resin for purification.The optimal column flow rate was 1.5 mL/min and 50%ethanol was used as eluent.In the next step,Sephadex G-50 column was used for further purification.The conditions for Sephadex G-50 column were 45 cm for column height and 2 mL/min for column flow rate.Three fractions,including F1,F2 and F3 were obtained and then analyzed by HPLC.Furthermore,structure of components in F1 was identified by infrared spectrometer,which its antioxidant activity was also assessed.It was found that there were three peaks in all the three fractions obtained from Sephadex G-50 column,indicating that melanodins had different molecular weights.According to infrared spectrum,it was deduced that the main functional groups of melanodins in lotus root filled with sticky rice were alkyl,alcoholic hydroxyl groups,amide groups,olefins,amines,carboxylic acid and tertiary alcohol.The fraction F1 had a high antioxidant activity with the total reducing power of 280%,DPPH radical scavenging capacity of 46.17%and OH radical scavenging capacity of 38.81%.Compared to the raw extract,melanoidins after purification had higher antioxidant ability.Excessive non-enzymatic browning can be controlled by reducing initial pH and addition of Ca²⁺ and Mg²⁺,both of which can inhibit the occurrence of non-enzymatic browning.The inhibition effect enhanced with the decrease of pH and the increase of addition of Ca²⁺ and Mg²⁺.However,excessively low pH can provide lotus root with sticky rice a sour taste,affecting its quality.Meanwhile,the addition of Ca²⁺ and Mg²⁺ should be strictly controlled.Orthogonal experiments were performed to select the optimal pH and addition of Ca²⁺ and Mg²⁺.It was found that when the pH was 5.5,and the additions of Ca²⁺ and Mg²⁺ were 0.063 and 0.6 mg/mL,the sensory score of lotus root filled with sticky rice was the highest.