Extraction And Purification Research Of Citrus Flavonoids And Its Inhibitory Mechanism On Acrylamide Formation In Foods

Acrylamide is a kind of harmful substances in fried and baked food. In order to decrease acrylamide content as far as possible, reduce possibility risk to human, the research studied the inhibitory effect of citrus flavonoids on acrylamide and analysed inhibitory pathway preliminarily under atmospheric frying system. Citrus flavonoids are abundant in raw material sources and have edible security. Therefore, this research has great significance in reducing the risk of food safety and utilizing citrus resources reasonably. The main research contents and conclusions are as follows:(1) Based on the single factor experiment, response surface methodology of four-factor and three-level was adopted to optimize the extraction technology by using ultrasonic-assisted ethanol to extract total flavonoids from mandarin peel. Results showed that the extraction amount of total flavonoids come to the highest under the condition of the particle size 60 mesh, ultrasonic power 225 W, ultrasonic temperature 79℃, ultrasonic treatment time 20 min, ethanol concentration 70%, solid-liquid ratio 1:45 (g/mL). Under these conditions, the extraction rate of total flavonoids reached 1.0438%, improved 14.77% than alcohol extraction method.(2) Using macroporous adsorption resin to purify total flavonoids extracts of mandarin peel. Selected five different macroporous adsorption resins, the results of adsorption and desorption performance testing showed that a resin type of AB-8 was the best for purification research. It determined the optimum adsorption conditions: the concentration of sample was about 0.4 mg/mL, the flow of injection liquid was 2 mL/min, the volume of sample was 6 BV; the best desorption condition:the leacheate was 4 BV distilled water, the eluent was 60% ethanol aqueous solution in volume concentration and elution velocity of 2 mL/min, dosage of eluent was 4 BV. Under these optimized conditions, the total flavonoids in mandarin peel through by purification, concentration and vacuum freeze-drying, the purity of flavonoids in final product was 53.56%, its reducing sugar content was 13.88%, compared the before purification (24.48%), has decreased 51.25%.(3) Set up a solid phase extraction with high performance liquid chromatography (HPLC) method for the determination of acrylamide, then use citrus flavonoids after purification as the research object, explore its inhibitory effect on acrylamide formation in fried chips. Results showed that the method of detecting acrylamide had high precision, good stability and high recovery under the condition of the mobile phase of methanol/water (5:95;V:V), the flow rate of 0.6 mL/min, column temperature 30℃, detection wavelength of 210 nm, when the sample was extracted with 0.1% formic acid in water, and cleaned up by Waters Oasis HLB SPE cartridge(200mg/3cc). It met the requirements of trace analysis. By analysis of multiple comparisons, determined that the citrus flavonoids had inhibitory effect on the formation of acrylamide in fried compound potato chips, its concentration-inhibition rate was nonlinear. When the concentration of citrus flavonoids was 0.05%, which was equivalent to add 200 milligrams citrus flavonoids per kilogram of potato powder, the acrylamide content of potato chips was 0.474 mg/kg, reached the maximum inhibition rate 62.20%.(4) Establishing atmospheric fried model reaction system of acrylamide, it investigated the frying temperature, frying time, the additive amount of asparagine and glucose/fructose/sucrose that influenced on the formation of acrylamide by response surface methodology. Results indicated that the acrylamide content was increased under the combined condition of low frying temperature and long frying time or high frying temperature and short frying time. According to the results of variance analysis, the factors of frying temperature and sugar levels played an important role in the formation of acrylamide. For the three reaction system with different sugar substrates, the response surface model of asparagine-glucose reaction system had the highest correlation coefficient (R2=0.9394), which could reflect the law of the formation of acrylamide better. Therefore, choose asparagine-glucose reaction system for subsequent research of inhibition kinetics of acrylamide. The reaction conditions of asparagine-glucose system were further optimized. Results showed that the level of acrylamide reached a maximum when equimolar asparagine and glucose of 0.6mmol were fried at 180℃ for 100s.(5) In asparagine-glucose atmospheric fried system, it studied the dose-effect relationship and inhibition kinetics of citrus flavonoids that inhibited acrylamide formation. According to the results, there was significant difference in acrylamide content between experimental group and blank control group within the scope of 1×10-7～lmg/mL of citrus flavonoids concentration (p<0.05), and its concentration-inhibition rate was nonlinear. When the concentration of citrus flavonoids was 0.001 mg/mL, which was equivalent to add 0.4 milligrams citrus flavonoids per kilogram of material, the acrylamide content of system was 0.511μmol, reached the maximum inhibition rate 58.42%. The results of acrylamide formation/eliminate kinetic research showed that acrylamide produced quickly within 0～180s, acrylamide content reached maximum at 180s for experimental group, at 220s for control group, then the acrylamide levels were declining. Kinetic curve and validation test showed that citrus flavonoids had significant influence on acrylamide formation, no significant influence on its elimination. Based on the kinetic curve, it established the first order kinetic model of acrylamide formation/elimination, the model of fitting degree was good (R2=0.961)that could describe acrylamide formation/eliminate kinetic in atmospheric fried system preferably.(6) Based on asparagine-glucose atmospheric fried system, it set up potato model system using atmospheric fried method, and studied the dose-effect relationship of five monomers of citrus flavonoids (hesperidin, neohesperidin, naringin, nobiletin, tangeretin) that inhibited acrylamide formation. The results showed that five monomers reached maximum inhibition rate (53.57%,42.86%, 39.29%,58.57%,55.71% respectively) at molarity of 10-8, 10-6,10-6,10-9,10-9mol/L respectively. Therefore, the sequence of inhibitory effect is:nobiletin>tangeretin> hesperidin>neohesperidin>naringin, further confirmed that the citrus flavonoids had inhibitory effect on the formation of acrylamide in fried chips. Meanwhile, it analyzed structure-activity relationship between molecular structure and inhibition of citrus flavonoid monomer. For three kinds of dihydrogen flavonoid glycosides (hesperidin, neohesperidin, naringin), the chemical structure of A ring with 5-hydroxyl,7-glycosyl and B ring with 3’-hydroxy1,4’-methoxyl had influence on inhibition rate of acrylamide. The glycosyl of rutinoserue had stronger inhibitory effect on acrylamide than neohesperidose. For two kinds of flavonoids (nobiletin, tangeretin), the chemical structure of A ring with 5,7-methoxy and B ring with 3’,4’-methoxy contributed to inhibiting acrylamide. The type of group where at 5, 7-position of A ring and 3,4’ -position of B ring in molecular structure of citrus flavonoid played an important role acrylamide inhibition by comprehensive analysis.(7) Select representative citrus flavonoids (hesperidin, nobiletin, tangeretin), then added each monomer with 10"8,10-9,10"9mol/L respectively into potato model system using atmospheric fried method. It studied the variation of asparagine, reducing sugar, melanoid, acrylamide content and inoxidizability in end product with different reaction time, drew their kinetic curve respectively. According to the results, the consumption rate of asparagine and reducing sugar was fast in the 300s, then became slowly. The content of melanoid and acrylamide increased quickly in the 120s, the change of acrylamide content was similar with melanoid content. The change of inoxidizability in end product was relative to acrylamide content. The stronger inoxidizability in this system, the greater inhibitory effect was. On the basis of each kinetic curve, mechanism of kinetic models was established, calculated kinetic parameters, and did significance test for each parameter. Results indicated that citrus flavonoid can significantly inhibit the formation of acrylamide from intermediate products such as Schiff. Citrus flavonoid can’t significantly inhibit intermediate products from reaction between asparagine and reducing sugar. Citrus flavonoid can’t significantly eliminate acrylamide that generated.