Preparation Of Proanthocyanidins With Different Structures And Their Effect On The Formation Of Acrylamide In Foods

Maillard reaction brings thermally processed foods with attractive color and flavor,but it produces toxics such as acrylamide and 5-hydroxymethylfurfural(HMF)at the same time.Acrylamide has drawn much attention because its neurotoxicity,carcinogenicity,and high concentrations in potato chips,cookies and other starch-based foods.Proanthocyanidins belong to flavonoids,and have antioxidant,anticancer,anti-inflammatory and other biological activities.They are rich in nature and lack of toxicity to humans.In recent years,the effect of polyphenols on the formation of contaminants during food processing has been investigated.However,the systematical and in-depth study about the effect of proanthocyanidins on the formation of acrylamide and HMF is still lack.In this dissertation,proanthocyanidins with different structures were prepared from three plant materials and then characterized.The relationship between their structures and inhibitory effects on the acrylamide and HMF formation was determined,and intervention mechanism was investigated.This study provides a basis for the development and utilization of proanthocyanidins.It provides new methods for controlling the contaminants during food processing and new ideas for the mechanism research.First,proanthocyanidins with different structures were prepared.Sorghum bran proanthocyanidins were extracted and then loaded onto a Sephadex LH-20 column.The column was eluted with 30%aqueous methanol,60%aqueous methanol,80%aqueous methanol,100%methanol and 70%aqueous acetone,successively.The 100%methanol fraction and 70%aqueous acetone fraction were designated as proanthocyanidin polymer-1 and polymer-2,respectively.Part of proanthocyanidin polymers were depolymerized with HCl and EC(or EGCG)followed with partition to prepare oligomer-1 and oligomer-2.The proanthocyanidins of cranberries were purified by ultrasonic-assisted solvent extraction and column chromatography.Then they were depolymerized in the acidic condition and partitioned to prepare oligomer-3.Oligomer-4 was separated from a grape seed proanthocyanidin extract commercial product.Meanwhile,the depolymerization of proanthocyanidin polymers to oligomers were investigated.It was found that the depolymerization followed a pseudo first-order kinetics model during the initial stage of reaction.The latter phase of reaction followed a slower and more complex kinetics.The reaction rate significantly increased at higher temperature.The influence of temperature on the depolymerization followed Arrhenius equation,and Ea was calculated as 83.0 kJ/mol.Lower pH favored the reaction rate as well.At a lower concentration of EC,adding more EC accelerated depolymerization.Reaction rate reached a plateau when sufficient amount of EC was added.A central composite design model with coefficient of determination R~2 of 0.98 was established.The optimized reaction condition was predicted as74?,pH 1.24,61 min and mass ratio of 0.89.The predicted yield was 87.0±4.0%which was close to the experimental yield of 86.0±1.2%.Afterwards,normal phase high performance liquid chromatography tandem mass spectrometry(NP-HPLC MS/MS),matrix-assisted laser-desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS)and thiolysis with cysteamine were used to characterize the structures of proanthocyanidin fractions.Results showed that oligomer-1,polymer-1,and polymer-2 were B-type procyanidins containing CAT/EC units,with mean degree of polymerization of 1.6,6.9 and 12.2,respectively.Oligomer-2 had subunits of CAT,EC,and EGCG(accounting for 62%).Oligomer-3 was consisted of CAT/EC units,and had 53.9%A-type linkage.The constitutional units of oligomer-4 were CAT/EC and CG/ECG(occupying10.7%).The mean degree of polymerization of oligomer-2,oligomer-3 and oligomer-4 were1.7,1.6 and 1.6,respectively.Cysteamine can be used as a thiolytic reagent in thiolysis.The thiolytic products were well separated in a reverse phase high performance liquid chromatography(RP-HPLC)with mobile phase of 0.1%aqueous TFA(trifluoroacetic acid)and acetonitrile.The monomers,A-type dimers and their cysteamine conjugates were identified based on their retention time in conjunction with mass spectrometry,including thioether of(epi)catechin,thioether of A-type dimer,and thioether of(epi)catechin gallate.Next,the inhibitory effects of proanthocyanidins on acrylamide and HMF formation in asparagine-glucose models and fried potato crisps were determined.Proanthocyanidins significantly and dose dependently mitigated acrylamide formation at concentrations of 50-200?g/mL in asparagine-glucose system.The inhibitory rates of monomeric flavan-3-ols for acrylamide were from 30%to 70%,and that for HMF were from 60%to 90%.Using the food model,the mitigation of acrylamide and HMF formation increased by higher immersion concentration of proanthocyanidins within the range of 0.01-0.1 mg/mL but descended when the addition level surpassed 0.1 mg/mL.Under the optimal concentration,about 40%acrylamide and 50%HMF were reduced.Effects of structure diversity of proanthocyanidins on their inhibitory activity were compared at same mass concentrations.B-type proanthocyanidins had stronger inhibitory activity for acrylamide and HMF than A-type.Higher degree of polymerization decreased their inhibition for HMF but had almost no effect for acrylamide.Proanthocyanidins with different unit composition had similar inhibitory activities on the formation of acrylamide.As for HMF,proanthocyanidins with subunits containing gallic acid esters showed lower inhibitory rates.Further,EC was used as a representative to study the intervention mechanism of proanthocyanidins on the formation of acrylamide and HMF.It was found that adding of EC resulted in a reduction of acrylamide and HMF but an increase of 3-deoxyglucosone(3-DG)in the asparagine-glucose model.In the asparagine-HMF model,more percentage of acrylamide than that in the asparagine-glucose model was inhibited and the residual of HMF was significantly lower in the presence of EC(p<0.05).It was found that EC adducted with HMF when they were heated together at 180?.The consumption of HMF,a carbonyl intermediate in Maillard reaction,through this pathway is the molecular mechanism of the inhibition of acrylamide formation by EC.Dimeric adducts composed of two CAT/EC units linked through an HMF bridge,and trimeric sandwich-type structures consisting of three CAT/EC bridged by two HMF molecules were identified.The major browning mechanism of the system is the interaction between EC and HMF,rather than the self-oxidation or self-condensation of EC.Finally,it was found that EC promoted the conversion from 3-aminopropanamide(3-APA)to acrylamide.A new pathway was proposed.EC adducted with 3-APA to form initial Schiff base,which is more easily to produce acrylamide.Five adducts in this pathway were identified.The error between the measured value and the theoretical value of their molecular weight is less than 1.4 mDa.The MS/MS spectra and kinetic curves matched the proposed structures and pathway as well.On the whole,EC not only consumed HMF to reduce the formation of acrylamide,but also promoted the conversion from 3-APA to acrylamide.The intervention of EC on the formation of acrylamide in Maillard reaction was a combined result of these two pathways.As a result,inhibitory effect for the acrylamide formation was observed.