Mechanism Exploration Of Okra (Abelmoschus Esculentus (L.)Moench) Polysaccharide Viscosity Reduction Induced By Ascorbic Acid

Okra（Abelmoschus esculentus（L.）Moench）,a new type of healthy vegetable with low fat and low calories,has various biological activities such as anti-oxidation,immune regulation and hypoglycemia.It is rich in polysaccharides,which are widely applied in functional foods and food additives（as emulsifier,thickener,etc.）and whose specific high viscosity characteristic plays important roles in quality and health effects of products.Ascorbic acid（AH₂）is an acidic polyhydroxy compound with six carbon atoms.AH₂ is frequently used in foods due to its unique anti-oxidation activity.However,it can be acted as a prooxidant,which possesses a certain degradation effect on polysaccharide viscosity.During the exploitation processes of healthy and functional foods based on the important component of okra pectic polysaccharide（OPP）,the possible adverse effects of AH₂on reduction in viscosity of OPP should be considered.In this study,influences of AH₂on viscosity,structural feature,digestive enzymes inhibitory effect and in vitro anti-oxidation activities were investigated.Moreover,the mechanism for AH₂ reducing viscosity of OPP was deeply explored through free radical reactions and chemical structure changes of the carbohydrate.Furthermore,the inhibitory effects of other compounds on reduction of AH₂ on viscosity of OPP were preliminarily analyzed.This study provides a theoretical basis for the future development of food with OPP and AH₂coexistence（for viscosity adjust and control）,and theoretical basis and references for the interaction between AH₂ and other polysaccharides.The main research contents and conclusions are as follows:（1）Effects of ascorbic acid treatment on viscosity of OPP.OPP was firstly extracted from okra peel,and its basic structural characteristics were analyzed.Then,influences of different antioxidants（AH₂,Erythorbic Acid,Dehydroascorbic acid）on viscosity of OPP were investigated.Results indicated that OPP was mainly composed of Rha（9.0%）,Gal（14.1%）and Gal A（11.9%）.It was belonged to RG-Ⅰ pectin and had high viscosity（behaved as weak gel characteristics when the concentration was≥1.0%）.AH₂ and Erythorbic Acid had the greatest effects on the viscosity of OPP.Increasing the temperature and extending the reaction time could strengthen the effect of AH₂ on OPP viscosity reduction.（2）Systematical investigation on influences of AH₂ on structural characteristics and in vitro functional activities of OPP.Structural characteristics of OPP before and after AH₂ treatment were analyzed by investigating polysaccharide molecular weight,monosaccharide composition and glycosidic bonds（polysaccharides treated by 0,0.5and 2 mM AH₂ were labeled as OPP-B,OPP-A-1 and OPP-A-2,successively）.Meanwhile,activities of OPP in vitro before and after AH₂ treatment mainly focused on antioxidation and amylase inhibition effects.Results revealed that OPP after AH₂treatment showed the characteristics of Newtonian fluid,and the decreased viscosity could not be recovered.After AH₂ treatment,the molecular weight of OPP was obviously decreased（M_wof OPP-B,OPP-A-1 and OPP-A-2 was 7.7×10⁶,7.3×10⁶,and 1.2×10⁶ g/mol）,and the conformational parameters（R_g、R_h、[η]）of solution were also altered.The main monosaccharides of OPP before and after AH₂ treatment were Rha,Gal and Gal A,and the relative proportion for these three monosaccharides were slightly altered.Moreover,the content of RG-Ⅰ branched pectin and the length of Gal side chain were essentially unchanged,and no significant changes were observed in the infrared spectroscopy.AH₂ treatment had no obvious effects on the main structure and NMR signal characteristics of OPP,as the main glycosidic types of OPP-B were1,2,4-Rhap（22.4%）,T-Galp（26.8%）and 1,4-Galp A（21.9%）,whose ratios were not obviously altered after the AH₂ treatment.However,the degree of methylation（16.6%,corresponding to 11.9%for OPP-B）and degree of acetylation values（90.5%,corresponding to 73.0%for OPP-B）of OPP-A-2 significantly increased.It could be speculated that galacturonic acid that has not been methylated or acetylated in OPP might be the action sites of AH₂.The scavenging abilities against hydroxyl radical,DPPH radical and superoxide anion radical OPP were not significantly improved before and after AH₂ treatment,whilst the inhibitory effects of OPP onα-amylase andα-glycosidase activities were notably decreased as the concentration of AH₂ increased.（3）Exploring the action mechanism of AH₂ on reduction of OPP viscosity based on the changes of chemical structure.Firstly,effects of multiple cycles of AH₂treatment on molecular weight and structural characteristic were analyzed.After that,polygalacturonic acid（PA）and pectic galactan（PG）having structural similarity to OPP were selected for exploring the effects of AH₂ treatment on their structure.The results indicated that multiple cycles of AH₂treatment could further reduce the molecular weight of OPP,but the reduction tendency is obviously diminished after the secondary treatment（the M_w after the fifth processing was 5.0×10⁵ g/mol）.Moreover,the ratios of monosaccharide compositions and the characteristics of infrared spectroscopy for OPP were slightly changed.However,the main solid morphology of OPP was changed from sheet structure to linear and granular structures.AH₂ treatment had little effect on structures of Rha,Gal and Gal A.No obvious alterations on the monosaccharide composition,characteristics of infrared spectroscopy,solid morphology and NMR signals of PA and PG were observed after AH₂treatment,but it was found that the NMR of PG-A-1 and PG-A-2 had some other weak signals which were not observed in that of PG-B,suggesting that other compounds might be produced.（4）Mechanism analysis concerning the reduction of AH₂ on viscosity of OPP by free radical reactions.Influences of alone or synergistic effects of Fe²⁺,Cu²⁺and H₂O₂on reduction of OPP on viscosity of OPP were systematically studied.Results implied that the concentration,reaction temperature and reaction time of H₂O₂ and metal ions（or combination）had a great influence on the viscosity of OPP+AH₂ system.Meanwhile,the synergistic effect of AH₂ with metal ions(Fe²⁺,Cu²⁺)or H₂O₂ could reduce the viscosity of OPP more effectively.The determination of radicals in systems using EPR experiment has shown that hydroxyl radical（·OH）was gradually generated,when ascorbic acid was added into OPP solution.A lot of·OH radicals were immediately produced in the solution,when AH₂was synergistically combined with Fe²⁺,Cu²⁺,or H₂O₂.By correlation analysis between·OH production and reduction of OPP,it could be inferred that AH₂ treatment induced·OH formation in OPP solution,thereby caused the decreases of solution viscosity and molecular weight.（5）Preliminary analysis and exploration on the effects of different molecule compounds on reduction of OPP viscosity.From the views of viscosity changes and free radical formation,the effectiveness of different compounds（organic acid,amino acids,quercetin,catalase and various sugars）and catalase in inhibiting reduction of AH₂on OPP viscosity were compared.Meanwhile,the possible mechanisms were Results reflected that various sugars,amino acids and catalase could inhibiting OPP viscosity reduction induced by AH₂,and the effectiveness depended on the type of compounds.Catalase had the best effect（the viscosity of polysaccharide system was118%of that of the control group when the shear rate was 10 1/s after 7 days storage）.Among them,sugars,amino acids and catalase showed the relatively stronger effectiveness in decreasing·OH formation.In a week of storage experiment,catalase was found to inhibit·OH formation by hydrolyzing H₂O₂ and had the best inhibitory effect（100%）on reduction of OPP viscosity.Sucrose and phenylalanine significantly slowed down the reduction of OPP viscosity,owing to their stronger scavenging activities on·OH（65%for sucrose,and 57%for phenylalaniney）.Quercetin,oxalic acid,citric acid and malic acid had the relatively weaker effectiveness.