Construction And Interaction Mechanism Of Whey Protein Concentrate Based Quercetin-coated System

Quercetin is an important dietary flavonoid,which is produced widely in fruits(e.g.,apple,grape),vegetables(e.g.,onion,broccoli),and herbal medicines(e.g.,Houttuynia cordata,dandelion).Quercetin is of interest for its anti-oxidation,anti-cancer,anti-inflammatory and other biological activities.However,quercetin is difficult to mix with water-soluble matrices because of its poor water solubility,which results in poor intestinal absorption.In addition,quercetin is susceptible to environmental factors,such as pH,light,heat and oxygen during storage and processing,and degrades rapidly in the intestinal environment,which severely limits its application in food and medicine.Therefore,a protective delivery system is needed to improve the defects of quercetin.Delivery systems,such as nanocapsules,nanoparticles,and emulsions,can encapsulate and protect bioactive molecules and improve their bioavailability,which is currently a hot research topic.However,the safety issues caused by the use of various additives and organic reagents in many studies have not been solved,so that the application of delivery systems in the food field is still limited.In this thesis,a safe and stable delivery system by physically modified whey protein concentrate is established,in order to improve the water solubility,chemical stability and bioavailability of quercetin.In this paper,the effect of pH on the interaction between unmodified whey protein concentrate(WPC)and quercetin(Q)was initially investigated to determine the acid-base condition which was favorable for their binding.Then,the composite system of pretreated WPC(preheating/pre-ultrasonic treatment)and quercetin was established and characterized.The interaction mechanism between WPC and quercetin and the digestibility of spray/vacuum freeze drying composite system were studied.The conclusions are as follows.(1)Effect of pH on the WPC-Q complex system.Fluorescence and infrared spectroscopy analyses demonstrated that quercetin bound to WPC through hydrophobic interaction and changed the secondary structure of WPC.Neutral and weakly basic conditions were more favorable for the formation of WPC-Q.At pH 7.4,the binding rate of quercetin was 36.79%,and the structural order of WPC increased significantly.WPC improved the stability and antioxidant properties of quercetin.The retention rate of quercetin was 42.30%(pH 7.4)when WPC-Q was maintained at37℃for 6 h.(2)The effect of preheating/pre-ultrasonic treatment on the WPC-Q composite system.The results showed that the pretreatment significantly changed the properties and structure of WPC-Q.The binding rate(61.68%)and water solubility(0.047mg/m L)of quercetin binding to whey protein concentrate preheated at 80℃for 30min(80WPC)were the highest.The complex(80WPC-Q)had smaller particle size(184.43 nm)and more negative surface charge(ζ-potential of-20.58 m V),which made it stable.The retention rate of quercetin in 80WPC-Q was 49.09%(4.54 times of free quercetin)at 37℃for 6 h and 30.25%(5.81 times of the free quercetin)at room temperature under natural light for 6 d.The binding of WPC and quercetin acted as an antagonist to the antioxidant properties of the complex system,confirming that WPC played a protective role to quercetin.(3)Mechanism of interaction between WPC and quercetin.Pretreatment increased the binding constant(K_a)and the number of binding site(n)values.The binding affinity of 80WPC and quercetin was strongest because of the largest binding constant(5.368×10~4L/mol),the largest energy transfer efficiency(0.92)and the shortest binding distance(1.41 nm).The pretreatment(preheating/pre-ultrasonic treatment)unfolded the structure of WPC and exposed the hydrophobic region,so that the free sulfhydryl content and surface hydrophobicity increased.After binding with quercetin,the free sulfhydryl content and surface hydrophobicity of WPC were significantly reduced,and the structure of WPC was more compact and tended to be ordered.Chemical dissociation to the interaction forces and molecular docking results revealed that hydrophobic interaction was the main driving force for WPC-Q complex formation,while hydrogen bond was also involved in the binding.The binding sites ofβ-Lg monomer,β-Lg dimer,α-La and BSA with quercetin are different.Quercetin was binding to the hydrophobic amino acids(Leucine,Isoleucine,Methionine,Alanine and Proline).Leucine(fromβ-Lg monomer,β-Lg dimer,α-La and BSA)bound to quercetin,and might be the key amino acid.(4)The digestibility of 80WPC-Q and the effect of drying methods.Spray-dried80WPC and 80WPC-Q were in the form of dried-out spheres(<10μm)with good dispersibility.The vacuum freeze-dried 80WPC and 80WPC-Q were large shaped blocks with pores formed by the accumulation of smaller particles.In vitro digestion showed that freeze-dried 80WPC and 80WPC-Q were more digestible than spray-dried ones.Quercetin made 80WPC-Q more resistant in the gastric digestion phase,while it did not affect the digestion of 80WPC-Q in the intestinal digestion phase.The retention rate of quercetin in 80WPC-Q digestive juice(freeze-dried:76.01%,spray-dried:64.14%)was higher than that of free quercetin(30.11%)after6 h of in vitro digestion.Both drying methods had no significant effect on the DPPH and ABTS radicals scavenging rate of digestive juice,while the scavenging rate of80WPC-Q digestive juice was greater than that of free quercetin.In summary,the binding to WPC(preheated at 80℃for 30 min)at pH 7.4significantly improved the binding rate,solubility,antioxidant activity and stability of quercetin.The main binding sites of WPC and quercetin were hydrophobic amino acids and the binding force was hydrophobic interaction.The retention rate of quercetin in 80WPC-Q digestive juice was high.DPPH and ABTS radicals scavenging rate of 80WPC-Q digestive juice was higher than that of free quercetin.The results provided a basis for the protection and delivery of quercetin.