Competitive Interaction Between Polyphenols And Proteins,Enzymes In Vitro Digestion And Its Effect On Functional Properties Of Polyphenols And Proteins

In recent years,with the cocoa milk,fruit milk,coffee milk being popular,the interaction between polyphenol and protein has been a hot topic.Most studies are mainly focused on the interaction between tea polyphenols and milk proteins which has effect on the functional properties of tea polyphenols or milk proteins.While for tea polyphenols,milk proteins and digestive enzymes in the digestive system,the effect of competition interactions on the nutrient and function of the complexes is not clear.Therefore,this study foucus on the competition interaction among tea polyphenols,milk proteins and digestive enzymes as well as the effect on the digestibility of milk proteins and the antioxidative activity of the system in the gastrointestinal pH environment.The results provide theoretical basis for enhancing the bioavailability and health effects of tea polyphenols as well as functional food processing.Based on the results of fluorescence quenching,circular dichroism,molecular docking and enzyme activity assay under simulated gastrointestinal digestion,tea polyphenols[(-)-epicatechin(EC),(-)-epigallocatechin(EGC)and(-)-epigallocatechin gallate(EGCG)]bound to milk proteins(?-casein,?-lactoglobulin)or digestive enzymes(pepsin,trypsin)in solution and the fluorescence quenching process was static quenching.In the gastric pH environment,the order of the interaction between tea polyphenols and protein was?-casein>?-lactoglobulin>pepsin.In the intestinal pH environment,the binding affinity of EGCG or EGC to protein decreased in the order ?-lactoglobulin>trypsin>?-casein and the order of binding affinity for EC was ?-casein>?-lactoglobulin>trypsin.The binding strength of tea polyphenols with protein was in the order EGCG>EC>EGC.In the gastrointestinal pH environment,the interaction of tea polyphenols and milk proteins or digestive enzymes had about one binding site.In the gastric pH environment,EGCG-pepsin/?-casein,EGC-?-lactoglobulin and EC-?-casein were bound by van der Waals forces or hydrogen bonds.In the reaction of EGC-?-casein,EGC/EC-pepsin or EGC/EC-?-lactoglobulin,the hydrophobic interaction was the main binding force.For the EGCG-?-lactoglobulin system,it was electrostatic and hydrophobic interaction.In the intestinal pH environment,the hydrophobic interaction was the main binding force between EGCG and trypsin,?-casein or?-lactoglobulin,EGC and ?-casein,EC and trypsin.EGC-trypsin,EGC-?-lactoglobulin,EC-?-casein and EC-?-lactoglobulin were bound by van der Waals forces or hydrogen bonds.CD spectra and molecular docking showed that the conformation of milk proteins and digestive enzymes were altered in the presence of tea polyphenols.In the gastrointestinal pH environment,the stability of tea polyphenols-?-lactoglobulin was stronger than tea polyphenols-enzymes and the binding strengths of tea polyphenols with ?-lactoglobulin or digestive enzymes was in the order EGCG>EC>EGC according to the principle of more stable complex with the lower energy.EGCG,EGC and EC inhibited the activity of pepsin and trypsin.The order of inhibitory intensity was EGCG>EC>EGC.The inhibition of trypsin was higher than pepsin.The inhibitions of pepsin and trypsin by EGCG were non-competitive and the inhibitions of digestive enzymes by EGC or EC were competitive.The effects of competitive interaction among tea polyphenols,milk proteins anddigestive enzymes on the bioavailability of tea polyphenols,digestibility of protein and antioxidant activity of the system were studied in vitro digestion.The results showed that tea polyphenols would first combine with milk proteins,followed by digestive enzymes to hydrolyze protein and tea polyphenols was released,so the competitive interactions would inprove the bioavailability of tea polyphenols when the binding affinity of tea polyphenols-milk proteins was greater than that of tea polyphenols-digestive enzymes.Conversely,tea polyphenols was combined with digestive enzymes,which inhibited the activity of digestive enzymes and protein hydrolysis,leading to tea polyphenols combine with protein to reduce the bioavailability of tea polyphenols.When adding ?-lactoglobulin,the bioavailability of tea polyphenols increased by 252.6%,85.0%,37.0%,respectively.After adding ?-casein,the bioavailability of EGCG and EGC decreased by 31.5% and 5.6%,while EC increased by 43.4%.Compared with tea polyphenols-digestive enzymes and the coexisting system of tea polyphenols,milk proteins and digestive enzymes,the scavenging ability of ABTS and iron-reducing ability of the three coexisting system were higher than tea polyphenols-digestive enzymes system.In gastrointestinal digestion,the antioxidant activity of EGCG/EGC-?-lactoglobulin-trypsin was higher than EGCG/EGC-?-casein-trypsin,while for EC,the result was opposite.The competition among tea polyphenols,milk proteins and digestive enzymes decreased the digestibility of ?-casein and ?-lactoglobulin in vitro environment.In gastric digestion,when adding EGCG,EGC or EC,it decreased by 20.64%,12.87%,17.69% and 34.21%,17.48%,24.31%,respectively.In intestinal digestion,it decreased by 22.27%,15.48%,20.16% and 36.26%,22.90%,27.92% respectively.So the degree of hydrolysis decline in the gastric digestion was smaller than in the intestine digestion.The inhibition order of tea polyphenols on the digestibility of ?-casein or ?-lactoglobulin was EGCG>EC>EGC and the same as the binding strengths of tea polyphenols with digestive enzymes.Thus,the competitive interaction among tea polyphenols,milk proteins and digestive enzymes would affect the digestion and absorption of milk proteins.