| ?-lactoglobulin(?-LG)is the main whey protein in cow's milk and has multiple hydrophobic binding sites.It is often used as an effective and innocuous carrier in food to transport active small molecules to the absorbing part and improve the biological absorption and utilization.Although the food system is multiple and complex,there are few reports on the mechanism of ?-LG interacting with hydrophilic and hydrophobic vitamins to form a ternary complex and its changes during digestion.Therefore,the spectroscopy methods including fluorescence spectrum,ultraviolet-visible spectrum,and circular dichroism spectrum were used to study the formation mechanism and structural characteristics of ternary complex formed by hydrophilic folic acid(FA),hydrophobic retinol(VA)and ?-LG.The digestion and release processes of ?-LG,FA and VA was studied by in vitro digestion simulation,and the effects of FA and VA on the thermal stability and antigenicity of ?-LG were characterized by DSC and indirect competition ELISA.The results were described as follow:1.FA and VA had strong fluorescence quenching to ?-LG,respectively.The quenching mechanism was mainly static quenching.The binding constant,the binding sites and the thermodynamic parameters indicated that ?-LG interacted with FA and VA form stable ternary complexes through hydrophobic interaction mainly.The binding constants(Ka)were greater than 104 and the binding sites were about 1 respectively.When comparing the degree of quenching of ?-LG fluorescence by different addition order of FA and VA,it could be seen that the presence of VA affected the affinity of FA to ?-LG,and the presence of FA did not affect the affinity of VA to ?-LG.2.In the process of FA and VA interacting with ?-LG to form a complex,the structural changes were mainly manifested as follow:After a binary complex between FA/VA and ?-LG formed,the original tertiary structure of ?-LG changes obviously,while the tertiary structure of ?-LG changed slightly after the ternary complexes ?-LG-FA-VA and ?-LG-VA-FA formed.VA obviouslyenhanced the hydrophobicity of ?-LG,and FA had little effect on the hydrophobicity of ?-LG.The order of the degree of hydrolysis between ?-LG and its complex was:?-LG-VA-FA>?-LG-VA>?-LG-FA-VA>?-LG-FA and ?-LG;Compared with ?-LG,the complexes ?-LG-FA-VA and ?-LG-VA had lower antigenicity,but the antigenicity of ?-LG-VA-FA was higher;The thermal denaturation temperatures of ?-LG-FA,?-LG-VA,?-LG-FA-VA and ?-LG-VA-FA were all lower than those of ?-LG.3.Simulated digestion in-vitro experiments revealed that different binding sequences of FA and VA had different effects on the structure of ?-LG.Therefore,the sensitivity of ?-LG to pepsin also was different,showing the different degree of hydrolysis than ?-LG.?-LG-FA had the lowest degree of hydrolysis during gastric hydrolysis,and the ternary complex ?-LG-FA-VA had the highest degree of hydrolysis.There was no significant difference of the degree of hydrolysis among the ?-LG-VA-FA,?-LG-VA and ?-LG.After further simulated intestinal hydrolysis,?-LG and the complex reached hydrolysis equilibrium at 120 minutes.The release rates of FA and VA in the complex were determined by HPLC.The results showed,in the gastric simulation process,that the release rates of FA in other complexes were less than 38%,except for the release rate of FA in ?-LG-FA-VA reached 50%.After further hydrolysis of the simulated intestine,the release rates of FA reached more than 83%.The release of VA in the complex during the enzymatic hydrolysis process was perplexing,and it could reach a high release rate in the stage of gastric enzymolysis.After further intestinal enzymolysis,the release rate was gradually decreased by the action of pH and trypsin.Indirect competitive enzyme-linked immunosorbent assay(ELISA)showed that the antigenicity of ?-LG-FA SD was not different from that of ?-LG SD after digestion in-vitro.The semi-inhibitory concentration(IC50)of ?-LG-FA-VA SD,?-LG-VA-FA SD and ?-LG-VA SD increased.In other words,the antigenicity of ?-LG-FA-VA SD,?-LG-VA-FA SD,and ?-LG-VA SD were lower than that of the original ?-LG SD.Therefore,?-LG interacted with FA and VA to form a stable ternary complex,and reducing the antigenicity of ?-LG after digestion. |
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