Study On The Interaction Of Milk Protein With Cyanidin-3-O-glucoside And Its Effect On Pigment Stability

As one of the most common anthocyanins in nature,cyanidin-3-O-glucoside（C3G）could provide bright colours and prossess many biological activities such as antioxidant properties.However,C3G is easily susceptible to degradation under varying physical and chemical factors during storage,leading to the discolourization and the degradation of its functional properties,as well as the shortened shelf life to the related commodities,thus,the application of anthocyanins has been limited.Nowadays,several studies has been reported in using proteins to bind the small molecules such as anthocyanins to form protein-ligand complexes and protect them based on protein’s ligand-binding property,however,limited data are available on the interaction between anthocyanins and preheated proteins at different temperatures.Moreover,single protein could only provide limited protection.Therefore,this dissertation adopted preheated proteins as carrier materials compound with antioxidants to protect anthocyanins,and we studied the interaction between milk proteins and bioactive components,investigated the effect of complex on the stability of C3G.This study will also provide guidance for the application of natural anthocyanin pigments in the food industry.Firstly,the interaction mechanism between preheated proteins under different preheated temperatures（55-90°C）,especiallyβ-lactoglobulin（β-Lg）andβ-casein（β-CN）at pH3.6 and pH6.3 was investigated via fluorescence quenching spectroscopy,Flourier transform infrared spectroscopy（FTIR）and circular dichroism（CD）.The effect of small molecules such as（-）-epigallocatechin gallate（EGCG）,gallic acid,Vitamin C on the binding of milk proteins and anthocyanins was also studied in this study.Fluorescence quenching spectroscopy data showed C3G quenched milk proteins’fluorescence strongly.Thermodynamic analysis revealed that C3G bound toβ-Lg mainly through hydrogen bonding and hydrophobic interactions,and their binding affinity increased gradually with increasing preheating temperature at pH 6.3,whereas it decreased at pH 3.6.Hydrogen bonding and van der Waals forces played the major roles in the interaction ofβ-CN with C3G,their affinity decreased with increasing preheating temperature at both pH values.Preheating of milk proteins did not change their major forces with C3G,however,it would affect the binding ability between anthocyanins and milk proteins.The combination of C3G and preheatedβ-Lg at 85°C had strongest binding affinity,with a K_A of 14.10（±0.33）×10⁵ L·M^-1（pH 6.3,298 K）.The interaction between C3G and milk protein altered the secondary structure of milk proteins.The effect of antioxidants on the complex of milk protein-C3G was also determined in this study,the results showed that the addition of EGCG,gallic acid,vitmamin C could form the protein-multiligand complex,the formation of protein-multiligand did change the secondary structure of milk protein.Then,milk proteins such as whey protein isolate and caseinate were used as protective carriers,the effect of milk proteins on the color quality and the reservation amount of C3G was studied via color measurement and high performance liquid chromatography（HPLC）.By the way,the effect of native and preheated milk proteins on the stability of C3G was investigated before and after heating（80°C）,oxidation（0.005%H₂O₂）and irradiation.The results showed that whey protein isolate and caseinate would inhibit the heating,oxidation and irradiation degradation and the best concentration of milk proteins was 40μM.The addition of milk proteins could increase the reservation amount of C3G from 47.9%/26.9%,41.3%/37.3%,14.6%/17.2%to 57.3%/44.7%,49.7%/62.7%,26.3%/31.6%（pH3.6/pH6.3）.The effect of protection between different preheated caseins showed no significant changes,however it could provide better protection with the preheaetd whey protein isolate at high temperatures.At pH 6.3,the addition of 40μM preheated whey protein isolate at 85°C could provide best protection and the reservation amount of C3G was at 51.9%,75.3%,58.3%.After that,the preheated whey protein isolate at 85°C was selected as the protective carrier,formulated with antioxidants（tea polyphenols,EGCG,gallic acid,vitmamin C）,and the stability of three-ligand complex under 25°C was investigated and the reservation amount of C3G was increased to 71.5%,68.7%,70.2%.Thus,the formulation of 85°C whey protein isolate-gallic acid-C3G could prolong the staility of pigment solution,and almost 36.8%C3G was still existed at pH6.3,and the complex could provide better protection than the others.