The Synergistic Effect Of Protein And Small Molecular Antioxidants On The Stability Of Melberry Anthocyanin Pigment

Mulberry anthocyanin extract(MAE)is a water-soluble natural pigment,however,it is easy to degrade and fade under the influence of environmental factors in aqueous solution,which affects the product quality and limits its application in food.At present,most of the protection methods of anthocyanins are single method,and the effect is limited.There are few reports on the combination of multiple methods.Protein can carry a variety of active components due to ligand-binding property.And it provides the possibility for multiple components to synergistically play the role of stabilizing anthocyanins in the proteinmultiligand complex system.In this paper,whey protein isolate(WPI)was used as a carrier to construct a ternary complex system that simultaneously carried small molecule antioxidants and anthocyanins.The effects of WPI and different small molecule antioxidants on the stability of MAE in binary and ternary systems were explored from the color difference(ΔE)of MAE solution and the degradation rate of total anthocyanins.And through a variety of techniques to study the interaction in the protein-small molecule antioxidant-anthocyanin system and the changes to the protein structure.The correlation between it and the stability of MAE was analyzed to provide theoretical basis and guidance for improving the stability of natural pigments such as MAE.First of all,three proteins: WPI,casein(CN),soy protein isolate(SPI),and nine small molecule antioxidants: gallic acid(GA),ferulic acid(FA),Marinic acid(RA),caffeic acid(CA),epigallocatechin gallate(EGCG),rutin(Ru),phlorizin(Phl),naringin(Nar),cysteine(Cys)were chosen as materials to stabilize MAE.According to their protective effects on the color stability of MAE solution at p H 6.3 and p H 3.6,the results showed that WPI and four small molecule antioxidants(FA,Phl,Nar,Cys),and WPI and six small molecule antioxidants(GA,FA,RA,CA,EGCG,Ru)showed protective effects on MAE.Secondly,the thermal stability of the binary system(MAE with different concentrations WPI)were tested at 80°C/2 h.The results showed that at p H 6.3 and p H 3.6,the optimal concentrations of WPI for inhibiting the fading of MAE and the degradation of anthocyanins were 1.6 mg/m L and 0.16 mg/m L,respectively.Subsequently,the thermal stability effects of the binary and ternary systems(WPI-MAE,small molecular antioxidant-MAE,and WPI-small molecule antioxidant-MAE)were compared at 80°C/2 h.Under the condition of p H 6.3,FA had the best stabilization effect,and the optimal concentration was 1.2 mg/m L.Compared with the binary system of WPI-MAE and FA-MAE,the ΔE of the ternary system was reduced by20.9% and 21.1%,and the degradation rate decreased by 38.00% and 39.3% respectively.The ternary system showed a more effective stabilization effect on the basis of the binary system.The presence of Cys in both ternary and binary systems improved the color stability of anthocyanins,but accelerated the degradation of anthocyanins.At p H 3.6,Ru has the best stabilization effect.When the concentration is 0.16mg/m L,compared with the binary system of WPI-MAE and Ru-MAE,the ΔE of the ternary system decreased by 40.0% and 23.8%,and the degradation rate of total anthocyanins decreased by 18.3% and 10.6%,respectively.And then the stability of MAE during accelerated storage at 40 °C was investigated for the WPI-FA-MAE system(p H 6.3)and the WPI-Ru-MAE system(p H 3.6).It was found that the ternary system could significantly prolong the protective effect of MAE solution.Finally,fluorescence spectroscopy,circular dichroism(CD),Fourier transform infrared spectroscopy(FTIR),UV-Vis Spectrum,particle size distribution,polyacrylamide gel electrophoresis(SDS-PAGE)were used to study the interaction among proteins,small molecule antioxidants and anthocyanins in different systems.The results show that β-lg can form binary and ternary complexes with C3 G and FA at p H 6.3.C3 G bound to β-lg mainly through the hydrophobic interaction and FA bound to β-lg mainly through electrostatic attraction.The presence of FA can enhance the binding ability of C3 G and β-lg.C3 G and FA had weak effect on the secondary structure of β-lg,but increased the particle size of the protein.The results of UV-Vis Spectrum indicated that WPI and FA could play a synergistic effect to further increase the chroma of anthocyanin solutions.In addition,β-lg can also form binary and ternary complexes with C3 G and Ru at p H 3.6.C3 G bound to β-lg mainly through the electrostatic attraction and Ru bound to β-lg mainly through hydrogen bond and van der Waals force.C3 G and Ru can change the secondary structure of β-lg,make it more open,and increase the particle size of protein particles.WPI and Ru can play the role of complexation and copigmentation simultaneously,bringing hyperchromic effect and bathochromic effect to anthocyanin solution.The SDS-PAGE results of the above two systems showed that the covalent binding between WPI and small molecules were not obvious.The synergistic effect of adding protein and small molecule antioxidants on the stability of anthocyanins during heat treatment and storage be closely related to the interaction of the protein-small molecule antioxidant-anthocyanins ternary system and the complexes formed.In addition,we preliminarily inferred by UV-Vis spectrum and UPLC-QTOF-MS that the there formed a covalent addition product between MAE and Cys during heat treatment.It explained the reason that the anthocyanins in the MAE-Cys system were degraded,but the color remained stable.