Study On The Interaction Mechanism Between Mulberry Anthocyanin And Proteins And Their Effects On Function

Mulberry anthocyanins(MA)is a kind of natural water-soluble pigment,with a variety of physiological activity of antioxidant and anti-cancer etc.However,the natural anthocyanins are unstable and easy to degrade,thus their application is limited.In food systems,studies have shown that some animal and plant originated proteins could interact with polyphenols and may have influence on their functional properties.It has been reported that protein macromolecules act as a protective carrier for anthocyanins and enhance their stability.This creates a new avenue for enhancing the stability of anthocyanins.However,the combination of anthocyanins and protein,to a certain extent,could also affect the structural and digestibility of proteins.The digestibility of protein as a carrier plays an important role in the use of the added value of the protein and in the sustained release of the delivered drug.The digestibility of protein as a carrier is of great significance to the use of the additional value of protein and to the effect of the sustained release effect of the drug.Based on the research background above,Cyanidin-3-glucoside(C3G),the highest content in MA,was used as anthocyanin material in this study.Proteins with unique carrier characteristics were selected,includingβ-lactoglobulin(β-lg),zein and soy protein insolate(SPI).Firstly,the interactions between C3G andβ-lg,Zein and SPI were investigated by means of fluorescence quenching spectrum,three-dimensional fluorescence spectrum,infrared spectrum,ultraviolet absorption spectrum and circular dichroism spectroscopy spectrum in depth.Secondly,effects of proteins on the stability of MA was investigated by high performance liquid chromatography(HPLC)at different temperatures;Finally,SDS-PAGE was used to analyze the effect of MA on protein’s in vitro digestibility.The results were as follows:(1)C3G has a strong static quenching effect onβ-lg.The binding constant of C3G toβ-lg is 3.14×10~4L/mol,and the binding site n is nearly1,which indicated that C3G andβ-lg were strongly bound to form a complex with a molar ratio of 1:1 for C3G andβ-lg.In addition,the thermodynamic parameters show that C3G andβ-lg are mainly bound by hydrophobic forces.In the process of interaction,the peptide chain ofβ-lg is dissociated and stretched,and the content of secondary structure changes correspondingly.In addition,the molecular docking results further indicate that C3G is mainly bound to the hydrophobic site inside theβ-barrel ofβ-lg and hydrogen bonding forces are also involved in the interaction.Meanwhile,the stability test shows thatβ-lg can effectively inhibit the thermal degradation of MA after heating at 80℃ and 95℃ respectively and acts as a protective role.During gastric and duodenal in vitro digestion,the addition of MA did not affect the hydrolysis ofβ-lg under the action of digestive enzymes,and had no significant effect on the digestibility ofβ-lg.(2)The quenching of zein by C3G is the result of the combined effect of dynamic quenching and static quenching.Zein has a strong binding affinity for C3G,C3G interacts with zein mainly through hydrogen bonding and van der Waals forces to form a stable complex with a molar ratio of 1:1.In addition,infrared spectroscopy,ultraviolet absorption spectroscopy,circular dichroism spectroscopy,etc.show that the binding of C3G lead to the decrease of theα-helix content and the increase of theβ-sheet content of zein.Furthermore,the results of scanning electron microscopy(SEM)showed that the particle size of zein molecules decreased significantly,indicating the interacting forces between Zein and C3G were changed during the process.The stability test shows that zein has no significant protective effect on the thermal degradation of MA at80℃ and 95℃.In vitro digestion,the combination of MA and Zein has no significant influence on its digestibility,which has a positive effect on Zein as a carrier of polyphenols.(3)C3G quenches the SPI,and its quenching mode is static quenching.The binding constants and binding sites for the interaction between SPI and C3G were 2.65×10~4L/mol and 1.01,respectively at 298 K.The calculation of the thermodynamic parameters of the interaction between C3G and SPI indicated that the strong interaction between C3G and SPI via hydrophobic forces resulted in the formation of a stable 1:1 molar ratio of complex.The results of synchronous fluorescence spectra showed that the polarity and the hydrophobicity of the SPI tyrosine residue microenvironment was changed during the interaction.The results of three-dimensional fluorescence spectroscopy,ultraviolet absorption spectroscopy and circular dichroism spectroscopy showed that the spatial structure of SPI changed during the interaction process with the increase ofβ-sheet and decrease ofα-helix and random coil.The stability experiments showed that SPI could effectively inhibit the thermal degradation of MA after heating at 80℃ for 80 min.In vitro digestion,MA promoted the digestibility of SPI in vitro,especially the hydrolysis of the major allergenα-subunit components in SPI.It is speculated that the addition of MA can improve the utilization rate of SPI and reduce its sensitization.In addition,the addition of MA did not affect the hydrolysis of SPI under the action of trypsin,and had no significant effect on the intestinal digestibility of SPI in vitro.