| China is rich in sweet potato leaf resources which an annual yield is about 500 million tons.Sweet potato leaves have a lot of polyphenols,it also has high antioxidant activity.However,the phenolic hydroxyl group of sweet potato leaves is active,and its stability is poor in the process of processing and storage,which limits their development and utilization.It has been shown that ?-lactoglobulin(?-Lg)can form complex with polyphenols.The complex can improve stability and antioxidant activity of polyphenols;High hydrostatic pressure treatment can promote forming.3,5-dicaffeoylquinic acid(3,5-CQA)which is main component of polyphenols in sweet potato leaves and ?-Lg were used as material in this thesis,Firstly,the antioxidant activity of 3,5-CQA:?-Lg complex were investigated under different ?-Lg concentration,temperature,pH value and salt ion concentration in vitro.On this basis,the effect of different pressure treatments on the antioxidant activity of 3,5-CQA:?-Lg complex under the same solvent condition was investigated;Then the effects of different pressure treatments on the surface hydrophobicity and particle size of 3,5-CQA:?-Lg complex were investigated by means of ANS fluorescence probe;Finally,the secondary structure,spatial conformation,and intermolecular forces of 3,5-CQA:?-Lg complex under different high hydrostatic pressure treatments were analyzed by means of circular dichroism,atomic force microscopy and high-pressure online fluorescence detection technology.The objective was ensure the effect of high hydrostatic pressure on the formation and antioxidant activity of complex,and provide theoretical support for the development and utilization of polyphenols from sweet potato leaves in food and other fields.The main results are as follows:(1)At atmospheric pressure,the antioxidant activity of 3,5-CQA:?-Lg complex under different molar ratios of 3,5-CQA to ?-Lg and solvent conditions(pH,temperature and NaCl solution concentration)was positively correlated with 3,5-CQA;The antioxidant activity of the complex was the strongest when the complex was prepared at the molar ratios of 3,5-CQA to ?-Lg at 5:1(pH7,35?)and without NaCl solution.ABTS free radical scavenging ability and Fe3+reducing ability of the complex were 15.82±0.52 ?g ACE/mL and 80.23±4.71 ?g TE/mL,respectively.On this basis,with the high hydrostatic pressure increase,the antioxidant activity of the complex improves.At 500 MPa(30min),the ABTS free radical scavenging ability and Fe3+reducing ability of the complex were 1.43 times and 1.28 times of that at atmospheric pressure,respectively.(2)At atmospheric pressure,the molar ratio of 3,5-CQA to ?-Lg and solvent conditions(pH,temperature,NaCl solution concentration)had significant effects on the physicochemical properties of complex.According to three physicochemical properties of solubility,surface hydrophobicity and particle size,Complex is stabilitity when 3,5-CQA to ?-Lg is 5:1(pH 7,35? and without NaCl solution).Different pressures and pressure times have significant effect on the physicochemical properties of the complex.When the pressure was 100 MPa(30min),the solubility of the complex can be increased from 428.73?g BSA/mL to 760.96 ?g BSA/mL,the surface hydrophobicity index decreased from 253.51 to 162.07,and the particle size decreased from 478.65 nm to 267.20 nm.(3)At atmospheric pressure,the molar ratio of 3,5-CQA to ?-Lg had slightly effect on the secondary structure of the complex.When the pH value is in the range of 3?7,with the increase of pH value,the?-sheet content of the complex decreases gradually,and the random coil content increase gradually.When the pH value is 9,the trend of the secondary structure of the complex is opposite.With the increase of temperature,the content of ?-sheet gradually increased,the content of random coil gradually decreased while the concentration of NaCl solution had no significant effect on the secondary structure of the complex;The results of fluorescence spectrum analysis show that the noncovalent complexes of 3,5-CQA and ?-Lg are mainly formed by hydrophobic interaction,electrostatic and hydrogen bonding at atmospheric pressure.With the increase of pressure,The ?-sheet content of complex decreased gradually,and the content of random coil increased gradually,which is conducive to the exposure of ?-Lg hydrophobic cavity(the binding site with small molecules),and its promote the formation of 3,5-CQA:?-Lg complex;The results of three-dimensional fluorescence spectrum shows that the high hydrostatic pressure treatment enhanced the hydrophobic interaction between phenol hydroxyl of 3,5-CQA and the hydrophobic amino acids in the hydrophobic cavity of ?-Lg.In conclusion,3,5-CQA:?-Lg complex have the highest solubility and antioxidant activity at pH7,35?.High hydrostatic pressure treatment makes the secondary structure of protein extend to more disordered direction,exposing more hydrophobic amino acids.Thus,the hydrogen bond and hydrophobic interaction between 3,5-CQA and hydrophobic amino acids of ?-Lg are enhanced,and a more stable and soluble noncovalent complex is formed. |
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