Glycation Of Milk Protein Under Dynamic High Pressure Microfluidization Treatment:Effects On IgE-Binding Ability And Conformation

In this study,spectroscopy was used to study the conformation structure and functional properties of milk proteins(α-lactalbumin and β-lactoglobulin)by dynamic high pressure microfluidization(DHPM)combined with glycation treatment.Orbitrap Fusion was used to study the effect of DHPM on the milk proteins glycation.Inhibition ELISA was used to investigate the Ig E-binding ability of milk proteins by DHPM combing with glycation treatment.The results are as following:(1)We studied structural and property changes of α-lactalbumin(α-LA)treated by DHPM treatments by analysis of SDS-PAGE,intrinsic fluorescence,surface hydrophobicity,circular dichroism spectra(CD),foaming property,emulsifying property,and antioxidant property.We found the molecular weight and secondary structure of α-LA remained unaffected by DHPM pretreatment.The decreased intrinsic fluorescence intensity and the increased surface hydrophobicity indicated that DHPM treatment induced tertiary structure changes of α-LA.The foaming properties and emulsifying properties of α-LA were found to be improved by DHPM treatment.The antioxidant properties remained unaffected by DHPM treatment.(2)We studied structural and property changes of α-lactalbumin(α-LA)glycated with galactose under DHPM treatments by analysis of SDS-PAGE,intrinsic fluorescence,surface hydrophobicity,CD,emulsifying property,antioxidant property,and immunoglobulin E(Ig E)binding ability.We found intrinsic fluorescence intensity and surface hydrophobicity of glycated α-LA under DHPM treatments was decreased.The contents of α-helix of α-LA was decreased,and the contents of β-strands of α-LA was increased.The emulsifying properties and antioxidant properties of α-LA were found to be improved by DHPM treatment.The Ig E binding ability was decreased.(3)The glycation extent of α-LA before and after DHPM was evaluated by MALDI-TOF and Orbitrap Fusion Mass Spectrometry.MALDI-TOF analysis showed there was no difference in molecular weight between native α-LA-galactose and DHPM treated α-LA-galactose.And the α-LA glycated by galactose with incorporation ratio was 10.Six galactose glycation sites have been identified by HCD/ETD Orbitrap Fusion mass spectrometry.DHPM pretreatment did not change the number of glycation site,however,the position of glycated amino acid was changed.The glycation degree of α-LA was improved by DHPM treatments.The results indicated the conformational of α-LA was partially changed by DHPM combined with glycation treatment.The results were agreement with the spectral results.(4)We studied structural and property changes of α-lactalbumin(α-LA)glycated with ribose under DHPM treatments by analysis of SDS-PAGE,MALDI TOF,intrinsic fluorescence,surface hydrophobicity,CD,emulsifying property,antioxidant property,and Ig E binding ability.The α-LA glycated by ribose with incorporation ratio was increased with the increased DHPM treatment pressures.We also found the incorportation ratio of α-LA glycated by ribose was bigger than the the incorportation ratio of α-LA glycated by galactose.We found intrinsic fluorescence intensity and surface hydrophobicity of glycated α-LA under DHPM treatments was decreased.The contents of α-helix of α-LA was decreased,and the contents ofβ-strands of α-LA was increased.The emulsifying properties and antioxidant properties of α-LA glycated by ribose were found to be improved by DHPM treatment.The Ig E binding ability was decreased.(5)We evaluated structural changes of β-lactoglobulin(β-LG)by DHPM at different pressures(80,120,and 160MPa)combined with glycation treatment by analysis of SDS-PAGE,thermal properties,protein surface hydrophobicity,and CD.We employed Inhibition ELISA to characterize the binding capacity of Ig E from patients’ sera with cow’s milk allergy on β-LG by DHPM at different pressures combing with glycation treatment.β-Lg treated after different DHPM methods and pressures yielded a significant discrepancy in Ig E-binding capacity.When β-Lg was pretreated by DHPM,the Ig E-binding capacity of β-Lg–galactose conjugates decreased with increasing pressure;However,the conjugates showed higher Ig E-binding capacity at 120 MPa than that at 80 and 160 MPa when theβ-Lg–galactose mixture was treated by DHPM.Results of thermal properties,intrinsic fluorescence spectroscopy,surface hydrophobicity,and CD spectra indicated the occurrence of protein unfolding,as well as the tertiary and secondary structural changes of β-Lg.The results suggested pretreatment by DHPM and glycation with galactose was a promising approach for eliminating the Ig E-binding capacity of β-Lg.(6)The high-resolution Oribitrap mass spectrometry was used to investigate the glycation sites and the extent of glycation,and the Ig E binding capacity was characterized by inhibition ELISA.The Ig E binding capacity of β-LG was reduced under DHPM combined with glycation treatment.DHPM pretreatment could promote the glycation reaction both in DSP value and the number of glycation sites.The addition of Lys60 and the increase of DSP values contributed to the decrease of Ig E binding capacity.Our results demonstrated that mass spectrometry analysis provided an overall picture of glycated β-LG for a better understanding of protein structure alteration.