Conformational Changes Of β-Lactoglobulin Induced By Dynamic High Pressure Microfluidization In Relation With Antigenicity

β-lactoglobulin (β-Lg) is a major whey protein found in the milk and is a valuable ingredient in food manufacturing due to its high content of essential amino acids and versatility in terms of functional properties (emulsifying ability, foaming ability, and gelling ability). However, β-Lg has been recognized as an allergenic component of bovine milk. In more than 82% of allergnic patiens, the β-Lg is the main elicitor of milk allergies for children and infants. The allergenic potential of milk protein depends in part on the integrity of conformation epitopes and linear epitopes. Processes in food industry have a significant effect on the modification of conformational epitopes but a little influence on the linear epitopes. Dynamic high pressure microfludizition (DHPM) used in this study is an emerging technology. An advantage of this new technology is that DHPM promised to extend the shelf-life of foods without altering their nutritional and sensory quality. DHPM has been applied to improvement of the quality of milk. Therefore, the aim of this work was to evaluate the aggregation and conformational changes of β-Lg subjected to DHPM and to relate these changes to the functional properties and antigenicity of β-Lg.Fistly, aggregation changes of whey protein induced by dynamic high pressure microfluidization treatment have been investigated in relation with their functional properties. Whey protein was treated with HPM under pressure from 40 to 160 MPa. Functional properties (solubility, foaming and emulsifying properties) of whey protein concentrate (WPC) ultrafiltered from fluid whey were evaluated. The results showed significant modifications in the solubility (30%-59%) and foaming properties (20%-65%) of WPC with increasing pressure. However, emulsifying property of WPC treated at different pressures was significantly worse than untreated sample. In order to better understand the mechanism of the modification by HPM, the HPM-induced aggregation changes were examined using particle size distribution, scanning electron microscopy, and hydrophobicity. It was indicated that HPM induced two kinds of aggregation changes on WPC:deaggregation and reaggregation of WPC, which resulted in the changes of functional properties of WPC modified by HPM.The antigenic response of β-lactoglobulin (β-Lg), treated by dynamic high pressure microfluidization (DHPM) at different temperatures, was determined by an indirect competitive enzyme-linked immunosorbent assay using polyclonal antibodies from rabbit serum. DHPM treatment causes changes in the protein structure and may influence the antigenicity of β-Lg. DHPM treatment of β-Lg at 90 ℃ showed significant effects with the antigenic response of 5.2 ug mL-1 (untreated), 45 ug mL-1 (40 MPa),79 ug mL-1 (80 MPa),132 ug mL-1 (120 MPa) and 158 ug mL-1 (160 MPa). In combination with temperature treatment (70℃-90℃), the antigenic response enhanced as the temperature increased at 160 MPa. The β-Lg antigenicities were about 14 ug mL-1,108 ug mL-1 and 158 ug mL-1 at 70,80 and 90℃, respectively. However, the influence of DHPM pressures on the antigenic response of β-Lg standards was different. DHPM modified β-Lg standards showed a remarkable increase of antigenicity when treated to 80 MPA. Above 80 MPa, the antigenic response decreased.The present study was undertaken in account previous observations in attempt to unravel the relationship between the aggregation changes of β-Lg and antigenicity. β-Lg was treated by dynamic high-pressure microfluidization (DHPM). The effect of DHPM on the microstructure of β-lactoglobulin was investigated by determining particle size, scanning electron microscope, and autio force morphological. The results were shown that the microstructure of untreated β-Lg was consisting of aggregates, which were compact in the texture. With DHPM pressure increasing, the microstructure became looser and the size of β-Lg was much smaller. However, as pressure was up to 100 MPa and 150 MPa,β-Lg reaggregated and formed different morphology.Our previous research indicated that dynamic high pressure microfluidization had a significant effect on the antigenicity of β-Lg. In this study, conformational changes subjected to DHPM (0.1-160 MPa) have been investigated in relation with the antigenicity. With DHPM pressure increasing from 0.1 to 80 MPa, it was indicated that the deaggregation of β-Lg samples and mildly-unfolding of its molecule were accompanied by the increase of β-Lg antigenicity, which was reflected in the decrease of particle size, increase of free sulphydrys (SH) contents and β-strands contents, as well as reflected in slight exposure of aromatic amino acid residues. As pressure was above 80 MPa, the reaggregation of β-Lg samples may contribute to the decrease of antigenicity, which was reflected by increase of particle size, the formation of aggregates, the decrease of SH contents and β-strands contents, as well as the slight changes of aromatic amino acid residues. Aggregation and conformational changes of β-Lg under DHPM was related to its antigenicity.In this study, conformational changes of β-Lg subjected to DHPM combined with temperatures have been investigated in relation with the antigenicity. It was indicated that β-Lg was partial unfolded after treated by DHPM and different temperatures. The results showed that the intensity of β-Lg induced DHPM at 208 nm was increased with the temperature increasing. In addition, DHPM combined with temperature treatments had a significant effect on the secondary structure and intrinsic fluorescence of β-Lg. Combined with the results of antigenicity in previous work, it was indicated that the antigenicity changes of β-Lg induced by DHPM and temperature were related to the conformational changes. DHPM and temperature treatment may weaken the convalence bond in the β-Lg molecule, for example S-S bond, which resulted in the unfolding of protein. Meanwhile, with the protein unfolding, the hidden epitopes may be exposed or some new epitopes may be formed, contributed to those epitopes gained access to the antibodies and the increase of antigenicity.Our previous research revealed that dynamic high pressure microfluidization increased the antigenicity of β-Lg below 80 MPa. It was related to the unfolding of protein, which may change proteolytic susceptibility of β-Lg and modulate its antigenicity druing the digestion. In this study, the steady-state kinetics of tryptic hydrolysis of β-Lg subjected to DHPM (0.1-80 MPa) have been investigated in relation with the antigenicity. According to the steady-state kenictics analysis, the improved digestion of β-Lg was accompanied with the obvious decrease of antigenicity during the hydrolysis with pressure increasing, reflected by the increase of kc, the decrease of Km, the increase of over-all catalytic efficiency (kc/Km), and the increase of the binding volume. It was indicated that although DHPM can increase the antigenicity of β-Lg, the enhanced digestibility of β-Lg at elevated pressure contributed to a decrease of antigenicity during the hydrolysis.