Bogahawaththa, Dimuthu (2018) Processing-induced modifications of native bovine milk proteins in relation to immunogenicity. PhD thesis, Victoria University.

Abstract

Bovine milk proteins are a source of high-quality proteins in the human diet. Raw milk is subjected to different processing treatments prior to human consumption to ensure food safety and extend the shelf life. However, the thermal processing including high-temperature short-time (HTST) pasteurization and ultra-high temperature (UHT) treatment and alternative nonthermal methods including application of high pressure (HP) appear to modify the native properties of milk proteins. The processing induced modifications in protein structure, mainly denaturation and aggregation, and associated changes in epitopes can modulate the immunogenicity and potential allergenicity of milk proteins. The severity of some processing conditions appears to alter the native minor proteins including immunoglobulins (Ig), which may otherwise exert immunomodulatory properties in such a way as to prevent occurrence of allergies. Bovine or cow’s milk protein allergy (CMPA) is an abnormal immunological reaction to one or more milk proteins and it is the most prevalent food allergy among infants globally. Hence, the overall aim of this study was to identify the modification of native milk proteins induced by selected thermal (heating at 72 for 15 s and 100 °C for 30 s) and nonthermal processing conditions (application of HP at 400, 500, or 600 MPa for 15 min at 30 °C) and to establish their impact on modulation of in vitro immunogenicity as a means of envisaging potential allergenicity. Processing induced changes in secondary structure of proteins were studied by Fourier transform infrared spectroscopy (FTIR), and protein denaturation and aggregation were mainly examined using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC). Changes in antigenicity of milk proteins followed by processing was studied by enzyme-linked immunosorbent assay (ELISA). Modulation of in vitro immunogenicity was assessed based on the concentration of several cytokines secreted by human peripheral blood mononuclear cells (PBMCs) in response to native and processed milk proteins. Thermal denaturation of bovine IgG was studied alone and in the presence of major whey proteins. The two heating regimes studied provided the simulated thermal effect compared to HTST (72 °C/ 15 s) and UHT (100 °C/ 30 s equivalent to 140 °C/ 5 s in terms of denaturation of β-lactoglobulin) conditions. Simulated HTST conditions least impacted on the secondary structure of IgG and other whey proteins when they were present either alone or in mixtures of whey proteins. The heating at 100 °C for 30 s caused formation of covalent complexes of IgG alone, as well as in the mixtures, mainly through thiol-disulfide reactions. Under 100 °C /30 s treatment, bovine serum albumin (BSA) did not interact with IgG through thiol-disulfide reactions in a binary mixture of proteins (IgG+BSA). α-Lactalbumin (ALA) appeared to preferentially lead denaturation of whey proteins over β-lactoglobulin (BLG), in a protein mixture (BLG+ALA+IgG+BSA), while native whey contains another component that can inhibit this effect. The presence of other whey proteins did not contribute to thermal stability of IgG at 100 °C for 30 s. Residual antigenicity of a processed protein is a marker of potential allergenicity. Other milk proteins affect thermal denaturation of bovine BLG and modulate its antigenicity. Denaturation of BLG and altered antigenicity were studied in protein mixtures during 72 °C/ 15 s and 100 °C/ 30 s treatments. BLG denaturation, affected by other proteins, correlated with altered antigenicity. The treatment at 72 °C/ 15 s enhanced antigenicity in BLG+ALA mixture possibly due to exposed epitopes in unfolded structure, while it did not affect other protein mixtures. The treatment at 100 °C/ 30 s resulted in BLG-led protein aggregation by thiol/disulphide interactions and declined antigenicity by fragmentation and masking of epitopes to a different extent depending on the mixture. IgG contributed to diminish antigenicity in BLG+ALA+IgG mixture at 100 °C/ 30 s. The protein denaturation governed by ALA over BLG in BLG+ALA+IgG+BSA mixture, was possibly catalysed by BSA at 100 °C/30 s, resulting in a higher retention of antigenicity than other mixtures. In vitro immunogenicity of various native and thermally processed (72 °C / 15 s and 100 °C /30 s) bovine milk protein fractions, their mixtures, whey, and skim milk, was studied by analysing the immune response of T helper (Th) cells in human PBMCs. The secretion of Th types cytokines induced by the protein stimulants was quantified, while determining the heat-induced protein denaturation. Purified whey proteins, caseins and whey fraction, and skim milk, provoked substantial immune responses at various degrees, indicating their potent immunogenicity. The protein mixtures prepared using the fractionated whey proteins with or without caseins appeared less immunogenic in both native and heat-treated forms, implying their potential of producing less immunogenic dairy products. The treatment at 100 °C/ 30 s significantly altered the immunogenicity of most of the potent protein stimulants, which mostly coincided with their levels of protein denaturation. The treatment at 72 °C / 15 s caused least protein denaturation but altered the immunogenicity of several protein stimulants notably including heat-stable caseins and ALA. High pressure processing (HPP), conducted at 400, 500 or 600 MPa for 15 min at 30 °C, of raw skim milk was studied in comparison to HTST pasteurization (72 °C/ 15 s), considering protein denaturation and in vitro immunogenicity. HTST pasteurization least impacted denaturation of native proteins leading to mostly unchanged milk immunogenicity. HPP resulted in denaturation of whey proteins, mostly BLG and IgG, and disturbed structure of the casein micelle. HPP at 600 MPa caused protein aggregation, involving mainly BLG and κ-casein, through thiol disulphide interactions. ALA was least denatured subjected to all HPP conditions. The balance between expression of Th1 and Th2 type cytokines, which is believed to regulate adverse immune response, was initially shifted toward Th1 with increase in HP, then the immunogenic capacity of milk proteins diminished at 600 MPa. This could be related to exposure of T cell-specific linear epitopes followed by unfolding of protein structure firstly and masking of them by protein aggregation subsequently with increase in high pressure. In overall, the conditions applied in raw milk processing should be further optimised in considering modifications of native milk proteins and subsequent modulation of their immunogenicity, in addition to ensuring the food safety, to make the final dairy product both hygienic and hypoallergenic. Mild heat treatments (< 72 °C) or combined mild processing, for instance application of HP below 400 MPa in combination with low temperature (< 50 °C), would be able to fulfil aforementioned requirements.

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