Effect of high hydrostatic pressure on the structural properties and bioactivity of immunoglobulins extracted from whey protein
Graphical abstract
Introduction
Whey proteins contain biologically active components such as immunoglobulins, which impart a protective shield against infectious diseases (Severin & Wenshui, 2005). Oral intake of bovine immunoglobulin concentrates (BIC) has been reported to protect the human body from several infections caused by microorganisms such as rotavirus, Escherichia coli, Cryptosporidium, Streptococcus mutants, Candida albicans, Helicobacter pylori, etc. (Mitra et al., 1995; Tacket et al., 1988). Whey proteins can be used as a potential resource to extract immunoglobulins for the preparation of infant formulae as well as other hyperimmune foods that are effective against infectious diseases affecting children and immunocompromised individuals. Natural antimicrobials have advantages over synthetic and semi-synthetic antibiotics due to their low cost, efficient activity against microbes based on their polyclonal conformation and by imparting less stress to the microbial gut ecology. From a commercial point of view, their entry into the market is faster due to less stringent regulatory issues being natural ingredients (Jan, 2001).
The main molecular fractions are immunoglobulin A (IgA), immunoglobulin M (IgM) and immunoglobulin G (IgG), with the latter constituting 80% of the total milk immunoglobulins. This major immunoglobulin is a monomeric peptide, approximately 150 kDa, consisting of two heavy polypeptide chains and two light chains linked by disulphide bonds (Indyk, Williams, & Patel, 2008). Successful supplementation of immunoglobulins into food and nutraceutical formulations requires sound knowledge on their stability to various processing conditions that are employed industrially.
Conventional heat treatments are used in the food industry to attain acceptable microbial safety hence extending the shelf life of products. Commonly, this leads to loss of bioactivity in several ingredients including vitamins, antioxidants and proteins. Regarding the protein of interest in this work, investigations have been carried out to examine the thermal stability of immunoglobulins. This class of proteins is considered to be thermolabile, as they undergo thermal denaturation at temperatures above 75 °C (Chen, Tu, & Chang, 2000; Domínguez, Perez, & Calvo, 1997; Li-Chan, Kummer, Losso, Kitts, & Nakai, 1995). Increasingly, high pressure processing (HPP) is considered as an alternative non-thermal processing technique to inactivate pathogenic and spoilage microorganisms without affecting the biological activity of food ingredients. For any protein, the expression (i.e. retention) of biological activity depends on the thermodynamic stability of its three-dimensional native conformation following processing (Balny, 2006; Price, 2000). Some studies have reported that immunoglobulins in low solid systems are sensitive to HPP at a pressure greater than 276 MPa. Under these conditions, HPP results in a twenty five percentage loss in bioactivity of bovine IgG while heat treatment yields a significant reduction (close to 100%) in immunoactivity (Li et al., 2006).
This work deals with the determination of the effect of hydrostatic pressure on the structural properties of condensed preparations of immunoglobulins (>60% solids) through thermomechanical analysis. A micromolecular investigation on their secondary state under both atmospheric and pressurised conditions using infrared spectroscopy is also carried out to reveal changes in the native conformation following processing (McClements, 2002). Detailed quantitative information is achieved by resolving the amounts of beta sheets and helical coils at each stage of the analysis for both types of materials. An index of physical significance (network Tg) for the rationalization of the mechanical properties of these high solid systems during vitrification is also developed and, as far as we are aware, this is the first report on rheological properties of immunoglobulins at atmospheric and high pressure conditions.
Section snippets
Materials
Whey protein isolate (WPI), for the extraction of immunoglobulins, was obtained from MG Nutritionals, Murray Goulburn Co-operative Co Ltd, Victoria, Australia. According to the supplier's information, the composition of WPI was reported as 91.3% protein, 0.7% fat, 3.5% moisture, 3.8% ash and 0.44% lactose.
Extraction of bovine immunoglobulins from whey proteins
Immunoglobulins were extracted from whey protein isolate using a procedure of selective precipitation with ammonium sulphate. Saturated ammonium sulphate solution (SAS) was prepared by
Structural functionality of pressurised and atmospheric immunoglobulins
Immunoglobulins are the major bioactive fraction of whey proteins along with lactoferrins and other glycoproteins, which are highly thermolabile materials. In an attempt to overcome the shortcomings of thermal processing, we obtained immunoglobulins from WPI through selective salt precipitation and carried out first a typical electrophoresis run in an SDS-PAGE gel to assess the quality of our protein extracts. Fig. 1 reproduces the picture of SDS-PAGE for native and thermally treated
Conclusions
Protein systems at high levels of solids remain underresearched and, consequently, molecular mechanisms responsible for their structural properties are poorly understood. The present work is part of an effort to provide a comprehensive picture of the structural behaviour of such systems by examining the behaviour of immunoglobulins at atmospheric conditions and following application of a high-pressure protocol. Mechanical properties of both types of immunoglobulin preparations reflect coherent
References (34)
Review: what lies in the future of high-pressure bioscience?
Biochimica et Biophysica Acta
(2006)- et al.
Interactions of milk proteins during heat and high hydrostatic pressure treatments—A review
Innovative Food Science and Emerging Technologies
(2007) - et al.
Effect of heat treatment on the antigen-binding activity of anti-peroxidase immunoglobulins in bovine colostrum
Journal of Dairy Science
(1997) - et al.
Diffusion of short chain alcohols from amorphous maltose-water mixtures above and below their glass transition temperature
Carbohydrate Research
(2000) - et al.
Glass transition and enthalpy relaxation of amorphous lactose glass
Carbohydrate Research
(2006) - et al.
Kinetics of combined thermal and pressure-induced whey protein denaturation in bovine skim milk
International Dairy Journal
(2005) - et al.
Pressure-induced conformational changes in an antigen and an antibody and the implications on their use for hyperbaric immunoadsorption
Biochimica et Biophysica Acta
(1992) - et al.
Analysis of denaturation of bovine IgG by heat and high pressure using an optical biosensor
International Dairy Journal
(2008) Relation between the structure of matrices and their mechanical relaxation mechanisms during the glass transition of biomaterials: a review
Food Hydrocolloids
(2012)- et al.
Vibrational spectroscopy and conformation of peptides, polypeptides, and proteins
Advances in Protein Chemistry
(1986)
Stability of bovine immunoglobulins to heat treatment and processing
Food Research International
New insights into protein secondary structure from resolution-enhanced infrared spectra
Biochimica et Biophysica Acta
The thermal stability of immunoglobulin: unfolding and aggregation of a multi-domain protein
Biophysical Journal
Rheological and thermal properties of narrow distribution poly (ethyl acrylate)s
Macromolecules
Effects of high pressure on proteins
Food Reviews International
Thermal stability of bovine milk immunoglobulin G (IgG) and the effect of added thermal protectants on the stability
Journal of Food Science
Unexpected high pressure effects on the structural properties of condensed whey protein systems
Biopolymers
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