Marija Stojadinovic

Binding affinity between dietary polyphenols and β-lactoglobulin negatively correlates with the protein susceptibility to digestion and total antioxidant activity of complexes formed.

Non-covalent interactions between β-lactoglobulin (BLG) and polyphenol extracts of teas, coffee and cocoa were studied by fluorescence and CD spectroscopy at pH values of the gastrointestinal tract (GIT). The biological implications of non-covalent binding of polyphenols to BLG were investigated by in vitro pepsin and pancreatin digestibility assay and ABTS radical scavenging activity of complexes formed. The polyphenol-BLG systems were stable at pH values of the GIT. The most profound effect of pH on binding affinity was observed for polyphenol extracts rich in phenolic acids. Stronger non-covalent interactions delayed pepsin and pancreatin digestion of BLG and induced β-sheet to α-helix transition at neutral pH. All polyphenols tested protected protein secondary structure at an extremely acidic pH of 1.2. A positive correlation was found between the strength of protein-polyphenol interactions and (a) half time of protein decay in gastric conditions (R(2)=0.85), (b) masking of total antioxidant capacity of protein-polyphenol complexes (R(2)=0.95).

Cross-linking of β-lactoglobulin enhances allergic sensitization through changes in cellular uptake and processing.

Cross-linking of proteins has been exploited by the food industry to change food texture and functionality but the effects of these manipulations on food allergenicity still remain unclear. To model the safety assessment of these food biopolymers, we created cross-linked bovine β-lactoglobulin (CL-BLG) by laccase treatment. The purpose of the present study was to compare the immunogenicity and allergenicity of CL-BLG with native BLG in a mouse model of food allergy. First, BALB/c mice were intragastrically sensitized and orally challenged with BLG or CL-BLG and BLG-specific serum antibodies and splenic leukocyte cytokine production and cell proliferation were measured. Hereafter, epithelial protein uptake was monitored in vitro and in vivo and the effects of BLG cross-linking on interactions with dendritic cells were analyzed in vitro. Sensitization of mice with CL-BLG resulted in higher levels of IgE, IgG1, and IgG2a. In contrast, a subsequent oral challenge with CL-BLG resulted in lower mast cell degranulation. Cross-linking of BLG reduced its epithelial uptake but promoted sampling through Peyers patches. Differences in endocytosis by dendritic cells (DCs) and in vitro endolysosomal processing were observed between BLG and CL-BLG. CL-BLG primed DCs induced higher Th2 response in vitro. Cross-linking of BLG increased its sensitizing capacity, implying that the assessment of highly polymerized food proteins is of clinical importance in food allergy. Moreover, manufacturers of foods or therapeutic proteins should pay considerate attention to the health risk of protein aggregation.

Interactions of epigallo-catechin 3-gallate and ovalbumin, the major allergen of egg white.

Polyphenols, the potent plant secondary metabolites, have beneficial effects on human health, but the mechanism(s) by which these effects are exerted is not well understood. Here, we present the detailed analysis of the interactions between the major green tea catechin, epigallo-catechin 3-gallate (EGCG), and the major dietary protein and allergen, ovalbumin (OVA). We show that EGCG binds to the pocket that partly overlaps with the previously identified IgE-binding region in OVA, and that this interaction induces structural changes in the allergen. Moreover, our ex vivo studies reveal that OVA binds IgE and stimulates degranulation of basophils, and that its uptake by monocytes proceeds at a slower rate in the presence of EGCG. This study provides further evidence in support of the proposed mechanism by which EGCG interactions with the food allergens contribute to its diverse biological activities and may impair antigen uptake by antigen-presenting cells.

One-step method for isolation and purification of native β-lactoglobulin from bovine whey.

BACKGROUND The major whey protein β-lactoglobulin (BLG) has been widely studied for its functional properties. The aim of this study was to develop an efficient, inexpensive and rapid one-step method for the isolation and purification of BLG while preserving its native structure. RESULTS BLG was purified from defatted whey obtained from raw cows milk by anion exchange chromatography. Protein purity and identity were determined using reverse phase high-performance liquid chromatography and mass spectrometry. Total BLG yield was 80% with protein purity from 97 to 99%. BLG isoforms A and B were separated into fractions of 91 and 99% purity respectively. The structure and native conformation of the isolated BLG were compared with those of standard commercial BLG by circular dichroism spectrometry, susceptibility to various crosslinking enzymes and enzyme-linked immunosorbent assay inhibition. CONCLUSION The proposed method is very useful for the rapid preparation of BLG suitable for studying antigenic and molecular characteristics of this protein, as well as the effect of food processing on these properties. The procedure requires only 1 day for the purification of about 300 mg of BLG from a single run using a small column (2.5 cm × 20 cm) of diethylaminoethyl Sephadex and has potential for scaling up.

The anti-cancer activity of green tea, coffee and cocoa extracts on human cervical adenocarcinoma HeLa cells depends on both pro-oxidant and anti-proliferative activities of polyphenols

It has been shown before that dietary polyphenols possess cancer chemopreventive effects. As cervical cancer is the second leading genital malignancy in women after breast cancer, the anti-cervical cancer effects of polyphenol extracts of commonly used beverages (green tea, coffee and cocoa) were tested and compared in HeLa cells. All the extracts induced apoptosis of HeLa cells, but green tea was the most potent. However, as opposed to green tea which induced a strong anti-proliferative response in HeLa cells, coffee and cocoa extracts promoted the proliferation of surviving cells. After short-term exposure, green tea and coffee extracts, but not cocoa, induced the formation of intracellular reactive oxygen species. Only the green tea extract increased the production of superoxide anion radicals and decreased reduced glutathione levels. Gene expression of Cu/Zn and Mn-superoxide dismutase or catalase was unaltered in cells treated with extracts, but green tea partially inhibited catalase activity. The cytotoxic activity of green tea and coffee extracts was partially inhibited by vitamin C. The in vitro anti-cervical cancer potency of tested polyphenol extracts is related to their pro-oxidant and anti-proliferative activities and are exhibited in the following order: green tea > coffee > cocoa, with only green tea showing both pro-oxidative and anti-proliferative action.

Hypoallergenic acid-sensitive modification preserves major mugwort allergen fold and delivers full repertoire of MHC class II-binding peptides during endolysosomal degradation

Modified allergens are a safer and more efficient alternative to natural allergens for specific immunotherapy. As the modification of an allergen can diminish its immunogenicity due to the alteration of T-cell epitopes, in this paper we study the effects of a reversible chemical modification of Art v 1, the main allergen of mugwort pollen, on its allergenicity and immunogenicity. Modification of Art v 1 by cis-aconitylation into a polyanionic derivative (CAA) did not result in any significant structural alteration. However, IgE-binding epitopes on CAA were blocked, resulting in a reduced IgE-binding and basophil activation. Both proteins induced proliferation of CD3+CD4+ T-cells in mugwort-allergic patients, but only unmodified allergens increased IL-4, IL-5 and IL-10 production. Rabbit and mouse anti-CAA antibodies exhibited cross-reactivity with native allergens and blocked human IgE-binding to Art v 1. Degradation of CAA by lysosomal fraction enzymes resulted in a similar set of peptides, harboring MHC class II-binding peptides, as unmodified proteins. Thus, cis-aconitylation modified Art v 1 had a significantly reduced allergenicity, whereas its immunogenicity was completely preserved. Acid-environment-responsive modification, which releases a full repertoire of native allergen epitopes within a particular site, can be considered a smart drug delivery system, which is able to deliver a therapeutically-effective dose in a controlled manner, and minimizes adverse side effects.

Application of Ion Exchanger in the Separation of Whey Proteins and Lactin from Milk Whey

Whey disposal represents a huge obstacle for dairy industry, being costly and problematic. On the other hand, it could be used as a starting material for isolation of some components that are valuable on the market. Whey processing is not an easy operation; it requires robust techniques of high volumetric throughput with minimal pretreatment of the starting material. For this purpose, ion exchangers can be used, due to their versatility, safety, and relative cheapness. Being a mixture of acidic and basic proteins, double-step ion exchange chromatography, involving both cation and anion exchangers, must be performed to obtain highly purified whey proteins. Development of new technologies, based on ion exchange, which can provide fast and efficient whey processing, provides maximal exploitation in environmentally safe way.