Maja Krstic

Sensitizing potential of enzymatically cross‐linked peanut proteins in a mouse model of peanut allergy

SCOPE The cross-linking of proteins by enzymes to form high-molecular-weight protein, aggregates can be used to tailor the technological or physiological functionality of food products. Aggregation of dietary proteins by food processing may promote allergic sensitization, but the effects of enzymatic cross-linking of dietary proteins on the allergenic potential of food are not known. In this study, the bioavailability and the sensitizing or tolerizing potential of peanut proteins (PE) cross-linked with microbial tyrosinase from Trichoderma reesei and mushroom tyrosinase from Agaricus bisporus, were investigated. METHODS AND RESULTS The impact of cross-linking of PE on the in vitro bioavailability of fluorescein isothiocyanate-labeled peanut proteins was tested in a Caco-2 cell monolayer and by competitive ELISA. The in vivo allergenicity or capacity to induce oral tolerance in mice were measured by serum levels of PE-specific antibodies and T cell cytokine production after exposure to PE and cross-linked PE. CONCLUSION Enzymatic processing of peanut proteins by the two tyrosinases increased the bioavailability of major peanut allergen Ara h 2, but did not significantly change the allergenic or tolerizing properties of peanut. Enzymatic treatment of peanut proteins yielded cross-linked proteins with preserved molecular and immunological features of peanut allergens.

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.

Digestion by pepsin releases biologically active chromopeptides from C-phycocyanin, a blue-colored biliprotein of microalga Spirulina.

C-phycocyanin, the major protein of cyanobacteria Spirulina, possesses significant antioxidant, anti-cancer, anti-inflammatory and immunomodulatory effects, ascribed to covalently attached linear tetrapyrrole chromophore phycocyanobilin. There are no literature data about structure and biological activities of released peptides with bound chromophore in C-phycocyanin digest. This study aims to identify chromopeptides obtained after pepsin digestion of C-phycocyanin and to examine their bioactivities. C-phycocyanin is rapidly digested by pepsin in simulated gastric fluid. The structure of released chromopeptides was analyzed by high resolution tandem mass spectrometry and peptides varying in size from 2 to 13 amino acid residues were identified in both subunits of C-phycocyanin. Following separation by HPLC, chromopeptides were analyzed for potential bioactivities. It was shown that all five chromopeptide fractions have significant antioxidant and metal-chelating activities and show cytotoxic effect on human cervical adenocarcinoma and epithelial colonic cancer cell lines. In addition, chromopeptides protect human erythrocytes from free radical-induced hemolysis in antioxidative capacity-dependant manner. There was a positive correlation between antioxidative potency and other biological activities of chromopeptides. Digestion by pepsin releases biologically active chromopeptides from C-phycocyanin whose activity is mostly related to the antioxidative potency provided by chromophore.

Voltammetric determination of an antipsychotic agent trifluoperazine at a boron-doped diamond electrode in human urine

A simple and efficient procedure is described for electrochemical determination of trifluoperazine (TFP), a prominent compound in a large group of phenothiazine derivatives with potent physiological activity. This method is based on the electrochemical oxidation of TFP in Britton–Robinson buffer solution in pH 6 at a boron-doped diamond electrode. Cyclic voltammetry provided a four well defined oxidation peaks at +0.65, +0.83, +1.06 and +1.35 V (vs. Ag/AgCl/3 M KCl electrode). Differential pulse voltammetry was applied as a very sensitive analytical technique for the determination of micromolar amounts of TFP. Two oxidation peaks on higher potentials were chosen for quantification of TFP. Under optimized conditions, the analytical curves obtained were linear in the TFP concentration range of 1.0 to 37.0 μmol L−1, with a detection limit of 0.7 and 0.6 μmol L−1, respectively. The effect of interfering agents (common urinary compounds) appeared to be negligible confirming the favourable selectivity of the method. The proposed method was analytically applied by determination of trifluoperazine content in model human urine samples with good accuracy (recoveries varied from 97% to 104%). The developed approach could be beneficial in analysis of TFP in biological samples using a boron-doped diamond electrode as an up-to-date electrochemical sensor and could represent an inexpensive analytical alternative to separation methods.

Peptidomics of an in vitro digested α-Gal carrying protein revealed IgE-reactive peptides

The mammalian carbohydrate galactose-α1,3-galactose (α-Gal) causes a novel form of food allergy, red meat allergy, where patients experience severe allergic reactions several hours after red meat consumption. Here we explored gastric digestion of α-Gal glycoproteins using an in vitro model. Bovine thyroglobulin (BTG), a typical α-Gal carrying glycoprotein, was digested with pepsin. The resulting peptides were characterised by SDS PAGE, immunoblot and ImmunoCAP using sera from 20 red meat allergic patients. During pepsinolysis of BTG, a wide range of peptide bands was observed of which 14 to 17 kDa peptides remained stable throughout the gastric phase. The presence of the α-Gal epitope on the obtained peptides was demonstrated by an anti-α-Gal antibody and IgE from red meat allergic patients. The α-Gal digests were able to inhibit up to 86% of IgE reactivity to BTG. Importantly, basophil activation test demonstrated that the allergenic activity of BTG was retained after digestion in all four tested patients. Mass spectrometry-based peptidomics revealed that these peptides represent mostly internal and C-terminal parts of the protein, where the most potent IgE-binding α-Gal residues were identified at Asn1756, Asn1850 and Asn2231. Thus allergenic α-Gal epitopes are stable to pepsinolysis, reinforcing their role as clinically relevant food allergens.