University of Vienna

Effects of Polypropylene and Polyethylene Terephthalate Microplastics on Trypsin Structure and Function.

Ingestion is one of the main exposure routes of humans and animals to microplastics (MPs). During digestion, MPs can interact with both gastrointestinal enzymes and food proteins. This study investigated the adsorption of trypsin onto polypropylene (PP) and polyethylene terephthalate (PET) MPs, the influence of MPs on trypsin structure and activity, and the in vitro trypsin digestibility of bovine meat extract (BME) sarcoplasmic proteins and BME α-Gal-carrying allergens (α-GalA) in the presence of PP and PET MPs. Trypsin, BME and α-GalA proteins interact with MPs, resulting in the formation of a soft (SC) and hard (HC) corona. This interaction is dynamic, leading to the adsorption and desorption of protein through time. Trypsin adsorption onto MPs results in slight structural changes in the SC and bulk solution, while a trypsin fraction residing in the HC loses most of its specific activity. The presence of MPs slightly slows down the digestibility of proteins with a mass of 38 kDa, while it does not affect the digestion of α-GalA. According to our results, it is unlikely that realistic concentrations of MPs in the intestine would have significant effects on meat extract proteins' and allergens' digestibility by trypsin. We confirmed that during trypsin digestion, the corona on PP and PET MP is composed of BME sarcoplasmic proteins and allergenic α-Gal-carrying proteins.

Bos d 13, A Novel Heat-Stable Beef Allergen.

Red meat, a staple food of Western diets, can also induce IgE-mediated allergic reactions. Yet, apart from the heat-labile protein serum albumin and the carbohydrate α-Gal, the molecules causing allergic reactions to red meat remain unknown. IgE reactivity profiles of beef-sensitized individuals are analyzed by IgE-immunoblotting with protein extracts from raw and cooked beef. Two IgE-reactive proteins are identified by peptide mass fingerprinting as myosinlight chain 1 (MYL1) and myosin light chain 3 (MYL3) in cooked beef extract and are designated Bos d 13 isoallergens. MYL1 and MYL3 are produced recombinantly in Escherichia coli. ELISAs proved their IgE reactivity and circular dichroism analysis showed that they represent folded molecules with remarkable thermal stability. In vitro gastrointestinal digestion experiments showed the higher stability of rMYL1 as compared to rMYL3. Exposure of a monolayer of Caco-2 cells to rMYL1 indicated that the molecule is able to cross intestinal epithelial cells without disturbing the integrity of the tight junctions, suggesting the sensitizing capacity of MYL1. MYLs are identified as novel heat-stable bovine meat allergens.

Effect of Dried Apple Pomace (DAP) as a Feed Additive on Antioxidant System in the Rumen Fluid.

The study aimed to evaluate the effect of dried apple pomace (DAP) as a feed additive on the enzymatic activity and non-enzymatic compounds belonging to the antioxidant system in cattle rumen fluid. The experiment included 4 Polish Holstein−Friesian cannulated dairy cows and lasted 52 days. The control group was fed with the standard diet, while in the experimental group, 6% of the feedstuff was replaced by dried apple pomace. After the feeding period, ruminal fluid was collected. The spectrophotometric technique for the activity of lysosomal enzymes, the content of vitamin C, polyphenols, and the potential to scavenge the free DPPH radical was used. The enzyme immunoassay tests (ELISA) were used to establish the activity of antioxidants enzymes and MDA. Among the rumen aminopeptidases, a significant reduction (p < 0.01) from 164.00 to 142.00 was observed for leucyl-aminopeptidase. The activity of glycosidases was decreased for HEX (from 231.00 to 194.00) and β-Glu (from 1294.00 to 1136.00), while a significant statistically increase was noticed for BGRD (from 31.10 to 42.40), α-Glu (from 245.00 to 327.00), and MAN (from 29.70 to 36.70). Furthermore, the activity of catalase and GSH (p < 0.01) was inhibited. In turn, the level of vitamin C (from 22.90 to 24.10) and MDA (from 0.36 to 0.45) was statistically higher (p < 0.01). The most positive correlations were observed between AlaAP and LeuAP (r = 0.897) in the aminopeptidases group and between β-Gal and MAN (r = 0.880) in the glycosidases group. Furthermore, one of the most significant correlations were perceived between SOD and AlaAP (r = 0.505) and AcP (r = 0.450). The most negative correlation was noticed between α-Gal and DPPH (r = −0.533) based on these observations. Apple pomace as a feed additive has an influence on lysosomal degradation processes and modifies oxidation−reduction potential in the rumen fluid. Polyphenols and other low-weight antioxidant compounds are sufficient to maintain redox balance in the rumen.

Gal d 7-a major allergen in primary chicken meat allergy.

Poultry meat can induce severe allergic reactions. So far, the molecules causing poultry meat allergy are largely unknown. Our aim was to identify and characterize poultry meat allergens. Profiles of patients' IgE reactivity to chicken muscle were analyzed in immunoblots, and proteins recognized by the majority of patients were subjected to peptide mass fingerprinting. A 23-kDa IgE-reactive protein was identified as myosin light chain 1, designated Gallus domesticus 7 (Gal d 7). Recombinant Gal d 7 was produced in Escherichia coli. The protein's IgE reactivity was analyzed in ELISA experiments, and cross-reactivity with allergens of other poultry species was assessed in inhibition immunoblots. Fold and thermal stability were evaluated by circular dichroism analysis, and enzymatic stability was investigated using in vitro gastrointestinal digestion assays. Recombinant Gal d 7 represents a properly folded, predominantly α-helical protein and displays IgE-binding activity comparable to that of its natural counterpart. IgE reactivity analysis in 28 patients allergic to chicken meat revealed that Gal d 7 is a major allergen for patients primarily sensitized to chicken meat. Furthermore, Gal d 7-cross-reactive allergens were also detected in other poultry species, suggesting that recombinant Gal d 7 can be used as a diagnostic marker allergen for poultry meat allergy. The high thermal stability, refolding capacity, and resistance to gastrointestinal enzymes might explain why Gal d 7 can act as a potent sensitizing agent. Gal d 7 represents a novel major chicken meat allergen. Recombinant Gal d 7 could be used for diagnosis of genuine poultry meat sensitization.

Only α-Gal bound to lipids, but not to proteins, is transported across enterocytes as an IgE-reactive molecule that can induce effector cell activation.

The oligosaccharide galactose-α-1,3-galactose (α-Gal), present in mammalian proteins and lipids, causes an unusual delayed allergic reaction 3 to 6 hours after ingestion of mammalian meat in individuals with IgE antibodies against α-Gal. To better understand the delayed onset of allergic symptoms and investigate whether protein-bound or lipid-bound α-Gal causes these symptoms, we analyzed the capacity of α-Gal conjugated proteins and lipids to cross a monolayer of intestinal cells. Extracts of proteins and lipids from beef were prepared, subjected to in vitro digestions, and added to Caco-2 cells grown on permeable supports. The presence of α-Gal in the basolateral medium was investigated by immunoblotting, thin-layer chromatography with immunostaining and ELISA, and its allergenic activity was analyzed in a basophil activation test. After addition of beef proteins to the apical side of Caco-2 cells, α-Gal containing peptides were not detected in the basolateral medium. Those peptides that crossed the Caco-2 monolayer did not activate basophils from an α-Gal allergic patient. Instead, when Caco-2 cells were incubated with lipids extracted from beef, α-Gal was detected in the basolateral medium. Furthermore, these α-Gal lipids were able to activate the basophils of an α-Gal allergic patient in a dose-dependent manner. Only α-Gal bound to lipids, but not to proteins, is able to cross the intestinal monolayer and trigger an allergic reaction. This suggests that the slower digestion and absorption of lipids might be responsible for the unusual delayed allergic reactions in α-Gal allergic patients and identifies glycolipids as potential allergenic molecules.

The significance of chicken meat allergy without sensitization to egg proteins.