alpha-chymotrypsin and Food-Hypersensitivity

We occasionally see egg-allergic children who develop contact urticaria to hen's egg despite the absence of the overt symptoms on ingestion. The mechanisms remain to be elucidated.. Twenty-one subjects with positive reactions to 20-min patch tests for egg-white antigens were divided into subgroups with positive (n = 10) and negative (n = 11) results to oral challenge tests by the same antigens. We measured IgE antibody for egg white and its components, and IgE-binding activities to digestive enzyme-treated ovomucoid by RAST inhibition.. There were no significant differences in IgE antibody titers to egg white (positive vs negative: 30.3% vs 15.3%, P=0.130), ovomucoid (21.5% vs 10.2%, P= 0.078), ovotransferrin (9.9% vs 3.7%, P = 0.105), and lysozyme (3.4% vs 2.9%, P=0.944), except ovalbumin (16.8% vs 5.6%, P=0.024), between the positive and negative subjects in the provocation tests. In contrast, the concentration (1.93 microg/ml) of pepsin-treated ovomucoid needed for 50% RAST inhibition in the challenge-positive subjects was significantly (P=0.0003) lower than that (114.9 microg/ml) of negative subjects. Similar but less significant differences were obtained when ovomucoid fragments treated with chymotrypsin (0.91 microg/ml vs 6.86 microg/ml, P=0.014) and trypsin (0.75 microg/ml vs 4.67 microg/ml, P= 0.041) were used as inhibitors.. We suggest that IgE antibodies from subjects showing contact urticaria despite the absence of reactions to the ingestion of egg white recognize the epitope(s) unstable to digestive enzymes.

Topics: Administration, Oral; Allergens; Animals; Child; Child, Preschool; Chymotrypsin; Conalbumin; Double-Blind Method; Egg White; Eggs; Female; Food Hypersensitivity; Humans; Immunoglobulin E; Infant; Male; Muramidase; Ovalbumin; Ovomucin; Pepsin A; Placebos; Radioallergosorbent Test; Trypsin; Urticaria

Food protein allergies are a major global concern. Hydrolysis of food proteins reduces their allergenicity, but another novel approach is the covalent attachment of polysaccharides to proteins via the Maillard reaction (i.e., glycation), which blocks some IgE binding epitopes on the protein allergen. We wanted to examine whether enzymatic hydrolysis, combined with glycation, could further reduce IgE binding for people with a cow milk protein allergy. Whey protein isolate (WPI) was hydrolyzed by immobilized trypsin and chymotrypsin to degree of hydrolysis (DH) values of 17 to 27%. Immobilized enzymes were used to avoid heat-treating the hydrolysate (to inactivate the enzymes, because heating could also affect the IgE binding ability of the protein). The resultant whey protein isolate hydrolysates (WPIH) were then glycated with 10-kDa dextran (DX) in aqueous solutions held at 62°C for 24 h. We analyzed the molar mass (M

Topics: Allergens; Animals; Cattle; Child; Chymotrypsin; Dextrans; Epitopes; Female; Food Hypersensitivity; Glycosylation; Humans; Hydrolysis; Immunoglobulin E; Maillard Reaction; Milk Hypersensitivity; Protein Hydrolysates; Trypsin; Whey Proteins

Many soybean protein products are processed by enzymatic hydrolysis to attain desirable functional food properties or in some cases to reduce allergenicity. However, few studies have investigated the effects of enzymatic hydrolysis on the allergenicity of soybean products. In this study the allergenicity of soybean protein isolates (SPI) hydrolyzed by Alcalase, trypsin, chymotrypsin, bromelain, or papain was evaluated by IgE immunoblots using eight soybean-allergic patient sera. The biological relevance of IgE binding was evaluated by a functional assay using a humanized rat basophilic leukemia (hRBL) cell line and serum from one subject. Results indicated that hydrolysis of SPI by the enzymes did not reduce the allergenicity, and hydrolysis by chymotrypsin or bromelain has the potential to increase the allergenicity of SPI. Two-dimensional (2D) immunoblot and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the chymotrypsin-hydrolyzed samples indicated fragments of β-conglycinin protein are responsible for the apparent higher allergenic potential of digested SPI.

Topics: Chymotrypsin; Food Hypersensitivity; Glycine max; Humans; Hydrolysis; Immunoglobulin E; Soybean Proteins; Subtilisins; Trypsin

This study was performed to examine how the characteristics of soybean 2S protein influence allergenicity after enzymatic hydrolysis. Soybean 2S protein was extracted and enzymatic hydrolysis was performed using pepsin and chymotrypsin. Allergenicity was observed using soybean-sensitive patients' sera.. Only 13.3% (6/45) of soybean-sensitive patients reacted to soybean Kunitz trypsin inhibitor (SKTI), known as the major allergen of soybean 2S protein. After peptic hydrolysis for 90 min at pH 1.2, the intensity of SKTI decreased to 25% but was still visible on SDS-PAGE. Chymotryptic hydrolysis following peptic hydrolysis at pH 8 for 60 min showed a limited hydrolytic effect on soybean 2S protein. Peptic hydrolysis of soybean 2S protein partially reduced the allergenicity of soybean 2S protein, while chymotryptic hydrolysis following peptic hydrolysis increased slightly the allergenicity.. Food allergy caused by soybean 2S protein occurred in part of the soybean-sensitive patients. SKTI was partially digested after peptic hydrolysis for 90 min. The allergenicity was decreased with peptic hydrolysis, while subsequent treatment of chymotrypsin increased slightly the allergenicity.

Topics: Allergens; Child, Preschool; Chymotrypsin; Female; Food Hypersensitivity; Glycine max; Humans; Hydrolysis; Infant; Male; Pepsin A; Protein Hydrolysates; Soybean Proteins; Trypsin Inhibitor, Kunitz Soybean; Trypsin Inhibitors

Fish roe, a nutritious food, is favored by consumers, but has also been confirmed to be allergenic in salmonid fish. However, little information is available in other fish species. To determine the allergen in the roe of large yellow croaker (Pseudosciaena crocea), crude extracts were incubated with sera of allergic patients. The major allergen was purified by column chromatography methods, revealing a single band with 16 kDa and was confirmed as β'-component (β'-c) by mass spectrometry. The results of physicochemical characterization showed that β'-c was a glycoprotein and was relatively stable following thermal or acid/alkali treatment. Furthermore, β'-c was easily degraded by pepsin, but was resistant to trypsin and α-chymotrypsin. After treatment with different processing methods, including Maillard reaction (MR), ultraviolet radiation (UVR), ultrasound-heat (UH), and retorting (RT), the IgG-binding activity of β'-c decreased obviously by MR, but decreased slightly by UVR and UH. Cross-immunoreactivity results of the allergens in the roes of different species revealed that β'-c was a specific allergen in teleostean, and the cross-immunoreactivity between the roe of large yellow croaker and other kinds of fish roe was relatively strong.

Topics: Adult; Allergens; Amino Acid Sequence; Animals; Antibody Formation; Chemical Phenomena; Chymotrypsin; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Female; Fish Proteins; Food Hypersensitivity; Humans; Hydrogen-Ion Concentration; Male; Molecular Sequence Data; Perciformes; Seafood; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Temperature; Ultraviolet Rays; Young Adult

Pulsed ultraviolet light (PUV), a novel technology most commonly used for microbial inactivation, has recently been employed to effectively mitigate food allergens in peanuts, soybean, shrimp, and almond. Putative mechanisms for the efficacy of PUV in reducing allergen reactivity include photothermal, photochemical, and photophysical effects. To date, there are no published data highlighting the effects of in vitro simulated gastric and intestinal digestion on the stability of PUV reduced allergen reactivity of food. In this study, PUV-treated shrimp extracts were subjected to simulated gastric fluid containing pepsin and simulated intestinal fluid containing trypsin and chymotrypsin, and then tested for changes in allergen potency. SDS-PAGE showed no major band deviation between undigested and digested PUV-treated shrimp extracts. IgE binding to tropomyosin remained markedly decreased as seen in Western blot analysis. Total shrimp allergen reactivity remained unchanged following in vitro peptic digestion and was markedly reduced following in vitro intestinal digestion as illustrated in indirect ELISA. The PUV reduced shrimp allergens remained at a low level under the in vitro simulated digestive conditions. The results inferred that PUV could be a potential method to create less allergenic shrimp products that would remain at a low allergen level under human gastric and intestinal digestive conditions.

Topics: Allergens; Animals; Arthropod Proteins; Blotting, Western; Chymotrypsin; Digestion; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Food Hypersensitivity; Immunoglobulin E; Penaeidae; Pepsin A; Seafood; Tropomyosin; Trypsin; Ultraviolet Rays

Non-specific lipid transfer proteins (ns-LTPs) are major food allergens of the Rosaceae family. The severity of allergic reactions often relates to resistance of the allergen to digestion. Thus, it is important to evaluate the digestibility of these proteins and characterise the peptides generated in the gastrointestinal tract.. Simulated gastrointestinal digestion of purified allergen Pru ar 3 was performed using pepsin for the gastric phase in aqueous HCl at pH = 2 and chymotrypsin and trypsin for the intestinal phase in aqueous NH(4)HCO(3) at pH = 7.8. The peptide mixture obtained was analysed by ultra-performance liquid chromatography/electrospray ionisation mass spectrometry (UPLC/ESI-MS). Peptide sequences were identified by comparing their molecular mass to that obtained by in silico digestion, and were confirmed by the ions obtained by in-source fragmentation. Semi-quantification was performed for the intact protein by comparison with internal standards.. The resistance to gastrointestinal digestion of Pru ar 3 allergen was evaluated to be 9%. This value is consistent with that found for grape LTP, but much lower than the resistance found for peach LTP (35%). All the peptides generated were identified by ESI-MS on the basis of their molecular mass and from the ions generated from in-source fragmentation. Apart from low molecular mass peptides, five high molecular mass peptides (4500-7000 Da) containing disulphide bridges were identified. ESI-MS of the intact protein indicated a less compact folded structure when compared to that of the homologous peach LTP.. An extensive characterisation of the peptides generated from the gastrointestinal digestion of Pru ar 3 allergen was performed here for the first time via UPLC/ESI-MS analysis. The digestibility of the allergen was evaluated and compared with that of other LTPs, demonstrating that only a small amount of undigested protein remains, and that specific proteolytic action involves immunodominant epitopes. These data might explain the lower allergenicity of apricot LTP compared to peach LTP, despite their high sequence homology.

Topics: Adult; Amino Acid Sequence; Antigens, Plant; Chromatography, High Pressure Liquid; Chymotrypsin; Female; Food Hypersensitivity; Gastric Acid; Humans; Hydrochloric Acid; Hydrogen-Ion Concentration; Immunoblotting; Immunoglobulin E; Male; Middle Aged; Molecular Sequence Data; Peptide Fragments; Protein Stability; Prunus; Sequence Alignment; Spectrometry, Mass, Electrospray Ionization; Trypsin; Young Adult

The specific effects of heat treatment and/or addition of low/high-methylated pectin (LMP/HMP) on the allergenicity of beta-lactoglobulin (beta-Lg) and its hydrolysis products were investigated through a two-step in vitro digestion approach. beta-Lg was first hydrolyzed by pepsin and then by a trypsin/chymotrypsin (T/C) mixture done in a dialysis bag with a molecular weight cutoff of 1000. The protein digestion was followed by SDS-PAGE electrophoresis performed on each digestion product, and their in vitro allergenicity was analyzed by immunoblotting. Such procedure was applied on beta-Lg samples mixed with the two kinds of pectin before or after heating (80 degrees C, 25 min) to determine the respective impact of heat treatment and pectin addition. Heat denaturation improved significantly the susceptibility of beta-Lg against the pepsin and the T/C. This effect, which was coupled to a reduction in immunoreactivity of the digested beta-Lg, appeared to be distinctively modulated by LMP and HMP. Through nonspecific interaction with the beta-Lg, pectin could reduce the accessibility of cleavage sites and/or epitope sequences. This mechanism of action is discussed in relation to the intra- and intermolecular interactions between beta-Lg and pectin initiated under the experimental conditions.

Topics: Allergens; Chymotrypsin; Food Hypersensitivity; Hot Temperature; Hydrolysis; Lactoglobulins; Methylation; Particle Size; Pectins; Pepsin A; Protein Denaturation; Trypsin

For application to the treatment of wheat-sensitive allergy, we developed a practical method for producing a haptenic peptide mixture, and evaluated its haptenic properties. Wheat flour was hydrolyzed with chymotrypsin to obtain a hydrolysate, and the diffusible fraction from the hydrolysate was subjected to gel-filtration. The resulting oligopeptide fraction could bind to wheat-specific IgE antibodies in the serum of patients allergic to wheat. Since this peptide mixture did not induce histamine release from basophils in these patients, it was concluded that the peptide mixture was composed of monovalent haptens. Histamine release from antigenstimulated basophils was almost completely inhibited when the basophils were preincubated with the haptenic peptide mixture. These results suggest that this haptenic peptide mixture can regulate the allergenic reaction in an epitope-specific manner.

Topics: Basophils; Chromatography, Gel; Chymotrypsin; Enzyme-Linked Immunosorbent Assay; Flour; Food Hypersensitivity; Haptens; Histamine Release; Humans; Immunoglobulin E; In Vitro Techniques; Peptide Fragments; Triticum

Twenty adult patients affected by atopic dermatitis (AD) were submitted to the p-aminobenzoic acid (PABA) test in order to evaluate the proteolytic pancreatic function. All but one showed a normal PABA test. These results seem to exclude a proteolytic maldigestion as a pathogenetic factor in AD.

Topics: 4-Aminobenzoic Acid; Adult; Chymotrypsin; Dermatitis, Atopic; Female; Food Hypersensitivity; Humans; Immunoglobulin E; Male; Pancreatic Diseases; Severity of Illness Index

Agarose gel electrophoresis (at pH 8.6) was used for qualitative determination of pancreatic enzymes in duodenal juice. The various enzymes were identified by staining techniques with specific chromogenic substrates, by quantitative determination of enzymes in eluates of gel slices, and by immunoelectrophoresis. The various protein bands corresponded to the following enzymes (from the anode to the cathode): chymotrypsin, trypsin, carboxypeptidase A, chymotrypsin, amylase (around the slit), lipase, elastase, and trypsin. The method was applied to a study of exocrine pancreatic function in 10 adults and 83 children suspected of having malabsorption. The duodenal juice, also analyzed for trypsin and amylase content, was collected in fasting condition and after a test meal of water. In patients with normal pancreatic function, all the enzyme bands were present and easy to recognize. In 87 patients carboxypeptidase A was present as two bands in 68 (80%), anodal trypsin as two bands in 39 (45%), and cathodal trypsin as two bands in 85 (97%). Electrophoresis of duodenal juice gave as much information from the fasting sample as after the test meal. Six children with pancreatic insufficiency (cystic fibrosis and Shwachmar's syndrome) had no or only faintly stained enzyme bands and a strongly stained albumin-containing band most anodally. The method is simple, rapid, and useful in routine work. The combination of this qualitative test with a quantitative one (e.g. trypsin determination) provides good information about exocrine pancreatic function.

Topics: Adolescent; Adult; Amylases; Carboxypeptidases; Celiac Disease; Child; Child, Preschool; Chymotrypsin; Duodenum; Electrophoresis; Electrophoresis, Agar Gel; Female; Food Hypersensitivity; Giardiasis; Humans; Immunoelectrophoresis; Infant; Malabsorption Syndromes; Male; Pancreatic Diseases; Pancreatic Elastase; Pancreatic Juice; Trypsin

Topics: 4-Aminobenzoic Acid; Animals; Chymotrypsin; Diarrhea; Dog Diseases; Dogs; Feces; Female; Food Hypersensitivity; Male; Pancreatitis; para-Aminobenzoates; Tyrosine; Xylose