Casein and rice protein hydrolysates: molecular weight distribution analysis and in vitro potential allergenicity assessment

Resumen del póster presentado al 43rd Annual Meeting of European Society for Paediatric Gastroenterology, Hepatology and Nutrition celebrado en Estambul (Turquia) del 9 al 12 de junio de 2010.-- et al.[Objectives and Study]: Infant Formulas for infants with cow's milk protein allergy (CMA) are be based on extensively hydrolysed milk protein or hydrolysed rice protein. The goal of this study was to characterize molecular weight distribution of two casein and two rice hydrolysates and its IgE reactivity [Methods]: Molecular size distribution was measured with HPLC Size exclusion chromatography (SEC) (GE Healthcare, Superdex 30 pg Column) with Phosphate Buffer 50mM NaCl 0,15 M, pH = 7,2, Flux 1 ml/min), and detection at 214 nm. Absolute molecular weight was determined by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry method (MALDI-TOF), a rapid and sensitive quantitative method with high resolution for peptides. The IgE reactivity was evaluated using sera of patients with clinically demonstrated allergy to cows milk proteins that contains at least 20 KU/L of specific IgE antibodies towards milk proteins, measured using FEIACAP System (Pharmacia diagnostics, Uppsala, Sweden). IgE binding was measured using an indirect ELISA coupled to a signal amplification system. [Results]: SEC and MALDI-TOF allowed us to characterize hydrolysates in terms of peptide size abundance distribution. The percentage of peptides with a molecular weight under 2.000 Daltons (Da) varied from 85% to 95% Absolute maximum molecular weights detected by MALDI-TOF ranged from 1.968 to 2.685 Da. Hydrolysates presented no reactivity against IgE in serum of allergic patients when they were used at concentrations similar to native protein (0.0025 mg/ml). However, when they were used in concentrated form (1000x) they showed different degrees of reactivity. [Conclusion]: Precise molecular weight characterization and IgE in vitro reactivity may be useful tools to evaluate the appropriateness of protein hydrolysates for formula feeding of infants with cow's milk protein allergy (CMA) as screening test previous to clinical trials in humans.Peer Reviewe

Hydrolysis under high hydrostatic pressure as a means to reduce the binding of β-lactoglobulin to immunoglobulin E from human sera

7 pages.-- PMID: 18680946 [PubMed].Cows' milk allergy is the most frequent food allergy in children, and β-lactoglobulin (β-Lg) is a major allergen. Milk-based hypoallergenic ingredients are manufactured by enzymatic hydrolysis, a process that could be improved by the application of high-pressure treatments. This study showed that the treatment of β-Lg dissolved in buffer with chymotrypsin and trypsin under high pressure for relatively short times accelerated proteolysis by leading to a rapid removal of the intact protein. The rapid proteolysis of the β-Lg substrate under pressure led to the production, in 20 min, of hydrolysates with lower immunoglobulin (Ig) G binding than those produced in 8 h (chymotrypsin) or 48 h (trypsin) at atmospheric pressure. However, those hydrolysates retained some residual IgE-binding properties that could be traced to the preferential release, during the initial stages of proteolysis, of peptides containing IgE epitopes, such as (Val-41-Lys-60), (Leu-149-Ile-162), and (Ser-21-Arg-40). The formation of these fragments was favored when proteolysis was conducted under high pressure due to the preferential hydrolysis of Arg-40 and Arg-148 by trypsin, and Tyr-42 and Leu-149 by chymotrypsin, all located at the dimer interface of β-Lg or very close to it. Although our results do not support that trypsin and chymotrypsin under high pressure selectively address the allergenic regions of β-Lg, it is possible to select the conditions that quickly produce hydrolysates with reduced potential allergenicity that could be used in hypoallergenic foods.Peer reviewe