In vivo methods for testing allergenicity show that high hydrostatic pressure hydrolysates of ß-lactoglobulin are immunologically inert.

The major milk allergen ß-lactoglobulin (ß-LG) exhibits an enhanced susceptibility to proteolysis under high hydrostatic pressure and this may be an efficient method to produce hypoallergenic hydrolysates. The aim of this work was to evaluate the in vivo allergenicity of 3 ß-LG hydrolysates produced under atmospheric pressure or high-pressure conditions. Hydrolysates were chosen based on previous experiments that showed that they provide a complete removal of intact ß-LG but differed in vitro IgE-binding properties that could be traced to the peptide pattern. The ability to trigger systemic anaphylaxis was assessed using C3H/HeJ mice orally sensitized to ß-LG. Outcome measures included symptom score, body temperature, serum mouse mast cell protease 1 (mMCP-1), and quantification of circulating basophils. Mast cell degranulation in vivo was assessed by passive cutaneous anaphylaxis. The 3 tested hydrolysates showed an abrogated allergenicity as revealed by the absence of anaphylactic symptoms and a decrease in body temperature. We demonstrated that the peptides present in the hydrolysates had lost their ability to cross-link 2 human IgE antibodies to induce mast cell degranulation, thus indicating that most of the peptides formed retain just one relevant IgE-binding epitope. The orally sensitized mouse model is a useful tool to address the in vivo allergenicity of novel milk formulas and demonstrates the safety of hydrolysates produced under high-pressure conditions.

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

Cows' milk allergy is the most frequent food allergy in children, and beta-lactoglobulin (beta-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 beta-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 beta-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 beta-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 beta-Lg, it is possible to select the conditions that quickly produce hydrolysates with reduced potential allergenicity that could be used in hypoallergenic foods.

Proteolytic pattern, antigenicity, and serum immunoglobulin E binding of beta-lactoglobulin hydrolysates obtained by pepsin and high-pressure treatments.

This study evaluates the use of high pressure to enhance pepsin hydrolysis of beta-lactoglobulin (beta-LG). The protein was subjected to high pressure before and during the proteolytic process. Analysis of remnant beta-LG, identification of the peptides produced, and evaluation of antigenicity (binding to commercial antibodies) and binding to IgE of allergic patients' sera were conducted in the hydrolysates. The results showed that the application of high pressure before the enzyme treatment slightly improved the proteolytic process but did not reduce the antigenicity or IgE binding of the hydrolysates. The application of high pressure during the enzymatic treatment enhanced the production of large intermediate fragments that were further proteolysed to smaller fragments as proteolysis proceeded for longer periods. At 400 MPa, all the intact protein was removed in minutes, simultaneously decreasing its antigenicity and serum IgE binding properties. However, for considerable reduction of the antigenicity and IgE binding of beta-LG, extending the incubation time with the enzyme was needed to reduce the amount of potentially allergenic intermediate peptides. Changes of beta-LG under pressure at acidic pH are discussed.