Rosa Chicón

Changes in the ovalbumin proteolysis profile by high pressure and its effect on IgG and IgE binding.

Egg proteins are responsible for one of the most common forms of food allergy, especially in children, and one of the major allergens is ovalbumin (OVA). With the aim to examine the potential of high pressure to enhance the enzymatic hydrolysis of OVA and modify its immunoreactivity, the protein was proteolyzed with pepsin under high-pressure conditions (400 MPa). Characterization of the hydrolysates and peptide identification was performed by reversed-phase high-performance liquid chromatography-tandem mass spectrometry (RP-HPLC-MS/MS). The antigenicity (binding to IgG) and binding to IgE, using the sera of patients with specific IgE to OVA, were also assessed. The results showed that, upon treatment with pepsin at 400 MPa, all of the intact protein was removed in minutes, leading to the production of hydrolysates with lower antigenicity than those produced in hours at atmospheric pressure. However, the exposure of new target residues only partially facilitated the removal of allergenic epitopes, because the hydrolysates retained residual IgG- and IgE-binding properties as a result of the accumulation of large and hydrophobic peptides during the initial stages of hydrolysis. These peptides disappeared at later stages of proteolysis, although reactivity toward IgG and IgE was not completely abolished. Some fragments identified in the hydrolysates (such as Leu124-Phe134, Ile178-Ala187, Leu242-Leu252, Gly251-Ile259, Lys322-Gly343, Phe358-Phe366, and Phe378-Pro385) carried previously identified IgE-binding epitopes. Because some of the peptides found, such as Phe358-Phe366, probably contain only one binding site for IgE, the possibility to use high pressure to tailor hydrolysates that contain mostly peptides with only one IgE-binding site, which may help the immune system to tolerate egg proteins, is suggested.

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

The major milk allergen β-lactoglobulin (β-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 β-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 β-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 β-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.

Influence of high hydrostatic pressure on the proteolysis of β-lactoglobulin A by trypsin

This work describes the effect of the hydrolysis time and pressure (0.1-400 MPa) on the proteolysis of beta-lactoglobulin A (beta-lg A) with trypsin, either conducting hydrolysis of beta-lg under pressure or hydrolysing beta-lg that was previously pressure treated. Pressurisation, before or during enzyme treatments, enhanced tryptic hydrolysis of beta-lg. Trypsin degraded pressure-modified beta-lg and pressure-induced beta-lg aggregates, favouring proteolysis to the intermediate degradation products: (Val(15)-Arg(40)), (Val(41)-Lys(69))S-S(Leu(149)-Ile(162)) and (Val(41)-Lys(70))S-S(Leu(149)-Ile(162)). These were further cleaved at the later stages of proteolysis to yield: (Val(15)-Tyr(20)), (Ser(21)-Arg(40)), (Val(41)-Tyr(60)), (Trp(61)-Lys(69))S-S(Leu(149)-Ile(162)) and (Trp(61)-Lys(70))S-S(Leu(149)-Ile(162)). Particularly, in the tryptic hydrolysates of pre-pressurized beta-lg, two other fragments linked by disulphide bonds: (Lys(101)-Arg(124))S-S(Leu(149)-Ile(162)) and (Tyr(102)-Arg(124))S-S(Leu(149)-Ile(162)), were found. These corresponded to rearrangement products induced by SH/SS exchange between the free thiol group of Cys(121) and Cys(160), that normally forms the disulphide bond Cys(66)-Cys(160). In the light of these results, structural modifications of beta-lg under high pressure are discussed.

Evaluation of allergenic potential of protein ingredients through in vitro methods

Protein hydrolysates are used in special infant formulas for dietary treatment of allergy to cow’s milk protein in infants. The aim of this study was to characterise two different milk casein hydrolysate (HCN-1 and HCN-2) and one whey protein hydrolysate (HWP), and to evaluate their potential for allergenicity in in vitro studies. The IgE reactivity was evaluated using sera of patients with clinically demonstrated allergy to cow’s milk proteins that contains ‡ 20 KU/l of specific IgE antibodies towards milk proteins, measured using FEIA-CAP System (Pharmacia diagnostics, Uppsala, Sweden). IgE binding was measured using an indirect ELISA coupled to a signal amplification system (ELAST ELISA amplification System, Perkin Elmer Life Sciences, Waltham, MA, USA. Each sample was also fractionated into three different fractions using Centriprep columns (Amicon): L-Fraction (>10 kDa), M-Fraction (<10 kDa–3 kDa) and S-Fraction (<3 kDa) and IgE reactivity was measured as previously described. In addition, reactivity against several commercial antibodies anti-casein, anti-b-lactoglobulin and anti-a-lactalbumin was measured in each product. 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 (1000 · ), HCN-2 showed the lower reactivity, whereas HCN-1 and HWP showed moderate reactivity (Fig. 1). Tests with protein fractions showed that reactivity in the low molecular weight fractions (<3000 Da) was lower than reactivity in the other two fractions. Again HCN-2 fractions also showed the lower IgE reactivity compared to CN-1 and HWP. HCN-1 showed reactivity against anti-caseins antibodies and HWP also showed reactivity against antibodies anti-b-lactoglobulin and anti-a-lactalbumin and to anti-caseins, whereas HCN-2 showed very low reactivity against anti-caseins. In conclusion, we have thus identified a casein hydrolysate that shows a very low in vitro reactivity (HCN-2). Even though clinical evidence is still a must, in vitro allergenic approach may help to predict reactivity in allergic individuals.