Josefina Belloque

Human immunoglobulin e (IgE) binding to heated and glycated ovalbumin and ovomucoid before and after in vitro digestion

This study focuses on the effect of heating and Maillard reaction (MR) on the in vitro digestibility and rabbit IgG- and human IgE-binding properties of ovalbumin (OVA) and ovomucoid (OM) to estimate the impact of processing on their allergenicity. With the human sera studied, heat treatment significantly reduced IgE binding to both OVA and OM, whereas MR reduced the IgE binding to OVA but increased IgE binding to OM. In contrast, heat treatment significantly favored OVA digestibility but glycation impaired it, and these treatments did not affect the digestibility of OM. The changes observed in the digestibility affected the immunogenicity of the digests accordingly, so that the higher the digestibility, the lower the antibody binding. Heat treatment and glycation by MR showed an influence on the potential allergenicity of the main egg white proteins that could be related to their resistance to denaturation and digestive enzymes.

Application of NMR spectroscopy to milk and dairy products

NMR spectroscopy is a technique in increasing use for dairy research. It provides us with unique information that can be applied to research or to quality control of dairy samples. In addition, it is a non-destructive and very versatile technique, providing data on the same sample under different parameters. This review intends to give an overview of the type of information that can be obtained, based on some results obtained in dairy research. It includes the application of NMR for qualitative and quantitative analysis, monitoring reactions in vivo, isotopic analysis, study of the physical state of milk fat and water, structural characterization and studies on the conformational and aggregation state of proteins.

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.

Release of galactose and N-acetylglucosamine during the storage of UHT milk

Abstract The changes in the concentration of monosaccharides and the influence of dephosphorylation of sugar phosphates thereon were studied in four batches of UHT milk, (two commercial and two experimental batches produced in a pilot plant) during storage at 10–30°C for 120 days. An increase in galactose and N-acetylglucosamine, together with a decrease in N-acetylglucosamine-1P and galactose-1P, were found, which were larger as the temperature of storage increased. The effects of microbial content and thermal treatment on these processes were evaluated. The contribution of dephosphorylation of the phosphorylated sugars to the total increment in N-acetylglucosamine and galactose was examined. While, in most cases, the monosaccharide increases found were consistent with the disappearance of phosphorylated sugars, changes in galactose during storage at 30°C seemed to be due to other processes, probably involving glycosidases, that may have potential detrimental consequences to milk stability.

Qualitative and quantitative analysis of phosphorylated compounds in milk by means of 31P-NMR

31P-NMR has been tested as a technique for determining the phosphorus content of milk samples in both the inorganic form (Pi) and bonded to caseins as phosphoserine (SerP). 31P-NMR made possible the simultaneous determination of Pi and SerP in approximately 1.7 h. The determination is based on measuring the area under the resonance in question and interpolating in a calibration curve prepared using external standards of known phosphorus contents and prepared in the same manner. The concentration area plots obtained were linear in the ranges 162-1993 mg P/l for Pi and 38-40.2 mg P/l for casein SerP. The repeatability of the method was good. When the same milk samples were analysed by both 31P-NMR and the classic colorimetric method the correlation was good. Polyphosphates added to commercial milk were also detected, as diphosphates. The NMR response for diphosphate was linear between 58 and 576 mg P/l and preliminary results indicated that quantitative analysis could be achieved in commercial milk.

Determination of the casein content in bovine milk by 31P-NMR.

The relative proportion of caseins to total protein is a parameter that can be used to control the protein quality in standardised milk, an increasing tendency in dairy industries. 31P-NMR was used to analyse the casein content of milk, by the quantitation of the area under the resonances belonging to SerP, and using methylenediphosphonic acid as internal standard. This procedure yielded good results, as similar values of caseins were obtained from N Kjeldahl and NMR analysis for slightly heated milk samples. Heating at 95 degrees C for 15 min did not alter the casein content results. Casein content of raw, pasteurised and UHT milks (25.6 +/- 1.4, 26.4 +/- 1.8, 25.5 +/- 1.6 g casein/l milk, respectively), obtained by NMR, were not significantly different, giving an average of 25.8 +/- 1.6 g casein/l for bulk liquid milk. This work concluded that 31P-NMR could be used as an alternative method to determine casein in raw, pasteurised, dry and UHT milks.

Changes in phosphoglyceride composition during storage of ultrahigh-temperature milk, as assessed by 31P-nuclear magnetic resonance: possible involvement of thermoresistant microbial enzymes.

Soluble phosphoglycerides were studied in ultrahigh-temperature (UHT) milk by 31P-nuclear magnetic resonance. It was shown that, during storage of UHT milk, manufactured from raw milk with poor microbial quality, glycerophosphocholine and glycerophosphoethanolamine disappeared in parallel with an increase in alpha-glycerophosphate (GP). Storage at 10, 20, and 30 degrees C showed a faster transformation as the temperature increased. UHT milk samples manufactured from raw milks with better microbial quality and submitted to severe heat processes did not display changes in phosphoglycerides during storage. Screening of commercial UHT milks showed variations regarding the presence of GP, while in pasteurized milk samples, the appearance of GP occurred when the commercial life had been exceeded. Inoculation of sterile milk with Pseudomonas fluorescens NCIB9046 and incubation at 10 degrees C supported that changes in phosphoglycerides could be the consequence of a phosphodiesterase activity of bacterial origin, able to survive UHT processing. A similar behavior was observed between this activity and proteolytic activity. The potential application of the detection of these compounds as spoilage predictor indices is discussed.

Degradation of natural phosphorylated compounds and added polyphosphates in milk by Pseudomonas fluorescens CECT378, Lactococcus lactis CECT539, and Kluyveromyces marxianus CECT10584.

The degradation of natural phosphorylated compounds (galactose-1-phosphate, N-acetyl-glucosamine-1-phosphate, glycerophosphoethanolamine, and glycerophosphocholine) and added phosphorylated compounds (diphosphate) in milk was investigated by phosphorus 31 nuclear magnetic resonance on the incubation of a sterile milk with Pseudomonas fluorescens CECT381, Lactococcus lactis CECT539, and Kluyveromyces marxianus CECT10584. This preliminary study showed that the degradation of these compounds was dependent on the compound, microorganism, and temperature of incubation. K. marxianus CECT10584 did not show any capability to degrade these compounds, and L. lactis CECT539 was only able to degrade diphosphate at its optimum growth temperature. P. fluorescens CECT381 was the most active strain and possessed more hydrolytic capabilities at 10 degrees C than at its optimum growth temperature. It is suggested that cold-induced enzymes are involved in the ability of P. fluorescens CECT381 to hydrolyze the natural phosphorylated compounds in milk. Consequent potential alterations of dairy products are discussed.