Modification of IgE binding during heat processing of the cow's milk allergen beta-lactoglobulin

The effect of heat treatment on the IgE binding ability of beta-lactoglobulin, as pure protein or in whole milk, was studied by inhibition of IgE antibody binding using FEIA-CAP inhibition. A slight but significant decreased IgE binding was seen between unheated and heat-treated beta-lactoglobulin solution at 74 degrees C (IC(50) = 2.03 and 3.59 microg/mL, respectively, p = 0.032). A more pronounced decrease was found at 90 degrees C with an IC(50) of 8.45 microg/mL (p = 0.014). The inhibition of IgE binding of milk after heat treatment at 90 degrees C was also significantly decreased (p = 0.007). However, at all heat treatments, a similar total amount of IgE antibodies could be inhibited at a sufficiently high concentration of beta-lactoglobulin. The inhibiting ability of beta-lactoglobulin was significantly impaired in some fermented acidified milk products such as yogurt as compared to that in nonfermented milk (p < 0.001). There was only a small difference of IgE binding between the native forms of genetic variants A and B.     

Riboflavin-sensitized photochemical changes in beta-lactoglobulin in an aqueous buffer solution as affected by ascorbic acid

The effects of ascorbic acid on the riboflavin-sensitized photochemical changes in beta-lactoglobulin in an aqueous buffer solution as determined by high performance gel permeation liquid chromatography (HPGPLC), insoluble protein content, and individual amino acid content during fluorescent light illumination were studied. The riboflavin-sensitized photochemical degradation of beta-lactoglobulin was effectively inhibited by ascorbic acid, and its inhibitory effectiveness was concentration dependent. The 0.1% ascorbic acid treatment showed 74.4% inhibition of beta-lactoglobulin degradation as determined by a HPGPLC during 6 h light illumination. Insolubility of beta-lactoglobulin in a buffer solution during light illumination was also effectively decreased by ascorbic acid treatment. The riboflavin-sensitized photochemical reduction of cysteine, histidine, lysine, methionine, and tryptophan in beta-lactoglobulin was high during 6 h fluorescent light illumination. The 0.1% ascorbic acid treatment exhibited 20.8% inhibition of total amino acid degradation in beta-lactoglobulin during 6 h light illumination, showing strong inhibitory activity against the degradation of arginine, aspartic acid, cystein, glycine, histidine, phenylalanine, proline, serine, and tryptophan.     

Changes in structures of milk proteins upon photo-oxidation

Changes in protein structures as a result of riboflavin-induced photo-oxidation were studied for six milk proteins: alpha-casein, beta-casein, kappa-casein, lactoferrin, alpha-lactalbumin, and beta-lactoglobulin. The milk proteins showed significant variability in sensitivity to photo-oxidation. After photo-oxidation, an increase in carbonyl content because of oxidation of tryptophan, histidine, and methionine, as well as formation of dityrosine, was observed for all proteins studied, although at very different levels. Generally, the increment was highest for alpha- and beta-casein and was lowest for lactoferrin. Loss of tryptophan because of photo-oxidation was well-correlated with the formation of the tryptophan oxidation products, N-formylkynurenine and kynurenine. Changes at the tertiary protein structure level were observed after photo-oxidation of the globular proteins, where tryptophan fluorescence emission indicated unfolding of alpha-lactalbumin and beta-lactoglobulin, whereas lactoferrin achieved a more compact tertiary structure. Changes in secondary structure were observed for alpha-lactalbumin and beta-lactoglobulin, whereas the secondary structure of lactoferrin did not change. Polymerization of alpha- and beta-casein and of lactoferrin was observed, whereas kappa-casein, alpha-lactalbumin, and beta-lactoglobulin showed little tendency to polymerize after photo-oxidation. Lability toward photo-oxidation is discussed according to the structural stabilities of the globular proteins.     

Homogenization conditions affect the oxidative stability of fish oil enriched milk emulsions: oxidation linked to changes in protein composition at the oil-water interface

Fish oil was incorporated into milk under different homogenization temperatures (50 and 72 degrees C) and pressures (5, 15, and 22.5 MPa). Subsequently, the oxidative stability of the milk and changes in the protein composition of the milk fat globule membrane (MFGM) were examined. Results showed that high pressure and high temperature (72 degrees C and 22.5 MPa) resulted in less lipid oxidation, whereas low pressure and low temperature (50 degrees C and 5 MPa) resulted in faster lipid oxidation. Analysis of protein oxidation indicated that especially casein was prone to oxidation. The level of free thiol groups was increased by high temperature (72 degrees C) and with increasing pressure. Furthermore, SDS-PAGE and confocal laser scanning microscopy (CLSM) indicated that high temperature resulted in an increase in beta-lactoglobulin adsorbed at the oil-water interface. This was even more pronounced with higher pressure. Less casein seemed to be present at the oil-water interface with increasing pressure. Overall, the results indicated that a combination of more beta-lactoglobulin and less casein at the oil-water interface gave the most stable emulsions with respect to lipid oxidation.     

Binding of 2-nonanone and milk proteins in aqueous model systems

Interactions of the model flavor compound 2-nonanone with individual milk proteins, whey protein isolate (WPI), and sodium caseinate in aqueous solutions were investigated. A method to quantify the free 2-nonanone was developed using headspace solid-phase microextraction followed by gas chromatography with flame ionization detection. Binding constants (K) and numbers of binding sites (n) for 2-nonanone on the individual proteins were calculated. The 2-nonanone binding capacities decreased in the order bovine serum albumin > beta-lactoglobulin > alpha-lactalbumin > alpha s1-casein > beta-casein, and the binding to WPI was stronger than the binding to sodium caseinate. All proteins appeared to have one binding site for 2-nonanone per molecule of protein at the flavor concentrations investigated, except for bovine serum albumin, which possessed two classes of binding sites. The binding mechanism is believed to involve predominantly hydrophobic interactions.     

Influence of high-intensity ultrasound and heat treatment in continuous flow on fat, proteins, and native enzymes of milk

The effect of continuous flow high-intensity ultrasound (with and without heat generation) on alkaline phosphatase, gamma-glutamyltranspeptidase, lactoperoxidase, whey proteins (alpha-lactalbumin and beta-lactoglobulin), casein, and fat was studied in milk. Results were compared with those obtained using a conventional heating system having similar processing conditions. Hardly any effect on enzymes was observed when ultrasound was applied without heat generation. The highest denaturation of enzyme and whey proteins was found in samples subjected to ultrasound and heat. At 61, 70, and 75.5 degrees C a synergistic effect between ultrasound and heat was observed for the inactivation of alkaline phosphatase, gamma-glutamyltranspeptidase, and lactoperoxidase, respectively. A noticeable synergism between ultrasound and heat was detected for alpha-lactalbumin and beta-lactoglobulin denaturation. No changes in the casein were observed after any of the conditions assayed. As a consequence of ultrasound effects, a substantial reduction (up to 81.5%) in the size of the fat globule was observed. When 70 and 75.5 degrees C were achieved during high-intensity ultrasonic homogenization, a better particle distribution was observed as compared to that obtained at lower temperatures. This work describes the influence of continuous flow high-intensity ultrasound on important milk components as a first step for future processing applications.     

Antioxidant impacts on volatile formation in high-pressure-processed milk

The effect of antioxidants on volatile formation in milk under high pressure was investigated. Raw milk samples with addition of either butylated hydroxyanisole (BHA), epicatechin, ascorbic acid, beta-carotene, or L-cystine were pressurized under 655 MPa at 75 degrees C for 3, 5, and 10 min. Formation of selected volatile compounds including aldehydes, ketones, and sulfur compounds was studied using headspace solid-phase microextraction and gas chromatography. BHA and epicatechin effectively inhibited the aldehyde formation. Ascorbic acid and beta-carotene also inhibited aldehyde formation but to a much lower extent. L-cystine was capable of inhibiting aldehyde and hydrogen sulfide formation. In general, the inhibition of volatile formation was proportional to the concentration of the added antioxidants. Reducing oxygen contents in milk also decreased aldehyde formation. Results suggested that the inhibition of volatile formation under high pressure could be similar to that under normal-pressure condition.     

Investigation of the lactosylation of whey proteins by liquid chromatography-mass spectrometry

Heat treatment of milk induces a reaction between the milk proteins and lactose, resulting in lactosylated protein species. The lactosylation of the two major whey proteins alpha-lactalbumin and beta-lactoglobulin was investigated by reversed phase liquid chromatography-mass spectrometry (LC-MS). Three sample series, consisting of aqueous model solutions of each whey protein separately and in mixture and whole milk, were heated for different time periods, and the progression of the lactosylation reaction was monitored. The observed degrees of lactosylation and the reaction kinetics showed that the lactosylation of beta-lactoglobulin was not influenced by the presence of other components, whereas the lactosylation of alpha-lactalbumin was enhanced in whole milk compared to the aqueous model systems. An in-depth evaluation of the LC-MS data yielded information regarding changes of physicochemical properties of the whey proteins upon lactosylation. Whereas retention time shifts indicated changes in hydrophobicity for both alpha-lactalbumin and beta-lactoglobulin, changes in the charge state distribution denoting conformational alterations were observed only for beta-lactoglobulin. The analysis of different liquid and solid milk products showed that the lactosylation patterns of the whey proteins can be used as indicators for the extent of heat treatment.     

Nuclear magnetic resonance spectroscopic study of beta-lactoglobulin interactions with two flavor compounds,gamma-decalactone and beta-ionone

Interactions between a well-characterized protein, beta-lactoglobulin, and two flavor compounds, beta-ionone and gamma-decalactone, were studied by 2D NMR spectroscopy. NMR spectra were recorded in aqueous solution (pH 2.0, 12 mM NaCl, 10% D(2)O) under conditions such that beta-lactoglobulin is present in a monomeric state. TOCSY and NOESY spectra were recorded on the protein and the complexes between protein and ligands. The spectra of the NH-CH(alpha) region showed the cross-signals due to the coupling between N- and C-bonded protons in the polypeptide backbone. The observed chemical shift variations in the presence of ligands can be assigned to changes in the protein conformation. It appears that the side chains of several amino acids are affected by binding of gamma-decalactone point into the central cavity (Leu46, Ile56, Met107, and Gln120), whereas binding of beta-ionone affects amino acids located in a groove near the outer surface of the protein (Leu104, Tyr120, and Asp129), as illustrated by molecular visualization. This NMR study provides precise information of the location of binding and confirms the existence of two different binding sites for aroma compounds on beta-lactoglobulin, which was suggested in previous competition studies by fluorometry or affinity chromatography and by structural information obtained from infrared spectroscopy.     

Identification of an IgE reactive peptide in hen egg riboflavin binding protein subjected to simulated gastrointestinal digestion

Riboflavin binding protein (RfBP) is a minor protein in hen egg; its potential involvement in egg allergy has seldom been studied. The aim of this work was to investigate the IgE binding capacity of RfBP before and after simulated gastrointestinal digestion. It was shown that digestion of RfBP mainly occurred during the gastric phase. The protein fragments resulting from the subsequent duodenal phase remained linked through disulfide bonds. Both the intact protein and its digests were subjected to inhibition ELISA with sera obtained from patients allergic to egg. The results revealed significant IgE binding to intact RfBP, whereas the digests showed reduced but substantial IgE binding levels, with serum-to-serum variability. The RfBP digests were then subjected to immunoblot with allergic patients' sera, and the IgE-reactive peptides were further analyzed by MALDI-TOF/TOF mass spectrometry for sequence determination. The RfBP sequence 41-84 was identified as a novel IgE binding peptide in patients allergic to egg.     

Effect of milk concentration on the irreversible thermal denaturation and disulfide aggregation of beta-lactoglobulin

The kinetics of beta-lactoglobulin (beta-LG) denaturation in reconstituted skim milk samples of various concentrations (9.6-38.4% total solids) over a wide temperature range (75-100 degrees C) was studied. The thermal denaturation of beta-LG had a reaction order of 1.5 at all milk solids concentrations and at all temperatures. The rate of denaturation of beta-LG was markedly dependent on the milk solids concentration and the heating temperature. At 75 degrees C, the thermal denaturation of beta-LG was retarded at higher milk solids concentrations. However, this retardation was less pronounced at higher temperatures so that a similar rate of denaturation was observed at all milk solids concentrations at 100 degrees C. From an examination of the level of disulfide-aggregated beta-LG, it was evident that most, but not all, of the denatured beta-LG was involved in disulfide-aggregated complexes, either with other denatured whey proteins or with the casein micelles. As with beta-LG denaturation, the rate of disulfide aggregation of beta-LG was markedly dependent on the milk solids concentration.     

Modification of bovine beta-lactoglobulin by glycation in a powdered state or in an aqueous solution: effect on association behavior and protein conformation

The effect of glycation with lactose on the association behavior and conformational state of bovine beta-lactoglobulin (beta-LG) was studied, using size exclusion chromatography, polyacrylamide gel electrophoresis, proteolytic susceptibility, and binding of a fluorescent probe. Two modification treatments were used, i.e., aqueous solution glycation and dry-way glycation. The results showed that the latter treatment did not significantly alter the nativelike behavior of the protein while the former treatment led to important structural changes. These changes resulted in a specific denatured beta-LG monomer, which covalently associated via the free thiol group. The homodimers thus formed and the expanded monomers underwent subsequent aggregation into a high molecular weight species, via noncovalent interactions. The association behavior of glycated beta-LG is discussed with respect to the known multistep denaturation/aggregation process of nonmodified beta-LG.     

Inhibition of bacteriophage m13 replication with esterified milk proteins

Esterified milk proteins [methylated (Met) or ethylated (Et) alpha-lactalbumin (ALA), beta-lactoglobulin (BLG), and beta-casein (BCN)], unmodified native milk proteins, and native basic proteins (calf thymus histone and hen egg white lysozyme) were tested for their antiviral activity against the bacteriophage M13 and for their influence on its replication (except BCN). All esterified milk proteins showed an antiviral activity against the bacteriophage M13, proportional to the extent of esterification and, hence, to the increased basicity of the modified proteins. Antiviral activity of 100% Met-BLG disappeared after its pepsinolysis but not after its trypsinolysis. The antiviral activity of Met-BLG was much higher than that of native basic proteins (histone and lysozyme). One hundred percent Met-BLG and 73% Et-BLG inhibited the replication of bacteriophage M13 completely, whereas 60% Met-ALA inhibited phage replication partially. Calf thymus histone inhibited the replication of bacteriophage M13 at a lower extent (20%) than Met- and Et-BLG (100% inhibition). Protein concentration, pH, and concentration of the Escherichia coli culture in the preincubation medium of the virus were other factors influencing antiviral activity. Interactions of esterified proteins with the phage DNA (phenol extracted) followed the same pattern as observed during studies of the inhibition of the phage replication: Met-BLG > Et-BLG > or = Met-ALA.     

Identification and site-specific relative quantification of beta-lactoglobulin modifications in heated milk and dairy products

During milk processing, proteins can be severely modified by oxidation, condensation, and Maillard reaction, leading to changes in their nutritional and technological properties. In this study, major modifications of beta-lactoglobulin, formed during the heating and processing of milk, were screened by mass spectrometry. For this purpose, beta-lactoglobulin was isolated from the milk samples by gel electrophoresis and analyzed by matrix-assisted laser desorption/ionization mass spectrometry after in-gel digestion with endoproteinase AspN. In heated milk, lactulosyllysine was detected at lysine 47 and 138 or 141 as well as methionine sulfoxide at methionine 7, 24, and 145. All these modifications increased gradually when raw milk was heated for 20, 40, and 60 min at 120 degrees C. The major modifications were also relatively quantified in dairy products, such as raw, high-temperature, ultra-high-temperature, sterilized, and condensed milk as well as infant formulas. The highest contents of lactulosyllysine at Lys47 were detected in powdered infant formulas, whereas lactulosyllysine at Lys138/141 was predominant in condensed milk samples. Methionine sulfoxide at Met7 and Met24 showed a trend toward higher modification rates in more severely processed products.     

Antioxidative activity of urate in bovine milk

The antioxidative effects of urate on peroxidase-induced protein oxidation and light-induced riboflavin degradation and lipid oxidation in whole milk were studied. In addition, experiments using ascorbate were conducted to directly compare the antioxidative activity of urate and ascorbate. The presence of urate and/or ascorbate (10-30 mg/L) lowered peroxidase-induced formation of dityrosine by 44-96% in unpasteurized whole milk. No synergistic effect of urate and ascorbate on peroxidase-induced dityrosine formation was registered, but merely an additive effect. Light exposure of pasteurized whole milk showed that ascorbate was oxidized at the expense of urate, which indicated ascorbate-mediated recycling of the urate radical. Moreover, both urate and ascorbate (30 mg/L) retarded light-induced lipid oxidation in pasteurized whole milk as measured by formation of lipid hydroperoxides with urate being the most effective (28% reduction in lipid hydroperoxides) compared with ascorbate (14%). Finally, addition of urate or ascorbate (300 mg/L) to pasteurized whole milk showed a slight protective effect against light-induced degradation of riboflavin with urate being the most effective.     

Determination of apparent binding constants for aroma compounds with beta-lactoglobulin by dynamic coupled column liquid chromatography

Apparent binding constants of aroma compounds limonene, alpha- and beta-ionone, and terpenyl acetate, with beta-lactoglobulin (BLG), were determined, using dynamic coupled column liquid chromatography, for pH values varying from 3 to 11. K(a) values varied from 2.61 to 3.21 x 10(3) M(-1) for limonene, indicating a strong interaction with BLG. Similarly, significant and close apparent binding constants were obtained for alpha- and beta-ionone, 1.7 x 10(2) and 4.5 to 5.4 x 10(2) M(-1), respectively. These data indicated that a similar mechanism is involved for the binding of these two molecules. The weaker values obtained at low pH, for alpha-ionone relative to beta-ionone, can be explained by the existence of steric hindrance. An increase of the apparent binding constant was observed, for all the compounds studied, when the pH was increased from 3 to 9. At this pH, an apparent binding constant was obtained for terpenyl acetate (1.04 x 10(2) M(-1)), whereas this determination was not possible at pH 3 and 6. The apparent binding constant increase was in agreement with the decrease of aroma compound relative activity coefficient in the presence of BLG, previously observed at this pH. It indicated a best accessibility to the same binding site. The binding constants of all the aroma compounds studied decreased at pH 11 as a result of the important release of the BLG structure previously reported.     

Comparison of different methods: static and dynamic headspace and solid-phase microextraction for the measurement of interactions between milk proteins and flavor compounds with an application to emulsions

Interactions between 10 aroma compounds from different chemical classes and 5 mixtures of milk proteins have been studied using static or dynamic headspace gas chromatography and solid-phase microextraction (SPME). Static headspace analysis allows the quantification of the release of only the most abundant compounds. Dynamic headspace analysis does not allow the discrimination of flavor release from the different protein mixtures, probably due to a displacement of headspace equilibrium. By SPME analysis and quantification by GC-MS (SIM mode) all of the volatiles were quantified. This method was optimized to better discriminate aroma release from the different milk protein mixtures and then from oil/water emulsions made with these proteins. The highest difference between the release in different proteins was observed for ethyl hexanoate, which has a great affinity for beta-lactoglobulin. Ethyl hexanoate is thus less released from models and emulsions containing this protein.     

Linking peanut allergenicity to the processes of maturation,curing, and roasting

The processes of peanut maturation, curing, and roasting are known to have an important role in peanut flavors. One of these processes (i.e., roasting) has been found to have an effect on allergenicity. To determine if the other processes (i.e., maturation and curing) affect allergenicity, mature and immature roasted peanuts and peanuts cured at different temperatures (35-77 degrees C) were, respectively, tested for IgE binding and advanced glycation end adducts (AGEs). Peanuts with and without stress proteins, which are associated with peanut maturation and curing, were also tested. Results showed that mature roasted peanuts exhibited a higher IgE binding and AGEs level than immature roasted peanuts. Curing temperatures between 35 and 60 degrees C gave no difference in the profiles. However, a higher curing temperature (i.e., 77 degrees C) exhibited a profile of higher levels of AGEs and IgE binding. These levels were higher in peanuts with stress proteins than without stress proteins. Roasting increased stress protein level and IgE binding. From these results, the processes of maturation and curing, in conjunction with roasting, may be associated with allergenicity, suggesting that these processes may lead to changes in the allergenic properties of peanuts.     

Esterified whey proteins can protect Lactococcus lactis against bacteriophage infection. Comparison with the effect of native basic proteins and L-polylysines

Inhibitory action of basic esterified milk whey proteins [methylated (Met) or ethylated (Et) beta-lactoglobulin (BLG) and alpha-lactalbumin (ALA)], basic native proteins (chicken egg white lysozyme and calf thymus histone), and basic protein-like substances (L-polylysines) against the activity and replication of lactococcal bacteriophages (bIL66, bIL67, and bIL170) was tested. Chemical interactions of these proteins with phage DNA were determined as well as their protective effect on the growth of a laboratory plasmid-cured Lactococcus lactis subjected to an infection by the bacteriophages. All the proteins studied showed inhibitory activity against the three bacteriophages as tested by marked reduction of their lytic activities and decreasing the replication of studied phages. Histone and Met-BLG were more active toward bIL66 and bIL67, respectively, while both proteins were highly and equally active toward bIL170. Lysozyme showed lower antiviral activity. Antiviral activity of Et-BLG was a little bit lower than that observed in the case of the Met derivative. Esterified ALA also showed considerable but slightly lower antiviral activity as compared to other proteins. L-polylysines also showed an antiviral effect against the three bacteriophages studied, their influence being highly dependent on their molecular size. The best effective size of L-polylysines was in the range 15-70 kDa. Replication of bIL67 was inhibited by the presence of esterified ALA or BLG and native basic proteins. Complete inhibition of replication of bIL67 occurred when using polylysines with molecular masses in the ranges 4-15, 15-30, and 30-70 kDa, while protein-like substrates with lower molecular masses had only a slight effect. The presence of histone and Met-BLG at a concentration of 0.13 mg/mL in the incubation medium protected L. lactis against lysis when it was subjected to an infection by bIL67 (10(5) pfu/mL). The same action was achieved by l-polylysine (15-30 kDa) used at a concentration of 0.03 mg/mL in the incubation medium.     

Front-face fluorescence spectroscopy allows the characterization of mild heat treatments applied to milk. Relations with the denaturation of milk proteins

Emission and excitation spectra of intrinsic fluorophores present in milk were used to evaluate changes in milk following thermal treatments in the 57-72 degrees C temperature range from 0.5 min up to 30 min. Alternatively, the concentrations of native alkaline phosphatase, lactoferrin, immunoglobulin G, bovine serum albumin, beta-lactoglobulin, and alpha-lactalbumin were determined in the same samples by enzymatic and immunochemical techniques. As principal component analysis applied to the normalized fluorescence spectra successfully discriminated different milk samples according to the temperature and time of thermal treatment, principal component regression was applied to predict the amounts of the native proteins investigated using fluorescence data. The results showed strong correlations between measured and predicted data for alkaline phosphatase and beta-lactoglobulin. This study has demonstrated that front-face fluorescence spectroscopy has a promising potential to become a rapid and nondestructive analytical technique for the evaluation of physicochemical changes in milk induced by low thermal treatment.