Elena Molina - Academia.edu (original) (raw)

Papers by Elena Molina

PLoS ONE, 2013

Although there is a wide consensus on the efficacy of paratuberculosis vaccination to limit econo... more Although there is a wide consensus on the efficacy of paratuberculosis vaccination to limit economic losses, its use has been restricted because of its interference in the diagnosis of tuberculosis. Data from a vaccine clinical trial in the Basque Country (Spain) has been evaluated in relationship with bovine tuberculosis intradermal test results. The trial included two herds applying a Test and Culling strategy and five applying an inactivated vaccine. The vaccine was applied to animals of all ages present in each vaccinated herd when joining the trial, and then to all the replacers within their first three months of life. Yearly testing done with the comparative intradermal test (CIT) was applied to all animals older than 6 weeks. Between 2005 and 2011, the study generated 2,033 records from Vaccinated Herds (VH) and 2,252 from Test and Cull herds (TC). Prevaccination positive results rate was 2.40% among the 7 herds in the single bovine intradermal tuberculin test (BSIT). Two years later it rose to 20.42% in the VH and remained below at 0.75% in the TC. Applying the CIT reduced these rates to only 0.58% in the VH and to 0.25% in the TC ons. Regarding time since each animal joined the program, the proportion of positives to BSIT was variable and, in some cases, significantly different between time points. With regard to the age of vaccination, no significant differences were found between vaccination within the first year of life and afterwards. Vaccinated animals showed seventeen times more reactions than the non-vaccinated in the BSIT, but only four times more in the CIT. In conclusion, comparative intradermal test can be a useful tool to differentiate paratuberculosis vaccine crossreactions from specific bovine tuberculosis reactions according to the European and Spanish legislation.

Annals of Allergy, Asthma & Immunology, 2015

Evidence of the efficacy of food oral immunotherapy (OIT) is not robust enough to change clinical... more Evidence of the efficacy of food oral immunotherapy (OIT) is not robust enough to change clinical practice from current standard management. Furthermore, the immunologic changes underlying food desensitization are unknown. To establish the immunologic basal status and differences between an egg-allergic group of children and a population of nonallergic children and to investigate the safety and efficacy of a specific egg OIT protocol to induce clinical desensitization and the associated immune responses. Children with or without egg allergy were recruited. Allergic subjects underwent an OIT protocol based on weekly doses of egg protein and a maintenance phase. Immune profile and changes in all subjects were investigated by measuring T-helper cells types 1 and 2 (TH1 and TH2) and T-regulatory cytokines and transcription factors and egg-specific IgE and IgG4 levels. At baseline, a significantly lower production of ovalbumin-specific interleukin (IL)-10 and tumor necrosis factor-α and a trend toward higher IL-5 and IL-13 were found in allergic children. The egg OIT protocol enabled 60% of them to ingest 32 mL of egg white. Significant increases in egg-specific IgG4 levels and IL-10 production, with a trend toward lower IL-5 and IL-13 and higher tumor necrosis factor-α and interferon-γ levels, and significant decreases in egg-specific IgE concentration were observed. Egg-allergic individuals display a bias toward TH2 type cytokine production and decreased TH1 and IL-10 responses compared with nonallergic individuals. The OIT protocol was safe and effective in inducing egg desensitization, leading to a shift in the immune profile of allergic individuals toward a nonallergic phenotype.

Journal of dairy science, 2004

A capillary electrophoresis method has been applied to the detection of illegal addition of milk ... more A capillary electrophoresis method has been applied to the detection of illegal addition of milk from goat and/ or cow in Halloumi cheese, traditionally made with sheep milk. The electrophoretic profiles of the casein from Halloumi cheeses have revealed that caprine para-kappa-casein and bovine alphas1-casein peaks point to the presence of low percentages of goat's and/or cow's milk added to Halloumi cheese. Stepwise multiple linear regression has been used to predict these percentages with a standard error of the estimation of 2.14%. The analytical method combined with the statistical application is valid for the prediction of percentages higher than 2% of goat's and percentages of 5% of cow's milk added to the cheese either in fresh or ripened cheese. The standard error of estimation was higher for the prediction of cow's milk than for goat's milk.

Journal of Dairy Science, 2003

The effects of pressure (up to 400 MPa), applied at room temperature, on native proteinase activi... more The effects of pressure (up to 400 MPa), applied at room temperature, on native proteinase activity of milk were investigated by means of plasmin activity, plasmin-derived activity after plasminogen activation and their distribution in different milk fractions, micelle microstructure, beta-LG denaturation, and casein susceptibility to proteolytic attack. The pressure conditions assayed did not lead to plasmin inactivation and only decreased around 20 to 30% total plasmin activity after plasminogen activation. However, pressure caused severe disruption of the micellar structure, releasing high levels of caseins, plasmin, and plasminogen to the soluble fraction of milk. High levels of soluble denatured beta-LG were also found in the ultracentrifugation supernatants of pressurized milks, particularly in those treated at 400 MPa. Probably as a result of micellar disintegration, caseins became more susceptible to proteolysis by exogenous plasmin. However, no enhanced proteolytic degradation was observed when we compared the evolution of pressurized and unpressurized milks during refrigerated storage. Serum-liberated plasmin may become more vulnerable to the action of proteinase inhibitors leading to a reduced proteolysis on refrigerated storage, despite the increased susceptibility of caseins to proteinase action.

Journal of Agricultural and Food Chemistry, 2010

Gastrointestinal digestion of ovalbumin (OVA) was simulated using an in vitro system in two steps... more Gastrointestinal digestion of ovalbumin (OVA) was simulated using an in vitro system in two steps, which mimicked digestion in the stomach and duodenum, to assess the effect of different gastric pHs, different concentrations of proteases, and the presence of surfactants, such as phosphatidylcholine (PC) and bile salts (BS). OVA was very resistant to pepsin action at an enzyme/substrate ratio that would resemble a physiological situation (1:20 w/w, 172 units/mg) at pH values equal or above 2. The presence of PC did not change the susceptibility of OVA to proteolysis with pepsin. Fluorescence experiments showed that OVA interacted with PC vesicles, particularly at acidic pH, but it is likely that the protein maintained a high degree of conformational stability, resisting pepsin action. The presence of BS at physiological concentrations considerably increased the proteolysis of OVA by a mixture of pancreatic enzymes. The addition of PC made OVA even more sensitive to proteolytic degradation, suggesting that OVA could associate with the surfactants under duodenal conditions, increasing its exposure to pancreatic proteinases. Immunoreactivity against IgE from sera of allergic patients was retained after in vitro gastric digestion, depending on the reactivity of the sera, but it decreased considerably after in vitro duodenal digestion.

Electrophoresis, 1995

A method that allows separation and quantitation of the main whey proteins by capillary electroph... more A method that allows separation and quantitation of the main whey proteins by capillary electrophoresis using uncoated capillaries is proposed. Separations are performed using 100 mM borate buffer, pH 8.2, containing 30 mM sodium sulfate. The use of high pH and high ionic strength buffer reduces adsorption of proteins on the capillary wall, making their separation possible. Reproducibility of migration times and areas of peaks are improved by optimizing the capillary equilibration protocol and by using an internal standard. Relative standard deviations ranging between 0.74 and 1.03% for migration times and 2.14 to 5.23% for areas of major peaks are obtained. Detection limits equal to or lower than 0.5 mg/100 mL are achieved. Linear relationships of peak area versus concentration have been used to quantitate bovine serum albumin (BSA), alpha-LA (alpha-lactalbumin), beta-LG A (beta-lactoglobulin A) and beta-LG B (beta-lactoglobulin B) in cow's milk subjected to different thermal treatments. Electrophoretic profiles of these milk samples show peaks from other peptides besides those from main proteins. Characteristic patterns for whey from different species are obtained.

International Dairy Journal, 2000

Pasteurized (65°C, 30min), pressurized (400MPa, 22°C, 15min) and pasteurized–pressurized milks we... more Pasteurized (65°C, 30min), pressurized (400MPa, 22°C, 15min) and pasteurized–pressurized milks were used for reduced-fat (approximately 32% of total solids) cheese production. Pressurization of milk increased the yield of reduced-fat cheese through an enhanced β-lactoglobulin and moisture retention. In addition, pressurisation of pasteurized skim milk improved its coagulation properties. The cheeses made from pasteurized–pressurized and pressurized milks showed a faster rate

Food Research International, 2014

ABSTRACT This work aimed to identify the IgE epitopes of the allergenic whey protein R-lactoglobu... more ABSTRACT This work aimed to identify the IgE epitopes of the allergenic whey protein R-lactoglobulin (beta-LG) following in vitro gastrointestinal digestion using human (HF) or simulated digestive fluids (SF). The IgE-binding of the digests was evaluated by inhibition ELISA with sera from milk allergic patients and the low molecular weight digestion products were identified by RP-HPLC-MS/MS. These peptides were then chemically synthesized and analyzed by a peptide microarray immunoassay. beta-LG resisted digestion during the gastric phase, while no residual beta-LG was observed at the end of the duodenal phase. The immunoreactivity of the digests was consistent with the levels of intact beta-LG present, showing almost negligible IgE binding after gastrointestinal digestion with both systems. There were similarities in the peptide patterns produced, showing, respectively, 21 and 18 identical peptides in the gastric and duodenal digests. Digestion products related to five high-frequency IgE-binding synthetic peptides: beta-LG (43-60), beta-LG (47-62), beta-LG (86-99), beta-LG (86-100) and beta-LG (135-147), which were released upon in vitro digestion, particularly with SF. These matched two very immunoreactive areas of the protein; 43-68 and 122-146, as mapped using a library of 36 20-amino add peptides corresponding to the primary sequence of beta-LG. However, no IgE-binding peptides comprising the residues 86-100 were detected by analyzing the 36-peptide library by microarray immunoassay.

Molecular Nutrition & Food Research, 2013

Scope: Besides its antimicrobial properties, lysozyme (LYS) is one of the major allergens from he... more Scope: Besides its antimicrobial properties, lysozyme (LYS) is one of the major allergens from hen egg. This paper addresses the identification of the peptides produced upon in vitro gastrointestinal digestion of LYS, together with their IgE-binding and biological activity as a contribution to the understanding of what makes it a relevant allergen. Methods and results: Simulated in vitro gastrointestinal digestion together with IgE binding, basophil degranulation, and peripheral blood mononuclear cells stimulation experiments were carried out. Identification of the fragments released was performed by HPLC-MS/MS and the immunoreactive products were analyzed by MALDI-TOF/TOF. Results showed that in vitro gastric and gastroduodenal digests of LYS maintained IgE binding, basophil activation capacity, and preserved T-cell immunogenicity. These biological activities could be attributed to either the persistence of intact LYS, due to incomplete gastric degradation and subsequent duodenal precipitation, the formation of fragment f(24-129) by chymotrypsin action on the soluble intact protein, or the release, upon combined gastric and pancreatic digestion, of immunoreactive peptides linked by disulphide bonds containing the epitopes f(57-83) and f(108-122). Conclusion: The pH of gastric hydrolysis greatly determined the extent of subsequent duodenal digestion of LYS and the disclosure of relevant epitopes that could increase its allergenic potential.

European Food Research and Technology, 2008

The correlation between chemical and sensory analysis of the water-soluble extract compounds with... more The correlation between chemical and sensory analysis of the water-soluble extract compounds with molecular weight less than 1,000 Da (WSE < 1,000 Da) from Manchego cheese at different stages of ripening, as well as the influence of the pasteurization processes, has been studied. Free amino acids, peptides and volatile compounds were more abundant in raw milk cheeses WSE < 1,000 Da. Further fractionation of the WSE < 1,000 Da was

Journal of Dairy Research, 2000

Casein fractions and their breakdown products in Iberico-type cheeses made from the milk of cows,... more Casein fractions and their breakdown products in Iberico-type cheeses made from the milk of cows, ewes or goats were analysed by capillary electrophoresis in order to characterize them. The actions of plasmin and chymosin on caseins were evaluated by comparing the electropherograms of caseins from milk and from cheese, both with and without treatment with plasmin. Characteristic capillary electrophoresis patterns were obtained for cheeses made from the milk of each of the three species, and the main components were identified. Caprine para-kappa-casein and bovine beta-caseins, eluting at the first and at the last part of the electropherogram respectively, were found to be indicative of the presence of the milks of these species.

International Dairy Journal, 1999

Fractions isolated by gel filtration from the water soluble extracts of cheeses made of cows&... more Fractions isolated by gel filtration from the water soluble extracts of cheeses made of cows&amp;amp;amp;amp;amp;amp;amp;#39;, ewes’ and goats’ milk, under the same manufacturing conditions, were subjected to organoleptic evaluation and chromatographic separation in order to elucidate the contribution of small peptides, free amino acids and volatile components to their sensory properties. Differences were found in intensity and predominance of individual

International Dairy Journal, 1999

Capillary electrophoresis was used to separate and analyze the casein fraction in mixtures of cow... more Capillary electrophoresis was used to separate and analyze the casein fraction in mixtures of cows', goats' and ewes' milk. The differences between the capillary electrophoresis patterns of the casein fraction from the whole milk of each species allowed identification ...

International Dairy Journal, 2011

A casein hydrolysis process was scaled up to produce the antihypertensive peptides a s1 -CN f(90e... more A casein hydrolysis process was scaled up to produce the antihypertensive peptides a s1 -CN f(90e94), with sequence RYLGY, and a s1 -CN f(143e149), with sequence AYFYPEL. Previously, the hydrolysis process was optimized under food-grade conditions to obtain a maximum amount of active peptides, quantified by high-performance liquid chromatographyemass spectrometry. Ultrafiltration as a strategy for enrichment of active peptides was also evaluated. The casein-derived ingredients prepared at large scale showed potent angiotensin converting enzyme-inhibitory activity in vitro. Furthermore, these products produced a significant decrease in the systolic blood pressure of spontaneously hypertensive rats after oral administration (200e800 mg kg À1 of body weight). The active peptides RYLGY and AYFYPEL were stable during the processes of atomization, homogenization and pasteurization. This hydrolysate was incorporated into liquid yoghurt and no significant reduction of either peptide was detected during the shelf-life of the product.

Food Research International, 2012

Whole hen egg produced a fine stable O/W emulsion. The presence of egg proteins as part of the em... more Whole hen egg produced a fine stable O/W emulsion. The presence of egg proteins as part of the emulsion did not change their IgE binding, but it slightly increased the digestibility of the main allergens present in the egg-white. The observation that egg white proteins, forming part of an emulsion system did not become a much more effective substrate for pepsin indicates that, in the case of egg white proteins, there were not adsorption-induced changes that would considerably increase their flexibility and proteinase susceptibility. The increased digestibility of the emulsion resulted in a slightly lower IgE-binding capacity of the in vitro gastric and duodenal digests compared to those obtained from the egg in solution.

Food Chemistry, 2013

Hen egg white comprises of a complex mixture of proteins, which greatly differ in their physicoch... more Hen egg white comprises of a complex mixture of proteins, which greatly differ in their physicochemical characteristics and relative abundance. We aimed to identify potential undiscovered egg allergens within the egg white proteome and investigated the existence of matrix effects on the proteolytic stability and resultant IgE-binding of the allergenic proteins. In addition to the main egg allergens: ovalbumin (OVA), ovomucoid (OM) and lysozyme (LYS), two minor egg white proteins, tentatively identified as ovoinhibitor and clusterin, were found to react with serum IgE from egg-allergic patients. Egg white exhibited residual immunoreactivity after gastrointestinal digestion due to the presence of intact OVA and LYS, as well as of several IgE-binding peptides derived from OVA. The presence of egg yolk slightly increased the susceptibility to hydrolysis of egg white proteins and abrogated bile salt-induced precipitation of LYS in the duodenal medium. However, the resultant immunoreactivity against IgE of egg white proteins after in vitro digestion was not significantly modified by the presence of yolk components.

Food Chemistry, 2012

S-OVA, a more thermostable form of ovalbumin (OVA), was formed from native OVA or egg white in vi... more S-OVA, a more thermostable form of ovalbumin (OVA), was formed from native OVA or egg white in vitro, by heating at high pH, and by storage at low temperatures. S-OVA showed a much lower reactivity against IgE than OVA, although this difference in IgE binding was minimized after simulated gastro intestinal digestion, despite S-OVA was more resistant to proteolysis, particularly to pepsin, than its native form. It is, therefore, likely that the transformation of OVA to S-OVA does not affect its ability to sensitise or trigger allergic reactions at the duodenal level. These results are discussed in the light of the described conformational changes reported to occur in the transition between OVA and S-OVA.

Food Chemistry, 2006

The effects of a previous heat treatment (60 and 80°C, 30min) and high-pressure (400MPa, 25 and 6... more The effects of a previous heat treatment (60 and 80°C, 30min) and high-pressure (400MPa, 25 and 60°C, 1h) on the subsequent lactosylation of β-lactoglobulin (50°C, 44% RH, 120h) were investigated. A control of native β-lactoglobulin was also stored under the afore-mentioned conditions. The structural changes caused during these treatments were studied by the loss of amino groups, SE-HPLC and native-PAGE and the degree of lactosylation was evaluated by means of furosine determination. After thermal and high-pressure treatments, the greatest structural changes were observed in the case of samples of β-lactoglobulin treated at 80°C, 30min and 400MPa, 60°C, 1h. During storage, the highest lactosylation degree was found in native β-lactoglobulin. In heat-treated samples, the increase of lactosylated lysines was lower than the decrease of free amino groups, probably due to the cross-linking reactions. A similar decrease of free amino groups of β-lactoglobulin was observed immediately after 400MPa, 60°C, 1h and 80°C, 30min; however, the level of lactosylation during the storage period was lower in the former, indicating different types of conformational changes in the two treatments. These differences lead to a higher effectiveness of heat-treatment than high-pressure in denaturating β-lactoglobulin for subsequent lactosylation under the tested conditions (of temperature, time, high-pressure and storage).

Zeitschrift f�r Lebensmittel-Untersuchung und -Forschung, 1995

Stepwise multiple linear regression (SMLR) and principal components regression (PCR) have been us... more Stepwise multiple linear regression (SMLR) and principal components regression (PCR) have been used to predict the percentages of cows&amp;amp;#39;, goats&amp;amp;#39; and ewes&amp;amp;#39; milk in ¿Iberico¿ cheese, using the results obtained by electrophoretic analysis (PAGE and IEF) of whey proteins, using standard cheeses. Similar predictions of the percentages of milks from the three species were obtained when either SMLR or PCR were applied to the electrophoretic data, i.e., the optical intensity of the electrophoretic bands (PAGE or IEF) of the whey proteins. The root mean square error of prediction in cross-validation (RMSEPCV) was lower than 4% in all cases.

Stability during digestion is considered an important feature in determining the allergenicity of... more Stability during digestion is considered an important feature in determining the allergenicity of food proteins. This study aimed to provide an immunological characterisation of the digestion products of the major cow&amp;amp;amp;amp;amp;amp;amp;amp;#39;s milk allergen β-casein (β-CN) produced by in vitro orogastrointestinal hydrolysis with simulated and human digestive fluids. β-CN was unaffected by oral digestion, but quickly broke down during the early stages of gastric digestion. The degradation with human fluids was faster than that with commercial enzymes. There were similarities in the peptide patterns of the hydrolysates produced in both models, showing 20 peptides in common after gastric digestion. After gastroduodenal digestion, the human fluids gave less numerous and shorter peptides. The IgE binding of most of the individual sera used to the hydrolysates produced with simulated and human fluids increased at the end of the gastric phase and decreased when the duodenal digestion was completed. Two IgE-binding synthetic peptides: β-CN (57-68) and β-CN (82-93), which matched fragments released by β-CN following in vitro digestion with simulated and human fluids, consisted of the most immunoreactive areas of the protein. The similarities found between the in vitro simulated digestion system and that using human digestive fluids suggest that the former would provide a reasonably good estimation of the potential allergenicity of protein digests.

PLoS ONE, 2013

Although there is a wide consensus on the efficacy of paratuberculosis vaccination to limit econo... more Although there is a wide consensus on the efficacy of paratuberculosis vaccination to limit economic losses, its use has been restricted because of its interference in the diagnosis of tuberculosis. Data from a vaccine clinical trial in the Basque Country (Spain) has been evaluated in relationship with bovine tuberculosis intradermal test results. The trial included two herds applying a Test and Culling strategy and five applying an inactivated vaccine. The vaccine was applied to animals of all ages present in each vaccinated herd when joining the trial, and then to all the replacers within their first three months of life. Yearly testing done with the comparative intradermal test (CIT) was applied to all animals older than 6 weeks. Between 2005 and 2011, the study generated 2,033 records from Vaccinated Herds (VH) and 2,252 from Test and Cull herds (TC). Prevaccination positive results rate was 2.40% among the 7 herds in the single bovine intradermal tuberculin test (BSIT). Two years later it rose to 20.42% in the VH and remained below at 0.75% in the TC. Applying the CIT reduced these rates to only 0.58% in the VH and to 0.25% in the TC ons. Regarding time since each animal joined the program, the proportion of positives to BSIT was variable and, in some cases, significantly different between time points. With regard to the age of vaccination, no significant differences were found between vaccination within the first year of life and afterwards. Vaccinated animals showed seventeen times more reactions than the non-vaccinated in the BSIT, but only four times more in the CIT. In conclusion, comparative intradermal test can be a useful tool to differentiate paratuberculosis vaccine crossreactions from specific bovine tuberculosis reactions according to the European and Spanish legislation.

Annals of Allergy, Asthma & Immunology, 2015

Evidence of the efficacy of food oral immunotherapy (OIT) is not robust enough to change clinical... more Evidence of the efficacy of food oral immunotherapy (OIT) is not robust enough to change clinical practice from current standard management. Furthermore, the immunologic changes underlying food desensitization are unknown. To establish the immunologic basal status and differences between an egg-allergic group of children and a population of nonallergic children and to investigate the safety and efficacy of a specific egg OIT protocol to induce clinical desensitization and the associated immune responses. Children with or without egg allergy were recruited. Allergic subjects underwent an OIT protocol based on weekly doses of egg protein and a maintenance phase. Immune profile and changes in all subjects were investigated by measuring T-helper cells types 1 and 2 (TH1 and TH2) and T-regulatory cytokines and transcription factors and egg-specific IgE and IgG4 levels. At baseline, a significantly lower production of ovalbumin-specific interleukin (IL)-10 and tumor necrosis factor-α and a trend toward higher IL-5 and IL-13 were found in allergic children. The egg OIT protocol enabled 60% of them to ingest 32 mL of egg white. Significant increases in egg-specific IgG4 levels and IL-10 production, with a trend toward lower IL-5 and IL-13 and higher tumor necrosis factor-α and interferon-γ levels, and significant decreases in egg-specific IgE concentration were observed. Egg-allergic individuals display a bias toward TH2 type cytokine production and decreased TH1 and IL-10 responses compared with nonallergic individuals. The OIT protocol was safe and effective in inducing egg desensitization, leading to a shift in the immune profile of allergic individuals toward a nonallergic phenotype.

Journal of dairy science, 2004

A capillary electrophoresis method has been applied to the detection of illegal addition of milk ... more A capillary electrophoresis method has been applied to the detection of illegal addition of milk from goat and/ or cow in Halloumi cheese, traditionally made with sheep milk. The electrophoretic profiles of the casein from Halloumi cheeses have revealed that caprine para-kappa-casein and bovine alphas1-casein peaks point to the presence of low percentages of goat's and/or cow's milk added to Halloumi cheese. Stepwise multiple linear regression has been used to predict these percentages with a standard error of the estimation of 2.14%. The analytical method combined with the statistical application is valid for the prediction of percentages higher than 2% of goat's and percentages of 5% of cow's milk added to the cheese either in fresh or ripened cheese. The standard error of estimation was higher for the prediction of cow's milk than for goat's milk.

Journal of Dairy Science, 2003

The effects of pressure (up to 400 MPa), applied at room temperature, on native proteinase activi... more The effects of pressure (up to 400 MPa), applied at room temperature, on native proteinase activity of milk were investigated by means of plasmin activity, plasmin-derived activity after plasminogen activation and their distribution in different milk fractions, micelle microstructure, beta-LG denaturation, and casein susceptibility to proteolytic attack. The pressure conditions assayed did not lead to plasmin inactivation and only decreased around 20 to 30% total plasmin activity after plasminogen activation. However, pressure caused severe disruption of the micellar structure, releasing high levels of caseins, plasmin, and plasminogen to the soluble fraction of milk. High levels of soluble denatured beta-LG were also found in the ultracentrifugation supernatants of pressurized milks, particularly in those treated at 400 MPa. Probably as a result of micellar disintegration, caseins became more susceptible to proteolysis by exogenous plasmin. However, no enhanced proteolytic degradation was observed when we compared the evolution of pressurized and unpressurized milks during refrigerated storage. Serum-liberated plasmin may become more vulnerable to the action of proteinase inhibitors leading to a reduced proteolysis on refrigerated storage, despite the increased susceptibility of caseins to proteinase action.

Journal of Agricultural and Food Chemistry, 2010

Gastrointestinal digestion of ovalbumin (OVA) was simulated using an in vitro system in two steps... more Gastrointestinal digestion of ovalbumin (OVA) was simulated using an in vitro system in two steps, which mimicked digestion in the stomach and duodenum, to assess the effect of different gastric pHs, different concentrations of proteases, and the presence of surfactants, such as phosphatidylcholine (PC) and bile salts (BS). OVA was very resistant to pepsin action at an enzyme/substrate ratio that would resemble a physiological situation (1:20 w/w, 172 units/mg) at pH values equal or above 2. The presence of PC did not change the susceptibility of OVA to proteolysis with pepsin. Fluorescence experiments showed that OVA interacted with PC vesicles, particularly at acidic pH, but it is likely that the protein maintained a high degree of conformational stability, resisting pepsin action. The presence of BS at physiological concentrations considerably increased the proteolysis of OVA by a mixture of pancreatic enzymes. The addition of PC made OVA even more sensitive to proteolytic degradation, suggesting that OVA could associate with the surfactants under duodenal conditions, increasing its exposure to pancreatic proteinases. Immunoreactivity against IgE from sera of allergic patients was retained after in vitro gastric digestion, depending on the reactivity of the sera, but it decreased considerably after in vitro duodenal digestion.

Electrophoresis, 1995

A method that allows separation and quantitation of the main whey proteins by capillary electroph... more A method that allows separation and quantitation of the main whey proteins by capillary electrophoresis using uncoated capillaries is proposed. Separations are performed using 100 mM borate buffer, pH 8.2, containing 30 mM sodium sulfate. The use of high pH and high ionic strength buffer reduces adsorption of proteins on the capillary wall, making their separation possible. Reproducibility of migration times and areas of peaks are improved by optimizing the capillary equilibration protocol and by using an internal standard. Relative standard deviations ranging between 0.74 and 1.03% for migration times and 2.14 to 5.23% for areas of major peaks are obtained. Detection limits equal to or lower than 0.5 mg/100 mL are achieved. Linear relationships of peak area versus concentration have been used to quantitate bovine serum albumin (BSA), alpha-LA (alpha-lactalbumin), beta-LG A (beta-lactoglobulin A) and beta-LG B (beta-lactoglobulin B) in cow&#39;s milk subjected to different thermal treatments. Electrophoretic profiles of these milk samples show peaks from other peptides besides those from main proteins. Characteristic patterns for whey from different species are obtained.

International Dairy Journal, 2000

Pasteurized (65°C, 30min), pressurized (400MPa, 22°C, 15min) and pasteurized–pressurized milks we... more Pasteurized (65°C, 30min), pressurized (400MPa, 22°C, 15min) and pasteurized–pressurized milks were used for reduced-fat (approximately 32% of total solids) cheese production. Pressurization of milk increased the yield of reduced-fat cheese through an enhanced β-lactoglobulin and moisture retention. In addition, pressurisation of pasteurized skim milk improved its coagulation properties. The cheeses made from pasteurized–pressurized and pressurized milks showed a faster rate

Food Research International, 2014

ABSTRACT This work aimed to identify the IgE epitopes of the allergenic whey protein R-lactoglobu... more ABSTRACT This work aimed to identify the IgE epitopes of the allergenic whey protein R-lactoglobulin (beta-LG) following in vitro gastrointestinal digestion using human (HF) or simulated digestive fluids (SF). The IgE-binding of the digests was evaluated by inhibition ELISA with sera from milk allergic patients and the low molecular weight digestion products were identified by RP-HPLC-MS/MS. These peptides were then chemically synthesized and analyzed by a peptide microarray immunoassay. beta-LG resisted digestion during the gastric phase, while no residual beta-LG was observed at the end of the duodenal phase. The immunoreactivity of the digests was consistent with the levels of intact beta-LG present, showing almost negligible IgE binding after gastrointestinal digestion with both systems. There were similarities in the peptide patterns produced, showing, respectively, 21 and 18 identical peptides in the gastric and duodenal digests. Digestion products related to five high-frequency IgE-binding synthetic peptides: beta-LG (43-60), beta-LG (47-62), beta-LG (86-99), beta-LG (86-100) and beta-LG (135-147), which were released upon in vitro digestion, particularly with SF. These matched two very immunoreactive areas of the protein; 43-68 and 122-146, as mapped using a library of 36 20-amino add peptides corresponding to the primary sequence of beta-LG. However, no IgE-binding peptides comprising the residues 86-100 were detected by analyzing the 36-peptide library by microarray immunoassay.

Molecular Nutrition & Food Research, 2013

Scope: Besides its antimicrobial properties, lysozyme (LYS) is one of the major allergens from he... more Scope: Besides its antimicrobial properties, lysozyme (LYS) is one of the major allergens from hen egg. This paper addresses the identification of the peptides produced upon in vitro gastrointestinal digestion of LYS, together with their IgE-binding and biological activity as a contribution to the understanding of what makes it a relevant allergen. Methods and results: Simulated in vitro gastrointestinal digestion together with IgE binding, basophil degranulation, and peripheral blood mononuclear cells stimulation experiments were carried out. Identification of the fragments released was performed by HPLC-MS/MS and the immunoreactive products were analyzed by MALDI-TOF/TOF. Results showed that in vitro gastric and gastroduodenal digests of LYS maintained IgE binding, basophil activation capacity, and preserved T-cell immunogenicity. These biological activities could be attributed to either the persistence of intact LYS, due to incomplete gastric degradation and subsequent duodenal precipitation, the formation of fragment f(24-129) by chymotrypsin action on the soluble intact protein, or the release, upon combined gastric and pancreatic digestion, of immunoreactive peptides linked by disulphide bonds containing the epitopes f(57-83) and f(108-122). Conclusion: The pH of gastric hydrolysis greatly determined the extent of subsequent duodenal digestion of LYS and the disclosure of relevant epitopes that could increase its allergenic potential.

European Food Research and Technology, 2008

The correlation between chemical and sensory analysis of the water-soluble extract compounds with... more The correlation between chemical and sensory analysis of the water-soluble extract compounds with molecular weight less than 1,000 Da (WSE < 1,000 Da) from Manchego cheese at different stages of ripening, as well as the influence of the pasteurization processes, has been studied. Free amino acids, peptides and volatile compounds were more abundant in raw milk cheeses WSE < 1,000 Da. Further fractionation of the WSE < 1,000 Da was

Journal of Dairy Research, 2000

Casein fractions and their breakdown products in Iberico-type cheeses made from the milk of cows,... more Casein fractions and their breakdown products in Iberico-type cheeses made from the milk of cows, ewes or goats were analysed by capillary electrophoresis in order to characterize them. The actions of plasmin and chymosin on caseins were evaluated by comparing the electropherograms of caseins from milk and from cheese, both with and without treatment with plasmin. Characteristic capillary electrophoresis patterns were obtained for cheeses made from the milk of each of the three species, and the main components were identified. Caprine para-kappa-casein and bovine beta-caseins, eluting at the first and at the last part of the electropherogram respectively, were found to be indicative of the presence of the milks of these species.

International Dairy Journal, 1999

Fractions isolated by gel filtration from the water soluble extracts of cheeses made of cows&... more Fractions isolated by gel filtration from the water soluble extracts of cheeses made of cows&amp;amp;amp;amp;amp;amp;amp;#39;, ewes’ and goats’ milk, under the same manufacturing conditions, were subjected to organoleptic evaluation and chromatographic separation in order to elucidate the contribution of small peptides, free amino acids and volatile components to their sensory properties. Differences were found in intensity and predominance of individual

International Dairy Journal, 1999

Capillary electrophoresis was used to separate and analyze the casein fraction in mixtures of cow... more Capillary electrophoresis was used to separate and analyze the casein fraction in mixtures of cows', goats' and ewes' milk. The differences between the capillary electrophoresis patterns of the casein fraction from the whole milk of each species allowed identification ...

International Dairy Journal, 2011

A casein hydrolysis process was scaled up to produce the antihypertensive peptides a s1 -CN f(90e... more A casein hydrolysis process was scaled up to produce the antihypertensive peptides a s1 -CN f(90e94), with sequence RYLGY, and a s1 -CN f(143e149), with sequence AYFYPEL. Previously, the hydrolysis process was optimized under food-grade conditions to obtain a maximum amount of active peptides, quantified by high-performance liquid chromatographyemass spectrometry. Ultrafiltration as a strategy for enrichment of active peptides was also evaluated. The casein-derived ingredients prepared at large scale showed potent angiotensin converting enzyme-inhibitory activity in vitro. Furthermore, these products produced a significant decrease in the systolic blood pressure of spontaneously hypertensive rats after oral administration (200e800 mg kg À1 of body weight). The active peptides RYLGY and AYFYPEL were stable during the processes of atomization, homogenization and pasteurization. This hydrolysate was incorporated into liquid yoghurt and no significant reduction of either peptide was detected during the shelf-life of the product.

Food Research International, 2012

Whole hen egg produced a fine stable O/W emulsion. The presence of egg proteins as part of the em... more Whole hen egg produced a fine stable O/W emulsion. The presence of egg proteins as part of the emulsion did not change their IgE binding, but it slightly increased the digestibility of the main allergens present in the egg-white. The observation that egg white proteins, forming part of an emulsion system did not become a much more effective substrate for pepsin indicates that, in the case of egg white proteins, there were not adsorption-induced changes that would considerably increase their flexibility and proteinase susceptibility. The increased digestibility of the emulsion resulted in a slightly lower IgE-binding capacity of the in vitro gastric and duodenal digests compared to those obtained from the egg in solution.

Food Chemistry, 2013

Hen egg white comprises of a complex mixture of proteins, which greatly differ in their physicoch... more Hen egg white comprises of a complex mixture of proteins, which greatly differ in their physicochemical characteristics and relative abundance. We aimed to identify potential undiscovered egg allergens within the egg white proteome and investigated the existence of matrix effects on the proteolytic stability and resultant IgE-binding of the allergenic proteins. In addition to the main egg allergens: ovalbumin (OVA), ovomucoid (OM) and lysozyme (LYS), two minor egg white proteins, tentatively identified as ovoinhibitor and clusterin, were found to react with serum IgE from egg-allergic patients. Egg white exhibited residual immunoreactivity after gastrointestinal digestion due to the presence of intact OVA and LYS, as well as of several IgE-binding peptides derived from OVA. The presence of egg yolk slightly increased the susceptibility to hydrolysis of egg white proteins and abrogated bile salt-induced precipitation of LYS in the duodenal medium. However, the resultant immunoreactivity against IgE of egg white proteins after in vitro digestion was not significantly modified by the presence of yolk components.

Food Chemistry, 2012

S-OVA, a more thermostable form of ovalbumin (OVA), was formed from native OVA or egg white in vi... more S-OVA, a more thermostable form of ovalbumin (OVA), was formed from native OVA or egg white in vitro, by heating at high pH, and by storage at low temperatures. S-OVA showed a much lower reactivity against IgE than OVA, although this difference in IgE binding was minimized after simulated gastro intestinal digestion, despite S-OVA was more resistant to proteolysis, particularly to pepsin, than its native form. It is, therefore, likely that the transformation of OVA to S-OVA does not affect its ability to sensitise or trigger allergic reactions at the duodenal level. These results are discussed in the light of the described conformational changes reported to occur in the transition between OVA and S-OVA.

Food Chemistry, 2006

The effects of a previous heat treatment (60 and 80°C, 30min) and high-pressure (400MPa, 25 and 6... more The effects of a previous heat treatment (60 and 80°C, 30min) and high-pressure (400MPa, 25 and 60°C, 1h) on the subsequent lactosylation of β-lactoglobulin (50°C, 44% RH, 120h) were investigated. A control of native β-lactoglobulin was also stored under the afore-mentioned conditions. The structural changes caused during these treatments were studied by the loss of amino groups, SE-HPLC and native-PAGE and the degree of lactosylation was evaluated by means of furosine determination. After thermal and high-pressure treatments, the greatest structural changes were observed in the case of samples of β-lactoglobulin treated at 80°C, 30min and 400MPa, 60°C, 1h. During storage, the highest lactosylation degree was found in native β-lactoglobulin. In heat-treated samples, the increase of lactosylated lysines was lower than the decrease of free amino groups, probably due to the cross-linking reactions. A similar decrease of free amino groups of β-lactoglobulin was observed immediately after 400MPa, 60°C, 1h and 80°C, 30min; however, the level of lactosylation during the storage period was lower in the former, indicating different types of conformational changes in the two treatments. These differences lead to a higher effectiveness of heat-treatment than high-pressure in denaturating β-lactoglobulin for subsequent lactosylation under the tested conditions (of temperature, time, high-pressure and storage).

Zeitschrift f�r Lebensmittel-Untersuchung und -Forschung, 1995

Stepwise multiple linear regression (SMLR) and principal components regression (PCR) have been us... more Stepwise multiple linear regression (SMLR) and principal components regression (PCR) have been used to predict the percentages of cows&amp;amp;#39;, goats&amp;amp;#39; and ewes&amp;amp;#39; milk in ¿Iberico¿ cheese, using the results obtained by electrophoretic analysis (PAGE and IEF) of whey proteins, using standard cheeses. Similar predictions of the percentages of milks from the three species were obtained when either SMLR or PCR were applied to the electrophoretic data, i.e., the optical intensity of the electrophoretic bands (PAGE or IEF) of the whey proteins. The root mean square error of prediction in cross-validation (RMSEPCV) was lower than 4% in all cases.

Stability during digestion is considered an important feature in determining the allergenicity of... more Stability during digestion is considered an important feature in determining the allergenicity of food proteins. This study aimed to provide an immunological characterisation of the digestion products of the major cow&amp;amp;amp;amp;amp;amp;amp;amp;#39;s milk allergen β-casein (β-CN) produced by in vitro orogastrointestinal hydrolysis with simulated and human digestive fluids. β-CN was unaffected by oral digestion, but quickly broke down during the early stages of gastric digestion. The degradation with human fluids was faster than that with commercial enzymes. There were similarities in the peptide patterns of the hydrolysates produced in both models, showing 20 peptides in common after gastric digestion. After gastroduodenal digestion, the human fluids gave less numerous and shorter peptides. The IgE binding of most of the individual sera used to the hydrolysates produced with simulated and human fluids increased at the end of the gastric phase and decreased when the duodenal digestion was completed. Two IgE-binding synthetic peptides: β-CN (57-68) and β-CN (82-93), which matched fragments released by β-CN following in vitro digestion with simulated and human fluids, consisted of the most immunoreactive areas of the protein. The similarities found between the in vitro simulated digestion system and that using human digestive fluids suggest that the former would provide a reasonably good estimation of the potential allergenicity of protein digests.