Lourdes Sánchez

University of Zaragoza, Zaragoza, Aragon, Spain

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Publications (75)149.98 Total impact

  • [show abstract] [hide abstract]
    ABSTRACT: The effect of high-pressure treatment (400, 500 and 650 MPa) on the structure and activity of bovine lactoferrin in different iron-saturation forms has been studied by several techniques. The structural changes produced in lactoferrin by high-pressure were analysed by differential scanning calorimetry and fluorescence spectroscopy, and the immunoreactivity by ELISA. The effect of high-pressure was also studied on some biological properties of lactoferrin, such as iron binding capacity, retention of the bound iron, and antibacterial activity against Escherichia coli O157:H7. Results obtained indicate that treatment at 400 MPa does not substantially modify the conformation of lactoferrin, meanwhile treatments at 500 and 650 MPa greatly affect some of its properties. With respect to the antibacterial activity, the apo and native forms of lactoferrin maintain that activity against Esch. coli only after 400 MPa treatment.
    Journal of Dairy Research 05/2013; · 1.34 Impact Factor
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    ABSTRACT: The effect of heat treatment on denaturation of Ara h1 protein, a major allergen from peanut, was studied using several techniques. Previously, Ara h1 protein was isolated from raw peanut using ammonium sulphate precipitation and chromatograpic techniques. Antibodies against Ara h1 protein were obtained in rabbits, conjugated with horseradish peroxidase and used to develop a sandwich ELISA. Denaturation of Ara h1 protein was estimated by the loss of reactivity with its specific antibodies by ELISA. Kinetic and thermodynamic parameters of the denaturation process of Ara h1 protein were determined over a temperature range of 82-90 ºC. Denaturation of Ara h1 was best described assuming a reaction order of 1.5. Thermal denaturation of Ara h1 protein was also studied by differential scanning calorimetry (DSC) using several heating rates. The maximum peak temperature and the enthalpy of denaturation obtained by extrapolation to a scan rate of 0 °C/min were 90.22 ºC and 1574 kJ/mol, respectively. Hydrophobicity of Ara h1 protein increased with the intensity of heat treatment and aggregates were formed when the protein was treated at 90 ºC for 10 min.
    Journal of Agricultural and Food Chemistry 03/2013; · 2.91 Impact Factor
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    ABSTRACT: It is over 60years since the discovery and isolation of the serum ferroxidase ceruloplasmin. In that time much basic information about the protein has been elucidated including its catalytic and kinetic properties as an enzyme, expression, sequence and structure. The importance of its biological role is indicated in genetic diseases such as aceruloplasminemia where its function is lost through mutation. Despite this wealth of data, fundamental questions about its action remain unanswered and in this article we address the question of how ferric iron produced by the ferroxidase activity of ceruloplasmin could be taken up by transferrins or lactoferrins. Overlapping peptide libraries for human ceruloplasmin have been probed with a number of different lactoferrins to identify putative lactoferrin-binding regions on human ceruloplasmin. Docking software, 3D-Garden, has been used to model the binding of human lactoferrin to human ceruloplasmin. Upon probing the human ceruloplasmin library with human lactoferrin, three predominantly acidic lactoferrin-binding peptides, located in domains 2, 5 and 6 of human ceruloplasmin, were identified. The docking software identified a complex such that the N-lobe of human apo-lactoferrin interacts with the catalytic ferroxidase centre on human ceruloplasmin. In vitro binding studies and molecular modelling indicate that lactoferrin can bind to ceruloplasmin such that a direct transfer of ferric iron between the two proteins is possible. A direct transfer of ferric iron from ceruloplasmin to lactoferrin would prevent both the formation of potentially toxic hydroxyl radicals and the utilization of iron by pathogenic bacteria.
    Biochimica et Biophysica Acta 03/2012; 1820(3):411-6. · 4.66 Impact Factor
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    ABSTRACT: Heat denaturation of recombinant human lactoferrin (rhLf) from rice with 3 different iron-saturation degrees, holo rhLf (iron-saturated), AsIs rhLf (60% iron saturation), and apo rhLf (iron-depleted), was studied. The 3 forms of rhLf were subjected to heat treatment, and the kinetic and thermodynamic parameters of the denaturation process were determined. Thermal denaturation of rhLf was assessed by measuring the loss of reactivity against specific antibodies. D(t) values (time to reduce 90% of immunoreactivity) decreased with increasing temperature of treatment for apo and holo rhLf, those values being higher for the iron-saturated form, which indicates that iron confers thermal stability to rhLf. However, AsIs rhLf showed a different behaviour with an increase in resistance to heat between 79 °C and 84 °C, so that the kinetic parameters could not be calculated. The heat denaturation process for apo and holo rhLf was best described assuming a reaction order of 1.5. The activation energy of the denaturation process was 648.20 kJ/mol for holo rhLf and 406.94 kJ/mol for apo rhLf, confirming that iron-depleted rhLf is more sensitive to heat treatment than iron-saturated rhLf.
    Biochemistry and Cell Biology 02/2012; 90(3):389-96. · 2.92 Impact Factor
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    ABSTRACT: Lactoferrin and lactoperoxidase are whey proteins with biological properties that may provide health benefits to consumers. These properties are vulnerable to potentially denaturing conditions during processing. High-pressure treatment is an appealing alternative to the traditional heat processing of foods because it exerts an antimicrobial effect without changing the sensory and nutritional quality of foods. In this work, the effect of high-pressure treatment on the denaturation of lactoferrin and lactoperoxidase present in skim milk and whey, and as isolated proteins in buffer, was studied over a pressure range of 450 to 700 MPa at 20°C. Denaturation of lactoferrin was measured by the loss of reactivity with their specific antibodies using a sandwich ELISA. Denaturation of lactoperoxidase was determined by measuring the loss of enzymatic activity using a spectrophotometric technique. No substantial inactivation of lactoperoxidase was observed in any treatment assayed. The concentration of the residual immunoreactive lactoferrin after each pressure treatment was determined, and the data were subjected to kinetic analysis to obtain D and Z values. Denaturation of lactoferrin increased with pressure and holding time, and D values were lower when lactoferrin was treated in whey than in milk, and lower in both whey and milk than in phosphate buffer. Thus, protein is denatured more slowly in buffer and in milk than in whey. Denaturation of lactoferrin in the 3 media was found to follow a reaction order of n=1.5. Volumes of activation of about -34.77, -24.35, and -24.09 mL/mol were obtained for lactoferrin treated in skim milk, whey, and buffer, respectively, indicating a decrease in protein volume under pressure.
    Journal of Dairy Science 02/2012; 95(2):549-57. · 2.57 Impact Factor
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    ABSTRACT: High pressure was applied to recombinant human lactoferrin obtained from rice (rhLF) and its effect was evaluated on the structure and activity of the protein. Treatments of 400, 500, and 650 MPa for 15 min (20 °C), were applied to rhLF at 2 mg/mL in three iron-saturation forms. The structural characteristics of the treated proteins were analyzed by differential scanning calorimetry (DSC) and by fluorometric analysis, and immunoreactivity by ELISA. Iron retention and binding properties and antibacterial activity against Escherichia coli O157:H7 were also studied. The results obtained indicate that the treatments at 400 and 500 MPa did not greatly modifiy the conformation of lactoferrin, meanwhile treatment at 650 MPa affected in different degrees the three forms of rhLF. With respect to antibacterial activity, only apo rhLF showed antibacterial activity against E. coli, activity that was maintained after treatment at 400 MPa, while holo and AsIs rhLF did not inhibit the growth of E. coli.
    Bioscience Biotechnology and Biochemistry 01/2012; 76(1):53-9. · 1.27 Impact Factor
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    ABSTRACT: Nowadays, the reference method for the detection of Clostridium tyrobutyricum in milk is the most-probable-number method, a very time-consuming and non-specific method. In this work, the suitability of the use of superparamagnetic beads coated with specific antibodies and peptides for bioseparation and concentration of spores of C. tyrobutyricum has been assessed. Peptide or antibody functionalized nanoparticles were able to specifically bind C. tyrobutyricum spores and concentrate them up to detectable levels. Moreover, several factors, such as particle size (200 nm and 1 μm), particle derivatization (aminated and carboxylated beads), coating method, and type of ligand have been studied in order to establish the most appropriate conditions for spore separation. Results show that concentration of spore is favored by a smaller bead size due to the wider surface of interaction in relation to particle volume. Antibody orientation, related to the binding method, is also critical in spore recovery. However, specific peptides seem to be a better ligand than antibodies, not only due to the higher recovery ratio of spores obtained but also due to the prolonged stability over time, allowing an optimal recovery of spores up to 3 weeks after bead coating. These results demonstrate that specific peptides bound to magnetic nanoparticles can be used instead of traditional antibodies to specifically bind C. tyrobutyricum spores being a potential basis for a rapid method to detect this bacterial target.
    Analytical and Bioanalytical Chemistry 01/2012; 402(10):3219-26. · 3.66 Impact Factor
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    Chockry Barbana, Lourdes Sánchez, María D Pérez
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    ABSTRACT: α-Lactalbumin (α-LA) is a whey protein that has been extensively studied for its folding properties and its ability to bind several cations. An interesting property of α-LA is its ability to interact with fatty acids, although this interaction requires the previous unfolding of the protein by removing the Ca(2+) bound. The main function of α-LA is to participate in lactose biosynthesis. However, other biological functions have been attributed to the protein in the last decade. It has been reported that a particular form of human and bovine apo-α-LA induces apoptosis in tumoral and immature cells though spares healthy differentiated cells. The conversion of α-LA to the active apoptotic form requires the unfolding of the protein and the binding of specific fatty acids, mainly unsaturated C18 fatty acids in the cis-conformation. Likewise, it has been shown that a folding variant of α-LA and also some peptidic fragments have a bactericidal activity. The proposed functions for α-LA open new perspectives for its use as a potential ingredient to be added in functional foods or in nutraceutical products. This review summarizes the current state of knowledge on the subject of the interaction of α-LA with fatty acids, and the consequences of this interaction on its bioactivity.
    Critical reviews in food science and nutrition 09/2011; 51(8):783-94. · 3.73 Impact Factor
  • C. Barbana, L. Sánchez, M.D. Pérez
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    ABSTRACT: Alpha-Lactalbumin (-LA) is a whey protein that has been extensively studied for its folding properties and its ability to bind several cations. An interesting property of -LA is its ability to interact with fatty acids, although this interaction requires the previous unfolding of the protein by removing the Ca2+ bound. The main function of -LA is to participate in lactose biosynthesis. However, other biological functions have been attributed to the protein in the last decade. It has been reported that a particular form of human and bovine apo--LA induces apoptosis in tumoral and immature cells though spares healthy differentiated cells. The conversion of -LA to the active apoptotic form requires the unfolding of the protein and the binding of specific fatty acids, mainly unsaturated C18 fatty acids in the cis-conformation. Likewise, it has been shown that a folding variant of -LA and also some peptidic fragments have a bactericidal activity. The proposed functions for -LA open new perspectives for its use as a potential ingredient to be added in functional foods or in nutraceutical products. This review summarizes the current state of knowledge on the subject of the interaction of -LA with fatty acids, and the consequences of this interaction on its bioactivity.
    Critical Reviews in Food Science and Nutrition. 01/2011; 51:783-794.
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    ABSTRACT: The aim of this work was to study the effect of milk supplementation with lactoferrin of different iron saturation on the manufacturing and characteristics of yogurt. Bovine lactoferrin was added at concentrations of 0.5, 1, and 2 mg/mL in the holo (iron saturated) and apo (without iron) forms. Some physicochemical properties, such as pH, concentration of lactic acid, and texture of supplemented yogurts, were determined throughout the shelf-life period storage (28 d) at 4°C. We also evaluated the stability of lactoferrin in supplemented yogurt throughout the storage time. The supplementation of milk with bovine lactoferrin did not greatly affect the physical properties of the yogurt, though apo-lactoferrin slightly delayed the decrease of pH. This could be attributed to the partial inhibition observed on the growth of Streptococcus thermophilus. The integrity and immunoreactive concentration of lactoferrin, determined by Western blotting and noncompetitive ELISA, respectively, remained constant throughout the shelf life of yogurt.
    Journal of Dairy Science 10/2010; 93(10):4480-9. · 2.57 Impact Factor
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    ABSTRACT: The effect of different heat treatments on the antimicrobial activity of bovine lactoferrin against Escherichia coli O157:H7, Salmonella enteritidis and Listeria monocytogenes has been studied. We have observed that the heat treatments lower than 85°C for 10 min did not affect the antibacterial activity of the protein. Hydrolysates of bovine lactoferrin were found to be more active than the native protein against the three pathogens. Moreover, the antibacterial effect of bovine lactoferrin was also assayed in milk and whey, and although we found a reduction in the number of viable cells, this reduction was lower than in culture media.
    International Journal of Dairy Technology 04/2010; 63(2):209 - 215. · 1.18 Impact Factor
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    ABSTRACT: The aim of this work was to study the in vitro digestion of Cry1A(b) protein by pepsin. To perform this work, a protein fraction purified from transgenic maize by immunoadsorption was employed. The undigested fraction showed several bands of molecular weight ranging between 14 and 70 kDa when assayed by SDS-PAGE. These bands were identified as corresponding to Cry1A(b) protein by immunochemical techniques and mass spectrometry. The rate of degradation of the purified fraction by pepsin estimated by ELISA was found to be about 75% within 30 min, and the protein concentration remained constant up to 4 h. In all treated samples, the full-length protein and fragments present in Cry1A(b) fraction were absent and peptides of less than 8.5 kDa were mainly found by SDS-PAGE and mass spectrometry. These peptides did not react with antiserum against Cry1A(b) protein by Western blotting. These results suggest that Cry1A(b) fraction purified from transgenic maize is rapidly and extensively degraded by pepsin, giving peptides of low molecular mass.
    Journal of Agricultural and Food Chemistry 02/2010; 58(4):2548-53. · 2.91 Impact Factor
  • Celia Conesa, Miguel Calvo, Lourdes Sánchez
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    ABSTRACT: Lactoferrin, the main iron-binding protein of milk, has biological activities that are essential for the newborn and are beneficial for adults. Given this beneficial effect, there is broad interest in exogenous sources of lactoferrin in human nutrition. Consequently, several transgenic approaches to produce lactoferrin have been achieved. However, the activity of heterologous lactoferrin cannot be assumed to identically mimic that of the homologous protein. Human lactoferrin obtained from yeast, transgenic cows, and rice has met the criteria of structural similarity, high yield, and ease of protein isolation. Human lactoferrin from Aspergillus awamori has been mainly directed to therapeutic uses with advanced phases of clinical trials currently in progress. In contrast, human lactoferrin produced in transgenic cows and rice brings the clear advantage of origins compatible with use in foods, although the approval for these applications is still in process.
    Biotechnology advances 01/2010; 28(6):831-8. · 8.25 Impact Factor
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    ABSTRACT: The possibility of using recombinant human lactoferrin from rice (rhLF) makes it necessary to study its differences from the protein of milk. In this work, the binding of different iron-saturated forms of rhLF to Caco-2 cells was studied. Iron-saturated rhLF bound in higher proportion than the apo-form, but, the data obtained for specific binding were not compatible with receptor-mediated binding. Competition assays showed the same binding capacity for human milk lactoferrin as for rhLF to Caco-2 cells. Another basic protein of milk, lactoperoxidase, was found to compete with rhLF for binding to Caco-2 cell membranes, suggesting an electrostatic interaction. The transport of iron ((59)Fe) bound to rhLF and to citrate and the transport of rhLF ((125)I-labeled) were studied on Caco-2 monolayers. Transport of iron was found to be significantly greater when bound to citrate than to rhLF. The amount of intact lactoferrin that traversed the Caco-2 monolayers was very low, suggesting degradation of it across these cells.
    Bioscience Biotechnology and Biochemistry 12/2009; 73(12):2615-20. · 1.27 Impact Factor
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    ABSTRACT: Clostridium tyrobutyricum is the main agent responsible for "late blowing" in cheese, which causes severe economic losses. Nowadays, the reference method for its detection is the Most-Probable-Number (MPN); however, it is time consuming and non-specific. Thus, in order to check milk contamination with spores of C. tyrobutyricum, a more specific and rapid method would be required. The objective of this work was to obtain a ligand to establish the basis to develop a biomagnetic separation method for detection of C. tyrobutyricum spores. This study describes the selection of thirteen highly affine peptides to C. tyrobutyricum spores from a phage-display peptide library. In order to test the ability of the peptides attached to a solid support to bind the spores, the most frequent peptide was synthesised and used to coat paramagnetic beads.
    Journal of microbiological methods 09/2009; 79(2):214-9. · 2.43 Impact Factor
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    ABSTRACT: The effect of pulsed electric field (PEF) processing on denaturation of bovine whey proteins has been studied. Protein concentration was determined by radial immunodiffusion and lactoperoxidase activity by a spectrophotometric technique. For the five proteins studied (beta-lactoglobulin, alpha-lactalbumin, lactoperoxidase, IgG and lactoferrin), no significant differences were found in the concentration of immunoreactive proteins between untreated milk or whey samples and those subjected to a treatment of 50, 100 and 200 pulses at 37.6 kV/cm. Likewise, lactoperoxidase activity did not change under PEF conditions tested. The effect of pasteurization treatments on whey proteins was also determined for comparison with PEF treatments. Treatment at 65 degrees C for 30 min and at 75 degrees C for 15 s markedly decreased concentration of lactoferrin to 53% and 58% of the initial value when heated in whey. Besides, both treatments caused a considerable decrease of concentration and enzymatic activity of lactoperoxidase treated in milk or whey. Heat treatments at 80 and 90 degrees C for 15 s produced a severe loss of immunoreactivity of IgG, lactoferrin and lactoperoxidase. The application of PEF treatments for milk preservation could prevent changes in structure and functionality of whey proteins with biological activity that are heat-sensitive.
    Milchwissenschaft 09/2009; 64:422-426. · 0.28 Impact Factor
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    ABSTRACT: Milk proteins have many functional properties, making them valuable food ingredients. However, they are a frequent cause of food allergy, especially in children. The aim of the present work was to develop and evaluate two immunoassays (indirect competitive and double antibody sandwich formats) for the detection of undeclared milk proteins in foods. Antisera raised to β-lactoglobulin was used in the competitive assay and antibodies isolated by immunoadsorption in the sandwich ELISA. Results obtained indicated that the sandwich format could detect lower percentages of powdered milk added to processed model foods than the competitive format. Likewise, the sandwich assay could discriminate better than the competitive assay between commercial foods with declared or non-milk ingredients. The antibody population used to recognize the target protein as well as the particularities of each ELISA format greatly influence the determination of immunoreactive β-lactoglobulin present in processed food.
    Food Control 07/2009; 20(7):643-647. · 2.74 Impact Factor
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    ABSTRACT: The present work investigates the feasibility of using flow cytometry (FCM) combined with fluorescent-labelled specific polyclonal antibodies for the detection and presumptive identification of Clostridium tyrobutyricum spores in bovine milk. Two fluorescent molecules (fluorescein isothiocyanate and Alexa Fluor 488) were conjugated to antispores polyclonal antibodies. Side scatter and forward scatter profiles of the Cl. tyrobutyricum spores marked with fluorescent antibodies permitted the detection of spores and differentiated them from other related microbial species. The detection limit of this method was 10(3) spores per 100 ml of milk, and results could be achieved in 2 h. FCM combined with fluorochrome-conjugated antibodies, especially Alexa Fluor, could be an efficacious means to detect and provide presumptive identification of Cl. tyrobutyricum spores, as well as differentiation from other Clostridium species that can also cause late blowing in cheese. This study describes the basis for the development of a method suitable for analysis of milk destined for cheese manufacture that would permit the detection of Cl. tyrobutyricum spores in a short period. This would enable the industry to use contaminated milk for dairy products other than cheese where Cl. tyrobutyricum does not cause a problem.
    Journal of Applied Microbiology 07/2009; 108(2):488-98. · 2.20 Impact Factor
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    ABSTRACT: The antibacterial activity of recombinant human lactoferrin from rice (rhLF) compared with that of human milk lactoferrin (hLF) was evaluated against Escherichia coli O157:H7, Salmonella Enteritidis and Listeria monocytogenes. The hydrolysates of rhLF and hLF were found to be more active than native proteins against E. coli O157:H7, and their activity was independent of their iron saturation. The effect of different heat treatments on the antibacterial activity of apo-rhLF was studied and compared with hLF. We observed that an HTST pasteurization treatment did not affect the antimicrobial activity of lactoferrin against the pathogens studied. Furthermore, the activity of apo-rhLF and hLF against E. coli O157:H7 and L. monocytogenes in UHT milk and whey was assayed, finding a decrease in the number of bacteria, although lower than that observed in a broth medium. This study shows the similar antibacterial activity of rhLF and hLF which is important in order to consider the addition of rhLF as a supplement in special products.
    Bioscience Biotechnology and Biochemistry 07/2009; 73(6):1301-7. · 1.27 Impact Factor
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    ABSTRACT: The activity of human milk on cell growth has been evaluated on two cell lines, MDCK and Caco-2. The proportion of human milk samples that reduced by half the growth of MDCK cells was of 36%. This inhibitory activity was associated with casein and not the whey fraction. Great variability was found in the degree of inhibitory activity depending on the milk sample. The susceptibility of Caco-2 cells to milk inhibitory activity was lower than that of MDCK. Bovine milk did not have any effect on cell growth, either as skimmed milk or as whey or casein. Morphology of cells incubated with active human casein showed abnormal features, such as chromatin condensation, reduced cellular volume and apoptotic bodies, and also fragmented DNA, which are all features of apoptosis.
    Journal of Dairy Research 06/2009; 76(3):308-16. · 1.34 Impact Factor

Publication Stats

559 Citations
149.98 Total Impact Points

Institutions

  • 1989–2013
    • University of Zaragoza
      • Faculty of Veterinary
      Zaragoza, Aragon, Spain
  • 2005–2006
    • Hospital Universitario Miguel Servet
      Caesaraugusta, Aragon, Spain
  • 1994–1996
    • University of Glasgow
      • Division of Immunology
      Glasgow, SCT, United Kingdom