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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; · 2.82 Impact Factor
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ABSTRACT: Immunoassays are used to screen for the presence of genetically modified organisms in raw materials. However, processing may
condition the usefulness of immunoassays to analyse genetically modified foods because it leads to protein denaturation that
affects recognition by antibodies. We studied the effect of processing on the detection of Cry1A(b) protein in model foods
prepared with transgenic maize using a sandwich ELISA. Nixtamalization at 100°C for 5min and at 85°C for 60min gave 40
and 70% loss of Cry1A(b) protein. In the preparation of porridge, the concentration of Cry1A(b) protein did not change until
the mixture reached 75°C, but it decreased by 90% after 3min at that temperature. Concentration of Cry1A(b) protein decreased
by 90% in tortillas griddled at 180°C for 20s, but no protein was detected in fried tortillas after 10s at 190°C. Cry1A(b)
protein is rapidly denatured by heat treatment resulting in a marked decline in concentration and decreased detection in processed
foods.
European Food Research and Technology 04/2012; 229(1):15-19. · 1.57 Impact Factor
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ABSTRACT: The antibacterial activity of human lactoferrin from milk (hLF), recombinant human lactoferrin from Aspergillus awamori (rhLF) and their hydrolysates obtained with pepsin was investigated against Escherichia coli O157:H7, Salmonella Enteritidis and Listeria monocytogenes. The minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC) were determined for all the
bacteria and the proteins assayed. Taking into account the MICs found for both lactoferrins studied, we can say that they
behave very similarly, except for L. monocytogenes for which rhLF was more active. We studied the effect that heat treatments exerted on the antibacterial activity of the two
types of lactoferrin and the only heat treatment that had a negative effect on that activity was 85°C for 10min. The activity
of hLF and rhLF in UHT milk and whey against E. coli O157:H7 and L. monocytogenes, was also assayed. Our results showed a reduction in the number of viable cells for both microorganisms when were incubated
with rhLF or hLF, but this decrease was lower than in broth media.
European Food Research and Technology 04/2012; 228(2):205-211. · 1.57 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. · 2.67 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. · 1.28 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. · 3.78 Impact Factor
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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
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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. · 1.11 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. · 2.82 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. · 1.28 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. · 1.28 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
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Food and Agricultural Immunology 12/2008; 19:339-350. · 0.71 Impact Factor
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ABSTRACT: Lactoferrin (LF) is an iron-binding glycoprotein found in different biological fluids of mammals and in neutrophils. It has been proposed to be involved in many functions, including protection from pathogens. In this work, purification of lactoferrin using an ion-exchange chromatography (SP-Sepharose) was attempted for the milk of the following animals: sheep (Ovis aries), goat (Capra hircus), camel (Camelus bactrianus), alpaca (Lama pacos), elephant (Elephas maximus) and grey seal (Halichoerus grypus), as well as human (Homo sapiens). Lactoferrin was identified in all the milks apart from that from grey seal. The thermal stability of the purified lactoferrins, in their native and iron-saturated forms, was studied by differential scanning calorimetry (DSC). Maximum temperature, onset temperature and enthalpy change of denaturation were higher when lactoferrins were saturated with iron than in their native form, indicating an increase in the stability of the protein structure upon iron-binding. Human lactoferrin was found to be the most heat-resistant and the other lactoferrins presented different degrees of thermoresistance, that of elephant being the least resistant. The antimicrobial activity of the different isolated lactoferrins was investigated against Escherichia coli 0157:H7. The minimal inhibitory concentrations (MICs) were determined by measuring the absorbance at 620 nm. The minimum bactericidal concentrations (MBCs) were also measured and it was found that camel lactoferrin was the most active lactoferrin against E. coli 0157:H7, whereas alpaca and human lactoferrins were the least active.
Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology 06/2008; 150(1):131-9. · 1.92 Impact Factor
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ABSTRACT: Recombinant human lactoferrin (rhLF) from Aspergillus awamori bound to Caco-2 cell membranes in a saturable manner. The dissociation constant for the apo form was (Kd)=2.2 x 10(-7) M; however, the specific binding of the iron-saturated rhLF and of lactoferrin from human milk (hLF) was too low to calculate the binding parameters. Recombinant human lactoferrin subjected to heat treatment did not lose the ability to bind to cell membranes except at high temperature and long time treatments (85 and 89 degrees C for 40 min) for which there was a slight decrease in the binding. No significant differences have been found in the transport of iron bound to rhLF or to hLF across Caco-2 cell monolayers. Nevertheless, the amount of iron-saturated hLF transported across Caco-2 monolayers was significantly higher than that of rhLF. For both lactoferrins, the amount of intact protein in the lower chamber was about 4.5% of the total radioactivity transported, indicating the degradation of lactoferrin in the passage across Caco-2 cells.
Journal of Agricultural and Food Chemistry 05/2008; 56(8):2831-7. · 2.82 Impact Factor
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ABSTRACT: Thermal denaturation of recombinant human lactoferrin from transgenic rice with different degrees of iron saturation has been studied by differential scanning calorimetry. The maximum temperature, enthalpy change, and activation energy of denaturation were higher when recombinant lactoferrin was more saturated with iron, indicating an increase in the stability of the protein structure. Maximum temperature and activation energy values for apo- and holo-lactoferrins were practically identical to those reported for the same forms of lactoferrin from human milk, which indicates a similar thermal stability. However, the value of enthalpy change for denaturation of the recombinant lactoferrin was 2.5-3-fold lower than that found for the human milk protein. This finding may reflect the influence that the different glycosylation pattern may have in the relationship between lactoferrin domains. Denaturation of recombinant lactoferrin in milk was compared with denaturation in phosphate buffer, and results indicated that the protein was more heat-sensitive when treated in milk than in buffer.
Journal of Agricultural and Food Chemistry 07/2007; 55(12):4848-53. · 2.82 Impact Factor
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ABSTRACT: This study was performed to contribute to the analysis of alpha-lactalbumin "molten globule" state by using spectral and proteolysis techniques. Samples of holo and apo alpha-lactalbumin in the presence of different concentrations of ethanol were analyzed. Results of fluorescence spectroscopy of both forms showed that as ethanol concentration increased, the tryptophanyl residues became more accessible to the solvent. Near circular dichroism spectra of holo alpha-lactalbumin indicated that its tertiary structure was maintained in 20% ethanol whereas it was altered in 30 and 40% ethanol. For apo alpha-lactalbumin, spectra were similar in all samples studied. Holo alpha-lactalbumin was resistant to trypsinolysis in 0% ethanol, whereas it was easily hydrolyzed in 20 and 30% ethanol. In the case of the apo form and in the absence of ethanol, 70% of the protein was degraded after 1 h. However, in the presence of 20 and 30% ethanol, the overall reaction rate was lowered. Peptides obtained after tryptic hydrolysis were identified by reversed-phase high-performance liquid chromatography coupled to mass spectrometry. Differences in population of produced peptides indicate the changes of folding intermediates present in the studied alpha-lactalbumin solutions. This study demonstrated that proteolytic enzymes are suitable tools to determine protein structure complementing physico-chemical studies.
Molecular Nutrition & Food Research 02/2006; 50(1):34-43. · 4.30 Impact Factor
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ABSTRACT: The effect of heat treatment on the denaturation of alpha-lactalbumin was studied, under different conditions, over a temperature range of 78-94 degrees C. The concentration of the residual immunoreactive protein after different treatments was determined by kinetic analysis, obtaining D and Z values. Thermodynamic parameters were also calculated. Denaturation of alpha-lactalbumin, measured by the loss of immunoreactivity, could be described as an order of reaction of n = 1.5. Results obtained indicated that alpha-lactalbumin was more heat-sensitive when treated in milk than in phosphate buffer. The protein was also denatured more rapidly in the apo form than in the calcium-saturated form. Besides, the thermal stability of apo-alpha-lactalbumin decreased with the binding of oleic acid.
Journal of Agricultural and Food Chemistry 01/2006; 53(25):9730-6. · 2.82 Impact Factor
