Miguel Calvo

Isolation of lactoferrin from milk of different species: calorimetric and antimicrobial studies

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.

Effect of β-lactoglobulin on the activity of pregastric lipase. A possible role for this protein in ruminant milk

The interaction of bovine beta-lactoglobulin with palmitic and oleic acids has been studied by a partition equilibrium method. Bovine beta-lactoglobulin displays only one high affinity binding site for fatty acids whose association constants for palmitic and oleic acids are 4.2 x 10(6) and 2.3 x 10(6) M-1, respectively. However, other binding sites with low affinity are also present. The existence of one high affinity binding site is in accordance with the amount of fatty acids naturally bound to beta-lactoglobulin isolated from milk. The effect of beta-lactoglobulin on ruminant pregastric lipases from a pharyngeal extract has been assayed. The activity of pharyngeal lipase on a triglyceride emulsion is increased about 200%, 250% and 190% in the presence of 10 mg/ml, 20 mg/ml and 40 mg/ml of beta-lactoglobulin, respectively, the last concentration representing that found physiologically in colostrum. Albumin, another ligand-binding protein, increases the activity of this enzyme to a lesser extent and high levels tend to inhibit enzyme action. These results indicate that beta-lactoglobulin could participate in the digestion of milk lipids during the neonatal period by enhancing the activity of pregastric lipase through removal of the fatty acids that inhibit this enzyme.

Interaction of bovine lactoferrin with other proteins of milk whey

The association between bovine lactoferrin and the major bovine whey proteins, beta-lactoglobulin, alpha-lactalbumin and albumin has been studied by immunochemical techniques, gel filtration and affinity chromatography in lacteal secretions and using purified proteins. Bovine lactoferrin is able to form non-covalent complexes with beta-lactoglobulin or albumin, with lactoferrin-protein molar ratios of 2:1 and 1:1 respectively. No association was detected with alpha-lactalbumin. Lactoferrin interacts with beta-lactoglobulin-Sepharose at low ionic strength, but not in the presence of 0.3 M NaCl, indicating that ionic interactions are important. The lack of association with alpha-lactalbumin suggests however a certain degree of specificity in this electrostatic interaction.

Comparison of the ability to bind lipids of β-lactoglobulin and serum albumin of milk from ruminant and non-ruminant species

The interaction of sheep, horse, pig, human and guinea-pig whey proteins with fatty acids has been studied. Using gel filtration and autoradiography, it was found that sheep beta-lactoglobulin and serum albumin from all species had the ability to bind fatty acids in vitro. Sheep beta-lactoglobulin, isolated from milk, had approximately 0.5 mol fatty acids bound per mol monomer protein, and albumin from sheep, horse and pig contained approximately 4.5, 2.9 and 4.7 mol fatty acids/mol protein respectively. However, beta-lactoglobulin from horse and pig milk had neither fatty acids physiologically bound nor the ability to bind them in vitro. Albumin was the only whey protein detected with bound fatty acids in these species as well as in human and guinea pig. This suggests that the ability of ruminant beta-lactoglobulin to bind fatty acids was not shared by the same protein of non-ruminants.

Fatty acids bound to α-fetoprotein and albumin during rat development

Abstract The time-course levels and composition of the fatty acids bound to rat α-fetoprotein (AFP) and albumin from several sources, were determined throughout development, and related to the intake of lipids from milk and the compositional changes in brain and liver fatty acids. The major fatty acids bound to AFP were polyunsaturated and mainly docosahexaenoic acid (22:6(n − 3)), either from fetal serum (23.1%) or whole fetuses (21.6%), whereas palmitic (34.1%) and oleic (29.9%) acids were the main acids bound to albumin from the same sources. Amniotic fluid AFP contained less fatty acids (0.8 mol/mol protein) than that of fetal serum (1.4 mol/mol protein), and especially noticeable was a reduced amount of 22:6 (9.6%). Both AFP-concanavalin A microforms showed identical fatty acid composition. Levels of 22:6 bound to AFP decreased quickly after birth until a minimum at 8–10 days, increasing moderately thereafter. This minimum is coincident in time with a maximal accumulation of this fatty acid by brain and a loss of 22:6 by liver. Except for colostrum, levels of 22:6 in milk lipids were low and fairly constant, but always greater than those of its precursor, linolenic acid (18:3 (n − 3)). These results support a specialized role of AFP in the plasma transport and tissue delivery of polyunsaturated fatty acids, and mainly docosahexaenoic acid.

Incorporation of radiolabelled alphafetoprotein in the brain and other tissues of the developing rat

The time course of uptake and autoradiographic localization of alphafetoprotein (AFP) were studied in the brain and other organs of fetuses, neonates and young rats injected with homologous radiolabelled AFP. Comparative data of radioactivity accumulation in the brain relative to that of several tissues (blood, liver, tongue, small intestine) showed bimodal patterns reflecting two periods of more active incorporation, with a maximum before the 16th day of fetal development. In brain autoradiographs, the strongest labelling was observed in 17-day-old fetuses 24 h after injection into the mother of [125I]AFP. The labelling included all regions of the brain. The results presented here give experimental support to the hypothesis that the presence of AFP in the developing nervous system of mammals and birds is primarily due to protein uptake rather than in situ synthesis.

Relations between vitamin D and fatty acid binding properties of vitamin D-binding protein

The interaction of fatty acids with bovine vitamin D-binding protein (DBP) was studied using a partition equilibrium method. This protein has one high affinity site for binding of fatty acids with an association constant Ka = 7 x 10(5) M-1 for palmitic acid and Ka = 6 x 10(5) M-1 for arachidonic acid. Competition experiments showed that palmitic acid hardly competes with 25-hydroxycholecalciferol for binding to DBP. However, arachidonic acid showed comparatively a stronger competition for binding to this protein. The great difference in competition of palmitic and arachidonic acids with 25-hydroxycholecalciferol may be related to changes in DBP conformation promoted by the binding of different ligands.

Recombinant human lactoferrin: A valuable protein for pharmaceutical products and functional foods

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.

Uptake and passage of β-lactoglobulin palmitic acid and retinol across the Caco-2 monolayer

Caco-2 cell line grown on collagen coated polycarbonate membranes in bicameral chambers has been used to study the effect of the binding of palmitic acid or retinol on the uptake and passage of iodinated beta-lactoglobulin and albumin across cell monolayers. The percentage of beta-lactoglobulin transported through the monolayer was higher than that of albumin, about 50% and 30% of the total protein after 24 h of incubation, respectively. In all cases, less than 1% of protein was retained intracellularly. No differences were found in the uptake and transport of beta-lactoglobulin or albumin in the presence or absence of ligands. Furthermore, uptake and passage across Caco-2 monolayer of retinol or palmitic acid added either bound to beta-lactoglobulin or to albumin have been compared. The percentage of retinol found in the lower chamber was about 35% of the total retinol after 24 h of incubation for both proteins. However, the amount of retinol associated to cells was higher when it was added bound to beta-lactoglobulin than to albumin, about 26% and 10%, respectively. This fact suggests that the metabolic processing of retinol by Caco-2 cells is the rate-limiting step for retinol transport. The percentage of palmitic acid that crossed the monolayer was about 7%, remaining approx. 90% in the cells for beta-lactoglobulin and albumin. These data support the hypothesis that palmitic acid internalized by Caco-2 cells is mainly destined to serve the structural and energy needs. These results show evidence of retinol and palmitic uptake by Caco-2 cells when beta-lactoglobulin or albumin are the donors, and indicate that the type of binding protein does not affect the transport of both ligands through Caco-2 monolayer.

Effect of retinol and fatty acid binding by bovine β-lactoglobulin on its resistance to trypsin digestion

Abstract β-Lactoglobulin has been reported to be quite resistant to proteolysis by digestive enzymes. In this work, the effect of the binding of retinol and fatty acids to β-lactoglobulin on the resistance of this protein to trypsin degradation has been studied. When delipidated β-lactoglobulin and β-lactoglobulin with bound retinol were treated with trypsin and subjected to HPLC gel filtration, no major differences were found in the degradation products. However, proteolysis decreased when β-lactoglobulin contained bound palmitic acid. After 4 h of trypsin incubation, less than 30% of delipidated β-lactoglobulin or β-lactoglobulin with bound retinol remained undegraded, while 60% of β-lactoglobulin with bound fatty acids was still intact. These results indicate that the binding of fatty acids to β-lactoglobulin increases its conformational stability to tryptic degradation.