M. Dolores Pérez

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.

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.

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.

Distribution of added lead and cadmium in human and bovine milk

Distribution of added lead and cadmium to bovine and human milk and whey has been studied. In bovine milk, about 97 and 89% of lead and cadmium, respectively, were recovered in the casein fraction obtained by enzymatic coagulation. However, only 6% of lead and 41% of cadmium were found in the same fraction separated by acid precipitation, indicating that the distribution of both metals is very different depending on the method used for milk fractionation. Moreover, gel filtration of bovine and human skimmed milk and whey after addition of lead and cadmium was carried out. Most of the lead was associated to the casein fraction after gel filtration of skimmed milk, whereas in the chromatography of whey, lead was eluted with the low molecular weight fraction in both species. However, a different pattern in the distribution of cadmium has been observed in the two species studied. In contrast to the binding of cadmium to the low molecular weight fraction in human skimmed milk and whey, it was mainly associated to a component of a molecular weight around 70,000 in bovine skimmed milk. This component was not present in bovine whey indicating that it is separated with casein during fractionation.