Luis Mata

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.

Evaluation of indirect competitive and double antibody sandwich ELISA tests to determine β-lactoglobulin and ovomucoid in model processed foods

Abstract Enzyme-linked immunosorbent assay (ELISA) kits (indirect competitive and sandwich formats) to determine either β-lactoglobulin or ovomucoid were evaluated in model foods. A cut-off value was established for each kit to consider food samples as positive for milk or egg addition. Sausage and bread were positive at lower percentages of added milk using the sandwich format (0.005 and 0.05%) than the indirect competitive format (0.05 and 0.25%) and pâté was positive at 0.25% milk addition for both formats. Sausage was positive at 0.005%, and bread at 0.05% added egg for indirect competitive and sandwich formats, whereas pâté was positive at 0.25% egg only by the indirect competitive assay. The concentration of added milk and egg to give a positive result depends on heat treatment, being higher for pâté (sterilised), followed by bread (baked) and sausage (pasteurised). The particularities of each format and the heat processing applied influenced the determination by ELISA of allergenic proteins in foods.

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.

CADMIUM UPTAKE BY CACO-2 CELLS. EFFECT OF SOME MILK COMPONENTS

The effect of some milk components on the cellular uptake of cadmium has been studied using a human intestinal cell line (Caco-2). Cadmium uptake by Caco-2 cells increased with the concentration of this metal in the culture medium, in a saturable way. These cells were exposed to different concentrations of cadmium and the synthesis of metallothionein was studied by a cadmium-saturation method. The levels of metallothionein increased with the cadmium concentration in the medium up to 20 microM of metal. Supplementation of the culture medium with 10% bovine milk caused a 25% decrease in the uptake of cadmium with respect to that internalized by the cells maintained in the culture medium alone. However, the uptake of cadmium from the medium supplemented with 10% human milk was similar to that with serum-free medium. beta-Lactoglobulin interacted with cadmium when studied by equilibrium dialysis, showing a stoichiometric binding constant of 5 x 10(4) l/mol. Interaction of lactoferrin with cadmium, however, was negligible. When Caco-2 cells were incubated in culture medium containing lactoferrin, cadmium uptake decreased with respect to that observed incubating the cells in a medium containing beta-lactoglobulin or in the free-protein medium. The inhibitory effect of lactoferrin on the uptake of cadmium might be due to a reduction of the cell surface charge, through its binding to the membrane.

Changes in the distribution of cadmium and lead in human and bovine milk induced by heating or freezing

The percentage of cadmium or lead present in the fat fraction of bovine milk is not affected by heating or freezing. In human skimmed milk, cadmium is mainly associated with a fraction with molecular weight lower than 10,000. Storage at -20°C for 10 days does not have any effect on the distribution of cadmium when milk is incubated with this metal before freezing. This treatment causes only a small increase in the amount of cadmium associated with the low molecular weight fraction when the metal is added after freezing. In bovine milk, 64% of cadmium is associated with a fraction with molecular weight above 70,000. Freezing causes a 37% decrease of the cadmium present in this fraction when the metal is added after thawing. When bovine milk was incubated with cadmium before freezing there was not a marked change in its distribution as when added after thawing. Heating at 63°C for 30 min caused a slight decrease in the amount of cadmium present in the casein fraction. The distribution change of cadmium after freezing or heating is probably due to the formation of complexes between the whey proteins and the metal, or to the disaggregation of the cadmium bound to casein micelles. Lead is mainly associated with caseins in bovine and human milk. No significant changes were caused by freezing or heating in the distribution of lead in human and bovine milk.

Validation of a rapid lateral flow method for the detection of cows’ milk in water buffalo, sheep or goat milk

ABSTRACT For many years, the adulteration of milk from sheep, goats or water buffalos with cows’ milk has been a widespread practice due to the higher cost of milk from those other species. Because of this, great concern has been shown by many Protected Designation of Origin councils that have to assure the quality and genuineness of the cheese produced by their associates. Therefore, the whole production chain needs analytical tools that allow the control of potential adulteration. Rapid methods to be used in the field are scarce and have not been validated according to international guidelines. The aim of this work has been to validate a rapid test based on lateral flow immunochromatography to detect cows’ milk in milk from other species, including buffalo’s milk, according to AOAC guidelines. No false-positive result was found after analysing 146 known negative samples from individual animals. The lowest level of adulteration with a Probability of Detection (POD) of 1.00 (confidence interval between 0.94 and 1.00) was found at 0.5% of cows’ milk. This level is below the current EU allowed level of cows’ milk, set at 1%. Variations in the time of assay, volume of the analysis buffer and different batches of the test were evaluated to detect any effect on the false-positive rate or on the limit of detection of the test. The effects of compositional factors (such as high level of fat, protein and somatic cell counts) were also evaluated. The new rapid test to detect cows’ milk in milk from other species is shown to be an adequate tool to control milk quality in routine analysis. This kind of test is very easy to use and it can be performed by untrained staff during milk collection at the farm or upon arrival at dairies.

Incidence of Antimicrobial Residues in Meat Using a Broad Spectrum Screening Strategy

Aims: The aim of this paper was to assess the incidence of antimicrobial residues in market muscle samples from different animal species (bovine, ovine, poultry and porcine) using a new screening strategy . Methodology: 4849 samples were evaluated with a methodology that combines a broad spectrum microbial test (Explorer) and a specific test for quinolones detection (Equinox). Supplementary tests were performed to achieve additional information about the nature of antimi crobials in positive samples. Results: In a first step, 355 samples (7.3%) showed a positive result in Explorer and/or Equinox tests. The highest incidence of positive samples was obtained in poultry (9.7%) while the lowest rate was found in porcine sample s (3.4%). Half of the positive screening samples (53%) showed also a positive result with supplementary tests indicating that tetracyclines, aminoglycosides sulphonamides and quinolones might be present in these samples. Aminoglycosides were the predominan t residues in poultry while tetracyclines were more frequent in bovine and porcine samples. Sulphonamides were the main family of residues found in ovine. Conclusion: Our results sugges t that the current strategies used for control of antimicrobial Original Research Article