María D. Pérez

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.

Bioactivity of α-Lactalbumin Related to its Interaction with Fatty Acids: A Review

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 Ca2+ 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.

Rapid immunoenzymatic method for detecting adulteration in ewe's milk

Abstract One of the main problems in the manufacture of ewes cheese is the adulteration of milk with milks from other species. We have designed an immunodotting assay to detect the presence of goats and/or cows milk in ewes milk. Anti-goat immunoglobulin antibodies are applied on a nitrocelullose membrane. The nitrocelullose is then incubated with the milk to be analysed and, afterwards, with peroxidase sheep anti-goat immunoglobulins. Finally, the membrane is developed with a peroxidase substrate. This assay takes only about two hours and is suitable for detection of lots of ewes milk adulterated with more than 0.5% of goats or cows milk. Furthermore, this assay is also suitable for detection of adulteration of ewes cheese as it is able to detect the presence of goats or cows milk proteins in ewes cheese.

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.

Comparison of the activity of human and bovine milk on two cell lines

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.

Presence and changes in the concentration of vitamin D-binding protein throughout early lactation in human and bovine colostrum and milk

Abstract Synthesis of vitamin D-binding protein (DBP) by explants of ovine mammary gland, and the changes in the concentration of this protein in human and bovine colostrum and milk throughout early lactation have been studied. The changes in the concentration of this protein were similar in human and bovine species. The highest concentration of DBP was found in the first milking (250 μg/ml. and 111 μ/mL for bovine and human colostrum, respectively). The levels of DBP then decreased sharply during the first days of lactation, reaching stable values during the second week postpartum (6 μg/mL and 16 μg/mL, respectively). Milk and plasma DBP were immunologically identical by immunodiffusion and there was no cross-reaction between the two species. DBP synthesis in mammary gland explants could not be detected. The ratio of DBP to albumin in mature milk and especially in colostrum was much higher than in plasma. This could be due to the existence of a specific mechanism in the mammary gland cells for the transference of DBP from plasma to milk.

Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow

Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme present in milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for newborn nutrition and development. Transgenic animals can offer a unique opportunity to the dairy industry, providing starting materials suitable to develop specific products with high added value.

Antirotaviral Activity of Bovine and Ovine Dairy Byproducts

Rotaviral gastroenteritis is associated with significant morbidity in developed countries and a high rate of infant mortality in developing countries. Diverse studies have demonstrated that a wide range of milk-derived fractions exhibit antirotaviral activity. The present study shows the antirotaviral activity of some bovine and ovine dairy byproducts, buttermilk, butter serum, and milk fat globule membrane (MFGM), and evaluates the effect of cream washing and heat treatment on that activity. Furthermore, the rotavirus-neutralizing activity was evaluated for some MFGM proteins, such as xanthine oxidase and lactophorin. Ovine and bovine buttermilk reached rotavirus-neutralizing values of 51.3 and 32.2%, at 1 mg/mL, respectively. The cream washing process led to a significant decrease in the antirotaviral activity of fractions. This activity was also influenced by heat treatment. Treatment at 75 °C for 20 s caused 24.6 and 36.1% decreases of activity in bovine and ovine buttermilk, respectively, and 85 °C for 10 min caused decreases of 80.9 and 79.0% in both fractions, respectively.

Pepsin Degradation of Cry1A(b) Protein Purified from Genetically Modified Maize (Zea mays)

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.

Study of the thermoresistance of the allergenic Ara h1 protein from peanut (Arachis hypogaea).

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 sulfate 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 °C. 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 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 °C and 1574 kJ/mol, respectively. The hydrophobicity of Ara h1 protein increased with the intensity of heat treatment, and aggregates were formed when the protein was treated at 90 °C for 10 min.