A New Method for Growth Factor Enrichment from Dairy Products by Electrodialysis with Filtration Membranes: The Major Impact of Raw Product Pretreatment.

This study is focused on fractionation of insulin-like growth factor I (IGF-I) and transforming growth factor-ß2 (TGF-ß2) using a new electro-based membrane process calledelectrodialysis with filtration membranes (EDFM). Before EDFM, different pretreatments were tested, and four pH conditions (4.25, 3.85, 3.45, and 3.05) were used during EDFM. It was demonstrated that a 1:1 dilution of defatted colostrum with deionized water to decrease mineral content followed by the preconcentration of GFs by UF is necessary and allow for these compounds to migrate to the recovery compartment during EDFM. MS analyses confirmed the migration, in low quantity, of only α-lactalbumin (α-la) and ß-lactoglobulin (ß-lg) from serocolostrum to the recovery compartment during EDFM. Consequently, the ratio of GFs to total protein in recovery compartment compared to that of feed serocolostrum solution was 60× higher at pH value 3.05, the optimal pH favoring the migration of IGF-I and TGF-ß2. Finally, these optimal conditions were tested on acid whey to also demonstrate the feasibility of the proposed process on one of the main by-products of the cheese industry; the ratio of GFs to total protein was 2.7× higher in recovery compartment than in feed acid whey solution, and only α-la migrated. The technology of GF enrichment for different dairy solutions by combining ultrafiltration and electrodialysis technologies was proposed for the first time.

Study on the interaction mechanism of whey protein isolate with phosphatidylcholine: By multispectral methods and molecular docking.

The elucidation of the interaction mechanism between phospholipids and milk proteins within emulsions is pivotal for comprehending the properties of infant formula fat globules. In this study, multispectral methods and molecular docking were employed to explore the relationship between phosphatidylcholine (PC) and whey protein isolate (WPI). Observations indicate that the binding constant, alongside thermodynamic parameters, diminishes as temperature ascends, hinting at a predominantly static quenching mechanism. Predominantly, van der Waals forces and hydrogen bonds constitute the core interactions between WPI and PC. This assertion is further substantiated by Fourier transform infrared spectroscopy, which verifies PC's influence on WPI's secondary structure. A detailed assessment of thermodynamic parameters coupled with molecular docking reveals that PC predominantly adheres to specific sites within α-lactalbumin, ß-lactoglobulin, and bovine serum albumin, propelled by a synergy of hydrophobic interactions, hydrogen bonding, and van der Waals forces, with binding energies noted at -5.59, -6.71, and -7.85 kcal/mol, respectively. An increment in PC concentration is observed to amplify the emulsification properties of WPI whilst concurrently diminishing the zeta potential. This study establishes a theoretical foundation for applying the PC-WPI interaction mechanism in food.

Competitive protein adsorption in isocratic anion exchange chromatography.

Protein chromatography is the dominant method of purification of biopharmaceuticals. Although all practical chromatography involves competitive absorption and separation of M. species, competitive protein absorption has remained inadequately understood. We previously introduced the measurement of equilibrium protein adsorption isotherms with all intensive variables held constant, including competitor concentration. In this work, we introduce isocratic chromatographic retention measurements of dynamic protein adsorption in the presence of a constant concentration of a competitor protein. These measurements are achieved by establishing a dynamic equilibrium with a constant concentration of competitor (insulin) in the mobile phase flowing through an ion exchange adsorbent column and following the behavior of a test protein (α-lactalbumin) injected into this environment. We observed decreased retention times for α-lactalbumin in presence of the competitor. The presence of competitor also reduces the heterogeneity of the sites available for adsorption of the test protein. This investigation provides an approach to fundamental understanding of competitive dynamics of multicomponent protein chromatography.

The mechanism of resveratrol stabilization and degradation by synergistic interactions between constituent proteins of whey protein.

Whey protein isolate (WPI) is mainly composed of ß-lactoglobulin (ß-LG), α-lactalbumin (α-LA) and bovine serum albumin (BSA). The aim of this study was to compare and analyze the influence of WPI and its three main constituent proteins, as well as proportionally reconstituted WPI (R-WPI) on resveratrol. It was found that the storage stability of resveratrol was protected by WPI, not affected by R-WPI, but reduced by individual whey proteins at 45°C for 30 days. The rank of accelerated degradation of resveratrol by individual whey proteins was BSA > α-LA > ß-LG. The antioxidant activity, localization of resveratrol and oxidation of carrier proteins were determined by ABTS, H2O2 assay, synchronous fluorescence, carbonyl and circular dichroism. The non-covalent interactions and disulfide bonds between constituent proteins improved the antioxidant activity of the R-WPI-resveratrol complex, the oxidation stability of the carrier and the solvent shielding effect on resveratrol, which synergistically inhibited the degradation of resveratrol in R-WPI system. The results gave insight into elucidating the interaction mechanism of resveratrol with protein carriers.

Modulating Whey Proteins Antigenicity with Lactobacillus delbrueckii subsp. bulgaricus DLPU F-36 Metabolites: Insights from Spectroscopic and Molecular Docking Studies.

Numerous studies have highlighted the potential of Lactic acid bacteria (LAB) fermentation of whey proteins for alleviating allergies. Nonetheless, the impact of LAB-derived metabolites on whey proteins antigenicity during fermentation remains uncertain. Our objective was to elucidate the impact of small molecular metabolites on the antigenicity of α-lactalbumin (α-LA) and ß-lactoglobulin (ß-LG). Through metabolomic analysis, we picked 13 bioactive small molecule metabolites from Lactobacillus delbrueckii subsp. bulgaricus DLPU F-36 for coincubation with α-LA and ß-LG, respectively. The outcomes revealed that valine, arginine, benzoic acid, 2-keto butyric acid, and glutaric acid significantly diminished the sensitization potential of α-LA and ß-LG, respectively. Moreover, chromatographic analyses unveiled the varying influence of small molecular metabolites on the structure of α-LA and ß-LG, respectively. Notably, molecular docking underscored that the primary active sites of α-LA and ß-LG involved in protein binding to IgE antibodies aligned with the interaction sites of small molecular metabolites. In essence, LAB-produced metabolites wield a substantial influence on the antigenic properties of whey proteins.

A novel proteomic approach for the identification and relative quantification of disulfide-bridges in the human milk proteome.

This study explores the disulfide bridges present in the human milk proteome by a novel approach permitting both positional identification and relative quantification of the disulfide bridges. Human milk from six donors was subjected to trypsin digestion without reduction. The digested human milk proteins were analyzed by nanoLC-timsTOF Pro combined with data analysis using xiSEARCH. A total of 85 unique disulfide bridges were identified in 25 different human milk proteins. The total relative abundance of disulfide bridge-containing peptides constituted approximately 5% of the total amount of tryptic-peptides. Seven inter-molecular disulfide bridges were identified between either α-lactalbumin and lactotransferrin (5) or αS1-casein and κ-casein (2). All cysteines involved in the observed disulfide bridges of α-lactalbumin and lactotransferrin were mapped onto protein models using AlphaFold2 Multimer to estimate the length of the observed disulfide bridges. The lengths of the disulfide bridges of lactotransferrin indicate a potential for multi- or poly-merization of lactotransferrin. The high number of intramolecular lactotransferrin disulfide bridges identified, suggests that these are more heterogeneous than previously presumed. SIGNIFICANCE: Disulfide-bridges in the human milk proteome are an often overseen post-transaltional modification. Thus, mapping the disulfide-bridges, their positions and relative abundance, are valuable new knowledge needed for an improved understanding of human milk protein behaviour. Although glycosylation and phosphorylation have been described, even less information is available on the disulfide bridges and the disulfide-bridge derived protein complexes. This is important for future work in precision fermentation for recombinant production of human milk proteins, as this will highlight which disulfide-bridges are naturally occouring in human milk proteins. Further, this knowledge would be of value for the infant formula industry as it provides more information on how to humanize bovine-milk based infant formula. The novel method developed here can be broadly applied in other biological systems as the disulfid-brigdes are important for the structure and functionality of proteins.

Using Capillary Electrophoresis to Investigate Protein Conformational and Compositional Heterogeneity.

Detailed insights into protein structure/function relationships require robust characterization methodologies. Free-solution capillary electrophoresis (CE) is a unique separation technique which is sensitive to the conformation and/or composition of proteins, and therefore provides information on the heterogeneity of these properties. Three unrelated, conformationally/compositionally-altered proteins were separated by CE. An electrophoretic mobility distribution was determined for each protein along with its conformational and/or compositional heterogeneity. The CE results were compared with molar mass distributions obtained from size-exclusion chromatography coupled to light scattering (SEC-MALS). Bovine serum albumin multimers and two monomeric species were separated, highlighting variations in conformational/compositional heterogeneity among the multimers. Analysis of yeast alcohol dehydrogenase resolved two monomeric conformers and various tetrameric species, illustrating the impact of zinc ion removal and disulfide bond reduction on the protein's heterogeneity. The apo (calcium-free) and holo forms of bovine α-lactalbumin were separated and differences in the species' heterogeneity were measured; by contrast, the SEC-MALS profiles were identical. Comparative analysis of these structurally unrelated proteins provided novel insights into the interplay between molar mass and conformational/compositional heterogeneity. Overall, this study expands the utility of CE by demonstrating its capacity to discern protein species and their heterogeneity, properties which are not readily accessible by other analytical techniques.

Modulation of the electrostatic potential around α-lactalbumin using oligoelectrolyte chains, pH and salt concentration.

In this study, we conducted a comprehensive computational investigation of the interaction between α-lactalbumin, a small globular protein, and strong anionic oligoelectrolyte chains with a polymerization degree from 2 to 9. Both the protein and oligoelectrolyte chains are represented using coarse-grained models, and their properties were calculated by the Monte Carlo method under constant pH conditions. We were able to estimate the effects of this interaction on the electrostatic potential around the protein. At acidic pH, the protein had a net positive charge; therefore, the electrostatic potential around it was also positive. To neutralize or reverse this electrostatic potential, oligoelectrolyte chains with a minimum size of six monomers were necessary. Simultaneously, low salt concentrations were required as elevated salt levels led to a significant attenuation of the electrostatic interactions and the corresponding electrostatic potential.

Myricetin and morin hydrate inhibit amyloid fibril formation of bovine α-lactalbumin (BLA).

Amyloid fibrils are self-assembled aggregates of proteins and peptides that can lead to a broad range of diseases called amyloidosis. So far, no definitive and approved treatment to target directly amyloid fibrils has been introduced. Nevertheless, the search for small molecules with ability to inhibit and suppress fibril formation is an active and promising area of the research. Herein, the binding interactions and inhibitory effects of myricetin and morin hydrate on the in vitro fibrillation of bovine α-lactalbumin (BLA) have been investigated. The intrinsic fluorescence of BLA was quenched by myricetin and morin hydrate through combination of the static and dynamic quenching along with non-radiative Förster energy transfer mechanisms. The binding of these two flavonoids to BLA were not accompanied by major alteration in the conformation of BLA as evidenced by CD studies. The results of the fluorescence quenching analyses indicated almost the same binding affinities of myricetin and morin hydrate toward BLA (Kb ~ 106 M-1). However, the results of thioflavin T (ThT) assays showed that myricetin is a stronger inhibitor against BLA fibrillation compared to morin hydrate.

Aggregation studies of alpha-lactalbumin induced by edible azo dyes with different sulfonyl group numbers: A comparative study.

As an opaque and complex colloidal mixture, milk is usually present as a positively charged colloid under acidic conditions. Adding negatively charged colloids can lead to protein aggregation in milk. Alpha-lactalbumin (α-La) is an essential component of whey protein and has good physicochemical properties for functional food development. We combined spectroscopy, computer simulations, and other techniques to comparative analyze the mechanisms and characteristics of isolated α-La aggregation induced by CI Acid Red 27 (C27)/CI Acid Red 14 (FB) containing different sulfonyl groups in vitro. The results showed that C27/FB (5.25 × 10-5 mol·L-1 to 3.15 × 10-4 mol·L-1) induced the formation of fibril-like aggregates under acidic conditions (pH 2.0 and 4.0) mainly benefit from hydrophobic and electrostatic forces. Weakening and redshift of α-La's characteristics negative peak were observed (208 nm to 218 nm) on circular dichroism. ß-Crosslinks self-assembly and reorganization of disulfide bonds occurred during protein fibrillation. Moreover, the different redshift intensity of Congo red binding to amyloid fibrils was observed to be induced by C27 (>551 nm) and FB (>536 nm), and the direct observation by TEM demonstrated the ability to induce protein fibrillation is C27 > FB. Edible azo dyes with more sulfonyl groups would possess a stronger ability to induce protein fibrillation.

The role of hydrophobic interactions in the molten globule state of globular protein modulated by surfactants.

In order to highlight the role of hydrophobic interactions in the molten globule (MG) state of globular protein modulated by surfactants, the interactions of bovine α-lactalbumin (α-LA) with alkyl trimethylammonium bromides (CnTAB, n = 10, 12, 14, and 16) have been studied by experimental and theoretical techniques. Isothermal titration calorimetry (ITC) showed that the enthalpy changes (ΔH) and area of the enthalpogram increased with increasing the chain length of CnTAB. The result of fluorescence, circular dichroism (CD) and 1H nuclear magnetic resonance (NMR) spectrum suggested that C10TAB and C12TAB unfolded α-LA partially, C14TAB reconstructed protein with a native-like secondary structure content, and C16TAB induced an MG state α-LA. The SAXS results confirmed that the tertiary structure of α-LA was disrupted by C16TAB forming an MG state complex with a micelle-like structure even at the surfactants concentrations below CMC. As indicated by MD results, the ß-domain and unstructured region(s) were involved in the MG state α-LA modulated by CnTAB. This work not only provides molecular insights into the role of hydrophobic interactions in the MG state of a globular protein but also helps understand the mechanism of preparing α-LA based biomacromolecule modulated by hydrophobic interactions.

Preparation of enzymatically cross-linked α-lactalbumin nanoparticles and their application for encapsulating lycopene.

High internal phase Pickering emulsions (HIPPEs) stabilized by protein nanoparticles have been widely reported, but the use of enzymatic methods for preparing these nanoparticles remains underexplored. Our hypothesis is that enzymatically crosslinked α-lactalbumin (ALA) nanoparticles (ALATGs) prepared using transglutaminase will demonstrate improved properties as stabilizers for HIPPEs. In this study, we investigated the physicochemical properties and microstructures of ALATGs, finding that enzymatic crosslinking could be enhanced by removing Ca2+ ions from ALA and preheating the proteins (85 °C, 15 min). The electrical charge, secondary structure, and surface hydrophobicity of ALATGs were found to depend on crosslinking conditions. HIPPEs formed with an ALA concentration of 10 mg/mL and an enzyme activity of 120 U/g exhibited the highest apparent viscosity and mechanical strength, as well as significantly improved loading capacity and photostability for the encapsulated lycopene. Overall, our results support the hypothesis that ALATG-nanoparticles show superior performance as emulsifiers compared to ALA-nanoparticles.

HAMLET in human milk is resistant to digestion and carries essential free long chain polyunsaturated fatty acids and oleic acid.

The oleic acid/alpha-lactalbumin complex HAMLET (human alpha-lactalbumin made lethal to tumors) is cytotoxic to various cancerous cell lines and is assembled from alpha-lactalbumin (ALA) and free oleic acid (OA). HAMLET is also cytotoxic to normal immature intestinal cells. It remains unclear if HAMLET, experimentally assembled with OA and heat, can spontaneously assemble in frozen human milk over time. To approach this issue, we used a set of timed proteolytic experiments to evaluate the digestibility of HAMLET and native ALA. The purity of HAMLET in human milk was confirmed by ultra high performance liquid chromatography coupled to tandem mass spectrometry and western blot to resolve the ALA and OA components. Timed proteolytic experiments were used to identify HAMLET in whole milk samples. Structural characterization of HAMLET was performed by Fournier transformed infrared spectroscopy and indicated a transformation of secondary structure with increased alpha-helical character of ALA upon binding to OA.

Interaction between a photoisomerizable azobenzene compound and alpha-lactalbumin: Spectroscopic and computational simulation studies.

The combination of light and photoresponsive compounds provides a peculiar way of regulating biological systems. Azobenzene is a classical organic compound with photoisomerization properties. Exploring the interactions between azobenzene and proteins can deepen the biochemical applications of the azobenzene compounds. In this paper, the interaction of 4-[(2,6-dimethylphenyl)diazenyl]-3,5-dimethylphenol with alpha-lactalbumin was investigated by UV-Vis absorption spectra, multiple fluorescence spectra, computer simulations, and circular dichroism spectra. Most critically, the interaction differences between proteins and the trans- and cis-isomer of ligands have been analyzed and compared. Results showed that both isomers of ligands were bound to alpha-lactalbumin to form ground state complexes and statically quenched the steady-state fluorescence of alpha-lactalbumin. The van der Waals forces and hydrogen bonding dominated the binding; the difference is that the binding of the cis-isomer to alpha-lactalbumin is more rapidly stabilized, and the binding strength is greater than the trans-isomer. These binding differences were modeled and analyzed by molecular docking and kinetic simulations, and we found that both isomers bind through the hydrophobic aromatic cluster 2 of alpha-lactalbumin. However, the bent structure of the cis-isomer is more closely aligned with the construction of the aromatic cluster and may have contributed to the above differences.

Cytotoxicity, structural, conformational, and techno-functional properties of α-lactalbumin nanostructured aggregates.

Protein nanostructures can be used in food applications to improve the techno-functional properties of a food formulation. This study aims to find the best conditions for the production and conformational change of α-lactalbumin nanostructured aggregates. The criteria to determine the best operating conditions to produce α-lactalbumin nanostructured aggregates were intensification of foaming and emulsification, techno-functional proprieties, cytotoxic, and antibacterial activity of nanostructures compared with native α-lactalbumin. Conformational alterations occurred in the α-helix and sheet-ß protein structures. The size obtained by dynamic light scattering was 163.84 nm with a polydispersity index of 0.29. The nano protein improved the techno-functional property compared to the native protein. Additionally, nanostructures had no cytotoxic effect and were innocuous to bacterial activity. Thus, this study presents the best conditions to produce α-lactalbumin nanostructured aggregates with improved properties that allow new food industry applications.

Purification and identification of xanthine oxidase inhibitory peptides from enzymatic hydrolysate of α-lactalbumin and bovine colostrum casein.

The development of food-derived Xanthine Oxidase (XO) inhibitors is critical to the treatment of hyperuricemia and oxidative stress-related disease. Few studies report on milk protein hydrolysates' XO inhibitory activity, with the mechanism of their interaction remaining elusive. Here, different commercial enzymes were used to hydrolyze α-lactalbumin and bovine colostrum casein. The two proteins hydrolyzed by alkaline protease exhibited the most potent XO inhibitory activity (bovine casein: IC50 = 0.13 mg mL-1; α-lactalbumin: IC50 = 0.28 mg mL-1). Eight potential XO inhibitory peptides including VYPFPGPI, GPVRGPFPIIV, VYPFPGPIPN, VYPFPGPIHN, QLKRFSFRSFIWR, LVYPFPGPIHN, AVFPSIVGR, and GFININSLR (IC50 of 4.67-8.02 mM) were purified and identified from alkaline protease hydrolysates by using gel filtration, LC-MS/MS and PeptideRanker. The most important role of inhibiting activity of peptides is linked to hydrophobic interactions and hydrogen bonding based on the results of molecular docking and molecular dynamics simulation. The enzymatic hydrolysate of α-lactalbumin and bovine colostrum casein could be a competitive candidates for hyperuricemia-resisting functional food.

Physical treatment synergized with natural surfactant for improving gas-water interfacial behavior and foam characteristics of α-lactalbumin.

The purpose of this study was to investigate effect of physical treatment (ultrasound, U/high pressure homogenization, H/combined treatment, UH or HU) and surfactant (Mogroside V, Mog) on air/water interface adsorption and foaming properties of α-lactalbumin (ALa). Firstly, the binding of Mog and all physical-treated ALa was a static quenching process. Mog had the greatest binding affinity for HU-ALa among all treated samples. U or H treatment could change surface hydrophobicity of ALa/Mog complex. Secondly, at the molar ratio (ALa:Mog) of 1:50, foaming ability (FA) of all ALa samples got the maximum. The sequence of FA in ALa and ALa/Mog complex was listed as follow: HU > U > H > UH. Moreover, foaming stability (FS) of HU-ALa was the highest, followed by H-ALa, U-ALa and UH-ALa. Meanwhile, low concentration Mog increased FS of ALa or UH-ALa, but it reduced FS of H-ALa, U-ALa and HU-ALa. Quartz crystal microbalance with dissipation monitoring (QCM-D) experiment indicated that ALa/Mog complex after U or H treatment was quickly absorbed at air/water interface, compared with the treated ALa, and HU-ALa/Mog had the largest frequency shift. In addition, HU-ALa had the thickest bubble membrane and the highest dissipation shift in all samples, indicating that the absorbed membrane thickness and viscoelasticity of samples was correlated with foam stability. Therefore, U and H treatment synergism with Mog was an effective approach to enhance foam properties of ALa, which indicated that HU-treated ALa/Mog complex could be viewed as the safe and efficient foaming agent applied in food processing.

Comparative analysis of the interaction between alpha-lactalbumin and two edible azo colorants equipped with different sulfonyl group numbers.

Alpha-lactalbumin (α-La) is a crucial active component in whey protein. It would be mixed with edible azo pigments during processing. Spectroscopic analyses and computer simulations were used here to characterize the interaction between acid red 27 (C27) /acidic red B (FB) and α-La. Fluorescence, thermodynamics, and energy transfer showed the binding mechanism is a static quenching with a medium affinity. This binding process occurred spontaneously and was mainly driven by hydrophobic forces. Conformation analysis showed FB led to a greater change in the secondary structure of α-La compared with C27. C27 increased and FB decreased the surface hydrophobicity of α-La. The spatial structures of complexes were visualized with computer aid. The azo colorant binds to α-La easily and deeply with a smaller space volume and dipole moment and thereby affecting the α-La conformation and functionality. This study provides a theoretical basis for the application of edible azo pigments.

Protein association on multimodal chromatography media.

The phenomenon of protein-protein association on multimodal chromatography resins was described for two different case study examples. The adsorption pattern of single-component solutions of calcium-rich alpha-lactalbumin (aLaCa) and calcium-depleted alpha-lactalbumin (aLa) and their mixtures with bovine serum albumin was determined on a multimodal anion-exchange chromatography medium. In single-component solutions, both aLaCa and aLa exhibited identical adsorption behavior at low resin loadings, whereas at high loadings the adsorption strength of aLa markedly exceeded that of alaCa. In binary mixtures, the adsorption of BSA enhanced at high concentrations of aLa or aLaCa in the adsorbed phase. The unusual adsorption patterns observed were attributed to the tendency of the proteins for molecular association in the adsorbed phase in single and binary solutions. The phenomena was examined for different pH of the solution: pH 6, 7, 8, and different solvent environments: phosphate buffer (PB), bis tris buffer (BT), 100 mM NaCl in BT and bis tris propane buffer (BTP). The strongest effect was observed for PB and for 100 mM NaCl in BT. Its occurrence was also evidenced for other case study example, i.e., adsorption of single-component solutions and binary mixtures of a monoclonal antibody (mAb) and lysozyme (LYZ) on a multimodal cation-exchange chromatography medium. The enhancement of adsorption of mAb was observed at high concentrations of LYZ in the adsorbed phase. To quantify the underlying effects, a mechanistic model was used, which accounted for both protein association and exclusion resulting from attractive and repulsive protein-protein iterations in the adsorbed phase.

Calcium bioaccessibility increased during gastrointestinal digestion of α-lactalbumin and ß-lactoglobulin.

Calcium bioaccessibility depends on the amount of soluble calcium under intestinal digestion. The changes in calcium during in vitro static digestion of α-lactalbumin and ß-lactoglobulin in presence of calcium chloride (0 mM, 20 mM and 50 mM) were followed by combining electrochemical determination of free calcium with the determination of soluble calcium by inductively coupled plasma optical emission spectroscopy. α-Lactalbumin and, more evident, ß-lactoglobulin were found to increase calcium bioaccessibility with increasing intestinal digestion time by around 5% and 10%, respectively, due to the complex binding of calcium to peptides formed from protein hydrolysis by gastrointestinal enzymes. In vitro digested samples of ß-lactoglobulin in presence of CaCl2 had nearly twice as much complex bound calcium as α-lactalbumin samples. The calcium bioaccessibility decreased significantly with the increasing concentration of added calcium chloride, although the amount of calcium chloride had little effect on the extension of digestion of α-lactalbumin and ß-lactoglobulin. Simulated digestion fluids were found to have a negative effect on calcium bioaccessibility, especially the presence of hydrogen phosphate, and the amount of precipitated calcium increased significantly with increasing amount of added calcium chloride. Based on analysis and visualization by sequences of the peptides formed during digestion of α-lactalbumin and ß-lactoglobulin, it was observed that peptides containing aspartic acid and glutamic acid acting as calcium chelators, may prevent precipitation of calcium in the intestines and increase calcium bioaccessibility. These results provide knowledge for the design of new dairy based functional foods to prevent calcium deficiency.