Functional diversity of Bisifusarium domesticum and the newly described Nectriaceae cheese-associated species.

Bisifusarium domesticum is among the main molds used during cheese-making for its "anticollanti" property that prevents the sticky smear defect of some cheeses. Previously, numerous cheese rinds were sampled to create a working collection and not only did we isolate B. domesticum but we observed a completely unexpected diversity of "Fusarium-like" fungi belonging to the Nectriaceae family. Four novel cheese-associated species belonging to two genera were described: Bisifusarium allantoides, Bisifusarium penicilloides, Longinectria lagenoides, and Longinectria verticilliformis. In this study, we thus aimed at determining their potential functional impact during cheese-making by evaluating their lipolytic and proteolytic activities as well as their capacity to produce volatile (HS-Trap GC-MS) and non-volatile secondary metabolites (HPLC & LC-Q-TOF). While all isolates were proteolytic and lipolytic, higher activities were observed at 12 °C for several B. domesticum, B. penicilloides and L. lagenoides isolates, which is in agreement with typical cheese ripening conditions. Using volatilomics, we identified multiple cheese-related compounds, especially ketones and alcohols. B. domesticum and B. penicilloides isolates showed higher aromatic potential although compounds of interest were also produced by B. allantoides and L. lagenoides. These species were also lipid producers. Finally, an untargeted extrolite analysis suggested a safety status of these strains as no known mycotoxins were produced and revealed the production of potential novel secondary metabolites. Biopreservation tests performed with B. domesticum suggested that it may be an interesting candidate for biopreservation applications in the cheese industry in the future.

Interactions between caseins and food-derived bioactive molecules: A review.

Caseins are recognized as safe for consumption, abundant, renewable and have high nutritional value. Casein molecules are found in different aggregation states and their multiple binding sites offer the potential for delivering biomolecules with nutritional and/or health benefits, such as vitamins, phytochemicals, fibers, lipids, minerals, proteins, peptides, and pharmaceutical compounds. In the present review, we highlight the interactions between caseins and food-derived bioactive molecules, with a special focus on the aggregation states of caseins and the techniques used to produce and study the particles used for delivering. Research on interactions between caseins-minerals and casein-pharmaceutical molecules are not included here. This review aims to support the development of new and innovative functional foods in which caseins can be used as designed delivery systems.

Precise Populations' Description in Dairy Ecosystems Using Digital Droplet PCR: The Case of L. lactis Group in Starters.

Lactococcus lactis group (composed of the lactis and cremoris subspecies, recently reassigned as two distinct species) plays a major role in dairy fermentations. Usually present in starter cultures, the two species enable efficient acidification and improve the organoleptic qualities of the final product. Biovar diacetylactis strains produce diacetyl and acetoin, aromas from the citrate metabolization. As these populations have distinct genomic and phenotypic characteristics, the proportions of each other will affect the final product. Today, there is no quantitative test able to distinguish between the two species and the biovar in dairy ecosystems. In this study, we developed a specific, reliable, and accurate strategy to quantify these populations using, species-, and diacetylactis-specific fluorescent probes in digital droplet PCR assays (ddPCR). Species were distinguished based on three single nucleotide polymorphisms in the glutamate decarboxylase gadB gene, and the citD gene involved in citrate metabolism was used to target the biovar. Used in duplex or singleplex, these probes made it possible to measure the proportion of each population. At 59°C, the probes showed target specificity and responded negatively to the non-target species usually found in dairy environments. Depending on the probe, limit of detection values in milk matrix ranged from 3.6 × 103 to 1.8 × 104 copies/ml. The test was applied to quantify sub-populations in the L. lactis group during milk fermentation with a commercial starter. The effect of temperature and pH on the balance of the different populations was pointed out. At the initial state, lactis and cremoris species represent, respectively, 75% and 28% of the total L. lactis group and biovar diacetylactis strains represent 21% of the lactis species strains. These ratios varied as a function of temperature (22°C or 35°C) and acidity (pH 4.5 or 4.3) with cremoris species promoted at 22°C and pH4.5 compared to at 35°C. The biovar diacetylactis strains were less sensitive to acid stress at 35°C. This methodology proved to be useful for quantifying lactis and cremoris species and biovar diacetylactis, and could complete 16S metagenomics studies for the deeply description of L. lactis group in complex ecosystems.

Effect of pH on the physicochemical characteristics and the surface chemical composition of camel and bovine whey protein's powders.

This study investigated the effect of pH on the denaturation extent, the surface chemical composition, the water sorption isotherm and the glass transition temperature of camel and bovine whey protein's powders. The LC-MS analysis indicated that the ß-Lactoglobulin was the most denatured protein in bovine whey powders regardless the pH value, while this protein was totally absent in camel whey. The α-Lactalbumin was relatively heat stable after drying and predominated the powder surface (X-ray photoelectron spectroscopy results) in both camel and bovine whey powders regardless the pH (neutral (6.7) or acidic (4.3 and 4.6)). Analysis of the water sorption isotherms indicated that decreasing the pH induced the increase of the water activity of lactose crystallization for camel and bovine whey powders. Finally, decreasing the pH led to the decrease of the glass transition temperature of camel and bovine whey powder (at 0.13, 0.23, and 0.33 of water activity).

The adhesion of homogenized fat globules to proteins is increased by milk heat treatment and acidic pH: Quantitative insights provided by AFM force spectroscopy.

The rheological properties and microstructure of dairy gels involve the connectivity between milk fat globules (MFG) and casein micelles that is affected by technological processes such as milk homogenization and heat treatment. The underlying mechanisms require further quantification of the interactions at the nanoscale level to be fully understood and controlled. In this study, we examined the adhesion of homogenized MFG to milk proteins and evaluated the role of ultra-high temperature (UHT) heat treatment and pH. The combination of physico-chemical analysis, rheology and microscopy observations at different scale levels associated to atomic force microscopy (AFM) force spectroscopy were used. AFM experiments performed at the particle scale level showed that adhesion of individual homogenized MFG to milk proteins (1) is increased upon acidification at pH 4.5: 1.4 fold for unheated samples and 3.5 fold for UHT samples, and (2) is enhanced by about 1.7 fold at pH 4.5 after UHT heat treatment of milk, from 176 pN to 296 pN, thanks to highly-reactive heat-denatured whey proteins located at the surface of MFG and caseins. The increased inter-particle adhesion forces accounted for more connected structures and stiffer UHT milk acid gels, compared to unheated-milk gels. Using a multiscale approach, this study showed that heat treatment of milk markedly affected the interactions occurring at the particle's surface level with consequences on the bulk structural and rheological properties of acid gels. Such findings will be useful for manufacturers to modulate the texture of fermented dairy products through the tailoring of heat-induced complexation of proteins and the connectivity of homogenized MFG with the protein network. This work will also contribute in a better understanding of the impact of process-induced changes on the digestibility and metabolic fate of proteins and lipids.

The surface properties of milk fat globules govern their interactions with the caseins: Role of homogenization and pH probed by AFM force spectroscopy.

The surface of milk fat globules consists of a biological membrane rich in polar lipids and glycoproteins. However, high shear stress applied upon homogenization disrupts the membrane and leads to the adsorption of casein micelles, as the major protein fraction of milk. These changes in the interface properties could affect the interactions between native or homogenized milk fat globules and the surrounding protein matrix, at neutral pH and upon acidification. In this study, macroscale rheometry, microscopic observations, nanoscale AFM-based force spectroscopy and physico-chemical analysis were combined to examine the interfacial composition and structure of milk fat globules and to evaluate their interactions with casein micelles. We showed that the surface properties of milk fat globules (biological membrane vs. caseins) and pH govern their interactions with casein micelles. The adhesion between individual fat globules and casein micelles was higher upon homogenization, especially at acid pH where the work of adhesion increased from 3.3 x 10-18 to 14 x 10-18 J for native and homogenized fat globules, respectively. Consequently, casein-coated homogenized fat globules yield stiffer milk acid gels. These findings cast light on the importance of colloidal particle's surface properties and pH on their connectivity with the surrounding matrix, which modulates the bulk microstructure and rheological properties with potential functional consequences, such as milk lipid digestion.

The phase and charge of milk polar lipid membrane bilayers govern their selective interactions with proteins as demonstrated with casein micelles.

The biological membrane surrounding fat globules in milk (milk fat globule membrane; MFGM) is an interface involved in many biological functions and interactions with the surrounding proteins or lipolytic enzymes in the gastro-intestinal tract during digestion. The MFGM exhibits lateral heterogeneities resulting from the different phase states and/or head-group charge of the polar lipids, which were both hypothesized to drive interaction with the casein micelles that is the major milk protein assembly. Atomic force microscopy (AFM) imaging was used to track the interactions of casein micelles with hydrated supported lipid bilayers of different composition, phase state and charge. Zeta-potential and Langmuir isotherms of the different polar lipids offered additional information necessary to interpret AFM observations. We showed that the negatively-charged casein micelles did not interact with milk sphingomyelin in the gel or liquid-ordered phases but did interact with polar lipids in the liquid-disordered phase (unsaturated polar lipids and milk sphingomyelin above its melting point). A wide intermolecular distance between polar lipids allowed protein adsorption on the membranes. However, the presence of the anionic polar lipids phosphatidylserine and phosphatidylinositol prevented any interaction with the casein micelles, probably due to electrostatic repulsion. These results open perspectives for the preparation of tailored emulsions covered by polar lipids able to modulate the interfacial interactions with proteins.

Dairy curd coagulated by a plant extract of Calotropis procera: Role of fat structure on the chemical and textural characteristics.

Milk is often subjected to technological treatments which have impacts on the structure of milk constituents and the characteristics of rennet curds. In this paper, the influence of the dairy fat structure on the biochemical and textural characteristics of curds coagulated by an extract of Calotropis procera leaves was studied. Standardized milks were reconstituted with the same contents in protein (35g·kg-1) and fat (35g·kg-1) but with different structures of fat i.e. homogenized anhydrous milk fat (HAMF), homogenized cream (HC) and non-homogenized cream (NHC). As expected, the size distributions of fat globules in the different milks were different. After their coagulations by the plant extract, the physico-chemical characteristics of the curds and respective wheys were determined. No difference was observed in the coagulation time between the three milks but the whey removed more quickly from HAMF and HC curds than NHC-curd. The biochemical analyses of curds revealed a lower content in dry matter and fat in the NHC-curd compared to HAMF- and HC-curds. Otherwise, the NHC-whey exhibited the highest amount of fat. Observations by confocal microscopy showed that the fat globules were homogenously distributed and well trapped in the protein networks of HAMF- and HC-curds. In the NHC-curd, the fat globules were located in whey pockets, with less connectivity with the protein network. The textural analysis showed that the NHC-curd was more elastic, soft and adhesive than HAMF- and HC-curds. Homogenization significantly reduced the loss of fat during cheese manufacturing and conferred specific textural characteristics to the curds coagulated by an extract of Calotropis procera.

Polar lipid composition of bioactive dairy co-products buttermilk and butterserum: Emphasis on sphingolipid and ceramide isoforms.

Bioactive lipids of the milk fat globule membrane become concentrated in two co-products of the butter industry, buttermilk and butterserum. Their lipid composition is detailed here with special emphasis on sphingolipid composition of nutritional interest, determined using GC, HPLC and tandem mass spectrometry. Butterserum was 2.5 times more concentrated in total fat than buttermilk, with 7.7±1.5vs 19.5±2.9wt% and even more concentrated in polar lipids, with 1.4±0.2vs 8.5±1.1wt%. Both ingredients constitute concentrated sources of sphingomyelin (3.4-21mg/g dry matter) and contained low amounts of bioactive ceramides in a ratio to sphingomyelin of 1:5mol% in buttermilk and 1:10mol% in butterserum. Compared to other natural lecithins, these two co-products are rich in long and saturated fatty acids (C22:0-C24:0), contain cholesterol and could have interesting applications in neonatal nutrition, but also as brain-protective, hepatoprotective and cholesterol lowering ingredients.

Bioaccessibility of four calcium sources in different whey-based dairy matrices assessed by in vitro digestion.

Numerous calcium sources are available to enrich food, but their behavior during digestion is still unknown. This study focused on the influence of the gastro-intestinal pH, the food structure and the calcium source on the bioaccessibility of the nutrient. Four calcium sources were studied: calcium carbonate, calcium citrate malate, calcium phosphate and calcium bisglycinate. These were added to dairy matrices, containing cream and whey proteins, of different forms (liquid or gel). The kinetics of solubility and ionic calcium concentration during in vitro digestion were studied, as function of gastro-intestinal pH. All calcium sources were almost fully soluble in the gastric compartment, and then became insoluble in the intestinal phase. The level of calcium insolubilisation in the intestinal phase was not significantly influenced by the matrix structure (liquid or gel), but was more dependent on the calcium source, this effect leading to different final calcium bioaccessibility from 36% to 20%.

Physico-chemical characterization of dairy gel obtained by a proteolytic extract from Calotropis procera - A comparison with chymosin.

Chymosin is the major enzyme used in cheesemaking but latex enzymes are also used. The aim of this work was to characterize the composition and the structure of dairy gel obtained by an extract of Calotropis procera leaves in comparison with those obtained by chymosin. The biochemical and mineral compositions of the curds and the cheese yields obtained by using Calotropis procera extract or chymosin were relatively similar. Quantitative and qualitative evaluations of proteolysis after milk coagulation, determined by the non-protein nitrogen content and chromatography coupled to mass spectrometry, indicated that Calotropis procera extract was more proteolytic than chymosin and that κ-casein was proteolyzed. The main consequence of proteolysis by Calotropis procera extract or chymosin was the formation of a similar and regular network with the presence of aggregates of casein micelles. These results support that Calotropis procera extract can be used as effective coagulant in cheesemaking.

Ser2 from Serratia liquefaciens L53: A new heat stable protease able to destabilize UHT milk during its storage.

The heat-stable protease Ser2 is secreted by the species Serratia liquefaciens, a psychrotrophic bacteria frequently found in raw milk. To understand the physicochemical modifications of casein micelles induced by Ser2 and to confirm its implication in UHT milk destabilization, the enzyme was purified and added to microfiltered raw milk before UHT treatment. UHT milk destabilization was investigated during 90days of storage. A visual destabilization appeared after 8days of storage with the presence of sediment. Zeta potential increase and formation of aggregates were observed during the storage. Using tandem mass spectrometry, numerous released peptides from the four caseins were identified at the end of storage. Caseins were hydrolyzed in the preferential order ß->αs1->κ->αs2. No specific peptidic hydrolysed bond was detected. The present study confirmed that the presence of the protease Ser2 in raw milk can be one of the main causes of UHT milk destabilization.

Proteomic profiling of camel and cow milk proteins under heat treatment.

Cow and camel milk proteins before and after heat treatment at 80°C for 60min were identified using LC/MS and LC-MS/MS following monodimensional electrophoresis. The database used for the identification of camel and cow proteins was set from http://www.uniprot.org/. The obtained results showed that, after heating, camel milk at 80°C for 60min, camel α-lactalbumin (α-la) and peptidoglycan recognition protein (PGRP) were not detected while camel serum albumin (CSA) was significantly diminished. When heating cow milk at 80°C for 60min, α-lactalbumin (α-la) and ß-lactoglobulin (ß-lg) were not significantly detected. Moreover, 19 protein bands from SDS-PAGE were analyzed and a total of 45 different proteins were identified by LC-MS/MS. Casein fractions were kept intact under a heat treatment of 80°C during 60min of both camel and cow milks. Camel and bovine whey proteins were affected by a heat treatment of 80°C for 60min.

Nutrient content of some Cameroonian traditional dishes and their potential contribution to dietary reference intakes.

Malnutrition is a serious public health problem in Cameroon. The research study was conducted to determine nutrient content of some Cameroonian traditional dishes and their potential contribution to dietary reference intakes. These dishes were Ekomba, prepared from maize flour with roasted peanuts paste; Ekwang, prepared from crushed cocoyam tubers and cocoyam leaves; Tenue militaire, prepared from dried maize flour and cocoyam leaves and Koki, prepared from dried crushed cowpea seeds. The samples were subjected to proximate, minerals, carotenoids, and amino acids analyses. Results showed that the protein content ranged between 1.4 and 5.4 g/100 g edible portion. The mineral content expressed in mg/100 g edible portion ranged between 13.4 and 38.9 (calcium), 12.9-30.7 (magnesium), 336.2-567.9 (sodium), 63.3-182.7 (potassium), 0.5-1.5 (iron), 0.3-1.1 (zinc), 0.1-0.2 (copper), and 0.3-0.4 (manganese). Vitamin A activity content ranged between 0.1 and 0.4 mg Retinol Activity Equivalents/100 g edible portion. Consumption of each dish (100 g) (Ekwang, Tenue militaire, and Koki) by children aged 1-2 years would meet more than 100% of their daily recommended intake for vitamin A. Except in Ekomba, essential amino acids in all dishes represented up to 33% of total amino acids, indicating a good equilibrium between amino acids. This up-to-date appropriate information will contribute for the calculation of accurate energy and nutrient intakes, and can be used to encourage the consumption of these dishes.

Development of casein microgels from cross-linking of casein micelles by genipin.

Casein micelles are porous colloidal particles, constituted of casein molecules, water, and minerals. The vulnerability of the supramolecular structure of casein micelles face to changes in the environmental conditions restrains their applications in other domains besides food. Thus, redesigning casein micelles is a challenge to create new functionalities for these biosourced particles. The objective of this work was to create stable casein microgels from casein micelles using a natural cross-linker, named genipin. Suspensions of purified casein micelles (25 g L(-1)) were mixed with genipin solutions to have final concentrations of 5, 10, and 20 mM genipin. Covalently linked casein microgels were formed via cross-linking of lysyl and arginyl residues of casein molecules. The reacted products exhibited blue color. The cross-linking reaction induced gradual changes on the colloidal properties of the particles. The casein microgels were smaller and more negatively charged and presented smoother surfaces than casein micelles. These results were explained based on the cross-linking of free NH2 present in an external layer of κ-casein. Light scattering and rheological measurements showed that the reaction between genipin and casein molecules was intramicellar, as one single population of particles was observed and the values of viscosity (and, consequently, the volume fraction of the particles) were reduced. Contrary to the casein micelles, the casein microgels were resistant to the presence of dissociating agents, e.g., citrate (calcium chelating) and urea, but swelled as a consequence of internal electrostatic repulsion and the disruption of hydrophobic interactions between protein chains. The casein microgels did not dissociate at the air-solution interface and formed solid-like interfaces rather than a viscoelastic gel. The potential use of casein microgels as adaptable nanocarriers is proposed in the article.

Polarized infrared reflectance spectra of brushite (CaHPO4·2H2O) crystal investigation of the phosphate stretching modes.

Polarized infrared (IR) reflectance measurements at near-normal incidence were recorded from the ac-plane of a monoclinic brushite (CaHPO4·2H2O) crystal in the 800-1200 cm(-1) spectral range (P-O stretching modes). The adjustment of these data, on the basis of a dispersion analysis (DA) model for monoclinic case, allowed the determination of oscillators parameters for the four P-O stretching observed modes of the phosphate group.

Quantitative and qualitative variability of the caseinolytic potential of different strains of Pseudomonas fluorescens: implications for the stability of casein micelles of UHT milks during their storage.

Pseudomonas fluorescens grows at low temperature and produces thermo-resistant protease(s) that can destabilize UHT (Ultra High Temperature) milk during its storage. The consequences of contamination of microfiltered milk with 9 strains of P. fluorescens on the stability of the corresponding UHT milk during storage had been investigated in this study. The strains were classified in two groups according to their ability to destabilize UHT milk. For the group of highly destabilizing strains, sedimentations of UHT milks, low values to phosphate test and the presence of aggregates were observed. Zeta potential and hydration of casein micelles decreased, whereas non casein nitrogen (NCN) and non protein nitrogen (NPN) contents increased. The analyses of NCN fraction by liquid chromatography coupled to mass spectrometry indicated that the different casein molecules were hydrolyzed in a similar way for the destabilizing strains suggesting that the same enzyme was implicated. For the group of slightly or not destabilizing strains no visual and biochemical alteration were found. This study showed that destabilization of UHT milk by P. fluorescens was highly variable and strain-dependent.

Effects of citrate and NaCl on size, morphology, crystallinity and microstructure of calcium phosphates obtained from aqueous solutions at acidic or near-neutral pH.

Precipitation of calcium phosphates occurs in dairy products and depending on pH and ionic environment, several salts with different crystallinity can form. The present study aimed to investigate the effects of NaCl and citrate on the characteristics of precipitates obtained from model solutions of calcium phosphate at pH 6·70 maintained constant or left to drift. The ion speciation calculations showed that all the starting solutions were supersaturated with respect to dicalcium phosphate dihydrate (DCPD), octacalcium phosphate (OCP) and hydroxyapatite (HAP) in the order HAP>OCP>DCPD. X-ray diffraction (XRD) and Fourier transform infrared (FTIR) analyses of the precipitates showed that DCPD was formed at drifting pH (acidic final pH) whereas poor crystallised calcium deficient apatite was mainly formed at constant pH (6·70). Laser light scattering measurements and electron microscopy observations showed that citrate had a pronounced inhibitory effect on the crystallisation of calcium phosphates both at drifting and constant pH. This resulted in the decrease of the particle sizes and the modification of the morphology and the microstructure of the precipitates. The inhibitory effect of citrate mainly acted by the adsorption of the citrate molecules onto the surfaces of newly formed nuclei of calcium phosphate, thereby changing the morphology of the growing particles. These findings are relevant for the understanding of calcium phosphate precipitation from dairy byproducts that contain large amounts of NaCl and citrate.

Milk and dairy products: a unique micronutrient combination.

Milk and dairy products contain micronutrients such as minerals and vitamins, which contribute to multiple and different vital functions in the organism. The mineral fraction is composed of macroelements (Ca, Mg, Na, K, P, and Cl) and oligoelements (Fe, Cu, Zn, and Se). From a physicochemical point of view, the chemical forms, the associations with other ions or organic molecules, and the location of macroelements such as Ca, Mg, Na, K, P, and Cl in milk are relatively well described and understood. Thus, it is admitted that these macroelements are differently distributed into aqueous and micellar phases of milk, depending on their nature. K, Na, and Cl ions are essentially in the aqueous phase, whereas Ca, P, and Mg are partly bound to the casein micelles. About one third of the Ca, half of the P, and two thirds of the Mg are located in the aqueous phase of milk. Dairy products are more or less rich in these different minerals. In cheeses, mineral content depends mainly on their processing. The Ca content is strongly related to the acidification step. Moreover, if acidification is associated with the draining step, the Ca content in the cheese will be reduced. Thus, the Ca content varies in the following increasing order: milks/fermented milks/fresh cheeses < soft cheeses < semi-hard cheeses < hard cheeses. The chemical forms and associations are less described than those present in milk. Concerning Ca, the formation of insoluble calcium phosphate, carbonate, and lactate is reported in some ripened cheeses. The NaCl content in cheeses depends on the salting of the curd. From a nutritional point of view, it is largely admitted that milk and dairy products are important sources of Ca, Mg, Zn, and Se. The vitamin fraction of milk and dairy products is composed of lipophilic (A, D, E, and K) and hydrophilic (B(1), B(2), B(3), B(5), B(6), B(8), B(9), B(12), and C) vitamins. Because of their hydrophobic properties, the lipophilic vitamins are mainly in the milk fat fraction (cream, butter). The hydrophilic vitamins are in the aqueous phase of milk. For one part of these vitamins, the concentrations described in the literature are not always homogenous and sometimes not in accordance between them; these discrepancies are due to the difficulty of the sample preparation and the use of appropriate methods for their quantification. However, there is no doubt of the significant contribution of milk and dairy products to the intake of vitamins. Milk and dairy are considered essential sources for vitamins. Key teaching points: Milk and dairy products are unique micronutrient combinations with recognized health benefits. The concentration, chemical forms, and location of different minerals are relatively well known and described. For example, Ca is present in dairy products in different forms: free, associated with citrate, inorganic and organic phosphates, and free fatty acids. Milk and dairy products are excellent sources of Ca, P, Mg, Zn, and Se. The concentration of vitamins in milk and dairy products is variable and depends on several factors such as biosynthesis, animal feeding, physicochemical conditions (heat, light, O(2), oxidant agents), and analytical methods for their determinations. Vitamins A, D, E, and K are mainly located in the lipid phase and vitamins of group B and C in the aqueous phase. Milk and dairy products are excellent sources of vitamins A, B(1), B(2), and B(12).

Determination of calcium-binding constants of caseins, phosphoserine, citrate and pyrophosphate: A modelling approach using free calcium measurement.

Dairy products contain large amount of calcium which is bound to caseins and different chelating agents like citrate and polyphosphates. The present study aimed to determine the calcium-binding capacities of phosphoserine (SerP), caseinophosphopeptide (CPP), ß-casein, caseinate, citrate and pyrophosphate in the same conditions of temperature, pH and ionic strength. The free calcium (Ca(2+)) was measured using a calcium ion-selective electrode and plotted as a function of total calcium concentration. The association constants and the number of calcium-binding sites were determined by fitting the experimental data to a theoretical model. The phosphate groups of caseins were the main binding sites with evidence for participation of carboxylate groups. The intrinsic association constants determined by the best fit of the data were in the order: pyrophosphate (557×10(3)M(-1))>citrate (20×10(3)M(-1))>ß-casein (5×10(3)M(-1))>caseinate, CPP and SerP (∼10(3)M(-1)). These findings may be of interest for the development of calcium-enriched products to overcome calcium deficiency in specific populations.