Calcium binding to milk components and perspective for calcium nutrition

Research output: Book/ReportPh.D. thesisResearch

Calcium is an essential nutrient required for many critical biological functions in living organisms to maintain good health. However, calcium deficiency is widespread due to insufficient intake and low bioavailability. Some molecules, such as amino acids, carbohydrates, and peptides, are known to bind calcium through complex formation and may increase calcium uptake from food. Other
molecules like the fatty acid anion palmitate, phosphate, and oxalate may hamper the uptake through precipitation of calcium during digestion. Milk and dairy products stand out as the best natural sources of calcium, which account for ~75% of dietary calcium in Europe and North America (Melse-Boonstra, 2020). Therefore, an increase of the absorption through calcium binding to milk components will improve bioavailability of calcium and deserves interest in relation to human nutrition and attention by the dairy companies and food industry in general.
Calcium binding to the amino acid o-phospho-L-serine (OPS), important for the structure of the milk protein casein was quantified using a calcium selective electrode and reported as association constants valid at various conditions of pH, ionic strength, and temperature using L-serine (Ser) as a reference compound. Calcium binding to OPS and Ser was found strongly dependent on pH, and binding increased with increasing pH for both OPS and Ser. pH-dependent binding of calcium ions was resolved, and calcium binding association constants were thus assigned to each acid/base forms of OPS and Ser. Results showed that calcium binding to mononegative OPS is endothermic,
while binding to dinegative and trinegative forms is exothermic. The shift from the endothermic binding of calcium to OPS at low pH to exothermic binding at higher pH, corresponding to a shift from hydrophobic interaction (phosphate ester) to ionic binding (carboxylate/amino chelation), was supported by quantum mechanical calculations within the framework of Density Functional
Theory (DFT).
Stoichiometry of calcium and sugar complex formation was investigated by conductivity measurements using Job's method of continuous variation. Calcium was found to bind lactose, galactose, glucose, and fructose in a 1:1 ratio, while calcium was found to bind the short-chain fructo-oligosaccharide inulin in a 2:1 (calcium: ligand) ratio. The association constant between calcium and sugars was studied both electrochemically using a calcium selective electrode as well
as by a titration method based on the solubility of calcium iodate in presence of the sugars. The ordering of calcium binding among the saccharides in the aqueous solution at 25.0 °C was found consistent by the two methods as glucose < fructose < galactose <lactose. Calcium binding to simple saccharides was found athermal by isothermal calorimetry and accordingly entropy controlled, while calcium binding to inulin was shown to be a moderately endothermic process. DFT calculations confirmed the ordering of the binding of calcium in sugar complexes in good agreement with the experimental results. In addition, the DFT result gives an explanation of why the β-anomer of lactose binds calcium stronger than the α-anomer, as found experimentally. An additional hydroxyl group was shown to be involved in calcium binding in the β-anomer compared to calcium binding to the α-anomer of lactose.
Thermodynamics of calcium binding to the peptides GlyTyr and TyrGly, both potential hydrolysis products of α-lactalbumin, were characterized including the effect of pH and temperature at physiological ionic strength of 0.16 M. Based on apparent pKa values, determined by potentiometric titration, the pH-dependent binding of calcium ions was resolved into binding of calcium to three forms of each dipeptide in acid-base equilibrium. It was found that calcium binding to GlyTyr is stronger for the pH condition of the intestine than to TyrGly. Moreover,
calcium binding to acid/base forms of GlyTyr and TyrGly dipeptides is at neutral pH strongly exothermic. An enthalpy-entropy compensation phenomenon was demonstrated, which may control the calcium binding to GlyTyr and TyrGly during the changing pH of digestion. DFT suggests that calcium binding to GlyTyr and TyrGly occurs through carboxylates and amides as well as supports an increasing affinity with increasing pH for both dipeptides. Calcium binding
was found to affect the antioxidant function of the peptides using assays based on oxygen consumption rate of peroxidising lipids and on scavenging of the semi-stable ABTS+• radical. Calcium binding was found to decrease both radical scavenging and antioxidative activity of GlyTyr and TyrGly.
Original languageEnglish
PublisherDepartment of Food Science, Faculty of Science, University of Copenhagen
Number of pages135
Publication statusPublished - 2021

ID: 290595128