Characterization of potato protein and its calcium binding capacity

Abstract: Calcium is essential for the human body as it has functions in the nervous system, bones and formation of teeth. Deficiency in this important nutrient can be detrimental to health and cause life-long issues. Calcium can be found in various foods, like dairy products, but certain plant-based alternatives to milk needs to be fortified with calcium. One of the complications with calcium supplements is that calcium ions interact with many compounds and can become insoluble. One of the solutions to keeping calcium soluble is to form soluble complexes with proteins, as calcium does with β-casein in milk. By fortifying the calcium with proteins, a higher total concentration calcium can be attained in a solution while the free calcium concentration is still low to minimize unwanted reactions. In this thesis, potato protein, a byproduct from the potato starch production, with the potential to act as a calcium binder in plant-based products was characterized. The potato protein’s potential capacity to form complexes with calcium was also assessed. The potato protein was also hydrolyzed using Alcalase in an attempt to improve its suitability as a calcium binder. The potato protein sample and hydrolysates of the proteins were analyzed with dry matter analysis and pH titration to characterize the composition, solubility and pI of the samples. Their size was investigated with gel electrophoresis and size exclusion chromatography. The calcium binding capacity was assessed by measuring free calcium concentration and monitoring changes in right-angle light scattering (RALS), absorbance at 254 nm and 280 nm, and fluorescence during calcium titration. To measure the free calcium concentration a procedure for a colorimetric calcium assay was established. Equilibrium dialysis was used when assessing the calcium binding capacity due to complications with the calcium assay. The sample of soluble potato protein (SPP) had high fraction of protease inhibitors and a lower fraction of patatin. The SPP was soluble in alkaline and highly acidic solutions, while insoluble at around pH 5. The hydrolysates had the highest insoluble fraction at pH 3, and higher soluble fractions at alkaline pH. The analysis of calcium binding capacity showed a decrease in free calcium concentration in samples with SPP and calcium, and changes in RALS, absorbance and fluorescence caused by protein aggregation and conformational changes that could indicate calcium binding. Kd-values, calculated using Hill-Langmuir equation, suggest low affinity binding. A 2 -factorial trial indicated that pH had a significant effect on SPP-calcium binding. The experiments were also done using a well-known calcium binder, β-casein, as a positive control.