Calcium Solubilization Ability and Anti-Inflammatory Effects of Hydrolyzed Casein

Abstract

This study performed to evaluate the applicability of functional dairy food materials by comparing the calcium solubilization ability and anti-inflammatory effects of hydrolyzed casein protein. Commercial enzyme (Alcalase^®; Neutrase^®; Protamex^®; Flavourzyme^®) was added to the 10% casein solution to prepare the casein hydrolysates. Samples obtained every hour [1:200 (w/v)]. According to results of measuring the degree of hydrolysis (DH), all of four enzymatic hydrolysates increased rapidly from 30 to 40 min, and after 150 min, there were no change. Protamex^® and Neutrase^® had the highest DH compared to others enzymatic hydrolysates. After that, peptides obtained throughout a preparative liquid chromatography system. In the calcium solubility experiments, neutrase fraction (NF) 4 and NF7 showed similar activities with casein phosphopeptide (CPP). In vitro cell experiments showed that no cytotoxicity except for NF6. Also, the production of nitric oxide (NO) inhibited as the concentration of fraction samples increased. The cytokine (IL-1α, IL-6, and TNF-α) production was lower than lipopolysaccharide (+) group significantly. Therefore, the possibility of anti-inflammatory activity found in the hydrolyzed samples. According to the above experiments, NF3 and Protamex Fraction (PF) 3 selected. Amino acids selected throughout an AccQ-Tag system. As a result, 17 species of amino acids and several species of unknown amino acids identified. Both fractions had the highest content of phenylalanine. This study identified the potential of biologically active and functional peptides derived from casein that affect the food and dairy industry.

Keywords: amino acids; anti-inflammatory effects; calcium solubilization ability; casein; milk protein.

Fig. 1.. Degree of hydrolysis (DH) of casein hydrolysates.

Reaction were carried out in a water bath (50°C, 120 rpm). All enzymes were applied at concentration 1:200 (w/v) to the casein solution. Alcalase^® 2.4 L; Neutrase^® 0.8 L; Flavourzyme^®; Protamex^® (Novozymes^TM).

Fig. 2.. Separations of casein hydrolysates using Neutrase^® in preparative LC system.

F#, fraction number in prep LC system.

Fig. 3.. Separations of casein hydrolysates using Protamex^® in preparative LC system.

F#, fraction number in prep LC system.

Fig. 4.. Calcium solubility of casein hydrolysate fractions using Neutrase^®.

^* p<0.05, ^** p<0.01, ^*** p<0.001; CPP vs. sample. CPP, casein phosphopeptide; NHC, non-hydrolyzed casein; NPC, non-purified casein; F#, fraction number in Prep LC system. All samples protein concentration were 200 μg/mL. All values were mean±SD of triplicates.

Fig. 5.. Calcium solubility of casein hydrolysate fractions using Protamex^®.

All samples protein concentration were 200 μg/mL. All values were mean±SD of triplicates. ^* p<0.05, ^** p<0.01, ^*** p<0.001; CPP vs. sample. CPP, casein phosphopeptide; NHC, non-hydrolyzed casein; NPC, non-purified casein; F#, fraction number in Prep LC system.

Fig. 6.. Effects of hydrolysate fractions on cell viability in RAW 264.7 cells.

All values were mean±SD of triplicates. ^* p<0.05, ^** p<0.01; CTR vs. NF# or PF#. CTR, control; CPP, casein phosphopeptide; NF#, Neutrase^® hydrolysate fraction number in Prep LC system; PF#, Protamex^® hydrolysate fraction number in Prep LC system; 200, 150, 100, 50 μg/mL: Samples protein concentration.

Fig. 7.. Effects of hydrolysate fractions on NO in LPS-stimulated RAW 264.7 cell.

All values were mean±SD of triplicates. ^* p<0.05, ^** p<0.01, ^*** p<0.001; CTR (–) vs. NF# or PF#. NO, nitric oxide; LPS, lipopolysaccharide; CTR, control; CPP, casein phosphopeptide; NF#, Neutrase^® hydrolysate fraction number in Prep LC system; PF#, Protamex^® hydrolysate fraction number in Prep LC system. 200, 150, 100, 50 μg/mL: Samples protein concentration.

Fig. 8.. Comparison of IL-1α production in LPS-stimulated RAW 264.7 cells.

All values were mean±SD of triplicates. ^* p<0.05, ^** p<0.01; LPS(+) vs. NF# or PF#. LPS, lipopolysaccharide; NPC, non-purified casein; NF#, Neutrase^® hydrolysate fraction number in Prep LC system; PF#, Protamex^® hydrolysate fraction number in Prep LC system.

Fig. 9.. Comparison of IL-6 production in LPS-stimulated RAW 264.7 cells.

All values were mean±SD of triplicates. ^* p<0.05, ^** p<0.01; LPS(+) vs. NF# or PF#. LPS, lipopolysaccharide; NPC, non-purified casein; NF#, Neutrase^® hydrolysate fraction number in Prep LC system; PF#, Protamex^® hydrolysate fraction number in Prep LC system.

Fig. 10.. Comparison of TNF-α production in LPS-stimulated RAW 264.7 cells.

All values were mean±SD of triplicates. ^* p<0.05, ^** p<0.01; LPS(+) vs. NF# or PF#. LPS, lipopolysaccharide; NPC, non-purified casein; NF#, Neutrase^® hydrolysate fraction number in Prep LC system; PF#, Protamex^® hydrolysate fraction number in Prep LC system.

Fig. 11.. HPLC chromatogram of amino acids in selected Neutrase^® hydrolysate fraction.

Fig. 12.. HPLC chromatogram of amino acids in selected Protamex^® hydrolysate fraction.