Preparation, Characterization and Iron Absorption by Caco-2 Cells of the Casein Peptides-Iron Chelate

Among 13 proteases from microbes and plants, bromelain was shown to hydrolyze casein to obtain the highest iron-chelating activity of the hydrolysate. The casein hydrolysate obtained by hydrolysis of casein with bromelain under optimized condition was ultrafiltrated to obtain the casein peptide fraction (CPF, < 1 kDa) with high iron-chelating activity (63.94%). Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of CPF indicated presence of 8 peptides with potential iron-binding glutamic acid, serine, phenylalanine and histidine residues. The casein peptides-iron chelate (CPIC) was prepared by reaction of CPF with ferrous sulfate. Characterization of CPIC in comparison with CPF showed significantly decreased intensity of the diffraction peak at 20 degrees with slight shift to higher diffraction angle (2 theta) by X-ray diffraction analysis. Fourier transform infrared spectrum analysis suggested that carboxyl, carbonyl and amino groups were the main chelating sites for iron ions. Thermogravimetry-differential scanning calorimetry analysis indicated that chelating with iron led to better structural and thermal stability of CPIC. Further iron absorption assay by a Caco-2 cells model indicated less toxicity of CPIC to the cells than that of ferrous sulfate specially at higher concentrations (5, 10 mg/mL), and significantly (p < 0.05) better iron absorption of the cells with CPIC than with ferrous sulfate. Western blot analysis confirmed significantly (p < 0.05) increased expression of iron transporter proteins such as divalent metal transporter 1 and ferroportin1. The results of this study suggests potential of CPIC as a dietary supplement to improve iron absorption.

Automated summary

The study found that bromelain can hydrolyze casein to obtain the highest iron-chelating activity, and the casein peptide fraction (CPF) obtained through ultrafiltration has high iron-chelating activity. Further analysis showed that the chelation of iron with CPF led to better structural and thermal stability, less toxicity to cells, and better iron absorption compared to ferrous sulfate. Western blot analysis also confirmed the increased expression of iron transporter proteins.