PREPARATION AND CHARACTERIZATION OF MILK PROTEIN HYDROLYSATE-IRON COMPLEX AND ITS APPLICATION IN MILK
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Date
2022
Authors
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Journal ISSN
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Publisher
ICAR-NDRI, KARNAL
Abstract
Iron is very important nutrient for human, but availability of iron from dietary
source has limitations and its simple fortification in food causes adverse
effect on quality of the food. Milk-derived bioactive peptides can be used to
overcome these challenges because of their mineral binding abilities. In the
present research milk protein concentrate was used as a precursor for
generating iron-binding peptides. On hydrolysis using alcalase, and
flavourzyme; alcalase showed high chelation (75.6%) and DH (35%) with
1150U/g protein. 10kDa fraction of hydrolysate showed maximum chelation
activity (80%), than lower molecular weight fractions. Metal chelating affinity
chromatography was used for further enrichment of iron binding peptides.
Based on SEC, molecular weight distribution of hydrolysate showed
presence of 43% low molecular weight peptide i.e., molecular weight less
than 0.5KDa but after in-vitro digestion percentage of this peptide increased
up to 77% due to degradation of peptides. Characterization of peptides
based on LCMS showed the presence of phosphorylated peptides and
other charged amino acid residues responsible for iron-binding
(QSEEQQQTEDE (B-CNf(34-44)), KFQSEEQQQTEDELQDK (B-CN f(32-
48), SQSKVLPVSQ (B-CNf(66-75)), KTKLTEEE αS2f(150-157),
VVRNANEEE αS2f(43-51), NSKKTVDMESTE αs2f(134-145), KNMAINPSKE
αs2f(25-33), AMEDIKQM αs1f(53-60), KVPQLEIVPN αs1f(105-114)). For the
preparation of the iron peptide complex alcalase hydrolysate under
optimized condition (pH 8 at 50°C for 2H in the 1:0.2ratio (hydrolysate: Fe)),
resulted in 43mg Fe/g of protein (corresponding to 60% of ref peptide-CPP
in iron-binding capacity). Characterization of iron peptide complex by using
particle size, Zeta potential, UV, Fluorescence, FTIR, SEM, EDX, VSM, and
XRD techniques showed that complex between iron and hydrolysate is
formed with high stability. The bioavailability of iron peptide complex was
2.3 times higher than free iron salt. The iron peptide complex was used in
milk fortification (with iron conc. 25ppm based on sensory acceptability).
Iron complex possessed high stability (resistance towards the release of
bound iron) during different processing conditions such as pH (6-8) and
heat treatment. Storage studies of fortified milk showed lesser changes in
physicochemical properties and better oxidative stability. Thus, the
methodology was standardized to prepare milk protein hydrolysate based
functional ingredient that has high iron chelation activity, high iron
bioavailability (81%), and stability under different milk processing
conditions.