First, an iron binding reaction was performed to produce complexes. Hydrolysates (fractions <5 kDa) with 1% (w/v) in protein content, pH adjusted to 5.5, were mixed with iron as FeSO4(s) 0.1% (w/v), both in aqueous solution. Incubation was performed in a shaking water bath for 30 min at 40 °C. Then, the solution was diluted 1:50 (v/v) in milli-Q water and dialyzed during 48 h at 26 °C, against milli-Q water, for the removal of free iron ions, using a Spectra/Por® dialysis membrane with Selleckchem XAV 939 a cut-off of 500 Da, (Spectrum Laboratories, Inc., CA, USA).

A blank without hydrolysates was run in parallel to samples and dialyzed. After dialysis, the retentate containing iron bound to peptides (complexes) was analysed for iron content by ICP. The percentage of iron binding capacity was calculated as: Iron binding capacity % = [iron content in complex (g)/total iron in solution before binding (g)]× 100. In vitro dialyzability was used to predict iron bioavailability

of hydrolysates (fraction <5 kDa). Dialyzability involves a two-stage (gastric and intestinal) simulated digestion and a dialysis. The procedure is similar to that described by Argyri, Birba, Miller, Komaitis, and Kapsokefalou (2009), a setup which allows the rapid and efficient application of the dialyzability method. The simulated gastrointestinal digestion occurs in six-well plates with inserts and Spectra/Por® dialysis membranes (cut-off of 6000–8000 Da) tightly held in place with elastic bands. The percentage of iron dialyzability was calculated as: [(dialyzable iron)/(total Everolimus iron)] × 100. Dialyzable iron was the iron that passed through the dialysis membrane during the in vitro digestion. Dialyzable iron was the iron content of the dialysate, and the total iron, the amount of iron added to the sample material prior to digesting (final concentration of 0.2 mM). A chromatographic column with IMAC Sepharose High Performance (IMAC-HP) resin (GE Healthcare Bio-Science AB, Sweden) installed why in a low pressure liquid chromatography system (FPLC) from Pharmacia

(Amersham Pharmacia Biotech) was used to separate the iron chelating peptides. The method of Lv et al. (2009) was followed with some modifications. A column was packed with IMAC-HP (10 mL) and charged with Fe3+ (5 mL of 200 mM FeCl3). After washing the unbound iron out of the column with milli-Q water (5 bed volumes), the nonspecific bound iron was removed with 50 mM sodium acetate-acetic acid buffer (NaAc/HAc), pH 3.6 (2–5 vol), and the column was equilibrated using the same buffer. Subsequently, 3 mL of yeast extract hydrolysate solution <5 kDa (20 mg/mL in protein content) was loaded onto the column. Peptides without affinity to immobilized iron in the column were eluted with the equilibration buffer (50 mM NaAc/HAc). Then, the bound peptides were eluted using 100 mM NH4H2PO4 solution, pH 4.5, and collected for further lyophilization. The absorbance of eluates was monitored at 280 nm.