Molecular Characterization of Whey Protein Hydrolysate Fractions with

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Molecular Characterization of Whey Protein Hydrolysate Fractions with Ferrous Chelating and Enhanced Iron Solubility Capabilities Ian B. O’Loughlin,†,§ Phil M. Kelly,† Brian A. Murray,† Richard J. FitzGerald,§ and Andre Brodkorb*,† †

Teagasc Food Research Centre, Moorepark, Fermoy, County Cork, Ireland Life Sciences Department, University of Limerick, Limerick, Ireland

§

S Supporting Information *

ABSTRACT: The ferrous (Fe2+) chelating capabilities of WPI hydrolysate fractions produced via cascade membrane filtration were investigated, specifically 1 kDa permeate (P) and 30 kDa retentate (R) fractions. The 1 kDa-P possessed a Fe2+ chelating capability at 1 g L−1 equivalent to 84.4 μM EDTA (for 30 kDa-R the value was 8.7 μM EDTA). Fourier transformed infrared (FTIR) spectroscopy was utilized to investigate the structural characteristics of hydrolysates and molecular interactions with Fe2+. Solid-phase extraction was employed to enrich for chelating activity; the most potent chelating fraction was enriched in histidine and lysine. The solubility of ferrous sulfate solutions (10 mM) over a range of pH values was significantly (P < 0.05) improved in dispersions of hydrolysate fraction solutions (10 g protein L−1). Total iron solubility was improved by 72% in the presence of the 1 kDa-P fraction following simulated gastrointestinal digestion (SGID) compared to control FeSO4·7H2O solutions. KEYWORDS: whey protein, enzymatic hydrolysis, iron-binding, simulated digestion, FTIR

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INTRODUCTION

transferred to the basolateral side of intestinal enterocytes and released into the interstitial space before absorption. The solubility of iron−peptide complexes can significantly affect the solubility/bioaccessibility of iron for luminal uptake. However, certain CPP iron complexes, for example, β-cn f(1−25), have been shown to be susceptible to further duodenal digestion.18 Iron chelating fractions of a WPI hydrolysate were investigated to gain some insight into the molecular nature of chelation and the solubility of iron-fortified hydrolysate fractions during subsequent simulated gastrointestinal digestion.

Whereas the major role of the milk proteins is to supply amino acids and nitrogen to the neonate, some milk proteins facilitate the uptake of key elements,1 for example, the Ca2+ chelating activity of β-lg.2 An estimated 2 billion people globally are affected by iron deficiency,3 and a number of iron-binding proteins exist within milk, including xanthine oxidase, lactoperoxidase, lactoferrin, and transferrin.4 Approximately 26% of the iron present in milk is distributed in the whey proteins.5 The low solubility, oxidation state, and reactivity of iron make direct fortification difficult, as iron needs to be in a soluble format to be effectively absorbed.6 Ferrous (Fe2+) salts are more utilized as fortificants than ferric (Fe3+), the former not requiring reduction prior to absorption.7 However, these Fe2+ salts can damage the walls of the gastrointestinal tract and be responsible for OH• radical formation.8 Enzymatic hydrolysates possess the possibility to facilitate the bioaccessibility of iron in humans.9 Generally, iron-binding peptides from milk have been casein-derived phosphopeptides (CPPs),10 where these phosphoseryl-containing peptides have a high content of negative charges for efficient binding of divalent iron.11 Chaud et al. utilized a casein hydrolysate hydrolyzed with pancreatin and Proteomix as a binder for Fe3+.12 Furthermore, the iron-binding capability of whey proteins and whey protein fractions has been studied previously;13 for example, enzymatic hydrolysates of β-lg have demonstrated increased iron chelating/binding ability compared to unhydrolyzed controls.14,15 Whey-derived caseinomacropeptide (CMP) has been proposed to facilitate effective iron absorption in infant formulas.16 The ferrous chelating capability of whey protein isolate (WPI) hydrolysate fractions has been previously correlated positively to the determined sample average molecular weight.17 Generally, gastrointestinal digestion extensively hydrolyzes proteins to di- or tripeptides. These peptides may be © 2015 American Chemical Society

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MATERIALS AND METHODS

Materials. Whey protein isolate (Isolac) was provided by Carbery Food Ingredients (Ballineen, Co. Cork, Ireland), and its composition was described previously.19 The porcine pancreatic preparation Corolase PP (EC 3.4.21.4) was from AB Enzymes GmbH (Darmstadt, Germany) with activities previously described.20 Acetonitrile (MeCN), analytical grade, was purchased from ThermoFisher Scientific (Waltham, MA, USA), and all further chemicals and reagents were purchased from Sigma-Aldrich (Dublin, Ireland). All experiments were conducted in duplicate unless otherwise stated. Hydrolysate fractions for ferrous chelation determination were produced as previously outlined by the authors.17 Briefly, control and heat-treated (80 °C, 10 min) dispersions (100 g L−1) of WPI were hydrolyzed to degree of hydrolysis (DH) values of 5 and 10%, whereupon they were subsequently fractionated via cascade membrane micro- and ultrafiltration (nondiafiltered in the case of the 30 kDa spiral membrane UF). Fractions were then evaporated and spray-dried, with the best and worst chelator samples selected on the basis of previous chelation experiments.17 These fractions were, namely, the 1 Received: Revised: Accepted: Published: 2708

December 1, 2014 February 20, 2015 February 26, 2015 February 26, 2015 DOI: 10.1021/jf505817a J. Agric. Food Chem. 2015, 63, 2708−2714

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Journal of Agricultural and Food Chemistry

Attenuated Total Reflectance−Fourier Transform Infrared (ATR-FTIR) Spectroscopy of WPI, Hydrolysates, and IronFortified Dispersions. Secondary structural changes were determined using ATR-FTIR spectroscopy. This was performed utilizing a Bruker Tensor 27 FTIR spectrometer (Bruker Optik GmbH, Ettlingen, Germany) with a thermally controlled BioATR Cell II. Sample solution (25 μL, 10 g L−1) was placed onto the ZnSe crystal, and a total of 180 scans were performed for each sample at a resolution of 4 cm−1. Interferograms were Fourier transformed and analyzed with Bruker OPUS 5.5 software, where both water vapor and CO2 compensations were performed and data were vector normalized. Simulated Gastrointestinal Digestion (SGID) of FerrousChelating Fractions. A static two-stage SGID process was performed on the iron-chelating fractions using a modification of the methodology of Walsh et al.24 The simulated gastric fluid contained pepsin (EC 3.4.23.1, calculated activity of 837 UHb mg−1 protein) dispersed in 150 mM NaCl at a concentration of 4 g L−1 (final pH 5.5). The simulated pancreatic juice (SPJ) was prepared according to the following protocol: 4.5 g of Corolase PP, 62.5 g of NaHCO3, and 30 g of bile salts per liter of dH2O modified from the method of Le et al.25 Iron-fortified samples (100 mL, 20 g protein L−1) were preincubated at 37 °C for 30 min under stirring (at 140 rpm). The pH of the solution was reduced to 3.5 at 37 °C with 1 N HCl, whereupon a 10 mL solution of simulated gastric fluid was added to give a final E:S of 1:100. The solution was then reduced to a final pH of 2 at 37 °C. Aliquots, 1 mL, were extracted at 1, 10, 30, 60, and 90 min after reaction. After 90 min, the pH was adjusted to pH 7.5 using 0.6 M Na2HPO4. SPJ (1:10 v/v of sample) was subsequently added to the solution, which was incubated at 37 °C for 150 min. Enzyme activity was terminated by heating at 80 °C for 20 min. Chromatographic Characterization of Control and HeatedTreated WPI Solutions. Reversed-phase (C18) HPLC was performed as previously described by O’Loughlin et al.19 Briefly, a Phenomenex Jupiter C18 column was used (Phenomenex, Cheshire, UK) under gradient elution conditions: solvent B, from 0 to 100% in 30 min, 100% for 5 min, from 100 to 0% in 5 min, and 0% for 5 min at a flow rate of 1 mL min−1, where solvent B was 80% MeCN and 0.1% TFA (v v−1). Solvent A was 0.1% TFA (v v−1). Size-exclusion chromatography (SEC) described by O’Loughlin et al.19 utilized a TSK Gel G2000SW, 7.8 mm × 600 mm, column (TosoHaas Bioscience GmbH, Stuttgart, Germany) using an isocratic gradient of 30% (v v−1) MeCN and 0.1% (v v−1) trifluoroacetic acid (TFA) at a flow rate of 0.5 mL min−1 over 60 min. Both C18 and SEC were performed on a Waters 2695 separation module and a Waters 2487 dual-wavelength absorbance detector running on Waters Empower software (Milford, MA, USA). Statistical Analysis. Statistical analysis was through the use of Minitab 15 software (Minitab Inc., State College, PA, USA). When necessary, data are presented as the mean ± SD for n = 2 determinations. The statistical difference of means was determined through the use of a one-way ANOVA followed by a Tukey test.

kDa permeate of the heat-treated 10% DH hydrolysate (86.6% protein via Kjeldahl, containing 9.2% ash), the 30 kDa retentate of the control 5% DH hydrolysate (87.5% protein via Kjeldahl, containing 8.7% ash), and their respective whole unfractionated hydrolysates (87.1 and 86.4% protein, respectively). For fortification of hydrolysate fractions, Fe2+ (139 mg of FeSO4· 7H2O) was added to 100 mL of 20 g protein L−1 solutions (initial concentration) of hydrolysate fractions adjusted to pH 5 with 1 N HCl. Total Iron Concentration, Ferrous Chelation Determination, and Solid Phase Extraction. Total iron was determined as previously described by O’Loughlin et al.17 modified from the method of Voillier et al.21 Briefly, test samples were dissolved in 0.5 M HCl and stored overnight. Test samples (200 μL, 1 g L−1) were diluted with 1.5 mL of dH2O, and 300 μL of 1.4 M hydroxylamine hydrochloride was subsequently added, whereupon the mixture was vortexed and allowed to stand for 30 min. Ferrozine reagent (200 μL), 0.01 M in 0.1 M ammonium acetate, was added along with 300 μL of 5 M ammonium acetate and the mixture allowed to stand for 3 min. The absorbance was read at 562 nm against a standard curve of FeSO4. The ferrous chelating capability of the WPI and whole and fractionated hydrolysates was determined using a modification of the method of Decker and Welch22 as previously described by O’Loughlin et al.17 Briefly, test samples (1 mL of 1 g L−1) were mixed with 1.35 mL of dH2O and 50 μL of 2 mM FeCl2. After vortexing, the reaction was initiated with 100 μL of ferrozine, vortexed again, and allowed to stand for 10 min. Samples were read at 562 nm, and EDTA-Na2 was used as a positive control for the assay. A standard curve for EDTA chelation of iron was prepared for 0.5−100 μM EDTA, giving the following linear relationship:

y(A562 ) = − 0.0105x + 1.1476 (R2 = 0.997)

(1)

The concentration of selected hydrolysates needed to chelate 50% of the ferrous iron (CC50) was determined by assaying and plotting various concentrations of hydrolysates against percentage chelation. Utilizing concentrations of the samples ranging from 0.5 to 10 g L−1, it was possible to determine the concentration of selected samples needed to chelate 50% of the ferrous iron (CC50) under the assay conditions with 2 mM FeCl2. Solid phase extraction (SPE) was carried out on Phenomenex Strata X cartridges (Phenomenex, Cheshire, UK) using the method of Nongonierma et al.23 Cartridges were conditioned with methanol followed by washing with Milli-Q water. Samples (5 g protein L−1) were loaded onto the cartridge, where flow-through was extracted under vacuum (≤−381 mmHg). Washes (four times) with Milli-Q water were pooled for analysis. Subsequently, 80% (v/v) acetonitrile (MeCN) was utilized (three times) to elute the bound fraction from the cartridge. Fractions were evaporated on a Büchi Rotavapor R-210 (Büchi Labortechnik AG, Switzerland) and freeze-dried on a Labconco 12 L FreeZone 12 plus freeze drier (Labconco, Kansas City, MO, USA) prior to analysis. Total and Free Amino Acid Analysis of Hydrolysate Fractions and Iron Solubility of Dispersions. Total and free amino acid analysis was performed as previously described by O’Loughlin et al.19 A JEOL JLC-500/V Amino Tac amino acid analyzer fitted with a JEOL Na+ high-performance cation-exchange column (JEOL (UK) Ltd., Herts, UK) was utilized, and deproteination was accomplished with 10% (v v−1) trichloroacetic acid (TCA) followed by centrifugation at 20000g for 20 min. The extracted supernatant was analyzed for amino acids through visible adsorption photometry after separation using ion exchange. Ninhydrin derivatization with norvaline was used as an internal standard. For total amino acid analysis samples were heated in 6 M HCl for 23 h under reflux at 105 °C before application to the JLC-500/V analyzer. For total iron solubility, test samples (10 g L−1) were centrifuged (Eppendorf 5810 R, Eppendorf GmbH, Hamburg, Germany) at 3000g for 20 min, whereupon the supernatant was extracted and diluted 1:9 using 0.5 N HCl and stored overnight. Total iron was determined using the previously mentioned methodology.

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RESULTS AND DISCUSSION Amino Acid Content and Ferrous-Chelating Capability of Whey Protein Isolate Hydrolysate Fractions. In a previous investigation by the authors, the ferrous-chelating capabilities of WPI hydrolysates were correlated with their respective average molecular weights (Mw).17 A total of 22 pilot-scale fractions produced during the study were the subject of further analysis of their iron-chelating characteristics. O’Loughlin et al. showed that a 30 kDa retentate from a 5% DH hydrolysate of unheated control WPI (WPI-C-5DH-30R) possessed a CC50 value of 4.500 ± 0.009 g protein L−1.17 Correspondingly, a 1 kDa permeate of a 10% DH hydrolysate of the prior heat-treated (80 °C, 10 min) WPI (WPI-HT10DH-1P) possessed a CC50 value of 0.846 ± 0.031 g protein L−1. The chelating capability of hydrolysate fractions was shown to increase with a reduction in the average Mw of the fraction.17 2709

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Journal of Agricultural and Food Chemistry The WPI-C-5DH-30R fraction possessed 57.5 ± 1.3% of material >30 kDa with the WPI-HT-10DH-1P fraction possessing 80.2 ± 1.1% of material 95% for all samples (control and fortified hydrolysate fractions) at pH 2−5. Thereafter, total iron solubility declined rapidly in the case of the control (aqueous dispersion), whereas that of the fortified 30 kDa retentate was reduced to 79.6 ± 1.2% at pH 7 and to 66.1 ± 1.0% at pH 8. Only a small (relatively) decrease was observed (to 95.8 ± 1.5% at pH 8) in the iron solubility with the 1 kDa hydrolysate permeate derived from the heat-treated WPI (Figure 3). This fraction was capable of sustaining the highest iron solubility of all fortified samples. WPI and hydrolysate samples were also analyzed for their inherent solubility over the pH range of 2−8 with the solubility curves for the 30 kDa retentate of the control 5% DH and the 1 kDa permeate of the 10% DH hydrolysate of heat-treated WPI shown in Figure 3 (inset). Generally, the solubility of the 1 kDa 2711

DOI: 10.1021/jf505817a J. Agric. Food Chem. 2015, 63, 2708−2714

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Journal of Agricultural and Food Chemistry permeate remained high (dropping to 90.2% at pH 5). Conversely, the solubility of the 30 kDa retentate sample was generally poor, dropping to 37.3% at pH 8 from a solubility maximum of 88.2% at pH 2 (Figure 3, inset). Oral ingestion of fortified hydrolysates may result in exposure to a range of gastrointestinal, luminal, and serum proteinases and peptidases, which may affect iron solubility/ bioaccessibility. Initial SGID demonstrated the selective proteolytic digestion of whey proteins (Supplementary Figure 2), with the peptic resistance of β-lg in agreement with previous work.41,42 Iron-fortified (139 mg of FeSO4·7H2O) samples (20 g L−1 protein) were subjected to SGID to ascertain iron solubility during and subsequent to digestion. Figure 4

Figure 5. C18 RP-HPLC chromatograms of simulated gastrointestinal digests of the whey protein isolate (WPI) hydrolysate ferrous-chelating fraction, WPI heat-treated, 10% DH−1 kDa permeate: (A) undigested sample at t0; (B) gastric digest at t+90 min; (C) intestinal digest at t+95 min; (D) intestinal digest at t+286 min. TIS, total iron solubility.

complete simulated digestion, serving to again highlight the relatively poor chelating characteristics of the 30 kDa retentate fraction (Figure 6). Tracking the course of SGID using C18 RP-HPLC (Figure 6) highlighted a reduction in the more hydrophobic material present toward the right of the

Figure 4. Iron solubility during simulated gastrointestinal digestion of 10 mM FeSO4 control and fortified (2.78 g FeSO4 L−1) whey protein isolate (WPI) hydrolysate dispersions (20 g L−1 (initial)): (A) gastric phase; (B) duodenal phase. WPI control, 5% DH−30 kDa retentate; WPI heat-treated, 10% DH−1 kDa permeate.

demonstrates the iron solubility of the 1 kDa permeate and the 30 kDa retentate samples over the course of gastric (marked A in Figure 4) and duodenal (marked B in Figure 4) digestion. From Figure 4, iron solubility remains generally high (≥94.3%) through fortification at pH 5 and subsequent gastric digestion at pH 2 in both samples. However, adjustment of the pH to 7.5 with dibasic sodium phosphate reduced the iron solubility of the 30 kDa retentate sample to 77.1%, although the solubility recovered to approximately 99% after 75 min of simulated duodenal digestion (Figure 4). The authors postulated that this was the result of both the higher ferrous chelating capacity of the 1 kDa sample (CC50 = 0.83 ± 0.03 g protein L−1) and also possible chelating capabilities of the constituents of the simulated intestinal fluid. From this, the chelating capacity (expressed as a CC50) of the bile extract was determined to be 1.24 ± 0.10 g L−1, which concurs with the known strong ferrous-chelating capability of porcine bile extract.43 The iron solubility under SGID of all samples is demonstrated in Supplementary Figure 3. The SGID of the fortified 1 kDa permeate (Figure 5) reveals relatively minor breakdown following termination of digestion (D, t+286 min) when compared to the undigested sample at time zero (A, t0). This is corroborated by the statistically insignificant (P < 0.05) differences in iron solubility of the samples determined at different reaction time points for gastric (B) and duodenal digestion (C and D, Figure 5). This is in contrast to the 30 kDa retentate sample, where the total iron solubility (TIS) decreased to 77.0 ± 1.2% at pH 7.5 and increased upon

Figure 6. C18 RP-HPLC chromatograms of simulated gastrointestinal digests of the whey protein isolate (WPI) hydrolysate ferrous chelating fraction WPI control, 5% DH−30 kDa retentate: (A) undigested sample at t0; (B) gastric digest at t+90 min; (C) intestinal digest at t+95 min; (D) intestinal digest at t+286 min. Increased level of hydrophobic proteinaceous material shown at “w”. 2712

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Lactose, Water, Salts and Minor Constituents, 3rd ed.; McSweeney, P. L. H., Fox, P. F., Eds.; Springer Science & Business Media: New York, 2009; pp 416−420. (6) Fidler, M. C.; Walczyk, T.; Davidsson, L.; Zeder, C.; Sakaguchi, N.; Juneja, L. R.; Hurrell, R. F. A micronised, dispersible ferric pyrophosphate with high relative bioavailability in man. Br. J. Nutr. 2004, 91, 107−112. (7) Fritz, J. C.; Pla, G. W.; Roberts, T.; Boehne, J. W.; Hove, E. L. Biological availability in animals of iron from common dietary sources. J. Agric. Food Chem. 1970, 18, 647−651. (8) Slivka, A.; Kang, J.; Cohen, G. Hydroxyl radicals and the toxicity of oral iron. Biochem. Pharmacol. 1986, 35, 553−556. (9) Ait-Oukhatar, N.; Peres, J. M.; Bouhallab, S.; Neuville, D.; Bureau, F.; Bouvard, G.; Arhan, P.; Bougle, D. Bioavailability of caseinophosphopeptide-bound iron. J. Lab. Clin. Med. 2002, 140, 290− 294. (10) Bouhallab, S.; Cinga, V.; Aít-Oukhatar, N.; Bureau, F.; Neuville, D.; Arhan, P.; Maubois, J. L.; Bouglé, D. Influence of various phosphopeptides of caseins on iron absorption. J. Agric. Food Chem. 2002, 50, 7127−7130. (11) Vegarud, G. E.; Langsrud, T.; Svenning, C. Mineral-binding milk proteins and peptides; occurrence, biochemical and technological characteristics. Br. J. Nutr. 2000, 84, S91−S98. (12) Chaud, M. V.; Izumi, C.; Nahaal, Z.; Shuhama, T.; Bianchi, M. d. L. P.; de Freitas, O. Iron derivatives from casein hydrolysates as a potential source in the treatment of iron deficiency. J. Agric. Food Chem. 2002, 50, 871−877. (13) Kim, S. B.; Seo, I. S.; Khan, M. A.; Ki, K. S.; Nam, M. S.; Kim, H. S. Separation of iron-binding protein from whey through enzymatic hydrolysis. Int. Dairy J. 2007, 17, 625−631. (14) Elias, R. J.; Bridgewater, J. D.; Vachet, R. W.; Waraho, T.; McClements, D. J.; Decker, E. A. Antioxidant mechanisms of enzymatic hydrolysates of β-lactoglobulin in food lipid dispersions. J. Agric. Food Chem. 2006, 54, 9565−9572. (15) Zhou, J.; Wang, X.; Ai, T.; Cheng, X.; Guo, H. Y.; Teng, G. X.; Mao, X. Y. Preparation and characterization of β-lactoglobulin hydrolysate-iron complexes. J. Dairy Sci. 2012, 95, 4230−4236. (16) Kelleher, S. L.; Chatterton, D.; Nielsen, K.; Lonnerdal, B. Glycomacropeptide and alpha-lactalbumin supplementation of infant formula affects growth and nutritional status in infant rhesus monkeys. Am. J. Clin. Nutr. 2003, 77, 1261−1268. (17) O’Loughlin, I. B.; Murray, B. A.; FitzGerald, R. J.; Brodkorb, A.; Kelly, P. M. Pilot-scale production of hydrolysates with altered biofunctionalities based on thermally-denatured whey protein isolate. Int. Dairy J. 2014, 34, 146−152. (18) Bouhallab, S.; Oukhatar, N. A.; Mollé, D.; Henry, G.; Maubois, J.-L.; Arhan, P.; Bouglé, D. L. Sensitivity of β-casein phosphopeptideiron complex to digestive enzymes in ligated segment of rat duodenum. J. Nutr. Biochem. 1999, 10, 723−727. (19) O’Loughlin, I. B.; Murray, B. A.; Brodkorb, A.; FitzGerald, R. J.; Robinson, A. A.; Holton, T. A.; Kelly, P. M. Whey protein isolate polydispersity affects enzymatic hydrolysis outcomes. Food Chem. 2013, 141, 2334−2342. (20) Mullally, M. M.; O’Callaghan, D. M.; FitzGerald, R. J.; Donnelly, W. J.; Dalton, J. P. Proteolytic and peptidolytic activities in commercial pancreatic protease preparations and their relationship to some whey protein hydrolyzate characteristics. J. Agric. Food Chem. 1994, 42, 2973−2981. (21) Viollier, E.; Inglett, P. W.; Hunter, K.; Roychoudhury, A. N.; Van Cappellen, P. The ferrozine method revisited: Fe(II)/Fe(III) determination in natural waters. Appl. Geochem. 2000, 15, 785−790. (22) Decker, E. A.; Welch, B. Role of ferritin as a lipid oxidation catalyst in muscle food. J. Agric. Food Chem. 1990, 38, 674−677. (23) Nongonierma, A. B.; Schellekens, H.; Dinan, T. G.; Cryan, J. F.; FitzGerald, R. J. Milk protein hydrolysates activate 5-HT2C serotonin receptors: influence of the starting substrate and isolation of bioactive fractions. Food Funct. 2013, 4, 728−737. (24) Walsh, D. J.; Bernard, H.; Murray, B. A.; MacDonald, J.; Pentzien, A. K.; Wright, G. A.; Wal, J. M.; Struthers, A. D.; Meisel, H.;

chromatogram (A, t0) and a general leftward shift in the chromatograms as digestion occurs with the increasing formation of peptide material in the 30 kDa retentate sample (Figure 6B−D). The high Mw more hydrophobic material present in the 30 kDa retentate sample (marked “w” in Figure 6) is reduced relatively quickly to more hydrophilic, smaller Mw material, upon gastric digestion with pepsin. This is in agreement with previous work on peptic digestion of aggregated/high Mw whey proteins.44 This study utilized enzymatic hydrolysis in conjunction with a prior heat treatment to facilitate the production, and subsequent fractionation, of a ferrous-chelating hydrolysate fraction from WPI. The 1 kDa permeate fraction possessed a ferrous-chelating capability equivalent to 84.4 μM EDTA and had a high content of certain aromatic and basic amino acid residues. ATR-FTIR spectroscopy demonstrated that there was a molecular level interaction between the 1 kDa permeate and the ferrous sulfate. Fractionation of the 1 kDa permeate via solid phase extraction yielded a hydrophilic fraction with increased chelation capability, with this fraction having a relatively high content of hydrophilic amino acids, namely, Arg, His, and Lys. The ferrous-chelating 1 kDa permeate fraction was utilized to stabilize iron during SGID, maintaining iron solubility ≥98% throughout digestion. This is exemplified by the 72% increase in the determined solubility of total iron at pH 7.5 subsequent to peptic digestion compared to a FeSO4· 7H2O control solution. It is postulated, therefore, that the 1 kDa hydrolysate fraction is a potential candidate ingredient for formulation into products to enhance iron bioaccessibility. Further investigation into the mechanism of iron chelation by the 1 kDa permeate hydrolysate fraction is warranted.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Tables 1 and 2 and Supplementary Figures 1− 3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(A.B.) Phone: +353-25-42222. Fax +353-25-42340. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the help and support offered by Paula O’Connor of Teagasc Food Research Centre, Moorepark, Ireland.

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REFERENCES

(1) Sharma, S.; Singh, R.; Rana, S. Bioactive peptides: a review. Int. J. BIO-autom. 2011, 15, 223−250. (2) O’Connell, J. E.; Fox, P. F. Effect of β-lactoglobulin and precipitation of calcium phosphate on the thermal coagulation of milk. J. Dairy Res. 2001, 68, 81−94. (3) Zimmermann, M. B.; Hurrell, R. F. Nutritional iron deficiency. Lancet 2007, 370, 511−520. (4) Fox, P. F. Milk: an overview. In Milk Proteins − from Expression to Food; Thompson, A., Boland, M., Singh, H., Eds.; Academic Press: New York, 2009; pp 8−43. (5) Hunt, C. D.; Nielsen, F. H. Nutritional aspects of minerals in bovine and human milks. In Advanced Dairy Chemistry − Vol .3. 2713

DOI: 10.1021/jf505817a J. Agric. Food Chem. 2015, 63, 2708−2714

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DOI: 10.1021/jf505817a J. Agric. Food Chem. 2015, 63, 2708−2714