Article pubs.acs.org/JAFC

Effect of Cross-Linking of Interfacial Sodium Caseinate by Natural Processing on the Oxidative Stability of Oil-in-Water (O/W) Emulsions Pui Yeu Phoon,†,§ Lake N. Paul,‡ John W. Burgner, II,‡,⊗ M. Fernanda San Martin-Gonzalez,*,† and Ganesan Narsimhan# †

Department of Food Science, 745 Agricultural Mall Drive, Purdue University, West Lafayette, Indiana 47907, United States Bindley Bioscience Center, 1203 West State Street, Purdue University, West Lafayette, Indiana 47907, United States # Department of Agricultural and Biological Engineering, 225 South University Street, Purdue University, West Lafayette, Indiana 47907, United States ‡

ABSTRACT: This study investigated how enzymatic cross-linking of interfacial sodium caseinate and emulsification, via highpressure homogenization, influenced the intrinsic oxidative stability of 4% (w/v) menhaden oil-in-water emulsions stabilized by 1% (w/v) caseinate at pH 7. Oil oxidation was monitored by the ferric thiocyanate perioxide value assay. Higher homogenization pressure resulted in improved intrinsic emulsion oxidative stability, which is attributed to increased interfacial cross-linking as indicated by higher weighted average sedimentation coefficients of interfacial protein species (from 11.2 S for 0 kpsi/0.1 MPa to 18 S for 20 kpsi/137.9 MPa). Moderate dosage of transglutaminase at 0.5−1.0 U/mL emulsion enhanced intrinsic emulsion oxidative stability further, despite a contradictory reduction in the antioxidant property of cross-linked caseinate as tested by the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. This implied the prominent role of cross-linked interfacial caseinate as a physical barrier for oxygen transfer, hence its efficacy in retarding oil oxidation. KEYWORDS: sodium caseinate, high-pressure homogenization, emulsion, oxidation, analytical ultracentrifugation

■

INTRODUCTION

The scientific motivation of the present study was to explore whether the cross-linking of interfacial sodium caseinate would improve emulsion oxidative stability. It was hypothesized that the cross-linking of interfacial caseinate would promote closer packing within an otherwise primarily loose, random coil conformation, thereby enhancing its physical barrier property. Two natural processing methods were considered to bring about interfacial cross-linking, namely, high-pressure homogenization (HPH) and the use of transglutaminase. The former is physical in nature, whereas the latter is enzymatic. The natural aspect of both studied methods may find appeal in today’s food industry, which is increasingly steering away from chemical modification of ingredients. Transglutaminase is an enzyme that, in proteins, catalyzes the reaction between a γcarboxyamide of glutamine residues and a lysine, forming an ε-(γ-glutamyl)lysine cross-link. Transglutaminases occur naturally in animal tissues, fish, and plants and are also produced by microorganisms. Commercial transglutaminase preparations are isolated from Streptoverticillium sp. and are active over a pH range9 from 5 to 9. In the food industry, transglutaminase is widely used in the manufacture of kamaboko to aid in the setting of salted ground fish paste9 and in sausage products to improve the texture of restructured meat products by strengthening the protein networks. Dairy caseins and caseinates are excellent substrates for transglutaminase cross-linking. Cross-linking of interfacial proteins by transglutaminase in

Sodium caseinate, which is made by acid precipitation of milk casein followed by the addition of sodium hydroxide for resolubilization, is an emulsifying ingredient extensively used in the food industry owing to its excellent emulsifying properties.1 With an unordered, flexible structure analogous to that of a complex linear copolymer2 along with a relatively high hydrophobicity, sodium caseinate can adsorb rapidly onto a lipid surface during emulsification such that the train segments in immediate contact form a physical barrier in the interface. Meanwhile, the high zeta potential of its protruding loops and tails at pH values away from the isoelectric point helps confer physical stability to the emulsion via electrostatic repulsion of the oil droplets. Some authors had previously studied the inhibitory effect of sodium caseinate (and casein) on lipid oxidation in oil-in-water emulsions, which they credited to the antioxidant and metal-chelating properties of the protein.3−5 On the other hand, the effectiveness of its interfacial film in inhibiting lipid oxidation from a physical standpoint is elusive. Horn et al.6 proposed that the characteristically thick interfacial layer of caseinate blocked transition metal ions in the continuous phase from coming into close proximity to the emulsified oil droplets, thereby retarding lipid oxidation. However, considering its loose, unordered structure and high water permeability7 at the interface, as compared to other proteins such as whey protein, which form tightly packed, ordered hydrogen-bonded networks that disfavor mass transfer across the oil−water interface,8 the efficacy of interfacial caseinate as a physical barrier toward dissolved oxygen is uncertain. © 2014 American Chemical Society

Received: Revised: Accepted: Published: 2822

July 25, 2013 January 27, 2014 March 7, 2014 March 7, 2014 dx.doi.org/10.1021/jf403285z | J. Agric. Food Chem. 2014, 62, 2822−2829

Journal of Agricultural and Food Chemistry

Article

casein-stabilized emulsions has been studied,10 and improved stability against creaming has been reported.11 However, studies on the oxidative stability of transglutaminase-crosslinked caseinate-stabilized emulsions are inconsistent. Whereas improved oxidate stability of flaxseed oil emulsions was recently reported when caseinate molecules were cross-linked prior to emulsification step,12 no improvement or decreased oxidative stability was reported for cross-linked caseinate-stabilized fish oil emulsions.13 In this work, analytical ultracentrifugation was used to gain insight into the state of aggregation in which sodium caseinate existed at the interface. Analytical ultracentrifugation is a versatile tool that can be used in the investigation of polymer size, polymer shape, molecular selfassociation, and molecular interactions.14,15 In particular for proteins, analytical centrifugation is one of the classical methods used for the determination of oligomeric states and macromolecular interactions.9 Due to the relatively large size of these macromolecules, the sedimentation profiles observed under gravitational fields lend this technique a high resolution.10 The objective of this work was to investigate the oxidative stability of transglutaminase cross-linked caseinatestabilized fish oil emulsions as a function of homogenization pressure.

■

Caseinate-Stabilized Emulsion Preparation. Emulsification via High-Pressure Homogenization. A 1% (w/v) sodium caseinate solution with 0.05 M sodium chloride and 0.05 M disodium phosphate was prepared at room temperature, during which the mixing time for ensuring complete solvation of the protein was at least 3 h. The pH was eventually adjusted to 7.0. A 4% (v/v) fish oil-in-water coarse emulsion was made using a VirtiShear homogenizer (SP Scientific, Gardiner, NY, USA) at 30000 rpm for 10 s, immediately followed by HPH using a Nano DeBEE 45 high-pressure homogenizer (BEE International) under three or eight passes at either 5 kpsi (34.5 MPa) or 20 kpsi (137.9 MPa). Samples from coarse emulsions were used as controls (∼0 kpsi). For temperature control during HPH, a countercurrent heat exchanger that was connected to a 2 °C water bath was positioned downstream of the emulsifying cell. Each emulsion sample batch consisted of 100 mL and was prepared in triplicate. Enzymatic Cross-Linking of Interfacial Protein in Emulsions. The transglutaminase enzyme product was added to emulsion samples prepared at 20 kpsi at different concentrations (0, 5, 10, and 25 mg/ mL of emulsion). Fifty milliliters of each emulsion batch added with transglutaminase was incubated for 1 h at 37 °C to avoid acceleration of oil oxidation. The emulsions were then immersed in boiling water for 4 min for enzyme inactivation and quickly cooled in water to room temperature. Recovery of Interfacial Protein for Analytical Ultracentrifugation Analysis. To analyze the mass distribution of protein at the oil/water interface, emulsions homogenized at 0, 5, and 20 kpsi were made using a volatile oil that could be removed from the emulsion. Thus, peppermint oil was used in place of fish oil. The emulsion samples were ultracentrifuged at 46000g for at least 3 h at 2 °C using an Optima XL-I ultracentrifuge (Beckman Coulter, Brea, CA, USA). Immediately after ultracentrifugation, the supernatant was removed with a syringe needle, and then distilled water was added to disperse the residual cream in the centrifugation tube. The dispersed cream was frozen in liquid nitrogen and freeze-dried at −55 °C and 0 mbar using a FreezeMobile freeze-dryer (The VirTis Co., Gardner, NY, USA), during which frozen peppermint oil and water sublimed. After freezedrying, the residual protein was redissolved in deionized water to a concentration of 2 mg/mL and subjected to overnight dialysis against a buffer of 0.05 M sodium chloride and 0.05 M disodium phosphate, at pH 7, using a Spectra/Por 4 standard regenerated cellulose dialysis membrane of 12−14 kDa molecular weight cutoff (Spectrum Laboratories, Inc., Rancho Dominguez, CA, USA). Characterization of Oils. The suitability of using peppermint oil to replace fish oil, in the sample preparation of interfacial caseinate for analysis by analytical centrifugation, lies in its similarity in its influence on protein interfacial behavior. Although oil viscosity influences emulsion drop size, it is not the critical factor at issue. Because protein interfacial behavior is influenced by hydrophobicity of oil, it was relevant to gain an understanding about the partition coefficient of peppermint oil and its interfacial tension with water compared with those of fish oil, to validate its appropriateness. Determination of Logarithm of Partition Coefficient. The solubility of oil in hexane could be determined by UV absorbance measurement.16 Both fish oil and peppermint oil were assayed. Oil (25 μL) was added to a mixture comprising 20 mL of hexane and 20 mL of deionized water and vortexed for 5 min. The mixture was left to stand for phase separation until both phases became clear. UV absorbance was read at 250 nm on a Beckman Coulter DU 700 series UV−vis spectrophotometer (Beckman Coulter, Brea, CA, USA) for the hexane phase in duplicates, against a standard curve of oil in hexane within a 0−0.125% (v/v) concentration range. The logarithm of the ratio of oil concentrations in hexane and deionized water, log Phexane/water, was calculated. Measurement of Interfacial Tension with Water. The interfacial tension of fish oil/water and peppermint oil/water interfaces was measured in triplicate by a platinum ring (diameter = 6.1 cm, ratio of radii of ring to wire = 55.6) attached to a CSC DuNouy tensiometer 70545 (CSC Scientific Co., Inc., Fairfax, VA, USA). Each measurement was taken 30 min after layering of oil above the water at room temperature, in a glass dish having inner diameter = 4.7 cm and height

MATERIALS AND METHODS

Chemicals. Sodium caseinate powder was provided by Tatua Cooperative Dairy Co. Ltd. (Tatuanui, New Zealand) and used in all experiments except for proton nuclear magnetic resonance (NMR) spectroscopy, in which sodium caseinate (Alanate 180) from New Zealand Milk Products (North America) Inc. (Santa Rosa, CA, USA) was used. Activa transglutaminase-T1 enzyme product, with maltodextrin and transglutaminase declared as main ingredients and with a commercially reported enzyme activity of 100 U/g, was provided by Ajinomoto North America, Inc. (Eddyville, IA, USA). The following were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA): 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) stock solution at 1.8 mM, 2-propanol, 30% hydrogen peroxide, ammonium thiocyanate, barium chloride dehydrate, disodium phosphate, ferric chloride hexahydrate, ferrous sulfate heptahydrate, isooctane, menhaden fish oil, potassium persulfate, sodium chloride, and triple-distilled peppermint oil. The following were purchased from Mallinckrodt Chemicals Inc. (St. Louis, MO, USA): butanol, hexane, and methanol. Ethyl acetate, glacial acetic acid, and sodium acetate trihydrate were purchased from J. T. Baker (Phillipsburg, NJ, USA). Iron powder was purchased from Acros Organics and hexane from EMD Chemicals (Philadelphia, PA, USA). Distilled water was used to prepare all aqueous solutions. Dilute solutions of hydrochloric acid and sodium hydroxide (≤5 N) were used for pH adjustments. Proton Nuclear Magnetic Resonance Spectroscopy. Proton NMR spectroscopy was carried out using a Varian 300 MHz NMR spectrometer (Varian, Inc., Palo Alto, CA, USA). Two percent (w/v) sodium caseinate solutions were either not treated by homogenization (0 kpsi, 0 MPa) or homogenized at 40 kpsi (276 MPa) under five passes using a Nano DeBEE 45 high-pressure homogenizer (BEE International, South Easton, MA, USA). Both solutions were freezedried and subsequently lyophilized from deuterium oxide of 99.9% isotopic purity (Sigma) twice. The samples were then dissolved to 2% (w/v) in deuterium oxide and analyzed. A homogenization pressure of 40 kpsi was selected for NMR studies because this is the highest pressure reached by the homogenizer. It is hypothesized that if protein interactions were promoted in protein solutions by the homogenization pressure, they should become evident after processing at highest homogenization pressures. However, in emulsion-making, a lower pressure (20 kpsi, 137.9 MPa) was selected because at higher homogenization pressures larger particle sizes were observed due to droplet coalescence promoted by the higher temperatures reached at 40 kpsi as compared to 20 kpsi. 2823

dx.doi.org/10.1021/jf403285z | J. Agric. Food Chem. 2014, 62, 2822−2829

Journal of Agricultural and Food Chemistry

Article

taken every 4 min over 8 h at 45000 rpm. The c(s) distributions were calculated using SEDFIT (v. 12.0b).22 To assess the formation of higher order species, the weighted average sedimentation coefficients of the species was obtained by integration of the entire c(s) distribution. The hydrodynamic movement of a particle can be described by the Svedberg equation: M = (RTs)/(D(1 − υ̅ρsolv)), where M is molar mass, R is the gas constant, T is the temperature, D is the solute diffusion coefficient, υ̅ is the partial specific volume of the particle, and ρsolv is the density of the solvent.23 The sedimentation and diffusion of a particle in a section-shaped cell under a centrifugation field is described by the Lamm equation:

= 2.9 cm). The reading was adjusted by a correction factor read from the manual of the tensiometer. Ferric Thiocyanate Peroxide Value Assay. To monitor oil oxidation, primary oxidation products were measured by the ferric thiocyanate peroxide value assay. Emulsions from each treatment, cross-linked at various transglutaminase concentrations homogenized at 0, 5, or 20 kpsi, were analyzed. The procedure for the assay was modified from that of Ogawa et al.,17 and the exact details were reported in our earlier work.18 Triplicate samples were removed over a period of 12 days for the determination of oxidative stability. Emulsion Drop Size Measurement. Triplicate emulsion samples were diluted 100-fold with deionized water, and each triplicate was measured twice for particle size (Z-average) at 25 °C by dynamic light scattering (DLS), using a Zetasizer Nano ZS90 with optical arrangement at 90° (Malvern Instruments Ltd., Worcestershire, UK). During size measurement, the refractive indices of water and oil were set at 1.33 and 1.46, respectively, whereas the dispersant viscosity was that of water at 25 °C (i.e., 0.89 cP). According to Thomas,19 and as previously reported in our earlier work,18 the average value of Z-average (Zav) was converted to the average Sauter mean diameter (d32) as av d32 = Zav × (1 + Q )2

⎤ ∂χ 1 ∂ ⎡ ∂χ = − sω2r 2χ ⎥ ⎢rD ⎦ ∂t r ∂r ⎣ ∂r

SEDFIT employs a numerical solution to the Lamm equation that generates a c(s) distribution describing the sedimenting solutes in the solution under a centrifugal field.24 Enzymatic Cross-Linking Turbidity Assay. It is expected that an increase in transglutaminase concentration will lead to an increase in cross-linking of caseinate. The turbidity assay was done as a quick qualitative verification of the relative extent of enzymatic cross-linking. Although the Zetasizer instrument may be used to measure particle size distribution in the sodium caseinate solutions to infer turbidity, the credibility of the results from the Zetasizer may be affected by the inherently high polydispersity at 1% (w/v). Hence, turbidity was analyzed by measuring the sample absorbance at 340 nm on a Beckman Coulter DU 700 series UV−vis spectrophotometer at room temperature. The transglutaminase enzyme product was added to 1% (w/v) sodium caseinate solutions (with 0.05 M sodium chloride and 0.05 M disodium phosphate, at pH 7) at 0, 5, 10, and 25 mg/mL and incubated in a 37 °C water bath for 1 h before being boiled for 4 min for enzyme inactivation. Sodium caseinate solutions with 5, 10, and 25 mg/mL of the enzyme product added and boiled immediately without an incubation step were prepared as positive controls. Triplicate measurements of the absorbance of the sodium caseinate solutions were taken. The mean increase in turbidity due to enzymatic crosslinking was reported by the subtraction of the mean absorbance of a positive control from the mean absorbance of the corresponding sample. Statistical analysis for mean comparison was done using the Tukey−Kramer test. ABTS Decolorization Assay. For the ABTS assay, sample solutions of 1% (w/v) sodium caseinate (with 0.05 M sodium chloride and 0.05 M disodium phosphate, at pH 7) were subjected to enzymatic cross-linking at 5, 10, and 25 mg/mL of the transglutaminase enzyme product (37 °C for 1 h) followed by enzyme inactivation (100 °C for 4 min). Positive controls were added with the corresponding amounts of the transglutaminase enzyme product but subjected only to boiling for 4 min. The negative controls consisted of the native protein solution. The assay is based on the quenching of the ABTS cationic radical, observed as the loss of blue-green color at 734 nm. The assay could be indicative of the scavenging capability of radicals such as reactive oxygen species or radical species formed from oil oxidation at the oil/ water interface. In particular, the assay was chosen because it can be set up such that the sample pH is not altered, and neither does it involve the use of organic solvents. As such, the assay is free of complications from pH- and solvent-induced changes in solubility, structure, and/or state of aggregation of the protein. Although antioxidative mechanisms may be diverse, in the work of Moure et al.25 involving protein analysis, the assay yielded results that generally agreed with the overall trend in antioxidant properties measured by different methods. The procedure for the assay was modified from that reported by Moure et al.25 Two parts volume of 1.8 mM ABTS stock solution and five parts volume of 2.45 mM potassium persulfate in 0.02 M disodium phosphate solution were mixed together, adjusted to pH 7, and incubated for 1 h at room temperature in the dark to generate the blue-green ABTS radical cation. Duplicate measurements of radical scavenging capacity were done by mixing 20 μL of the sample or control with 2 mL of the radical solution and, after 15 min, reading the

(1)

where Q was the average experimentally determined polydispersity obtained during particle size measurement. Intrinsic Rate of Oxygen Depletion Calculation. It must be emphasized that the average emulsion drop size has a strong implication on the total interfacial area per volume subjected to contact with dissolved oxygen in the emulsion. Hence, the intrinsic rate of oxidation, which takes into account the interfacial area, is a more accurate indicator of the rate of oxidation in the average emulsion drop than the change of peroxide value of the emulsion over time. The intrinsic rate of oxidation has an especially strong bearing on the oxidative stability of emulsions with very fine drops, which are often desirable due to the slowing of creaming/sedimentation according to Stokes’ law. The calculation steps were reported in our earlier work.18 The total rate of oxygen depletion during oxidation of oil was proportional to (∂(Abst − Abs0))/(∂t) and in turn (6/d32) × intrinsic rate of oxygen depletion per unit of interfacial area of the emulsion drops, where Abs0 was the average absorbance reading at 510 nm of an emulsion sample from peroxide value assay corrected by that of the blank at time = 0; Abst was the absorbance at 510 nm from peroxide value assay at time = t; and (6/d32t) represented the ratio of the total surface area of the emulsion drops to total volume of the drops at time = t. Hence, the relative intrinsic rate of oxygen depletion per unit of interfacial area of the emulsion drops was calculated as d ∂(Abst − Abs0 ) × 32t ∂t 6

(2)

Analytical Ultracentrifugation. Sedimentation velocity experiments were performed in an Optima XL-A ultracentrifuge (Beckman CoulterS). The samples analyzed included protein dissolved directly as it was in buffer, as well as freeze-dried protein recovered from emulsions after the sublimation of peppermint oil and redissolved in buffer. The final protein concentration ranged from 0.5 to 2.0 mg/mL as determined by the Lowry protein assay. Solvent densities and viscosities, along with the partial specific volume, were calculated with SEDNTERP (v. 1.09). The estimation of the partial specific volume (υ)̅ was done according to the method of Cohn and Edsall:20 υ̅ = (∑υ̅iniMi)/(∑niMi), where ni is the number of moles of ith component and M is its molecular weight. Solvent densities are extrapolated from the polynomial fit to

Δρc = a + bC1/2 + cC + dC 2 + eC 3 + fC 4 − ρwater

(4)

(3)

where Δρc is the density increment at a certain concentration, a−f are coefficients, C is the concentration of the solute, and ρwater is the density of water.21 Boundary positions were determined using the absorbance (280 nm) and interference (675 nm) optics, with scans 2824

dx.doi.org/10.1021/jf403285z | J. Agric. Food Chem. 2014, 62, 2822−2829

Journal of Agricultural and Food Chemistry

Article

absorbance at 734 nm on a Jasco V-650 spectrophotometer (Jasco Spectroscopic Co., Easton, MD, USA). The relative antioxidant capacity of the samples was inferred by comparing the mean absorbance values using the Tukey−Kramer method.

■

RESULTS Figure 1 shows that the homogenizing pressure in HPH had no effect on the molecular structure of sodium caseinate in

Figure 1. One-dimensional NMR spectra of 2% (w/v) sodium caseinate solutions that were not treated by homogenization (0 kpsi) and homogenized at 40 kpsi under five passes.

Figure 3. Plots of the relative intrinsic rate of oxygen depletion in fish oil per unit interfacial area, as influenced by (1) homogenization pressure and number of passes (P) and (2) use of different concentrations of the transglutaminase (Tgase) enzyme product at homogenization conditions of 20 kpsi and 3P. Error bars indicate the standard deviation from the mean of oxygen depletion. (Insets) Plots of the variation of the saunter mean diameter d32 of emulsions with time, with error bars indicating the standard deviation from the mean of six measurements from triplicates.

solution. Thus, it could be established that during emulsification by HPH, the homogenizing pressure would not exert any influence on the chemical antioxidant property of sodium caseinate in the continuous phase. Despite this, emulsions made by HPH (5 and 20 kpsi) showed slower initial rates of formation of primary oxidation products compared to a coarse emulsion (0 kpsi), as shown by the slope of the oxidation curves in Figure 2. The mean peroxide value (PV) of the coarse

lower intrinsic state of oxidation than those formed at 5 kpsi. The difference in the intrinsic rate of oxidation between 5 and 20 kpsi homogenizing pressures is much more pronounced (Figure 3.1) than that in the PV values (Figure 2). This can be explained by the difference in the interfacial area available for oxidation. Compared to emulsions prepared at 5 kpsi, those made at 20 kpsi had significantly (p < 0.05) smaller mean d32 and thus larger total interfacial area per volume available for reaction with dissolved oxygen. The addition of transglutaminase to emulsions did not lead to any significant difference in drop size, as shown in the inset of Figure 3.2. No sedimentable aggregates were formed. Even though the total interfacial area per volume was similar, there was improved emulsion oxidative stability at 5 and 10 mg/mL dosage of the enzyme product compared to no dosage. However, when the enzyme product concentration increased further (25 mg/mL), the beneficial effect of the use of transglutaminase apparently decreased. The general trend in the results suggested that there was an optimal enzyme level for emulsion oxidative stability. Peppermint oil seemed to be a reasonable replacement of fish oil, as suggested by the results displayed in Table 1. From Table 1, the mean logarithm of partition coefficient of the peppermint oil was within 24% difference from that of the fish oil. The mean oil/water interfacial tension of the peppermint oil was within 32% difference from that of the fish oil. Figure 4 shows how homogenizing pressure affected the state of aggregation in solvated and interfacial caseinate. As seen in Figure 4.1, there was a slight increase in the mass of caseinate aggregates in the continuous phase of emulsions with increasing pressure. However, for interfacial caseinate, higher homogenizing pressure led to increased formation of larger species, as

Figure 2. Plots showing the effect of high-pressure homogenization on the peroxide value with increasing incubation time (number of passes = 3) of emulsion samples. A decrease in PV at longer times is due to the conversion of primary to secondary oxidation products. Error bars indicate the standard deviation from the mean of triplicate readings.

emulsion declined considerably at longer incubation time (from days 7 to 12), reflecting further aggravated oil oxidation with the conversion of primary to secondary oxidation products. Between homogenizing pressures, the intrinsic rate of oxidation in emulsions at 20 kpsi was slower than for the emulsions made at 5 kpsi, as shown in Figure 3.1 by a steeper slope of the oxidation curve at 5 kpsi and early incubation times. By day 4 of incubation, emulsions formed at 20 kpsi were at a significantly 2825

dx.doi.org/10.1021/jf403285z | J. Agric. Food Chem. 2014, 62, 2822−2829

Journal of Agricultural and Food Chemistry

Article

Table 1. Physical Properties of Fish Oil and Peppermint Oila oil

log Phexane/water

oil/water interfacial tension (mN/m)

fish peppermint

1.37 ± 0.02 (SD) 1.05 ± 0.04 (SD)

12.0 ± 0.2 (SD) 15.8 ± 0.3 (SD)

a

SD stands for the standard deviation based on duplicate measurements for the logarithm of the partition coefficient, log Phexane/water, and triplicate measurements for oil/water interfacial tension.

Figure 5. Results of spectrophotometric assays of 1% (w/v) sodium caseinate (Cas) solutions: (1) Turbidity measured at 340 nm as a function of concentration of the transglutaminase (Tgase) enzyme product during enzymatic cross-linking. Error bars indicate the standard deviation from the mean values derived from three measurements. (2) Absorbance measurements at 734 nm in the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. Prior to the ABTS assay, the samples (which contained Tgase) were subjected to treatment at 37 °C for 1 h followed by 100 °C for 4 min, whereas the positive controls (which also contained Tgase) were subjected to treatment at 100 °C for 4 min. Means with different letters are significantly different from one another at p < 0.01, according to the Tukey−Kramer test. Error bars indicate the standard deviation from the mean absorbance values derived from two measurements.

Figure 4. Sedimentation coefficient distribution plots for (1) sodium caseinate solutions homogenized under three passes (P) at 0, 5, and 20 kpsi, which served as controls, and (2) interfacial protein isolated from sodium caseinate emulsions made under three passes at 0, 5, and 20 kpsi. The weighted average sedimentation coefficient, s, of each sample is indicated.

indicated in Figure 4.2 and by an upward shift in the weighted average sedimentation coefficient (from 11.2 S for 0 kpsi to 18 S for 20 kpsi). Figure 5.1 depicts the results of the turbidity assay, with increase in transglutaminase concentration causing an increase in the cross-linking of caseinate. Figure 5.2 shows the results of the ABTS decolorization assay. The native sodium caseinate solution had the lowest mean absorbance reading at 734 nm, inferring higher antioxidant capacity than any other treated sample. Heat treatment at 37 °C and boiling of the native caseinate solution did not change the absorbance and antioxidant capacity significantly. Boiled protein samples containing the enzyme product had much higher absorbance at 734 nm overall due to the presence of maltodextrin in the commercial product. Generally, the higher the dosage of the enzyme product, the higher was the absorbance. However, at every enzyme product concentration, an additional 60 min of incubation at 37 °C (followed by boiling) led to even higher absorbance, indicating the reduction of chemical antioxidant capacity due to enzymatic cross-linking of caseinate.

particle size would be expected to increase its susceptibility to react with oxygen present in the continuous phase, when oxygen concentration is not limiting. This was supported by Lethuat et al.,26 who measured the rate of formation of conjugated diene in sunflower oil emulsions stabilized by bovine serum albumin. However, Let et al.27 reported that milkstabilized fish oil emulsions with smaller drops (and therefore larger interfacial area) were less oxidized than emulsions with larger drops. Similarly, higher lipid oxidative stability of caseinate-stabilized emulsion with smaller droplets was also reported by Ries et al.28 In the present study, oxidative stability increased with decreasing emulsion droplet size. Figure 2 shows that the increase in PV versus time for the 20 kpsi emulsion was not significantly (p < 0.05) faster than that for the 5 kpsi emulsion. However, when particle size is taken into account, as shown in Figure 3.1, the lower the homogenizing pressure, the lower was the intrinsic emulsion oxidative stability. From the d32 results in the different test scenarios, the varying total interfacial areas per volume of emulsion could be evaluated. It was estimated that regardless of the total interfacial area in each test scenario, there was excessive caseinate at 1% (w/v) to permit maximum protein load at the plateau region of the interfacial adsorption isotherm, which was deduced to be around 3 mg/m2 oil according to Tornberg’s work.29 As such, the protein load per unit interfacial area in each test scenario

■

DISCUSSION The poor oxidative stability exhibited by coarse emulsions (0 kpsi) was expected because these emulsions are prone to creaming, due to the large particle size, which causes the oil droplets to become directly exposed to oxygen in the headspace. On the other hand, in emulsions made by homogenization, the increase in interfacial area due to smaller 2826

dx.doi.org/10.1021/jf403285z | J. Agric. Food Chem. 2014, 62, 2822−2829

Journal of Agricultural and Food Chemistry

Article

MPa and mild temperature (40 °C), whereas deaggregation was observed at similar pressure levels but lower temperatures.35 Cross-Linking of Caseinate Was Associated with Decreased Chemical Antioxidant Property. The samples in the present study were all controlled for the starting level of dissolved oxygen by using the same source of water. To understand how cross-linking of caseinate would influence its inherent antioxidant property, the ABTS decolorization assay at 734 nm was performed on sodium caseinate solution samples dosed with different concentrations of the transglutaminase product, which also contained maltodextrin. When activated, transglutaminase catalyzes the formation of a covalent bond between the free ε-amine group of a protein-bound lysine and the γ-carboxyamide group of a protein-bound glutamine.36 It was acknowledged that during enzyme inactivation by boiling, the process might cause the removal of dissolved oxygen and/ or trigger the Maillard reaction between caseinate and maltodextrin; hence, the impact of boiling was also tested in the ABTS assay. In all of the samples, absorbance due to turbidity was checked and verified to be negligible at 734 nm and hence would not significantly interfere with true absorbance due to the blue-green ABTS radical. In light of this, Figure 5.2 clearly shows that transglutaminase-induced cross-linking caused a reduction in antioxidant capacity, across the three enzyme product concentrations tested (5, 10, and 25 mg/mL dosage of the enzyme product or 50, 100, and 250 U/g sodium caseinate). Despite this, the emulsion oxidative stability in all three cases was better than without the use of transglutaminase (Figure 3.2). Because the improvement in emulsion oxidative stability could not be explained from a chemical standpoint, the cross-linked interfacial caseinate layer must have played a dominant role in terms of physical barrier property against oxidation. Considering that Kellerby et al.37 did not observe improvement in the oxidative stability of sodium caseinatebased menhaden oil-in-water emulsions at a transglutaminase activity of 10 U/g protein, the present finding suggests a minimum enzyme activity needed to boost the physical barrier property of interfacial caseinate. The results in Figure 3.2 also suggested a maximum in emulsion oxidative stability at an optimum transglutaminase activity, similar to the findings of Bao et al.38 for spray-dried microalgal oil encapsulated by cross-linked sodium caseinate (with transglutaminase) as a function of the cross-linking time. Protein cross-linking could possibly reduce the solvent accessibility of some amino acids with antioxidant activity, such as tryptophan, tyrosine, histidine, methionine, and cysteine; however, the decline in emulsion oxidative stability from 10 to 25 mg/mL dosage of the enzyme product did not seem to be a case of cross-linking induced reduction in solvent accessibility of antioxidant amino acids prevailing over the interfacial physical barrier property. This was because as the dosage of the enzyme product increased, the reduction in antioxidant property owing to cross-linking decreased (see shaded regions in Figure 5.2). There was no significant difference (p > 0.05) in the average emulsion drop size between the 10 and 25 mg/mL dosed emulsions (inset in Figure 3.2), thus ruling out the possibility of different interfacial areas for oxygen transfer affecting the oxidation rates. The early stages of milk protein−carbohydrate Maillard reaction had been reported by Calligaris et al.39 to generate oxidative species. Puscasu and Birlouez-Aragon40 reported on how intermediary and/or advanced Maillard reaction products formed from milk protein

could be reasonably assumed the same. Thus, it was not probable that the more oxidatively stable emulsions had significantly higher protein loads. It was unlikely that they had significantly thicker interfacial layers either, because the thickness of adsorbed caseinate (and casein) was found to be insensitive to adsorption conditions but constant at ∼10−15 nm.30−32 The next section discusses the disparity in the observed emulsion oxidative stability from a molecular viewpoint (of how cross-linking might alter the chemical antioxidant property) and a physical standpoint (of how crosslinked interfacial caseinate might serve as a physical barrier against oil oxidation). Analytical Ultracentrifugation Findings Show Higher State of Interfacial Protein Aggregation at Higher Homogenizing Pressure. Sodium caseinate in water at pH 7 is translucent in appearance at as low a concentration of 0.5% (w/v) and thus not completely soluble. Owing to this physical character, attempts to analyze the conformation of interfacial caseinate were met with obstacles. FTIR for studying protein secondary structure was not considered in this case, because the protein in question essentially lacks any defined secondary structure. Khun and Cunha33 used sodium dodecyl sulfate− polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing and reducing conditions to demonstrate the formation of disulfide linkages in interfacial whey protein with increase in homogenizing pressure. This method is, however, not applicable for the study of interfacial sodium caseinate because, first, sodium caseinate does not possess substantial sulfur amino acid residues and, second, the method dissociates any electrostatic and hydrophobic interactions that could have contributed to interfacial caseinate polymerization. In a preliminary experiment, native PAGE assay on a 15% criterion precast Tris-HCl gel (from Bio-Rad) was found not useful for the analysis of sodium caseinate either, as it was impossible to separate sodium caseinate into bands due to the size of molecular aggregates being >200 kDa. In another previous instance, sodium caseinate at pH 7 was found not soluble enough to be analyzed by multiangle laser light scattering-size exclusion chromatography (MALLS-SEC). To our knowledge, the present study was the first time analytical ultracentrifugation was demonstrated to be a viable method for experimentally examining the state of protein aggregation at an oil−water interface. The analytical ultracentrifugation results of interfacial caseinate elucidated an upward shift in the order of oligomer species and molecular weight, when the homogenizing pressure increased. These results were in agreement with the trend in the highperformance liquid chromatography (HPLC) results published by Lee et al.,34 which showed dimensionally larger aggregates in interfacial whey protein from emulsions subjected to higher homogenizing pressure. The increased aggregation in interfacial caseinate was an indication of cross-linking in its random coil structure. Slight increases in sedimentation coefficient patterns were observed in homogenized protein solutions, with values gradually increasing with increase in homogenization pressure (Figure 4.1). However, protein recovered for the interface dramatically increased the values for sedimentation coefficient with increasing pressure, presumably due to protein aggregation promoted by interactions between pressure and temperature. Similar observations have been reported for casein micelles subjected to static high-pressure treatments, where aggregation of casein micelles was reported for skim milk exposed to 200 2827

dx.doi.org/10.1021/jf403285z | J. Agric. Food Chem. 2014, 62, 2822−2829

Journal of Agricultural and Food Chemistry

Article

phase. As such, a tighter packing in the foremost subinterfacial layer to the oil, comprising a higher density of train segments, could be envisioned for HPH at 20 versus 5 kpsi. At sufficiently high surface coverage, the closer local proximity among the train segments could have promoted intermolecular crosslinking and thus led to the analytical ultracentrifugation finding of more interfacial protein oligomers of higher species during HPH at 20 kpsi. The postulated denser subinterfacial layer of train segments probably provided additional resistance to oxygen diffusion from the continuous phase to the oil, thus increasing the physical interfacial barrier property against oxidation. Furthermore, densification of a β-casein cross-linked adsorbed onto polystyrene beads was recently reported by Partanen et al.46

(α-lactalbumin) and lactose exhibited pro-oxidant property. Albeit scarce, and although the antioxidative property of Maillard reaction products is more widely recognized in general, reports on the pro-oxidant property of milk protein− carbohydrate Maillard reaction products do exist. In Figure 5.2, the increase in absorbance of the boiled-only protein− carbohydrate samples (without the 37 °C incubation) with increasing enzyme product dosage might be attributed to (i) a correspondingly higher formation of pro-oxidant Maillard reaction products and/or (ii) increasing protein−maltodextrin conjugation, leading to lessening solvent accessibility of amino acids with antioxidant activity. Hence, a plausible reason for the decline in emulsion oxidative stability in the 25 mg/mL dosed emulsion was that either or both the aforementioned points started to offset the effect of interfacial barrier resistance to oxygen transfer as a result of greater cross-linking in the interfacial caseinate. Cross-Linking of Interfacial Caseinate Might Promote Tighter Packing and Hence Enhance Emulsion Oxidative Stability. The influential role of cross-linked interfacial caseinate in improving emulsion oxidative stability, as implied by the results of the ABTS assay on transglutaminase-treated systems, could similarly be inferred when interfacial crosslinking was induced by HPH. Such a view is substantiated by the works of Lefèvre and Subirade41 and Lee et al.34,42 on whey protein adsorbed at the oil/water interface. These authors reported the formation of intermolecular β-sheet, which led to protein aggregation in the interfacial membranes. Lee et al.34 also detected an increase in intermolecular β-sheet in the interfacial protein of whey protein-stabilized emulsions subjected to higher homogenizing pressure, further demonstrating how HPH could promote condensed interfacial packing that could boost the physical interfacial barrier property against oxidation. The ability to analyze the density of the HPH-induced interfacial caseinate in situ was met with paramount technical challenge, because the dynamics of conformational behavior accompanying interfacial adsorption during HPH needed to be captured. Neutron scattering and dual polarizing interferometry are both advanced techniques suitable for analyzing the thickness of the interfacial film or the variation of density along the film thickness, but only under static experimental conditions when the film is preformed or immobilized. Molecular dynamics simulations may hold the key to better understanding of the conformational dynamics. The following proposes what might have happened at a higher homogenizing pressure of 20 kpsi as opposed to 5 kpsi, such that it promoted higher emulsion oxidative stability: For simplicity, sodium caseinate at pH 7 could be molecularly viewed as a polyelectrolyte chain. At 20 kpsi, there was a higher convection rate and thus greater kinetic energy accompanying the turbulence in HPH, which could better overcome the adsorption energy barriers43 arising from increasing repulsive electrostatic and/or steric interaction forces of protein molecules already adsorbed,44 whereas free interfacial area continued to diminish. According to the simulation study by Aguilera-Granja and Kikuchi,45 the polyelectrolyte chains approaching the adsorbent surface experienced such strong electrostatic repulsion that in avoidance they preferentially adsorbed almost as a monolayer with a dominantly high train fraction rather than the loops and tails combined because such a conformation considerably lowered the free energy in the system by exposing hydrophobic functional groups to the oil

■

AUTHOR INFORMATION

Corresponding Author

*(M.F.S.M.G.) E-mail: [email protected]. Phone: +1 (765) 496-1140. Fax: +1 (765) 494-7953. Present Addresses §

(P.Y.P.) Sensient Flavors LLC, 5600 W. Raymond St., Indianapolis, IN 46241, USA. ⊗ (J.W.B.) 3024 Newquay Lane, Richmond, VA 23236, USA. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors would like to thank Ajinomoto North America, Inc., for providing the transglutaminase enzyme used in this study.

■

REFERENCES

(1) Dickinson, E.; Rolfe, S. E.; Dalgleish, D. G. Competitive adsorption of αs1-casein and β-casein in oil-in-water emulsions. Food Hydrocolloids 1988, 265, 395−405. (2) Dickinson, E. Properties of emulsions stabilized with milk proteins: overview of some recent developments. J. Dairy Sci. 1997, 80, 2607−2619. (3) Allen, J. C.; Wrieden, W. L. Influence of milk-proteins on lipid oxidation in aqueous emulsion: I. Casein, whey protein and αlactalbumin. J. Dairy Res. 1982, 49, 239−248. (4) Hu, M.; McClements, D. J.; Decker, E. A. Lipid oxidation in corn oil-in-water emulsions stabilized by casein, whey protein isolate, and soy protein isolate. J. Agric. Food Chem. 2003, 51, 1696−1700. (5) Faraji, H.; McClements, D. J.; Decker, E. A. Role of continuous phase protein on the oxidative stability of fish oil-in-water emulsions. J. Agric. Food Chem. 2004, 52, 4558−4564. (6) Horn, A. F.; Nielsen, N. S.; Jensen, L. S.; Horsewell, A.; Jacobsen, C. The choice of homogenisation equipment affects lipid oxidation in emulsions. Food Chem. 2012, 134, 803−810. (7) Ho, B. Water Vapour Permeabilities and Structural Characteristics of Casein Films and Casein-Lipid Emulsion Films. M.S. thesis, University of California, Davis, CA, 1992. (8) Yang, L.; Paulson, A. T. Effects of lipids on mechanical and moisture barrier properties of edible gellan film. Food Res. Int. 2000, 33, 571−578. (9) Kuraishi, C.; Yamazaki, K.; Susa, Y. Transglutaminase: its utilization in the food industry. Food Rev. Int. 2001, 17, 221−246. (10) Dickinson, E.; Yamamoto, Y. Rheology of milk protein gels and protein-stabilized emulsion gels cross-linked with transglutaminase. J. Agric. Food Chem. 1996, 44, 1371−1377. (11) Faergemand, M.; Murray, B. S.; Dickinson, E. Cross-linking of milk proteins with transglutaminase at the oil-water interface. J. Agric. Food Chem. 1998, 45, 2514−2519. 2828

dx.doi.org/10.1021/jf403285z | J. Agric. Food Chem. 2014, 62, 2822−2829

Journal of Agricultural and Food Chemistry

Article

(32) Helstad, K. M. Nanorheological Studies of Caseins. Ph.D. thesis, Lund University, Lund, Sweden, 2007. (33) Kuhn, K. R.; Cunha, R. L. Flaxseed oil−whey protein isolate emulsions: effect of high pressure homogenization. J. Food Eng. 2012, 111, 449−457. (34) Lee, S.-H.; Lefèvre, T.; Subirade, M.; Paquin, P. Effects of ultrahigh pressure homogenization on the properties and structure of interfacial protein layer in whey protein-stabilized emulsion. Food Chem. 2009, 113, 191−195. (35) Anema, S. G.; Lowe, E. K.; Stockman, R. Particle size changes and casein solubilization in high-pressure treated skim milk. Food Hydrocolloids 2005, 19, 257−267. (36) Folk, J. E.; Finlayson, J. S. The ε-(γ-glutamyl) lysine crosslink and the catalytic role of transglutaminases. Adv. Protein Chem. 1977, 31, 1−133. (37) Kellerby, S. S.; Gu, Y. S.; McClements, D. J.; Decker, E. A. Lipid oxidation in a menhaden oil-in-water emulsion stabilized by sodium caseinate cross-linked with transglutaminase. J. Agric. Food Chem. 2006, 54, 10222−10227. (38) Bao, S.-S.; Hu, X.-C.; Zhang, K.; Xu, X.-K.; Zhang, H.-M.; Huang, H. Characterization of spray-dried microalgal oil encapsulated in cross-linked sodium caseinate matrix induced by microbial transglutaminase. J. Food Sci. 2011, 76, E112−E118. (39) Calligaris, S.; Manzocco, L.; Anese, M.; Nicoli, M. C. Effect of heat-treatment on the antioxidant and pro-oxidant activity of milk. Int. Dairy J. 2004, 14, 421−427. (40) Puscasu, C.; Birlouez-Aragon, I. Intermediary and/or advanced Maillard products exhibit prooxidant activity on Trp: in vitro study on α-lactalbumin. Food Chem. 2002, 78, 399−406. (41) Lefèvre, T.; Subirade, M. Formation of intermolecular β-sheet structures: a phenomenon relevant to protein film structure at oilwater interfaces of emulsions. J. Colloid Interface Sci. 2003, 263, 59−67. (42) Lee, S.-H.; Lefèvre, T.; Subirade, M.; Paquin, P. Changes and roles of secondary structures of whey protein for the formation of protein membrane at soy oil/water interface under high-pressure homogenization. J. Agric. Food Chem. 2007, 55, 10924−10931. (43) McClements, D. J. Food Emulsions: Principles, Practices, and Techniques, 2nd ed.; CRC Press: Boca Raton, FL, USA, 2004; p 190. (44) Mohan, S.; Narsimhan, G. Coalescence of protein-stabilized emulsions in a high-pressure homogenizer. J. Colloid Interface Sci. 1997, 192, 1−15. (45) Aguilera-Granja, F.; Kikuchi, R. Polymer statistics IV. Simulation of adsorption of polymers and polyelectrolytes on surfaces. Physica A 1992, 189, 108−126. (46) Partanen, R.; Forssell, P.; Mackie, A.; Blomberg, E. Interfacial cross-linking of β-casein changes the structure of the adsorbed layer. Food Hydrocolloids 2013, 32, 271−277.

(12) Ma, H.; Forssell, P.; Kylli, P.; Lampi, A. M.; Buchert, J.; Boer, H.; Partanen, R. Transglutaminase catalyzed cross-linking of sodium caseinate improves oxidateive stability of flaxseed oil emulsion. J. Agric. Food Chem. 2012, 60, 6223−6229. (13) Kellerby, S. S.; Gu, Y. S.; McClements, D. J.; Decker, E. Lipid oxidation in a menhaden oil-in-water emulsion stabilized by sodium caseinate cross-linked with transglutaminase. J. Agric. Food Chem. 2006, 54, 10222−10227. (14) Schuck, P. On the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation. Anal. Biochem. 2003, 320, 104−124. (15) Schuck, P.; Perugini, M. A.; Gonzales, N. R.; Howlett, G. J.; Schubert, D. Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems. Biophys. J. 2002, 82, 1096−1111. (16) Kulkarni, A. R.; Soppimath, K. S.; Aminabhavi, T. M. Effect of cosolvent and non-ionic surfactant on partition coefficient of Azadirachta indica A. Juss. (neem) seed oil in water-hexane at (298.15, 303.15, 308.15, and 313.15) K. J. Chem. Eng. Data 2000, 45, 75−77. (17) Ogawa, S.; Decker, E. A.; McClements, D. J. Influence of environmental conditions on the stability of oil in water emulsions containing droplets stabilized by lecithin-chitosan membranes. J. Agric. Food Chem. 2003, 51, 5522−5527. (18) Phoon, P. Y.; Narsimhan, G.; San Martin-Gonzalez, M. F. Effect of thermal behaviour of β-lactoglobulin on the oxidative stability of menhaden oil-in-water emulsions. J. Agric. Food Chem. 2013, 61, 1954−1967. (19) Thomas, J. C. The determination of log normal particle size distributions by dynamic light scattering. J. Colloid Interface Sci. 1986, 117, 187−192. (20) Cohn, E. J.; Edsall, J. T. Proteins, Amino Acids and Peptides as Ions and Dipolar Ions; Reinhold: New York, 1943; Chapter 4, p 157. (21) Laue, T. M.; Shah, B. D.; Ridgeway, T. M.; Pelletier, S. L. Analytical Ultracentrifugation in Biochemistry and Polymer Science; Royal Society of Chemistry: London, UK, 1992; pp 90−125. (22) Schuck, P. Size distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys. J. 2000, 78, 1606−1619. (23) Schuck, P.; MacPhee, C. E.; Howlett, G. J. Determination of sedimentation coefficients for small peptides. Biophys. J. 1998, 74, 466−474. (24) Brown, P. H.; Schuck, P. A new adaptive grid-size algorithm for the simulation of sedimentation velocity profiles in analytical ultracentrifugation. Comput. Phys. Commun. 2007, 178, 105−120. (25) Moure, A.; Domínguez, H.; Parajó, J. C. Antioxidant properties of ultrafiltration-recovered soy protein fractions from industrial effluents and their hydrolysates. Process Biochem. 2006, 41, 447−456. (26) Lethuaut, L.; Métro, F.; Genot, C. Effect of droplet size on lipid oxidation rates of oil-in-water emulsions stabilized by protein. J. Am. Oil Chem. Soc. 2002, 79, 425−430. (27) Let, M. B.; Jacobsen, C.; Sørensen, A. D. M.; Meyer, A. S. Homogenization conditions affect the oxidative stability of fish oil enriched milk emulsions: lipid oxidation. J. Agric. Food Chem. 2007, 55, 1773−1780. (28) Ries, D.; Ye, A.; Haisman, D.; Singh, H. Antioxidant properties of caseins and whey proteins in model oil-in-water emulsions. Int. Dairy J. 2010, 20, 72−78. (29) Tornberg, E. Functional characterisation of protein stabilised emulsions: emulsifying behaviour of proteins in a valve homogenizer. J. Sci. Food Agric. 1978, 29, 867−879. (30) Caldwell, K. D.; Li, J.; Li, J.-T.; Dalgleish, D. G. Adsorption behaviour of milk proteins on polystyrene latex: a study based on sedimentation field-flow fractionation and dynamic light scattering. J. Chromatogr., A 1992, 604, 63−71. (31) Leaver, J. D.; Brooksbank, D. V.; Horne, D. S. Influence of glycosylation and surface charge on the binding of κ-casein to latex particles. J. Colloid Interface Sci. 1994, 162, 463−469. 2829

dx.doi.org/10.1021/jf403285z | J. Agric. Food Chem. 2014, 62, 2822−2829