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High Hydrostatic Pressure Affects Flavor-bindingProperties of Whey Protein ConcentrateXXXXXIAOMINGIAOMINGIAOMINGIAOMINGIAOMING L L L L LIUIUIUIUIU, J, J, J, J, JOSEPHOSEPHOSEPHOSEPHOSEPH R. P R. P R. P R. P R. POWERSOWERSOWERSOWERSOWERS, B, B, B, B, BARRYARRYARRYARRYARRY G. S G. S G. S G. S G. SWANSONWANSONWANSONWANSONWANSON, H, H, H, H, HERBERTERBERTERBERTERBERTERBERT H. H H. H H. H H. H H. HILLILLILLILLILL, , , , , ANDANDANDANDAND S S S S STEPHANIETEPHANIETEPHANIETEPHANIETEPHANIE C C C C CLARKLARKLARKLARKLARK

ABSTRAABSTRAABSTRAABSTRAABSTRACTCTCTCTCT: : : : : The effects of high hyThe effects of high hyThe effects of high hyThe effects of high hyThe effects of high hydrdrdrdrdrostatic prostatic prostatic prostatic prostatic pressuressuressuressuressure (HHP) on flave (HHP) on flave (HHP) on flave (HHP) on flave (HHP) on flavororororor-binding pr-binding pr-binding pr-binding pr-binding properoperoperoperoperties of whey prties of whey prties of whey prties of whey prties of whey protein concentrotein concentrotein concentrotein concentrotein concentrateateateateate(((((WPC) wWPC) wWPC) wWPC) wWPC) wererererere detere detere detere detere determined with benzaldehymined with benzaldehymined with benzaldehymined with benzaldehymined with benzaldehydedededede, heptanone, heptanone, heptanone, heptanone, heptanone, octanone, octanone, octanone, octanone, octanone, and nonanone, and nonanone, and nonanone, and nonanone, and nonanone. After HHP tr. After HHP tr. After HHP tr. After HHP tr. After HHP treatment (600 MPeatment (600 MPeatment (600 MPeatment (600 MPeatment (600 MPa,a,a,a,a,50 °C, for 0-, 10-, or 30-min holding time), flav50 °C, for 0-, 10-, or 30-min holding time), flav50 °C, for 0-, 10-, or 30-min holding time), flav50 °C, for 0-, 10-, or 30-min holding time), flav50 °C, for 0-, 10-, or 30-min holding time), flavororororor-binding pr-binding pr-binding pr-binding pr-binding properoperoperoperoperties of ties of ties of ties of ties of WPC wWPC wWPC wWPC wWPC wererererere studied be studied be studied be studied be studied by intry intry intry intry intrinsic fluorinsic fluorinsic fluorinsic fluorinsic fluorescenceescenceescenceescenceescencetitrtitrtitrtitrtitration and static headspace analysisation and static headspace analysisation and static headspace analysisation and static headspace analysisation and static headspace analysis. . . . . The HHP trThe HHP trThe HHP trThe HHP trThe HHP treatments increatments increatments increatments increatments increased the number of binding sites and the appareased the number of binding sites and the appareased the number of binding sites and the appareased the number of binding sites and the appareased the number of binding sites and the appar-----ent dissociation constants of ent dissociation constants of ent dissociation constants of ent dissociation constants of ent dissociation constants of WPC for benzaldehyWPC for benzaldehyWPC for benzaldehyWPC for benzaldehyWPC for benzaldehydedededede. HHP tr. HHP tr. HHP tr. HHP tr. HHP treatment of eatment of eatment of eatment of eatment of WPC for 0 min incrWPC for 0 min incrWPC for 0 min incrWPC for 0 min incrWPC for 0 min increased the number ofeased the number ofeased the number ofeased the number ofeased the number ofbinding sites of binding sites of binding sites of binding sites of binding sites of WPC for heptanone and octanoneWPC for heptanone and octanoneWPC for heptanone and octanoneWPC for heptanone and octanoneWPC for heptanone and octanone. As obser. As obser. As obser. As obser. As observvvvved bed bed bed bed by headspace analysisy headspace analysisy headspace analysisy headspace analysisy headspace analysis, HHP tr, HHP tr, HHP tr, HHP tr, HHP treatments did noteatments did noteatments did noteatments did noteatments did notrrrrresult in significant changes in the flavesult in significant changes in the flavesult in significant changes in the flavesult in significant changes in the flavesult in significant changes in the flavor ror ror ror ror retention for benzaldehyetention for benzaldehyetention for benzaldehyetention for benzaldehyetention for benzaldehyde in de in de in de in de in WPC solutionsWPC solutionsWPC solutionsWPC solutionsWPC solutions. F. F. F. F. Flavlavlavlavlavor ror ror ror ror retention of 100 ppmetention of 100 ppmetention of 100 ppmetention of 100 ppmetention of 100 ppmand 200 ppm heptanone and octanone in HHPand 200 ppm heptanone and octanone in HHPand 200 ppm heptanone and octanone in HHPand 200 ppm heptanone and octanone in HHPand 200 ppm heptanone and octanone in HHP-tr-tr-tr-tr-treated (10 min) eated (10 min) eated (10 min) eated (10 min) eated (10 min) WPC was significantly loWPC was significantly loWPC was significantly loWPC was significantly loWPC was significantly lowwwwwer than for untrer than for untrer than for untrer than for untrer than for untreatedeatedeatedeatedeatedWPC and HHPWPC and HHPWPC and HHPWPC and HHPWPC and HHP-tr-tr-tr-tr-treated eated eated eated eated WPC for 0 min or 30 min. FWPC for 0 min or 30 min. FWPC for 0 min or 30 min. FWPC for 0 min or 30 min. FWPC for 0 min or 30 min. For flavor flavor flavor flavor flavor ror ror ror ror retention of nonanoneetention of nonanoneetention of nonanoneetention of nonanoneetention of nonanone, significant decr, significant decr, significant decr, significant decr, significant decreases weases weases weases weases wererererere onlye onlye onlye onlye onlyobserobserobserobserobservvvvved at 100 ppm when ed at 100 ppm when ed at 100 ppm when ed at 100 ppm when ed at 100 ppm when WPC solutions wWPC solutions wWPC solutions wWPC solutions wWPC solutions wererererere HHPe HHPe HHPe HHPe HHP-tr-tr-tr-tr-treated for 10 min. eated for 10 min. eated for 10 min. eated for 10 min. eated for 10 min. While use of HHP trWhile use of HHP trWhile use of HHP trWhile use of HHP trWhile use of HHP treatment of eatment of eatment of eatment of eatment of WPC hasWPC hasWPC hasWPC hasWPC haspotential in real food systems, these findings demonstrate the importance of careful selection of HHP treatmentpotential in real food systems, these findings demonstrate the importance of careful selection of HHP treatmentpotential in real food systems, these findings demonstrate the importance of careful selection of HHP treatmentpotential in real food systems, these findings demonstrate the importance of careful selection of HHP treatmentpotential in real food systems, these findings demonstrate the importance of careful selection of HHP treatmenttimes and flavor concentrations for desired outcomes in food applications.times and flavor concentrations for desired outcomes in food applications.times and flavor concentrations for desired outcomes in food applications.times and flavor concentrations for desired outcomes in food applications.times and flavor concentrations for desired outcomes in food applications.

Keywords: whey protein concentrate, high hydrostatic pressure, flavor binding, fluorescence, gas chroma-Keywords: whey protein concentrate, high hydrostatic pressure, flavor binding, fluorescence, gas chroma-Keywords: whey protein concentrate, high hydrostatic pressure, flavor binding, fluorescence, gas chroma-Keywords: whey protein concentrate, high hydrostatic pressure, flavor binding, fluorescence, gas chroma-Keywords: whey protein concentrate, high hydrostatic pressure, flavor binding, fluorescence, gas chroma-tographytographytographytographytography

Introduction

The acceptability of a food depends mainly on its sensory qual-ities and in particular on its flavor (Casimir 1998). Concentra-

tion of aroma compounds and aroma perception during eatingdepend on the nature and concentration of the volatiles present inthe food as well as on their availability for perception (Harrison1997). Availability is influenced in part by the process of eating,such as mastication, temperature, and the effect of saliva, butmainly by interactions between aroma compounds and non-vola-tile food constituents, such as fats, proteins, and carbohydrates(Bakker and others 1996). Thus, the composition of a food productgreatly influences the performance of a flavoring and therefore thesensory quality. Modifications of a food matrix require changes offlavorings to optimize their performance (Harrison and Hills 1997).

Although fat is important for sensory qualities such as flavor,color, texture, and mouthfeel, manufacturers have made it a prac-tice to substitute fat with fat replacers to create products that meetthe demands of health-conscious consumers (Casimir 1998). As fatsubstitutions are made, the flavor challenges are significantly in-creased, and aroma chemicals may be perceived as harsh and un-balanced (Hatchwell 1994).

In addition to fats, proteins belong to another important class ofcomponents in food systems that are capable of influencing flavorrelease. The market for functional protein-rich ingredients is ex-panding and is currently supplied by various proteins. Whey pro-tein concentrate (WPC) represents a potentially significant sourceof functional protein ingredients for many traditional and novelfood products. Its utilization as a flavor carrier, besides its otherproperties such as emulsifying and gelation properties, could be in-teresting for the food industry (Buhr and others 1999). It is suggest-

ed that �-lactoglobulin (�-LG), the major whey protein, could beengineered to bind and protect a wide range of volatile and unsta-ble flavors during food manufacturing or to release them in more orless controlled ways by chemical or physical modifications (Bound-aud and Dumount 1996).

High hydrostatic pressure (HHP) presents unique advantagesover both chemical and thermal processing for food product mod-ifications, including application at low temperatures, which has lit-tle effect on food quality (Knorr 1995). Studies have been done tounderstand the effect of HHP on some of the functional propertiesof WPC or isolates, such as gel formation (Famelart and others1998), emulsifying capacity (Galazka and others 1996) and foam-ability (Ìbanoglu and Karatas 2001). However, little work has beendone on the effects of HHP on flavor binding by WPC.

The objective of this research was to investigate the bindingproperties of HHP-treated and untreated WPC with selected flavorcompounds. Intrinsic fluorescence titration and static headspaceanalysis were used in the present research.

Materials and Methods

MaterialsMaterialsMaterialsMaterialsMaterialsRT-80 Grade A whey protein concentrate (WPC RT-80) was provid-

ed by Main St. Ingredients (La Crosse, Wis., U.S.A.). WPC RT-80 withthe same lot number was used throughout the experiments. Theproduct contained 84.9% protein, 3.9% fat, 3.4% ash, 3.5% lactose,and 3.7% moisture. The pH of a 0.2% solution of WPC at 20 °C was 6.4.All of the chemicals used were of analytical grade and obtained fromFisher Chemicals (Fairlawn, N.J., U.S.A.) unless otherwise specified.

Heat treatmentHeat treatmentHeat treatmentHeat treatmentHeat treatmentWPC solutions, at the protein concentration of 0.2% (w/v) in so-

dium phosphate buffer (0.01 M, pH 7.0), were heated at 50 °C for 30min. The 50 °C temperature was selected because using the sametemperature for preheating as used for HHP treatments, would

MS 20050378 Submitted 6/24/05, Revised 8/15/05, Accepted 8/25/05. AuthorsLiu, Powers, Swanson, and Clark are with Dept. of Food Science and Hu-man Nutrition, Washington State Univ., Pullman, WA 99164-6376. AuthorHill is with Dept. of Chemistry, Washington State Univ., Pullman, Wash.Direct inquiries to author Clark (E-mail: StephClark@wsu.edu).

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indicate whether this mild heat treatment causes denaturation ofthe proteins.

High-pressure treatmentHigh-pressure treatmentHigh-pressure treatmentHigh-pressure treatmentHigh-pressure treatmentWPC solutions, at concentrations of 0.2% in sodium phosphate

buffer (0.01 M, pH 7.0), were treated with HHP of 600 MPa at 50 °Cfor holding times of 0, 10, or 30 min in a warm isostatic press withyoke (Engineered Pressure Systems, Inc., Haverhill, Mass., U.S.A.)with a cylindrical pressure chamber (height = 0.25 m, dia = 0.10 m).The 50 °C temperature was selected to decrease the time requiredfor HHP treatments. Samples were equilibrated in the chamber for5 min to reach 50 °C. The come-up time (4.5 min) is the compressiontime required to reach a pressure of 600 MPa. Pressure release iswithin 30 s. Three batches of WPC solutions were used for all HHPtreatments. After exposure to high pressure, WPC solutions werestudied immediately, or stored at 4 °C for less than 1 mo.

Size exclusion chromatographySize exclusion chromatographySize exclusion chromatographySize exclusion chromatographySize exclusion chromatographyWPC solutions were filtered through a polyvinylidene difluoride

membrane (pore size 0.45 �m) and fractionated by size exclusionchromatography (SEC) on a Protein-Pack SW 300 Glass column (8× 300 mm, Waters Corp., Milford, Mass.). The elution buffer wascomposed of 0.05 M sodium phosphate (pH 7.0). The flow rate was0.5 mL/min, and the absorbance of the eluate was monitored by aWaters 440 UV/VIS detector at 280 nm, with a run time of 28 min.

Individual whey proteins in the chromatogram were identifiedby means of a calibration curve with the logarithm of the molecu-lar weight of standards as a function of the retention time. Differentstandards of lyophilized proteins were used: �-lactalbumin (�-LA,14 kDa, L-5385), �-lactoglobulin (�-LG, dimers of variants A and B,37.2 kDa, L-2506), bovine serum albumin (BSA, 66 kDa, A-2153),and bovine immunoglobulin G (IgG, 152 kDa, I-9640). Under theprocess conditions studied, linear separation with high resolutionwas possible for proteins with a molecular weight between 14 and66 kDa. The relationship between the molecular weight (Mw, in Da)of the protein and the retention time (tR, in min) within this molec-ular weight range was calculated as (r2 = 0.96):

log (Mw) = –0.159 tR + 7.688

For �-LG and �-LA, quantitative measurements were obtainedby SEC using regression lines in the concentration range 0 to 10 g/L for �-LG (r2 = 0.99), and in the concentration range 0 to 5 g/L for �-LA (r2 = 0.99). Each SEC analysis was performed in triplicate.

Fluorescence binding of flavor compoundsFluorescence binding of flavor compoundsFluorescence binding of flavor compoundsFluorescence binding of flavor compoundsFluorescence binding of flavor compoundsBenzaldehyde, heptanone, octanone, and nonanone were flavor

compounds selected to bind with WPC. Benzaldehyde has a char-acteristic almond flavor. Heptanone, octanone, and nonanone aretypical flavors developed during fermentation in yogurt.

The binding of flavor compounds to WPC was evaluated by fol-lowing the quenching of intrinsic tryptophan fluorescence (Dufourand Haertlé 1990; Marin and Relkin 1998). In the binding study, 4�L solutions of benzaldehyde, heptanone, octanone, or nanonone(125 �M in absolute ethanol) were titrated to 2 mL of the treated oruntreated or HHP-treated WPC solution (0.02%) to reach a final fla-vor concentration of 2.5 �M. In other words, 4 �L flavor solutionswere added continuously to WPC to reach a final flavor concentra-tion of 2.5 �M. Following each titration of flavor compound, thesystem was thoroughly mixed and then allowed to equilibrate for30 min before the recording of the fluorescence intensity. At theend of the titrations, the total added solvent (ethanol) volume didnot exceed 3%. Intrinsic fluorescence was measured using an exci-

tation wavelength of 295 nm and observing an emission wave-length of 350 nm. To eliminate the dilution effect upon WPC solu-tion by the added flavor solution and tryptophan fluorescencechanges induced by alcohol, a blank containing WPC solutions ti-trated with ethanol was monitored as described above. The fluores-cence intensity changes of the blank were subtracted from fluores-cence intensity measurements of the flavor/protein complexes forevery considered titration point. In all cases, tryptophan fluores-cence intensity at 350 nm was normalized to 1, and the fluorescenceintensity was expressed as arbitraty units (a.u.).

Flavor-binding properties were evaluated with the Cogan meth-od (Cogan and others1976). The numbers of accessible bindingsites and apparent dissociation constants of flavor compounds withWPC are calculated with the equation

P0� = (1/n)(L0�/(1 – �)(K�d/n),

where P0 is protein concentration, L0 is a given ligand concentration,n is the number of binding sites per molecule of protein, K�d is theapparent dissociation constant, and � is the fraction of bindingsites remaining free, assuming � = (FImax – FI)/FImax. FImax is definedas the fluorescence intensity when protein molecules are saturat-ed by flavor compounds.

Headspace analysisHeadspace analysisHeadspace analysisHeadspace analysisHeadspace analysisBenzaldehyde, heptanone, octanone and nonanone were chosen

to investigate the effects of HHP on flavor retention. Analyses weredone in triplicate in amber flasks (40 mL), closed with mininert valves(Supelco, Bellefonte, Pa., U.S.A.). Two aroma concentrations (100 and200 ppm) and 1 WPC concentration (0.2%, 5 mL) were tested for eachflavor compound. Analyzed solutions, with or without WPC, werestirred and equilibrated at 37 °C for 30 min. Vapor phase samples (1mL) were taken with a gastight syringe and injected onto a Carlo Erba8000 gas chromatograph equipped with a DB-Wax column (J & W Sci.,Folson, Calif., inner dia 0.32 mm, 30 m, film thickness = 0.5 �m).Temperature of injector and detector were 250 °C and 260 °C, respec-tively. The H2 carrier gas velocity was 1.9 mL/min.

Statistical analysisStatistical analysisStatistical analysisStatistical analysisStatistical analysisAll experiments and analyses were done in triplicate. The analysis

of variance test for significant effects of treatments and assay sam-ples were determined using the General Linear Model procedure(PROC GLM) in SAS (SAS Inst., Cary, N.C., U.S.A.). Main effect dif-ferences were considered significant at the P < 0.05 level. Meansseparations were determined by Fisher’s Least Significant Differ-ence (LSD) for multiple comparisons (SAS Inst. 1993).

Results and Discussion

Size exclusion chromatographySize exclusion chromatographySize exclusion chromatographySize exclusion chromatographySize exclusion chromatographyThe SEC chromatograms of WPC samples were characterized by

3 protein peaks (Figure 1). On the basis of whey protein standards,peaks with retention times of 22.37 ± 0.03 and 19.56 ± 0.02 min werecharacterized as the �-LA monomer with a molecular weight of 14kDa, and as the �-LG dimer with a molecular weight of 36.5 kDa,respectively. Also, IgG and BSA were present in a protein fractionwith retention time 16.98 (± 0.01) min, which corresponds to a mo-lecular weight range 66.3 to 152 kDa. IgG and BSA were not in thevoid volume, but eluted as 1 peak.

Information on the pressure sensitivity of individual whey pro-teins may be gathered by the use of SEC. Based on Figure 1b, mildheat treatments of WPC, such as 50 °C, did not result in significantprotein denaturation or aggregation in 0.01 M sodium phosphate

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buffer (pH 7.0). Increases in the amount of aggregates and decreas-es in the amounts of individual whey proteins were observed afterHHP treatments (Figure 1c). �-LG is more sensitive to HHP treat-ments than �-LA (Nakamura and others 1993), and a significantreduction in the amount of �-LG occurred after pressurization.Thirty minutes of HHP treatment of the 0.2% WPC solution in 0.01M sodium phosphate buffer resulted in a 77% reduction in theamount of �-LG (pH 7.0), and a 3% reduction in the amount of �-LA. At the same time, the amount of aggregates doubled.

Flavor compound binding fluorescenceFlavor compound binding fluorescenceFlavor compound binding fluorescenceFlavor compound binding fluorescenceFlavor compound binding fluorescenceThe binding curves for benzaldehyde, heptanone, octanone,

and nonanone are presented in Figures 2a through 2d. The fluo-rescence emission spectra of WPC solutions were studied as a func-tion of added compounds, and the observed tryptophan fluores-cence quenching, because changes of the polarity in theneighborhood of indoles (Lakowicz 1999), is indicative of the forma-tion of complexes. The addition of benzaldehyde, heptanone, oc-tanone, and nonanone to WPC solutions all produced fluorescencequenching, suggesting that these compounds bind to whey pro-teins or interfere with whey protein tryptophans. For untreated WPC,the maximum fluorescence quenching was obtained at a flavor-protein ratio of 1:5 to 1:3, which is lower than 1:1 reported for earlier�-LG and flavor-binding studies (Marin and others 1998; Guichardand Langourieux 2000). The lower number of binding sites in WPCthan in �-LG may indicate that some binding sites in whey proteinswere blocked during processing of the WPC, possibly by the forma-tion of aggregates, during filtration, evaporation, or drying.

�-LG binds structurally different molecules such as fatty acids,retinol (Diaz de Villegas and others 1987), and alkanone flavors(O’Neill and Kinsella 1987). The affinity of �-LG for a flavor com-pound or a ligand is dependent on the molecular structure of theflavor compound or ligand (Damodaran and Kinsella 1980; Reinersand others 2000). There are at least 2 distinct binding sites permonomer of �-LG for a variety of ligands (Sawyer and others 1998;Wu and others 1999). The primary hydrophobic binding site is lo-cated within the calyx, formed by 8 strands of antiparallel �–sheets,and a 2nd hydrophobic binding site lies in a cleft between the helixand an edge of the barrel (Wu and others 1999).

Marin and others (1998) studied the effect of heat treatment onthe binding property of �-LG with benzaldehyde. Although theplateau value obtained from the intrinsic fluorescence study wasreached for a 1:1 molar ratio in both untreated and heat-treated �-LG, the percentage of quenching was higher with previously heatedprotein solutions, indicating that the binding capacity of the pro-tein was increased by heating (pH 6, 75 °C, 10 min). O’Neill andKinsella (1988) reported that heating of solutions of �-LG (in 20 mMphosphate buffer, pH 7.6) at 75 °C for 10 or 20 min decreased thebinding affinity of �-LG for benzaldehyde with a concomitant in-crease in the number of low affinity, nonspecific binding sites. In

our study with benzaldehyde, HHP treatment for 30 min increasedthe number of binding sites from 0.20 to 0.36, and the apparentdissociation constant from 2.7 × 10–8 M to 4.7 × 10–8 M (Table 1),showing similarity with the heat treatment results reported byO’Neill and Kinsella (1988).

Three aliphatic methyl ketones (2-heptanone, 2-octanone, and 2-nonanone) were used to evaluate the flavor-binding properties ofWPC. Their apparent dissociation constants for WPC were 2.5 × 10–8

M, 2.2 × 10–8 M, and 1.9 × 10–8 M, respectively (Table 1). The interac-tions between �-LG and methyl ketones are hydrophobic (O’Neilland Kinsella 1987) because the affinity constants increase with in-creasing hydrophobic chain length (Sostmann and Guichard 1998).

HHP treatment of 0 min resulted in an increase in the number ofbinding sites of WPC from 0.23 to 0.39 per molecule of protein forheptanone, and from 0.21 to 0.40 for octanone (Table 1). Sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)results showed that during the come-up time (4.5 min) to reach 600MPa of HHP treatment, dissociation of aggregates occurred (Liu2005), which may have exposed more binding sites for heptanoneand octanone. However, no changes in the number of binding sitesof WPC for nonanone occurred during HHP treatments. Frapin andothers (1993) studied the interactions of fatty acids with porcine andbovine �-LG. One �-LG fatty acid binding pocket can accommodatebest an aliphatic fatty acid chain constituted by 16 carbon atoms.Apparently, the new binding site of WPC for methyl ketones gener-

Table 1—Apparent dissociation constants (K�d) and thenumber of flavor binding sites (n) of whey protein concen-trate (WPC) and WPC after high hydrostatic pressure (HHP)treatment (600 MPa and 50 °C) for come-up time (HP0, 4.5min to reach 600 MPa) and holding time of 10 or 30 min(HP10 and HP30)a

Ligands WPC n K�����d (M)

Benzaldehyde Untreated 0.20 a 2.7 × 10–8 M a

HP 0 0.20a 5.2 × 10–8 Mb HP 10 0.25a 6.2 × 10–8 Mc HP 30 0.36b 4.7 × 10–8 MbHeptanone Untreated 0.24 a 2.5 × 10–8 Ma HP 0 0.39b 1.9 × 10–8 Ma HP 10 0.27a 3.9 × 10–8 Mb HP 30 0.29a 1.8 × 10–8 MaOctanone Untreated 0.21 a 2.2 × 10–8 Ma HP 0 0.40b 1.8 × 10–8 Ma HP 10 0.25a 3.1 × 10–8 Mb HP 30 0.26a 2.0 × 10–8 MaNonanone Untreated 0.20 a 1.9 × 10–8 Ma HP 0 0.19a 2.1 × 10–8 Ma HP 10 0.22a 2.7 × 10–8 Mb HP 30 0.23a 1.8 × 10–8 MaaData are means of 3 analyses calculated using method by Cogan and others(1976). Means with different letters in the column are significantly different(P < 0.05).

Figure 1—Size exclusion chromatogra-phy chromatograms of soluble proteinsobtained from untreated, heat-treated,and high hydrostatic pressure (HHP)-treated whey protein concentrate(WPC) solutions (0.2%, pH 7.0) in 0.01 Msodium phosphate buffer. From left toright: (a) untreated WPC; (b) heat-treated WPC (50 °C, 30 min); (c) HHP-treated WPC (600 MPa, 50 °C, 30 min).1 = �-lactalbumin (�-LA); 2 = �-lactoglo-bulin (�-LG); 3 = aggregates.

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ated by HHP during 4.5 min come-up time can accommodate analiphatic chain consisting of no more than 8 carbon atoms.

HHP treatment for 10 min resulted in an increase in the apparentdissociation constants of WPC from 2.5 × 10–8 M to 3.9 × 10–8 M forheptanone, from 2.2 × 10–8 M to 3.1 × 10–8 M for octanone, and from1.9 × 10–8 M to 2.7 × 10–8 M for nonanone. The increases in the appar-ent dissociation constants of WPC for the methyl ketones may bedue to the formation of aggregates in WPC, which affects the acces-sibility of the binding sites. However, HHP treatments for 30 minresulted in decreases in the apparent dissociation constants of WPCfor heptanone, octanone, and nonanone. Dissociation constantsreturned close to the values of the untreated WPC for these 3 me-thyl ketones. It seems that additional conformational changes oc-curred during 30 min of HHP treatment, which compensated for theeffects of aggregates on the binding affinity.

Headspace analysisHeadspace analysisHeadspace analysisHeadspace analysisHeadspace analysisThe volatility of the flavor compounds decreased in the presence

of WPC (Figure 3a through 3d), mainly due to hydrophobic interac-tions between the compounds and the proteins (Guichard 2000).The current findings are consistent with other studies, showingincreases in flavor retention in the presence of individual wheyproteins (Guichard and others 2000). Androit and others (1999)reported that addition of �-LG to aqueous solutions reduced theperceived aroma intensity of methyl ketones and increased theretention of methyl ketones. Marin and others (1999) observed anincrease in the retention of benzaldehyde in �-LG solution.

The retention of flavors by WPC in decreasing order were:nonanone > octanone > heptanone > benzaldehyde for flavor con-centrations of both 100 and 200 ppm. The percentage of retentionof 200 ppm nonanone, octanone, heptanone, and benzaldehyde

Figure 3—Staticheadspace analysis offlavors (200 ppm) and(100 ppm) in wheyprotein concentrate(WPC) or high hydro-static pressure (HHP)-treated WPC (600 MPaand 50 °C) with 0, 10,or 30 min holding time(H0, H10, and H30). (a)BEN = benzaldehyde;(b) HEP = heptanone;(c) OCT = octanone; (d)NON = nonanone.

Figure 2—Fluorescencetitration curves of wheyprotein concentrate (WPC)( × ) and high hydrostaticpressure (HHP)-treated(600 MPa and 50 °C) WPCfor 0 (�), 10 (�) or 30 min(�) holding time (H0, H10,and H30) with (a) benzalde-hyde, (b) heptanone, (c)octanone, (d) nonanone.

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were 40.5%, 38.5%, 27.9%, and 18.5%, respectively. The retention of100 ppm nonanone, octanone, heptanone, and benzaldehydewere higher, at 50.7%, 38.6%, 32.2%, and 19.3%, respectively. Thepercentage of retention of benzaldehyde and the methyl ketonesis consistent with previously reported results for the retention offlavors in �-LG solutions (Charles and others 1996; Jouenne andCrouzet 1996; Roozen and Legger 1997).

For benzaldehyde, at both 100 ppm and 200 ppm, HHP treat-ments did not result in significant changes in the flavor retentionin WPC solutions (Figure 3a), although the results from the fluores-cence titration showed decreases in the dissociation constants ofWPC for benzaldehyde (Table 1). The amount of volatiles releasedto the gaseous phase is influenced by many factors of the flavorcompounds, such as vapor pressure, solubility, concentration, par-titioning of volatiles between air and water phase, and interactionswith other food constituents (Kinsella 1990; Landy and others 1996).The discrepancy may also arise from the fact that headspace sam-ples of flavor and WPC solutions were equilibrated at 37 °C insteadof room temperature, which was used for the fluorescence titrationexperiments.

Flavor retentions of 200 ppm heptanone, octanone, andnonanone in WPC solutions HHP-treated for 10 min were signifi-cantly lower than values measured for untreated WPC and WPCHHP-treated for 0 min and 30 min (Figure 3b through 3d). In con-trast, no significant differences in flavor retentions of nonanone at100 ppm were observed among the untreated WPC and HHP-treat-ed WPC solutions. The decreases in retention of the concentratedmethyl ketones in WPC solutions after 10 min of HHP treatmentare consistent with the results from the fluorescence titration (Table1). The findings demonstrate the importance of carefully selectingHHP treatment times and flavor concentrations for desired exper-imental outcomes. The decreases in retention of the methyl ke-tones in HHP-treated (10 min) WPC solutions may have beencaused by conformational changes of whey proteins or formationof aggregates, which could decrease the binding affinity of themethyl ketones for WPC.

Static headspace analysis uses a sealed system that allows equi-librium to be attained. This approach simplifies analysis, but it isdoubtful whether equilibrium is actually achieved when food is eat-en (Taylor and Linforth 1996). Time-intensity assessment of flavorrelease should provide more useful information regarding flavorperception (Bakker and others 1996). Research is currently under-way to document the potential of HHP treatment to improve theflavor-binding and flavor release properties of WPC in reduced fatice cream.

Conclusions

HHP treatments affect the flavor-binding properties of WPC, asshown by changes in the number of binding sites and the ap-

parent dissociation constants. Because the effects depend on thestructure of the flavor compounds and HHP treatment conditions,tailored research is needed to investigate the effects of HHP on dif-ferent flavor compounds and a variety of applications. Careful selec-tion of flavor concentrations and HHP treatment times will be criti-cal to determining the potential of HHP treatment on flavor-bindingand flavor release properties of WPC in a variety of food applications.

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