efecto de la extrusion sobre proteinas del suero de leche

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der with a high protein content (>90% wt/wt) normallymanufactured by ion exchange chromatography or mi-crofiltration, followed by spray drying, a well-establishedindustrial method for converting liquid feed materialsinto a dry powder form (Masters, 1987). During thespray-drying process, the liquid feed is exposed to hot

gas and evaporation takes place to yield dried particles,which are subsequently separated from the gas streamby a variety of methods. Even though possible loss inmany functional properties of WPI (notably, solubilityand gelling) occurs due to high-temperature processing,spray drying is still the preferred method for produc-ing whey protein in powder form (Morr and Ha, 1993;Foegeding et al., 2002; Anandharamakrishnan et al.,2008).

Extensive research (Dannenberg and Kessler, 1988;Roefs and De Kruif, 1994; Law and Leaver, 2000; dela Fuente et al., 2002; Havea et al., 2002; Hong andCreamer, 2002) has been done on heat-induced changesin whey proteins in model solutions as well as in wheyand milk systems under a wide range of experimentalconditions (e.g., concentration, pH, ionic strength). Itis generally recognized that in the heated mixture of-LA and -LG at neutral pH, both physical changes(e.g., ionic, van der Waals, and hydrophobic interac-tions) and chemical reactions (e.g., thioldisulfide bondscramble and exchange on the intra- and intermolecularlevels) occur, leading to denaturation and aggregation(Gezimati et al., 1997; Schokker et al., 2000; de la Fuenteet al., 2002; Foegeding et al., 2002; Havea et al., 2004;Oldfield et al., 2005). The sensitive behavior of whey

proteins upon heating is of particular interest becausewhen experimental conditions are properly controlled,protein denaturation and aggregation often result innovel materials with many potential uses (Zuniga etal., 2010).

Advances in processing technology provided optionsin applying alternative treatments of whey such as ex-trusion to produce protein-enriched food and nonfoodproducts, which has led to new and economically viableways of increasing whey utilization. By using a twin-screw extruder, Onwulata et al. (2001, 2003) success-fully incorporated whey protein (in the form of WPI)into snack products to increase the protein content

(up to 20%) and extend the nutritive value. Duringextrusion, high temperature and high shear force areapplied to produce a product with unique physical andchemical characteristics. Product characteristics of ex-trudates can vary considerably depending on the extru-sion processing conditions such as barrel temperature,die geometry, extruder type, feed composition, feedmoisture content, feed particle size, feed rate, screwconfiguration, and screw speed (Purwanti et al., 2010).For example (Onwulata et al., 2003), extrusion of WPI

at 35 or 50C increased gel strength, which was almostlost when WPI was extruded at 75 or 100C.

Despite the increased use of extrusion processingon whey proteins to create and improve the desiredfunctional properties of whey protein dispersions asstructuring agents in dairy protein-based foods, it is

still difficult to predict any structures, texture, or func-tionality resulting from a extrusion process (Purwantiet al., 2010). The available knowledge is scarce on theeffect of process parameters, including extrusion tem-perature, moisture content of the feed, and shearingspeed. Little is known about the chemical reactionstaking place during extrusion, and the structures,textures, and nutritional implications associated withthese changes (Camire, 2000; Purwanti et al., 2010;Verbeek and van den Berg, 2010) due to the diversemolecular compositions of proteins and the broad andintricate macromolecular interactions that could oc-cur during the extrusion process (Aras, 1992; AbdEl-Salam et al., 2009). Research is in the early stageon the effect of twin-screw extrusion variables on thestructural properties, functionalities, and enzymaticand nutritional activity survival of some vegetable pro-teins, such as wheat flour protein (Li and Lee, 1996; DePilli et al., 2009), soybean protein isolate (Crowe andJohnson, 2001; Chen et al., 2010), and legume stor-age proteins (Hood-Niefer and Tyler, 2010; Lazou andKrokida, 2010). We recently conducted studies (Qi andOnwulata, 2011) on the effect of extrusion temperatureon protein solubility, molecular structure, and proteinquality of extruded WPI in an effort to understand that

certain functional properties of the extrudates may becontrolled by varying the extrusion temperature.The objective of the present work was to investigate

the effect of moisture content of the feed while maintain-ing a constant extrusion temperature on protein solu-bility, protein distribution, protein structures on boththe secondary and tertiary levels, and protein quality togain further insight on the relationship between extru-sion conditions and protein structures leading to theprotein functionality and quality of the extrudates.

materIaLS anD metHODS

Materials

All reagents, including NaCl, 5,5-dithio-bis(2-ni-trobenzoic acid) (DTNB), and ninhydrin reagent usedin these studies, unless otherwise noted, were of theanalytical grade or ACS certified from Sigma-Aldrich(St. Louis, MO). Tris Ultra Pure was purchased fromICN Biomedicals Inc. (Cleveland, OH).

The WPI (Provon 190) in both frozen liquid andspray-dried powder forms used in this work was pur-

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chased from Glanbia Nutritionals Inc. (Twin Falls, ID).The components of the spray-dried WPI, according tothe manufacturers specifications, were 2.8% moisture,89.6%, protein, 2.5% fat, and 3.3% ash. The frozenliquid form was freeze-dried upon arrival and stored at4C for use as a control.

Extrusion Texturization of WPI

Extrusion texturization process for whey proteinshas been described previously by Pordesimo andOnwulata (2008). The WPI powder was extruded ina model ZSK30 twin-screw extruder (Krupp, Werner& Pfleiderer Co., Ramsey, NJ) consisting of 9-barrelzones, each with individual temperature control. Thescrew elements were selected to provide low shear at300 rpm; the screw profile was described by Onwulataet al. (1998). Feed was conveyed into the extruder witha Series 6300 digital feeder, type T-35 twin-screw volu-metric feeder (K-tron Corp., Pitman, NJ). The feedscrew speed was set at 600 rpm, corresponding to a rateof 3.50 kg/h. Texturization temperature was varied at50, 75, and 100C, and moisture content was controlledat 20, 30, 50, and 75% for each extrusion temperature.Water was added into the extruder with an electro-magnetic dosing pump (Milton Roy, Acton, MA). Atdifferent water pump settings ranging from 20 to 70(1.3 to 6 L/h), water input rates were 1.556 L/h (pumpset). Samples were collected after 25 min of processing,freeze-dried overnight in a VirTis Freeze Mobile 12XLResearch Scale Freeze Dryer (Virtis, Gardiner, NY),

and stored at 4.4C until analyzed. The experimentswere performed in triplicate.

Protein Solubility

Protein solubility was determined according to amodified Bradford method (Basch et al., 1985) using-LG as a standard. Total protein content in WPIsamples was determined using a Leco Protein Analyzer(Model FP-2000, Leco Corp., St. Joseph, MI), and thenitrogen conversion factor 6.38 was used for whey pro-teins. Average solubility (%) was obtained over triplerepeated sets of assays.

Gel Electrophoresis

Sodium dodecyl sulfate-PAGE of the WPI sampleswas carried out on a Phast System (Pharmacia, Pis-cataway, NJ) with a Phast homogeneous gel containing20% acrylamide. Protein samples at appropriate con-centrations from 5.0 mg/mL (for extrudates obtainedat 50 and 75C extrusion) to 20.0 mg/mL (for 100Cextruded samples) were solubilized in a solvent sys-

tem containing 10 mM Tris-HCl, 1 mM EDTA, 2.5%SDS, 0.01% bromophenol blue dye, pH 8.0, and 5.0%-mercaptoethanol when needed for reduction. Algel solutions were spun down to remove any insolublefractions before being loaded onto the gel. Gel wasstained with Coomassie blue dye for 15 min followed

by destaining in a solution containing 30% methanoand 10% acetic acid until the desired color density levewas reached.

Reversed-Phase HPLC

An analytical HPLC, Varian ProStar 230 (VarianPalo Alto, CA), unit equipped with a Varian UV-Vis325 ProStar detector and a binary pump was usedfor the reversed-phase (RP)-HPLC analysis. A 20-Lsample was auto-injected into a polymeric RP columncontaining a polystyrene-divinylbenzene copolymer-based packing (column length 250 mm, diameter 4.6mm, particle size 3.5 m, pore size 30 nm). Gradientelution was carried out with a mixture of 2 solventsEluant A contained 100% acetonitrile and 0.1% trifluo-roacetic acid, and eluant C contained 100% water and0.1% trifluoroacetic acid. The elution gradient was setas follows: 0 min: 30% A; 030 min: 3050% A; 3040min: 5090% A; and 5 min for column re-equilibrationat 90% A. The flow rate was 0.8 mL/min and the col-umn temperature was maintained at 30C. All HPLCruns were monitored and fractions were collected at 2wavelengths of 214 and 280 nm.

Determination of Primaryand Secondary Amine Contents

The assay method using the ninhydrin reagent solu-tion (2%; Sigma Aldrich N7285; Starcher, 2001) wasused to determine the concentration of the primary andsecondary amines in the soluble fraction of the textur-ized WPI samples at concentration of approximately0.5 mg/mL for all assay reactions. Leucine (50 M) in0.05% glacial acetic acid was used to build the standardcurve, and the absorbance was recorded at 570 nm.

Determination of Free Sulfhydryl Content

The total concentration of sulfhydryl (SH) groupsand disulfide bonds was determined according to themethod of Thannhauser et al. (1987). Extruded andheated WPI (Provon 190) samples at concentrationsfrom 20 to 100 mg/mL, depending on the solubilitywere centrifuged at 10,000 g for 30 min at roomtemperature after a 30-min vigorous vortex in 0.10 Msodium phosphate (pH 7.50) buffer containing 2.0%SDS. A total of 100 to 200 L of extract of extrudates

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(approximatey 2.0 mg/mL) was pipetted into 3 mL ofthe 1.0 mMDTNB assay soution. The reaction mixturewas incubated in the dark for 25 min at room tempera-ture (20C), and the absorbance was then recorded at412 nm against a bank of 3 mL of DTNB assay soutionand the appropriate amount of sovent. l-Cystine was

used as the standard for the determination of disufidebonds. The content of free sufhydry groups was de-termined by using Emans reagent (Eman, 1959), bymixing 100 L of extract with 100 L of assay reagent(4 mg/mL Emans reagent in 0.1 Nsodium phosphatebuffer, pH 8.0) and 5 mL of 0.1 N sodium phosphatebuffer (pH 8.0). The reaction mixture was aowed tostand for 15 min, and the absorbance was read at 412nm.

Circular Dichroism Spectroscopy

Extruded WPI sampes (approximatey 10.0 mg/mL) were initiay suspended in 33 mM sodium phos-phate buffer at pH 6.75 (50 mM ionic strength) androom temperature, and then centrifuged at 10,000 g to remove any undissoved precipitate. The soubeportion was fitered through a 0.45-m-pore regener-ated ceuose fiter and used in the far-UV circuar di-chroism (CD) experiments. Appropriate diutions weremade to obtain absorbance vaues of approximatey1.0 on the UV-visibe spectrophotometer. Successivemeasurements of 5 repetitive scans in the far UV re-gion (190250 nm) were competed with overappingsampes at 2 to 70C. A sovents for CD measurements

were first fitered through a Miipore 0.22-m porefiter (Miipore, Bierica, MA). The CD spectra wererecorded on an Aviv mode 60DS spectrophotometer(Aviv Associates, Lakewood, NJ) using cuvettes with a0.5-mm path ength and a scan time of 4.0 s/nm. The

jacketed ces were attached to a circuating constanttemperature bath. The time for equiibrating sampeswas cacuated to be 30 min for a 30C change in thebath temperature. A spectra were normaized at anabsorbance unit of 1.0 at 190 nm, corrected for soventcontributions, and expressed in reative eipticity (far-UV; mdegrees) versus waveength.

Fluorescence Spectroscopy

Fuorescence spectra were recorded with a SPEXFuoroLog-3 fuorescence spectrometer (JY Horiba,Edison, NJ) in a quartz ce with a 1-cm path ength.Temperature was changed and controed by an F-3004Petier controer. A intrinsic tryptophan (Trp) fuo-rescence experiments were carried out using the soubeportion of WPI protein sampes in 33 mM sodiumphosphate buffer (ionic strength 50 mM) at pH 6.75

at simiar concentrations as measured by UV-visibeabsorption.

An excitation waveength of 295 nm was used toavoid absorption from phenyaanine residues, and fuo-rescence emission was recorded from 300 to 450 nm.

Atomic Force Microscopy

Extruded WPI sampes, 35 mg/mL uness otherwisenoted, were vortexed with deionized water before ap-propriate diutions and appications for atomic forcemicroscopy (AFM) anaysis. Sampe aiquots of 3.0 L,diuted 1:200, were appied to a freshy ceaved musco-vite mica (Peco Internationa, Redding, CA) substrateand kept at room temperature for about 30 min. Themica surface was then rinsed with Miipore-fiteredwater (100 mL) to remove oosey bound protein anddried in the air. This procedure was repeated 4 times.The sampe was then imaged immediatey using aNanoscope IIIa controer (Veeco Metroogy Inc., SantaBarbara, CA) with a mutimode scanning probe micro-scope equipped with an E-scanner. A measurementswere carried out in the tapping mode under ambientconditions using singe-beam siicon cantiever probes.Nomina tip radius of correction was 1 to 5 nm. Particesize anaysis was accompished using Adobe PhotoshopCS2 software (Adobe Systems Inc., San Jose, CA) withthe Fovea Pro 4.0 (Reindeer Graphics Inc., Ashevie,NC) pug-in instaed.

reSuLtS anD DISCuSSIOn

Recognizing the compex and chaotic nature of theeffect of extrusion conditions combined with prob-ems associated with heterogeneous distributions oftemperature, shear, and eongation rates within theextruder die during extrusion, we made our best effortin carefuy controing a reproducibe extrusion processand coected sampes ony upon ensuring an adequateequiibrium has been estabished.

Effect of Extrusion Moisture Content

on the Protein Solubility of WPI

The tota protein content of a the extruded WPIsampes used was anayzed using a standard nitrogenanayzer based on the micro-Kjedah method. A nitro-gen conversion factor of 6.38 was used for cacuation ofwhey protein contents (AOAC, 1991). The tota proteincontent for a sampes was greater than 89%.

The Bradford assay (Dougas et a., 1982; Lonnerdaet a., 1987) has ong been used as an effective andquantitative method of determining the tota amountof soube protein in dairy sampes. In this work, we


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used laboratory-purified -LG from bovine milk (90%by SDS-PAGE) as a standard to determine the proteinsolubility of texturized WPI samples in water. The re-sults are shown in Figure 1. As reported in our previouswork (Qi and Onwulata, 2011), increasing the extru-sion temperature resulted in a significant reduction in

protein solubility, most noticeably when temperaturewas increased from 50 to 75C and the solubility de-

creased from 55.0 to 65.0% to approximately 3.0 to13.0%. When the temperature reached 100C, proteinsolubility declined to a negligible level, approximately1.0 to 3.0%.

At each extrusion temperature used in this work(50, 75, and 100C), increasing the moisture content

of the feed (Figure 1), however, appeared to cause theprotein water solubility to increase, particularly at the

Figure 1. Protein solubility (%) of whey protein isolate (WPI) in water determined by the Bradford assay method. The samples extruded at50, 75, and 100C and various moisture contents (%) used as indicated were represented by black bars. The freeze-dried WPI sample was usedas a control (white bar). The gray bar represents the pre-extruded WPI. All data were collected in triplicate and averaged.

Figure 2. Comparison of SDS-PAGE under nonreduced and reduced (with -mercaptoethanol) conditions. Lanes were loaded as follows: 1= freeze-dried whey protein isolate (WPI); 2 = pre-extruded WPI (Provon 190, Glanbia Nutritionals Inc., Twin Falls, ID); 3 = WPI extrudedat 50C and 30% moisture; 4 = WPI extruded at 50C and 50% moisture; 5 = WPI extruded at 75C and 30% moisture; 6 = WPI extruded at75C and 50% moisture.


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low (50C) and medium (75C) temperatures, withthe increase averaging approximately 5.0% for every10% increase in moisture content. For the extrudateobtained at 100C, the protein solubility was only 3.0%,even at the highest moisture content used (50%). Pastresearch on vegetable proteins (Akdogan, 1999; Chenet al., 2010) revealed a loss of protein dispersibility orsolubility for extrudates obtained at high moisture con-tents (>40%) relative to low moisture content extrusion(1030%), perhaps as a result of the increased level ofprotein denaturation when moist protein is heated, nor-mally at much higher temperature than that was usedin this work, and extruded (Cheftel et al., 1992; Ver-beek and van den Berg, 2010). Because of the relativelylow temperature (75C) used for WPI extrusion in thiswork, when the majority of the proteins (-LG and-LA) remain in their native state, the higher moisturecontent may help disperse and solubilize proteins in

the extrudates, while low moisture level can producelocalized chemical modification that would lower thesolubility, in a similar way that the solubility of ex-truded defatted lung flour was affected by moisturelevel (Campos and Aras, 1993).


on the Protein Composition of WPI Extrudates

Sodium dodecyl sulfate-PAGE techniques under bothreduced (with -mercaptoethanol) and nonreduced

conditions were applied to study changes in the proteinprofile and composition because of varying extrusionconditions, moisture contents, and temperature. Figure2 compares the gels of various extruded WPI samples.Large molecular weight species were clearly visible inthe nonreduced gel as samples migrated into the run-ning gel, indicating the formation of large protein ag-gregates during extrusion, which can be significantly orcompletely eliminated by using a reducing agent.

As expected, -LG constitutes the major fraction ofthe protein in both the pre-extruded and freeze-driedWPI (Provon 190) control samples. -Lactoglobulinand -LA were found to be approximately 65 and 35%of the total proteins for the freeze-dried WPI samplein both reduced and nonreduced gels by densitometryanalysis. The contents of -LG and -LA in the pre-extruded WPI samples were determined to be approxi-mately 60 and 28%, and 70 and 25%, in the reduced

and nonreduced gels, respectively. This corresponds to10% increase in the amount of -LG in the pre-extruded(spray-dried) WPI, an indication of heat-induced dena-turation and aggregation due to possible formation ofthe intermolecular S-S disulfide bonds formed throughthe single free Cys residue in -LG even before any fur-ther processing treatment (Daemen and van der Stege,1982; Anandharamakrishnan et al., 2008), includingheating and extrusion, as reported in our previous work(Qi and Onwulata, 2011) and in this work.

Figure 3. Analysis by HPLC of -LA (black bars) and -LG (light gray bars) content (%) in extruded whey protein isolate (WPI) as a func-tion of extrusion temperature (50, 75, or 100C) and moisture content of the feed. Control samples: pre-extruded WPI is represented by the blackbar (-LA) and gray bar (-LG), and freeze-dried WPI is represented by the gray bar (-LA) and white bar (-LG). All runs were performed3 times and the average values were taken.


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Quantitative protein content analysis was carried outby analytical RP-HPLC techniques as shown in Figure3. It must be pointed out that this analysis can only beperformed using the soluble portion of each sample forseparation and peak identification. Because of the poorsolubility of the extruded WPI samples in the HPLC

solvent, acetonitrile, the results may be only applicableto the soluble protein portion; these results neverthe-less yield useful information about the relative distribution of each protein component, particularly the 2 mainwhey proteins, -LG and -LA, and the changes theyunderwent as a function of extrusion conditions.

Figure 4. (A) Free primary and secondary amine concentration (mM) determined by ninhydrin assay method. (B) Free sulfhydryl (SH)content (mM) determined by 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB) method. The level of free SH in pure -LG was determined to be 67.4

4.5 M (2.0% wt/wt) and was used as an independent control of the method. The extruded whey protein isolate (WPI) samples obtained at50, 75, and 100C and varying moisture contents are represented by the black bars. Pre-extruded and freeze-dried WPI samples are representedin dark gray and white bars, respectively. All data were collected in triplicate with standard deviations plotted.


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Figure 5. (A) Far-UV circular dichroism (CD) spectra of -LA and -LG at room temperature, 33 mMphosphate buffer (ionic strength = 50mM), pH 6.75. Ellipticity was normalized as per residue. (B) Far-UV CD spectra of various whey protein samples at room temperature, 33 mMphosphate buffer (ionic strength = 50 mM), pH 6.75. Black solid line = pre-extruded whey protein isolate (WPI); gray solid line = freeze-driedWPI; gray dotted line = WPI extruded at 50C and 20% moisture; black dotted line = WPI extruded at 50C and 50% moisture; gray dashedline = WPI extruded at 75C and 20% moisture; black dashed line = WPI extruded at 75C and 50% moisture; gray dotted-dashed line = WPIextruded at 100C and 20% moisture; and black dotted-dashed line = WPI extruded at 100C and 50% moisture.


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In Figure 3, the content of -LG in WPI extruded at50C (a relatively low extrusion temperature) decreasedby approximately 10% (from 60 to 50 to 40%) as themoisture content was increased accordingly (from 30

to 50%), indicative of the sensitive nature of -LG asa function of moisture content. It appears that thisobserved loss of -LG at increasing moisture contentis contradictory to the results from protein solubility(Figure 1). It did, however, reflect the fact that par-tially denatured or disulfide cross-linked proteins (oftenreversible), -LG in this case, can remain soluble insolution (water in this case). The -LA content re-mained relatively unchanged with extrusion at 50C,approximately 25% for all extrusion moisture levels, asexpected. Both major proteins in the extrudates ob-tained at 75C did not change as a function of moisture,with -LA remaining at 22 to 28% (similar to that

extruded at 50C) and -LG decreasing to below 10%at all moisture levels, almost one-fifth of the value inWPI extruded at 50C. This is clearly consistent withthe protein solubility results and confirms that the lossof -LG is attributable to the overall reduction in pro-tein solubility as temperature was increased from 50to 75C due to an increase in irreversible denaturationand intermolecular disulfide bond formation. Extrusionat 100C produced extrudates containing a greatly de-

creased level of -LA (10%) and an almost undetect-able level of -LG at all moisture contents, which isstill slightly higher than the protein solubility obtainedusing Bradford assay methods (5.0%).


on the Protein Quality of WPI Extrudates

Chemical methods including DTNB and ninhydrinassays were used to measure available free SH groupsand primary and secondary amine concentrations, re-spectively, and to assess the effect of moisture contentof the feed on protein quality of the WPI extrudatesobtained with extrusion at 50, 75, and 100C. The re-sults are shown in Figure 4A and 4B. Spray dryingclearly caused losses in the levels of available primaryand secondary amines as well as free SH groups com-

pared with freeze-dried WPI. Moisture content did notappear to affect the overall protein quality as measuredby free primary and secondary amines and free SH con-centrations in the final extrudates obtained at a giventemperature. Temperature reduced the protein qualityof the extrudates overwhelmingly, as reported in ourdata published elsewhere (Qi and Onwulata, 2011).

The effect of moisture content on the free SH con-centration of the extrudates seemed to be slightly more

Figure 6. Tryptophan intrinsic fluorescence spectra of pre-extruded whey protein isolate (WPI; light gray lines) compared with the texturized WPI sample extruded at 100C and 50% moisture (black lines) as a function of increasing temperature (T) in the direction of the arrow.


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Figure 7. Maximum Trp intrinsic fluorescence of whey protein isolate (WPI) samples as a function of increasing temperature (t). (A)Relative fluorescence intensity versus temperature, and (B) maximum wavelength of relative fluorescence versus temperature. Solid black circlesand dark line = pre-extruded WPI sample; open circles and light gray line = freeze-dried WPI sample; open downward triangles and light graydotted line = WPI extruded at 50C and 30% moisture; solid downward triangles and black dotted line = WPI extruded at 50C and 50%moisture; open squares and gray dashed line = WPI extruded at 75C and 30% moisture; solid squares and dark dashed line = WPI extruded at75C and 50% moisture; open triangles and light gray dotted-dashed line = WPI extruded at 100C and 30% moisture; and solid triangles anddark dotted-dashed line = WPI extruded at 100C and 50% moisture.


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Figure 8. Atomic force microscopy (AFM) images of extruded whey protein isolate (WPI) samples obtained at varying moisture contentsand temperatures: (A) freeze-dried WPI; (B) pre-extruded WPI (Provon 190, Glanbia Nutritionals Inc., Twin Falls, ID); (C) WPI extruded a50C and 30% moisture; (D) WPI extruded at 50C and 50% moisture; (E) WPI extruded at 75C and 30% moisture; and (F) WPI extrudedat 75C and 50% moisture.


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pronounced than on the available amine concentrations,following a similar direction as the effect on solubilityalthough temperature was still a determining factor.

Effect of Extrusion Moisture Content on the

Secondary Protein Structures of WPI Extrudates

Changes in the collective secondary structures of WPIwere assessed by CD spectroscopy using pure -LA and-LG (90%) as a reference (Figure 5A). Figure 5Brepresents the far-UV CD spectra of the WPI samplesextruded at 50, 75, and 100C and various moisture con-tents of the feed compared with those of pre-extrudedand freeze-dried WPI samples at room temperature.The far-UV CD spectrum of -LA at pH 6.75 (Fig-ure 5A) exemplifies a protein containing a primarily-helical structure with double negative CD peaks at206 nm and 222 nm, and that of -LG indicates a-sheet-dominant protein with a broad band spanningin the area from 208 to 222 nm. The far-UV CD spectraof pre-extruded and freeze-dried WPI approximate anappropriate mixture of -LA and -LG with doublenegative peaks at 205 nm and 222 nm, similar but lesswell defined (especially at 222 nm) than that of pure-LA. Extrusion at 50C (dotted lines) induced fewchanges in the secondary structure of WPI relative tothe pre-extruded WPI. Reduction of ellipticity at 222nm along with the simultaneous increase in magnitudeand blue shift of the negative peak at 205 nm (for pre-extruded WPI) to 201 nm in the WPI sample extrudedat 75C (dashed lines) indicated significant loss in

protein secondary structural content. The far-UV CDspectra of the extrudates at 100C extrusion (dotted-dashed lines), with its almost nonexistent 222 nm peakand even larger negative blue shifted to approximately199 nm (from 205 nm), manifested a protein with al-most complete random coil structure.

At each extrusion temperature (50, 75, and 100C),varying moisture content did not seem to exert a sig-nificant effect on the protein secondary structures ofthe WPI extrudates, although high moisture extrusion(50%) can somewhat prevent the loss of the secondarystructural elements compared with low moisture extru-sion of WPI.

Effect of Extrusion Moisture Content on the

Tertiary Structures of WPI Extrudates

Six tryptophan residues are present in the 2 majorwhey proteins, 4 for -LA and 2 for -LG, making itpossible to use intrinsic Trp fluorescence to monitorthe effect of extrusion on the tertiary structural levelof WPI. In this work, we applied heat as an externalsource of perturbation to study changes in the environ-

ment of the Trp residues of the extrudates obtainedat varying temperatures and moisture contents. Fig-ure 6 shows changes in the Trp fluorescence spectraof WPI sample extruded at 100C and 50% moisture(black lines) as a function of increasing temperature(5 to 75C) compared with those of pre-extruded WPI

sample (light gray lines).First, the intensity of the Trp fluorescence was

greatly reduced at a given temperature (Figure 6) forthe extruded sample than for the pre-extruded WPIsample, coupled with much longer wavelength andbroader emission peak at 350 nm versus 336 nm (forProvon 190), suggesting an increased level of mobilityand solvent exposure of the Trp residues. Second, in-creasing temperature caused severe loss in the intensityof maximum fluorescence emission peak for both sam-ples, which was more drastic for the pre-extruded WPIsample than for the extruded WPI. Third, increasingtemperature caused the maximum fluorescence emis-sion peak position to move to a longer wavelength (redshift), from 336 nm to 360 nm for pre-extruded WPIand from 350 nm to 360 nm for the extrudate, indicat-ing near-complete loss of tertiary structural contacts inthe Trp residues for both samples.

The detailed analysis of the Trp fluorescence as afunction of temperature for the extrudates obtained atvarying extrusion moisture content is shown in Figure7A and 7B. Clearly, the WPI samples extruded at100C and moisture contents at 30 and 50% showedthe least amount of residual tertiary structural contactscompared with the extrudates obtained at 50C (30 and

50%) and 75C (30 and 50%), and thus underwent theleast amount of further loss as a function of tempera-ture in our denaturation experiments using heat as aperturbation. High extrusion moisture content of thefeed (50%) appeared to accentuate temperature-inducedloss in the tertiary structural contacts, compared withthe lower moisture content (30%)

Atomic Force Microscopy

In this study, AFM was used to visualize the particu-late WPI aggregates to gain insights into how extrusionmoisture content of the feed has changed the shape

and distribution of these aggregates using freeze-driedand pre-extruded WPI as references, see Figure 8. Thetopography of heat-induced aggregation of -LG andWPI at pH 2 to 7 was investigated by Ikada and Morris(2002) using AFM. It was found that fine-stranded ag-gregates were formed at pH 2, the diameter of strandsbeing approximately 10 nm for WPI, and at pH 7, ag-gregates were mainly composed of ellipsoidal particles,which is in close agreement with what was observed forfreeze-dried WPI (native whey protein aggregates) in


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our AFM studies (Figure 8A), with approximately 40%of particles measuring 10 nm and 30% measuring 4 to5 nm. Spray drying caused the formation of larger par-ticles, almost doubled in diameter, but fewer were seen(Figure 8B). Low temperature (50C) and low mois-ture extrusion (30%, Figure 8C) decreased both the

size and the number of these larger particles comparedwith high moisture (Figure 8D), which is in agreementwith the results from protein solubility experiments.Extrusion texturization at medium temperature (75C;Figure 8E and 8F) and high moisture, on the otherhand, produced more densely packed and uniform par-ticles that were larger, approximately 20 to 25 nm. Theextrudate obtained at the same temperature but lowmoisture produced similarly sized and shaped particles,although less densely populated. A closer examinationof this image revealed bead-like and necklace-like struc-tures among larger and smaller particles, presumablythrough the formation of S-S bonds of the buildingunits. The combined effects of heat level and shearingduring extrusion cooking appear to have produced aunique class of particulate aggregates and may haveimplications for potential use as a biomaterial.

Taken together with our results on solubility, compo-sition, protein quality, and molecular structures, AFMprovided us with a convenient and useful tool for directvisualization and allowed us to gain insight on the ef-fects of extrusion temperature and moisture content onthe properties of final WPI extrudates.

COnCLuSIOnS

Unlike temperature (Qi and Onwulata, 2011), ex-trusion moisture content showed an attenuated effecton protein solubility, protein quality, and molecularstructure. At a constant extrusion temperature, thehigh moisture content of the feed could provide limitedprotection for WPI extrudates against losses in proteinwater solubility, quality, and molecular structure causedby the multiple parameters involved in the complexextrusion process.

aCKnOWLeDGmentS

The authors greatly appreciate the technical assis-tance of Edward D. Wickham (Wyndmoor, ERRC, ARS,USDA) in performing protein solubility, RP-HPLC,CD, fluorescence spectroscopy, and all chemical assayexperiments. We thank Winnie Ye (Wyndmoor, ERRC,ARS, USDA) for her work on AFM, and acknowledgeZerlina Muir (Wyndmoor, ERRC, ARS, USDA) for hertechnical assistance in running gel electrophoresis.

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