Properties of Whey Protein Isolates Extruded under Acidic and Alkaline Conditions
J.Dairy Sci.89:71–81
?American Dairy Science Association,2006.
Properties of Whey Protein Isolates Extruded under Acidic and Alkaline Conditions1
C.I.Onwulata,*2S.Isobe,?P.M.Tomasula,*and P.H.Cooke*
*USDA-ARS,Eastern Regional Research Center,600East Mermaid Lane,Wyndmoor,PA19038
?Food Engineering Division,National Food Research Institute,Tsukuba,Ibaraki,Japan
ABSTRACT
Whey proteins have wide acceptance and use in many products due to their bene?cial nutritional properties. To further increase the amount of whey protein isolates (WPI)that may be added to products such as extruded snacks and meats,texturization of WPI is necessary. Texturization changes the folding of globular proteins
to improve interaction with other ingredients and create new functional ingredients.In this study,WPI pastes (60%solids)were extruded in a twin-screw extruder at 100°C with4pH-adjusted water streams:acidic(pH2.0±0.2)and alkaline(pH12.4±0.4)streams from2N HCl and2N NaOH,respectively,and acidic(pH2.5±0.2)and alkaline(pH11.5±0.4)electrolyzed water streams;these were compared with WPI extruded with deionized water.The effects of water acidity on WPI solubility at pH7,color,microstructure,Rapid Visco Analyzer pasting properties,and physical structure were determined.Alkaline conditions increased insolubility caused yellowing and increased pasting properties sig-
ni?cantly.Acidic conditions increased solubility and de-creased WPI pasting properties.Subtle structural changes occurred under acidic conditions,but were more pronounced under alkaline conditions.Overall,alkaline conditions increased denaturation in the extruded WPI resulting in stringy texturized WPI products,which could be used in meat applications.
Key words:pH,extrusion,whey protein isolates,elec-trolyzed water
INTRODUCTION
Extruders provide mechanical and thermal energy for mixing,cooking,melting,and forming biomaterials.A demonstrated bene?t of extrusion processing is the abil-Received June28,2005.
Accepted September14,2005.
1Mention of trade names or commercial products in this publication
is solely for the purpose of providing speci?c information and does not imply recommendation or endorsement by U.S.Department of Agriculture.
2Corresponding author:conwulata@https://www.wendangku.net/doc/5810323362.html,
71ity to change the molecular structure of food ingredients (Batterman-Azcona and Hamaker,1998).Because ex-trusion processing offers versatility in forming materials for various functional uses,there has been a steady in-crease in food products created by this technology.These products include breakfast cereals,snacks,meal replace-ment bars,and confectionery candies(Breitenbach, 2002).
The use of extrusion to impart?brous texture to plant proteins for use as meat extenders has been practiced for many years(Atkinson,1970).Rhee et al.(1981)re-viewed the processes for texturization of soy proteins. High moisture(≥60weight%)and temperatures above 150°C are needed for texturizing and forming?brous structures by extrusion from soy isolates(Kitabatake et al.,1985).Extrusion shear-induced?brous networks are formed through formation of disul?de bonds,and cross-linking of protein chains through amide bonds between free-carboxyl and amino side groups(Harper,1981).Ad-justment of pH through acid or alkali treatment in?u-ences conformation,molecular interactions of proteins, and development of structure in soy proteins(Dahl and Villota,1991).Whey proteins can be modi?ed using chemicals,heat,or shear in extrusion processes.Chemi-cal treatment alone alters the reactive groups of the amino acids,resulting in changes in the noncovalent forces that in?uence conformation,such as van der Waals forces,electrostatic interactions,hydrophobic in-teractions,and hydrogen bonding(Kester and Richard-son,1984).
For whey proteins,texturization is more dif?cult to accomplish.Whey proteins are degraded by prolonged heat treatment above140°C(Walstra et al.,1999).Ther-mal denaturation of the2major protein fractions in whey protein isolates,β-lactoglobulin(50%)andα-lactalbumin (22%),takes place between50and75°C and is accompa-nied by unfolding and unmasking of the SH groups(Lin-den and Lorient,1999).When the whey proteins are denatured,they become insoluble,and aggregate(Wals-tra et al.,1999).The extent of denaturation is deter-mined by proportion of protein insoluble at pH7and depends on heating temperature,time,and the pH of whey at heating(Ennis and Mulvihill,2000).Moreover, adjusting the acidity(H+)or alkalinity(OH?)of whey
ONWULATA ET AL. 72
proteins while heating increases loss of solubility,and denaturation(Harwalker,1979).However,during tex-turization in the extruder,insoluble cross-linked protein aggregates are aligned by?ow and complex shearing action of the extruder screws(Queguiner et al.,1992). Heating and shear alter the conformational structure of the whey protein through partial denaturation of the protein,thereby exposing groups that are normally con-cealed in the native protein(Kim and Maga,1987).By varying extrusion temperatures and denaturing whey proteins at temperatures below100°C,various textured protein products were created that retained their physi-cal functionality such as foaming and digestibility(Hale et al.,2002;Onwulata et al.,2003;Onwulata and Toma-sula,2004).Differences in the interaction of denatured whey depending on pH have been reported in milk(An-ema et al.,2004).Anema et al.(2004)showed,for in-stance,that at pH6.5,about70%of the denaturedβ-LG andα-LA were associated with casein,whereas at pH6.7,only about30%were associated with casein. Electrolyzed water has been used as an adjunct for enhanced food quality and safety in Japan,and its use has been reported to improve kneading quality of wheat ?our and dough alone without using other food additives (Kato et al.,2001).Improvement in the textural proper-ties of Japanese rice cooked with electrolyzed water(pH 9to10)was also reported by the same authors.Kobay-ashi et al.(1996)reported increased volume and texture, as measured by the adhesiveness and hardness ratio, signifying the effect of pH on protein structure and func-tion.Therefore,the use of electrolyzed water in affecting structural changes in proteins was worth investigating. Because small shifts in pH affect milk protein structure, this study was conducted to determine the effect of pH with extrusion shear on texturization of whey protein isolates and the resulting effects on physical properties of the extrudates.
MATERIALS AND METHODS
Materials
Whey protein isolate(WPI)Provon190purchased from Glanbia Ingredients(Glanbia Foods Inc.,Rich?eld, ID)was used for this work.Electrolyzed water for the acid or alkaline extrudates was generated with an Amano800electrolyzer(Amano USA Inc.,Roseland,NJ) at pH of2.0±0.2and11.5±0.2,respectively,for compari-son studies.Nonelectrolyzed pH-adjusted water was also prepared.For acid,water was adjusted to pH of1.4±0.2with2N HCl,and alkali to pH12.5±0.2with2N NaOH.Electrolyzed water was generated by electrolysis of sodium chloride solution producing low pH water at the anode(HCl)and hypochlorous(OCl?)at the cathode. Acidic electrolyzed water(AcEW)was generated using Journal of Dairy Science Vol.89No.1,2006a?ow type electrolysis apparatus(ROX-20TA,Hoshizaki Electric Co.Ltd.,Toyoake,Aichi,Japan).The current passing through the electrolysis apparatus was set at 16A,and the voltage between the electrodes was set at 18V.Acidic electrolyzed water(40ppm free available chlorine)was prepared within the anode compartment of an electrolytic cell and alkaline electrolyzed water (AlEW)was prepared within the cathode compartment. The properties of each solution were determined,includ-ing pH and free available chlorine concentration.The pH of the tested solution was measured with a pH meter (HM-11P,Toa Electronics Ltd.,Tokyo,Japan).Within 1h,the initial concentration of the free available chlorine was determined with EPA-approved chlorine test kits (Hach Co.,Loveland,CO).Estimated pH values were 2.6±0.1,11.4±0.1,and6.8±0.1for AcEW,AlEW, and deionized water(DW),respectively.Free available chlorine concentration of AcEW and chlorinated water was40.3±1.5ppm.
Extrusion Processing
The screw pro?le of the Krupf Werner P?eiderer ZSK30twin-screw extruder used for this study was re-ported earlier(Onwulata et al.,2001).The screw speed of the extruder was maintained at200rpm.The screw elements were selected to provide low shear at200rpm. The barrel temperature pro?le for extrusion was35,35, 35,50,100,100,100,100,and100°C,from the feed section to the die.Water input to the extruder was at the rate of23g/min.The pH-adjusted water was injected with an electromagnetic dosing pump(Milton Roy,Ac-ton,MA)to bring the moisture content of the feed to approximately20g of H2O/100g of mixture(wet basis). Feed rate of WPI was35g/min and WPI extrudates assayed were coded as follows:NEXT=nonextruded WPI(control);HACI=WPI extruded at pH2.0±0.2(pH was adjusted with2N HCl);NEUT(extrudate control)= WPI extruded at pH6.8±0.2with DW;EACI=WPI extruded at pH2.5±0.4with AcEW;EALK=WPI extruded at pH11.5±0.2with AlEW;and NALK= WPI extruded at pH11.5±0.4with2N NaOH.Extruded WPI samples were collected,freeze-dried in a laboratory freeze dryer for5min,and stored at4.4°C until analyzed. Moisture content was measured using a vacuum oven (method#925.09;AOAC,1998).
Buffering
To understand the buffering effect of the WPI,nonex-truded pastes of WPI and pH-adjusted water streams were made by adding a speci?ed acid or alkali to deion-ized water,and the pastes were made using a Hobart commercial mixer(The Hobart Mfg.Co.,Troy,OH).Five
EXTRUDED WHEY PROTEIN ISOLATES
73
different pH water streams were prepared as follows:pH 2.6adjusted with 1N HC1;pH 1.4adjusted with 2N HCl;pH 6.8,deionized water;pH 11.6adjusted with 1N NaOH;and pH 13adjusted with 2N NaOH.Whey protein isolate pastes were made at the ratio of 60g of WPI/40mL of the appropriate pH-adjusted water stream,and pH was measured every 15s for 2min.The maximum residence time of pH-adjusted pastes in the extruder was recorded.Solubility
Solubility of extruded WPI was determined with a 1.0-g sample of extrudate stirred with 90mL of deionized water at 125rpm and pH 7.0for 2h.Samples were then centrifuged at 5,000×g for 20min.The supernatant was freeze-dried overnight.The nitrogen/protein conversion factor was 6.38and percentage protein solubility was calculated as described by Kilara (1984).Insolubility or denaturation was calculated as (100?solubility).Color
Tristimulus color measurements of WPI extrudates were measured instrumentally with a Hunter Labora-tory (ColorQuest XE model)infrared spectrophotometer (Hunter Associates Laboratory,Inc.,Reston,VA).Extru-dates were scanned through a small-angle port (2°).Hunter L*,a*,b*values were used to calculate total color difference (ΔE),de?ned as the square root of (L 2+a 2+b 2).Standard reference tile values were L =93.5;a =?0.89;and b =1.01,where L =lightness,a =redness,and b =yellowness.
Rapid Visco Analyzer Pasting Viscosity
Post-extrusion,pastes of texturized extrudates were made as described,to determine their viscosity.Analysis of the pasting behavior was conducted with a Rapid Visco Analyzer (RVA ;model RVA-3D,Foss North America,Eden Prairie,MN)equipped with Thermocline for Win-dows software.Pasting properties,a measure of WPI paste viscosity,were determined by RVA Application Method No.48,using a 28-g specimen,13.5%wet basis (Parkes et al.,1998).
Stereo Fluorescence and Confocal Microscopy Short (1cm)segments of extruded ribbons were cut perpendicular to the axis of extrusion with a stainless steel razor blade by hand and sectioned surfaces were illuminated with either white light from a ?ber-optic illuminator (model 190,Dolan-Jenner Industries,Inc.,Woburn,MA)or violet light (425to 440nm)from a high-
Journal of Dairy Science Vol.89No.1,2006
pressure Hg lamp.Images were obtained with a stereo ?uorescence microscope,equipped with a color CCD and digital imaging hardware and software (model MZ FLIII,Leica Microsystems,Inc.,Bannockburn,IL).For confocal microscopy,the cut faces of ribbon segments were ap-plied to the glass bottom of microwell dishes (MatTek Corp.,Ashland,MA)and illuminated with blue light (488nm)from an argon laser.Maximum projection images of green auto?uorescence (510to 550nm)within approxi-mately 30?m-thick stacks of optical sections were col-lected using a 10×lens mounted in an optical microscope integrated with a spectrophotometric confocal micro-scope system (model IRBE,Leica Microsystems,Inc.,Exton,PA).The distribution of intensity in digital im-ages was analyzed using Fovea Pro3.0plug-ins (Rein-deer Graphics,Asheville,NC)with Photoshop v7.0(Adobe Systems Inc.,San Jose,CA).Transmission Electron Microscopy
Small segments of extruded ribbons in glutaraldehyde ?xative solution were trimmed into small blocks (～1×2mm)and washed in 0.1M imidazole buffer.Then,the blocks were immersed in 2%osmium tetroxide solution buffered with imidazole buffer for 2h,washed in distilled water,and gradually dehydrated in a series of ethanol solutions.After several changes of absolute ethanol,blocks were immersed in propylene oxide and trans-ferred to a 1:1mixture of propylene oxide and epoxy resin mixture and slowly agitated to promote in?ltration of the plastic into the sample blocks.Following in?ltra-tion,the blocks were transferred to epoxy resin mixture and cured for 2d at 55°C.Thin sections of the cured blocks were stained with solutions of uranyl acetate and lead citrate,and then examined in the bright-?eld im-aging mode using a electron microscope (model CM12,FEI Co.,Inc.,Hillsboro,OR).Images were recorded on photographic ?lm at an instrumental magni?cation of 45,000×.
RESULTS AND DISCUSSION
The buffering capacities of WPI pastes under different pH conditions are shown in Figure https://www.wendangku.net/doc/5810323362.html,ing the same amount of WPI (60g)and pH-adjusted water (40mL),a paste with the same consistency as the paste that leaves the extruder was created and the buffering time depended on the initial https://www.wendangku.net/doc/5810323362.html,paratively,1M HCl of lower molarity weaker acids or bases took a shorter time,15to 30s,to buffer whereas high molarity acids and bases took a little longer,30to 45s.This time is within the range of the average residence time of materials in the barrels of the extruder (30to 75s).The buffering effect is seen with both acid and alkali;although the
ONWULATA ET AL.
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Figure 1.The buffering properties of extruded whey protein isolates adjusted with 5different pH-adjusted waters as follows:1N HCl (pH 2.62),2N HCl (pH 1.4),deionized water (pH 7.2),1N NaOH (pH 11.6),and 2N NaOH (pH 13.2).
materials start with different pH conditions,they end up close to the normal pH of WPI,pH 6.2±0.2.The short time (<30s)to complete buffering means that all reactions are carried out within the extruder,allowing for maximum short-term effect with minimal long-term alteration of the proteins.Buffering capacity of milk is due to the presence of protein and salts (Walstra et al.1999).In general,whey proteins have maximum buff-ering capacity between pH 3and 4for proteins with acidic residues and pH >8for those with alkaline resi-dues (Salaun et al.,2005).For this experiment,we used acid-precipitated whey proteins,which reportedly have higher buffering capacity due to higher inorganic min-eral content,particularly phosphates.Heat treatment of whey proteins has been shown to increase buffering capacity (Lucey et al.,1993).The combination of shear and heat treatment explains the short buffering times observed in this study.
There was a signi?cant (P <0.05)difference in solubil-ity between nonextruded pastes (NEXT)and the pH-adjusted extrudates.The solubility of extruded WPI was affected by pH of the water (Table 1).There was a slight increase (～18to 20%)in solubility associated with acid extrudates over the extruded control (NEUT).The differ-ence between EACI and HACI extrudates was not sig-ni?cant,but both were higher in solubility than NEUT.The electrolyzed alkali extrudates were similar to the control,but signi?cantly more denatured than the other pH-adjusted products.
The effect of pH on yellowness values and total color difference (ΔE)of WPI extrudates is shown in Table 1.
Journal of Dairy Science Vol.89No.1,2006
On the color scale,the b coordinate is a measure of yellowness.All pH-adjusted extruded samples were sig-ni?cantly (P <0.05)more yellow and darker than the NEXT paste.The yellowness order was NEXT (pH 6.8) Table 1.The effect of pH of extruded whey protein isolates (WPI)on the solubility and color of WPI Color Extrusion Solubility Color difference 2condition 1 (%)(b)(ΔE)EACI (pH 2.5)22.3b 10.9b 89.9b HACI (pH 1.4)21.8b 9.1bc 89.8b NEUT (pH 6.8)18.4c 9.1bc 86.9bc EALK (pH 11.5)18.6c 12.1b 90.3b NALK (pH 13.2)15.3d 15.7a 73.8c NEXT (pH 6.8) 73.5a 6.7c 94.7a a–d Means within a column with the same letter are not signi?cantly different (P <0.05).1 EACI =WPI Extruded at pH 2.5with electrolyzed acid water;HACI =WPI extruded at pH 1.4with 2N HCl-adjusted water;NEUT =WPI extruded at pH 6.8with deionized water;EALK =WPI extruded at pH 11.5with electrolyzed alkali water;NALK =WPI extruded at pH 13.2with 2M NaOH-adjusted water;NEXT =nonextruded WPI paste.2 ΔE is the total color difference. EXTRUDED WHEY PROTEIN ISOLATES 75 Figure 2.Photograph showing the effect of pH on extruded whey protein isolates.A)HACI (extruded at pH 1.4with electrolyzed acid water);B)NEUT (extruded at pH 6.8with deionized water);and C)NALK (extruded at pH 13.2with 2N NaOH-adjusted water). color difference of EALK extrudate was brighter than NALK,indicating less effect.Yellowness may be the ef-fect of ammonium sul?de,olfactorally perceptible from NALK extrudates. Rapid Visco analysis is a time-temperature pro?le or pasting viscogram of a material that show the points of Journal of Dairy Science Vol.89No.1,2006 Table 2.The effect of pH of whey protein isolates (WPI)on the Rapid Visco Analyzer pasting properties of extruded WPI Peak Final Setback Extrusion viscosity viscosity viscosity condition 1 (cP)(cP)(cP)EACI (pH 2.5)34d 23b 14b HACI (pH 1.4)108b 268a 171a NEUT (pH 6.8)96bc 263a 175a EALK (pH 11.5)128b 320a 202a NALK (pH 13.2) 208a 381a 186a a–d Means within a column with the same letter are not signi?cantly different (P <0.05).1 EACI =WPI Extruded at pH 2.5with electrolyzed acid water;HACI =WPI extruded at pH 1.4with 2N HCL-adjusted water;NEUT =WPI extruded at pH 6.8with deionized water;EALK =WPI extruded at pH 11.5with electrolyzed alkali water;NALK =WPI extruded at pH 13.2with 2M NaOH-adjusted water;NEXT =nonextruded WPI paste. peak,?nal,and setback viscosities (Almeida-Dominguez et al.,1994).Peak viscosity is the ?rst peak in the swell-ing region that characterizes viscous properties.Pasting properties reveal important information about a prod-uct’s degree of denaturation,and the effect of processing conditions on degree of denaturation (DesRochers,1998).The RVA peak viscosities for gels of pH-adjusted textur-ized WPI (Table 2)show the NALK extrudate to be sig-ni?cantly more viscous than the rest of the samples.The rest of the samples were similar to the neutral extrudate,but EACI was less viscous than its control (NEUT).Final and setback viscosities were similar for all extrudates except EACI,which was signi?cantly lower (P <0.05).The effect of pH-adjustment on protein pasting proper-ties depended on the molar strength of the solution used to adjust pH. Differences in structure of the WPI extrudates pro-duced under different pH conditions were probed by mac-roscopic and microscopic imaging.Cross-sections of ex-truded ribbons illustrate several differences,examined by stereomicroscopy (Figure 3,panels A,B and C).At pH 1.4,the ribbons were narrow and dry,and frequently split horizontally along the midline,while the outer edges were smooth.At neutral pH 6.8,the ribbons were slightly thicker and moist with rough or ?uted edges;and at alkaline pH,the ribbons were thickest,moist,and the edges were irregular on a submillimeter scale.When the edges of the ribbons were examined by ?uo-rescence confocal microscopy,the differences in struc-ture were apparent,especially at the edges.Following extrusion,the ribbons were immersed in a glutaralde-hyde solution;the reaction of glutaraldehyde with pro-tein typically produces auto?uorescence in the visible light spectrum,which can be imaged with a confocal laser scanning microscope or other epi?uorescence im-aging system.When excited with blue light (488nm)from an argon laser,the protein-glutaraldehyde com- ONWULATA ET AL. 76 Figure 3.Two sets of images illustrating the effects of pH on extruded whey protein isolate ribbons.A (HACI;extruded at pH 1.4with electrolyzed acid water),B (NEUT;extruded at pH 6.8with deionized water),and C (NALK;extruded at pH 13.2with 2N NaOH-adjusted water)are cross-sections of extruded ribbons imaged by re?ected light illustrating general features and variations in color following ?xation in glutaraldehyde solution;D (HACI),E (NEUT),and F (NALK)are confocal microscope images including edges of the cross sections demonstrating the differences in breadth of the dense bands and granularities that correspond to intense auto?uorescence. plexes in the WPI extrudates emitted visible ?uores-cence extending from 500to >600nm.When maximum projection images at the cross-sectioned edges of the rib-bons were made,distinct differences in the distribution of ?uorescence were apparent,depending on the pH of Journal of Dairy Science Vol.89No.1,2006 the extrusion process.At pH 1.4,a narrow region of the edge (<250?m)was most intense,but the whole edge area was composed of numerous irregular,?uorescent granules,with dimensions up to about 50?m in diameter (Figure 3D).Similarly,ribbons extruded at neutral pH EXTRUDED WHEY PROTEIN ISOLATES 77 Figure 4.Plots of the horizontally averaged relative intensities in Figure 3D,E and F,showing the edge effects at the different pH values. also had a thin bright edge,including the super?cial irregularities (<250?m)but the matrix of the ribbon contained nearly uniform ?uorescence that was not re-solved in the form of granules compared with the acid pH ribbons (Figure 3E).Notably,at alkaline pH,the increased ?uorescence intensity at the edge was much deeper or broader than that of the others,extending more than 500?m from the edges,and the intensity decreased gradually,rather than abruptly as in the acid and neutral pH samples (Figure 3F).For comparison,pro?les of the ?uorescence intensities from each type of ribbon were made by averaging the intensity along the horizontal axis of each image,and the computed line pro?les for the maximum projection images are illus-trated in Figure 4.The comparison reveals that at acid and neutral pH,the intensities of ?uorescence are maxi-mal at short distances (<250?m)from the edge,and they drop sharply to a baseline level.The intensity peak at alkaline pH also occurs at a depth of less than 250?m,but the intensity does not drop rapidly to low levels;instead,?uorescence extends at more than one-half the peak height to depths of more than 500?m into the matrix of the ribbon.These results suggest that the pH conditions of extrusion affect the aggregation and confor-mation of the WPI proteins in the ribbons,as expressed by a differential reaction with glutaraldehyde,producing different patterns of auto?uorescence.Averaged intensi-ties along the horizontal axes of the images are compared in Figure 4.A maximum projection image of the ?uores-cent surface of the 3extrudates shows different degrees of aggregation,and that HACI extrudates were most aggregated (see Figure 3D).The HACI extrudates were aggregated into the most discrete,granular-shaped ?u-orescent material,delineated by narrow spaces with no Journal of Dairy Science Vol.89No.1,2006 ?uorescence.The NEUT extrudates had the most contin-uous,and least well-de?ned ?uorescence (see Figure 3E)and in the alkaline extrudates,the ?uorescent material was aggregated into particles or clumps (Figure 3F).Compared with NEXT (pH 6.8;Figure 5A)and NEUT (Figure 5B)pastes,the folding of the acid and alkali samples are more extensive.The ?uorescence images of the surfaces of HACI and NALK extrudates (Figures 5C and 5D)show different folding patterns or aggregation from the control extrudates.The NALK extrudate has large folds,whereas the HACI folds are smaller with spaces in the matrix.The increased folding with HACI and NALK extrudates suggests greater insolubility or denaturation.This was con?rmed by the solubility anal-ysis (Table 1).Under acid conditions,the auto?uores-cence was clustered into the form of small,well-de?ned particles ranging from less than 1to 2?m in diameter and under neutral conditions the distribution of inten-sity was similar.Alkaline conditions produced broader distribution of sizes and less well-de?ned shapes of ?uo-rescent aggregates. The pH of the electrolyzed water created the visible differences in the organization of the ?ne structures of products extruded with electrolyzed water and may have been the result of electrolysis products.The auto?uores-cence from the 2electrolyzed water extrudates,EACI and EALK,showed different degrees of aggregation,with acid extrudates being more aggregated (Figure 6A)than the alkali extrudates (Figure 6B).These images show the ?uorescence variation due to degree of aggrega-tion and differences in clumping.Extruded samples con-tain dense aggregates of material with irregular shapes and sizes,interspersed with electron-lucent areas with smaller aggregates (Figure 7).Ultrastructure in the dense clumps resembles the ?ne granularity (5nm)found in the fully denatured materials examined pre-viously (Onwulata et al.,2003).Control samples (NEXT and NEUT)did not contain the irregular dense clumps of material;instead the ultrastructure is a uniform dis-persion of loosely packed small clumps,containing very ?ne granularity (5nm)unlike the long periodicity (200to 300nm)found in the fully hydrated “gel”.When exam-ined by transmission electron microscopy of stained thin sections,nonextruded WPI paste was homogeneous,con-sisting of ?ne granules around 5nm in diameter and short threads about 15to 25nm long (Figure 7A).At neutral pH (Figure 7B),the ultrastructure of the extru-date contained regular,circular or ellipsoidal electron-dense aggregates ranging from under 100to over 300nm in diameter,and the aggregates were separated by less electron-dense areas containing randomly oriented threads,around 5nm wide and 100nm long in the interstices that merge with the surfaces of the close-packed aggregates.At acid and alkaline pH (Figure 7C ONWULATA ET AL. 78 Figure 5.Confocal ?uorescence images illustrating the effects of pH on the microstructure of whey protein isolate (WPI)ribbons:A)diffuse ?uorescence in NEXT (nonextruded WPI paste);B)diffuse clusters of ?uorescence in NEUT (WPI extruded at pH 6.8with deionized water);C)and D)well de?ned,cumulative clusters of ?uorescence in HACI (WPI extruded at pH 1.4with 2N HCl-adjusted water)and NALK (WPI extruded at pH 13.2with 2N NaOH-adjusted water),respectively. and D),the ultrastructure of extrudates contained very large,irregular electron-dense aggregates,separated by equally large and irregular areas of low electron density ?lled with short threads that were con?uent with the surfaces of the aggregates,similar to those found at neutral pH. Although it is known that pH buffering (the resistance to change in pH on addition of acid or base)retards any physical modi?cation of WPI (Fox and McSweeney,1998),we observed in this study that buffering of WPI was more effective with comparatively weaker acids and bases,but not with strong acids and bases (Figure 1).Buffering capacity can be altered depending on the sever-ity of heat treatment of various milk protein fractions (Salaun et al.,2005).Heat treatment lower than 100°C Journal of Dairy Science Vol.89No.1,2006 for 10min induced a slight increase in buffering capacity at pH 5of milk,whereas intense heat treatment at 120°C for 10min increased its buffering capacity intensely and shifted maximum peak pH (Lucey et al.,1993).Gezimati et al.(1998)showed that combining heating and shear alters the conformational structure of the protein by exposing groups that are normally hidden away in the native protein. We have reported here that extrusion induces shear-generated structural changes leading to loss of solubility with increasing pH in a short time (<40s).Previously,Kavanagh et al.(2000)showed that mixtures of α-LA and β-LG formed coupled network gels at pH 3and 7,and ?ne-stranded ?ber-like aggregates at pH 2,in the presence of 0.1M NaCl,and that aggregation of globules EXTRUDED WHEY PROTEIN ISOLATES 79 Figure 6.Confocal ?uorescence images illustrating the effects of pH on the microstructure of whey protein isolate (WPI)ribbons show-ing well de?ned,cumulative clusters of ?uorescence:A)diffuse ?uo-rescence in EACI (WPI extruded at pH 2.5with electrolyzed acid water);and B)diffuse clusters of ?uorescence in EALK (WPI extruded at pH 13.2with 2N NaOH-adjusted water). occurred at pH 7.They showed also that aggregation occurs more with acid because of modest increases in intermolecular β-sheet with shoulder,which develops in the carbonyl-stretching amide I region.Our results Journal of Dairy Science Vol.89No.1,2006 (Figures 5and 6)con?rm the formation of aggregates,and shows further varying degrees of aggregation in-duced by shear and acidic conditions,and demonstrated that NALK samples produced with water adjusted with 2N NaOH at pH 13were most aggregated. The network structure in a heat-induced globular gel depends strongly on the balance between the attractive and repulsive forces during aggregation.If the pH is far from the isoelectric point,and the ionic strength is suf?ciently low,then intermolecular electrostatic repul-sion is dominant.Structural transitions are re?ected by the macroscopic properties—?ne-stranded gels are translucent,whereas particulate gels are opaque and syneretic.A ?ne-stranded gel formed at neutral pH is rubbery and deformable to a large strain without fractur-ing (Ikeda and Morris,2002).At acidic pH,intramolecu-lar disul?de bonding is unlikely to occur;the structures are brittle.Our results show brittleness of HACI extru-date pH (1.4)with fractures (Figure 3A).Bilayer ?uores-cent patterns of protein and glutaraldehyde complexes observed through confocal scanning laser microscopy (Figure 4)agree with the observation that heat induces gelation of globular proteins at low pH (2to 2.5),and at the molecular level,rod-like structures are formed (Onwulata et al.,2003),whereas aggregation of globules occurs at pH 7.Gosal et al.(2004a)showed that β-LG formed aggregates with worm-like ?brils when heated in various alcohol-water mixtures at pH 2,and that ther-mally induced aggregation occurred more cooperatively in a nucleation and growth process.At higher concentra-tions,heated β-LG self-assembled in a uniform ?brillar pattern (Gosal et al.,2004b).It is reasonable to consider that whey protein aggregates generated under extrusion at high temperature would also contain more aligned ?brillar structures.Finally,Pelegrine and Gasparetto (2005)have shown that strong links between pH and temperature affect solubility of whey proteins in the pH range 3.5to 7.8and temperature range of 40to 60°C and that denaturation occurs.We show that extrusion texturization occurs in the short time frame of 15to 60s within the extruder;therefore,pH-adjusted extrusion-texturized WPI will maintain their characteristic quality after treatment,and provide added functionality due to newly induced conformation. CONCLUSIONS Denaturation and aggregation of whey protein isolates are pH dependent,with strong alkalis producing the more textural effect.Denaturation at the molecular level produces rod-like microstructures and forms the basis for ?ne-stranded ?ber-like structures in texturized prod-ucts.We have demonstrated that whey protein can be texturized by high shear at different pH values to form ONWULATA ET AL. 80 Figure 7.Thin sections of embedded whey protein isolate (WPI)ribbons illustrating the ultrastructure of the protein aggregates:A)uniform,?ne granularity of EACI (WPI extruded at pH 2.5with electrolyzed acid water);B)micrometer-and submicrometer-diameter circular dense aggregates surrounded by ?ne ?laments in the interstices in NEUT (WPI extruded at pH 6.8with deionized water);C)and D)irregular dense aggregates and interstitial ?laments in EALK (WPI extruded at pH 11.5with electrolyzed alkali water)and NALK (WPI extruded at pH 13.2with 2N NaOH-adjusted water),respectively. stringy products.Whey proteins denatured under acidic and alkaline conditions form small primary aggregates that combine to form large clusters and are aligned by extrusion shear to form ?brous structures.Fibrous pro-tein extrudates may be spun into meat alternatives simi-lar to textured soy products. 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