Con fnSI. Food Sci. Technol. J. Vol. 21, No. 3, pp. 305·311,1988
RESEARCH
Acid Coagulation of Skimmilk Powderby Extrusion Processingl
V.L. Barraquio, J. Fichtali, K.F. Ng-Kwai-Hang2
andF.R. van de Voort
Department of Food Science and Agricultural ChemistryMacdonald College of McGill University
21111 Lakeshore Rd., Ste. Anne de Bellevue, Quebec H9X ICO
AbstractUsing the extruder as a continuous reactor, skimmilk powder was
mixed with acid to produce an acid-coagulated product. The extrusion process was studied by response surface methodology to determine the influence of the extrusion parameters on selected extrudate physico-chemical responses including total and soluble protein,non protein nitrogen (NPN), lactose, and pH. Analysis of the highheat skimmilk powder extrudates showed that total and soluble protein, NPN and lactose were not affected by any of the extrusionparameters, while pH was significantly affected by moisture andscrew speed. When medium heat skimmilk powder was used, totalprotein and lactose levels were significantly affected by temperature. The final pH values of extrudates from both powders weresignificantly affected by moisture or acid level, however polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulphate(SDS)PAGE electrophoresis of the extruded product did not show anyalteration in the caseins of the original skimmilk powders. Extrusion processing appears to be a potential means of preparing an acidcoagulated product from skimmilk powder as a preliminary stepto the production of sodium caseinate.
ResumeA I'aide d'un extrudeur utilise comme reacteur en continu, un
produit coagule a l'acide a ete fabrique a partir d'un melange delait ecreme en poudre et d'acide. Le procede d'extrusion a ete etudie par la methodologie des effets apparents pour determinerI'influence des parametres d'extrusion sur certaines reactions physicochimiques de I'extrudat dont les proteines totales et solubles, I'azotenon proteique (NPN), le lactose et le pH. L'analyse d'extrudats depoudre haute temperature (high heat) a montre que les parametresd'extrusion ont ete sans effet sur les proteines totales et solubles,I'azote non proteique et le lactose, tandis que le pH a ete influencesignificativement par I'humidite et la vitesse de la vis. En utilisantde la poudre de lait ecreme de temperature moyenne (medium heat),les niveaux de proteines totales et de lactose furent affectes significativement par la temperature. Les pHs des extrudats de l'une ouI'autre des poudres furent affectes significativement par le niveaud'humidite ou d'acide. Toutefois, aucune alteration des caseines despoudres originales ne fut mise en evidence par electrophorese duproduit extrude sur gel de polyacrylamide (PAGE) ou sur gel desulfate dodecyl de sodium (SOS) - PAGE. 1I semblerait que le procede al'extrusion pourrait etre un bon moyen de preparer le coagulat acide de poudre de lait ecreme comme etape preIiminaire ala production de caseinate de sodium.
I Presented at the annual CIFST Meeting, held May 17-20, 1987in Hamilton, Ontario.
2 Department of Animal Science.
IntroductionCanada, the United States, and the European Eco
nomic Community (EEC) have a chronic oversupplyof milk of which substantial amounts are convertedinto skimmilk powder. The EEC manages its skimmilkpowder from surplus milk by its subsidized use in themanufacture of calf milk replacers and its subsidizedexport as nonfat dry milk (Norton, 1987). In 1985-86,69.9 thousand tonnes of Canada's skimmilk powderwas mostly exported as is, while 11.8 thousand tonneswere exported in the form of whole milk products toreduce surplus butter stocks (Canadian Dairy Commission, 1986). As early as 1978, one option consideredin Canada has been the diversion of this oversupplyof skimmilk powder into casein production (ACP Marketing, 1978). The production of 9.1 million kg ofcasein could divert 31.8 million kg of skimmilk powder into a functionally useful food ingredient and savethe Canadian Dairy Commission one to three milliondollars per year (Canadian Dairy Commission, 1983).The United States imports about 59 million kg ofcasein and caseinate annually and uses roughly 60070of this amount in formulated food applications (Morr,1985a). The largest amounts of casein and caseinatesare used for manufacturing imitation cheese (33%) andcoffee whitener (10%). The major non-food usesinclude glues, adhesives, paints, rubber and leatherproducts, paper sizing, cleaning agents, and lubricants.About 13% of the total casein and caseinates are utilized in pet foods (Morr, 1985b).
Traditionally, acid casein has been produced byacidification, curd formation, washing, and drying(Morr, 1985a). The process is based on the neutralization of the negative charge on the surface of thecasein micelles by the acid (Bloomfield and Mead,1975; Schmidt, 1980; Swaisgood, 1982; West, 1986)and the resulting acid casein is then further neutralized to yield a functionally useful caseinate (Southward, 1985). The processes presently used or proposed
Copyrighl 0 1988 Canadian Institute of Food Science and Technology
305
to date for acid casein and/or caseinate manufactureare cumbersome and expensive because of the equipment and energy requirements of the process. It maybe possible to manufacture acid casein more efficientlywith a minimum of unit operations from surplus skimmilk powder by extrusion processing. Extrusionprocessing, where suitable, is being utilized or is replacing a variety of thermal processes because it combinesa number of unit operations into a single process whichis generally more energy efficient (Smith, 1976;Scheuler, 1986). The extruder is basically a screw pumpin which food material is conveyed, mixed and subjected to heat and/or pressure before being dischargedthrough a die (Harper, 1981) and recently, this processhas been used for the conversion of acid casein tocaseinates (Millauer et al., 1984; Boulle, 1986; Linkoet al., 1986). Extrusion processing, if feasible, couldshorten the traditional process by four steps: batchcoagulation of liquid skimmilk, cutting of the coagulum, cooking, and whey drainage.
This study was undertaken to determine the possibility of using the extruder for the acid coagulationof skimmilk powder as a preliminary step in theproduction of an acid casein to be subsequently converted to sodium caseinate.
Materials and MethodsA pilot scale Creusot-Loire BC-45 twin screw
extruder was used for this work and the equipment has
been described in earlier publications (Owusu-Ansahet al., 1983; van de Voort et al., 1984). The extruder(Figure Ia) was equipped with thermocouples, a pressure transducer, plus sensors to follow screw speed,amperage (load), and pump flow rate. These data werecontinually monitored by a Campbell CR7 data logger, collected on magnetic tape and later transferredto an IBM PC for analysis. The moisture and the rawmaterial delivery systems were carefully calibrated toinsure that defined acid/skimmilk powder (SMP)ratios were being introduced into the extruder. A special, multi-orifice die was assembled (Figure lc) toreduce pressure and increase the surface area of theresultant product. Commercially available high andmedium heat SMP were obtained from Fedeco(Cooperative Federee du Quebec, Montreal, Que.) andused for all the extrusion runs.
Preliminary laboratory coagulation studies showedthat a 0.2 M HCI solution was adequate to providethe variable acid or moisture levels required to studythe coagulation of skimmilk powder based on theskimmilk powder/moisture or acid ratios. A screwprofile (Figure Ib) was assembled, consisting ofapproximately three equal (a) feed, (b) mixing andheating, and (c) metering sections, respectively. Thisprofile generated sufficient pressure to prevent backflow, yet maximized mixing and residence time.
Two extrusion runs were conducted. The first, withhigh heat SMP using a central composite 2' + 2k + 1
Fig. I. (a) The Creusot-Loire twin screw extruder/data acquisitionsystem, (b) the extrusion screw profile, (c) the die and (d)a typical acid coagulated extrudate.
306 / Barraquio et al. J. /nSl. Can. Sci. Technol. A/imef1l. Vol. 21, No. 3. 1988
Table I. Proximate analysis of high and medium heat SMP (as isbasis).
To evaluate whether heat and/or shear in theextruder altered the proteins in any significant manner, two electrophoretic methods were used to evaluate the extrudates. Polyacrylamide gel electrophoresis (PAGE) using gels prepared for casein and wheyproteins as described by Ng-Kwai-Hang and Kroeker(1984) were carried out to compare standard caseinpatterns to those of the extrudates. Commercialsodium caseinate (Champlain Industries Ltd., Quebec,Canada), Baker reagent grade casein, and acid caseinprepared from raw milk and from reconstituted SMPwere used as standards. Acid casein from raw milk andreconstituted (10% w/w) SMP were prepared as suggested by Ng-Kwai-Hang and Kroeker (1984) and theresulting wheys from each were also subjected toPAGE. In addition, sodium dodecyl sulfate (SDS)PAGE electrophoresis (Weber and Osborn, 1969) wasalso carried out to make similar comparisons including commercially available molecular weight markers(Biorad Laboratories Canada, Ltd). All the electrophoretic runs used O.Olmg total protein for eachwell.
The extrusion variables (temperature, moisture,screw speed, and feed rate) were studied in relationto the physico-chemical responses (total protein, soluble protein, NPN, lactose, and pH) to determine whichextrusion parameters significantly affected the composition of the acid coagulum. The response surfacedata for high heat SMP were analyzed using the SAS
design (Cochran and Cox, 1957) consisted of 25 treatment combinations which included three levels of temperature (50, 70, 90°C), screw speed (50, 70, 90 rpm),feed rate setting (0.5, 0.75, 1.0), and moisture(0.2 M HCI) levels (25, 30, 35070). Feed rate settingsof 0.5, 0.75 and 1.0 corresponded to 143, 204 and265 g of SMP delivered per minute. Based on theresults of the first extrusion run, a 2" + n factorialdesign of seven treatment combinations was followedfor the extrusion of medium heat SMP. In this case,only temperature (74, 84, 94°C) and moisture (26, 32,38070) were tested while screw speed and feed rate weremaintained at 70 rpm and 0.75, respectively.
Representative extrudate samples were collectedfrom each extrusion run, freeze-dried, ground to 0.5mm (40 mesh) using a Wiley mill and subjected to aseries of chemical analyses. Both high and mediumheat SMP and the extrudates were reconstituted to 8%w/w solution, the SMP in water and the extrudate in0.03M NaOH, and subsequently analyzed for fat, protein and lactose using the Multispec MKl infrared milkanalyzer (van de Voort, 1980; Mills and van de Voort,1982). The Multispec was calibrated using AOAC(1984) procedures based on preanalyzed calibrationmilks supplied by the Guelph Central Milk TestingLaboratory. Moisture and ash of the skimmilk powders were determined by AOAC (1984) methods andthe pH of the powders and the resulting extrudateswere measured as pastes (1:2 sample/water) using anHI 8417 pH meter. For soluble protein determinations,5% extrudate solutions were prepared in distilledwater, stirred gently for 1 h and then centrifuged at13,000 x g for 30 min. The supernatant was assayedfor total soluble nitrogen by the AOAC (1984) microkjeldahl method and non protein nitrogen (NPN) wasdetermined after protein precipitation with TCA (Haggett, 1976). The soluble protein was calculated by subtracting NPN from the total soluble nitrogen and multiplying the result by a factor of 6.38.
Table 2. Regression analysis for high heat SMP extrusion.
Component
Protein (010)Lactose (0J0)Fat (0J0)Moisture (0J0)Ash (0J0)pH
High Heat SMP
31.052.10.85.88.26.1
Medium Heat SMP
30.654.00.84.28.06.2
Regression Coefficients
Parameters Total Soluble NPNProtein Protein
Intercept -16.4566 10.1914 -0.1793xla 0.3270 0.0171 -0.0002X2 1.3260 -0.5112 0.0084X3 46.3253 -4.1398 0.2412X4 0.2578 0.0539 0.0009XIXI -0.0050 0.0001 0.0000X2X1 0.0079 -0.0006 0.0000X2X2 -0.0098 0.0045 -0.0001X3X1 0.2567 0.0068 -0.0006X3X2 -1.4517* 0.2380* -0.0029X3X3 -11.9006 0.0503 -0.0628X4X1 -0.0003 -0.0001 -0.0000X4X2 -0.0019 0.0012 -0.0000X4X3 -0.0043 -0.0428 -0.0003X4X4 -0.0012 -0.0003 0.0000
R 0.82 0.84 0.83
ax l , Temperature; X2' Moisture; X3, Feedrate; X4' Screw speed* Significant at P < 0.05** Significant at P <0.01
Can. Insl. Food 5ci. Technol. J. Vol. 21. No. 3, 1988
Lactose pH
48.2690 5.6445**-0.6759 0.0071
1.2038 0.0123- 3.8388 0.6574
0.1855 -0.01010.0068* -0.0001
-0.0053 0.0000-0.0187 -0.0002-0.1998 -0.0007
0.0743 0.002916.5383 -0.3056
-0.0003 -0.0000-0.0048 -0.0002*-0.0603 -0.0032-0.0023 0.0001*
0.87 0.92*
Barraquio et al. / 307
Table 3. Quadratic response surface models for total protein (VI), soluble protein (V2) and pH (V5) of high heat SMP extrudates.
YI = - 37.0159 + 3.4230X2+ 73.8554X} -0.0413Xi - 24.5058Xl- 1. 1379X}X2+ €
Y2 = 12.3117 - 0.4095X2- 6.167IX} + 0.0035Xi - 0.3459X}2 + 0.245IX}X2+ €
Y3 = 5.4977 + 0.0514X2-O.0082X4 -0.0008Xi + 0.OOOIXi-0.0002X4X2+ €
Fig. 2. (a) PAGE patterns in whey gels for (I) BSA, (2) alpha lactalbumin, (3) beta lactoglobulin, (4) whey from raw milk,(5) whey from high heat SMP, (6-8) extrudates from runs#18-20. (b) PAGE patterns in whey gels of (I) raw milkwhey, (2) medium heat SMP whey, (3) medium heat SMPacid casein, and (4-8) extrudates from runs #1-5.
Response Surface regression (RS) procedure, then asecond RS regression was done on the parameterswhich were significant to obtain the coefficients forplotting the response surfaces (Table 3). The GeneralLinear Model (SAS Institute Inc., Cary, NC) was usedfor the medium heat SMP data analysis.
Results and DiscussionThe proximate analysis results obtained for the high
and medium heat powders are presented in Table 1.Preliminary runs were carried out to determine thebasic operating limits of the extrusion process; temperatures beyond 100°C, especially at low moisturelevels, caused extensive browning via the Maillard reaction due to the reaction of lactose with the milk proteins. High moisture contents, beyond 30%, lubricated
13- casein
K- casein
t><- casein
. ... . . - - ;- • - - ~ <......
2 3 4 5 6 7 8 9 10 11
Fig. 3. PAGE patterns in casein gels (1-7) of extrudates from runs#1-7, acid casein from high heat SMP (8), acid casein fromraw milk (9), commercial sodium caseinate (10), and Bakerreagent grade casein (11).
High Heat SMPHigh heat SMP was chosen for the initial study to
restrict the assessment of protein changes to thecaseins, since most of the whey proteins would bedenatured. To confirm that this assumption was thecase, whey from reconstituted SMP and whey fromraw milk were subjected to PAGE on whey gels alongwith bovine serum albumin (BSA), alpha lactalbuminand beta lactoglobulin standards. Figure 2a illustratesthat none of the whey proteins from high heat SMPentered the gel, indicating denaturation and/or polymerization. Heating beta-lactoglobulin above 60°C,can unfold the protein, exposing thiol groups for reaction and intermolecular disulfide interchange can takeplace inducing polymerization above 70°C (Walstraand Jenness, 1984). Figure 3 compares the PAGE patterns of Baker reagent grade casein, commerciallyavailable sodium caseinate, acid casein from raw milk,acid casein from high heat SMP, and the extrudatesfrom runs #1 to 7. As can be seen, the classic alpha,beta and kappa bands were present in the patterns ofBaker reagent grade casein, commercial sodiumcaseinate and raw milk acid casein. In the case of acid
the SMP to the point that positive pumping action wasinadequate to feed the barrel using a conventional lowto high pressure screw profile. By manipulating thescrew profile, specifically using high pressure screwsin the feed section (Figure Ib), a maximum moisturelevel of 40070 could be tolerated without the productbacking up. Screw speed was limited to a maximumof 120 rpm on this older model extruder and a rangeof 40 to 120 rpm could be utilized. The extrudate(Figure Id) exiting from the die under the extrusionconditions imposed, varied from a dry, brittle productto one which was wet, crumbly and showed markedsyneresis.
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C-Lg
«-La
308 / Barraquio et al. J. InSI. Can. 5ci. Technol. Alimem. Vcl. 21, No. 3, 1988
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speed (X4X4) had a positive effect. From its initial pHof 6.1 (Table 1), high heat SMP pH dropped to therange 5.78 to 5.95 (Figure 5c) upon extrusion, valueswhich are substantially higher than pH 4.6 commonlyassociated with the isoelectric point of casein. Thehigher extrudate pH values could be due to the highbuffering capacity of milk solids in the range of pH5.0 to 6.0 (Whittier, 1929) or due to changes in thesalt balance caused by the combination of the highsolids and pH. Titratable acidity and hydrogen ionconcentration both increase in direct proportion toconcentration, and a slight increase in the acidity ofmilk can cause a decrease in the heat stability of casein,since any change in pH is magnified by concentration(Knipschildt, 1986). Heat may also have played a role(Riel, 1985). Regardless of the actual mechanism,coagulation does take place at higher pH values in theextruder under high solids conditions and pH was theonly significant physico-chemical response due to the
casein from high heat SMP and the extrudateshowever, only two bands are visible, indicating the lossof kappa casein. Kappa casein and beta lactoglobulinassociation upon the heating of milk from 70 to 90°Chas been reported previously (Sawyer, 1969; Elfgamand Wheelock, 1977; Smits and Brouwershover, 1980).Figure 4 presents the SDS-PAGE patterns of themolecular weight standards relative to acid casein fromraw milk, acid casein from high heat SMP and extrudates from extrusion runs #23 and 24. The extrudateproteins and the acid caseins from the high heat SMPand raw milk migrated as one band with a molecularweight of around 34,000 similar to the results reportedby Burk and Greenberg (1930). Our results indicatethat extrusion did not cause any alteration in the proteins of the high heat SMP.
None of the extrusion parameters (temperature,moisture, screw speed, and feed rate) significantlyaffected the total protein, soluble protein, NPN or lactose concentrations in the end product. The pHhowever, was affected by the moisture delivered(P ::50.01) and screw speed (P ::50.05). These resultscould be anticipated since a change in moisture ineffect, affects the amount of acid delivered to the SMPand the screw speed affects the subsequent mixing.There are, however, effects due to the interaction ofthe extrusion parameters as indicated in Table 2. Thenegative interaction effect of feed rate x moisture(X3X2) indicates that higher or lower feed rate settings and moisture levels decreased the protein content in the end product (Figure 5a). Factor combinations that yielded optimum protein were 30.45070moisture at a feed rate setting of 0.80 (216 g/min). Theopposite effect was seen for soluble protein (Figure5b), while NPN was not affected at all. This impliesthat as feed rate and moisture levels are increased, acidcoagulation is not as efficient based on the observedincrease in soluble protein and, in all likelyhood, therewas insufficient time for the reaction between the acidand SMP to equilibrate. The lactose content wasaffected in a positive quadratic fashion by temperature, while screw speed x moisture (X4X2) had a negative effect on pH (Figure 5c), but the square of screw
2 3 456
5.•
5.70
ClZl31»
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; 5.12
soybean trypsin inhibitor
lysozyme
carbonic anhydrase
ovalbumin
phosphorylase BBSA
... .. .... ...~l ~f" "~. , ••
it ... If " ••t,
Fig. 4. SDS·PAGE patterns of extrudates from runs #23 and 24(1-2), acid casein from high heat SMP (3), acid casein fromraw milk (4), and molecular weight standards (5-6).
Fig. 5. Response surface plots of (a) total protein vs moisture andfeedrate, (b) soluble protein vs moisture and feedrate, and(c) pH vs moisture and screwspeed.
Can. Ins/. Food Sci. Technol.1. Vo1. 21, No. 3,1988 Barraquio et al. / 309
Table 4. Regression analysis for medium heat SMP extrusion.
Regression Coefficients
Parameters Total Soluble NPNProtein Protein
Lactose pH
InterceptTemperatureMoisture
R
* Significant at P :s0.05** Significant at P :s0.01
12.46130.2880**0.0999
0.95*
5.0765-0.0755
0.0938
0.43
0.36310.0001
-0.0043
0.51
87.6134**-0.3358*-0.0684
0.91*
6.4499**-0.0010-0.0181**
0.98**
process. Ninety-two per cent of the variation in pHwas accounted for by the extrusion parameters indicating that acid coagulation of high heat SMP in theextruder is mainly a function of acid delivered, witha minor screw speed effect, being essentially independent of temperature and feed rate over the rangestested.
Medium Heat SMPBased on the work carried out on high heat SMP,
a simplified factorial design (2X + n) was chosen usingmoisture and temperature as the two main variablesto assess the five chosen responses, temperature beingincluded as a variable because of its potential effecton the whey proteins. Whey gel electrophoresis confirmed that undenatured whey proteins were presentin the medium heat SMP and that these whey proteinswere denatured by extrusion (Figure 2b). PAGE andSOS-PAGE patterns of acid casein from medium heatSMP were similar to those of its extrudates as werethose of high heat SMP. Total protein was significantlyaffected by temperature and pH by moisture, both atP ~ 0.01 level and lactose was also affected by temperature (P ~ 0.05). The regression coefficientsobtained are presented in Table 4, with the modelsaccounting for 95, 98, and 91070 of the variation in totalprotein, pH, and lactose, respectively. Temperatureaffected the protein content positively because the protein was being modified by heat denaturation, coprecipitation and syneresis. Increases in moisturedecrease the pH as noted earlier, by delivering moreacid to the SMP, however, the decrease observed inlactose with temperature was not observed in the highheat SMP. This observation implies that lactose maybe bound to the whey proteins, either physically, viathe Maillard reaction or degraded to organic acids suchas formic and lactic acids as a result of heating (Blaiset al., 1985).
ConclusionThis preliminary study has indicated that SMP may
be extruded to produce an acid-coagulated product.A response surface methodology approach was takenin this research to obtain a comprehensive view of theeffect of the extrusion variables on selected physicochemical responses associated with the end product.In essence, only moisture was shown to be a significant factor because it carried the coagulating acid.
310 / Barraquio et al.
Both high or medium heat skimmilk powders appearto work equally well as a raw material, with mediumheat powder being more susceptible to the extrusionvariables in general. The additional steps and furtherprocessing required to convert the acid casein-likecoagulum to a more functional sodium caseinate-likeproduct by washing and further extrusion using base(Millauer et al., 1984; Linko et al., 1986) is underinvestigation and will be the subject of future publications.
AcknowledgementsThe authors would like to acknowledge the Natural
Sciences and Engineering Research Council of Canadafor their financial support of this research and Mr. S.Khanizadeh for his assistance with the statistical analysis.
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Submitted December 8, 1987Revised January 28, 1988Accepted February 11, 1988
Barraquio et al. / 311