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Food Research International 41 (2008) 286–294

Physicochemical characterization of soymilk afterstep-wise centrifugation

Amir Malaki Nik a, Susan Tosh b, Vaino Poysa c, Lorna Woodrow c, Milena Corredig a,*

a Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G2W1b Agriculture and Agri-Food Canada, Food Research Program, 93 Stone Road West, Guelph, Ontario, Canada N1G5C9

c Agriculture and Agri-Food Canada, Greenhouse and Processing Crops Research Centre, 2585 Country Road 20, Harrow, Ontario, Canada N0R 1G0

Received 16 July 2007; accepted 10 December 2007

Abstract

The effect of heat treatment at 95–100 �C for 5 min and homogenization on the physicochemical properties of soymilk was studied,determining the particle size distribution and the amount and type of protein present after step-wise centrifugation. Differential scanningcalorimetry of soy protein showed three thermal transitions for unheated soymilk at 54, 70 and 94 �C, which were attributed to 2S, 7Sand 11S, respectively. These thermal transitions were absent from heated (and homogenized) soymilk. Unheated soymilk showed a largeaverage particle size, a broad size distribution, and significant protein precipitation with centrifugation. Heating of soymilk decreased theparticle size distribution and improved its stability. Homogenization also resulted in a decrease in particle size, with a narrower size dis-tribution compared to heated soymilk. During step-wise centrifugation changes in the ratios of 11S (glycinin) and 7S (b-conglycinin) inthe supernatants were noted, and they depended on the treatments applied to soymilk. Transmission electron microscopy observationsshowed the distribution of the colloidal particles in soymilk and helped further identify the differences after heating and heating withhomogenization.� 2008 Elsevier Ltd. All rights reserved.

Keywords: Soy milk; Soy proteins; Colloidal characterization

1. Introduction

Production and consumption of soymilk is rising notonly because of the increasing consumer interest for thisprotein beverage, but also because of its utilization as abase in other food products. Although several studies havebeen reported on soy protein isolates, their functionalproperties and processing behaviour (Lakemond, Jongh,Gruppen, & Voragen, 2002; Nielsen et al., 1989; Thanh& Shibasaki, 1978), very little information is available onthe physicochemical properties of soymilk.

Soymilk is a turbid and colloidal dispersion extractedfrom soaked soybeans, containing a large proportion of

0963-9969/$ - see front matter � 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.foodres.2007.12.005

* Corresponding author. Tel.: +1 (519) 824 4120x56101; fax: +1 (519)824 6631.

E-mail address: mcorredi@uoguelph.ca (M. Corredig).

the original components in soybean (Guo, Tomotada, &Masayuki, 1997). Soymilk, after its separation from thefiber (okara), is composed of about 2% lipid, 4–6% protein,2% carbohydrate, 0.6% ash and 8–10% total solids (Cruzet al., 2007; Iwuoha & Umunnakwe, 1997). Glycinin(11S) and b-conglycinin (7S) are the main proteins presentin soybean, constituting about 40% and 30% of the totalsoy protein, respectively. The amount of these two majorsoy proteins and their ratios varies among soybean varie-ties and affects the quality of the final food product(Murphy, Chen, Hauck, & Wilson, 1997; Tezuka, Taira,Igarashi, Yakasaki, & Ono, 2000). Glycinin (11S) is aheterogeneous hexamer with molecular weight of approxi-mately 320–380 kDa. It is composed of five subunits, eachmade up of an acidic (A) and a basic (B) polypeptide linkedby a disulfide bond (Nielsen et al., 1989) and the differentsubunits have been divided into group I (A1aB1b; A1bB2;

A. Malaki Nik et al. / Food Research International 41 (2008) 286–294 287

A2B1a), group IIa (A5A4B3) and group IIb (A3B4) (Nielsenet al., 1989; Yagasaki, Kaizuma, & Kitamura, 1996). Thesecond main component of soy protein, b-conglycinin(7S), is a trimeric glycoprotein with molecular weight ofapproximately 180 kDa consisting of three subunits a0, a,and b (Thanh & Shibasaki, 1978; Zarkadas et al., 2006).

Heating is commonly applied during the soymilk pro-cess, mostly to ensure food safety and extend the shelf lifeof the product. Heat inactivates enzymes such as lipoxy-genases, and other proteins such as trypsin inhibitors whichcan negatively impact the quality and nutritional propertiesof soymilk (Kumar, Rani, Tindwani, & Jain, 2003). Heattreatment causes the dissociation, denaturation and aggre-gation of both 7S and 11S globulins. The dissociated sub-units of 7S and 11S interact with each other, formingsoluble macrocomplexes (Utsumi, Damodaran, & Kinsella,1984). Soy proteins show a different denaturation tempera-ture; 65–75 �C for 7S and 85–95 �C for 11S have beenreported depending on the soybean variety and environ-mental conditions (German, Damodaran, & Kinsella,1982; Lakemond et al., 2002; Zhang, Takenada, & Isobe,2004).

Homogenization can also be applied in soymilk process-ing to improve the product quality. Recent research hasfocused on the effect of static high pressure and ultra highpressure homogenization treatments on soy milk (Cruzet al., 2007; Lakshmanan, De Lamballerie, & Jung, 2006;Zhang, Li, Tatsumi, & Isobe, 2005). However, very littleis understood on the physicochemical changes occurringto soymilk after heating and after combining heat andhomogenization treatments.

Particle size distribution is one of the properties in soy-milk and soymilk-based products that can help identifychanges occurring during processing. It has been previ-ously reported that large particles are present in unheatedsoymilk and that they are disrupted during heat treatment(Ono, Choi, Ikeda, & Odagiri, 1991) as well as after highpressure homogenization (Cruz et al., 2007), however, adetailed study on the characterization of the soymilkdispersions has never been reported.

The objective of this study was to examine the effect oftwo treatments, heated, and heated with homogenization(and compare them to the unheated control), on proteinparticles in soymilk, using the analytical techniques ofintegrated light scattering, differential scanning calorimetry(DSC), electrophoresis, and transmission electronmicroscopy.

2. Materials and methods

Soybean seed (Harovinton variety) was harvested at theGreenhouse and Processing Crops Research Centre ofAgriculture and Agri-Food Canada in Harrow, Ontario,in 2005. All chemicals were purchased from Sigma Chemi-cal (St. Louis, MO) or Fisher Scientific (Mississauga, ON,Canada). Ultrapure water was used for preparation ofsoymilk and buffer solutions.

2.1. Soymilk preparation

Soymilk was prepared according to the procedure ofMullin et al. (2001) with slight modifications. Soybeanswere weighed (100 g) and soaked with excess ultrapurewater overnight at room temperature, drained and rinsedwith cold water and drained again, then reweighed to deter-mine the amount of water absorbed by the beans. Thewater uptake was calculated by dividing the weight of thesoaked beans by the initial weight of the dry beans. Theamount of additional water needed to obtain a ratio of18:1 water to protein (as in Mullin et al., 2001) was thencalculated by subtracting the amount of absorbed water.

Approximately half of the additional water needed wasthen added to the beans at 20 �C and blended (commercialblender, WARING, New Hartford, CT) at high speed for3.5 min. The remaining water was heated to 60 �C andadded to the slurry for better protein extraction, and thewhole mixture was blended at high speed for another30 s. A two step filtration was then carried out to removethe coarse material (okara, which is mainly composed offiber material): the slurry was filtered through a juiceextractor (Juiceman, professional series 211, Korea) andthe okara collected and passed through the juice extractoragain. The soymilk obtained from the juice extractor wasfiltered through cheese cloth to remove fines (Mullinet al., 2001). The soymilk was then collected for analysis(unheated soymilk).

A portion of soymilk was then divided in test tubes(10 ml each tube) and placed in boiling water (95–100 �C)for 7 min, following a previously published procedure(Ono et al., 1991) and then cooled to room temperaturein an ice-waterbath (heated soymilk). A portion of theheated soymilk was passed through a valve homogenizer(Emulsiflex C5, Avestin, Ottawa, Canada) at 69 MPa atroom temperature for four passes (heated-homogenizedsoymilk).

A step-wise centrifugation procedure was applied tosoymilk from each of these treatments to obtain fourdifferent supernatant phases by centrifuging the soymilkat 8000g (SN1), 15,000g (SN2), 40,000g (SN3), and122,000g (SN4) at 20 �C for 30 min using a refrigeratedultracentrifuge (Optima LE-80K Beckman Coulter, CA,USA). These centrifugation speeds were chosen as inprevious literature reports (Ono et al., 1991). The proteincontent in soymilk and the various supernatant fractionswas measured by the Dumas method using a NitrogenAnalyzer (LECO, FP-528, Mississauga, ON, Canada)with EDTA as a standard for calibration of the instru-ment. The protein concentration was calculated using aconversion factor of 6.25. The amount of total solidspresent in the soymilk and the various supernatant frac-tions was determined with the forced air oven method.The soymilk was weighted in dry aluminum panscontaining dried sea-sand as a dispersing agent (FisherScientific) and dried for 24 h in a forced air oven (FisherScientific).

288 A. Malaki Nik et al. / Food Research International 41 (2008) 286–294

2.2. Differential scanning calorimetry (DSC)

Thermograms of soymilk samples before and afterheating and homogenization were obtained by differen-tial scanning microcalorimetry (Microcal Incorporated,NorthHampton, MA). The enthalpy (DH) and temperatureof denaturation of the different soymilk samples weredetermined by heating the samples from 20 to 115 �C ata rate 1 K/min and cooling at 20 �C. The microcalorimetricanalysis of the samples was carried out using the corre-sponding serum for each soymilk samples in the referencecell. The serum sample was obtained by centrifuging10 ml of soymilk in ultrafiltration centrifuge tube (Macro-sep 10K Omega, Pall Corporation, NY, USA) at 10,000gfor 2 h (Beckman Coulter, J2-21, CA, USA).

2.3. Particle size distribution

The laser diffraction instrument (Mastersizer S, MalvernSouthborough, MA) was employed to determine the parti-cle size distribution ðd3;2 ¼ ð

Pnid

3i =P

nid2i ÞÞ and average

particle diameter ðd4;3 ¼ ðP

nid4i =P

nid3i ÞÞ, of the soy milk

and the various supernatants, using 1.46 as refractive indexof the scatterers in soymilk and 1.333 as a refractive indexof dispersant (water).

2.4. Electrophoresis

Sodium dodecyl sulphate polyacrylamide gel electro-phoresis (SDS–PAGE) was carried out in a vertical slabgel of 1.5 mm thickness with 12.5% acrylamide runninggel and 4% stacking gel in a Bio-Rad mini-protein electro-phoresis system (Bio-Rad Labratories, Hercules, CA) at aconstant voltage of 200 V. Samples (100 ll) were mixedwith 250 ll of extraction buffer containing 50 mM Tris–HCl, 5 M Urea, 1% SDS, and 4% 2-mercaptoethanol. After1 h of incubation at room temperature, 450 ll of electro-phoresis sample buffer containing 125 mM Tris–HCl, 5 MUrea, 1% SDS, 20% glycerol and 1% bromophenol bluewere added. The solution was heated at 95 �C for 5 minand centrifuged at 5000g for 4 min using an Eppendorf cen-trifuge (Brinkmann Instruments, Westbury, NY). Aliquots(6 ll) were then loaded to the gel. After the run, gels wereimmediately stained using Coomassie blue R-250 for30 min and destained with a destaining solution composedof 45% ultrapure water, 45% methanol and 10% acetic acid,for 2 h with two changes, and then destained overnightwith a destaining solution containing 22.5% methanoland 5% acetic acid. Gels were scanned using a SHARPJX-330 scanner (Amersham Biosciences, Quebec) and thebands were analyzed using image analysis software (Image-Master� 1D, Version 2.0, Amersham Biosciences).

2.5. Electron microscopy

Transmission electron microscopy (TEM) was also car-ried out on the soymilk samples. The samples were encap-

sulated in agar sleeves, prepared in capillary tubes (5 llvolume). The samples were fixed with 1.4% glutaraldehydesolution in 0.07 M Sorenson’s (sodium and potassium)phosphate buffer (pH 6.8) by immersion at room tempera-ture overnight (in the fume hood). Samples were thenwashed three times with Sorenson’s buffer. Post fixationwas carried out by immersing samples in 0.5% osmiumtetroxide at room temperature overnight and then washedthree times with imidazole buffer (0.2 M) (5 min for eachwash).

Samples were dehydrated using an anhydrous ethanolseries with 70% (10 min), 90% (10 min), and 100%(3 � 10 min) and then washed with 100% propylene oxidefor 15–20 min. The propylene oxide was replaced withSPURRS (a resin composed of ERL 4206 vinyl cyclohexendioxide, diglycidyl ether of polypropylene glycol, nonenylsuccinic anhydrate and dimethyl amino ethanol, ElectronMicroscopy Sciences, Fort. Washington, PA, USA) witha series of exchanges, 3:1, 1:1, 1:3 propylene oxide:SPURRS (30 min each exchange) and 100% SPURRS for1 h, finally incubated overnight with 100% SPURRS. Thesamples were placed in molds and polymerized in the ovenat 60 �C for 24 h. The blocks were then sectioned (70–90 nm thick) using a microtome (Reichert Ultracut S, LeicaMicrosystems, Vienna, Austria), and sections were col-lected on copper grids (300 mesh). The grids were stainedwith saturated uranyl acetate in 50% ethanol for 7 min, fol-lowed by Reynold’s lead citrate for 5 min. The electronmicroscope (Hitachi H – 7100, Nissei Sangyo, Tokyo,Japan) was operated at 75 kV. Images were acquired usingAnalySIS (Soft imaging system, CO) digital image capturesystem.

2.6. Statistical analysis

All results presented are the average of at least three rep-licates. Significant differences were determined by the gen-eral linear model (GLM) and Duncan test (SAS version9.1). All differences discussed in the paper were significantat p < 0.05.

3. Results and discussion

The amount of solids and residual protein in thesupernatant after the different centrifugation steps is sum-marized in Fig. 1. Unheated soymilk contained approxi-mately 5% protein and 10% solids, similarly to previouslypublished data (Lakshmanan et al., 2006; Mullin et al.,2001). The pH of the soymilk was 6.7 and did not changeafter heating or homogenization, confirming previousreports (Lakshmanan et al., 2006). It is understood thatsoymilk contains complexes between the various compo-nents (oil fraction, protein aggregates, complex carbohy-drates, phytates), however, only a limited amount of datais available in the literature so, it was considered necessaryto characterize fractions of soymilk separated by consecu-tive centrifugations. The amount of residual solids and

0

2

4

6

8

10

0.01 0.1 1 10 100

Particle Size (μm)

Vo

lum

e (%

)

Fig. 3. Particle size distribution as determined by light-scattering ofunheated (d), heated (N), and heated-homogenized (s) soymilk samples.

0

2

4

6

8

10

12

SM SN1 SN2 SN3 SN4

To

tal S

olid

s (%

)

0

1

2

3

4

5

6

SM SN1 SN2 SN3 SN4

Pro

tein

(%

)

a a

b

d

b bb

d

a

cc

de e e

a

a a a a a

b

a a

b b b bc c

d

Fig. 1. Total solids (A) and protein content in soymilk (SM) heated-homogenized (solid bar), heated (empty bar), and unheated (gray bar) andthe various supernatants after the centrifugation steps (8000g (SN1),15,000g (SN2), 40,000g (SN3), and 122,000g (SN4), see methods fordetails). Bars indicated standard deviations of three replicate experiments.

-0.018-0.016-0.014-0.012-0.010-0.008-0.006-0.004-0.002

40 50 60 70 80 90 100 110 120

Temp (oC)

Cp

(ca

l/K)

Unheated Heated-Homogenized Heated

Fig. 2. Thermograms of unheated, heated, and heated-homogenizedsoymilk.

A. Malaki Nik et al. / Food Research International 41 (2008) 286–294 289

proteins after the various centrifugation steps depended onthe treatment applied. After the first centrifugation at8000g unheated soymilk already showed a significant lossof both solids and protein. On the other hand, heatedand heated-homogenized samples did not show a signifi-cant amount of protein loss after this centrifugation step.These results may suggest that the heating step denaturesproteins or solubilizes proteins from carbohydrate-proteincomplexes, and a much lower amount of protein co-precip-itates after centrifugation in heated and heated-homoge-nized samples than in unheated soymilk. Unheatedsoymilk was the most unstable of the treatments, as indi-cated by both losses of solids and protein. On the otherhand, for heated and heated-homogenized samples, the lossof protein was more gradual during the various centrifuga-tion steps. The lowest residual protein in SN4 (after122,000g) was in the heated sample, while heated-homoge-nized and unheated soymilk showed a similar amount ofprotein.

Microcalorimetric analysis was carried out on soymilksamples to determine the denaturation behavior of the pro-teins (Fig. 2). In the unheated soymilk sample three ther-mal transition peaks were clearly identified at 53.3, 69.4and 93.3 �C. These peaks correspond to the denaturation

peaks for 2S, 7S (b-conglycinin) and 11S (glycinin), respec-tively. These results are in agreement with published dataon the thermal denaturation of the soy proteins (Lake-mond et al., 2002; Zhang et al., 2005). On the other hand,no thermal transition peaks were shown for the heated orthe heated-homogenized soymilk, indicating that the heattreatment at 95–100 �C caused denaturation of the soy pro-teins. These results contrasted those reported by Cruz et al.(2007), where the base soymilk (heated at 80 �C andhomogenized) showed only a small transition peak for11S glycinin.

Fig. 3 summarizes the effect of heating and homogeniza-tion on the size distribution of the soy protein particles insoymilk. Unheated soymilk showed the largest averageparticle size of all the samples analyzed. The unheated soy-milk had a bimodal distribution with a large population ofparticles >1 lm. This soymilk was not subjected to homog-enization. There were significant differences in the particlesize distribution of the two treatments (heating and heatingwith homogenization) and compared to the unheatedcontrol sample. Heated soymilk showed a monomodaldistribution of sizes with a D3,2 of 0.20 ± 0.01 lm andheated-homogenized soymilk showed a monomodaldistribution of sizes with a significantly smaller D3,2 of0.130 ± 0.003 lm. It was concluded that a significant por-tion of the native protein in unheated soymilk was presentin large protein aggregates of sizes >1 lm. This was thecause of the large loss of protein in the precipitate after cen-trifugation at 8000g. During heating, the proteins under-went dissociation from very large complexes and showeddenaturation (i.e. the lack of thermal transition peaks in

290 A. Malaki Nik et al. / Food Research International 41 (2008) 286–294

Fig. 2). After heating the average size distribution of theprotein particles was <1 lm. As expected, homogenizationfurther decreased the size of the protein particles. Theseresults are somewhat in disagreement with other recentlypublished data (Cruz et al., 2007). In the present work,there was a significant decrease in the size with heating,while other authors previously concluded that homogeniza-tion applied to the UHT soymilk did not produce furtherreduction in particle size. However, the base soymilk inthat case was already passed through a colloid mill andextensive denaturation of the soy protein was noted usingDSC. In the present work, the soymilk was mildly treatedand therefore the results may not be fully comparable tothose of the previous literature. Nonetheless, it is clearfrom Fig. 3 that both heating and homogenization signifi-cantly decrease the size of the particles present in soymilk.

0

2

4

6

8

10

12

0.01 0.1 1 10 100

0.01 0.1 1 10 100

Particle Size (μm)

Particle Size (μm)

0

5

10

15

20

Vo

lum

e (%

)V

olu

me

(%)

Fig. 4. Particle size distribution of soymilk (N), SN1 (d), and SN2 (D) ofsoymilk heated (A) and heated and homogenized (B).

α’

α

β A3

A1,A2,A4

B

1 2 3 4 5 1 2

Fig. 5. SDS–PAGE electrophoresis of unheated (A), heated (B) and heated-hoSN2 (lane3), SN3 (lane4), SN4 (lane5). 11S and 7S subunits are indicated.

The particle size distribution of the supernatant frac-tions resulting from the subsequent centrifugation steps isshown in Fig. 4 for heated and heated-homogenized sam-ples. In the unheated samples, SN2 and SN3 showed lowsignal to noise ratio, and could not be measured using lightscattering. For heated and heated-homogenized samples,SN2 still showed sufficient scattering. This is importantto note, as in the heated and heated-homogenized soymilkthe protein present in SN2 was in aggregated form. Thiswould be in agreement with previous reports on the dena-turation and aggregation of soy protein during heating(German et al., 1982; Ono et al., 1991).

In heated soymilk, SN1 and SN2 showed a monomodaldistribution of sizes with an average at 0.2 lm (Fig. 4A).The particle size distribution of the heated samples sub-jected to homogenization was always monomodal, withan average size at about 0.2 lm (Fig. 4B), however the dis-tribution was narrower after the first and subsequent cen-trifugation steps. No significant difference in the particlesize was shown between SN1 and SN2.

When comparing these results with the amount of pro-tein recovered in the various supernatants of heated andheated-homogenized samples (Fig. 1), it was possible tosuggest that very little protein was lost during the firsttwo centrifugation steps and most of the protein particleswere present as aggregates after heating and homogeniza-tion. The amount of residual protein after the fourth centri-fugation step was quite high, and it was considerednecessary to determine whether differences in the polypep-tide composition of the supernatants could be foundbetween the various treatments.

Fig. 5 summarizes the results from SDS–PAGE electro-phoresis for soymilk and the corresponding four superna-tants. The 7S and 11S subunits were clearly separated inthe electrophoresis gel. Soymilk and all the supernatantscontained all the subunits, even after centrifugation at122,000g, suggesting that the 7S and 11S proteins were

3 4 5 1 2 3 4 5

mogenized soymilk (lane 1) and corresponding supernatants: SN1 (lane2),

A. Malaki Nik et al. / Food Research International 41 (2008) 286–294 291

present in a polydispersed forms from soluble native toaggregated forms.

When comparing the density ratios between the differentsubunit bands (Fig. 6) it was clear that there was a differ-ence in the ratio of 7S/11S subunits depending on the treat-ment applied. In soymilk (Fig. 6A), there were nosignificant differences among unheated, heated, andheated-homogenized milk. After the first centrifugation at

0% 20% 40%

UH

H

H-H

0% 20% 40%

UH

H

H-H

0% 20% 40%

UH

H

H-H

α’ α β A3 acid

α’ α β A3

α’ α β A3

α’ α β A3

α’ α β A3

α’ α β A3

α’ α β A3

α’ α β

α’ α β A3

Fig. 6. Densitometric analysis of 7S (white) and 11S (gray) fractions in soymilksamples. Data are reported as % of total 7S and 11S combined; the various su

8000 g, SN1 of heated soymilk showed a decrease in the7S fraction, (Fig. 6B). After centrifugation at 122,000g,SN4 of heated and heated-homogenized samples showeda similar ratio of 7S to 11S, when compared to the SN1(Fig. 6B and C). In unheated samples, the 11S fraction con-tinues to decrease with subsequent centrifugations, show-ing a higher ratio of 7S to 11S present in the SN4compared to SN1 and the original soymilk. These results

60% 80% 100%

60% 80% 100%

60% 80% 100%

ic basic

acidic basic

acidic basic

acidic basic

acidic basic

acidic basic

acidic basic

A3 acidic basic

acidic basic

(A), SN1 (B) and SN4 (C) for unheated, heated and heated-homogenizedbunits are indicated in the figure.

Fig. 7. Transmission electron micrographs of unheated (UH), heated (H) and heated-homogenized (HH) soymilk and the corresponding SN1 and SN3.Bar = 2000 nm. Protein (P) and oil droplets (O) are indicated.

292 A. Malaki Nik et al. / Food Research International 41 (2008) 286–294

were in agreement with previous work which reported thatthe larger particles in unheated soymilk are composed of11S protein and these particles precipitate by centrifuga-tion (Ono et al., 1991). On the other hand, heated-homog-enized samples showed a very similar protein patternbetween soy milk and the fourth step of centrifugation(Fig. 6A and C) suggesting that homogenization decreasedthe precipitation of glycinin subunits. This is an importantpoint as it may suggest that two different mechanisms areinvolved in decreasing the particle size during heatingand with homogenization. With heating, rearrangementof the protein structures occurs, with formation of largesoluble aggregates. Homogenization seemed to cause a fur-ther rearrangement of 11S subunits.

Figs. 7 and 8 show representative micrographs of soy-milk, SN1 (after the first centrifugation step) and SN3(after the third centrifugation step at 40,000g). Unheatedsamples showed large aggregated structures containingprotein, which appear gray, and oil droplets, which appearblack. After centrifugation, oil droplets were still present inthe supernatant; even after centrifugation at 40,000g theydid not cream. It was clear that after heating, the large

aggregates were disrupted, and the protein structures wereless densely stained than in unheated samples (see Figs. 7and 8). Homogenization after heating caused further dis-ruption of the aggregates. In SN1 and SN3 of heated andheated-homogenized soymilk samples more darkly stainedprotein structures were shown than in unheated samples,suggesting the formation of heat-induced aggregates ofsoy proteins. The distorted shapes of the oil droplets sug-gest that interactions between the aggregated proteins arestronger than surface tension forces.

4. Conclusions

The sequential centrifugation allowed a better character-ization of the physicochemical properties of soymilk.Results suggest that heating of soymilk disrupts largeaggregates of soy proteins and causes a decrease in the par-ticle size distribution. After centrifugation at 122,000g,both 7S and 11S subunits are still present in the solubleform. While heating of soymilk decreases the solubility of7S, the large 11S subunits are disrupted and more 11S sub-units are recovered in the supernatants of heated soymilk.

Fig. 8. Transmission electron micrographs of unheated (UH), heated (H) and heated-homogenized (HH) soymilk and the corresponding SN1 and SN3.Bar = 1000 nm. Protein (P) and oil droplets (O) are indicated.

A. Malaki Nik et al. / Food Research International 41 (2008) 286–294 293

Homogenization further improved the particle size distri-bution of the soymilk particles, and electron micrographsshowed much smaller protein-fat globule complexes inheated-homogenized samples than in heated or unheatedsoymilk samples.

Acknowlegements

The authors would like to thank Ms. Mannephan Keer-ati-u-rai for her help with the DSC experiments, Dr. Marc-ela Alexander with her advice during refractive indexexperiments and Dr. Alexandra Smith for her help withmicroscopy. This work was carried out thanks to the finan-cial support of the Ontario Soybean Board, the HannamSoybean Utilization Fund and Agriculture and Agri-FoodCanada.

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