LWT - Food Science and Technology 42 (2009) 1245–1252

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LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Effect of soy protein subunit composition and processing conditions on stability

and particle size distribution of soymilk
Amir Malaki Nik a, Susan M. Tosh b, Lorna Woodrow c, Vaino Poysa c, Milena Corredig a, *
a
Department of Food Science, University of Guelph, Guelph, Ontario N1G2W1, Canada
b
Agriculture and Agri-Food Canada, Guelph Food Research Center, 93 Stone Road West, Guelph, Ontario N1G5C9, Canada
c
Agriculture and Agri-Food Canada, Greenhouse and Processing Crops Research Centre, 2585 Country Road 20, Harrow, Ontario N0R 1G0, Canada

a r t i c l e i n f o a b s t r a c t

Article history: The influence of soy protein subunit composition on the particle size distribution and solid content of
Received 17 May 2008 soymilk and various supernatant fractions was investigated. A well-established seed variety (Har-
Received in revised form ovinton), containing all protein subunits, and eleven null soybean genotypes lacking specific glycinin and
26 February 2009
b-conglycinin subunits were investigated, to determine the effect of protein composition on the physico-
Accepted 2 March 2009
chemical characteristic of soymilk. Soymilk made from Harovinton and soybean lines null in glycinin
showed significantly higher total solid yields than the other genotypes evaluated. Soymilks prepared
Keywords:
from soybeans null for glycinin or lacking glycinin’s group I (A1, A2) showed a smaller particle size
Soymilk
Soy protein composition distribution compared to soybean lines containing glycinin. In general, unheated soymilk made from
Particle size distribution lines having glycinin showed a bimodal size distribution with large particles, with a decrease in the
Heat treatment particle size distribution after heating and heating with homogenization. The results of this work will
Homogenization help in the evaluation of breeding lines with a specific protein composition tailored to a specific pro-
cessing functionality.
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction however, about the physico-chemical changes occurring in soymilk

The distribution of particle size in a colloidal system such as
soymilk has remarkable effects on its stability and quality. Several
studies have demonstrated that the processing conditions such as
heat treatment (Kwok, Liang, & Niranjan, 2002; Kwok & Niranjan,
1995) have a significant effect on soy protein colloidal properties.
Heat treatment can cause denaturation and aggregation of soy
proteins (Kitabatake, Tahara, & Doi, 1990; Malaki Nik, Tosh, Poysa,
Woodrow, & Corredig, 2008a; Zhang, Takenaka, & Isobe, 2004);
however, soy protein aggregates still show stability in solution
(German, Damodaran, & Kinsella, 1982). It has been previously
reported that raw soymilk contains a majority (about 0.4 vol/vol) of
large particles (>120 nm), 0.2 vol/vol medium particles
(120–40 nm), and particles smaller than <40 nm, and that heat
treatment can disrupt these large particles and decrease the
particle size diameter (Ono, Choi, Ikeda, & Odagiri, 1991). In addi-
tion to heat treatment, static high pressure alters soymilk structure
(Cruz et al., 2007; Kajiyama, Isobe, Uemura, & Noguchi, 1995;
Zhang, Li, Tatsumi, & Isobe, 2005). Very little is understood,
* Corresponding author. Tel.: þ1 519 824 4120x56101; fax: þ1 519 824 6631.
E-mail address: mcorredi@uoguelph.ca (M. Corredig).
depending on the original bean’s protein composition.
Glycinin and b-conglycinin are the most important soy
proteins, as in combination they represent more than 70 g/100 g
of the total soy proteins (Liu, 1997). The structure of these two
proteins has been extensively studied (Adachi et al., 2003; Adachi,
Takenaka, Gidamis, Mikami, & Utsumi, 2001; Maruyama et al.,
2001; Nielsen et al., 1986; Nielsen et al., 1989; Staswick, Her-
modson, & Nielsen, 1984; Tumer, Thanh, & Nielsen, 1981; Utsumi,
Matsumura, & Mori, 1997). Glycinin is generally in hexameric
form, and each monomer unit consist of one acidic and one basic
polypeptide linked together by one disulfide bond (Nielsen et al.,
1986; Utsumi et al., 1997). Glycinin subunits have been divided to
three groups; group I (A1aB1b, A1bB2, A2B1a), group IIa (A5A4B3),
and group IIb (A3B4) (Adachi et al., 2003; Yagasaki, Takagi, Sakai, &
Kitamura, 1997). b-conglycinin is a trimeric glycoprotein with
a molecular mass of 180 kDa consisting of three subunits a0 , a,
b (Yamauchi, Yamagishi, & Iwabuchi, 1991) linked by hydrophobic
interactions and hydrogen bridging (Liu, 1997; Thanh & Shibasaki,
1978).
It has been previously demonstrated that the processing
quality of soy products is affected not only by processing and
environmental conditions, but also by protein composition
(Bhardwaj et al., 1999; Poysa & Woodrow, 2002; Poysa, Woodrow,

0023-6438/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2009.03.001
1246 A. Malaki Nik et al. / LWT - Food Science and Technology 42 (2009) 1245–1252

Table 1
Glycinin and b-conglycinin subunit composition of Harovinton and null soybean genotypes.

Subunits absent

Soybean genotypes b-conglycinin Glycinin Grouping

Line Designations Acidic polypeptide Protein (g/100 g) 11S/7S ratio

V1 Harovinton 43.50 1.3
V2 SQ98-0110-3-1 A3 IIb 41.50 0.87
V3 SQ97-0263-54-1-5 a0 A4, A5 a0 , IIa 44.60 1.18
V4 SQ98-0105-6-1 a0 A3 a0 ,IIb 44.40 1.5
V5 SQ97-0263-71-1-3 A1, A2, A4, A5 I, IIa 41.90 0.49
V6 SQ98-0105-1-1b A3, A4, A5 IIa, IIb 43.30 0.47
V7 SQ97-0263-21-7-2 a0 A3, A4, A5 a0 , IIa, IIb 43.00 0.76
V8 SQ97-0263-3-10-1 a0 A1, A2, A3 a0 ,I, IIb 45.40 0.73
V9 SQ97-0252-S17-2-1 A1, A2, A3, A4, A5 11S 42.90 0.21
V10 SQ97-0252-S17-2-3 A1, A2, A3, A4, A5 11S 42.00 0.27
0
V11 SQ97-0263-3-1a a A1, A2, A3, A4, A5 a0 , 11S 42.70 0.27
V12 SQ98-0112-S7-1 A1, A2 I 45.50 0.65

& Yu, 2006; Tezuka, Ono, & Ito, 1995). New soybean lines with
different seed compositions have been developed, including lines
containing low levels of antinutritional components such as
trypsin inhibitor and lines with improved flavour due to reduced
lipoxygenase levels (Kitamura, Davies, Kaizuma, & Nielsen, 1983;
Kobayashi, Tsuda, Hirata, Kubota, & Kitamura, 1995; Kumar, Rani,
Pandey, & Chauhan, 2006). The ratio of glycinin to b-conglycinin
has an important effect on the quality of soymilk and soy protein-
based products (i.e. yield, stability) (Ji, Cai, & Chang, 1999; Tezuka,
Taira, Igarashi, Yagasaki, & Ono, 2000; Tezuka, Yagasaki, & Ono,
2004). In particular, for soymilk, samples containing group I
(A1, A2) of glycinin have more particles than those without group I
(Tezuka et al., 2000).
Little is known about the effect of soybean’s subunit composi-
tion on the physico-chemical properties of soymilk. The aim of this
study was to first confirm the effect of protein composition on the
structure of soymilk made from different soybean genotypes, and
then to highlight the effect of heating and homogenization on the
stability and particle size distribution of soymilk.
2. Materials and methods
All chemicals and reagents were purchased from Sigma Chem-
ical (St Louis, MO, USA) or Fisher Scientific (Mississauga, ON,
Canada). Ultrapure water (Barnstead International, Model 02622,
Chicago, USA) was used for the preparation of soymilk and all buffer
solutions.
2.1. Selected soybean genotypes
Harovinton and eleven soybean lines lacking specific glycinin
and b-conglycinin subunits used in this study were provided by
Harrow Research Centre (Harrow, Ontario) of Agriculture and Agri-
Food Canada (Poysa et al., 2006). The subunit null soybean geno-
types were developed from crosses and back-crosses between
Harovinton (Buzzell, Anderson, Hamill, & Welacky, 1991) and
‘‘Iwate-1’’ which lacks all glycinin subunits and ‘‘Iwate-3’’ which
lacks the a0 subunit of b-conglycinin and all glycinin subunits.
Table 1 summarizes the differences in protein subunit composition,
and the ratio of glycinin to b-conglycinin in the twelve soybean
lines. The values were obtained from Near Infra-Red (NIR) and
SDS-PAGE electrophoresis analysis (Poysa & Woodrow, 2002).
Soy protein patterns of Harovinton and eleven null soybean
genotypes were determined by SDS-PAGE electrophoresis (Fig. 1)
using 4 g/100 g stacking gel and 12.5 g/100 g running gel in a Bio-
Rad mini-protein electrophoresis system (Bio-Rad Labratories,
Hercules, CA) at a constant voltage of 200 V. The proteins were
extracted from defatted soybean samples (6 mg) in (420 mL) buffer
containing 50 mmol/L Tris-HCl, 5 mol/L Urea, 10 g/L SDS, and 40 g/L
2-mercaptoethanol. After 1 h of incubation at room temperature,

V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12

Lipoxygenase
α'
α

β
A3
A4
A1, A2

Basic

Fig. 1. SDS-PAGE electrophoresis gel of Harovinton and eleven null soybean genotypes differing in their protein subunit composition. The lanes are marked at the top as follows: V1
(Harovinton); V2 (null A3); V3 (null a0 , A4, A5); V4 (null a0 , A3); V5 (null A1, A2, A4, A5); V6 (null A3, A4, A5); V7 (null a0 , A3, A4, A5); V8 (null a0 , A1, A2, A3); V9 (null 11S); V10 (null 11S);
V11 (null a0 , 11S); V12 (null A1, A2).
 A. Malaki Nik et al. / LWT - Food Science and Technology 42 (2009) 1245–1252 1247

420 mL of electrophoresis sample buffer containing 125 mmol/L Tris-

4.53  0.47c,d,e
5.86  0.10a,b,c

4.61  0.16a,b,c

6.73  0.28c,d*

5.29  0.27d,e
5.45  0.31a,b

5.94  0.18d,e
6.27  0.15b,c

5.80  0.18b,c
6.54  0.66b*
7.08  0.20a*

5.24  0.45b

8.20  0.02e
8.19  0.14d
8.25  0.20f
V12: A1, A2
HCl, 5 mol/L urea, 10 g/L SDS, 200 g/L glycerol and 10 g/L bromo-
phenol blue were added. The solution was heated at 95  C for 5 min
and centrifuged at 5000 g for 4 min using an Eppendorf centrifuge
(Brinkmann Instruments, Westbury, NY). Aliquots (6 mL) were then

4.45  0.44a,b,c,d

4.67  0.40b,c,d,e
loaded onto the gels.
8.70  0.25c,d,e

6.78  0.38c,d*

5.12  0.32d,e
6.54  0.24a,b

8.81  0.29b,c

5.89  0.28b,c
5.78  0.07b,c

8.80  0.37c,d

5.92  0.12d,e
5.50  0.11a,b

6.77  0.60b*

4.89  0.24b*
7.39  0.33a*
After the run, gels were immediately stained using Coomassie
V11: a0 , 11S

blue R-250 for 30 min and destained with a destaining solution

composed of 45 mL/100 mL ultrapure water, 45 mL/100 mL meth-
anol and 10 mL/100 mL acetic acid, for 2 h with two changes, and
then destained overnight with a destaining solution containing

5.59  0.34c,d,e
5.36  0.22a,b,c

6.29  0.11c,d,e
7.09  0.07b,c*
6.56  0.28a,b

6.08  0.59b,c

9.09  0.22b,c
4.88  0.31a,b

5.32  0.41a,b
8.93  0.12b,c

7.02  0.34b*
7.36  0.29a*

5.53  0.49b
9.18  0.32b
6.17  0.26a

22.5 mL/100 mL methanol and 50 mL/100 mL acetic acid. Gels were

V10: 11S

scanned using a SHARP JX-330 scanner (Amersham Biosciences,

Quebec, QC, Canada).
8.60  0.12c,d,e,f

6.27  0.20c,d,e
5.90  0.28a,b,c

5.20  0.43a,b,c
5.13  0.17a,b,c

7.09  0.23b,c*

5.90  0.46c,d
6.28  0.24c,d

8.67  0.07c,d

5.93  0.09b,c

8.75  0.19c,d
7.22  0.35a*

6.80  0.10b*

2.2. Soymilk preparation

5.55  0.43b
4.97  0.38a
V9: 11S

Soymilk was prepared according to the procedure of Mullin et al.

(2001) with slight modifications (Malaki Nik et al., 2008a). Washed
soybeans were weighed (100 g) and soaked in excess ultrapure
V8: a0 , A1, A2, A3

5.86  0.09a,b,c
6.23  0.08c,d*

water overnight at room temperature. After draining and rinsing

8.35  0.25d,e

4.37  0.45d,e
8.43  0.25c,d
5.45  0.19a,b

4.26  0.16c,d
6.76  0.23b*

6.35  0.31d*

5.00  0.26e*
8.39  0.15e,f

5.59  0.02c*
7.07  0.14a*

4.95  0.34b

5.72  0.13e

with cold water and draining off excess water, soaked soybeans
were re-weighed to determine the amount of water absorbed by
Total solids contents (g/100 g) of soymilk (SM) and different supernatant fractions made from Harovinton and eleven null soybean genotypes.

the beans. The water uptake was calculated by dividing the weight
of the soaked beans by the initial weight of the dry beans. The
Within a genotype and a treatment, means with * are significantly different at p < 0.05 (compared to the previous centrifugation step).
V7: a0 , A3, A4, A5

amount of additional water needed to obtain a ratio of 18:1 water to

6.38  0.36b,c,d
9.05  0.27b,c,d

8.68  0.42c,d,e

4.47  0.47c,d,e
5.29  0.20d,e*
4.36  0.28c,d
5.83  0.41c,d

6.09  0.51b,c
4.67  0.35e,f

6.92  0.68b*
6.13  0.13b*

5.43  0.64b

protein was then calculated by subtracting the amount of absorbed

9.17  0.33b

7.53  0.35b
5.30  0.14d

water. The ratio was calculated on a protein basis, so the soymilk

samples would be equal in protein, and corresponded to a water to
bean ratio in the range from 7.5 to 8.2. The amount of protein
present in the soymilk depended on the genotype (Malaki Nik,
5.07  0.44a,b,c,d
8.54  0.17c,d,e,f

4.97  0.09c,d,e

6.12  0.36c,d,e
8.73  0.27c,d,e
4.57  0.15a,b,c

6.72  0.30c,d*
V6: A3, A4, A5

5.82  0.29c,d

5.82  0.52c,d
8.67  0.07c,d

Tosh, Poysa, Woodrow, & Corredig, 2008b), but had an average of

6.20  0.52b*

6.30  0.46b*
5.36  0.02d

5.30  0.22b
5.56  0.36c

Duncan multiple range test. Means along a row with different superscripts are significantly different p < 0.05.

46 g/L with 49 g/L for Harovinton, the variety with the highest
protein content.
Approximately half of the additional water needed was then
added to the soybeans at 20  C and blended (commercial blender,
V5: A1, A2, A4, A5

WARING, New Hartford, CT) at high speed for 3.5 min. The
8.88  0.24b,c,d
8.75  0.28c,d,e

7.28  0.44b,c*

6.60  0.20a,b
6.86  0.05b,c
5.99  0.18a,b

8.87  0.14c,b

6.13  0.17b,c
7.05  0.66b*

5.47  0.61a*
7.49  0.18a*

remaining water was heated to 60  C and added to the slurry for

5.49  0.09b
5.64  0.06a
6.95  0.60a

4.99  0.29a

better protein extraction, and the whole mixture was blended at

high speed for another 30 s. A two-step filtration was then carried
out to remove the coarse material (okara, which is mainly
composed of fiber material): the slurry was filtered through a juice
6.07  0.22c,d,e*
5.63  0.29c,d,e
8.47  0.16d,e,f

4.19  0.07c,d*

6.80  0.12c,d*
5.69  0.18b,c*

8.38  0.39d,e
8.64  0.35c,d
4.37  0.34f,g

6.59  0.20b*

4.08  0.01e*
4.94  0.19b*

extractor (Juiceman, professional series 211, Korea) and the okara

5.03  0.17c*

4.56  0.25e
4.78  0.17e
V4: a0 , A3

collected and passed through the juice extractor again. The soymilk
Genotypes lacking various glycinin and b-conglycinin subunits

obtained from the juice extractor was filtered through cheesecloth

to remove fines (Mullin et al., 2001). The soymilk was then
Data are the average of at least three independent experiments.

collected for analysis (unheated soymilk).

4.59  0.42b,c,d,e*
6.38  0.28b,c,d
8.52  0.24c,d,e

A portion of soymilk was then divided in test tubes (10 mL each

6.97  0.26b,c*
8.41  0.26c,d

5.96  0.57b,c
5.90  0.21b,c
V3: a0 , A4, A5

8.33  0.32e,f

6.63  0.35b*
4.77  0.04c*

3.97  0.16d*
5.38  0.39b
4.19  0.22g
4.65  0.07e
4.53  0.11e

tube) and heated in boiling water (95–100  C) for 7 min, according

to a previously published procedure (Ono et al., 1991) and cooled
immediately to room temperature in an ice-waterbath (heated
soymilk). A portion of the heated soymilk was passed through
5.68  0.61c,d,e*
5.12  0.28b,c,d

a valve homogenizer (Emulsiflex C5, Avestin, ON, Canada) at

4.67  0.38a,b,c

5.19  0.22a,b,c
9.34  0.27a,b
6.18  0.25b*

7.17  0.46b*

7.61  0.19b*
5.61  0.24d
9.19  0.05b

9.29  0.28b

6.96  0.38b
6.25  0.35b
5.37  0.24b
5.39  0.10d

69 MPa at room temperature for four passes (heated-homogenized

soymilk).
V2: A3

Selective centrifugation steps were applied to obtain four

different supernatant phases by centrifuging the soymilk at 8000 g
SN4 4.84  0.20b,c,d,e*
Heated–homogenized

(SN1), 15,000 g (SN2), 40,000 g (SN3), and 122,000 g (SN4)

V1: Harovinton

SN4 4.81  0.35a,b*

SN4 4.84  0.24d,e
SN2 5.78  0.36c,d

following a previously published procedure (Ono et al., 1991) at

SN1 5.92  0.38b*

SN1 8.13  0.50a*

SN3 5.6  0.32c,d
9.71  0.43a

9.80  0.30a
SN1 8.56  0.56a
SN2 7.98  0.81a
SN3 7.30  0.59a
9.83  0.37a

SN3 6.67  0.13a

SN2 7.66  0.17a
Soybean Cultivars

20  C for 30 min using a refrigerated ultracentrifuge (Optima LE-

80 K Beckman Coulter, CA, USA). The amount of total solids present
Unheated

in the soymilk and the various supernatant fractions were deter-

Table 2

Heated

mined with the forced air oven method. The soymilk was weighed
SM

SM

SM

in dry aluminum pans containing dried sea-sand as a dispersing

1248 A. Malaki Nik et al. / LWT - Food Science and Technology 42 (2009) 1245–1252

agent (Fisher Scientific) and dried for 24 h in a forced air oven
(Fisher Scientific, Mississauga, ON, Canada) at 102  C.
2.3. Particle size distribution
The particle size distribution and average particle diameter
d3,2 ¼ (S nid3i /S nid2i ) and d4,3 ¼ (S nid4i /S nid3i ), of the soymilks and
various supernatant fractions were measured with a laser light
scattering instrument (Mastersizer S, Malvern Southborough, MA),
using 1.46 as refractive index of the scatterers in soymilk and 1.333
as a refractive index of dispersant (water).
2.4. Statistical analysis
General linear model (GLM) and Duncan multiple range test
(SAS version 9.1) were carried out to determine significant differ-
ences. All results presented are the average of at least three inde-
pendent replicates. The statistical significance of genotypes,
treatments, centrifugation steps, and their two-way interactions
was tested using the three-way interaction (genotypes 
treatments  centrifugation) as the error term. Differences were
considered significant at p < 0.05.
3. Results and discussions
3.1. Seed composition
The electrophoretic results (Fig. 1) clearly demonstrate the
differences in the protein composition of the twelve lines (see also
Table 1). Harovinton (V1) contains all glycinin and b-conglycinin
subunits, while the five null lines V3, V4, V7, V8, and V11 lack the
a0 subunit of b-conglycinin. The null lines V5, V8, and V12 do not
contain the polypeptide chains of group I (A1, A2), while lines V3,
V5, V6, and V7 are missing the group IIa (A4, A5). The lines V2, V4,
V6, V7 and V8 lack group IIb (A3); lines V9, V10, and V11 lack all
glycinin subunits.
3.2. Total solids content
Genotype, treatment, and centrifugation each had a significant
effect on the total solids content (Table 2). The genotype by treat-
ment and treatment by centrifugation effects were much larger
than the genotype by centrifugation effect (data not shown). The
soymilks prepared from Harovinton and null soybean lines had
a total solids content ranging from 9.8 (g/100 g) for Harovinton to
8.2 (g/100 g) for V12 (null A1, A2). Soymilks made from Harovinton
and V2 (null A3) had higher total solids content compared to other
lines. The variation of the total solids content of soymilks could be
due to differences in hydration of the soybeans during soaking, or
due to different solubility and extractability of some of the
components. It is important to note that soymilk was produced on
18:1 water to protein ratio, and lines with less protein may contain
more non-protein dry matter. For all genotypes, there were no
significant effects of treatment (unheated, heated, and heated–
homogenized) on the total solids contents of soymilks before
centrifugation (Table 3, first row). For lines V6 (null A3, A4, A5), V9
and V10 (both null 11S), there was also no significant treatment
effect at any centrifugation step, while for most other lines treat-
ment significantly affected total solids content after the first and
second centrifugation steps.
The variability among genotypes for loss of total solids with
centrifugation was much greater for unheated soymilk than for
heated or heated–homogenized samples (Fig. 2). This was found at
each centrifugation step, although it was greatest after the first
centrifugation. The variability among genotypes for protein losses
with centrifugation has been previously reported (Malaki Nik et al.
2008b).
Unheated soymilks containing group I (A1, A2) (Harovinton, V2,
V3, V4, and V7) showed larger losses in total solids after each
centrifugation compared to lines without group I (Fig. 2). After the
third centrifugation, however, there was no further significant
decrease in the amount of residual solids in unheated soymilk for
any genotype (Table 2). An increase in solids recovered in the
supernatant after centrifugation of heated and heated–
homogenized samples was observed for soymilks having group I of
glycinin, compared to those genotypes lacking these subunits.
There were significant differences in the amount of solids recovered
depending on genotype after the fourth centrifugation step, in SN4,
regardless of treatment (Table 2 and Fig. 2D).
It is important to note that the first centrifugation at 8000 g
precipitates not only large protein aggregates, but also insoluble
carbohydrate material.
After the third centrifugation step (Fig. 2C) in most cases, heated
samples had larger percent losses in total solids content compared
to heated–homogenized samples from the same genotypes.
Genotypic effects on colloidal stability were greater in unheated
and heated–homogenized soymilks, compared to heated soymilks
after the third centrifugation step (Fig. 2C).
Fig. 2D illustrates the effect of genotype and treatment on the
soluble fraction remaining after the fourth centrifugation step
(at 122,000 g). While within each treatment (unheated, heated and
heated–homogenized) the protein composition (genotype) had
a significant effect, treatment effects were significant only for V8,
V11, and V12 (Table 3). In addition, unheated samples made from
glycinin-null (V9, V10, V11) or group I null (V5, V8, V12) genotypes
showed a higher solids recovery compared to lines containing
glycinin.
3.3. Particle size distribution
To better characterize the colloidal stability of the samples, and
explain the differences in protein losses depending on the subunit
composition, particle size analysis was performed. There were
major differences in the particle size distribution of unheated,
heated, and heated–homogenized soymilks obtained from Har-
ovinton and eleven null soybean genotypes before centrifugation

Table 3
Mean square values for treatment effects (unheated, heated, and heated–homogenized) on total solid content for the Harovinton and eleven null genotypes.

Genotypes lacking various glycinin and b-conglycinin subunits

V1: V2: V3: V4: V5: V6: V7: V8: V9: V10: V11: V12:
Harovinton A3 a0 , A4, A5 a0 , A3 A1, A2, A4, A5 A3, A4, A5 a0 , A3, A4, A5 a0 , A1, A2, A3 11S 11S a0 , 11S A1, A2
SM 0.921 ns 0.728 ns 0.647 ns 0.624 ns 0.763 ns 0.496 ns 0.293 ns 0.908 ns 0.476 ns 0.497 ns 0.900 ns 0.864 ns
SN1 0.001* 0.004* <0.0001* <0.0001* 0.562 ns 0.367 ns 0.024* 0.028* 0.184 ns 0.322 ns 0.236 ns 0.361 ns
SN2 0.004* 0.007* 0.000* 0.001* 0.084 ns 0.214 ns 0.365 ns 0.000* 0.103 ns 0.373 ns 0.021* 0.039*
SN3 0.005* 0.563 ns 0.005* 0.005* 0.000* 0.180 ns 0.893 ns 0.009* 0.524 ns 0.151 ns 0.009* 0.093 ns
SN4 0.987 ns 0.157 ns 0.099 ns 0.272 ns 0.191 ns 0.136 ns 0.628 ns 0.005* 0.383 ns 0.208 ns 0.024* 0.029*

ns: non-significant and *: significant at p < 0.05, respectively.

heated–homogenized soymilk was less than half the size of the

largest average particle size (D3,2) among the genotypes (Table 4).
For both these genotypes, average particle size (D3,2) for heated or

(Fig. 3). Unheated soymilk made from Harovinton and V4 had the

samples).
ences p < 0.05 (means compared within unheated, heated and heated–homogenized
fourth centrifugation step (SN4) (D). Different superscripts identify significant differ-
second centrifugation step (SN2) (B), the third centrifugation step (SN3) (C) and the
homogenized (hatched bar) samples, after the first centrifugation step (SN1) (A), the
supernatant fractions of unheated (solid bar), heated (white bar) and heated–
Fig. 2. Loss in total solids (as g/100 g of the initial total solids in the soymilk) for

A
Total Solid Loss (g/100g) Total Solid Loss (g/100g) Total Solid Loss (g/100g) Total Solid Loss (g/100g)

10

20

30

40

50

60

10
15
20
25
30
35
40
45
50

10
15
20
25
30
35
40
45
50

10
15
20
25
30
35
40
45
0

0
5

0
5

0
5
a aa

a
b

V1
V1

V1
V1

c
d

b
c

b
d

a
b

b
bb

V2
V2

V2
V2

b
a

a
b
b

a
a

a
V3
V3

V3
V3

b
a

b
d

a
b

a
V4
a

V4

V4
V4

a
b

b
aa

a
d

d
d

V5
c

V5

V5
V5

b
b
c

a
d
c

b
c

ca
V6
b

V6

V6
V6

a
b
b

a
c

a
c

b bb

ba
V7

b
V7
a

V7
V7

b
a

A. Malaki Nik et al. / LWT - Food Science and Technology 42 (2009) 1245–1252
b

b
b

c
d

V8

d
V8
c

V8
V8

b
a
bb
a

a
a
b

c
d

V9 V10 V11 V12

d
V9 V10 V11 V12
c

V9 V10 V11 V12

V9 V10 V11 V12

b
c
c

b
c

a
c

c
d

d
c

a
b b

b
b

a
c

c
d

d
c

aa
a

ba
a

a
b

c
d

d
c

a
c b

b
cb

a
Table 4
Average particle size (D3,2, mm) values of soymilk and different supernatant fractions made from Harovinton and eleven null soybean genotypes.

Soybean Cultivars

Genotypes lacking various glycinin and b-conglycinin subunits

V1: Harovinton V2: A3 V3: a0 , A4, A5 V4: a0 , A3 V5: A1, A2, A4, A5 V6: A3, A4, A5 V7: a0 , A3, A4, A5 V8: a0 , A1, A2, A3 V9: 11S V10: 11S V11: a0 , 11S V12: A1, A2
Unheated
SM 0.38  0.08a 0.26  0.01b 0.28  0.03b 0.35  0.03a 0.18  0.01c,d 0.19  0.01c,d 0.21  0.02c 0.15  0.01d 0.16  0.01d 0.18  0.02c,d 0.14  0.00d 0.16  0.02d
SN1 _ 0.15  0.01b _ _ 0.17  0.02a 0.12  0.02c 0.13  0.02c 0.12  0.00c 0.12  0.01c 0.12  0.00c 0.12  0.00c 0.12  0.00c

Heated
SM 0.17  0.00a 0.17  0.00a 0.16  0.00b 0.17  0.00a 0.15  0.00c 0.15  0.00c 0.16  0.00b 0.13  0.00e 0.14  0.00d 0.14  0.00d 0.13  0.00e 0.13  0.02e
SN1 0.11  0.00b 0.11  0.00b 0.11  0.00b 0.12  0.01a 0.12  0.00a 0.12  0.00a 0.12  0.00a 0.11  0.00b 0.12  0.00a 0.11  0.00a 0.12  0.00a 0.12  0.00a

Heated–homogenized
SM 0.13  0.00a 0.13  0.01a 0.13  0.00a 0.13  0.01a 0.12  0.01b 0.12  0.01b 0.12  0.01b 0.12  0.00b 0.12  0.00b 0.12  0.00b 0.12  0.00b 0.12  0.00b
SN1 0.11  0.01b 0.14  0.03a 0.12  0.01b 0.12  0.00b 0.12  0.00b 0.12  0.00b 0.11  0.00b 0.12  0.00b 0.12  0.00b 0.11  0.01b 0.12  0.01b 0.12  0.00b

Data are the average of at least three independent experiments.

Duncan multiple range test. Within a rows, means with different superscripts are significantly different at p < 0.05.

1249
1250 A. Malaki Nik et al. / LWT - Food Science and Technology 42 (2009) 1245–1252

10 10
V1 V2
8 8

Volume (%)

Volume (%)
6 6

4 4

2 2

0 0
0.01 0.1 1 10 100 0.01 0.1 1 10 100
Particle Size (µm) Particle Size (µm)

10 10
V3 V4
8 8
Volume (%)

Volume (%)
6 6

4 4

2 2

0 0
0.01 0.1 1 10 100 0.01 0.1 1 10 100
Particle Size (µm) Particle Size (µm)

10 10
V5 V6
8 8
Volume (%)

Volume (%)

6 6

4 4

2 2

0 0
0.01 0.1 1 10 100 0.01 0.1 1 10 100
Particle Size (µm) Particle Size (µm)

Fig. 3. Particle size distribution of unheated (,), heated (C), and heated–homogenized (6) soymilks made from Harovinton and eleven null soybean genotypes. The soybean
genotypes are marked as follows: V1 (Harovinton); V2 (null A3); V3 (null a0 , A4, A5); V4 (null a0 , A3); V5 (null A1, A2, A4, A5); V6 (null A3, A4, A5); V7 (null a0 , A3, A4, A5); V8 (null a0 , A1,
A2, A3); V9 (null 11S); V10 (null 11S); V11 (null a0 , 11S); V12 (null A1, A2).

unheated soymilk. The average particle size of the supernatants
after the first centrifugation step at 8000 g consistently decreased
relative to the soymilk, with the effect being greatest for the
unheated treatment (Table 4). Supernatant from subsequent
centrifugations did not show enough scattering signal, therefore
could not be measured.
Unheated soymilks showed in general a larger average particle
size than heated and heated–homogenized samples. The subunit
composition had a significant effect on the particle size distribu-
tion. Unheated soymilks made from V1 (Harovinton), V4 (null a0 ,
A3), V3 (null a0 , A4, A5), and V2 (null A3) showed a significantly
different bimodal distribution of particles, with a diameter (D3,2)
ranging from 0.38  0.08 mm to 0.26  0.01 mm and a population
>1 mm. Unheated samples made from V5 (null A1, A2, A4, A5), V8
(null a0 , A1, A2, A3), V9 (null 11S), V10 (null 11S), V11 (null a0 , 11S)
and V12 (null A1, A2) showed a monomodal distribution of particles
with a significantly smaller D3,2 about 0.16 mm. Unheated soymilks
obtained from V6 (null A3, A4, A5), and V7 (null a0 , A3, A4, A5)
showed an average D3,2 of about 0.2 mm, with a monomodal
distribution. Subunit composition and heat treatment had a signif-
icant effect on the average particle size (D3,2) values of soymilks
(Table 4). Soymilks made from soybean seeds lacking glycinin (V9,
V10, V11) and group I (A1, A2) of glycinin (V8, V12) show a signifi-
cantly smaller particle distribution compared to soymilks con-
taining group I (Harovinton, V3). Among glycinin subunits, group I
(A1, A2) has the most significant effect on the particle size distri-
bution of unheated soymilk followed by group IIb and group IIa.
Unheated samples prepared from soybean lines containing group
IIb show a larger particle size distribution compared to the samples
containing group IIa.
Heat treatment has a significant effect on average particle size
and particle size distribution of soymilks made from lines con-
taining glycinin or group I of glycinin. Heated soymilk had signifi-
cantly smaller particle size distribution (monomodal) with D3,2
values of about 0.15–0.17 mm. However, heated soymilks made
from soybean seeds lacking glycinin (V9, V10, V11) or group I (V5,
V8, V12) of glycinin did not show a significant difference in size
compared to the unheated sample of same line. These results
confirm that heat treatment solubilizes the large native protein
aggregates (protein bodies) present in the soymilks made from
lines rich in glycinin, and for this reason, the particle size decreases
significantly after heating. The effect of heating on the decrease in
the average particle size was previously shown (Malaki Nik et al.,
2008a), however, this work demonstrates that genotypic differ-
ences also play a major role in the particle size distribution. Based
on the unheated soymilk data, group I of glycinin (11S) strongly
affects the size and solubility of the protein bodies.
For certain soybean lines, the particle size distribution of soy-
milk can be decreased even further with subsequent homogeni-
zation. Heated–homogenized soymilk samples show in most cases
a slight shift in the average particle size, and a monomodal particle
size distribution with an average diameter (D3,2) of about 0.12 mm
for all soymilks. The particle size distribution caused by genotype
differences could be minimized by processing.
 A. Malaki Nik et al. / LWT - Food Science and Technology 42 (2009) 1245–1252 1251

10 10
V7 V8
8 8

Volume (%)

Volume (%)
6 6

4 4

2 2

0 0
0.01 0.1 1 10 100 0.01 0.1 1 10 100
Particle Size (µm) Particle Size (µm)

10 10
V9 V10
8 8
Volume (%)

Volume (%)
6 6

4 4

2 2

0 0
0.01 0.1 1 10 100 0.01 0.1 1 10 100
Particle Size (µm) Particle Size (µm)

10 10
V11 V12
8 8
Volume (%)

Volume (%)

6 6

4 4

2 2

0 0
0.01 0.1 1 10 100 0.01 0.1 1 10 100
Particle Size (µm) Particle Size (µm)

Fig. 3. (continued).

The particle size distribution of the residual fractions after the
first centrifugation at 8000 g (SN1) is summarized in Table 4. In
unheated samples, SN1 obtained from soymilks made of Har-
ovinton, V3 (null a0 , A4, A5), and V4 (null a0 , A3) had a low signal to
noise ratio and could not be measured by light scattering, meaning
that most of the aggregates were lost during centrifugation.
Whereas the unheated samples made from null genotypes V2 (null
A3) and V5 (null A1, A2, A4, A5) showed a monomodal particle
distribution with a D3,2 of about 0.16 mm. All the other samples
showed a monomodal distribution of sizes with average diameter
(D3,2) of about 0.13  0.02 mm for unheated, heated and heated–
homogenized samples. In all samples, particles >1 mm present in
soymilk were lost during centrifugation at 8000 g. This may suggest
that these larger particles may be responsible for product
sedimentation.
The results obtained in the sequential centrifugation steps are
in agreement with those previously published by Ono et al. (1991).
They have reported that unheated soymilk consists of larger
particles and heat treatment can decrease the particle size
distribution. However, soymilk prepared from Harovinton variety
was the only soymilk containing all the protein subunits, and
therefore the only sample comparable to previous literature. In
addition, it is important to note that the particle size distribution
reported earlier is much smaller than that measured in all the
samples shown in Fig. 3 and Table 4. This might be due to the use
by Ono et al. (1991) of differential centrifugation to determine
particle size distribution while light scattering technique was
carried out in this study.
Particle size distribution, however, does not fully explain the
solid losses seen during the centrifugation experiments, indicating
that although of similar size, the composition of the particles also
affects the colloidal stability of the soymilk.
4. Conclusions
The results presented in this study clearly demonstrate that
genotypic changes in protein subunit composition strongly affect
the particle size distribution and, most likely the stability of soy-
milk. Heat treatment and homogenization also have a significant
effect on physico-chemical properties of soymilk, and in all cases
improve the particle size distribution of soymilk. Soymilks made
from lines containing glycinin or group I (A1, A2) of glycinin showed
larger losses of solids compared to lines null in group I (A1, A2).
Unheated soymilks made from glycinin-null genotypes showed
smaller and more uniform particle size distribution, while soymilks
made from genotypes having glycinin or group I (A1, A2) of glycinin
showed a larger average particle size and in some cases a bimodal
particle distribution. The effect of heat treatment was greater for
lines containing group I, Harovinton (V1), V2, V3, V4, V6, and V7. On
the other hand, samples containing high glycinin/b-conglycinin
ratios showed a decrease in particle size distribution after heating.
The use of different soybean lines with a wide range of subunit
composition to study the effect of protein composition on pro-
cessing is of great importance, as it will result in the development
of genotypes tailored for specific processing applications. However,
beyond the industrial application, this research also reveals a large
1252 A. Malaki Nik et al. / LWT - Food Science and Technology 42 (2009) 1245–1252
amount of information can be collected about the behavior of
glycinin and b-conglycinin depending on their subunit composi-
tion. By studying the effect of composition directly from the seed, it
is in fact possible to eliminate any effects caused by purification
procedures and processing history on the proteins.
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