LWT - Food Science and Technology 85 (2017) 58e65

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LWT - Food Science and Technology

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Effects of organic acid coagulants on the physical properties of and

chemical interactions in tofu
Feng-Hong Cao, Xing-Jiang Li, Shui-Zhong Luo, Dong-Dong Mu, Xi-Yang Zhong,
Shao-Tong Jiang, Zhi Zheng*, Yan-Yan Zhao**
School of Food Science and Engineering, Key Laboratory for Agricultural Products Processing of Anhui Province, Hefei University of Technology, Hefei
230009, China

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

Article history: To improve tofu quality, the effects of using 0.12e0.18 g/100 mL citric acid, l-(-)-malic acid, or tartaric acid
Received 16 January 2017 as coagulation agents were investigated. The results showed that storage modulus, hardness, water-
Received in revised form holding capacity, non-freezable water content, and in-gel hydrophobic interactions were optimal
1 July 2017
when tofu was prepared with 0.14 g/100 mL organic acids, but deteriorated when more than 0.16 g/
Accepted 3 July 2017
Available online 4 July 2017
100 mL was used. Slower acidication during gelling in the presence of 0.14 g/100 mL organic acid
promoted proteineprotein hydrophobic interactions, resulting in a higher elastic modulus. Moreover, the
tofu products showed higher water-holding capacity and non-freezable water content, as well as more
Keywords:
Citric acid
compact gel microstructures. Notably, tofu prepared with citric or l-(-)-malic acid was of better quality
l-(-)-malic acid than tofu prepared with tartaric acid. Collectively, the results demonstrate that soft or rm tofu with
Tartaric acid desirable physicochemical properties can be prepared with organic acids if acidication is adequately
Tofu controlled during gelation.
Gelation 2017 Elsevier Ltd. All rights reserved.

1. Introduction rapid release of magnesium chloride typically results in a coarse

Tofu has historically been popular in Asia as an excellent source
of protein and essential amino acids, and has been growing in
popularity worldwide (Cai, Chang, Shih, Hou, & Ji, 1997; Zhu, Wu,
Saito, Tatsumi, & Yin, 2016). Accordingly, tofu has been exten-
sively studied for many years as a form of soybean protein matrix.
Tofu is produced by soaking, milling, ltering, cooking, coagulating,
and pressing soybeans, with coagulation being the most important
step (Rui et al., 2016; Zhang, Li, Feng, & Dong, 2013). During
coagulation, soy proteins aggregate via proteineprotein and pro-
teinewater crosslinking into gels with a random or a regular hon-
eycomb structure (Zhang et al., 2013). To produce rm, silken, and
soft tofu, coagulation is induced using salt, enzymes, and acid,
respectively.
Calcium sulfate and magnesium chloride are widely used as salt
coagulants, although residual undissolved calcium sulfate in tofu or
* Corresponding author. School of Food Science and Engineering, Hefei University
of Technology, 193 Tunxi Road, Hefei City 230009, Anhui Province, China.
** Corresponding author.
E-mail addresses: zhengzhi@hfut.edu.cn (Z. Zheng), zhaoyanyan@hfut.edu.cn
(Y.-Y. Zhao).
texture, an indicator of poor quality (Meng et al., 2015; Ting, Kuo,
Lien, Li, & Sheng, 2009). Microbial transglutaminase is typically
used to enzymatically crosslink soy proteins into tofu with low gel
hardness, although this process is time-consuming (Chang, Shiau,
Chen, & Lin, 2011; Tang, Li, Wang, & Yang, 2007). Finally, glu-
cono-d-lactone (GDL) has been widely studied as an acidifying
agent to produce tofu with a homogeneous network (Bi, Li, Wang, &
Adhikari, 2013; Chang, Li, Wang, Bi, & Adhikari, 2014; Schuldt, Raak,
Jaros, & Rohm, 2014). Of note, proteins gel at pH 5.6 to 5.8
depending on the soy variety used, regardless of the GDL concen-
tration (Malaki Nik, Alexander, Poysa, Woodrow, & Corredig, 2011).
Acid-induced gelation is driven by interactions that are formed
when hydrophobic amino acids in soy proteins are exposed
following the formation of hydronium ions that neutralize repul-
sive charges and reduce steric repulsion (Kohyama, Sano, & Doi,
1995; Grygorczyk & Corredig, 2013). These interactions are pre-
dominantly noncovalent and include salt bridges, hydrogen bonds,
and hydrophobic and Van der Waals interactions (Kohyama et al.,
1995; Lee & Kuo, 2011). However, little attention has been paid to
organic acids, including citric acid, l-(-)-malic acid, and tartaric acid,
as mild, nutritionally harmless, and water-soluble coagulation
agents. These organic acids were initially identied in fruits and

http://dx.doi.org/10.1016/j.lwt.2017.07.005
0023-6438/ 2017 Elsevier Ltd. All rights reserved.
 F.-H. Cao et al. / LWT - Food Science and Technology 85 (2017) 58e65
vegetables and are currently extensively used in the food industry
(Huang, Xu, Zhang, Xue, & Chen, 2007; DeNobili et al., 2016).
Organic acids induce signicant gelation at concentrations suitable
for relatively slow acidication, a desirable property, as accelerated
acidication typically results in weaker gels (Jacob, No bel, Jaros, &
Rohm, 2011).
The objective of this study was to investigate the effects of
organic acid (citric acid, l-(-)-malic acid, and tartaric acid) treat-
ments on the soymilk gelation process and tofu quality. The relative
contribution of various chemical interaction types, including ionic
bonds, hydrogen bonds, and hydrophobic interactions, to tofu
properties was evaluated. In addition, the feasibility of organic acid
in the production of tofu with desirable physicochemical properties
was assessed. Texture, water-holding capacity, and water distri-
bution were assessed to measure gel strength, rigidity, and water
state. pH, rheological properties, and in-gel chemical interactions
were analyzed to investigate gel nucleation and formation. The gel
microstructure was evaluated to investigate its relationship with
gel macro-properties to allow quality control of organic acid-
induced tofu gel.
2. Materials and methods
2.1. Materials
Zhonghuang 13 soybeans, determined to contain 45.80% total
protein, 18.66% crude fat, 8.80% moisture, and 4.65% ash by AOAC
methods were purchased from Xiangfeng Seed Station (Shandong,
China), and stored at room temperature under 8e9% humidity.
Food-grade citric acid, l-(-)-malic acid, tartaric acid, and GDL were
purchased from Luyu Foods Industry Co. (Shanghai, China), while
food-grade anti-foaming agent was obtained from Yingli Foods
Industry Co. (Shanghai, China). All other chemicals were analytical
grade.
2.2. Preparation of soymilk
Soymilk was prepared according to Kamizake, Silva, and
Prudencio (2016), with some modication. Soybeans were rinsed,
soaked in distilled water for 18 h at 4
C at a ratio of 1:3 g/g, and
ground for 3 min with distilled water at a ratio of 1:7 g/g in a JS30-
230 mill (SUPOUR, Zhejiang, China). The raw milk was mixed with
0.3 g anti-foaming agent, boiled for 5 min with constant stirring on
an electromagnetic oven (MAZUBA Co. Ltd., Jiangsu, China), ltered
through a 100-mesh screen, and quickly cooled to room tempera-
ture in an ice bath. Finally, the milk was diluted to obtain standard
soymilk with 8
brix and pH 6.6 0.1, and stored for 12 h at 4
C
prior to analysis.
2.3. Preparation of tofu
Soymilk (100 mL) was poured into a 200-mL beaker, heated to
80
C in a water bath, and coagulated for 30 min at 80
C, using
0.30 g/100 mL GDL or 0.12, 0.14, 0.16, or 0.18 g/100 mL citric acid, l-
(-)-malic acid, or tartaric acid as a coagulation agent. Tofu curds
were immediately transferred to a 7  7  7-cm mold lined with
cheese cloth and pressed at 8 g/cm2 for 30 min using an in-house
press to remove whey. Products were then stored at 4
C for 12 h
prior to analysis. pH was measured every 5 min using a handheld
pH meter in parallel samples processed and kept in identical
conditions.
2.4. Texture analysis
Tofu samples were cut into 1  1  1-cm cubes using a razor and
59
analyzed on a TA-XT texture analyzer (Stable Micro System Co. Ltd.,
Godalming, UK) according to Zhang et al. (2013). The samples were
compressed twice to 50% deformation using a P/36R probe, with
trigger force 5 g, pretest speed 5.0 mm/s, test speed 1.0 mm/s, and
post-test speed 10.0 mm/s. Hardness, springiness, cohesiveness,
and gumminess were determined (Zhang et al., 2013).
2.5. Determination of water-holding capacity
Approximately 3 g of each tofu sample was used to measure
water-holding capacity according to Hu et al. (2013), with some
modication. Samples were cut into 5  5  5-mm cubes, weighed
(Wt), and centrifuged at 5632 g for 20 min at 4
C in a CR21
centrifuge (Hitachi, Japan). Supernatants were discarded and re-
sidual liquids were carefully removed with dry lter paper. The
removed water was weighed (Wr), and water-holding capacity was
calculated according to equation (1):
WHC Wt  Wr =Wt  100 % (1)
2.6. Differential scanning calorimetry
Freezable water content was measured by differential scanning
calorimetry on a DSC-Q200 system (TA Instruments, USA) accord-
ing to Chen, Wei, and Zhang (2010), with some modication. A
small sample (5e10 mg) was heated from 60
C to 50
C at 5
C/
min under a 50-mL/min ow of nitrogen gas. Freezable water (Wf)
was calculated according to Yoshida, Hatakeyama, and Hatakeyama
(1993) as the ratio of the melting enthalpy per gram of wet sample
at 0
C to that of pure water. Non-freezable water (Wnf) was
determined using equation (2):
Wnf Wt  Wf (2)
where Wt is the total water content as measured by the oven-
heating method (Yoshida et al., 1993).
2.7. Dynamic rheological properties
Rheological behavior was analyzed according to Chang et al.
(2014), with some modication, using a DHR-3 rheometer (TA In-
struments, Leatherhead, UK) equipped with parallel 40-mm-thick
plates at 1-mm distance and thermo-controlled by a circulation
system. Samples were analyzed with strain amplitude sweeps to
determine the linear viscoelastic region, and storage (G0 ) moduli
were recorded at a constant frequency of 1 Hz and a strain ampli-
tude of 0.5%.
Soymilk (10 mL) was prepared at room temperature and mixed
with 0.12e0.18 g/100 mL organic acid or 0.30 g/100 mL GDL. A 2-mL
sample of the mixture was immediately injected between the
plates and equilibrated for 2 min. Gelation was monitored for
30 min using a small-amplitude oscillatory time sweep at 80
C.
Once completely gelled, the sample was cooled from 80
C to 25
C
at 5
C/min. Data were collected in triplicate, and a solvent trap was
used to cover sample edges and minimize evaporation during
measurements.
2.8. Chemical interactions
The contributions of various chemical interactions to gelation
and gel structure were assessed using modied procedures
described by Wang et al. (2017), in which powder samples are
dissolved in reagents that break formation of ionic bonds, hydrogen
60 F.-H. Cao et al. / LWT - Food Science and Technology 85 (2017) 58e65

bonds, or hydrophobic interactions. Briey, freeze-dried tofu

powder (0.6 g) was homogenized in 10 mL of reagent, stirred at 4
C
for 1 h, and centrifuged at 8800 g at 4
C for 20 min. Soluble
protein content was determined by the Bradford assay and
expressed as g/L of soluble protein in the homogenate. The reagents
used to break certain types of bonds were as follows: 0.05 moL/L
NaCl (PA); 0.6 moL/L NaCl (PB); 0.6 moL/L NaCl 1.5 moL/L urea
(PC); and 0.6 moL/L NaCl 8 moL/L urea (PD). Ionic bonds were
calculated as the difference between soluble tofu protein in PA and
PB, hydrogen bonds were calculated as the difference between
soluble tofu protein in PB and PC, and the hydrophobic interactions
were calculated as the difference between soluble tofu protein in PC
and PD. All reagents were prepared in 0.05 moL/L phosphate buffer
(pH 7.0).

2.9. Gel microstructure

Tofu gel microstructure was examined on a JSM-6490LV scan-

ning electron microscope (JEOL Ltd., Tokyo, Japan) according to a
modied method described by Lee and Kuo (2011). Samples were
cut into 3  3  1-mm cubes, freeze-dried, mounted to an SEM
plate using conductive carbon tabs, coated with gold, and imaged at
an accelerating voltage of 20 kV.

2.10. Statistical analysis

Data were collected in triplicate, and are reported as the

mean standard deviation (SD). Error bars in gures represent SDs.
Statistical signicance was assessed by ANOVA (p < 0.05) in SPSS
version 19.0. Figures were generated in Origin version 8.6.

3. Results and discussion

3.1. Acid-induced gelation

As can be seen in Fig. 1, the nal pH in soymilk acidied with

0.12e0.18 g/100 mL citric, l-(-)-malic, or tartaric acid, was reached
within 10 min, and was 5.60e5.32, 5.65e5.34, and 5.56e5.23,
respectively. The nal pH of soymilk gelled with 0.30 g/100 mL GDL
was 5.42, which is consistent with the ndings of Kohyama and
Nishinari (2002), who reported a pH range of 5.0e5.5 for soymilk
gels prepared with GDL. Similar data have been previously reported
for soymilk matrixes (Grygorczyk & Corredig, 2013; Liu, Chien, &
Kuo, 2013). Values were comparable among all samples, suggest-
ing that the nal soymilk pH was immaterial to the properties of
the nal tofu product, although the acidication rate was critical.
We note, however, that signicant differences in nal pH may
indeed result in differential properties of gels prepared with the
same organic acid (Grygorczyk & Corredig, 2013), and that different
acids lower the pH to various extents, even at the same
concentration.

3.2. Rheological properties of tofu prepared with organic acids

Dynamic rheological data were collected to assess gel formation

as well as gelation ability (Yoon, Gunasekaran, & Park, 2006).
Gelation was similar in the presence of organic acids or GDL, and
the storage modulus (G0 ) rapidly increased in early stages to reach a
Fig. 1. Acidication of soymilk with 0.12 (-), 0.14 (C), 0.16 (:) and 0.18 (;) g/
plateau (Fig. 2). Similar gelation curves for acid-induced gel for- 100 mL organic acids or 0.30 g/100 mL glucono-d-lactone (A). (a) Citric acid, (b) l-
mation during time sweeping have been previously reported (Lee & (-)-malic acid, (c) tartaric acid, and glucono-d-lactone was used as a reference.
Kuo, 2011; Schuldt et al., 2014). Strikingly, G'max values clearly
changed with increasing acid concentration, signifying changes in
proteineprotein and proteinewater interactions. The aggregation
of soymilk during acidication involves short-range attractions
among glycinin subunits, these short-range interactions contribute
 F.-H. Cao et al. / LWT - Food Science and Technology 85 (2017) 58e65 61

Fig. 2. Storage modulus (G0 ) in soymilk spiked with 0.14 g/100 mL citric acid (-), l-
(-)-malic acid (C) and tartaric acid (:) or 0.30 g/100 mL glucono-d-lactone (;) (a).
Maximum storage modulus (G'max) in soymilk spiked with 0.12e0.18 g/100 mL citric
acid (gray column), l-(-)-malic acid (white column), tartaric acid (hatched column) or
0.30 g/100 mL glucono-d-lactone (dark gray column), and incubated at 80
C for
30 min (b). Different letters on the top of bars indicate signicant differences
(P < 0.05).

to gel network formation, thus increasing the storage modulus

(Grygorczyk & Corredig, 2013). In general, G'max was highest when
0.14 g/100 mL organic acid was used as coagulant, reaching
127.11 Pa, 87.80 Pa, and 60.31 Pa in tofu prepared with citric, l-
(-)-malic, and tartaric acid, respectively. In tofu cured with 0.30 g/
100 mL GDL, G'max was 134.34 Pa. Acids induce protein gelling by
isoelectric precipitation, a process for in which G'max is considered
to be indicative of the optimal network conguration that in-
corporates the highest number of protein molecules (Jacob et al.,
2011). Indeed, a steep drop in pH reduces the time available for
proteineprotein and proteinewater bonds to form, ultimately
resulting in lower G'max, as noted by Grygorczyk and Corredig
(2013) and by Kuiper, van Koningsveld, Alting, Driehuis, Gruppen,
and Voragen (2005). In addition, Schuldt et al. (2014) demon-
Fig. 3. Storage modulus (G0 ) in soymilk spiked with 0.12 (-), 0.14 (C), 0.16 (:) and
strated by SDS-PAGE that all soy proteins were incorporated into
0.18 (;) g/100 mL organic acids or 0.30 g/100 mL glucono-d-lactone (A). (a) Citric
gels at pH 4.5, but glycinin, a hydrophilic protein, was excluded acid, (b) l-(-)-malic acid, (c) tartaric acid, and glucono-d-lactone was used as a refer-
ence, and cooled from 80
C to 25
C.
62 F.-H. Cao et al. / LWT - Food Science and Technology 85 (2017) 58e65

from gels at pH 3.6, thereby reducing G'max.

After the time sweeps, the gels were cooled from 80
C to 25
C
at 5
C/min, and G0 values obtained are plotted in Fig. 3. G0 increased
drastically as the temperature decreased, perhaps indicating
recombination or rearrangement in the gel structure, or incorpo-
ration of more proteins into the gel (Qin et al., 2016). The nal G0
values were 433.78, 264.43, 129.79 Pa in tofu gelled with 0.14 g/
100 mL citric, l-(-)-malic, and tartaric acid, respectively. In tofu
coagulated with 0.30 g/100 mL GDL, the nal G0 was 412.98 Pa. Of
note, Sun and Arnteld (2012) demonstrated that increased hy-
drophobic and hydrogen bonding sustains and reinforces the gel
structure as it cools.

3.3. Texture and water-holding capacity of tofu gelled with organic

acids

Texture is a critical indicator of tofu quality, the results of tofu

prepared with organic acids and GDL are summarized in Table 1.
Maximum hardness, a measure of resistance to destructive force
and of solid content, was 200.46, 188.32, and 176.98 g in tofu
coagulated with citric, l-(-)-malic, and tartaric acid, respectively,
and was comparable to that for GDL-cured tofu (189.41 g). How-
ever, hardness decreased at organic acid concentrations higher
than 0.14 g/100 mL. A similar observation has been reported by Rui
et al. (2016), who found that gel hardness decreased at higher
concentrations of GDL. Trends in gumminess were consistent with
those in hardness. Based on springiness, tofu gels cured with citric
and l-(-)-malic acid were more rigid and elastic than tofu prepared
with tartaric acid. Finally, values of cohesiveness, which is an in-
dicator of the strength of internal gel bonds, suggested that acid-
cured tofu gels were generally weak (Liu et al., 2013).
The water-holding capacity, a measure of proteinewater in-
teractions, is another important indicator of tofu quality (Lee & Kuo,
2011; Li et al., 2015). In tofu prepared with citric, l-(-)-malic, and
tartaric acid, the water holding capacity was highest (79.91%,
77.91%, and 77.90%, respectively) when 0.14 g/100 mL acid was used
(Fig. 4). In comparison, water-holding capacity was 80.26% in GDL-
coagulated tofu (data not shown). In tofu prepared with citric and l-
(-)-malic acid, water-holding capacity increased with acid con-
centration from 0.12 to 0.14 g/100 mL, but decreased at concen-
trations from 0.14 to 0.18 g/100 mL. While in tofu prepared with
tartaric acid, water-holding capacity increased at acid concentra-
tions of 0.12e0.14 g/100 mL, decreased at concentrations of
0.14e0.16 g/100 mL, and increased again at 0.16e0.18 g/100 mL.
These differences in behavior appear to reect differences in acid-
ity, acidication rate, and aggregation rate. The higher water-
Fig. 4. Water-holding capacity (WHC) of tofu prepared with 0.12e0.18 g/100 mL citric
acid (gray column), l-(-)-malic acid (white column), or tartaric acid (hatched column).
Different letters on the top of bars indicate signicant differences (P < 0.05).
holding capacity at 0.12e0.14 g/100 mL is likely attributable to
the total shielding of protein surface charges, a process that also
amplies the effects of van der Waals and hydrophilic forces (Lee &
Kuo, 2011). On the other hand, a coarser gel network that holds less
water might have been formed as a result of accelerated aggrega-
tion at higher acid concentrations (Saowapark, Apichartsrangkoon,
& Bell, 2008). Li et al. (2015) reported that more rapid gel formation
resulted in a coarser network structure and lower water-holding
capacity. Conversely, the higher water-holding capacity of tofu
prepared with 0.18 g/100 mL tartaric acid may be due to the lower
total water content of the gel. Of note, the texture and water
holding capacity of acid-cured tofu correlated with viscoelastic
properties (Figs. 2 and 3).
3.4. Water distribution in tofu prepared with organic acids
Differential scanning calorimetry is a standard technique used
to characterize water mobility and distribution in polymer systems
(Chen et al., 2010; Tananuwong & Reid, 2004). Water distribution
profoundly affects gel texture and stability. Water molecules can
bind to protein groups or hold up in the network pores of gel sys-
tems (Liu et al., 2013). Tofu prepared with the different coagulants
at various concentrations presented different water states as shown

Table 1
Texture of tofu prepared with organic acids.

Coagulants Usage level Hardness Springiness Cohesiveness Gumminess

(g/100 mL) (g) (-) (g)

CA 0.12 159.15 10.95b 0.93 0.01a 0.56 0.07a 89.44 8.29ab

CA 0.14 200.46 17.31a 0.90 0.02ab 0.54 0.03a 108.5314.16a
CA 0.16 154.89 8.50b 0.89 0.03ab 0.60 0.06a 93.01 9.51ab
CA 0.18 137.06 6.15b 0.88 0.03b 0.56 0.02a 76.63 5.85b
MA 0.12 167.74 5.35b 0.92 0.01a 0.60 0.03a 101.14 7.47ab
MA 0.14 188.32 7.67a 0.93 0.02a 0.59 0.08a 109.86 10.53a
MA 0.16 153.92 3.50c 0.87 0.01b 0.56 0.06a 86.90 10.24bc
MA 0.18 139.29 9.74d 0.88 0.02b 0.56 0.02a 78.30 8.32c
TA 0.12 163.25 7.90b 0.89 0.01a 0.56 0.03a 91.43 7.73ab
TA 0.14 176.98 7.10a 0.90 0.03a 0.56 0.08a 98.67 10.78a
TA 0.16 161.26 9.29b 0.89 0.01a 0.58 0.05a 98.67 10.79a
TA 0.18 142.69 13.17b 0.88 0.01a 0.53 0.01a 98.67 10.80b
GDL 0.30 189.41 15.86 0.94 0.03 0.51 0.01 96.76 7.51

Values are the mean SD from triplicates. Different letters within a column indicate signicant differences (P < 0.05). CA, citric acid; MA, l-(-)-malic acid; TA, tartaric acid; GDL,
glucono-d-lactone.
 F.-H. Cao et al. / LWT - Food Science and Technology 85 (2017) 58e65 63

Fig. 5. Total water (Wt) (a) and non-freezable water (Wnf) (b) in tofu prepared with
0.12e0.18 g/100 mL citric acid (gray column), l-(-)-malic acid (white column), or tar-
taric acid (hatched column), and pressed at 8 g/cm2 for 30 min. Different letters on the
top of bars indicate signicant differences (P < 0.05).

in Fig. 5. The Wt of citric acid-, l-(-)-malic acid-, and tartaric acid-

cured gels was 82.43e79.73%, 82.10e80.20% and 80.83e75.68%,
respectively. Notably, Wnf exhibited trends similar to those in
water-holding capacity (Fig. 4), with the highest Wnf value of
13.04% noted for tofu prepared with citric acid, followed by 12.80%
in tofu prepared with l-(-)-malic acid, and 10.37% in tofu produced
with tartaric acid. Hayashi, Hayakawa, and Fujio (2010) analyzed
the water state in soybean protein isolate by measuring freezable
water content and found that non-freezable water molecules
bound to protein or other macromolecules. Accordingly, Chen et al.
(2010) reported that water molecules bonded tightly, resulting in
high water-holding capacity, and presented in the non-freezable
form through hydrophilic-group interactions. Indeed, higher Wt
and Wnf values generally correlated with higher water-holding
capacity, and with higher stiffness and tightness in the underly-
ing gel structure (Table 1, Fig. 4).

3.5. In-gel chemical interactions in tofu produced with organic acid

Fig. 6. Contributions of intermolecular forces in tofu prepared with (a) citric acid, (b) l-
(-)-malic acid, and (c) tartaric acid. Ionic bonds (gray column), hydrogen bonds (white
In acid-induced soymilk gelation, the gel network results from column), hydrophobic interactions (hatched column). Different letters on the top of
bars indicate signicant differences (P < 0.05).
64 F.-H. Cao et al. / LWT - Food Science and Technology 85 (2017) 58e65

non-covalent interactions, including ionic bonds, hydrogen bonds,
and hydrophobic interactions (Tay & Perera, 2010; Wang et al.,
2015). These contributions of these interactions in the acid-cured
tofu are summarized in Fig. 6; hydrophobic interactions contrib-
uted to gelation and gel structure more signicantly than hydrogen
bonds or ionic bonds. Several studies have indicated that hydro-
phobic interactions occur after charge neutralization (Guo & Ono,
2010; Kohyama & Nishinari, 2002). Hydrogen bonds and hydro-
phobic interactions are the main contributors to gel-like structure
(Wang et al., 2015). Moreover, Wang et al. (2017) suggested that
ionic and hydrogen bonds are generally disrupted by heating to
90
C, highlighting the relatively low contribution of these bonds to
chemical interactions in tofu prepared at 80
C. In addition, acidi-
cation may also have interfered with the formation of ionic and
hydrogen bonds.
Hydrophobic interactions contributed 6.07 g/L, 5.40 g/L, and
5.15 g/L in tofu coagulated with 0.14 g/100 mL citric acid, l-(-)-malic
acid and tartaric acid, respectively. In comparison, hydrophobic
interactions contributed 6.18 g/L in tofu prepared with 0.30 g/
100 mL GDL, likely as a result of increased total protein in the gel
matrix as well as moderated acidication. Remarkably, these values
correlated with Wnf values, and thus, may be further interpreted in
terms of hardness, water-holding capacity, and G'.
3.6. Microstructure of organic acid-induced tofu
Changes in the physicochemical properties of organic acid-
induced tofu corresponded with different microstructures (Lee &
Kuo, 2011; Wang et al., 2017). Scanning electron micrographs of
tofu coagulated with organic acids are presented in Fig. 7. A
honeycomb-like structure with varying size of pores were observed
in tofu prepared with various organic acids at different concen-
trations. The surface charge of soy protein were screened gradually
by protons, the hydrophobic interactions among proteins were
favored, resulting in the lamentous and porous network (Lee &
Kuo, 2011; Liu et al., 2013). Tofu prepared with lower coagulant
concentrations (0.12e0.14 g/100 mL) exhibited with denser
honeycomb-like topography than that prepared with higher acid
coagulant concentrations (0.16e0.18 g/100 mL). In addition, tofu
prepared with GDL exhibited a rmer structure with small pores, in
accordance with its higher springiness and hardness (Table 1).
Tofu prepared with citric acid formed a higher number of small

Fig. 7. Scanning electron micrographs of tofu prepared with (A) citric acid, (B) l-(-)-malic acid, and (C) tartaric acid at (a) 0.12 g/100 mL, (b) 0.14 g/100 mL, (c) 0.16 g/100 mL, and (d)
0.18 g/100 mL. Tofu prepared with (D) 0.30 g/100 mL glucono-d-lactone was used as a reference. Scale bars, 100 mm.
 F.-H. Cao et al. / LWT - Food Science and Technology 85 (2017) 58e65
pores (Fig. 7Aa-Ad) than that prepared with l-(-)-malic (Fig. 7Ba-
Bd) or tartaric acid (Fig. 7Ca-Cd), which was in accordance with its
higher storage modulus and gel hardness. This might have resulted
from differences in acidication rate and pH. Indeed, the small
pores in the former appear to have contributed to higher water-
holding capacity and hardness. The random and coarse structure
of tofu prepared with 0.16 or 0.18 g/100 mL acid indicates the lower
quality of these gels.
4. Conclusion
The use of different types and concentrations of organic acids as
coagulant resulted in textural and microstructural differences in
tofu. Rheological properties, pH, and in-gel chemical interactions
suggested that accelerated acidication reduces the amount of time
available for ordered aggregation of soy proteins, thus lowering
tofu quality.
Tofu acidied with 0.14 g/100 mL organic acids exhibited
excellent physical properties, as indicated by high storage modulus,
hardness, water-holding capacity, and non-freezable water content.
In addition, these gels were formed mostly because of hydrophobic
interactions. On the other hand, tofu prepared with 0.12 and 0.16 g/
100 mL citric acid were comparable in hardness, although the
former was more elastic. Similar trends were observed for tofu
prepared with 0.12 and 0.16 g/100 mL L-(-)-malic acid. However,
tofu prepared with tartaric acid had comparatively poorer physical
properties and less robust chemical interactions. Finally, tofu pre-
pared with 0.18 g/100 mL organic acid was of low quality, as indi-
cated by low elastic modulus, hardness, water-holding capacity,
non-freezable water content, and coarse gel structure. Collec-
tively, the data indicated that the quality of tofu prepared with
organic acids is comparable to that prepared with GDL. Our study
revealed that different types tofu (soft or rm) can be produced in a
straightforward manner by using suitable types and concentrations
of organic acids.
Acknowledgements
This work was funded by National High Technology Research
and Development Program (863 Program) of China (No.
2013AA102201), and by Key Scientic and Technological Project of
Anhui Province of China (No. 1301031031 and 15czz03096).
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