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International Journal of Food Science and Technology 2002, 37, 277–283 277

Evaluation of physico-chemical characteristics and

microstructure of tofu containing high viscosity chitosan

Meera Kim1* & Jin-suk Han2

1 Department of Food Science and Nutrition, Kyungpook National University, Taegu 702–701, Korea
2 Department of Food and Nutrition, Seoul National University, Seoul 151–742, Korea
(Received 21 August 2000; Accepted in revised form 4 April 2001)

Summary Tofu, containing high viscosity chitosan dissolved in a d-gluconolactone solution, was
prepared and physico-chemical properties, microstructure, textural properties and sensory
characteristics were investigated. Moisture content and pH of the chitosan tofu were
slightly lower than those of the control tofu. The textural properties of tofu such as
hardness, cohesiveness, gumminess and chewiness measured by an instrumental method
were not significantly changed by the addition of chitosan to tofu. Springiness of chitosan
tofu, however, was significantly higher than that of the control tofu. All characteristics
except the roasted nutty aroma and yellowness in the sensory evaluation, did not exhibit
significant differences between the chitosan tofu and control tofu. Therefore, the quality of
tofu was little affected by the addition of the chitosan content employed in this experiment.
Keywords Chitosan, microstructure, sensory characteristics, textural properties, tofu.

soybean curds stored at 4.5 °C pretreated with

microwave heating to 65, 80, and 95 °C had
Tofu is a nutritious and digestible food that has shelf-lives of 16, 21 and 27 days, respectively.
been widely consumed in the Orient. However, Champagene et al. (1991) reported that pasteur-
tofu is highly perishable even under refrigeration ization was effective in lowering the bacterial
because of its relatively high pH (5.8–6.2) and counts and the addition of lactic acid (reducing pH
moisture content (80–88%) (Lim et al., 1990; Shen to 5.5) to tofu helped reduce gas production by
et al., 1991). Some researches have studied quality about 50%.
improvement and shelf-life extension of tofu. To Chitin and chitosan, natural amino polysaccha-
extend the shelf-life of tofu, microwave treatment, rides, are the most intriguing new functional
coagulation with organic acid and pH adjustment materials (Cho, 1989) and are the major wastes
of immersion solutions have been tried (Wu & of the shellfish-processing industry. Chitinous
Salunkhe, 1977; Pontecorvo & Bourne, 1978; polymers have functions such as hypocholestero-
Champagene et al., 1991). Pontecorvo & Bourne lemic activity, lipid binding properties, antibiotic
(1978) conducted an experiment to extend the activities, water conservation, emulsion stability
storage stability including immersion in aqueous and dietary fiber (Knorr, 1982, 1984; Yang et al.,
solutions, smoking and combinations of the 1992; Muzzarelli & De Vincenzi, 1997). Moreover,
above, and reported that the shelf-life of tofu they are not only naturally abundant but also
was extended to 10–15 days by smoking without nontoxic and biodegradable. Therefore, chitin and
refrigeration. Wu & Salunkhe (1977) reported that chitosan have high potential as additives to foods
for various purposes. However, some additives
used in foods may impair the quality of the foods
*Correspondent: Fax: 82-53-950-6229; such as the sensory characteristics and structural
e-mail: properties. Therefore, it is important to ensure

Ó 2002 Blackwell Science Ltd

278 Evaluation of physico-chemical characteristics and microstructure of chitosan tofu Meera Kim & Jin-suk Han

that the additive does not give undesirable proper- 20 mL of 2% d-gluconolactone solution. This
ties to the foods before it is used. Chitosan has the chitosan solution was added to 1700 mL of
potential to be used as an additive to tofu for the soymilk and then calcium chloride solution (9 g
purpose of shelf-life extension because it has of CaCl2 in 20 mL of distilled water) was added
antimicrobial activity (Muzzarelli & De Vincenzi, slowly while stirring until the soymilk started to
1997; Yalpani et al., 1992). Therefore, in this coagulate. After settling for 10 min, the curd was
paper, we evaluated the effect of high viscosity transferred to a cheesecloth-lined wooden box
chitosan on tofu qualities such as physico-chem- (13 · 9 · 7 cm) and pressed using a 1-kg weight
ical properties, microstructure, textural properties placed on the top for 30 min. The tofu was then
and sensory characteristics as a preliminary study removed from the box and immersed in water for
before we would conduct preservation experi- 30 min and packed in the polyethylene container
ments using chitosan in tofu. with 300 mL of soaking water. The control tofu
was also prepared using the same procedure
without the addition of chitosan and d-glucono-
Materials and methods
Physico-chemical properties of tofu
Soybeans that were grown in Chungbook Prov-
ince, Korea, were purchased from Nong-Hyup The moisture content of the tofu was determined
market. High viscosity chitosan (viscosity 400– by the AOAC method (1995). Yield was calcu-
450 cp) was obtained from RC Bio Chemicals lated as the wet weight of fresh tofu obtained
(Pusan, Korea). Calcium chloride and d-glucono- from 300 g of soybean. The colour of tofu,
lactone were obtained from Sigma Chemical Co. expressed in L, a, and b values, was measured
(St Louis, MO, USA). using a Whiteness checker RF-1 colorimeter
(Nippon Denshoku Kogyo Co., Osaka, Japan).
The pH of the soaking solution of the tofu
Preparation of tofu
was determined using a Toldedo 340 pH meter
Tofu was prepared using the procedure of Wang (Mettler, Leicester, UK).
et al. (1983). Soybeans (300 g) were washed and
soaked in water at room temperature for 12 h. The
Structure of tofu
soaked beans were drained and blended for 10 min
in a blender (A76, Moulinex, Paris, France) with The tofu samples were serially dehydrated with 50,
water to give a water-dry beans ratio of 10:1 60, 70, 80, 90 and 95% ethanol for 15 min,
(weight basis). The mash was cooked for 15 min at respectively, and the samples were immersed into
boiling temperature with occasional stirring. The isoamyl acetate for 1 h at room temperature. The
hot mash was then filtered through double layers of samples were treated with propylene oxide,
cheesecloth and the soymilk was cooled to about embedded in epon 812 (embed-812, EMS Co.
80 °C. Tofu samples were prepared by mixing Ltd, Washington, USA) sliced by ultramicrotome
chitosan, d-gluconolactone and CaCl2 (Table 1). (Sapernova, Leica, Austria), and electrostained
First, 0.1 or 0.2 g of chitosan was dissolved in with 2% uranyl acetate and 2% lead acetate.

Table 1 Amount of the various

Soymilk High viscosity 2% Glucono- CaCl2 solution† compounds in tofu
Tofu (mL) chitosan* (g) d-lactone (mL) (mL)

Control tofu 1700 0 0 20

Zero chitosan tofu 1700 0 20 20
Low chitosan tofu 1700 0.1 20 20
High chitosan tofu 1700 0.2 20 20

*Viscosity was 400–450 cp.

CaCl2 (9 g) was dissolved in 20 mL of distilled water.

International Journal of Food Science and Technology 2002, 37, 277–283 Ó 2002 Blackwell Science Ltd
Evaluation of physico-chemical characteristics and microstructure of chitosan tofu Meera Kim & Jin-suk Han 279

Microstructures of the tofu were observed using a one time. Tofu samples were evaluated using the
transmission electron microscope (TEM) (Hitachi- line-scale method with a 15-cm line anchored from
H-7100, Nackashi, Japan). none to extremely intense for aroma, taste, texture
and appearance of tofu. The overall eating quality
of the tofu was evaluated separately during
Instrumental analysis of tofu texture
another session. The partitioned booths with green
The texture of the tofu was evaluated using a lights were used to test the aroma, taste, flavour,
rheometer (COMPAC-100, Sun Scientific, Tokyo, and texture, while the appearance was evaluated
Japan) with the two-bite compression test mode. under fluorescent light.
Cylindrical samples (2.5 cm diameter · 2.0 cm
height) were prepared from the central portion
Statistical analysis
of tofu with a stainless steel boring tube and a wire
cutter. The samples were compressed by the The physico-chemical properties of the tofu were
cylindrical plunger with a 2.5 cm diameter to measured in triplicate and the instrumental meas-
30% deformation using a 10 kg load cell and urements of the tofu texture were measured six
300 mm min–1 crosshead speed. Each kind of times. Sensory evaluation was performed with
sample was measured with six replications. four replications for each sample. The data was
analyzed using the Statistical Analysis System
(SAS Institute Inc., 1995). The results were repor-
Sensory evaluation
ted as mean values with a standard deviation.
The tofu was stored at 4 °C for 30 min prior to Two-way analysis of variance (ANOVA) and Dun-
serving. Tofu blocks were cut into 3 · 4 · 1 cm to can’s multiple range tests were used to determine
evaluate the sensory attributes. All samples were whether or not a significant difference existed in
served in Petri dishes with covers. The reference the means. All tests of significance were at the 5%
sample (control tofu) was labelled and the significance level.
unknown samples were coded with random
three-digit numbers using a random number table
(Meilgaard et al., 1991). Sensory evaluations were
by ten trained panelists who were graduate The physico-chemical properties of the tofu sam-
students at Kyungpook National University. ples are presented in Table 2. The yields of tofu
Three samples and one reference were served at ranged from 1.40 to 1.50 (g/g) and were not

Table 2 Physico-chemical proper-

ties of tofu Control Zero chitosan Low chitosan High chitosan
Properties tofu tofu tofu tofu

Yield (g/g) 1.47 ± 0.14*,a 1.50 ± 0.20a 1.43 ± 0.15a 1.40 ± 0.17a
Moisture content (%) 83.40 ± 0.39a 81.50 ± 1.25b 80.63 ± 0.16b 80.68 ± 0.09b
pH 5.70 ± 0.03a 5.65 ± 0.01b 5.68 ± 0.01ab 5.67 ± 0.01ab

*Values are the means of three replications. Means with a row followed by same letter
are not significantly different (P < 0.05).

Table 3 Hunter colour values of

tofu Control Zero chitosan Low chitosan High chitosan
tofu tofu tofu tofu

L 79.80 ± 0.10*,a 79.83 ± 0.06a 76.43 ± 0.06c 79.03 ± 0.12b

a 1.67 ± 0.06b 1.57 ± 0.15b 2.33 ± 0.15a 2.17 ± 0.06a
b 10.57 ± 0.06c 11.10 ± 0.17b 11.07 ± 0.11b 11.57 ± 0.06a

*Values are the means of three replications. Means with a row followed by same letter
are not significantly different (P < 0.05).

Ó 2002 Blackwell Science Ltd International Journal of Food Science and Technology 2002, 37, 277–283
280 Evaluation of physico-chemical characteristics and microstructure of chitosan tofu Meera Kim & Jin-suk Han

Figure 1 Transmission electron

microscope images (·10 000) of
tofu. (a) Control tofu;
(b) Zero chitosan tofu; (c) Low
chitosan tofu; (d) High chitosan

significantly different among the tofu samples. The well-developed protein aggregations and connec-
moisture content of the fresh tofu varied from tions between protein granules that surrounded oil
80.63 to 83.40% and significantly decreased when droplets. The zero chitosan tofu (b) also had well-
d-gluconolactone solution was added to the tofu. developed protein aggregations, but the connec-
The pH of the tofu was lower than 6.0 and there tions between the protein granules were less
was no significant pH difference between the developed when compared with those of the
control tofu and the low or high chitosan tofu. control tofu (a). However, the aggregations of
The Hunter’s colour values of the tofu containing protein granules and the connections between
high viscosity chitosan are shown in Table 3. The protein granules decreased in the tofu containing
L values of the control tofu and zero chitosan tofu high viscosity chitosan (c, d). High chitosan tofu
were higher than those of the low and high showed fewer aggregations and connections than
chitosan tofu, and the a value increased when low chitosan tofu. The connections between pro-
chitosan was added to the tofu. tein granules were loose and intermittent and oil
The TEM image of tofu (Fig. 1) shows the droplets were not effectively surrounded by pro-
network structures constructed with small protein tein granules in the tofu containing chitosan.
granules (0.05–0.1 lm diameter) and oil drops The results obtained by the instrumental
(0.1–1 lm diameter). The control tofu (a) had measurement of textural properties of tofu are

Table 4 Textural properties of tofu

Control Zero chitosan Low chitosan High chitosan
Properties tofu tofu tofu tofu

Hardness (g cm)2) 352 ± 45.97*,a 406 ± 37.75a 391 ± 63.22a 342 ± 29.98a
Cohesiveness (%) 95 ± 5.53a 93 ± 4.75a 96 ± 5.85a 93 ± 3.66a
Springiness (%) 96 ± 2.51b 95 ± 1.70b 96 ± 1.21ab 98 ± 1.77a
Gumminess (%) 317 ± 56.16a 366 ± 42.51a 359 ± 69.45a 295 ± 27.49a
Chewiness (%) 305 ± 58.92a 351 ± 44.86a 348 ± 70.02a 291 ± 26.73a

*Values are the means of six replications. Means with a row followed by same letter are
not significantly different (P < 0.05).

International Journal of Food Science and Technology 2002, 37, 277–283 Ó 2002 Blackwell Science Ltd
Evaluation of physico-chemical characteristics and microstructure of chitosan tofu Meera Kim & Jin-suk Han 281

Table 5 Sensory attributes of tofu

Control Zero chitosan Low chitosan High chitosan
Attributes tofu tofu tofu tofu

Roasted nutty 8.15 ± 1.93*,a 6.70 ± 1.92b 7.10 ± 2.10ab 7.04 ± 1.88ab
Bean 6.29 ± 2.73a 6.03 ± 2.78a 5.69 ± 2.89a 5.82 ± 3.11a

Roasted nutty 7.86 ± 2.44a 7.09 ± 2.84a 6.61 ± 2.75a 6.84 ± 3.04a
Bean 6.87 ± 2.73a 5.53 ± 2.86a 6.13 ± 3.14a 6.03 ± 3.04a
Sour 5.54 ± 3.15a 5.34 ± 2.44a 5.72 ± 3.18a 6.85 ± 3.96a

Hardness 6.70 ± 1.92a 6.60 ± 1.63a 6.60 ± 2.56a 7.09 ± 2.06a
Springiness 7.34 ± 2.28a 6.89 ± 2.02a 8.25 ± 2.23a 8.05 ± 1.87a
Adhesiveness 6.96 ± 2.27a 6.96 ± 2.03a 6.87 ± 2.50a 7.17 ± 2.72a

Yellowness 8.67 ± 1.70b 8.27 ± 1.51b 8.32 ± 1.62b 8.78 ± 1.85a
Smoothness 8.41 ± 2.29a 8.14 ± 1.88a 8.52 ± 1.58a 7.92 ± 1.73a
Homogeneity 8.13 ± 2.14a 7.93 ± 2.16a 7.97 ± 1.79a 7.83 ± 1.38a

Overall eating quality 8.50 ± 2.32a 8.47 ± 41.98a 8.07 ± 2.12a 8.48 ± 2.07a

*Values are the means of responses for ten panelists with four replications. Means with a
row followed by same letter are not significantly different (P < 0.05).

summarized in Table 4. This demonstrates that the springiness for the high and low chitosan tofu
the textural properties of tofu with chitosan were were also higher than those for the control tofu in
similar to those of the tofu without chitosan the sensory tests, although there was no statisti-
except for its springiness, although zero chitosan cally significant difference among the tofu sam-
tofu had the highest value of hardness while high ples. High chitosan tofu was more yellow than the
chitosan tofu rated the lowest. The springiness of control tofu. This is the same as the result
the tofu ranged from 95.68 to 98.80%. There was measured by the colorimeter. The overall eating
no significant difference between the control tofu quality was not significantly different among the
and zero chitosan tofu in springiness, but the tofu samples.
addition of chitosan to the tofu increased sprin-
giness. Moreover, the springiness of the tofu
increased with an increasing amount of chitosan.
Sensory attributes of the control and chitosan High viscosity chitosan is insoluble in water
tofu by sensory evaluation were compared and the because of its high molecular weight, but it is
results are shown in Table 5. Sensory panelists soluble in dilute hydrochloric and organic acids
noted that zero chitosan tofu had a less roasted- (Knorr, 1984). Therefore, the d-gluconolactone
nutty aroma than the control tofu. The following solution (2%), which has a milder acid taste than
tastes that were evaluated, such as roasted nutty, other acids and acts as a coagulant, was used for
bean and sour, were not significantly different, dissolving high viscosity. The two kinds of tofu,
although the mean values of the roasted nutty the tofu based on water (control tofu) and the tofu
taste decreased and the bean and sour tastes based on d-gluconolactone (zero chitosan tofu),
slightly increased in the tofu containing high were prepared in this experiment to evaluate the
viscosity chitosan in d-gluconolactone. Texture addition effect of high viscosity chitosan and
properties, as described by sensory evaluation, did d-gluconolactone on tofu.
not show significant differences among the tofu Tofu is actually a precipitated soy protein and
samples. The springiness of the tofu measured by it starts coagulating at about pH 6.0 (Knorr,
the instrument was rated high in the chitosan tofu 1984; Lu et al., 1980). Therefore, the pH of tofu
as previously mentioned, and the mean values of can be related to its textural properties. It was

Ó 2002 Blackwell Science Ltd International Journal of Food Science and Technology 2002, 37, 277–283
282 Evaluation of physico-chemical characteristics and microstructure of chitosan tofu Meera Kim & Jin-suk Han

reported that pH strongly affects the extent of charges able to neutralize the negatively charged
Ca2+ binding because hydrogen ions compete proteins were sufficiently provided by the CaCl2
with calcium ions for the same binding sites on the and d-gluconolactone, it was determined that
protein molecule (Kroll, 1984). They explained chitosan did not play a great role in coagulating
that the affinity of the binding sites for calcium the protein. Rather, excess positive charges
ions was shown to increase as pH increased over induced by chitosan might inhibit the protein
the pH range of 4–9, as the binding constant aggregations which occur via hydrophobic inter-
increased with increasing pH. Between pH 3 and actions.
7, a small change in pH results in a large change The textural properties of the tofu with chitosan
in the amount of Ca2+ bound. Therefore, it was as measured by sensory evaluation were similar to
considered that the calcium-binding affinity in those of the tofu without chitosan. In addition, the
the control tofu was stronger than that in the other sensory attributes were not greatly affected
zero chitosan tofu because of the pH decrease by adding high viscosity chitosan to the tofu. This
by adding d-gluconolactone to the zero chitosan result implies that the addition of high viscosity
tofu. chitosan used in this study does not greatly affect
The fine structure of tofu can be classified by the sensory properties of the tofu.
network density, protein aggregation and coagu-
late size (Saio, 1979). Although the hardness of
tofu was not significantly different among the tofu
samples using the instrumental analysis, the zero The addition of high viscosity chitosan to tofu
chitosan tofu showed a higher hardness than the scarcely affected the physico-chemical properties
control tofu (Table 4). This result agrees with the of tofu such as yield, moisture content and pH,
report by Shen et al. (1991). Additionally, the low but the colour of tofu became slightly more
and high chitosan tofu had smaller protein aggre- yellow. The TEM image showed that high visco-
gates and looser connections between protein sity chitosan affected the interactions between
aggregates than zero chitosan tofu (Fig. 1). There- protein molecules, but the instrumental textural
fore, this structural difference could cause the low properties of the chitosan tofu did not greatly
and high chitosan tofu to have a lower inner differ from those of the control tofu. The sensory
hardness and to be more fragile than zero chitosan characteristics also showed that high viscosity
tofu. Chun et al. (1999) also reported that chito- chitosan only slightly influenced the quality of the
san tofu has a low failure stress and lower stress tofu. Therefore, these results suggest that high
relaxation values than tofu coagulated with CaCl2. viscosity chitosan would be useful as an additive
The gelation of tofu is formed by protein for extending the shelf-life of tofu because its
denaturation, mainly caused by heat, coagulation quality did not severely deteriorate with the
is also promoted by cations (Kohyama et al., addition of high viscosity chitosan.
1995). The hydrophobic regions of the native
protein molecules are exposed to the solvent by
heat denaturation (Koshiyama et al., 1981). As the
denatured soybean protein is negatively charged AOAC (1995). Official Methods of Analysis, 16th edn.
(Kohyama & Nishinari, 1993), the protons pro- Chapter 32, P. 22. Arlington: Association of Official
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