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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.
Chitosan, microstructure, sensory characteristics, textural properties, tofu.

Keywords

Introduction

Tofu is a nutritious and digestible food that has been widely consumed in the Orient. However, tofu is highly perishable even under refrigeration because of its relatively high pH (5.8–6.2) and moisture content (80–88%) (Lim et al., 1990; Shen et al., 1991). Some researches have studied quality improvement and shelf-life extension of tofu. To extend the shelf-life of tofu, microwave treatment, coagulation with organic acid and pH adjustment of immersion solutions have been tried (Wu & Salunkhe, 1977; Pontecorvo & Bourne, 1978; Champagene et al., 1991). Pontecorvo & Bourne (1978) conducted an experiment to extend the storage stability including immersion in aqueous solutions, smoking and combinations of the above, and reported that the shelf-life of tofu was extended to 10–15 days by smoking without refrigeration. Wu & Salunkhe (1977) reported that

*Correspondent: Fax: 82-53-950-6229; e-mail: meerak@knu.ac.kr

soybean curds stored at 4.5 °C pretreated with microwave heating to 65, 80, and 95 °C had shelf-lives of 16, 21 and 27 days, respectively. Champagene et al. (1991) reported that pasteurization was effective in lowering the bacterial counts and the addition of lactic acid (reducing pH to 5.5) to tofu helped reduce gas production by about 50%. Chitin and chitosan, natural amino polysaccharides, are the most intriguing new functional materials (Cho, 1989) and are the major wastes of the shellfish-processing industry. Chitinous polymers have functions such as hypocholesterolemic activity, lipid binding properties, antibiotic activities, water conservation, emulsion stability and dietary fiber (Knorr, 1982, 1984; Yang et al., 1992; Muzzarelli & De Vincenzi, 1997). Moreover, they are not only naturally abundant but also nontoxic and biodegradable. Therefore, chitin and chitosan have high potential as additives to foods for various purposes. However, some additives used in foods may impair the quality of the foods such as the sensory characteristics and structural properties. Therefore, it is important to ensure

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that the additive does not give undesirable properties to the foods before it is used. Chitosan has the potential to be used as an additive to tofu for the purpose of shelf-life extension because it has antimicrobial activity (Muzzarelli & De Vincenzi, 1997; Yalpani et al., 1992). Therefore, in this paper, we evaluated the effect of high viscosity chitosan on tofu qualities such as physico-chemical properties, microstructure, textural properties and sensory characteristics as a preliminary study before we would conduct preservation experiments using chitosan in tofu.
Materials and methods

20 mL of 2% d-gluconolactone solution. This chitosan solution was added to 1700 mL of soymilk and then calcium chloride solution (9 g of CaCl2 in 20 mL of distilled water) was added slowly while stirring until the soymilk started to coagulate. After settling for 10 min, the curd was transferred to a cheesecloth-lined wooden box (13 · 9 · 7 cm) and pressed using a 1-kg weight placed on the top for 30 min. The tofu was then removed from the box and immersed in water for 30 min and packed in the polyethylene container with 300 mL of soaking water. The control tofu was also prepared using the same procedure without the addition of chitosan and d-gluconolactone. Physico-chemical properties of tofu The moisture content of the tofu was determined by the AOAC method (1995). Yield was calculated as the wet weight of fresh tofu obtained from 300 g of soybean. The colour of tofu, expressed in L, a, and b values, was measured using a Whiteness checker RF-1 colorimeter (Nippon Denshoku Kogyo Co., Osaka, Japan). The pH of the soaking solution of the tofu was determined using a Toldedo 340 pH meter (Mettler, Leicester, UK). Structure of tofu The tofu samples were serially dehydrated with 50, 60, 70, 80, 90 and 95% ethanol for 15 min, respectively, and the samples were immersed into isoamyl acetate for 1 h at room temperature. The samples were treated with propylene oxide, embedded in epon 812 (embed-812, EMS Co. Ltd, Washington, USA) sliced by ultramicrotome (Sapernova, Leica, Austria), and electrostained with 2% uranyl acetate and 2% lead acetate.
Table 1 Amount of the various compounds in tofu

Materials Soybeans that were grown in Chungbook Province, Korea, were purchased from Nong-Hyup market. High viscosity chitosan (viscosity 400– 450 cp) was obtained from RC Bio Chemicals (Pusan, Korea). Calcium chloride and d-gluconolactone were obtained from Sigma Chemical Co. (St Louis, MO, USA). Preparation of tofu Tofu was prepared using the procedure of Wang et al. (1983). Soybeans (300 g) were washed and soaked in water at room temperature for 12 h. The soaked beans were drained and blended for 10 min in a blender (A76, Moulinex, Paris, France) with water to give a water-dry beans ratio of 10:1 (weight basis). The mash was cooked for 15 min at boiling temperature with occasional stirring. The hot mash was then filtered through double layers of cheesecloth and the soymilk was cooled to about 80 °C. Tofu samples were prepared by mixing chitosan, d-gluconolactone and CaCl2 (Table 1). First, 0.1 or 0.2 g of chitosan was dissolved in

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

Soymilk (mL) 1700 1700 1700 1700

High viscosity chitosan* (g) 0 0 0.1 0.2

2% Gluconod-lactone (mL) 0 20 20 20

CaCl2 solution† (mL) 20 20 20 20

*Viscosity was 400–450 cp. † CaCl2 (9 g) was dissolved in 20 mL of distilled water.

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Microstructures of the tofu were observed using a transmission electron microscope (TEM) (HitachiH-7100, Nackashi, Japan). Instrumental analysis of tofu texture The texture of the tofu was evaluated using a rheometer (COMPAC-100, Sun Scientific, Tokyo, Japan) with the two-bite compression test mode. Cylindrical samples (2.5 cm diameter · 2.0 cm height) were prepared from the central portion of tofu with a stainless steel boring tube and a wire cutter. The samples were compressed by the cylindrical plunger with a 2.5 cm diameter to 30% deformation using a 10 kg load cell and 300 mm min–1 crosshead speed. Each kind of sample was measured with six replications. Sensory evaluation The tofu was stored at 4 °C for 30 min prior to serving. Tofu blocks were cut into 3 · 4 · 1 cm to evaluate the sensory attributes. All samples were served in Petri dishes with covers. The reference sample (control tofu) was labelled and the 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 students at Kyungpook National University. Three samples and one reference were served at

one time. Tofu samples were evaluated using the line-scale method with a 15-cm line anchored from none to extremely intense for aroma, taste, texture and appearance of tofu. The overall eating quality of the tofu was evaluated separately during another session. The partitioned booths with green lights were used to test the aroma, taste, flavour, and texture, while the appearance was evaluated under fluorescent light. Statistical analysis The physico-chemical properties of the tofu were measured in triplicate and the instrumental measurements of the tofu texture were measured six times. Sensory evaluation was performed with four replications for each sample. The data was analyzed using the Statistical Analysis System (SAS Institute Inc., 1995). The results were reported as mean values with a standard deviation. Two-way analysis of variance (ANOVA) and Duncan’s multiple range tests were used to determine whether or not a significant difference existed in the means. All tests of significance were at the 5% significance level.
Results

The physico-chemical properties of the tofu samples are presented in Table 2. The yields of tofu ranged from 1.40 to 1.50 (g/g) and were not

Table 2 Physico-chemical properties of tofu

Properties Yield (g/g) Moisture content (%) pH

Control tofu 1.47 ± 0.14*,a 83.40 ± 0.39a 5.70 ± 0.03a

Zero chitosan tofu 1.50 ± 0.20a 81.50 ± 1.25b 5.65 ± 0.01b

Low chitosan tofu

High chitosan tofu

1.43 ± 0.15a 1.40 ± 0.17a 80.63 ± 0.16b 80.68 ± 0.09b 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
L a b

Control tofu 79.80 ± 0.10*,a 1.67 ± 0.06b 10.57 ± 0.06c

Zero chitosan tofu 79.83 ± 0.06a 1.57 ± 0.15b 11.10 ± 0.17b

Low chitosan tofu 76.43 ± 0.06c 2.33 ± 0.15a 11.07 ± 0.11b

High chitosan tofu 79.03 ± 0.12b 2.17 ± 0.06a 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).

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Figure 1 Transmission electron

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

significantly different among the tofu samples. The moisture content of the fresh tofu varied from 80.63 to 83.40% and significantly decreased when d-gluconolactone solution was added to the tofu. The pH of the tofu was lower than 6.0 and there was no significant pH difference between the control tofu and the low or high chitosan tofu. The Hunter’s colour values of the tofu containing high viscosity chitosan are shown in Table 3. The L values of the control tofu and zero chitosan tofu were higher than those of the low and high chitosan tofu, and the a value increased when chitosan was added to the tofu. The TEM image of tofu (Fig. 1) shows the network structures constructed with small protein granules (0.05–0.1 lm diameter) and oil drops (0.1–1 lm diameter). The control tofu (a) had

well-developed protein aggregations and connections between protein granules that surrounded oil droplets. The zero chitosan tofu (b) also had welldeveloped protein aggregations, but the connections between the protein granules were less developed when compared with those of the control tofu (a). However, the aggregations of protein granules and the connections between protein granules decreased in the tofu containing high viscosity chitosan (c, d). High chitosan tofu showed fewer aggregations and connections than low chitosan tofu. The connections between protein granules were loose and intermittent and oil droplets were not effectively surrounded by protein granules in the tofu containing chitosan. The results obtained by the instrumental measurement of textural properties of tofu are

Properties Hardness (g cm)2) Cohesiveness (%) Springiness (%) Gumminess (%) Chewiness (%)

Control tofu 352 95 96 317 305 ± ± ± ± ± 45.97*,a 5.53a 2.51b 56.16a 58.92a

Zero chitosan tofu 406 93 95 366 351 ± ± ± ± ± 37.75a 4.75a 1.70b 42.51a 44.86a

Low chitosan tofu 391 96 96 359 348 ± ± ± ± ± 63.22a 5.85a 1.21ab 69.45a 70.02a

High chitosan tofu 342 93 98 295 291 ± ± ± ± ± 29.98a 3.66a 1.77a 27.49a 26.73a

Table 4 Textural properties of tofu

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

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Table 5 Sensory attributes of tofu Attributes Aroma Roasted nutty Bean Taste Roasted nutty Bean Sour Texture Hardness Springiness Adhesiveness Appearance Yellowness Smoothness Homogeneity Overall eating quality

Control tofu

Zero chitosan tofu

Low chitosan tofu

High chitosan tofu

8.15 ± 1.93*,a 6.29 ± 2.73a 7.86 ± 2.44a 6.87 ± 2.73a 5.54 ± 3.15a 6.70 ± 1.92a 7.34 ± 2.28a 6.96 ± 2.27a 8.67 ± 1.70b 8.41 ± 2.29a 8.13 ± 2.14a 8.50 ± 2.32a

6.70 ± 1.92b 6.03 ± 2.78a 7.09 ± 2.84a 5.53 ± 2.86a 5.34 ± 2.44a 6.60 ± 1.63a 6.89 ± 2.02a 6.96 ± 2.03a 8.27 ± 1.51b 8.14 ± 1.88a 7.93 ± 2.16a 8.47 ± 41.98a

7.10 ± 2.10ab 5.69 ± 2.89a 6.61 ± 2.75a 6.13 ± 3.14a 5.72 ± 3.18a 6.60 ± 2.56a 8.25 ± 2.23a 6.87 ± 2.50a 8.32 ± 1.62b 8.52 ± 1.58a 7.97 ± 1.79a 8.07 ± 2.12a

7.04 ± 1.88ab 5.82 ± 3.11a 6.84 ± 3.04a 6.03 ± 3.04a 6.85 ± 3.96a 7.09 ± 2.06a 8.05 ± 1.87a 7.17 ± 2.72a 8.78 ± 1.85a 7.92 ± 1.73a 7.83 ± 1.38a 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 textural properties of tofu with chitosan were similar to those of the tofu without chitosan except for its springiness, although zero chitosan tofu had the highest value of hardness while high chitosan tofu rated the lowest. The springiness of the tofu ranged from 95.68 to 98.80%. There was no significant difference between the control tofu and zero chitosan tofu in springiness, but the addition of chitosan to the tofu increased springiness. Moreover, the springiness of the tofu increased with an increasing amount of chitosan. Sensory attributes of the control and chitosan tofu by sensory evaluation were compared and the results are shown in Table 5. Sensory panelists noted that zero chitosan tofu had a less roastednutty aroma than the control tofu. The following tastes that were evaluated, such as roasted nutty, bean and sour, were not significantly different, although the mean values of the roasted nutty taste decreased and the bean and sour tastes slightly increased in the tofu containing high viscosity chitosan in d-gluconolactone. Texture properties, as described by sensory evaluation, did not show significant differences among the tofu samples. The springiness of the tofu measured by the instrument was rated high in the chitosan tofu as previously mentioned, and the mean values of

the springiness for the high and low chitosan tofu were also higher than those for the control tofu in the sensory tests, although there was no statistically significant difference among the tofu samples. High chitosan tofu was more yellow than the control tofu. This is the same as the result measured by the colorimeter. The overall eating quality was not significantly different among the tofu samples.
Discussion

High viscosity chitosan is insoluble in water because of its high molecular weight, but it is soluble in dilute hydrochloric and organic acids (Knorr, 1984). Therefore, the d-gluconolactone solution (2%), which has a milder acid taste than other acids and acts as a coagulant, was used for dissolving high viscosity. The two kinds of tofu, the tofu based on water (control tofu) and the tofu based on d-gluconolactone (zero chitosan tofu), were prepared in this experiment to evaluate the addition effect of high viscosity chitosan and d-gluconolactone on tofu. Tofu is actually a precipitated soy protein and it starts coagulating at about pH 6.0 (Knorr, 1984; Lu et al., 1980). Therefore, the pH of tofu can be related to its textural properties. It was

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reported that pH strongly affects the extent of Ca2+ binding because hydrogen ions compete with calcium ions for the same binding sites on the protein molecule (Kroll, 1984). They explained that the affinity of the binding sites for calcium ions was shown to increase as pH increased over the pH range of 4–9, as the binding constant increased with increasing pH. Between pH 3 and 7, a small change in pH results in a large change in the amount of Ca2+ bound. Therefore, it was considered that the calcium-binding affinity in the control tofu was stronger than that in the zero chitosan tofu because of the pH decrease by adding d-gluconolactone to the zero chitosan tofu. The fine structure of tofu can be classified by network density, protein aggregation and coagulate size (Saio, 1979). Although the hardness of tofu was not significantly different among the tofu samples using the instrumental analysis, the zero chitosan tofu showed a higher hardness than the control tofu (Table 4). This result agrees with the report by Shen et al. (1991). Additionally, the low and high chitosan tofu had smaller protein aggregates and looser connections between protein aggregates than zero chitosan tofu (Fig. 1). Therefore, this structural difference could cause the low and high chitosan tofu to have a lower inner hardness and to be more fragile than zero chitosan tofu. Chun et al. (1999) also reported that chitosan tofu has a low failure stress and lower stress relaxation values than tofu coagulated with CaCl2. The gelation of tofu is formed by protein denaturation, mainly caused by heat, coagulation is also promoted by cations (Kohyama et al., 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 (Kohyama & Nishinari, 1993), the protons produced by d-gluconolactone or calcium ions neutralize the net charge of the protein. Thus the hydrophobic interaction of the neutralized proteins becomes more predominant and induces aggregation (Kohyama et al., 1995). The tofu containing high viscosity chitosan had less protein aggregation than control tofu in this study (Fig. 1). Chitosan might act as a coagulant because chitosan has a positive charge from amine and amide groups. However, because positive

charges able to neutralize the negatively charged proteins were sufficiently provided by the CaCl2 and d-gluconolactone, it was determined that chitosan did not play a great role in coagulating the protein. Rather, excess positive charges induced by chitosan might inhibit the protein aggregations which occur via hydrophobic interactions. The textural properties of the tofu with chitosan as measured by sensory evaluation were similar to those of the tofu without chitosan. In addition, the other sensory attributes were not greatly affected by adding high viscosity chitosan to the tofu. This result implies that the addition of high viscosity chitosan used in this study does not greatly affect the sensory properties of the tofu.
Conclusions

The addition of high viscosity chitosan to tofu scarcely affected the physico-chemical properties of tofu such as yield, moisture content and pH, but the colour of tofu became slightly more yellow. The TEM image showed that high viscosity chitosan affected the interactions between protein molecules, but the instrumental textural properties of the chitosan tofu did not greatly differ from those of the control tofu. The sensory characteristics also showed that high viscosity chitosan only slightly influenced the quality of the tofu. Therefore, these results suggest that high viscosity chitosan would be useful as an additive for extending the shelf-life of tofu because its quality did not severely deteriorate with the addition of high viscosity chitosan.
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