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The effect of chitosan on the gel properties of tofu (soybean curd)
Journal of Food Engineering 57 (2003) 315–319 www.elsevier.com/locate/jfoodeng
The effect of chitosan on the gel properties of tofu (soybean curd) K.L.B. Chang, Y.-S. Lin, R.H. Chen
*
Department of Food Science, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung 202, Taiwan Received 16 November 2001; accepted 12 August 2002
Abstract Chitosan was added to tofu prepared with different curdling agents. The properties of tofu depended on the curdling agents, chitosan content, and the degree of deacetylation (DD) of chitosan. The addition of 2% chitosan increased the gel strength of tofu for 5–305% and its shelf life for 2–10 days. Chitosan changed the water content of tofu slightly. The gel strength of tofu containing chitosan decreased with increasing DD. The shelf life of tofu increased with increasing DD of chitosan. Chitosan caused the smallest change in the gel properties of tofu prepared with glucono-d-lactone, but it affected the tofu prepared with acetic acid the most. Ó 2003 Elsevier Science Ltd. All rights reserved. Keywords: Chitosan; Tofu; Curdling agent; Gel strength; Shelf life
1. Introduction Chitosan is a cationic polysaccharide prepared from the deacetylation of chitin, the second most abundant biopolymer. The natural resources for chitin include shells of crustacean and shellfish, squid pen, and fungi (Knorr, 1984). Chitin and chitosan are biodegradable and biocompatible. As a consequence, extensive research has been conducted to explore their potential applications in various industries. Chitosan was an effective coagulant for removing proteins and lipids from wastewater (Hwang & Damodaran, 1995; Jun, Kim, No, & Meyers, 1994; SelmerOlsen, Ratnaweera, & Pehrson, 1996). The coagulating, lipid-binding, and antimicrobial properties make chitosan useful in a lot of food and nutritional applications (Shahidi, Arachchi, & Jeon, 1999). For instance, chitosan was found to help lower the blood cholesterol content in animal and human. It could therefore help reduce overweight (Muzzarelli, 1996). Chitosan has antibacterial as well as antifungal characteristics. Chitosan spray or coating could extend the storage life and quality of fresh produce such as tomatoes and strawberries (El Ghaouth, Arul, Ponnampalam, & Boulet, 1991; El Ghaouth, Ponnampalam, Castaigne, & Arul, 1992). Recently, more findings regarding the effect of chitosan on the properties of pro*
Corresponding author. Fax: +8862-2463-1977. E-mail address: [email protected] (R.H. Chen).
cessed foods have been reported. Inaba, Hoshizawa, and Fujiwara (1995) reported that chitin was less effective in modifying the textural properties of starch gels than collagen. Roller and Corvill (1999) reported that chitosan lengthened the lag phase and reduced the growth rate of many fungal species in apple juice. When combined with acetic acid and refrigerated storage, chitosan glutamate was able to inhibit the growth of several bacteria and yeast in mayonnaise and mayonnaise-based salad (Roller & Corvill, 2000). Although the antimicrobial properties of chitosan have been frequently investigated, little research has been done on the effect of chitosan on food gels. The objective of this study was to examine the gel properties of tofu containing chitosan. The effect of the addition of chitosan on the physical and storage characteristics of tofu prepared by different curdling agents from soy proteins was compared.
2. Materials and methods 2.1. Materials Shrimp (Solenocera prominentis) shells were collected from a local fish market (Keelung, Taiwan) and stored under frozen temperature ()20 °C) until further processing. Soybean was purchased from a local supermarket and stored at ambient temperature (25 °C). Chemicals (sodium chloride, oxalic acid, potassium permanganate, hydrochloric acid, sodium acetate, potassium
0260-8774/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 0 - 8 7 7 4 ( 0 2 ) 0 0 3 4 5 - X
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K.L.B. Chang et al. / Journal of Food Engineering 57 (2003) 315–319
bromide, etc.) were reagent grade products supplied by Merck (Darmstadt, Germany). Wako Pure Chemical Inc. (Osaka, Japan) supplied the acetic acid (99–100%). Food grade gypsum (calcium sulfate) and glucono-dlactone (GDL) were purchased from local food ingredient suppliers.
The specific viscosity data ðgsp =cÞ was plotted and regressed against the concentration to obtain the intrinsic viscosity ðgÞ. The Mark–Howink equation constants reported by Wang, Bo, Li, and Qin (1991) were used to calculate the molecular weight ðMw Þ of chitosan. That is, a ½g ¼ kðMw Þ , where k ¼ 1:64 1030 DD14:0 , a ¼ 1:82 1:02 102 DD.
2.2. Preparation of chitosan Shrimp shells were soaked in sodium hydroxide solution for 6 h at room temperature. The alkaline solution was drained and the shells were rinsed with tap water until neutrality. After drying, the shells were ground to 40–60 mesh size. The shrimp shell powder was demineralized in 2 N hydrochloric acid solution for 2 h. After washing the drained sample with water, we put the powder into 2 N sodium hydroxide solution at 80 °C to remove protein. The sample was washed with water until neutrality. Then it was discolored with potassium permanganate for 1 h, and reacted with oxalic acid for 1 h. The sample was then rinsed with water and dried at 50 °C to obtain white chitin powder. Chitin powder was added into 50% (w/w) sodium hydroxide solution to form 5% suspension. The suspension was heated to 100 °C for 90 min. Then the sample was rinsed till neutrality and dried at 50 °C to obtain chitosan sample with low degree of deacetylation (DD). A portion of the chitosan sample was reacted in 50% NaOH solution at 140 °C for 60 min to prepare chitosan with medium DD. After similar washing and drying, half of the sample was further deacetylated in 50% NaOH solution at 140 °C for another 180 min to obtain chitosan with high DD. 2.3. Degree of deacetylation Infrared spectrometer (BioRad FTS-155, FT-IR spectrometer, USA) was used to determine the DD of chitosan samples. Chitosan sample was mixed with potassium bromide (1:1000) and compressed into pellets. The absorption values at 1655 and 3450 cm1 were used to calculate the DD according to that described by Baxter, Dillon, Taylor, and Roberts (1992). That is, DD ð%Þ ¼ 100 ðA1655 =A3450 Þ115. 2.4. Molecular weight calculation Chitosan powder was dissolved in a solvent of 0.2 M acetic acid/0.1 M sodium acetate to form solution with concentrations of 0.01%, 0.025%, 0.05%, 0.1%, 0.2% (w/v). Each of the solution was poured into a capillary viscometer (Cannon-Fenske, No. 100), placed in a thermostat (Tamson, TMV 40, Sweden) at 30 0:1 °C to measure the retention time. The retention time data was then used to calculate the specific viscosity ðgsp Þ.
2.5. Preparation of tofu 2.5.1. GDL tofu––prepared using GDL as the curdling agent Soybeans were rinsed and soaked in water for 12 h. The beans are then pulverized with boiling water (850 ml water/240 g wet soybean, prepared by soaking 100 g dry beans with water) in a commercial soymilk grinder. The resultant mash was filtered to remove the particles. After the filtration, approximately 800 ml of soymilk remained. Distilled water (50 ml) was added to each 200 ml of soymilk. Then the diluted 250 ml of soymilk was heated with boiling water to 95 °C for 10 min to denature the antinutritive factors such as trypsin inhibitor. To the control sample 0.75 g of GDL was added. For the other samples, additional amount of 0.13, 0.26, 0.39, or 0.52 g of chitosan was included. The sample was left undisturbed for 5 min. Then it was cooled with 5 °C chilled water for 1 h to form GDL tofu containing 0–2% chitosan. 2.5.2. Gypsum tofu––prepared using gypsum as the curdling agent Soymilk was prepared as described above. To each 400 ml of soymilk, 400 ml of distilled water was added. The diluted sample was heated with boiling water to 95 °C for 10 min. A small amount of gypsum (1.5 g) was added to the control. For other samples, additional amount of 0.26, 0.58, 0.72, or 1.04 g of chitosan was added. The whey was separated from the curd. The curd was left undisturbed for 2 min. Then it was compressed with a force of 1.379 kPa (0.2 psia) to form gypsum tofu samples containing 0–2% chitosan. The resulting tofu was immersed into a container of 5 °C cold water and allowed to sit for another hour. 2.5.3. Acetic acid tofu––prepared with acetic acid as the curdling agent Soymilk was prepared as described above. To each 300 ml of soymilk, 200 ml of distilled water was added. The diluted sample was heated with boiling water to 95 °C for 10 min. A small volume of 5% acetic acid (6 ml) was added to the control. For other samples, additional amount of 0.22, 0.44, 0.66, or 0.88 g of chitosan was added. The rest of the procedure was the same as that described for gypsum tofu.
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2.6. Gel properties of tofu Tofu samples were cut into pieces of weight 5–8 g. Each sample was placed in a crucible, vacuum dried at 105 °C for 4 h. Then quickly take the samples out of the dryer and put them into a desiccator for 30 min. The difference between the initial weight and final dry weight was used to calculate the water content (AOAC, 1984). The shelf life of tofu was determined by performing a sensory test to samples stored at 4 °C refrigerator. A group of five experienced panelists was asked to examine the samples to see if there was noticeable change in color or flavor, or microbial growth on sample surface. The shelf life was reported as the days in storage that significant microbial growth on samples was found by the panelists. To measure the gel strength, tofu was cut into samples with a thickness of 15 mm. Each sample was placed on the anvil of a Sun Rheometer (model CR-300, Japan). A spherical probe (5 mm diameter) traveled downward (6 cm/min) to penetrate the samples. The breaking force (g) and breaking strain (mm) were recorded. The gel strength (g mm) was calculated as the product of breaking force and breaking strain.
3. Results and discussion 3.1. Characteristics of chitosan The yield of recovering chitin from shrimp shells was 31%. After deacetylation, the DD of chitosan was 54%, 73%, and 91% respectively (Table 1). Reacting in alkaline solution at higher temperature or for longer period resulted in chitosan samples with higher DD. This result agreed with a previous literature report for chitosan preparation (Chang, Tsai, Lee, & Fu, 1997). The molecular weight ðMw Þ of chitosan decreased from 2780 kDa for 54% DD to 189 kDa for 91% DD. This was because that deacetylation at elevated temperature in
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alkaline solution also broke down the glycosidic linkages in chitosan molecules. As a consequence, the intrinsic viscosity and Mw of chitosan decreased with increasing DD. 3.2. Influence of chitosan on the gel strength of tofu Table 2 shows the effect of 2.0% chitosan on the gel properties of tofu. Among the control samples prepared with three different methods, those coagulated with gypsum had the highest gel strength. On the contrary, GDL tofu was the softest. This was because that calcium ion in gypsum contributed to the ionic binding with acidic amino acids of soy proteins in tofu gels. In contrast, GDL became hydrolyzed into gluconic acid in water and formed relatively weaker hydrogen bonds. Regardless of the method of preparation, the gel strength of tofu was increased (for 5–305% versus control samples) by the addition of 2% chitosan. In addition, the increase in gel strength decreased with rising DD (Fig. 1). This indicated that higher Mw chitosan strengthened the gel structure of tofu more than lower Mw molecules did. High Mw chitosan apparently bound to a higher extent with soy protein. More entanglements could have occurred between the polysaccharide and protein molecules. Thus resulted in more stable gel network and firmer texture. Acetic acid tofu was most sensitive to the Table 1 The DD and molecular weight ðMw Þ of chitosan prepared under different conditions NaOH (%, w/w)
Temperature (°C)
50 50 50
100 140 140
Time (h) 1.5 1.0 3.0
DD (%)
Mw a (Dalton)
54 73 91
2:78 106 7:20 105 1:89 105
a The molecular weight calculated for the 54% DD samples was extrapolated to a level beyond the experimental range in the original reference.
Table 2 Changes in the gel strength, water content, and shelf life of different types of tofu caused by the addition of 2% chitosan Control
54% DD
73% DD
91% DD
GDL as the curdling agent Gel strength (g mm) Water content (%) Shelf lifea (days)
56 2.1 80 0.30 5
108 0 84 0.66 7
75 0 81 1.4 8
59 0 82 0.21 13
Gypsum as the curdling agent Gel strength (g mm) Water content (%) Shelf life (days)
71 7.2 74 0.71 8
210 30 79 7.2 13
158 2.4 82 0.97 13
121 12 80 0.65 18
Acetic acid as the curdling agent Gel strength (g mm) Water content (%) Shelf life (days)
66 2.3 79 0.84 12
267 0 78 0.22 15
166 2.8 78 0.22 20
90 8.9 76 2.1 22
a
The total days of storage in 4 °C refrigerator until significant mold growth and staled flavor were observed.
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Fig. 1. The gel strength of acetic acid tofu after adding chitosan with different degrees of deacetylation: (r) 54%; (j) 73%; (N) 91%.
GDL tofu from 5 to 7–13 days. The increase in shelf life (by 2% chitosan) for gypsum or acetic acid tofu was from 8 to 13–18 days, from 12 to 15–22 days respectively. Apparently chitosan had a good preserving effect when it was incorporated in the gel matrix of tofu. The shelf life increased with increasing DD. This might be due to that higher DD chitosan generally had higher solubility and more positive charges in acidic environment. The DD of chitosan would affect its charge density (at pH < 6:3), ion chelating ability and water binding capability. The current theory is that one or more of these three factors contribute to the antimicrobial property of chitosan (Shahidi et al., 1999). During refrigerated storage, the tofu samples first lost the pleasant fresh flavor, then its color turned darker. Eventually molds grew on the surface and tofu samples emitted a strong sour and staled odor (Table 3). The addition of 91% DD chitosan significantly improved the storage stability. Even the lowest addition level (0.5%) increased the shelf life by 3 (GDL tofu), 6 (gypsum tofu), and 8 (acetic acid tofu) days, respectively. The shelf life increased with increasing chitosan content. Nevertheless, the increase in the shelf life of tofu samples by the addition of 1.5% or 2.0% chitosan was nearly the same. 3.4. Influence of chitosan on the water content of tofu
Fig. 2. The effect of 54% DD chitosan on the gel strengths of tofu prepared by different curdling agents: (r) GDL; (j) Gypsum; (N) Acetic acid.
addition of chitosan (Fig. 2). This might be due to that chitosan was more soluble in soymilk containing acetic acid. This would allow more entanglements or interactions to occur between chitosan and soy protein molecules. The gel strength of tofu increased with increasing concentration of chitosan (Figs. 1 and 2). The gel strength nearly doubled when the level of 54% DD chitosan was raised from 0.5% to 2%. These results suggested that chitosan is quite effective in modifying the gel strength of tofu. 3.3. Influence of chitosan on the shelf life of tofu Acetic acid tofu had the longest shelf life, followed by gypsum and GDL. During refrigerated storage at 4 °C, the tofu samples prepared by different curdling agents showed different rates of quality deterioration. Among the control samples, GDL tofu had the shortest shelf life, the smallest gel strength and the largest amount of water (Table 2). For the same type of tofu, the shelf life increased with decreasing water content. The addition of 2% chitosan with different DD increased the shelf life of
The water content of tofu depended on the methods of preparation (Table 2). GDL tofu contained the largest amount of water, while gypsum tofu had the Table 3 The influence of 91% DD chitosan on the sensory changes of tofu samples prepared by different curdling agents during refrigerated storage at 4 °C Days until significant changes in sensory attributes were observed Chitosan content (%)
Loss of pleasant flavor
Color difference
Mold growth
Staled odor
GDL as the curdling agent 0 3 0.5 6 1.0 6 1.5 8 2.0 11
4 7 7 10 12
5 8 8 11 13
5 8 8 11 13
Gypsum as the curdling agent 0 6 0.5 12 1.0 12 1.5 15 2.0 15
7 13 13 16 17
8 14 14 17 18
8 14 14 17 18
Acetic acid as the curdling agent 0 10 11 0.5 17 19 1.0 19 20 1.5 19 21 2.0 20 21
12 20 21 22 22
12 20 21 22 22
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smallest amount of water. The addition of chitosan increased the water content slightly for tofu prepared using GDL and gypsum. But it decreased the water content of the tofu prepared with acetic acid by 1–3%. Consequently, for the samples that contained chitosan, the acetic acid tofu had the lowest water content. These results indicated that chitosan bound with more water in the presence of GDL and calcium ions. However, in an environment containing acetic acid, it became much more hydrophobic and excluded more water from the gel. The water content of samples differed slightly for samples containing chitosan of different DD. These results suggest that one order of magnitude difference in the Mw of chitosan (from 106 to 105 ) did not cause a significant change in its water holding capability. Water content of tofu samples seemed to have less influence on their gel strengths than the DD of chitosan. However, the lower water content acetic acid tofu had the highest gel strength and longest shelf life. This suggested that water content also had substantial influence on the gel properties of tofu.
4. Conclusions The gel properties of tofu depended on the curdling agent and method used for its preparation. For the same type of tofu, the addition of chitosan increased its gel strength and storage stability. Chitosan changed the water holding of tofu gel structure slightly. The effect on the gel properties of tofu showed a strong relationship with the DD of chitosan.
Acknowledgements The authors express their thanks to the National Science Council, ROC, for supporting this research (Grant: NSC 86-2815-C-019-017-B).
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References AOAC (1984). Official methods of analysis (14th ed.). Washington, DC: Association of Official Analytical Chemists (1141 p). Baxter, A., Dillon, M., Taylor, K. D. A., & Roberts, G. A. F. (1992). Improved method for i.r. determination of the degree of Nacetylation of chitosan. International Journal of Biological Macromolecules, 14(6), 166–169. Chang, K. L. B., Tsai, G., Lee, J., & Fu, W. R. (1997). Heterogeneous N-deacetylation of chitin in alkaline solution. Carbohydrate Research, 303(3), 327–332. El Ghaouth, A., Arul, J., Ponnampalam, R., & Boulet, M. (1991). Chitosan coating effect on storability and quality of fresh strawberries. Journal of Food Science, 56(6), 1618–1620, 1631. El Ghaouth, A., Ponnampalam, R., Castaigne, F., & Arul, J. (1992). Chitosan coating to extend the storage life of tomatoes. Horticultural Science, 27(9), 1016–1018. Hwang, D. C., & Damodaran, S. (1995). Selective precipitation and removal of lipids from cheese whey using chitosan. Journal of Agricultural and Food Chemistry, 43(1), 33–37. Inaba, H., Hoshizawa, M., & Fujiwara, A. (1995). Textural properties of starch gels filled with collagen and chitin. Journal of Texture Studies, 26(5), 577–586. Jun, H. K., Kim, J. S., No, H. K., & Meyers, S. P. (1994). Chitosan as a coagulant for recovery of proteinaceous solids from tofu wastewater. Journal of Agricultural and Food Chemistry, 42(8), 1834–1838. Knorr, D. (1984). Use of chitinous polymers in food. Food Technology, 38(1), 85–97. Muzzarelli, R. A. A. (1996). Chitosan-based dietary foods. Carbohydrate Polymers, 29(4), 309–316. Roller, S., & Corvill, N. (1999). The antifungal properties of chitosan in laboratory media and apple juice. International Journal of Food Microbiology, 47(1), 67–77. Roller, S., & Corvill, N. (2000). The antimicrobial properties of chitosan in mayonnaise and mayonnaise-based shrimp salads. Journal of Food Protection, 63(2), 202–209. Selmer-Olsen, E., Ratnaweera, H. C., & Pehrson, R. (1996). A novel treatment process for dairy wastewater with chitosan produced from shrimp-shell waste. Water Science and Technology, 34(11), 33–40. Shahidi, F., Arachchi, J. K. V., & Jeon, Y. J. (1999). Food applications of chitin and chitosans. Trends in Food Science and Technology, 10(1), 37–51. Wang, W., Bo, S., Li, S., & Qin, W. (1991). Determination of the Mark–Houwink equation for chitosans with different degrees of deacetylation. International Journal of Biological Macromolecules, 13(5), 281–285.
The effect of chitosan on the gel properties of tofu (soybean curd) K.L.B. Chang, Y.-S. Lin, R.H. Chen
*
Department of Food Science, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung 202, Taiwan Received 16 November 2001; accepted 12 August 2002
Abstract Chitosan was added to tofu prepared with different curdling agents. The properties of tofu depended on the curdling agents, chitosan content, and the degree of deacetylation (DD) of chitosan. The addition of 2% chitosan increased the gel strength of tofu for 5–305% and its shelf life for 2–10 days. Chitosan changed the water content of tofu slightly. The gel strength of tofu containing chitosan decreased with increasing DD. The shelf life of tofu increased with increasing DD of chitosan. Chitosan caused the smallest change in the gel properties of tofu prepared with glucono-d-lactone, but it affected the tofu prepared with acetic acid the most. Ó 2003 Elsevier Science Ltd. All rights reserved. Keywords: Chitosan; Tofu; Curdling agent; Gel strength; Shelf life
1. Introduction Chitosan is a cationic polysaccharide prepared from the deacetylation of chitin, the second most abundant biopolymer. The natural resources for chitin include shells of crustacean and shellfish, squid pen, and fungi (Knorr, 1984). Chitin and chitosan are biodegradable and biocompatible. As a consequence, extensive research has been conducted to explore their potential applications in various industries. Chitosan was an effective coagulant for removing proteins and lipids from wastewater (Hwang & Damodaran, 1995; Jun, Kim, No, & Meyers, 1994; SelmerOlsen, Ratnaweera, & Pehrson, 1996). The coagulating, lipid-binding, and antimicrobial properties make chitosan useful in a lot of food and nutritional applications (Shahidi, Arachchi, & Jeon, 1999). For instance, chitosan was found to help lower the blood cholesterol content in animal and human. It could therefore help reduce overweight (Muzzarelli, 1996). Chitosan has antibacterial as well as antifungal characteristics. Chitosan spray or coating could extend the storage life and quality of fresh produce such as tomatoes and strawberries (El Ghaouth, Arul, Ponnampalam, & Boulet, 1991; El Ghaouth, Ponnampalam, Castaigne, & Arul, 1992). Recently, more findings regarding the effect of chitosan on the properties of pro*
Corresponding author. Fax: +8862-2463-1977. E-mail address: [email protected] (R.H. Chen).
cessed foods have been reported. Inaba, Hoshizawa, and Fujiwara (1995) reported that chitin was less effective in modifying the textural properties of starch gels than collagen. Roller and Corvill (1999) reported that chitosan lengthened the lag phase and reduced the growth rate of many fungal species in apple juice. When combined with acetic acid and refrigerated storage, chitosan glutamate was able to inhibit the growth of several bacteria and yeast in mayonnaise and mayonnaise-based salad (Roller & Corvill, 2000). Although the antimicrobial properties of chitosan have been frequently investigated, little research has been done on the effect of chitosan on food gels. The objective of this study was to examine the gel properties of tofu containing chitosan. The effect of the addition of chitosan on the physical and storage characteristics of tofu prepared by different curdling agents from soy proteins was compared.
2. Materials and methods 2.1. Materials Shrimp (Solenocera prominentis) shells were collected from a local fish market (Keelung, Taiwan) and stored under frozen temperature ()20 °C) until further processing. Soybean was purchased from a local supermarket and stored at ambient temperature (25 °C). Chemicals (sodium chloride, oxalic acid, potassium permanganate, hydrochloric acid, sodium acetate, potassium
0260-8774/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 0 - 8 7 7 4 ( 0 2 ) 0 0 3 4 5 - X
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K.L.B. Chang et al. / Journal of Food Engineering 57 (2003) 315–319
bromide, etc.) were reagent grade products supplied by Merck (Darmstadt, Germany). Wako Pure Chemical Inc. (Osaka, Japan) supplied the acetic acid (99–100%). Food grade gypsum (calcium sulfate) and glucono-dlactone (GDL) were purchased from local food ingredient suppliers.
The specific viscosity data ðgsp =cÞ was plotted and regressed against the concentration to obtain the intrinsic viscosity ðgÞ. The Mark–Howink equation constants reported by Wang, Bo, Li, and Qin (1991) were used to calculate the molecular weight ðMw Þ of chitosan. That is, a ½g ¼ kðMw Þ , where k ¼ 1:64 1030 DD14:0 , a ¼ 1:82 1:02 102 DD.
2.2. Preparation of chitosan Shrimp shells were soaked in sodium hydroxide solution for 6 h at room temperature. The alkaline solution was drained and the shells were rinsed with tap water until neutrality. After drying, the shells were ground to 40–60 mesh size. The shrimp shell powder was demineralized in 2 N hydrochloric acid solution for 2 h. After washing the drained sample with water, we put the powder into 2 N sodium hydroxide solution at 80 °C to remove protein. The sample was washed with water until neutrality. Then it was discolored with potassium permanganate for 1 h, and reacted with oxalic acid for 1 h. The sample was then rinsed with water and dried at 50 °C to obtain white chitin powder. Chitin powder was added into 50% (w/w) sodium hydroxide solution to form 5% suspension. The suspension was heated to 100 °C for 90 min. Then the sample was rinsed till neutrality and dried at 50 °C to obtain chitosan sample with low degree of deacetylation (DD). A portion of the chitosan sample was reacted in 50% NaOH solution at 140 °C for 60 min to prepare chitosan with medium DD. After similar washing and drying, half of the sample was further deacetylated in 50% NaOH solution at 140 °C for another 180 min to obtain chitosan with high DD. 2.3. Degree of deacetylation Infrared spectrometer (BioRad FTS-155, FT-IR spectrometer, USA) was used to determine the DD of chitosan samples. Chitosan sample was mixed with potassium bromide (1:1000) and compressed into pellets. The absorption values at 1655 and 3450 cm1 were used to calculate the DD according to that described by Baxter, Dillon, Taylor, and Roberts (1992). That is, DD ð%Þ ¼ 100 ðA1655 =A3450 Þ115. 2.4. Molecular weight calculation Chitosan powder was dissolved in a solvent of 0.2 M acetic acid/0.1 M sodium acetate to form solution with concentrations of 0.01%, 0.025%, 0.05%, 0.1%, 0.2% (w/v). Each of the solution was poured into a capillary viscometer (Cannon-Fenske, No. 100), placed in a thermostat (Tamson, TMV 40, Sweden) at 30 0:1 °C to measure the retention time. The retention time data was then used to calculate the specific viscosity ðgsp Þ.
2.5. Preparation of tofu 2.5.1. GDL tofu––prepared using GDL as the curdling agent Soybeans were rinsed and soaked in water for 12 h. The beans are then pulverized with boiling water (850 ml water/240 g wet soybean, prepared by soaking 100 g dry beans with water) in a commercial soymilk grinder. The resultant mash was filtered to remove the particles. After the filtration, approximately 800 ml of soymilk remained. Distilled water (50 ml) was added to each 200 ml of soymilk. Then the diluted 250 ml of soymilk was heated with boiling water to 95 °C for 10 min to denature the antinutritive factors such as trypsin inhibitor. To the control sample 0.75 g of GDL was added. For the other samples, additional amount of 0.13, 0.26, 0.39, or 0.52 g of chitosan was included. The sample was left undisturbed for 5 min. Then it was cooled with 5 °C chilled water for 1 h to form GDL tofu containing 0–2% chitosan. 2.5.2. Gypsum tofu––prepared using gypsum as the curdling agent Soymilk was prepared as described above. To each 400 ml of soymilk, 400 ml of distilled water was added. The diluted sample was heated with boiling water to 95 °C for 10 min. A small amount of gypsum (1.5 g) was added to the control. For other samples, additional amount of 0.26, 0.58, 0.72, or 1.04 g of chitosan was added. The whey was separated from the curd. The curd was left undisturbed for 2 min. Then it was compressed with a force of 1.379 kPa (0.2 psia) to form gypsum tofu samples containing 0–2% chitosan. The resulting tofu was immersed into a container of 5 °C cold water and allowed to sit for another hour. 2.5.3. Acetic acid tofu––prepared with acetic acid as the curdling agent Soymilk was prepared as described above. To each 300 ml of soymilk, 200 ml of distilled water was added. The diluted sample was heated with boiling water to 95 °C for 10 min. A small volume of 5% acetic acid (6 ml) was added to the control. For other samples, additional amount of 0.22, 0.44, 0.66, or 0.88 g of chitosan was added. The rest of the procedure was the same as that described for gypsum tofu.
K.L.B. Chang et al. / Journal of Food Engineering 57 (2003) 315–319
2.6. Gel properties of tofu Tofu samples were cut into pieces of weight 5–8 g. Each sample was placed in a crucible, vacuum dried at 105 °C for 4 h. Then quickly take the samples out of the dryer and put them into a desiccator for 30 min. The difference between the initial weight and final dry weight was used to calculate the water content (AOAC, 1984). The shelf life of tofu was determined by performing a sensory test to samples stored at 4 °C refrigerator. A group of five experienced panelists was asked to examine the samples to see if there was noticeable change in color or flavor, or microbial growth on sample surface. The shelf life was reported as the days in storage that significant microbial growth on samples was found by the panelists. To measure the gel strength, tofu was cut into samples with a thickness of 15 mm. Each sample was placed on the anvil of a Sun Rheometer (model CR-300, Japan). A spherical probe (5 mm diameter) traveled downward (6 cm/min) to penetrate the samples. The breaking force (g) and breaking strain (mm) were recorded. The gel strength (g mm) was calculated as the product of breaking force and breaking strain.
3. Results and discussion 3.1. Characteristics of chitosan The yield of recovering chitin from shrimp shells was 31%. After deacetylation, the DD of chitosan was 54%, 73%, and 91% respectively (Table 1). Reacting in alkaline solution at higher temperature or for longer period resulted in chitosan samples with higher DD. This result agreed with a previous literature report for chitosan preparation (Chang, Tsai, Lee, & Fu, 1997). The molecular weight ðMw Þ of chitosan decreased from 2780 kDa for 54% DD to 189 kDa for 91% DD. This was because that deacetylation at elevated temperature in
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alkaline solution also broke down the glycosidic linkages in chitosan molecules. As a consequence, the intrinsic viscosity and Mw of chitosan decreased with increasing DD. 3.2. Influence of chitosan on the gel strength of tofu Table 2 shows the effect of 2.0% chitosan on the gel properties of tofu. Among the control samples prepared with three different methods, those coagulated with gypsum had the highest gel strength. On the contrary, GDL tofu was the softest. This was because that calcium ion in gypsum contributed to the ionic binding with acidic amino acids of soy proteins in tofu gels. In contrast, GDL became hydrolyzed into gluconic acid in water and formed relatively weaker hydrogen bonds. Regardless of the method of preparation, the gel strength of tofu was increased (for 5–305% versus control samples) by the addition of 2% chitosan. In addition, the increase in gel strength decreased with rising DD (Fig. 1). This indicated that higher Mw chitosan strengthened the gel structure of tofu more than lower Mw molecules did. High Mw chitosan apparently bound to a higher extent with soy protein. More entanglements could have occurred between the polysaccharide and protein molecules. Thus resulted in more stable gel network and firmer texture. Acetic acid tofu was most sensitive to the Table 1 The DD and molecular weight ðMw Þ of chitosan prepared under different conditions NaOH (%, w/w)
Temperature (°C)
50 50 50
100 140 140
Time (h) 1.5 1.0 3.0
DD (%)
Mw a (Dalton)
54 73 91
2:78 106 7:20 105 1:89 105
a The molecular weight calculated for the 54% DD samples was extrapolated to a level beyond the experimental range in the original reference.
Table 2 Changes in the gel strength, water content, and shelf life of different types of tofu caused by the addition of 2% chitosan Control
54% DD
73% DD
91% DD
GDL as the curdling agent Gel strength (g mm) Water content (%) Shelf lifea (days)
56 2.1 80 0.30 5
108 0 84 0.66 7
75 0 81 1.4 8
59 0 82 0.21 13
Gypsum as the curdling agent Gel strength (g mm) Water content (%) Shelf life (days)
71 7.2 74 0.71 8
210 30 79 7.2 13
158 2.4 82 0.97 13
121 12 80 0.65 18
Acetic acid as the curdling agent Gel strength (g mm) Water content (%) Shelf life (days)
66 2.3 79 0.84 12
267 0 78 0.22 15
166 2.8 78 0.22 20
90 8.9 76 2.1 22
a
The total days of storage in 4 °C refrigerator until significant mold growth and staled flavor were observed.
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Fig. 1. The gel strength of acetic acid tofu after adding chitosan with different degrees of deacetylation: (r) 54%; (j) 73%; (N) 91%.
GDL tofu from 5 to 7–13 days. The increase in shelf life (by 2% chitosan) for gypsum or acetic acid tofu was from 8 to 13–18 days, from 12 to 15–22 days respectively. Apparently chitosan had a good preserving effect when it was incorporated in the gel matrix of tofu. The shelf life increased with increasing DD. This might be due to that higher DD chitosan generally had higher solubility and more positive charges in acidic environment. The DD of chitosan would affect its charge density (at pH < 6:3), ion chelating ability and water binding capability. The current theory is that one or more of these three factors contribute to the antimicrobial property of chitosan (Shahidi et al., 1999). During refrigerated storage, the tofu samples first lost the pleasant fresh flavor, then its color turned darker. Eventually molds grew on the surface and tofu samples emitted a strong sour and staled odor (Table 3). The addition of 91% DD chitosan significantly improved the storage stability. Even the lowest addition level (0.5%) increased the shelf life by 3 (GDL tofu), 6 (gypsum tofu), and 8 (acetic acid tofu) days, respectively. The shelf life increased with increasing chitosan content. Nevertheless, the increase in the shelf life of tofu samples by the addition of 1.5% or 2.0% chitosan was nearly the same. 3.4. Influence of chitosan on the water content of tofu
Fig. 2. The effect of 54% DD chitosan on the gel strengths of tofu prepared by different curdling agents: (r) GDL; (j) Gypsum; (N) Acetic acid.
addition of chitosan (Fig. 2). This might be due to that chitosan was more soluble in soymilk containing acetic acid. This would allow more entanglements or interactions to occur between chitosan and soy protein molecules. The gel strength of tofu increased with increasing concentration of chitosan (Figs. 1 and 2). The gel strength nearly doubled when the level of 54% DD chitosan was raised from 0.5% to 2%. These results suggested that chitosan is quite effective in modifying the gel strength of tofu. 3.3. Influence of chitosan on the shelf life of tofu Acetic acid tofu had the longest shelf life, followed by gypsum and GDL. During refrigerated storage at 4 °C, the tofu samples prepared by different curdling agents showed different rates of quality deterioration. Among the control samples, GDL tofu had the shortest shelf life, the smallest gel strength and the largest amount of water (Table 2). For the same type of tofu, the shelf life increased with decreasing water content. The addition of 2% chitosan with different DD increased the shelf life of
The water content of tofu depended on the methods of preparation (Table 2). GDL tofu contained the largest amount of water, while gypsum tofu had the Table 3 The influence of 91% DD chitosan on the sensory changes of tofu samples prepared by different curdling agents during refrigerated storage at 4 °C Days until significant changes in sensory attributes were observed Chitosan content (%)
Loss of pleasant flavor
Color difference
Mold growth
Staled odor
GDL as the curdling agent 0 3 0.5 6 1.0 6 1.5 8 2.0 11
4 7 7 10 12
5 8 8 11 13
5 8 8 11 13
Gypsum as the curdling agent 0 6 0.5 12 1.0 12 1.5 15 2.0 15
7 13 13 16 17
8 14 14 17 18
8 14 14 17 18
Acetic acid as the curdling agent 0 10 11 0.5 17 19 1.0 19 20 1.5 19 21 2.0 20 21
12 20 21 22 22
12 20 21 22 22
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smallest amount of water. The addition of chitosan increased the water content slightly for tofu prepared using GDL and gypsum. But it decreased the water content of the tofu prepared with acetic acid by 1–3%. Consequently, for the samples that contained chitosan, the acetic acid tofu had the lowest water content. These results indicated that chitosan bound with more water in the presence of GDL and calcium ions. However, in an environment containing acetic acid, it became much more hydrophobic and excluded more water from the gel. The water content of samples differed slightly for samples containing chitosan of different DD. These results suggest that one order of magnitude difference in the Mw of chitosan (from 106 to 105 ) did not cause a significant change in its water holding capability. Water content of tofu samples seemed to have less influence on their gel strengths than the DD of chitosan. However, the lower water content acetic acid tofu had the highest gel strength and longest shelf life. This suggested that water content also had substantial influence on the gel properties of tofu.
4. Conclusions The gel properties of tofu depended on the curdling agent and method used for its preparation. For the same type of tofu, the addition of chitosan increased its gel strength and storage stability. Chitosan changed the water holding of tofu gel structure slightly. The effect on the gel properties of tofu showed a strong relationship with the DD of chitosan.
Acknowledgements The authors express their thanks to the National Science Council, ROC, for supporting this research (Grant: NSC 86-2815-C-019-017-B).
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