Calcinated Shell Powder from Corbicula fluminea as a Natural Antimicrobial Agent for Soybean Curd (Tofu) Preservation

Abstract

The waste clam shells from Corbicula fluminea is a problem that needs to be solved in Taiwan. We aimed to develop the shell powder for use as antimicrobial agent in soybean curd (tofu) preservation. The calcined clam shell powder (CCSP) characteristics, antimicrobial ability and influence on tofu quality were studied. The results have shown the significantly inhibitoratory effect on Aspergillus niger, Bacillus cereus, Kloeckera apiculata were found. Tofu (1.0 and 1.5 g/L CCSP addition) exhibited dimensional reticular structure, and the sensory evaluation of tofu which added with 0.5 and 1.0 g/L of CCSP showed higher mouthfeel, texture, and lower beany-flavor. The shelf-life of tofu could be extending for 3 d when added with 1.5 g/L of CCSP. The CCSP was firstly reported as an antimicrobial agent in the paper. On the basis of the overall observations, CCSP can be regarded as a natural antimicrobial agent on tofu processing and preservation.

Introduction

Corbicula fluminea, often called golden clam or golden freshwater clam, one of popular edible freshwater clam in Taiwan and Asian countries. A number of studies of clam extract has reported be beneficial to the health function (Chijimatsu et al., 2012; Huang et al., 2013; Soliman, 2011). In recent years, the nutritional value of clam has been accepted by consumer, so it has promoted the development of its nutritional supplements and diversified products, such as clam oriyen and clam essence. However, tens of thousands of tons of clam shells are stacked on the side of pool in clam harvesting area, and increase the costs of waste management. Therefore, the new development and novel application for clam shell are required.

Previous studies have shown that shell powder of shellfish exist antibacterial ability after calcination, such as the heated scallop-shell powder (HSSP) possesses superior antibacterial activity (Sawai et al., 2001a). In fact, the shell powder exhibited highly bactericidal action after calcining above 700 °C (Sawai et al., 2001b). Bae et al. (2006) indicated that CaO from scallop shell may substitute for synthetic chemical agents in foods.

Soybean curd (tofu) could be an excellent substitute for meat, and it is a highly favorable medium for microorganisms due to its high-protein and high-moisture content. Prevention of microbial growth in tofu is considered an important research topic to ensure food safety. Contamination of tofu usually occurs during transportation and manufacturing processes, and the temperature fluctuations is a major influencing factor. Though refrigeration is common nowadays, but it does not eliminate undesirable microorganisms without the addition of preservatives (Anbarasu et al., 2007; Ezekiel et al., 2010). Therefore, freshly made tofu without food preservatives added under tropical conditions can be stored for only 1–2 d before the deterioration or corruption occurs (Lim and Foo, 1993). Even though, tofu can only control the total microbes below 10⁵ CFU/g about 48 h in refrigerated storage conditions (5–10 °C). Therefore, it is necessary to find a manufacturing method or effective and natural additive to extend the shelf life of tofu or inhibit corruption. Many researchers tried to find additive to prolong the shelf life of tofu. Chen et al. (2014) enhanced shelf life of tofu by using bacteriocinogenic Weissella hellenica D1501 was isolated from Chinese fermented meat. Purba et al. (2014) suggested that liquid smoke and chitosan can be used as a natural preservative in tofu and replaced synthetic preservatives and to extend the life of tofu for three days. Lee and Jung (2017) reported that used licorice powder as supplement on tofu can get a better shelf life. Regarding shell powder, previous studies also reported that quality and shelf life of kimchi could be enhanced by adding oyster shell powder (OSP) (Choi et al. 2006), and addition of OSP (0.05 and 0.1%) for tofu manufacturing can improve the sensory evaluation and prolong the shelf life of above 2 days longer (Kim et al., 2007). Beside the above-mentioned aquatic waste, the uncontaminated shell from cultured of Corbicula fluminea was adopted as an alternative source of antimicrobial substances in the present study. And, the calcined clam shell powder (CCSP) was firstly investigated the antimicrobial activity and quality of CCSP-containing tofu in the research.

Materials and Methods

Materials    Soybean (hundred seed weight was 19.44 g) was purchased from Chi-Sheng Co., Ltd. (R.O.C.). Clam shell was obtained from Li Chuan Aquafarm (R.O.C.). Commercial OSP was obtained from Micro-Tech Foods Ingredients, Inc. (R.O.C.). The 8-mm diameter disc was obtained from Advantec Toyo Kaisha, Ltd. (Japan). Calcium oxide was obtained from Nippon Kogyo K.K. Co., Ltd. (Japan). Food-grade MgCl₂ was purchased from Ako Kasei Co., Ltd. (Japan).

Microorganisms and cultivation media    Microorganism strains were obtained from Bioresource Collection and Research Center (BCRC, Food Industry Research and Development Institute, Hsinchu, R.O.C.), inclusive of Aspergillus niger ATCC 15475 (BCRC 30204), Bacillus cereus ATCC 14579 (BCRC 10603), Escherichia coli ATCC 25922 (BCRC 11509), Kloeckera apiculata ATCC 9774 (BCRC 20539), Salmonella enterica serovar typhimurium ATCC 13311 (BCRC 12947), Staphylococcus aureus ATCC12600 (BCRC 10780) and Vibrio parahaemolyticus ATCC 17802 (BCRC 10806). Yeast peptone dextrose, potato dextrose agar, plate count agar, potato dextrose broth, nutrient agar, nutrient broth, tryptone soy agar trypticase soy agar and trypticase soy broth mediums were purchased from Difco Co., Ltd. (USA).

Preparations of CCSP    The attachments and residual meat were removed after pre-washing and then coarsely ground, which were soaked in 0.1 M HCl solution for 48 h to eliminate the stratum corneum and then immersed in 1.5 volumes of 3 M H₂O₂ for 24 h. Washed by tap water for 1 h and then ultrasonic vibration for 90 min on 30 kHz to remove impurities. Thereafter, they were crude crushed after dried in oven at 80 °C for 20 h, and calcined at different temperature (600, 800 and 1 000 °C) then ground into finely by a blender RT-02B from Rong Tsong Precision Technology Co., Ltd. (R.O.C.), after that the ground CCSP (particle size < 109 µm) stored at 25 °C in vacuum desiccator until use.

Water activity, Moisture content, Color analysis of CCSP and tofu    Water activity (aw) of sample (3 g) was measured by CX-2 AQUA LAB from Decagon Devices, Inc. (USA) Moisture content of shell powder and tofu (5 ± 0.5 g) was measured by Infrared moisture analyzer MX-50 from A&D Co., Ltd. (Japan). The evaluation of color in sample of shell powder and tofu were performed by a color difference meter CDM-08 from Laiko Co., Ltd. (Japan). There is a standard white tile (reflectance values of X = 80.30, Y = 81.65 and Z = 91.39) used as a reference. Sample (2 g) was placed in quartz container (diameter 3 cm × height 1.5 cm) for analyzing. Sample (2 g) was placed in quartz container (diameter 3 cm × height 1.5 cm) for analyzing and recording hunter L*, a*, and b*, namely, lightness (L* = 0, black; L* = 100, whiteness), green/red color component (a* > 0, red; a* < 0, green), (b* > 0, yellow; b* < 0, blue), ΔE is the total color difference and was calculated as [(ΔL*)² + (Δa*)² + (Δb*)²]^1/2. Each sample has five replicated measurements.

Antimicrobial activity test of shell powder    The disk diffusion method (Salem et al., 2013) was employed to determine the in vitro antimicrobial of the CCSP against one fungal strain, one yeast strain, and five bacterial strains. Activated microorganisms inoculated in Yeast peptone dextrose broth (A. niger), nutrient broth (B. cereus, E. coli), trypticase soy broth (S. aureus, V. parahaemolyticus, S. enterica serovar typhimurium), potato dextrose broth (K. apiculate), and incubated at 37 °C for 24 ± 1 h. The strains (1 × 10⁷ CFU/mL) were counted by bacteria counting chamber, and overnight culture poured into the plate of nutrient agar (B. cereus) trypticase soy agar (E. coli, S. aureus, V. parahaemolyticus and S. enterica serovar typhimurium) and potato dextrose agar (A. niger). The 8-mm diameter discs were sterilized and immersed in the 0.5, 1.0, and 1.5 g/L CCSP solution for 5 min, and the disks were transferred to the surface on the plate, respectively. After incubated at 37 °C for 24 ± 1 h, inhibition zone was measured based on the diameter of the clear zone formed around the well. A negative control was prepared using sterilized water.

Processing of tofu    Tofu was made followed the method recommended by Park et al. (2003) with little modification. Soybeans (300 g) washed and soaked in reverse osmosis water (volume: 2 L, temperature: 20 – 25 °C) for 12 h. After soaking, the soybeans were grinding with 3000 mL reverse osmosis water for 3 min in the blender by using high speed. Soy milk (1 000 mL) was extruded through a filter cloth and boiled and then the 18 g/L of MgCl₂ or 0.5, 1.0, and 1.5 g/L of CCSP were added (alone or together) at 85 ± 1 °C, and pH (6.8, 8.3, 9.8) of soy milk when CCSP (0.5, 1.0, and 1.5 g/L) were added respectively, followed the soy milk was coagulated after 15 min. The curd was poured into a wood container (16 cm length × 12 cm width × 9 cm depth) and pressed for 20 min by using 8-lb iron plate. Tofu was moved to clean cloth and drained for 30 min at room temperature (25 °C) after immersing under reverse osmosis running water for 30 min, and then the tofu was removed for scanning electron microscopy (SEM) analysis and sensory evaluation. Furthermore, tofu prepared after clean cheese cloth for draining 5 min was weighed to calculate the yield of tofu.

Morphology observation of shell powder and tofu    Tofu sample (10 × 10 × 2 mm) was prepared followed the modified method (Onodera et al., 2009). Tofu was fixed with 1% glutaraldehyde for 1 h, and freeze-dried by liquid nitrogen after wash with deionized water. The clam shell powder (CSP), CCSP and freeze-dried tofu were mounted on specimen stub, sputter-coated with platinum in an ion sputter E-1010, Hitachi Co Ltd. (Japan) prior to the observation. The microstructure samples were examined by a field emission scanning electron microscope S-3000N from Hitachi Co Ltd.

Instrumental texture profile analysis (TPA) of tofu    The instrumental TPA of tofu was performed by using an Instron Universal Testing Machine 4464 from Instron Corporation Canton (USA), which measured the objective texture of the tofu during storage. A cylinder with a diameter of 6.3 mm compresses the tested samples (15 mm length × 15 mm width × 15 mm height) to 50% of their original height with an inspection speed of 120 mm/min in double adaptor compression test. The adaptor was repeated once for completing the measurements. The methods of hardness, cohesiveness, springiness, gumminess, chewiness and adhesiveness of tofu were tested according to the definitions given by Bourne (1982). Each test was repeated ten times.

Storage experiment of tofu    Tofu was cut into cube sample (15 × 15 × 15 mm) and then put in a container (12 × 8 × 12 cm) filled with 50 mL of sterilized distilled water at 10 °C for analysis. The tofu was sealed in the container was stored at 10 °C for 14 days. The tofu were homogenized with immersion solution together 1 min by using Osterizer Homogenizer K106-1T from Sunbeam Product, Inc. (USA) and centrifuged at 2 500 × g for 5 min, and the supernatant was diluted with 0.1% peptone water to determine total viable bacterial counts by using plate count agar. All experimental groups incubated at 35 °C for 48 ± 2 h, and the colony forming unit (CFU) means each colony that can be counted per gram. Each test was repeated triplicated.

Sensory evaluation of tofu    The tofu added with different concentrations of CCSP subjected to recognize and score with sensory evaluation test by twenty panelists who were trained after 20 h of sensory training courses and 10 h practical operation. After cutting, the tofu cubes (2 × 2 × 2 cm) were steamed on a dish and coded in a randomized arrangement before the test. The results were presented by using the 9-point scale method (i.e., the lower (higher) the point corresponds the poorer or weaker (better or stronger) attribute).

Statistical analysis    The collected data were analyzed by ANOVA (analysis of variance) procedure of the SPSS (Statistical Package for Social Sciences, SPSS Inc., Chicago, USA). In this study, all the experiments were executed in triplicate at least and all data were expressed mean ± standard deviation.

Results and Discussion

The color, aw, and moisture content of shell powder and CCSP-containing tofu    The moisture content and aw of CCSP significantly reduced by removing partial water of crystallization after calcination. The aw values of CCSP (0.31) and OSP (0.22) were respectively lower than that of CSP (0.54) and the moisture content of CCSP decreased drastically to 0.28% (Table 1), and CCSP-containing tofu exhibit high level moisture content and aw values, which were not significantly different among the groups. Interestingly, CCSP exhibited the highest brightness value (L*) and ΔE suggesting that it was brighter than those of commercial OSP and CSP. The CCSP attained the second low a* and lowest b* values. This is because the yellow keratin on the clam shell was removed by hydrogen peroxide, and the residual elements (e.g., magnesium, phosphorus) in CSP had been oxidized after calcination at 1 000 °C; hence, the brightness was markedly increased. This is a beneficial property for food additives and hint it does not easy to cause color changes during food processing. Moreover, the results showed that there was no significant difference in water activity and water content of all the tofu samples. Tofu prepared without CCSP showed a slightly higher L* and a* values, but lower in b* value than the tofu prepared with CCSP (Table 1). By increasing the addition of CCSP, there were trends of decrease for L* and a* value, but there were no significant differences in L* value which were observed among the addition of CCSP. Although the ΔE had a significant difference, the color of all the tofu samples had light yellow. And, tofu of good quality is generally white or light yellow in color. These results were consistent according to Kim et al. (2007).

Table 1. The color, aw, and moisture content of shell powder and CCSP-containing tofu.

Powder types	aw	Moisture content (%)	L* Value	a* Value	b* Value	ΔE
CSP	0.54 ± 0.00a	0.53 ± 0.02a	90.4 ± 0.0c	1.70 ± 0.30a	4.06 ± 0.05a	0.12 ± 0.03c
CCSP	0.31 ± 0.01b	0.28 ± 0.01b	93.7 ± 0.1a	0.75 ± 0.04b	3.51 ± 0.01b	3.40 ± 0.07a
OSP	0.22 ± 0.01c	0.21 ± 0.02c	93.1 ± 0.0b	0.04 ± 0.02c	4.11 ± 0.01a	3.12 ± 0.04b
CCSP-Containing tofu (g/L)
0	0.94 ± 0.02a	83.8 ± 0.7a	87.3 ± 0.2a	3.06 ± 0.04a	10.78 ± 0.04c	–
0.5	0.91 ± 0.03a	84.1 ± 1.2a	87.2 ± 0.2a	2.53 ± 0.04b	11.21 ± 0.11b	0.88 ± 0.07c
1	0.92 ± 0.02a	84.3 ± 0.9a	87.0 ± 0.2a	2.34 ± 0.04b	11.39 ± 0.13b	1.14 ± 0.13b
1.5	0.94 ± 0.01a	83.3 ± 0.9a	86.8 ± 0.5a	2.22 ± 0.05c	11.60 ± 0.13a	1.58 ± 0.09a

Values within a column, the different common lowercase letter means significantly different (p < 0.05). Data are shown as means of measurements ± standard error (n = 5).

aw = water activity, L*: lightness, a*: green to red color, b*: blue to yellow color, ΔE: the total color difference.

“–” represents “Δ of tofu prepared in the presence of single-use of MgCl₂”.

Semblance and SEM images of CSP and CCSP    According to Fig. 1, CCSP produced aggregated on particles surface (indicated by arrow) after calcination at 600 °C (Fig. 1B) as opposed to CSP (Fig. 1A), and particles began to collapse (indicated by arrow) at 800 °C. (Fig. 1C); furthermore, the particles exhibited bumpy wrinkle (indicated by arrow), which were evenly distributed after calcination at 1 000 °C (Fig. 1D). The surface of CCSP particle was aggregated and collapsed on the after calcination and some particles form part small particles and detached from the surface (Fig. 1B). Moreover, the collapse of particle resulted in the surface area increases dramatically (Fig. 1C), and it is beneficial to release antimicrobial activity of CCSP. Previous studies indicated that a non-calcified white clam shell was composed of three layers of CaCO₃ and exhibited a sheet structure similar to that of the surface and fracture layers of an oyster shell (Yoon et al., 2003). A higher antibacterial activity of wild surf clam shell powder after heat treatment was found due to its strong alkalinity (Oikawa et al., 2000). In this study, the CCSP was oxidized and disintegrated into CaO at above 600 °C and the changes of particle characteristic can be observed by SEM. The smaller particles size of CCSP and increased surface area exerts a greater effect on pH and spurs physiological activities such as the release of reactive oxygen species (ROS). For example, administer the release of intracellular substances by an adequate particle size and pH of CaO to improve the antibacterial activity (Yamamoto et al., 2010; Jin and He, 2011). The disintegration and the conversion of CCSP particles were conductive to the release of bacteriostatic substances.

Fig. 1.

SEM photographs of clam shell powder. A; CSP magnifications at 4 000x, B; CCSP 600 °C magnifications at 4 000x, C; CCSP 800 °C magnifications at 4 000x, D; CCSP 1 000 °C magnifications at 4 000x.

Microstructure of tofu    The vary CCSP levels affected the tofu structure. The control group (without CCSP) exhibited aggregates with a bumpy surface (Fig. 2A). The microstructure of tofu was out of shape after the addition of 0.5 g/L CCSP (Fig. 2B), and a web-like structure was formed when added 1.0 g/L CCSP (Fig. 2C); the pores and free spaces within the web-like structure increased in size after the addition of 1.5 g/L CCSP (Fig. 2D). Finally, the granules link with each other to form a colloid and reticular structure as Guo et al. (1999) and Wang et al. (2015) illustrated. It means that the Ca²⁺ could be an important factor to change the structure formation in the tofu coagulant system. The Ca²⁺ of CCSP induced the web-like reticular when treated with CCSP at a concentration of above 1.0 g/L.

Fig. 2.

SEM photographs of tofu with coagulant (MgCl₂) and CCSP. A; 18 g/L of MgCl₂ magnifications at 1 500x, B; 0.5 g/L CCSP-containing magnifications at 1 500x, C; 0.1 g/L CCSP-containing magnifications at 1 500x, D; 0.15 g/L CCSP-containing magnifications at 1 500x.

Microbial inhibition effects of CCSP    The inhibition of common food microorganisms by CCSP and indicates a positive correlation between inhibition capacity and CCSP levels. As shown in Table 2, CCSP has excellent inhibition against A. niger, while CCSP revealed good inhibition (16–20 mm on 1.5 g/L) against the B. cereus and K. apiculate. Moreover, it exhibits less inhibition (13.7 mm on 1.5 g/L) against the S. enterica serovar Typhimurium, E. coli, S. aureus and V. parahaemolyticus showed resistance to CCSP. The findings showed that CCSP can be used as a broad-spectrum antimicrobial agent, which has a strong inhibitory effect on fungi and revealed good resistance in Gram-positive bacterium. CaCO₃ is the main component of CSP, and it was converted to CaO after calcination at 1 000 °C. Bae et al. (2006) discovered that of the viable count of several bacteria in solution were killed within in 10 min by adding 0.05% scallop shell powder. The bacterial inhibition capacity of CaO may have been induced by high pH level (Sawai 2003, and Sawai et al. 1996) or released ROS level (Ohshima et al., 2015; Kubo et al., 2013; Sawai, 2003). Other studies demonstrated that antibiotic mechanisms involving metal oxides damage bacterial DNA by releasing ROS, which induces the denaturation of proteins and damages cell walls to cause cell death (Kwon et al., 2004; Cabiscol et al., 2000; Ezraty et al., 2017; Stankic et al., 2016). In general, the antimicrobial mechanism of CCSP may be similar to that of shell powder or other metal oxides against pathogens, i.e., induction of ROS release.

Table 2. Antimicrobial activity and spectrum of CCSP solution on pathogens determined.

Conc. of CCSP (g/L)	0	0.5	1	1.5	Sensitivity
Microorganism	Inhibition zone of Diameter (mm)
Aspergillus niger ATCC 15475	−	15.3 ± 0.6Ca	19.3 ± 0.6Ba	24.3 ± 0.6Aa	+ + +
Bacillus cereus ATCC 14579	−	11.0 ± 0.5Bb	11.8 ± 0.3Bb	18.3 ± 0.6Ab	+ +
Escherichia coli ATCC25922	−	10.3 ± 0.3Cbc	11.5 ± 0.5Bbc	14.7 ± 0.6Acd	+
Kloeckera apiculata ATCC 9774	−	9.2 ± 0.3Cd	10.8 ± 0.3Bc	19.3 ± 0.6Ab	+ +
Salmonella enterica serovar typhimurium ATCC 13311	−	10.0 ± 0.5Cc	11.2 ± 0.3Bbc	13.7 ± 0.6Ad	+
Staphylococcus aureus ATCC 12600	−	10.5 ± 0.5Bbc	11.3 ± 0.3Bbc	14.3 ± 0.6Acd	+
Vibrio parahaemolyticus ATCC 17802	−	10.2 ± 0.3Cc	11.2 ± 0.3Bd	15.3 ± 0.6Ac	+

Values within a column or row followed by the different lowercase letters are significantly different (p < 0.05). Data are shown as means of measurements ± standard error (n = 3).

Within a column, the different common lowercase letter means significantly different (p < 0.05).

Within a row, the different common uppercase letter means significantly different (p < 0.05).

Inhibition zone of diameter (mm): “−” represents “no inhibition”; Conc.: concentration.

Sensitivity of antimicrobial activity: “+ + +” excellent activity (21–25 mm), “+ +” good activity (16–20 mm), “+” significant activity (10–15 mm).

Bacterial inhibitory effects of CCSP addition to tofu    Spoilage during tofu storage is caused owing to contamination by incomplete sterilization or packaging process. Therefore, the contamination of tofu is of great concern to us all. As shown in Fig. 3, the total bacterial count in the sample without CCSP increased rapidly at 10 °C. The 1.5 g/L CCSP group demonstrated the most optimal inhibition capacity, and after 14 days, a bacterial count below the statutory requirement (10⁵ CFU/g) in the sample was observed. The bacterial growth in the 1.0 g/L CCSP sample group was significantly inhibited during the first 7 days, but the count was not significantly different from that of the control group after 14 days. The bacterial count in the 0.5 g/L CCSP group reached 10⁵ CFU/g on the third day, which was not significantly different from that in the control group. By contrast, addition of CCSP effectively inhibits the bacterial growth in tofu, and the total bacterial count decreases with increasing CCSP concentration. Previous studies have shown that scallop shell powder can reduce the bacterial counts of L. monocytogenes and S. enteritidis on chicken wings, but does not affect the color and taste (Cagri-mehmetoglu, 2011). Kim et al. (2007) confirmed that 0.2% oyster shell powder could prolong the shelf life of tofu for 10 days at 10 °C by inhibiting the total bacterial count to < 10⁷ CFU/g, thereby indicating that ROS might be the main component affecting the total bacterial count in tofu. This finding is consistent with the mechanism proposed for the inhibition of pathogen (Sawai, 2001a). Rossi et al. (2016) suggested that a high efficiency heat treatment can prolong the shelf life of fresh tofu by inhibit lactic acid bacteria species. According to the findings in Table 2, high concentration (1.5 g/L) of CCSP has a significant resistance to Gram-positive bacterium, and previous studies have shown that B. cereus is the main microorganism in tofu. Therefore, CCSP inhibit significantly the bacteria growth of tofu and prolong the shelf life. The enhanced inhibition might be attributable to the increased surface area of the material exerting a greater effect on the release of metal ions and ROS.

Fig. 3.

The inhibitory effect of CCSP on total microbes of tofu. Each point represents the mean of three independent experiments and error bars indicate ± standard error

Yield, TPA and sensory evaluation of CCSP-containing tofu    The tofu sample with 1.0 g/L CCSP weight was 489.7 g whereas the control group, 0.5 g/L and 1.5 g/L CCSP were about 401.7, 415.0 and 373.7 g (Table 3), respectively, suggesting a higher weight (increase about 22%). This result was consistent with Kim et al. (2007) reported that a high yield of tofu can be obtained when the shell powder concentration increased to 1.0 g/L. Compared to tofu prepared with a single use of MgCl₂, the yield (Table 3) and higher moisture (Table 1) in tofu prepared with CCSP is probably due to the differences in the gel network and the water-holding capacity of the soy protein gels (Wang and Hesseltine, 1982). The rheological property of tofu and its microstructure can be modified by selective glucono-δ-lactone (GDL) treatment, which in turn affects the texture, characteristics, and sensory properties of tofu (Lin et al., 2016). The SEM images (Fig. 2) showing the three-dimensional tofu structure indicate that higher yield might be attributable to its water retention property.

Table 3. Yield and texture profile analysis of tofu treated with CCSP.

CCSP (g/L)	Yield (g)	Hardness (N)	Gumminess (N)	Chewiness (N × mm)	Springiness (mm)	Adhesiveness (N × mm)	Cohesiveness
0	401.7 ± 9.50b	4.51 ± 0.23c	0.37 ± 0.02d	1.85 ± 0.03d	5.37 ± 0.17b	0.019 ± 0.001a	0.86 ± 0.03a
0.5	415.0 ± 13.2b	8.48 ± 0.22a	0.74 ± 0.02a	4.52 ± 0.11a	5.06 ± 0.11c	0.020 ± 0.003a	0.79 ± 0.03b
1	489.7 ± 5.50a	6.93 ± 0.36b	0.60 ± 0.04b	4.02 ± 0.09b	5.73 ± 0.16a	0.019 ± 0.002a	0.83 ± 0.02b
1.5	373.7 ± 14.3c	2.68 ± 0.25d	0.56 ± 0.03c	2.81 ± 0.22c	5.30 ± 0.14b	0.019 ± 0.003a	0.82 ± 0.04b

Values within a column followed by the different lowercase letters are significantly different (p < 0.05). Data are shown as means of measurements ± standard error (n = 10).

Table 3 shows the degree of hardness (i.e., 8.48 and 6.93 N), gumminess (i.e., 0.74 and 0.60 N), and chewiness (i.e., 4.52 and 4.02 N × mm) of the tofu samples on addition of 0.5 g/L and 1.0 g/L CCSP, respectively; these values were significantly higher than those in the sample without CCSP addition. Additionally, 1.0 g/L CCSP-containing tofu attained the maximum level of springiness, whereas 0.5 g/L CCSP-containing tofu exhibited the least level of springiness. CCSP reduced cohesiveness but did not affect adhesiveness of tofu. The results verified that addition of low CCSP concentrations (i.e., 0.5 g/L or 1.0 g/L) effectively enhances the hardness, gumminess, and chewiness of tofu. Compared with the results obtained, Kim et al. (1997) also observed that calcium ions tend to form a GDL-calcium mixture in a coagulant system to increase the hardness of tofu. Bernal et al. (1987) indicated that a decrease in intermolecular interactions would form a larger amount of homogeneous web structures to increase tofu hardness. In soymilk, GDL and Ca²⁺ induced the formation of colloids, and addition of GDL and Ca²⁺ offsets and covers the surface charges of denatured proteins. Wang et al. (2015) confirmed that Ca²⁺ ions react with soluble proteins to form 7S-rich protein granules, moreover, the protein granules were more sensitive to Ca²⁺ than non-proteinic granules and thereby formed tofu with increased hardness under low Ca²⁺ concentrations (Guo and Ono, 2005). Therefore, Ca²⁺ ion concentration in CCSP and the amount of CCSP added to tofu influence the hardness level of tofu; calcium-binding mechanism and interaction with proteins are the main factors that affect the structure and properties of tofu.

Kim et al. (2007) reported that the quality and sensory evaluation of tofu can be improved by adding OSP. The main sensory evaluation levels (Table 4) showed that the CCSP significantly impacted the taste of tofu based on its concentration. The results revealed that mouthfeel decreased significantly when excessive amounts of CCSP (1.0 g/L and 1.5 g/L) were added to tofu. By contrast, the mouthfeel for tofu without CCSP and that containing low CCSP concentration (0.5 g/L) were not significantly different. The lowestscore for beany-flavor was achieved for tofu sample containing 0.5 g/L CCSP, and the difference in this score compared with other sample groups was also statistically significant. Ideally, high CCSP levels result in high alkalinity of the solution, which likely inhibits the activity of lipoxygenase in soybean to produce the beany-flavor during tofu processing. There were nonsignificant difference among 1.0 > 0.5 > 1.5 > 0 g/L CCSP-containing tofu in firmness and elasticity levels. Overall acceptability of the sample groups of 0; 0.5; 1.0 g/L CCSP were more than 6 points without any significant differences. However, 1.5 g/L CCSP got the lowest score which was 4.25 points. Table 4 shows that overall acceptability of tofu added with 1.0 g/L CCSP was better than tofu added with 1.5 g/L CCSP. However, tofu added with 1.0 g/L CCSP had worse antimicrobial activity compare with 1.5 g/L CCSP. According to the results of the study, the addition of 1.5 g/L CCSP will be better improvement agent for tofu.

Table 4. Effect of CCSP on the sensory quality of tofu.

CCSP (g/L)	Mouthfeel	Beany-flavor	Firmness	Elasticity	Overall acceptability
0	6.90 ± 0.79a	5.85 ± 0.67a	4.35 ± 0.93b	4.60 ± 1.05a	6.45 ± 0.69a
0.5	7.05 ± 0.69a	4.40 ± 0.60c	4.80 ± 0.95ab	4.85 ± 0.59a	6.80 ± 0.70a
1	5.70 ± 0.66b	4.95 ± 0.83b	5.15 ± 0.88a	4.65 ± 0.75a	6.60 ± 0.88a
1.5	4.55 ± 0.51c	5.15 ± 0.67b	4.60 ± 0.94ab	4.70 ± 0.86a	4.25 ± 0.97b

For each type of tofu, the same lowercase latter in same column are not significantly different (p > 0.05). Data are shown as means of measurements ± standard error (n = 20). Score scale of 1.9 with 1 = very poor (weak) and 9 = very good (strong).

Conclusion

In summary, clam is a common shellfish aquaculture waste in Taiwan and Asia, but it is not fully utilized though it is clean than oyster shell. CCSP can effectively inhibit the pathogen, prolong the shelf life and improve the properties of tofu. The results confirmed that CCSP is a novel and effective antimicrobial material for tofu preservation and ROS released might be the main factor of the antimicrobial activity. Compared with chemical preservation agents commonly used currently, CCSP is friendlier and lower pollution for environment, and it could be better natural material to replace chemical antimicrobial agent. Moreover, CCSP is a biological material source that can be developed to many products, such as food cleaning agents, food preservative, nutritional supplement and biomedical materials. The extended application and potential of CCSP will be the one of the research directions in the future.

Acknowledgements    The authors gratefully acknowledge Li Chuan Aquafarm (R.O.C.) for providing the clam shell. This work is connected to the scientific programme of the “Popularize natural antibacterial agent in the food industry applications” project. This project is supported by Council of Agriculture, Executive Yuan, R.O.C. [Project ID: 104AS-16.3.1-ST-a2].

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