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Removal of cadmium from fish sauce using chelate resin
Food Chemistry 173 (2015) 375–381
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Analytical Methods
Removal of cadmium from fish sauce using chelate resin Tetsuya Sasaki a, Ryohei Araki b, Toshihide Michihata a, Miyuki Kozawa c, Koji Tokuda c, Takashi Koyanagi b, Toshiki Enomoto b,⇑ a
Chemistry/Food Department, Industrial Research Institute of Ishikawa, Kanazawa, Ishikawa 920-0223, Japan Department of Food Science, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, Japan c Shata Shuzo Co. Ltd., Hakusan, Ishikawa 924-0823, Japan b
a r t i c l e
i n f o
Article history: Received 4 February 2014 Received in revised form 1 August 2014 Accepted 9 August 2014 Available online 7 October 2014 Keywords: Fish sauce Cadmium Chelate resin Tannin
a b s t r a c t Fish sauce that is prepared from squid organs contains cadmium (Cd), which may be present at hazardous concentrations. Cd molecules are predominantly protein bound in freshly manufactured fish sauce, but are present in a liberated form in air-exposed fish sauce. In the present study, we developed a new method for removing both Cd forms from fish sauce using chelate resin and a previously reported tannin treatment. Sixteen-fold decreases in Cd concentrations were observed (0.78–0.05 mg/100 mL) following the removal of liberated Cd using chelate resin treatment, and the removal of protein-bound Cd using tannin treatment. Major nutritional components of fish sauce were maintained, including free amino acids and peptides, and angiotensin I-converting enzyme inhibitory and antioxidant activities. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Fish sauce is a traditional seasoning, and is produced by fermenting fish under high sodium chloride (NaCl) concentrations for 1–2 years. It is mainly produced and consumed in southeast Asian provinces such as Nampla in Thailand, Noucnam in Vietnam, Shotturu and Ishiru in Japan, Patis in the Philippines, Budu in Malaysia, Ketjapikan and Bakasang in Indonesia, Toeuk Trey in Cambodia, Yuilu in China, and Ngapi in Myanmar. Fish sauce is added to various foods for its complex and characteristic taste. It contains large quantities of free amino acids and peptides (Gasaluck, Yokoyama, Kimura, & Sugahara, 1996; Lopetcharat, Choi, Park, & Daeschel, 2001; Michihata, Sado, Yano, & Enomoto, 2000; Park et al., 2001) and has been shown to contain beneficial antioxidants, and angiotensin I-converting enzyme (ACE) inhibitors (Aoshima & Ooshima, 2009; Ichimura, Hu, Aita, & Maruyama, 2003; Taniguchi, Enomoto, & Michihata, 2009). Moreover, many studies of fish sauces have characterised odors (Fukami et al., 2002; Michihata, Yano, & Enomoto, 2002), microbial composition (Chuon et al., 2014; Saisithi, Kasermsarn, & Dollar, 1966; Yoshikawa et al., 2010), and heavy metal and histamine contents (Nakazato et al., 2000; Sanceda, Suzuki, Ohashi, & Kurata, 1999; Shozen et al., 2012). Fish sauce contains heavy metals, including cadmium (Cd), arsenic, lead (Pb), and mercury (Hg; Nakazato et al., 2000), and ⇑ Corresponding author. Tel./fax: +81 76 227 7452. E-mail address: [email protected] (T. Enomoto). http://dx.doi.org/10.1016/j.foodchem.2014.08.134 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
due to bioaccumulation of Cd from sea water in squid organs such as the liver (Ichihashi, Kohno, Kannan, Tsumura, & Yamasaki, 2001; Kim, Kang, & Kim, 2008) fish sauce from squid organs may contain hazardous concentrations of Cd, which may lead to poor marketability. However, few studies have developed methods for removing heavy metals from fish sauces. In our previous report, we developed a simple Cd removal method using tannin, which did not affect the nutritional components or beneficial activities of fish sauce (Sasaki et al., 2013). This method involves the addition of tannin to fish sauce and subsequent removal of the resulting precipitate, which contains protein-bound Cd. Accordingly, heavy metals are removed without using strong acids, and in shorter time periods than previously reported methods (Ghimire et al., 2008; Obara et al., 1999; Sakuta et al., 2000; Seki, Maruyama, Kawabe, & Nakade, 2006). Metal-bound proteins such as metallothionein have thiol group metal-binding sites (Giles et al., 2003; Klaassen, Liu, & Choudhuri, 1999). Because oxidative stress converts thiols to disulphide and causes conformational changes in proteins (Holmgren, 1995), exposure of fish sauce to air caused release of Cd from thiol proteins and reduced Cd removal efficiency. In corresponding experiments, exposure of fish sauce to air for 2 months decreased the Cd removal rate from 97% to only 15%. Hence, removal of both protein bound and liberated Cd is required. The purpose of this study was to develop a method for removing both liberated and protein-bound Cd using agents that do not affect the nutritional and functional components of fish sauce. Chelate resins selectively remove heavy metal ions from aqueous
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solutions, and some have been approved as food additives. Indeed, chelate resins have been used to recover waste water (Kolodynska, 2010) and soil (Dermont, Bergeron, Mercier, & Richer-Lafleche, 2008), and in highly sensitive analyses of metal ions (Furusho et al., 2008). In the present study, we removed liberated Cd from tannin-treated fish sauce using chelate resin, which did not influence nutritional constituents or beneficial functions. Resins with iminodiacetic acid or ethylenediamine functional groups were selected for static and dynamic experiments. Additionally, we clarified the influences of major fish sauce components such as NaCl and amino acids on Cd removal using chelate resin. Subsequently, Cd forms were identified using gel filtration chromatography and removal of both liberated and protein-bound Cd was confirmed after treatments with both chelate resin and tannin. 2. Materials and methods 2.1. Removal of Cd from fish sauce using a batch method with chelate resin Commercial fish sauce from squid liver was purchased from a local market in Japan. Fish sauce samples were exposed to air for 1 month and were treated with the commercial chelate resins DIAION CR11 and CR20 (Mitsubishi Chemical Co., Tokyo, Japan), which are approved as food additives in Japan. Chelating groups on CR11 and CR20 include iminodiacetic acid and ethylene diamine, respectively. Prior to experiments, resins were conditioned using the standard procedures described in associated operation manuals. Briefly, chelate resins (7 g) were washed with 2-M hydrochloric acid (30 mL), water (30 mL, 2 times), 2-M sodium hydroxide (30 mL), and then water (30 mL, 3 times) in sequence. Tannin treatment was performed as described previously (Sasaki et al., 2013). Briefly, 1% aqueous tannin (10 mL) was added to fish sauce samples (100 mL), and supernatants were separated from precipitates by centrifugation (8000g, 10 min). Tannin-treated fish sauce (20 mL) samples were mixed with CR11 or CR20 (700 mg) and were shaken for 1 h. Mixtures were then filtered with coffee filter paper to separate supernatants from chelate resins. 2.2. Removal of Cd from fish sauce using a column method with chelate resin A glass column (90 4 mm interior diameter) was packed with CR11 or CR20 (700 mg). Tannin-treated fish sauce (50 mL) samples were then eluted through these columns at 5 mL/h, and 10-mL fractions were collected every 2 h. 2.3. Measurement of Cd and other mineral concentrations in fish sauce Fish sauce (1 mL) samples were diluted in 3-M nitric acid (20 mL) and were incubated at 200 °C for 8 h in a conical beaker using a hot plate. Samples were then diluted to 100 mL with distilled water and were analysed using an inductively coupled plasma analysis emission spectrometer (ICP-AES; IRIS Advantage/SSEA, Jarrell-Ash Co. Ltd., Tokyo, Japan). Results are presented as the mean ± standard error of three independent experiments. 2.4. Analyses of chemical components and functional properties of fish sauce Total nitrogen contents of fish sauce were determined using the Kjeldahl method with a nitrogen/protein analyser (KJEL-Auto DTD4; Mitamura Riken Kogyo Co. Ltd., Tokyo, Japan). Free amino acid
concentrations were analysed using an amino acid autoanalyser (L-8900; Hitachi High-Technologies Corporation, Tokyo, Japan). Sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), zinc (Zn), and copper (Cu) concentrations were determined using ICP– AES as described above. Results are presented as the mean ± standard error of three independent experiments. Antioxidant activities were evaluated using the method of Suda (2000) with the radical scavenger 1,1-diphenyl-2-picrylhydrazyl (DPPH) and were expressed relative to standard gallic acid solutions. ACE inhibitory activities were estimated as half maximal inhibitory concentrations (IC50) of rabbit lung ACE by determining hippuryl-L-histidyl-L-leucine concentrations using HPLC according to the methods of Horiuchi, Fujimura, Terashima, and Iso (1982) and Ohta, Iwashita, Sakai, and Kawamura (1997). Results are presented as the mean of three independent measurements.
2.5. Removal of Cd from model samples at various NaCl concentrations To evaluate the influence of NaCl concentrations on Cd removal by chelate resins, NaCl aqueous solutions of varying concentrations up to that of fish sauce were prepared, and were used as standard model samples for Cd removal using the batch method. These model samples contained 0, 5, 10, 15, 20, and 25 g of NaCl/ 100 mL, and Cd concentrations were adjusted to 2.0 mg/100 mL using a Cd standard solution (Cd-1000; Kanto Chemical Co. Inc., Tokyo, Japan). Moreover, pH was adjusted to 6 using 0.1-M sodium phosphate buffer according to reported pH values for the present fish sauce (pH 5.8) and other fish sauces (pH 5.6–6.2, Park et al., 2001). Subsequently, 20-mL aliquots were mixed with CR11 or CR20 (70 mg) and were shaken for 24 h. Cd concentrations were then analysed using ICP–AES. Results are presented as the mean of three independent measurements.
2.6. Removal of Cd from model samples at various amino acid concentrations The effects of amino acids on Cd removal by chelate resins were examined using aqueous amino acid solutions as model samples. Amino acids concentrations were adjusted to 0, 2000, 4000, 6000, 8000, and 10,000 mg of alanine, glutamic acid, and lysine at a ratio of 3:2:2 in 100 mL. These are the three major amino acids in fish sauce, and their concentration in reference samples was prepared according to that found in fish sauce (7460 mg/100 mL). As with NaCl samples, Cd concentrations were adjusted to 2.0 mg/ 100 mL, and pH was adjusted to 6 using 0.1-M sodium phosphate buffer. Samples (20 mL) were then mixed with CR11 (70 mg) or CR20 (70 mg) and were shaken for 24 h. Cd concentrations were analysed using ICP–AES. Results are presented as the mean of three independent measurements.
2.7. Removal of Cd from fish sauce for comparison with reference samples To compare inhibitory effects of NaCl and amino acids on Cd binding of chelate resin in fish sauce and reference samples, initial Cd concentrations in fish sauce were adjusted to that of reference samples (2.0 mg/100 mL) by adding 1000 ppm Cd standard solution to tannin- and CR11-treated fish sauce. Subsequently, fish sauce samples (20 mL) were mixed with CR11 (70 mg) or CR20 (70 mg) and were shaken for 24 h. Cd concentrations were then analysed in treated and untreated fish sauce samples using ICP– AES. Results are presented as the mean ± standard error of three independent experiments.
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2.8. Gel filtration chromatography of fish sauce
3. Results and discussion 3.1. Removal of Cd from fish sauce using the batch method with chelate resin
Cd concentration in fish sauce (mg/100 mL)
A
0.8
0.6
0.4
0.2
0.0 Untreated
Tannin
CR11
CR20
Removal methods
0.4
Cd concentrations in fish sauce (mg/100 mL)
Cd forms in fish sauce were identified using gel filtration chromatography according to the method of Takeuchi, Inari, and Morimoto (2001) with some modifications. Untreated fish sauce, tannin-treated fish sauce, CR11-treated fish sauce, and standard solution were compared. CR11-treated fish sauce was prepared using the batch method. Standard solutions were prepared by dissolving 100 mg of human albumin (Wako Pure Chemical Industries Ltd., Tokyo, Japan), 100 mg of alanine (Wako Pure Chemical Industries Ltd.), and 100 lL of 1000-ppm Cd standard solution in 10 mL of distilled water. Samples (2 mL) were then applied to an equilibrated Sephadex G-15 gel (GE Healthcare Bio-Science, Uppsala, Sweden) filtration column (50 1.5 cm internal diameter) and were eluted at 1 mL/ min with 0.1-M aqueous NaCl solution. Fractions were collected every 4 min. Cd concentrations and absorbance (280 nm) of fractions were analysed using ICP–AES and spectrophotometry (UV1800; Shimadzu Corporation), respectively. Amino acids and peptides were analysed in 80-lL fractions after mixing with 80 lL of 1 g/100 mL ninhydrin solution and 1.84 mL of distilled water and heating in boiling water for 15 min (Takeuchi et al., 2001). Absorbance was then measured at 570 nm using a spectrophotometer.
1.0
B
0.3
0.2
0.1
0.0
Cd in fish sauce was initially removed with tannin according to a previously described method (Sasaki et al., 2013), and Cd concentrations were reduced from 0.78 ± 0.01 to 0.28 ± 0.01 mg/100 mL (Fig. 1A). Two-thirds of total Cd contents were removed from fish sauce by tannin treatment, presumably corresponding to organic molecule-bound Cd. Subsequently, tannin-treated fish sauce was treated with chelate resin using the batch method, which reduced Cd concentrations to 0.05 ± 0.01 mg/100 mL (CR11) and 0.06 ± 0.01 mg/100 mL (CR20), which were less than one-fifth of that in tannin-treated fish sauce (Fig. 1A). Hence, residual Cd in fish sauce after tannin treatment was removed using the chelate resin, presumably representing the liberated form of Cd. The Cd binding ability of chelate resin is affected by pH, which was 5.8 in the present fish sauce samples, and is generally between 5.6 and 6.2 in other fish sauces (Park et al., 2001). Because iminodiacetic acid and polyamine functional groups are known to exhibit strong chelating ability for heavy metal ions at pH 4–7, the present chelate resins are likely to remove Cd from most fish sauces (Li et al., 2012; Ueda, Teshima, Sakai, Joichi, & Motomizu, 2010).
1
2
3
4
5
Fraction number Fig. 1. Cd concentrations in fish sauce treated with chelate resins using the batch and the column method. Data from the column method are present in (A). Data from untreated fish sauce, tannin-treated fish sauce (before chelate resin treatment), and CR11- and CR20-treated fish sauce after tannin treatment are shown. Data are presented as the mean ± standard error of three independent samples. Data from the column method are present in (B). Tannin-treated fish sauce was eluted through columns packed with CR11 (d) or CR20 (h) chelate resins, and 10 mL fractions were collected every 2 h. Initial Cd concentrations in fish sauce were 0.33 mg/100 mL. Data are presented as the mean ± standard error of three independent samples.
fractions (from 0.05 ± 0.01 to 0.07 ± 0.01 mg/100 mL), those after treatment with CR20 increased linearly with elution volume (from 0.04 ± 0.003 to 0.17 ± 0.01 mg/100 mL). These increasing eluent concentrations reflect gradual saturation of binding sites (Popuri, Vijaya, Boddu, & Abburi, 2009), and indicate that the Cd removal ability of CR11 was higher than that of CR20 when using the column method.
3.2. Removal of Cd from fish sauce with chelate resin using the column method
3.3. Chemical components and functionality of fish sauces treated with CR11 using the column method
Columns packed with chelate resin are suitable for fish sauce manufacturing factories, because unlike the batch method, both fish sauce and conditioning solution are passed through the column, and chelate resins do not require further conditioning. Thus, we evaluated the Cd removal efficiency of columns packed with the chelate resins CR11 and CR20 (Fig. 1B). Tannin-treated fish sauce contained Cd at 0.33 ± 0.01 mg/100 mL. Fig. 1B shows Cd concentrations in fractions eluted from chelate resin columns. Cd concentrations in the first fractions were 0.05 ± 0.01 mg/100 mL (CR11) and 0.04 ± 0.003 mg/100 mL (CR20), indicating effective Cd removal by both chelate resins. Whereas Cd concentrations after treatment with CR11 were constant across the first five
Using the column method, CR11 had superior Cd removal efficacy to that of CR20. Thus, we evaluated chemical components and functionality of fish sauce after treatment with CR11-packed columns (Table 1). Total nitrogen and free amino acid contents did not differ between untreated fish sauce and the first fraction (1.8 ± 0.04 vs. 1.8 ± 0.02 g/100 mL and 10,600 ± 200 vs. 10,200 ± 300 mg/100 mL, respectively) and did not change in subsequent fractions. Thus, nutritional peptides and free amino acids remained in fish sauce samples after Cd removal using CR11. In subsequent metal ion analyses, Cu and Zn concentrations in untreated fish sauce were 1.4 ± 0.01 and 2.4 ± 0.04 mg/100 mL and were decreased to approximately 0.9 and 0.1 mg/100 mL
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Table 1 Chemical components and functionality of fish sauces treated with CR11 using the column method.
Chemical component Total nitrogen (g/100 mL) Total Free amino acid (mg/100 mL) Cu (mg/100 mL) Zn (mg/100 mL) Mg (mg/100 mL) Ca (mg/100 mL) Na (g/100 mL) K (mg/100 mL) Taurine (mg/100 mL) Functionality DPPH radical scavenging activity as gallic acid (lmol/mL) ACE inhibitory activity (IC50: lL/mL)
Untreated
Fr. 1
Fr. 2
Fr. 3
Fr. 4
Fr. 5
1.8 ± 0.04 10,600 ± 200 1.4 ± 0.01 2.4 ± 0.04 33 ± 1.3 11 ± 0.1 7.5 ± 0.01 103 ± 4 720 ± 10
1.8 ± 0.02 10,200 ± 300 0.7 ± 0.02 0.4 ± 0.01 21 ± 0.6 4.5 ± 0.2 7.5 ± 0.02 103 ± 2 700 ± 1
1.8 ± 0.02 10,500 ± 600 0.9 ± 0.02 0.2 ± 0.02 33 ± 0.1 10 ± 0.1 7.5 ± 0.02 104 ± 1 720 ± 22
1.8 ± 0.01 10,500 ± 500 0.9 ± 0.03 0.1 ± 0.01 33 ± 0.1 11 ± 0.1 7.5 ± 0.02 103 ± 1 710 ± 13
1.8 ± 0.02 10,500 ± 600 0.9 ± 0.03 0.1 ± 0.01 33 ± 0.5 11 ± 0.1 7.5 ± 0.01 104 ± 1 720 ± 9
1.8 ± 0.08 10,700 ± 200 1.0 ± 0.04 0.1 ± 0.01 33 ± 0.2 11 ± 0.1 7.5 ± 0.02 104 ± 1 720 ± 11
0.96 ± 0.08 9.5 ± 0.7
0.96 ± 0.08 9.8 ± 0.9
1.0 ± 0.07 9.9 ± 0.6
1.1 ± 0.02 9.3 ± 1.0
0.97 ± 0.01 8.8 ± 0.8
1.0 ± 0.07 9.3 ± 0.6
Experiments were performed on three independent samples, and data are shown as the mean ± standard error.
A
Cd concentration (mg/100 mL)
2.0
1.5
1.0
0.5
0.0 0
5
10
15
20
25
NaCl concentration (g/100 mL)
B
2.0 Cd concentration (mg/100 mL)
following CR11 treatment, respectively. Divalent ions such as Cu2+ and Zn2+ are known to bind iminodiacetic acid. However, Mg and Ca concentrations were maintained at approximately 33 and 11 mg/100 mL, respectively, following CR11 treatment, but were decreased to half of those in untreated fish sauce, potentially reflecting low binding constants of these metals. Because Cu, Zn, Mg, and Ca concentrations are low in untreated fish sauce, these decreases may not have a significant impact on the quality of fish sauce, although their adsorption by CR11 may competitively inhibit Cd removal. Na and K concentrations in fish sauce were 7.5 ± 0.01 g/100 mL and 103 ± 4 mg/100 mL, respectively. Because alkaline metals do not bind iminodiacetic acid with high affinity, Na and K concentrations were not significantly altered after resin treatments. Na is predominantly present as NaCl in fish sauce and is an important preservative that inhibits microbial contamination. Thus, maintenance of Na concentrations is critical for the quality of fish sauce. Fish sauce contains beneficial antioxidants and ACE inhibitors, and fish sauce prepared from squid contains high concentrations of taurine, which facilitates recovery from fatigue (Michihata et al., 2000; Taniguchi et al., 2009). Taurine contents in untreated fish sauce and in treated first fractions were 720 ± 10 and 700 ± 1 mg/100 mL, respectively. DPPH radical scavenging activity was also determined using gallic acid as a standard radical scavenger, and did not differ between untreated fish sauce and first fractions (0.96 ± 0.08 vs. 0.96 ± 0.08 lmol/mL, respectively). Moreover, ACE IC50 values did not differ between untreated fish sauce and first fractions (9.5 ± 0.7 vs. 9.8 ± 0.9 lL/mL, respectively). These major functionalities remained in all subsequent fractions, confirming that CR11 did not remove organic compounds.
1.5
1.0
0.5
0.0 0
2000
4000
6000
8000
10000
12000
Amino acid concentration (mg/100 mL)
3.4. Influences of NaCl on Cd removal by chelate resin Fish sauce contains large amounts of NaCl and amino acids (Michihata et al., 2000; Park et al., 2001), and although chelate resins are effective in diluted solutions such as waste water, these fish sauce constituents may inhibit Cd removal. Thus, we initially investigated the influence of NaCl on the Cd removal abilities of chelate resins in aqueous reference samples. These samples were prepared using NaCl in aqueous phosphorous buffer, and the concentrations of NaCl ranged from 0 to that of fish sauce. Fig. 2A shows the influence of NaCl concentrations on the removal of Cd from model samples using chelate resins (CR11, CR20) with the batch method. Because fish sauce contains NaCl at 18–23 g/100 mL (Park et al., 2001), reference samples contained NaCl in the range of 0–25 g/100 mL. After treatment with CR11, Cd concentrations in these standards increased with NaCl concentrations from 0.20 ± 0.050 to 1.7 ± 0.005 mg/100 mL. Because Na+ is
Fig. 2. The influence of NaCl and amino acids on Cd removal from reference samples using chelate resin. Data from NaCl are presented in (A). Aqueous solutions containing NaCl at 0–25 g/100 mL and Cd at 2.0 mg/100 mL in phosphate buffer (pH 6) were used as reference samples, and were treated with CR11 (d) or CR20 (h) chelate resins using the batch method. Data are presented as the mean ± standard error of three independent samples. Data from amino acids are presented in (B). Aqueous solutions containing amino acids at 0–10,000 mg /100 mL and Cd at 2.0 mg/100 mL in phosphate buffer (pH 6) were used as reference samples and were treated with CR11 (d) or CR20 (h) chelate resins using the batch method. Data are presented as the mean ± standard error of three independent samples.
known to occupy iminodiacetic acid groups (Pramanik, Dhara, & Chattopadhyay, 2004; Takaku, Kudo, Kimura, Hayashi, & Ota, 2002), NaCl was expected to inhibit binding of Cd to CR11. However, Cd2+ may bind iminodiacetic acids more strongly than Na+, reflecting differences in binding modes and spatial structures. Nonetheless, Cd removal by CR11 decreased with increasing NaCl
A
1.0
Amino Acids, Peptides
Cd
0.6 Cd 0.5
0.4
0.2
0.0 0
10
20
30
3.0
Ultraviolet absorbance (280 nm)
Cd concentration (mg/100 mL)
0.8
2.0
1.0
0.0 40
0.0
1.0
3.0
Absorbance of ninhydrin reaction (570 nm)
T. Sasaki et al. / Food Chemistry 173 (2015) 375–381
Cd concentration (mg/100 mL)
Cd
0.6
0.4
0.5
0.2
0.0 0
10
20
30
2.0
1.0
0.0 40
0.0
1.0
3.0
Absorbance of ninhydrin reaction (570 nm)
B
0.8
Ultraviolet absorbance (280 nm)
Fraction
Cd concentration (mg/100 mL)
0.6 Cd 0.5
0.4
0.2
0.0 0
10
20
30
2.0
1.0
0.0 40
0.0
3.5
0.6
Absorbance of ninhydrin reaction (570 nm)
C
0.8
Ultraviolet absorbance (280 nm)
Fraction
Cd concentration (mg/100 mL)
6
Liberated Cd 3.0
Alanine Albumin
5
2.5
4
2.0
3
1.5
2
1.0
1
0.5
0 0
10
20
30
0.0 40
0.5 0.4 0.3 0.2 0.1 0.0
Absorbance of ninhydrin reaction (570 nm)
D
7
Ultraviolet absorbance (280 nm)
Fraction
Fraction Fig. 3. Gel filtration chromatography of fish sauce and reference solutions. Data from (A) untreated fish sauce, (B) tannin-treated fish sauce, (C) chelate-treated fish sauce, and (D) reference solutions are presented. Reference solutions contained albumin, alanine, and liberated Cd. Cd concentrations (d), absorbance at 280 nm (h), and absorbance at 570 nm after ninhydrin reactions (N) were measured in each fraction.
concentrations, presumably because concentrations of Na+ were much higher than those of Cd2+. After treatment with CR20, Cd concentrations did not change significantly with increasing NaCl concentrations. It is known that
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ethylene diamine forms Cd2+ complexes with nitrogen atoms, which provide electron pairs that form stable coordination covalent bonds with metal ions. Moreover, non-ionic ethylene diamine groups may not interact with Na+. Hence, although CR11 had greater efficacy than CR20, the two chelate resins had equal efficacy in the presence of NaCl at 20 g/100 mL, and under high salt conditions, the Cd removal ability of CR11 was decreased to the same extent as that of CR20. Because NaCl decreased the removal of Cd by CR11 in reference samples, we evaluated the inhibitory effect of NaCl on Cd binding of CR11 in fish sauce. Samples were prepared by addition of 1000 ppm Cd standard solution to fish sauce samples after Cd removal by tannin treatment, and initial Cd concentrations were adjusted to those in reference samples (2.0 mg/100 mL), and Cd removal tests were performed using the same method as for reference samples. In these experiments, Cd concentrations in fish sauce were decreased to 1.0 ± 0.1 mg/100 mL. In comparison with Cd concentrations (1.6 ± 0.01 mg/100 mL) in reference samples containing NaCl at 20 g/100 mL, fish sauce samples (NaCl, 19.8 g/ 100 mL) contained lower Cd concentrations. These data indicate that the inhibitory effect of NaCl on Cd binding of chelate resins was reduced by fish sauce components. In addition to NaCl and amino acids (Michihata et al., 2000; Nakazato et al., 2000; Park et al., 2001), these components may have included organic acids such as lactic acid and pyroglutamic acid, polyamines such as histamine and tyramine, minerals such as Cu and Mg. Thus, numerous interactions with fish sauce components may contribute to decreased inhibitory properties of NaCl. Nonetheless, NaCl strongly inhibited Cd binding by CR11 in fish sauce, as confirmed by higher Cd concentrations in CR11-treated fish sauce (0.2 ± 0.01 mg/ 100 mL) than in CR11-treated reference samples (NaCl at 0 g/ 100 mL). 3.5. Influences of amino acids on Cd removal using chelate resin The influences of amino acids on Cd removal by chelate resins were evaluated as described above for NaCl. Fig. 2B shows Cd removal from reference samples containing amino acids at 0– 10,000 mg/100 mL. Fish sauces contain amino acids at 6000– 10,000 mg/100 mL (Park et al., 2001), and predominantly comprise alanine, glutamic acid, and lysine at a 3:2:2 ratio in squid fish sauce. In the presence of CR11, Cd concentrations in amino acid reference solutions remained constant at approximately 0.2 mg/ 100 mL, and were impervious to amino acid concentrations. In contrast, remaining Cd concentrations in the reference samples treated with CR20 were increased in proportion to amino acid concentrations (from 1.3 ± 0.09 to 1.9 ± 0.02 mg/100 mL), indicating that amino acids inhibit binding of Cd to CR20. Previous reports indicate that some carboxylic acid compounds form complexes with polyamines (Kimura, 2002; Kimura, Sakonaka, Yatsunami, & Kodama, 1981), suggesting that interactions between amino acids and polyamine may decrease Cd binding by CR20. In subsequent experiments, the inhibitory effects of amino acids on Cd binding ability of CR20 were determined in fish sauce samples containing Cd at 2.0 mg/100 mL). Treatment with CR20 reduced Cd concentrations in fish sauce (total free amino acid concentration, 7460 mg/100 mL) to 1.0 ± 0.03 mg/100 mL, which was lower than in treated standard solutions (1.9 ± 0.02 mg/100 mL) containing amino acids at 8000 mg/100 mL. This result indicates that inhibition of CR20–Cd binding by amino acids was ameliorated by other fish sauce components. 3.6. Determination of Cd forms in fish sauce To compare the effects of chelate resin and tannin treatments, Cd forms were determined in untreated, tannin-treated, and
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CR11-treated (without tannin treatment) fish sauces using gel filtration chromatography (Fig. 3). Cd concentrations in the untreated fish sauce were 0.76 ± 0.01 and decreased following treatment with tannin and CR11 to 0.47 ± 0.01 and 0.24 ± 0.01 mg/100 mL, respectively. After treatments with both tannin and chelate resin, Cd was almost completely removed from fish sauce (0.05 ± 0.004 mg/100 mL). In chromatographic analyses of standard solutions (D), albumin, alanine, and liberated Cd were detected in fractions 9, 13, and 17, respectively. Chromatograms of untreated fish sauce (A) had two main Cd peaks in fractions 9 and 18. Comparisons of untreated fish sauce and reference solutions indicated that the peaks at fractions 9 and 18 corresponded with protein-bound and liberated forms of Cd in untreated fish sauce, respectively. Chromatograms of untreated fish sauce samples also had small peaks at fraction 23, indicating that liberated Cd may be eluted following interactions with small molecules such as phenethyl amine, which was detected in fraction 23 using HPLC (data not shown). After tannin-treatment of fish sauce, the first chromatographic Cd peak disappeared, whereas the second Cd peak was retained, indicating that tannin treatments removed protein-bound Cd but not liberated Cd. In contrast, in chromatographic analyses of the CR11-treated fish sauce, the first Cd peak remained and the second Cd peak disappeared. Thus, CR11 treatment removed liberated Cd but did not remove protein-bound Cd. Taken together, the present data show that tannin treatments remove protein-bound Cd, and CR11 treatments remove liberated Cd. Hence, the combined application of tannin and CR11 treatments may reliably remove almost all Cd from fish sauce. 4. Conclusion We have developed a new method for removal of Cd from fish sauce using a combination of chelate resins that are approved as food additives and the previously reported tannin method. This combined method removed both liberated and protein-bound Cd from fish sauce, and solved the limitations of tannin treatments. The iminodiacetic acid type chelate resin (Diaion CR11) was more suitable for the removal of Cd from fish sauce than the ethylene diamine type (Diaion CR20). Accordingly, CR11 removed Cd more effectively than CR20 in the column method. Moreover, Cd–CR11 binding was strongly inhibited by NaCl, although other fish sauce components reduced this effect. Finally, chemical components with nutritional value or known beneficial functions were retained after tannin and chelate resin treatments. Thus, the present method is safe and effective and has promising applications in fish sauce factories. References Aoshima, H., & Ooshima, S. (2009). Anti-hydrogen peroxide activity of fish and soy sauce. Food Chemistry, 112, 339–343. Chuon, M., Shiomoto, M., Koyanagi, T., Sasaki, T., Michihata, T., Chan, S., et al. (2014). Microbial and chemical properties of cambodian traditional fermented fish products. Journal of the Science of Food and Agriculture, 94, 1124–1131. Dermont, G., Bergeron, M., Mercier, M., & Richer-Lafleche, M. (2008). Soil washing for metal removal: A review of physical/chemical technologies and field applications. Journal of Hazardous Materials, 152, 1–31. Fukami, K., Ishiyama, S., Yaguramaki, H., Masuzawa, T., Nabeta, Y., Endo, K., et al. (2002). Identification of distinctive volatile compounds in fish sauce. Journal of Agricultural and Food Chemistry, 50, 5412–5416. Furusho, Y., Ono, M., Yamada, M., Ohashi, K., Kitade, T., Kuriyama, K., et al. (2008). Advanced solid phase extraction for inorganic analysis and its applications. Bunseki Kagaku, 57, 969–989. Gasaluck, P., Yokoyama, K., Kimura, T., & Sugahara, I. (1996). Some chemical and microbiological properties of Thai fish sauce and paste. Journal of Antibacterial and Antifungal Agents, 24, 385–390. Ghimire, K. N., Kai, H., Inoue, K., Ohto, K., Kawakita, H., Harada, H., et al. (2008). Heavy metal removal from contaminated scallop waste for feed and fertilizer application. Bioresource Technology, 99, 2436–2441.
Giles, N. M., Watts, A. B., Giles, G. I., Fry, F. H., Littlechild, J. A., & Jacob, C. (2003). Review metal and redox modulation of cysteine protein function. Chemistry & Biology, 10, 677–693. Holmgren, A. (1995). Thioredoxin structure and mechanism: Conformational changes on oxidation of the active-site sulfhydryls to a disulfide. Structure, 3, 239–243. Horiuchi, M., Fujimura, K., Terashima, T., & Iso, T. (1982). Method for determination of angiotensin-converting enzyme activity in blood and tissue by highperformance liquid chromatography. Journal of Chromatography, 233, 123–130. Ichihashi, H., Kohno, H., Kannan, K., Tsumura, A., & Yamasaki, S. (2001). Multielemental analysis of purpleback flying squid using high resolution inductively coupled plasma-mass spectrometry (HR ICP-MS). Environmental Science and Technology, 35, 3103–3108. Ichimura, T., Hu, J., Aita, D. Q., & Maruyama, S. (2003). Angiotensin I-converting enzyme inhibitory activity and insulin secretion stimulative activity of fermented fish sauce. Journal of Bioscience and Bioengineering, 96, 496–499. Kim, G. B., Kang, M. R., & Kim, J. W. (2008). Specific accumulation of heavy metals in squid collected from offshore Korean waters: Preliminary results for offshore biomonitoring and food safety assessment. Fishers Science, 74, 882–888. Kimura, E. (2002). From new molecular science to new supramolecular science with macrocyclic polyamines. The Pharmaceutical Society of Japan, 122, 219–236. Kimura, E., Sakonaka, A., Yatsunami, T., & Kodama, M. (1981). Macromonocyclic polyamines as specific receptors for tricarboxylate-cycle anions. Journal of American Chemical Society, 103, 3041–3045. Klaassen, C. D., Liu, J., & Choudhuri, S. (1999). Metallothionein: An intracellular protein to protect against cadmium toxicity. Annual Review of Pharmacology and Toxicology, 39, 267–294. Kolodynska, D. (2010). 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Nakazato, M., Tateishi, Y., Kobayashi, C., Yamajima, Y., Ohno, I., Kawai, Y., & Yasuda, K. (2000). Hygienic studies on imported fish sauce (II) -contents of 9 elements. Annual Report of Tokyo Metropolitan Research Laboratory of Public Health, 51, 155–159. Obara, T., Sawaya, T., Hokari, K., Umehara, Y., Mizukami, M., & Tomita, F. (1999). Removal of cadmium from scallop hepatopancreas by microbial processes. Bioscience, Biotechnology, and Biochemistry, 63, 500–505. Ohta, T., Iwashita, A., Sakai, S., & Kawamura, Y. (1997). Antihypertensive action of the orally administered protease hydrolysates of chum salmon head and their angiotensin I-converting enzyme inhibitory peptides. Food Science and Technology International, 3, 339–343. Park, J. N., Fukumoto, Y., Fujita, E., Tanaka, T., Washio, T., Otsuka, S., et al. (2001). Chemical composition of fish sauces produced in SouthEast and East Asian countries. Journal of Food Composition, 14, 113–125. Popuri, S. R., Vijaya, Y., Boddu, K., & Abburi, K. (2009). Adsorptive removal of copper and nickel ions from water using chitosan coated PVC beads. Bioresource Technology, 100, 194–199. Pramanik, S., Dhara, P. K., & Chattopadhyay, P. (2004). A chelating resin containing bis(2-benzimidazolymethyl)amine: Synthesis and metal-ion uptake properties suitable for analytical application. Talanta, 63, 485–490. Saisithi, P., Kasermsarn, B., & Dollar, A. M. (1966). Microbiology and chemistry of fermented fish sauce. Journal of Food Science, 31, 105–110. Sakuta, Y., Nagano, N., Tomita, K., Wakasugi, M., Saitou, T., Shimakage, K., et al. (2000). Developments of cadmium removal systems for residues wasted from scallop processing manufacture by an electrochemical method. Haikibutsu Shigen Jyunkan Gakkai Ronbunshi, 11, 145–154. Sanceda, N. G., Suzuki, E., Ohashi, M., & Kurata, T. (1999). Histamine behavior during the fermentation process in the manufacture of fish sauce. Journal of Agricultural and Food Chemistry, 47, 3596–3600. Sasaki, T., Michihata, T., Katsuyama, Y., Take, H., Shizuo, N., Aburatani, M., et al. (2013). Effective removal of cadmium from fish sauce using tannin. Journal of Agricultural and Food Chemistry, 61, 1184–1188. Seki, H., Maruyama, H., Kawabe, M., & Nakade, N. (2006). Removal of cadmium from squid liver by competitive adsorption method. Aquaculture Science, 54, 449–453. Shozen, K., Mori, M., Harada, Y., Yokoi, K., Satomi, M., & Funatsu, Y. (2012). Histamine reduction in fish sauce with bentonite. Nippon Shokuhin Kagaku Kogaku Kaishi, 59, 17–21. Suda, I. (2000). Kousanka kinou. In Shinohara, T. Suzuki, & K. Kaminogawa (Eds.). Shokuhin Kinou Kenkyuhou (pp. 218–220). Tokyo: Kourin Publishing Co., Ltd. Takaku, Y., Kudo, Y., Kimura, H., Hayashi, T., & Ota, I. (2002). Determination of rareearth elements in environmental water using chelating resin disk separation/ ICP-MS. Bunseki Kagaku, 51, 539–544. 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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Removal of cadmium from fish sauce using chelate resin Tetsuya Sasaki a, Ryohei Araki b, Toshihide Michihata a, Miyuki Kozawa c, Koji Tokuda c, Takashi Koyanagi b, Toshiki Enomoto b,⇑ a
Chemistry/Food Department, Industrial Research Institute of Ishikawa, Kanazawa, Ishikawa 920-0223, Japan Department of Food Science, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, Japan c Shata Shuzo Co. Ltd., Hakusan, Ishikawa 924-0823, Japan b
a r t i c l e
i n f o
Article history: Received 4 February 2014 Received in revised form 1 August 2014 Accepted 9 August 2014 Available online 7 October 2014 Keywords: Fish sauce Cadmium Chelate resin Tannin
a b s t r a c t Fish sauce that is prepared from squid organs contains cadmium (Cd), which may be present at hazardous concentrations. Cd molecules are predominantly protein bound in freshly manufactured fish sauce, but are present in a liberated form in air-exposed fish sauce. In the present study, we developed a new method for removing both Cd forms from fish sauce using chelate resin and a previously reported tannin treatment. Sixteen-fold decreases in Cd concentrations were observed (0.78–0.05 mg/100 mL) following the removal of liberated Cd using chelate resin treatment, and the removal of protein-bound Cd using tannin treatment. Major nutritional components of fish sauce were maintained, including free amino acids and peptides, and angiotensin I-converting enzyme inhibitory and antioxidant activities. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Fish sauce is a traditional seasoning, and is produced by fermenting fish under high sodium chloride (NaCl) concentrations for 1–2 years. It is mainly produced and consumed in southeast Asian provinces such as Nampla in Thailand, Noucnam in Vietnam, Shotturu and Ishiru in Japan, Patis in the Philippines, Budu in Malaysia, Ketjapikan and Bakasang in Indonesia, Toeuk Trey in Cambodia, Yuilu in China, and Ngapi in Myanmar. Fish sauce is added to various foods for its complex and characteristic taste. It contains large quantities of free amino acids and peptides (Gasaluck, Yokoyama, Kimura, & Sugahara, 1996; Lopetcharat, Choi, Park, & Daeschel, 2001; Michihata, Sado, Yano, & Enomoto, 2000; Park et al., 2001) and has been shown to contain beneficial antioxidants, and angiotensin I-converting enzyme (ACE) inhibitors (Aoshima & Ooshima, 2009; Ichimura, Hu, Aita, & Maruyama, 2003; Taniguchi, Enomoto, & Michihata, 2009). Moreover, many studies of fish sauces have characterised odors (Fukami et al., 2002; Michihata, Yano, & Enomoto, 2002), microbial composition (Chuon et al., 2014; Saisithi, Kasermsarn, & Dollar, 1966; Yoshikawa et al., 2010), and heavy metal and histamine contents (Nakazato et al., 2000; Sanceda, Suzuki, Ohashi, & Kurata, 1999; Shozen et al., 2012). Fish sauce contains heavy metals, including cadmium (Cd), arsenic, lead (Pb), and mercury (Hg; Nakazato et al., 2000), and ⇑ Corresponding author. Tel./fax: +81 76 227 7452. E-mail address: [email protected] (T. Enomoto). http://dx.doi.org/10.1016/j.foodchem.2014.08.134 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
due to bioaccumulation of Cd from sea water in squid organs such as the liver (Ichihashi, Kohno, Kannan, Tsumura, & Yamasaki, 2001; Kim, Kang, & Kim, 2008) fish sauce from squid organs may contain hazardous concentrations of Cd, which may lead to poor marketability. However, few studies have developed methods for removing heavy metals from fish sauces. In our previous report, we developed a simple Cd removal method using tannin, which did not affect the nutritional components or beneficial activities of fish sauce (Sasaki et al., 2013). This method involves the addition of tannin to fish sauce and subsequent removal of the resulting precipitate, which contains protein-bound Cd. Accordingly, heavy metals are removed without using strong acids, and in shorter time periods than previously reported methods (Ghimire et al., 2008; Obara et al., 1999; Sakuta et al., 2000; Seki, Maruyama, Kawabe, & Nakade, 2006). Metal-bound proteins such as metallothionein have thiol group metal-binding sites (Giles et al., 2003; Klaassen, Liu, & Choudhuri, 1999). Because oxidative stress converts thiols to disulphide and causes conformational changes in proteins (Holmgren, 1995), exposure of fish sauce to air caused release of Cd from thiol proteins and reduced Cd removal efficiency. In corresponding experiments, exposure of fish sauce to air for 2 months decreased the Cd removal rate from 97% to only 15%. Hence, removal of both protein bound and liberated Cd is required. The purpose of this study was to develop a method for removing both liberated and protein-bound Cd using agents that do not affect the nutritional and functional components of fish sauce. Chelate resins selectively remove heavy metal ions from aqueous
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solutions, and some have been approved as food additives. Indeed, chelate resins have been used to recover waste water (Kolodynska, 2010) and soil (Dermont, Bergeron, Mercier, & Richer-Lafleche, 2008), and in highly sensitive analyses of metal ions (Furusho et al., 2008). In the present study, we removed liberated Cd from tannin-treated fish sauce using chelate resin, which did not influence nutritional constituents or beneficial functions. Resins with iminodiacetic acid or ethylenediamine functional groups were selected for static and dynamic experiments. Additionally, we clarified the influences of major fish sauce components such as NaCl and amino acids on Cd removal using chelate resin. Subsequently, Cd forms were identified using gel filtration chromatography and removal of both liberated and protein-bound Cd was confirmed after treatments with both chelate resin and tannin. 2. Materials and methods 2.1. Removal of Cd from fish sauce using a batch method with chelate resin Commercial fish sauce from squid liver was purchased from a local market in Japan. Fish sauce samples were exposed to air for 1 month and were treated with the commercial chelate resins DIAION CR11 and CR20 (Mitsubishi Chemical Co., Tokyo, Japan), which are approved as food additives in Japan. Chelating groups on CR11 and CR20 include iminodiacetic acid and ethylene diamine, respectively. Prior to experiments, resins were conditioned using the standard procedures described in associated operation manuals. Briefly, chelate resins (7 g) were washed with 2-M hydrochloric acid (30 mL), water (30 mL, 2 times), 2-M sodium hydroxide (30 mL), and then water (30 mL, 3 times) in sequence. Tannin treatment was performed as described previously (Sasaki et al., 2013). Briefly, 1% aqueous tannin (10 mL) was added to fish sauce samples (100 mL), and supernatants were separated from precipitates by centrifugation (8000g, 10 min). Tannin-treated fish sauce (20 mL) samples were mixed with CR11 or CR20 (700 mg) and were shaken for 1 h. Mixtures were then filtered with coffee filter paper to separate supernatants from chelate resins. 2.2. Removal of Cd from fish sauce using a column method with chelate resin A glass column (90 4 mm interior diameter) was packed with CR11 or CR20 (700 mg). Tannin-treated fish sauce (50 mL) samples were then eluted through these columns at 5 mL/h, and 10-mL fractions were collected every 2 h. 2.3. Measurement of Cd and other mineral concentrations in fish sauce Fish sauce (1 mL) samples were diluted in 3-M nitric acid (20 mL) and were incubated at 200 °C for 8 h in a conical beaker using a hot plate. Samples were then diluted to 100 mL with distilled water and were analysed using an inductively coupled plasma analysis emission spectrometer (ICP-AES; IRIS Advantage/SSEA, Jarrell-Ash Co. Ltd., Tokyo, Japan). Results are presented as the mean ± standard error of three independent experiments. 2.4. Analyses of chemical components and functional properties of fish sauce Total nitrogen contents of fish sauce were determined using the Kjeldahl method with a nitrogen/protein analyser (KJEL-Auto DTD4; Mitamura Riken Kogyo Co. Ltd., Tokyo, Japan). Free amino acid
concentrations were analysed using an amino acid autoanalyser (L-8900; Hitachi High-Technologies Corporation, Tokyo, Japan). Sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), zinc (Zn), and copper (Cu) concentrations were determined using ICP– AES as described above. Results are presented as the mean ± standard error of three independent experiments. Antioxidant activities were evaluated using the method of Suda (2000) with the radical scavenger 1,1-diphenyl-2-picrylhydrazyl (DPPH) and were expressed relative to standard gallic acid solutions. ACE inhibitory activities were estimated as half maximal inhibitory concentrations (IC50) of rabbit lung ACE by determining hippuryl-L-histidyl-L-leucine concentrations using HPLC according to the methods of Horiuchi, Fujimura, Terashima, and Iso (1982) and Ohta, Iwashita, Sakai, and Kawamura (1997). Results are presented as the mean of three independent measurements.
2.5. Removal of Cd from model samples at various NaCl concentrations To evaluate the influence of NaCl concentrations on Cd removal by chelate resins, NaCl aqueous solutions of varying concentrations up to that of fish sauce were prepared, and were used as standard model samples for Cd removal using the batch method. These model samples contained 0, 5, 10, 15, 20, and 25 g of NaCl/ 100 mL, and Cd concentrations were adjusted to 2.0 mg/100 mL using a Cd standard solution (Cd-1000; Kanto Chemical Co. Inc., Tokyo, Japan). Moreover, pH was adjusted to 6 using 0.1-M sodium phosphate buffer according to reported pH values for the present fish sauce (pH 5.8) and other fish sauces (pH 5.6–6.2, Park et al., 2001). Subsequently, 20-mL aliquots were mixed with CR11 or CR20 (70 mg) and were shaken for 24 h. Cd concentrations were then analysed using ICP–AES. Results are presented as the mean of three independent measurements.
2.6. Removal of Cd from model samples at various amino acid concentrations The effects of amino acids on Cd removal by chelate resins were examined using aqueous amino acid solutions as model samples. Amino acids concentrations were adjusted to 0, 2000, 4000, 6000, 8000, and 10,000 mg of alanine, glutamic acid, and lysine at a ratio of 3:2:2 in 100 mL. These are the three major amino acids in fish sauce, and their concentration in reference samples was prepared according to that found in fish sauce (7460 mg/100 mL). As with NaCl samples, Cd concentrations were adjusted to 2.0 mg/ 100 mL, and pH was adjusted to 6 using 0.1-M sodium phosphate buffer. Samples (20 mL) were then mixed with CR11 (70 mg) or CR20 (70 mg) and were shaken for 24 h. Cd concentrations were analysed using ICP–AES. Results are presented as the mean of three independent measurements.
2.7. Removal of Cd from fish sauce for comparison with reference samples To compare inhibitory effects of NaCl and amino acids on Cd binding of chelate resin in fish sauce and reference samples, initial Cd concentrations in fish sauce were adjusted to that of reference samples (2.0 mg/100 mL) by adding 1000 ppm Cd standard solution to tannin- and CR11-treated fish sauce. Subsequently, fish sauce samples (20 mL) were mixed with CR11 (70 mg) or CR20 (70 mg) and were shaken for 24 h. Cd concentrations were then analysed in treated and untreated fish sauce samples using ICP– AES. Results are presented as the mean ± standard error of three independent experiments.
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2.8. Gel filtration chromatography of fish sauce
3. Results and discussion 3.1. Removal of Cd from fish sauce using the batch method with chelate resin
Cd concentration in fish sauce (mg/100 mL)
A
0.8
0.6
0.4
0.2
0.0 Untreated
Tannin
CR11
CR20
Removal methods
0.4
Cd concentrations in fish sauce (mg/100 mL)
Cd forms in fish sauce were identified using gel filtration chromatography according to the method of Takeuchi, Inari, and Morimoto (2001) with some modifications. Untreated fish sauce, tannin-treated fish sauce, CR11-treated fish sauce, and standard solution were compared. CR11-treated fish sauce was prepared using the batch method. Standard solutions were prepared by dissolving 100 mg of human albumin (Wako Pure Chemical Industries Ltd., Tokyo, Japan), 100 mg of alanine (Wako Pure Chemical Industries Ltd.), and 100 lL of 1000-ppm Cd standard solution in 10 mL of distilled water. Samples (2 mL) were then applied to an equilibrated Sephadex G-15 gel (GE Healthcare Bio-Science, Uppsala, Sweden) filtration column (50 1.5 cm internal diameter) and were eluted at 1 mL/ min with 0.1-M aqueous NaCl solution. Fractions were collected every 4 min. Cd concentrations and absorbance (280 nm) of fractions were analysed using ICP–AES and spectrophotometry (UV1800; Shimadzu Corporation), respectively. Amino acids and peptides were analysed in 80-lL fractions after mixing with 80 lL of 1 g/100 mL ninhydrin solution and 1.84 mL of distilled water and heating in boiling water for 15 min (Takeuchi et al., 2001). Absorbance was then measured at 570 nm using a spectrophotometer.
1.0
B
0.3
0.2
0.1
0.0
Cd in fish sauce was initially removed with tannin according to a previously described method (Sasaki et al., 2013), and Cd concentrations were reduced from 0.78 ± 0.01 to 0.28 ± 0.01 mg/100 mL (Fig. 1A). Two-thirds of total Cd contents were removed from fish sauce by tannin treatment, presumably corresponding to organic molecule-bound Cd. Subsequently, tannin-treated fish sauce was treated with chelate resin using the batch method, which reduced Cd concentrations to 0.05 ± 0.01 mg/100 mL (CR11) and 0.06 ± 0.01 mg/100 mL (CR20), which were less than one-fifth of that in tannin-treated fish sauce (Fig. 1A). Hence, residual Cd in fish sauce after tannin treatment was removed using the chelate resin, presumably representing the liberated form of Cd. The Cd binding ability of chelate resin is affected by pH, which was 5.8 in the present fish sauce samples, and is generally between 5.6 and 6.2 in other fish sauces (Park et al., 2001). Because iminodiacetic acid and polyamine functional groups are known to exhibit strong chelating ability for heavy metal ions at pH 4–7, the present chelate resins are likely to remove Cd from most fish sauces (Li et al., 2012; Ueda, Teshima, Sakai, Joichi, & Motomizu, 2010).
1
2
3
4
5
Fraction number Fig. 1. Cd concentrations in fish sauce treated with chelate resins using the batch and the column method. Data from the column method are present in (A). Data from untreated fish sauce, tannin-treated fish sauce (before chelate resin treatment), and CR11- and CR20-treated fish sauce after tannin treatment are shown. Data are presented as the mean ± standard error of three independent samples. Data from the column method are present in (B). Tannin-treated fish sauce was eluted through columns packed with CR11 (d) or CR20 (h) chelate resins, and 10 mL fractions were collected every 2 h. Initial Cd concentrations in fish sauce were 0.33 mg/100 mL. Data are presented as the mean ± standard error of three independent samples.
fractions (from 0.05 ± 0.01 to 0.07 ± 0.01 mg/100 mL), those after treatment with CR20 increased linearly with elution volume (from 0.04 ± 0.003 to 0.17 ± 0.01 mg/100 mL). These increasing eluent concentrations reflect gradual saturation of binding sites (Popuri, Vijaya, Boddu, & Abburi, 2009), and indicate that the Cd removal ability of CR11 was higher than that of CR20 when using the column method.
3.2. Removal of Cd from fish sauce with chelate resin using the column method
3.3. Chemical components and functionality of fish sauces treated with CR11 using the column method
Columns packed with chelate resin are suitable for fish sauce manufacturing factories, because unlike the batch method, both fish sauce and conditioning solution are passed through the column, and chelate resins do not require further conditioning. Thus, we evaluated the Cd removal efficiency of columns packed with the chelate resins CR11 and CR20 (Fig. 1B). Tannin-treated fish sauce contained Cd at 0.33 ± 0.01 mg/100 mL. Fig. 1B shows Cd concentrations in fractions eluted from chelate resin columns. Cd concentrations in the first fractions were 0.05 ± 0.01 mg/100 mL (CR11) and 0.04 ± 0.003 mg/100 mL (CR20), indicating effective Cd removal by both chelate resins. Whereas Cd concentrations after treatment with CR11 were constant across the first five
Using the column method, CR11 had superior Cd removal efficacy to that of CR20. Thus, we evaluated chemical components and functionality of fish sauce after treatment with CR11-packed columns (Table 1). Total nitrogen and free amino acid contents did not differ between untreated fish sauce and the first fraction (1.8 ± 0.04 vs. 1.8 ± 0.02 g/100 mL and 10,600 ± 200 vs. 10,200 ± 300 mg/100 mL, respectively) and did not change in subsequent fractions. Thus, nutritional peptides and free amino acids remained in fish sauce samples after Cd removal using CR11. In subsequent metal ion analyses, Cu and Zn concentrations in untreated fish sauce were 1.4 ± 0.01 and 2.4 ± 0.04 mg/100 mL and were decreased to approximately 0.9 and 0.1 mg/100 mL
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Table 1 Chemical components and functionality of fish sauces treated with CR11 using the column method.
Chemical component Total nitrogen (g/100 mL) Total Free amino acid (mg/100 mL) Cu (mg/100 mL) Zn (mg/100 mL) Mg (mg/100 mL) Ca (mg/100 mL) Na (g/100 mL) K (mg/100 mL) Taurine (mg/100 mL) Functionality DPPH radical scavenging activity as gallic acid (lmol/mL) ACE inhibitory activity (IC50: lL/mL)
Untreated
Fr. 1
Fr. 2
Fr. 3
Fr. 4
Fr. 5
1.8 ± 0.04 10,600 ± 200 1.4 ± 0.01 2.4 ± 0.04 33 ± 1.3 11 ± 0.1 7.5 ± 0.01 103 ± 4 720 ± 10
1.8 ± 0.02 10,200 ± 300 0.7 ± 0.02 0.4 ± 0.01 21 ± 0.6 4.5 ± 0.2 7.5 ± 0.02 103 ± 2 700 ± 1
1.8 ± 0.02 10,500 ± 600 0.9 ± 0.02 0.2 ± 0.02 33 ± 0.1 10 ± 0.1 7.5 ± 0.02 104 ± 1 720 ± 22
1.8 ± 0.01 10,500 ± 500 0.9 ± 0.03 0.1 ± 0.01 33 ± 0.1 11 ± 0.1 7.5 ± 0.02 103 ± 1 710 ± 13
1.8 ± 0.02 10,500 ± 600 0.9 ± 0.03 0.1 ± 0.01 33 ± 0.5 11 ± 0.1 7.5 ± 0.01 104 ± 1 720 ± 9
1.8 ± 0.08 10,700 ± 200 1.0 ± 0.04 0.1 ± 0.01 33 ± 0.2 11 ± 0.1 7.5 ± 0.02 104 ± 1 720 ± 11
0.96 ± 0.08 9.5 ± 0.7
0.96 ± 0.08 9.8 ± 0.9
1.0 ± 0.07 9.9 ± 0.6
1.1 ± 0.02 9.3 ± 1.0
0.97 ± 0.01 8.8 ± 0.8
1.0 ± 0.07 9.3 ± 0.6
Experiments were performed on three independent samples, and data are shown as the mean ± standard error.
A
Cd concentration (mg/100 mL)
2.0
1.5
1.0
0.5
0.0 0
5
10
15
20
25
NaCl concentration (g/100 mL)
B
2.0 Cd concentration (mg/100 mL)
following CR11 treatment, respectively. Divalent ions such as Cu2+ and Zn2+ are known to bind iminodiacetic acid. However, Mg and Ca concentrations were maintained at approximately 33 and 11 mg/100 mL, respectively, following CR11 treatment, but were decreased to half of those in untreated fish sauce, potentially reflecting low binding constants of these metals. Because Cu, Zn, Mg, and Ca concentrations are low in untreated fish sauce, these decreases may not have a significant impact on the quality of fish sauce, although their adsorption by CR11 may competitively inhibit Cd removal. Na and K concentrations in fish sauce were 7.5 ± 0.01 g/100 mL and 103 ± 4 mg/100 mL, respectively. Because alkaline metals do not bind iminodiacetic acid with high affinity, Na and K concentrations were not significantly altered after resin treatments. Na is predominantly present as NaCl in fish sauce and is an important preservative that inhibits microbial contamination. Thus, maintenance of Na concentrations is critical for the quality of fish sauce. Fish sauce contains beneficial antioxidants and ACE inhibitors, and fish sauce prepared from squid contains high concentrations of taurine, which facilitates recovery from fatigue (Michihata et al., 2000; Taniguchi et al., 2009). Taurine contents in untreated fish sauce and in treated first fractions were 720 ± 10 and 700 ± 1 mg/100 mL, respectively. DPPH radical scavenging activity was also determined using gallic acid as a standard radical scavenger, and did not differ between untreated fish sauce and first fractions (0.96 ± 0.08 vs. 0.96 ± 0.08 lmol/mL, respectively). Moreover, ACE IC50 values did not differ between untreated fish sauce and first fractions (9.5 ± 0.7 vs. 9.8 ± 0.9 lL/mL, respectively). These major functionalities remained in all subsequent fractions, confirming that CR11 did not remove organic compounds.
1.5
1.0
0.5
0.0 0
2000
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Amino acid concentration (mg/100 mL)
3.4. Influences of NaCl on Cd removal by chelate resin Fish sauce contains large amounts of NaCl and amino acids (Michihata et al., 2000; Park et al., 2001), and although chelate resins are effective in diluted solutions such as waste water, these fish sauce constituents may inhibit Cd removal. Thus, we initially investigated the influence of NaCl on the Cd removal abilities of chelate resins in aqueous reference samples. These samples were prepared using NaCl in aqueous phosphorous buffer, and the concentrations of NaCl ranged from 0 to that of fish sauce. Fig. 2A shows the influence of NaCl concentrations on the removal of Cd from model samples using chelate resins (CR11, CR20) with the batch method. Because fish sauce contains NaCl at 18–23 g/100 mL (Park et al., 2001), reference samples contained NaCl in the range of 0–25 g/100 mL. After treatment with CR11, Cd concentrations in these standards increased with NaCl concentrations from 0.20 ± 0.050 to 1.7 ± 0.005 mg/100 mL. Because Na+ is
Fig. 2. The influence of NaCl and amino acids on Cd removal from reference samples using chelate resin. Data from NaCl are presented in (A). Aqueous solutions containing NaCl at 0–25 g/100 mL and Cd at 2.0 mg/100 mL in phosphate buffer (pH 6) were used as reference samples, and were treated with CR11 (d) or CR20 (h) chelate resins using the batch method. Data are presented as the mean ± standard error of three independent samples. Data from amino acids are presented in (B). Aqueous solutions containing amino acids at 0–10,000 mg /100 mL and Cd at 2.0 mg/100 mL in phosphate buffer (pH 6) were used as reference samples and were treated with CR11 (d) or CR20 (h) chelate resins using the batch method. Data are presented as the mean ± standard error of three independent samples.
known to occupy iminodiacetic acid groups (Pramanik, Dhara, & Chattopadhyay, 2004; Takaku, Kudo, Kimura, Hayashi, & Ota, 2002), NaCl was expected to inhibit binding of Cd to CR11. However, Cd2+ may bind iminodiacetic acids more strongly than Na+, reflecting differences in binding modes and spatial structures. Nonetheless, Cd removal by CR11 decreased with increasing NaCl
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Ultraviolet absorbance (280 nm)
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Cd concentration (mg/100 mL)
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Fraction
Cd concentration (mg/100 mL)
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C
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Cd concentration (mg/100 mL)
6
Liberated Cd 3.0
Alanine Albumin
5
2.5
4
2.0
3
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2
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1
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0 0
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0.0 40
0.5 0.4 0.3 0.2 0.1 0.0
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D
7
Ultraviolet absorbance (280 nm)
Fraction
Fraction Fig. 3. Gel filtration chromatography of fish sauce and reference solutions. Data from (A) untreated fish sauce, (B) tannin-treated fish sauce, (C) chelate-treated fish sauce, and (D) reference solutions are presented. Reference solutions contained albumin, alanine, and liberated Cd. Cd concentrations (d), absorbance at 280 nm (h), and absorbance at 570 nm after ninhydrin reactions (N) were measured in each fraction.
concentrations, presumably because concentrations of Na+ were much higher than those of Cd2+. After treatment with CR20, Cd concentrations did not change significantly with increasing NaCl concentrations. It is known that
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ethylene diamine forms Cd2+ complexes with nitrogen atoms, which provide electron pairs that form stable coordination covalent bonds with metal ions. Moreover, non-ionic ethylene diamine groups may not interact with Na+. Hence, although CR11 had greater efficacy than CR20, the two chelate resins had equal efficacy in the presence of NaCl at 20 g/100 mL, and under high salt conditions, the Cd removal ability of CR11 was decreased to the same extent as that of CR20. Because NaCl decreased the removal of Cd by CR11 in reference samples, we evaluated the inhibitory effect of NaCl on Cd binding of CR11 in fish sauce. Samples were prepared by addition of 1000 ppm Cd standard solution to fish sauce samples after Cd removal by tannin treatment, and initial Cd concentrations were adjusted to those in reference samples (2.0 mg/100 mL), and Cd removal tests were performed using the same method as for reference samples. In these experiments, Cd concentrations in fish sauce were decreased to 1.0 ± 0.1 mg/100 mL. In comparison with Cd concentrations (1.6 ± 0.01 mg/100 mL) in reference samples containing NaCl at 20 g/100 mL, fish sauce samples (NaCl, 19.8 g/ 100 mL) contained lower Cd concentrations. These data indicate that the inhibitory effect of NaCl on Cd binding of chelate resins was reduced by fish sauce components. In addition to NaCl and amino acids (Michihata et al., 2000; Nakazato et al., 2000; Park et al., 2001), these components may have included organic acids such as lactic acid and pyroglutamic acid, polyamines such as histamine and tyramine, minerals such as Cu and Mg. Thus, numerous interactions with fish sauce components may contribute to decreased inhibitory properties of NaCl. Nonetheless, NaCl strongly inhibited Cd binding by CR11 in fish sauce, as confirmed by higher Cd concentrations in CR11-treated fish sauce (0.2 ± 0.01 mg/ 100 mL) than in CR11-treated reference samples (NaCl at 0 g/ 100 mL). 3.5. Influences of amino acids on Cd removal using chelate resin The influences of amino acids on Cd removal by chelate resins were evaluated as described above for NaCl. Fig. 2B shows Cd removal from reference samples containing amino acids at 0– 10,000 mg/100 mL. Fish sauces contain amino acids at 6000– 10,000 mg/100 mL (Park et al., 2001), and predominantly comprise alanine, glutamic acid, and lysine at a 3:2:2 ratio in squid fish sauce. In the presence of CR11, Cd concentrations in amino acid reference solutions remained constant at approximately 0.2 mg/ 100 mL, and were impervious to amino acid concentrations. In contrast, remaining Cd concentrations in the reference samples treated with CR20 were increased in proportion to amino acid concentrations (from 1.3 ± 0.09 to 1.9 ± 0.02 mg/100 mL), indicating that amino acids inhibit binding of Cd to CR20. Previous reports indicate that some carboxylic acid compounds form complexes with polyamines (Kimura, 2002; Kimura, Sakonaka, Yatsunami, & Kodama, 1981), suggesting that interactions between amino acids and polyamine may decrease Cd binding by CR20. In subsequent experiments, the inhibitory effects of amino acids on Cd binding ability of CR20 were determined in fish sauce samples containing Cd at 2.0 mg/100 mL). Treatment with CR20 reduced Cd concentrations in fish sauce (total free amino acid concentration, 7460 mg/100 mL) to 1.0 ± 0.03 mg/100 mL, which was lower than in treated standard solutions (1.9 ± 0.02 mg/100 mL) containing amino acids at 8000 mg/100 mL. This result indicates that inhibition of CR20–Cd binding by amino acids was ameliorated by other fish sauce components. 3.6. Determination of Cd forms in fish sauce To compare the effects of chelate resin and tannin treatments, Cd forms were determined in untreated, tannin-treated, and
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CR11-treated (without tannin treatment) fish sauces using gel filtration chromatography (Fig. 3). Cd concentrations in the untreated fish sauce were 0.76 ± 0.01 and decreased following treatment with tannin and CR11 to 0.47 ± 0.01 and 0.24 ± 0.01 mg/100 mL, respectively. After treatments with both tannin and chelate resin, Cd was almost completely removed from fish sauce (0.05 ± 0.004 mg/100 mL). In chromatographic analyses of standard solutions (D), albumin, alanine, and liberated Cd were detected in fractions 9, 13, and 17, respectively. Chromatograms of untreated fish sauce (A) had two main Cd peaks in fractions 9 and 18. Comparisons of untreated fish sauce and reference solutions indicated that the peaks at fractions 9 and 18 corresponded with protein-bound and liberated forms of Cd in untreated fish sauce, respectively. Chromatograms of untreated fish sauce samples also had small peaks at fraction 23, indicating that liberated Cd may be eluted following interactions with small molecules such as phenethyl amine, which was detected in fraction 23 using HPLC (data not shown). After tannin-treatment of fish sauce, the first chromatographic Cd peak disappeared, whereas the second Cd peak was retained, indicating that tannin treatments removed protein-bound Cd but not liberated Cd. In contrast, in chromatographic analyses of the CR11-treated fish sauce, the first Cd peak remained and the second Cd peak disappeared. Thus, CR11 treatment removed liberated Cd but did not remove protein-bound Cd. Taken together, the present data show that tannin treatments remove protein-bound Cd, and CR11 treatments remove liberated Cd. Hence, the combined application of tannin and CR11 treatments may reliably remove almost all Cd from fish sauce. 4. Conclusion We have developed a new method for removal of Cd from fish sauce using a combination of chelate resins that are approved as food additives and the previously reported tannin method. This combined method removed both liberated and protein-bound Cd from fish sauce, and solved the limitations of tannin treatments. The iminodiacetic acid type chelate resin (Diaion CR11) was more suitable for the removal of Cd from fish sauce than the ethylene diamine type (Diaion CR20). Accordingly, CR11 removed Cd more effectively than CR20 in the column method. Moreover, Cd–CR11 binding was strongly inhibited by NaCl, although other fish sauce components reduced this effect. Finally, chemical components with nutritional value or known beneficial functions were retained after tannin and chelate resin treatments. Thus, the present method is safe and effective and has promising applications in fish sauce factories. References Aoshima, H., & Ooshima, S. (2009). Anti-hydrogen peroxide activity of fish and soy sauce. Food Chemistry, 112, 339–343. Chuon, M., Shiomoto, M., Koyanagi, T., Sasaki, T., Michihata, T., Chan, S., et al. (2014). Microbial and chemical properties of cambodian traditional fermented fish products. Journal of the Science of Food and Agriculture, 94, 1124–1131. Dermont, G., Bergeron, M., Mercier, M., & Richer-Lafleche, M. (2008). Soil washing for metal removal: A review of physical/chemical technologies and field applications. Journal of Hazardous Materials, 152, 1–31. Fukami, K., Ishiyama, S., Yaguramaki, H., Masuzawa, T., Nabeta, Y., Endo, K., et al. (2002). Identification of distinctive volatile compounds in fish sauce. Journal of Agricultural and Food Chemistry, 50, 5412–5416. Furusho, Y., Ono, M., Yamada, M., Ohashi, K., Kitade, T., Kuriyama, K., et al. (2008). Advanced solid phase extraction for inorganic analysis and its applications. Bunseki Kagaku, 57, 969–989. Gasaluck, P., Yokoyama, K., Kimura, T., & Sugahara, I. (1996). Some chemical and microbiological properties of Thai fish sauce and paste. Journal of Antibacterial and Antifungal Agents, 24, 385–390. Ghimire, K. N., Kai, H., Inoue, K., Ohto, K., Kawakita, H., Harada, H., et al. (2008). Heavy metal removal from contaminated scallop waste for feed and fertilizer application. Bioresource Technology, 99, 2436–2441.
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