A rapid immunochromatographic (ICG) strip based on a conjugate of colloidal gold and monoclonal antibody (mAb) was developed for the rapid, sensitive, and on-site detection of sulfamethazine in meat and egg samples. The detection limit of the ICG strip is 2 ng/mL, and the assay can be completed in 10 min. A cross-reactivity test indicated that the ICG strip was highly specific to sulfamethazine with no cross-reaction with sulfonamide compounds and other antibiotics. The results of the recovery test from meat and egg samples spiked with sulfamethazine were in good agreement with those obtained by the indirect competitive enzyme-linked immunosorbent assay. These results demonstrated that the ICG strip can be used as a rapid and qualitative tool for on-site screening of sulfamethazine in meat and egg samples.
The present immunochromatographic strip could be used as a rapid and on-site screening tool for sulfamethazine in livestock products.
Sulfamethazine belongs to the group of sulfonamides, which are widely used in veterinary as well as human medicine (Sashenbrecker and Fish 1980). Sulfonamides are antimicrobial agents used for prophylactic, therapeutic, and growth-promoting purposes (Ko and others 2000; Moreno and others 2001; Shelver and others 2008). In cases of improper or prolonged use in animals, residues of sulfonamides can be found in meat and processed meat products, leading to the presence of illegal levels. Among the sulfonamides, sulfamethazine has been identified as the major agent in 95% of all sulfonamide violations in animal tissues (Renson and others 1993), and the safety of its use as an antibiotic has been considered because it has potential toxic effects such as allergenic effects, carcinogenicity, and antibiotic resistance of microorganisms.
Stockbreeders in many countries have used sulfamethazine when breeding animals, and approximately 42 to 165 tons of this antibiotic are being used annually in South Korea. Since many researchers have reported that sulfamethazine has been detected in milk, meat, and other animal food products (Clark and others 2005; Kim and others 2008), many countries have set regulations for sulfamethazine to prevent problems with consumers. In the case of Europe, Canada, the United States of America, and the Republic of Korea, the maximum residue limit (MRL) for the total amount of sulfonamides in edible tissues is set at 100 μg/kg, while Japan and the WHO have set an MRL of 20 and 25 μg/kg, respectively, for meat and milk products (Cliquet and others 2003; Zhang and Wang 2009).
The detection of sulfamethazine in meat and milk products was generally carried out using instrumental methods such as high-performance liquid chromatography/mass spectrometry (HPLC/MS) (Furusawa 2003; Tamošiūnas and Padarauskas 2008) and gas chromatography/mass spectrometry (GC/MS) (Takatsuki and Kikuchi 1990; Reeves 1999), which are accepted as official methods for sulfamethazine detection in food-based meat and milk. Although the methods are highly accurate, sensitive, specific, and reliable, they are time-consuming and labor intensive and require expensive equipments and skilled analysts (Yang and others 2007). Thus, these methods are unsuitable for routine screening of large sample numbers. Therefore, rapid, cost-effective, and easy-to-use methods for on-site screening of sulfamethazine are strongly required.
Typical immunochemical assays based on an antibody–antigen interaction, such as the enzyme-linked immunosorbent assay (ELISA) (Cliquet and others 2003), fluorescence polarization immunoassay (Gasilova and Eremin 2010), and immunosensor (Conzuelo and others 2012), can be a simple, rapid, and economical method used as an alternative to instrumental methods. However, these methods still require a long incubation time and multiple steps. Furthermore, the utilization of such immunoassays has been confined to laboratories equipped with special devices (washer and reader, and so on) (Nagatani and others 2006; Kolosova and others 2008).
Many studies have attempted to develop a simple and rapid method for on-site analysis of antibiotics in major livestock products, and these efforts have been realized through the concept of the immunochromatographic (ICG) assay, based on the inhibition (competitive) assay (Guo and others 2010; Byzova and others 2011). An ICG strip assay combines several benefits such as user-friendly format, long-term stability, and cost-effectiveness. These properties still make the ICG attractive for on-site screening by untrained personnel. In this study, we present the development of an ICG for the rapid, sensitive, and on-site detection of sulfamethazine, and validated the ICG by analyzing meat (pork and chicken) and egg samples spiked with known concentrations of sulfamethazine.
Materials and Methods
Chemicals, materials, and apparatus
Sulfamethazine, other related antibiotics (sulfadimethoxine, sulfamerazine, sulfamethoxypyridazine, sulfamethoxazole, sulfathiazole, sulfadiazine, sulfaquinoxaline, sulfadoxine, albendazole, thiamphenicol, thiambendazole, oxytetracycline, and olaquindox), keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), tetrachloroauric acid (HAuCl4), sodium citrate, N,N-dicyclohexylcarbodiimide, sucrose, sodium azide, dextran, goat antimouse IgG, peroxidase-conjugated goat antimouse IgG and 2,2-azinobis (3-ethylbenzthiazoline-6sulfonic acid) (ABTS) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, Mo., U.S.A.). Sodium chloride was obtained from SK Chemicals (Ulsan, Korea). Protein G agarose for the purification of monoclonal antibody (mAb) was purchased from Bioprogen (Daejeon, Korea). Sulfamethazine-ovalbumin (OVA) conjugate used as a capture reagent in the ICG strip and the mAb to sulfamethazine were prepared in our laboratory (Yang and others 2007). All other chemicals and organic solvents were analytical grade.
The nitrocellulose membrane and pads (sample, conjugate, and absorbent pads) were purchased from Millipore Co. (Bedford, Mass., U.S.A.). Semi-rigid polyethylene sheets were obtained from a local market.
Microtiter plates (96 wells) and a 12-channel microplate washer were obtained from Nunc Intl. (Rockilde, Denmark). The Multiskan* FC Microplate Photometer for ELISA reading was purchased from Thermo Fisher Scientific Inc. (Waltham, Mass., U.S.A.). The centrifuge (Micro 17R) was obtained from Hanil Science Industrial Co. (Gangneung, Korea).
Monoclonal antibody (mAb) specific to sulfamethazine
The monoclonal hybridoma named 1H11, producing an mAb specific to sulfamethazine, was developed by cell fusion using myeloma cells and spleen cells obtained from mice immunized with sulfamethazine-KLH conjugate as an antigen. The hybridoma was grown in a culture medium and intraperitoneally injected into BALB/c mice that had been pretreated with an intraperitoneal injection of 0.5 mL of pristane. After 1 wk, ascite fluid from the mice was obtained and purified by precipitation with saturated ammonium sulfate followed by affinity chromatography on a protein G-agarose column. In a previous paper by the current author (Yang and others 2007), the mAb was already confirmed to be highly specific to sulfamethazine by ELISA.
Preparation of mAb-colloidal gold probe
Nano-size colloidal gold particles (40-nm diameter) used as a marker in the ICG strip were synthesized according to the method of Frens (1975). Briefly, 100 mL of 0.01% HAuCl4 was boiled in a glass beaker while stirring, and then 2.0 mL of 1% sodium citrate was added rapidly. After the color of the solution changed to wine red, the mixture was boiled for an additional 5 min while stirring. Then, the colloidal gold solution was cooled down to room temperature and stored at 4 °C.
Conjugation of the gold nanoparticles and purified mAb was conducted using the method of Roth (1982). Briefly, 80 μg of the mAb was added to 20 mL of the gold nanoparticle solution previously adjusted to pH 8.5 with 0.1 M K2CO3. The mixture was stirred for 60 min, and 2.0 mL of 10% (w/v) BSA solution was added and stirred for a further 60 min. The mixture was centrifuged at 12000 × g at 4 °C for 10 min, and the supernatants were removed. The pellets were washed 3 times with 2 mM borate buffer (pH 7.2), and the final pellets were suspended in 1 mL of 2 mM borate buffer (pH 7.2) containing 1% BSA, 1% sucrose, and 0.05% sodium azide, and stored at 4 °C until use.
Three pads (sample, conjugate, and absorbent pads) and a nitrocellulose membrane were gradually attached to semirigid polyethylene sheets as shown in Figure 1. Before assembly, the sample pads were soaked with 50 mM borate buffer (containing 1% BSA, 0.5% Tween 20, 5% sucrose, 5% dextran, and 0.05% sodium azide, pH 7.4) and dried at 60 °C for 1 h. The mAb-gold probe (5 μL), diluted 5-fold in 20 mM borate buffer (pH 8.2) containing 1% BSA and 1% sucrose, was treated on the conjugate pad and dried at 37 °C for 30 min. The absorbent pads were not treated. Sulfamethazine-OVA conjugate (0.7 μL, 0.2 mg/mL in phosphate buffer saline [PBS, pH 7.4]) and goat antimouse IgG (0.7 μL, 0.05 mg/mL in PBS) were sprayed onto the bottom and top of the nitrocellulose membrane as test and control lines, respectively, using a manual dispenser fabricated in our laboratory and dried for 30 min at 37 °C. The distance of both the test and control lines was 5 mm. The treated and dried conjugate pad and nitrocellulose membrane were kept in a desiccator at room temperature. Assembly of the strip test was conducted as described in a previous paper by the current authors (Shim and others 2009).
Performance of the ICG strip
To perform the ICG strip developed in this study, 200 μL of extracts from unknown samples or standard solutions were added to the wells of the microtiter plate, and the sample pad of the ICG strip was placed into the wells. The sample pad was immersed, and the liquids then migrated up to the conjugated pad, nitrocellulose membrane, and absorbent pad in consecutive order. Results were obtained within 10 min and evaluated visually. Positive samples or standards produce one red line at the control zone because the mAb-gold on the conjugate pads first reacts to the sulfamethazine, and the complexes (sulfamethazine-mAb-gold) do not bind to the sulfamethazine-OVA treated at the test zone and pass the test zone. The complexes finally bind to the goat antimouse IgG immobilized at the control zone. Meanwhile, the mAb-gold with the sulfamethazine-free samples or standards sufficiently reacts to both the test and control zones and creates 2 red lines at both zones. If there is no color development at the control region, analysis by the ICG strip is invalid, and an additional test is required.
Validation of the ICG strip
The ICG strip developed in this study was validated by determining the detection limit, reproducibility, and cross-reactivity with sulfonamide compounds. To determine the detection limit of the ICG strip, sulfamethazine standards at concentrations of 0, 1, 2, 4, 6, 8, and 10 ng/mL in phosphate buffer saline (PBS, pH 7.4) containing 10% acetone (working buffer) were tested. The sulfamethazine standards described above were analyzed 10 times for the intraday assay (n = 10) and daily for 10 successive days for the interday assay (n = 10). To determine cross-reactivity, the sulfonamide compounds (sulfadimethoxine, sulfamerazine, sulfamethoxypyridazine, sulfamethoxazole, sulfathiazole, sulfadiazine, sulfaquinoxaline, and sulfadoxine) and other antibiotics (albendazole, thiamphenicol, thiambendazole, oxytetracycline, and olaquindox) at a concentration of 100 ng/mL were prepared in the working buffer and applied to the ICG strip.
Indirect competitive ELISA (IC-ELISA)
IC-ELISA reported in our previous paper (Yang and others 2007) was used to compare the results obtained from the analysis of the ICG strip. Briefly, 100 μL of sulfamethazine-OVA in carbonate-bicarbonate buffer (0.05 M, pH 9.6) was added to wells of the microtiter plate (100 ng/well) and kept at 4 °C overnight. After the wells were washed 3 times with PBS containing 0.05% Tween 20 (PBST), 200 μL of 1% skim milk in PBS was added and incubated at 37 °C for 1 h, and the wells were washed 4 times with PBST. Fifty microliter of the standards (0, 0.1, 1, 10, 100, and 1000 ng/mL) or samples and 50 μL of the mAb (diluted 1:1000 in PBS) were sequentially added to the wells and left to react at room temperature for 1 h. After washing 5 times with PBST, 100 μL of peroxidase-conjugated goat antimouse IgG (diluted 1:10000 in PBS) was added to the wells, incubated at 37 °C for 1 h and washed 6 times again. The washed wells were incubated with 100 μL of substrate solution (2 mg/mL ABTS, 0.03% (v/v) H2O2 in 0.1 M citrate buffer, pH 4.0) at 37 °C for 30 min. Absorbance was measured at 405 nm using an ELISA reader (Multiskan FC, Thermo Fisher Scientific Inc).
Meat (beef, pork, and chicken) and egg samples were tested with both the ICG strip and IC-ELISA. Briefly, the samples (2 g) were homogenized in a blender, except for the egg. The homogenized samples were mixed with 4 mL of ethyl acetate and extracted for 5 min. The mixture was centrifuged at 2000 × g for 10 min, and the supernatant (400 μL) was taken and evaporated under nitrogen at 60 °C. The residue was dissolved in 200 μL of 10% acetone in PBS or 10% methanol in PBS for the ICG strip and IC-ELISA, respectively.
Analyses of meat and egg samples spiked with sulfamethazine
Samples of meat (beef, pork, and chicken) and egg were purchased from supermarkets located in Jinju, South Korea and treated as mentioned above. All samples were tested the HPLC method reported in our previous paper (Yang and others 2007). Among the samples, pork, beef, chicken, and egg confirmed as sulfamethazine-free samples by HPLC were chosen for the preparation of sulfamethazine positive samples by spiking with known concentrations of sulfamethazine. Two gram or milliliter of the sulfamethazine-free samples was artificially contaminated with sulfamethazine at concentrations of 10, 50, 100, and 200 ng/g (or ng/mL), extracted as mentioned above and subjected to analysis by the ICG strip and IC-ELISA. SkanIt Software (Version 2.5.1) connected to Multiskan FC reader (Thermo Fisher Scientific Inc.) was used for data processing of the IC-ELISA results. The detection limits of the HPLC were 8 ng/g for all meat and egg samples, and samples with ≤8 ng of sulfamethazine were considered and used as the sulfamethazine-free samples.
The results obtained from the ICG strip tests were estimated by naked eyes and recorded by taking pictures. The data of the IC-ELISA were transferred from the data log sheets into Excel 2007. The IC-ELISA standard curves were obtained by plotting absorbance against the analyte concentrations. The coefficient of variation (CV) was used to compare the variation among the 3 replicates from the different standard and sample means. Data were graphed using SigmaPlot 12.0 (Systat Software Inc., San Jose, Calif., U.S.A.).
Results and Discussion
Colloidal gold-monoclonal antibody
In ELISA assays, enzymes (horseradish peroxidase and alkaline phosphatase) have been generally used as markers or tracers. Meanwhile, colloidal gold nanoparticles were usually used as a marker in the ICG assay. In our previous paper, we reported that the volume of 1% sodium citrate directly affected the size of the colloidal gold particles, and increasing the volume of sodium citrate uniformly decreased the diameter of the colloidal gold particles (Shim and others 2006). Colloidal gold particles with a 40-nm diameter have been considered a suitable marker in the development of the ICG strip because they generally provide maximum visibility. In this study, colloidal gold nanoparticles of approximately 40 nm in diameter were produced by adding 2 mL of 1% sodium citrate to 100 mL of 0.01% HAuCl4, before boiling and using for conjugation to the mAb.
Bare nano-size colloidal gold nanoparticles are unstable in a salt solution such as NaCl, whereas gold nanoparticles coupled with an appropriate amount of protein and DNA are stable in salt solutions. Thus, the optimal amount of MAb specific to sulfamethazine required to stabilize the colloidal gold nanoparticles under 10% NaCl was determined. The mAb was directly adsorbed on the surface of the colloidal gold particles by van der Waals force and hydrophobic interactions (Sperling and Parak 2010), and gold nanoparticles conjugated with a sufficient amount of mAb are stable in 10% NaCl. To get a strong adsorption between the mAb and gold nanoparticles, a titration was performed to check whether the gold nanoparticles were saturated with mAb by adding a different amount of the mAb (0 to 20 μg) to 1 mL of the colloidal gold solution. As shown in Table 1, 8 μg of mAb was confirmed as the minimum mAb amount required to stabilize the colloidal gold solution. However, we chose 9 μg mAb per 1 mL of gold solution for the conjugation since 8 μg mAb per 1 mL gold solution often caused coagulation of the gold nanoparticles during conjugation. To validate the suitability of the gold particle-mAb in the development of the ICG strip, the result of the ICG strip assembled with a conjugate pad treated with the gold particle-antisulfamethazine mAb was compared with that of the ICG strip with the gold particle-BSA conjugate used as a control test. No signal on the nitrocellulose membrane, which is appropriate, was obtained from the control test. This means that using BSA as a blocking agent for the gold particles did not cause nonspecific binding to the sulfamethazine-OVA and antimouse IgG treated on the test and control zones, respectively. Meanwhile, the ICG strip with the gold particle-mAb showed 2 red spots and one red spot for negative (0 ng/mL) and positive (10 ng/mL) samples, respectively (Figure 2). These results indicate that the conjugate of the mAb and gold particles can be used as a probe in the development of the ICG strip.
Table 1. Determination of mAb amount required to stabilize the colloidal gold nanoparticles.
mAb concentration (μg/mL)
Optimization of the ICG strip
ICG strips have been well known as an on-site screening tool because of their simplicity, user-friendliness, and rapidity. The ICG strip for the analysis of sulfamethazine is based on the inhibition assay format. Therefore, appropriate amounts of detector reagent (gold particle-mAb) and capture reagent (sulfamethazine-OVA) should be estimated to get the highest sensitivity because an increase in the concentration of the reagents decreases the sensitivity of the assay. In this study, a checkerboard titration was performed using different dilutions of gold-mAb (1/5, 1/10, 1/15, and 1/20) in the dilution buffer and a different amount of sulfamethazine-OVA, to determine the optimal amounts of the reagents. Figure 3 shows the results of the checkerboard titration. Five microliter of gold-mAb diluted 5 times and 200 ng of sulfamethazine-OVA were chosen as appropriate amounts to give a clear, stable, and sufficient color intensity at the test zone for negative samples, as well as having higher sensitivity.
Generally, organic solvents have been used to extract antibiotics from samples but they coextract substances existing in complex matrices and inhibit the antibody–antigen interaction in immunoassays. Therefore, sulfamethazine-positive (100 ng/mL) and -negative (0 ng/mL) standards prepared with 10% organic solvent (acetone or ethanol, methanol, and acetonitrile) in PBS (10% organic solvent/PBS) were tested using the ICG strip. The sulfamethazine-free standard solution in 10% acetone/PBS provided 2 red lines at the test and control zones, which showed sufficient color intensity to be distinguished from positive results, but positive samples in 10% acetone/PBS presented one clear red line at the control zone. On the other hand, positive and negative standards in 10% other organic solvents/PBS (ethanol, methanol, and acetonitrile) all showed faint red lines at the test and control zones. Thus, in this study, 10% acetone was used as the working solution for preparing standards and samples in further experiments.
Properties of the ICG strip
The ICG strip was validated by determining the detection limit, reproducibility, and cross-reactivity. First, the detection limit and reproducibility were validated by analyzing sulfamethazine standards (200 μL) at concentrations of 0, 1, 2, 4, 6, 8, and 10 ng/mL in 10% acetone/PBS. Results from the ICG strips were obtained within 10 min, after samples were loaded at the sample pads, and estimated by the naked eye. The visual detection limit of the assay could be defined as the minimum analyte concentration producing no red color at the test line (Zhang and others 2011). According to the triplicate tests with standard solutions using the ICG strip, the visual detection limit of the assay was estimated to be 2 ng/mL of sulfamethazine in the working buffer since a faint red line appeared at the test zone with 1 ng/mL of sulfamethazine (Figure 4). The reproducibility results are summarized in Table 2. The results from the interday and intraday assays were all identical. This means that the ICG strip developed is a stable and reproducible tool for sulfamethazine analysis. To determine the specificity of the ICG strip, negative (0 ng/mL) and positive (100 ng/mL) samples of other antibiotics in 10% acetone/PBS were prepared and analyzed using the ICG strip. No cross-reaction with other antibiotics was observed and this demonstrates that the assay is highly specific to sulfamethazine (data not shown). In our previous paper, the mAb against sulfamethazine showed a weak cross-reaction with sulfamerazine (5.4%) by IC-ELISA (Zhang and others 2011), but this weak cross-reaction was not observed in the ICG strip format.
Table 2. Results of intra- and inter-assays for the strip of sulfamethazine.
(n = 10)
(n = 10)
aNegative for sulfamethazine.
cPositive for sulfamethazine.
−a, −, −, −, −, −, −, −, −, −
−, −, −, −, −, −, −, −, −, −
Analysis of meat and egg samples artificially spiked with sulfamethazine
Sulfamethazine-positive beef, pork, chicken, and eggs (spiked with sulfamethazine concentrations of 10, 50, 100, and 200 ng/g or ng/mL) were prepared as previously described and analyzed by both the ICG strip and IC-ELISA based on the same mAb. The IC-ELISA used in this study was also specific to sulfamethazine and its detection limit (10% inhibition) was 0.43 ng/mL. The IC50 value (concentration resulting in half-maximum inhibition) was calculated to be 5.39 ng/mL and the detection range was from 0.5 to 100 ng/mL (Figure 5). The results of the sulfamethazine positive samples (beef, pork, chicken, and egg spiked with sulfamethazine standards at concentrations of 10, 50, 100, and 200 ng/g or ng/mL) by the ICG strip were compared to those of IC-ELISA, and Table 3 shows the results of the analysis. The recoveries of sulfamethazine by IC-ELISA were from 73.4% to 92.9% with CVs of 4.0% to 7.8%, from 87.2% to 96% with CVs of 5.9% to 7.7%, from 96.6% to 112.1% with CVs of 1.9% to 4.4%, and from 78.5% to 125.6% with CVs of 3.7% to 7.3% for beef, pork, chicken, and egg, respectively. All samples contaminated with sulfamethazine at ≥10 ng/g (or ng/mL), an antibiotic level that exceeds the regulations set by the Korean government for meats and eggs, were classified as positive by the ICG strip. Moreover, the results from the ICG strip were in good agreement with those obtained from IC-ELISA. These results indicated that the developed ICG strip can be used as a reliable method for rapid screening of sulfamethazine in meats and eggs. Li and others (2009) presented results about an ICG strip used for the detection of sulfamethazine in swine urine, and its detection limit was 8 ng/mL. We consider that the ICG strip developed in this study possesses a higher sensitivity than the one developed by them. To extract sulfamethazine from meat and egg, the samples should be diluted with extraction solvents, and this step causes concentrations of sulfamethazine in real samples to be lower than the initial concentration in the samples. The sample preparation method used in this study did not dilute the original sulfamethazine levels in the meat and egg samples. Therefore, the sensitivity of the ICG strip is sufficient to detect samples contaminated with sulfamethazine to levels greater than the MRL set by not only Korea but also Japan and the WHO (Cliquet and others 2003; Zhang and Wang 2009).
Table 3. Comparison of results of sulfamethazine analysis of samples by ICG strip and IC-ELISA.
IC-ELISA (n = 3)
Spiked level (ng/mL)
ICG strip (n = 3)
Mean ± SD (ng/mL)
aNegative, red color on test line.
bPositive, no color on test line.
7.4 ± 1.2
39.6 ± 5.5
+, +, +
96.7 ± 6.2
+, +, +
191.3 ± 1.8
−, −, −
+, +, +
6.6 ± 2.3
+, +, +
39.3 ± 3.2
+, +, +
90.2 ± 6.4
+, +, +
184.7 ± 6.
−, −, −
+, +, +
11.6 ± 3.3
+, +, +
37.6 ± 4.1
+, +, +
77.9 ± 2.1
+, +, +
165.5 ± 5.5
−, −, −
+, +, +
8.9 ± 4.2
+, +, +
37.8 ± 4.0
+, +, +
73.8 ± 2.4
+, +, +
150.5 ± 6.9
With the results of the analysis of the meat and egg samples artificially spiked with known concentrations of sulfamethazine, we believe that the ICG strip developed is suitable for on-site determination and possesses sufficient sensitivity to be applied to real meat and egg samples. However, further research to simplify sample preparation is required to establish more rapid and easy-to-use methods.
In conclusion, the rapid and user-friendly ICG strip based on a colloidal gold-mAb probe for sulfamethazine detection in meats and eggs was developed, optimized, and validated in this study. The developed ICG strip can detect sulfamethazine residue of greater than 10 ng/g or (ng/mL) in real samples, and the results from the ICG strip for sulfamethazine-positive samples were the same as those for IC-ELISA. Thus, the results demonstrate that the assay for sulfamethazine possesses sufficient sensitivity (detection limit in standard solutions: 2 ng/mL, cutoff value in real samples: 10 ng/g or ng/mL) to classify positive samples, which are contaminated above acceptable levels established by several countries. Although IC-ELISA was found to have a higher sensitivity than the ICG strip, the ICG strip is better within the framework of on-site detection because the ICG strip is simple, accurate, and easy-to-perform and can produce results in 10 min without the need for multiple steps requiring washing and expensive equipment.
This study was supported by grant from Advanced Production Technology Development Program, Korean Ministry of Agriculture, Food and Rural Affairs and by grant of Korean Ministry of Food and Drug Safety.