Kyeong-Yeol Kim

Development and validation of a gold nanoparticle immunochromatographic assay (ICG) for the detection of zearalenone.

A monoclonal antibody (mAb)-based gold nanoparticle immunochromatographic assay (ICG) for zearalenone detection was developed, optimized, and validated. The detection limits of ICG optimized with appropriate amounts of zearalenone-bovine serum albumin and gold nanoparticle-mAb to zearalenone were 2.5 ng/mL and 30 μg/kg for the standard solution and spike sample, respectively, and a weak cross-reaction for α-zearalenol and β-zearalenol was observed. The assay required only 15 min to obtain results and one step to perform the assay. In validation, the results obtained from spiked corn (10, 20, 30, 50, and 100 μg/kg) and naturally contaminated corn samples by the ICG were in good agreement with those obtained by direct competitive enzyme-linked immunosorbent assay (DC-ELISA) and high-performance liquid chromatography (HPLC). Therefore, the results obtained in this study could be used as basic research for the development of zearalenone-ICG, and the ICG developed could be a useful on-site screening tool for the rapid detection of zearalenone in corn without special instrumentation.

Rapid detection of enterotoxigenic Clostridium perfringens in meat samples using immunomagnetic separation polymerase chain reaction (IMS-PCR).

Rapid detection of enterotoxigenic Clostridium perfringens in meat samples was accomplished with an immunomagnetic separation polymerase chain reaction (IMS-PCR). First, a monoclonal antibody (mAb) specific to C. perfringens was generated. The antibody showed strong binding to C. perfringens and no binding to non- Clostridia bacteria, except a weak cross-reaction to Staphylococcus aureus based on the enzyme-linked immunosorbent assay (ELISA). Then, magnetic beads were coated with the mAb, and the IMS-PCR system was developed. With the optimized conditions, the IMS-PCR assay was capable of detecting as few as 10 colony forming units (CFU)/g of C. perfringens cells in the meat sample within 10 h. Of the 116 collected samples (26 chicken samples, 20 beef samples, 30 pork samples, 20 fish samples, and 20 processed meat samples) examined with IMS-PCR, 36 (31%) were C. perfringens -positive samples and 2 (1.7%) were enterotoxigenic C. perfringens -positive samples. The IMS-PCR results gave a good agreement with the results obtained by conventional culture methods. In comparison to conventional culture methods, the IMS-PCR is a rapid and specific method and has potential use as a screening tool for enterotoxigenic C. perfringens in food samples.

Screening of gamma irradiated spices in Korea by using a microbiological method (DEFT/APC)

Irradiation can be used to control the microbiological spoilage and pathogen microorganisms of foods. Microbiological changes associated with food irradiation include the reduction or elimination of spoilage and pathogenic microorganisms, insect disinfestation and parasite disinfection. The effects on the microbial populations of irradiated foods have been used to develop detection methods for irradiated foods. The method used in this study was direct epifluorescent filter technique/aerobic plate count (DEFT/APC), which is based on the difference between DEFT counts and APC counts. The samples were imported spices and domestic spices produced in Korea. These samples were irradiated at 1.0, 3.0, 5.0, 7.0, and 10 kGy to reduce the spoilage organisms. Irradiation doses of 3.0 kGy or over eliminated viable microorganisms effectively, and the logDEFT/APC ratio gradually increased with dose increment in all the samples. It could be suggested that if the logDEFT/APC ratio is 2.5 or over for peppers in Korea, the samples can be suspected as irradiated at a dose level of at least 3.0 kGy. The DEFT/APC method could be used for screening for the irradiation treatment of spices produced in Korea.

Occurrence of aflatoxins in herbal medicine distributed in South Korea.

The objective of this study was to investigate the occurrence of aflatoxins in herbal medicines distributed in South Korea. A total of 700 herbal medicine samples (10 samples each for 70 types of herbal medicine) were analyzed by an enzyme-linked immunosorbent assay (ELISA) for aflatoxin B(1) (AFB(1)), and levels of total aflatoxins were quantified and confirmed by liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). The levels of recovery of the methods were 84.30 to 102.68% (ELISA for AFB(1)) and 72.17 to 90.92% (LC-MS/MS for total aflatoxins). Fifty-eight (8.29%) of 700 samples were AFB(1) positive by ELISA, and 17 (2.43%) of them were finally confirmed as positive for total aflatoxins by LC-MS/MS. Total aflatoxin levels in the herbal medicines were from 4.51 to 108.42 μg/kg. Among the 17 samples, the AFB(1) content of 6 samples (11.95 to 73.27 μg/kg) and the total aflatoxin content of 10 (12.12 to 108.42 μg/kg) samples exceeded the legal limits set by the Korea Food and Drug Administration for AFB(1) (10 μg/kg) and by the European Commission for total aflatoxins (10 μg/kg), respectively. These results demonstrate the risk to consumers of herbal medicine contamination by aflatoxins and encourage further studies to investigate the transfer rate of mycotoxins to decoction, which is the final product for consumption.

Development of fluorescence polarization immunoassay for the rapid detection of 6-chloronicotinic acid: main metabolite of neonicotinoid insecticides.

A fluorescence polarization immunoassay (FPIA) for the quantitative determination of 6-chloronicotinic acid (6-CNA) using polyclonal antibody was developed. The 6-CNA-protein (bovine serum albumin and soybean trypsin inhibitor) conjugates and fluorescein-labeled 6-CNA derivative (tracer) were prepared and used as the immunogens and tracer, respectively. The synthesized tracer was purified by thin layer chromatography (TLC) and showed a good binding to antiserum (73/5) which was obtained from the immunized rabbit (No. 73) with 6-CNA-BSA conjugate. The detection limit (10% inhibition) of FPIA was 4 microg/mL, and IC(50) value was 32 microg/mL. The FPIA showed a cross-reaction for 5-amino-2-chloropyridine (60%), but no cross-reaction for other pesticides was observed. Recoveries for spiked apple, urine, soil, and water samples (5, 50, and 500 ppm) averaging between 78.6 +/- 8.8 and 114 +/- 18% were reasonable and in good agreement with the amounts spiked. Although the developed FPIA possesses low sensitivity, this assay is more simple and quick than other analytical methods, such as high performance liquid chromatography and gas chromatography. Thus, the developed FPIA method could be a useful tool for express screening 6-CNA in agricultural, environmental, and biological samples.

Fumonisins B1 and B2 in Agricultural Products Consumed in South Korea : An Exposure Assessment

To survey fumonisins B1 (FB1) and B2 (FB2) in agricultural products consumed in South Korea and provide an exposure assessment, ground samples were extracted (80% MeOH), filtered (0.2 microm), and cleaned up. After evaporation, dry residues were reconstituted in 50% MeOH, and a 50-micro1 aliquot of this sample was mixed with 200 micro1 of o-phthaldialdehyde for derivatization. The derivatives were analyzed with a high-performance liquid chromatography system equipped with a fluorescence detector. For validation of the detection procedure, linearity, accuracy, precision, detection limit, and quantification limit were determined. The validated detection method was then used to survey fumonisins in white rice, brown rice, barley, barley tea, beer, wheat flour, millet, dried corn, corn flour, corn tea, canned corn, popcorn, and breakfast cereal. Retention times for FB1 and FB2 standards were 7 and 18 min, respectively. Linearity (R2 = 0.99995 to 0.99998), accuracy (81.47 to 108.83%), precision (2.35 to 5.77), detection limit (25 ng/g or ng/ml), and quantification limit (37 ng/g or ng/ml) indicated that this procedure is capable of quantifying fumonisins in agricultural products. Only FB1-positive samples (5.12%, three dried corn samples and five corn flour samples) were found at 90.89 to 439.67 ng/g. According the survey results, an estimated daily intake of FB1 and FB2 in Korea was 0.087 ng/kg of body weight per day. These results indicate that continuous monitoring of these mycotoxins is necessary to establish appropriate risk assessment, and the maximum tolerable daily intake of fumonisins in Korea is lower than the 2 microg/kg set by the Joint Food and Agriculture Organization-World Health Organization Expert Committee.

Microbiological Hazard Analysis for Strawberry Farms at the Harvest Stage to Establish Good Agricultural Practices (GAP) Model Based on Principle of HACCP

This study assessed hazards at the harvest stage of strawberry farms which may cause risk to humans. A total of 216 samples were collected from 6 strawberry farms (soil culture farms: A, B, C; nutriculture farms: D, E, F) located in Western Gyeongnam. The collected samples were subjected for sanitary indicator bacteria (aerobic plate count, coliforms and Escherichia coli), major foodborne pathogens (E. coli O157:H7, Listeria monocytogenes, Salmonella spp., Staphylococcus aureus and Bacillus cereus), and fungi. The levels of APC and coliform in the soil culture farms were 1.0-6.9 and 0.4-4.6 log CFU/g (leaf, mL, hand or 100 ), respectively. The samples obtained from the nutriculture farms were contaminated with the levels of 0.8-4.9, and 0.2-2.6 log CFU/g (leaf, mL, hand or 100 ) of APC and coliform. However, E. coli was not detected in any samples. In major foodborne pathogens, S. aureus was detected at the level of 3.3 log CFU/hand in workers` hand samples and B. cereus was detected at the levels of 0.4-4.1 log CFU/g (hand or 100 ) in soil, plants and workers` hygiene. L. monocytogenes, E. coli O157:H7 and Salmonella spp. were not detected. Fungi were detected at the levels of 1.0-5.2 and 0.2-4.4 log CFU/g (leaf, mL, hand or 100 ) in soil culture and nutriculture farms, respectively.

Development of a Simultaneous Lateral Flow Strip Test for the Rapid and Simple Detection of Deoxynivalenol and Zearalenone

UNLABELLED The objective of this study was to develop a 1-step simultaneous lateral flow strip test for the rapid and simple detection of deoxynivalenol (DON) and zearalenone (ZEA) in grains. Two monoclonal antibodies (MAbs) against DON and ZEA were respectively conjugated with gold nanoparticles and used to develop a lateral flow strip test for a single toxin and multiple toxins. First, individual lateral flow strips for a single toxin were optimized, and their conditions were used to develop a simultaneous lateral flow strip for multiple toxins. Limits of detection of both lateral flow strip tests for DON and ZEA were the same (DON: 50 ng/mL, ZEA: 1 ng/mL). Both methods showed cross-reactivity for α-zearalenol and β-zearalenol, but no cross-reaction to other mycotoxins. The results can be completed obtained within 15 min. The cut-off values of the simultaneous lateral flow strip for the spiked rice and corn were 500 and 10 ng/g for DON and ZEA, respectively. The results demonstrated that the developed simultaneous lateral flow strip test offers a rapid, easy-to-use, and portable analytical system and can be used as a convenient qualitative tool for the on-site detection of DON and ZEA in food and agricultural commodities. PRACTICAL APPLICATION Simultaneous lateral strip test is useful for a rapid detection of DON and ZEA at a time in food and grain samples.

Microbiological Hazard Analysis on Perilla Leaf Farms at the Harvesting Stage for the Application of the Good Agricultural Practices(GAP)

The purpose of this study was to analyze microbiological hazards for plants, cultivation environments and personal hygiene of perilla leaf farms at the harvesting stage. Samples were collected from three perilla leaf farms(A, B, C) located in Gyeongnam, Korea and tested for sanitary indications, fungi and pathogenic bacteria(Escherichia coli O157:H7, Listeria monocytogens, Salmonella spp., Staphylococcus aureus and Bacillus cereus). As a result, total bacteria and coliform in perilla leaf were detected at the levels of 4.4~5.2 and 3.4~4.3 log CFU/g, respectively, but E. coli was not detected in all samples. Among the pathogenic bacteria, B. cereus(perilla leaf: 2.0~2.4 log CFU/g, stem: 1.4~2.1 log CFU/g, water: 0.7 log CFU/ml, soil: 4.2~5.0 log CFU/g, hands: 3.0 log CFU/ hand, gloves: 2.1~2.4 log CFU/100 , glothes: 1.5~2.8 log CFU/100 ) and S. aureus(3.4 log CFU/hand) were detected in all samples and worker`s hand from farm A, respectively. However, other pathogenic bacteria were not detected. This study demonstrates that perilla leaf at the harvesting stage was significantly contaminated with microbial hazards.

Determination of Total Aflatoxins in Foods by Parallelism of ELISA and LC/MS/MS

High performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC/MS) have been widely used to quantify aflatoxins in food, but these methods are expensive, time-consuming, unsuitable for analysis of the routine screening of large sample numbers and require derivatization and high level techniques to perform. The objective of this study is to detect aflatoxins in a large number of foods by a high efficient analytical system of combined enzyme linked immunosorbent assay (ELISA) for screening and LC/MS/MS for confirmation. The samples spiked individually with aflatoxin (0.5 and 1.0 ng/g) and total aflatoxins (10 ng/g) were analyzed by ELISA and LC/MS/ MS, and the recoveries for ELISA and LC/MS/MS were 71.8~119.2% and 70.8~135.3%, respectively. A total of 378 samples (grains, nuts, soybean and fermented soybean foods, pepper and fermented pepper foods) were purchased from the six major cities in Korea and analyzed by ELISA-LC/MS/MS system. Twenty two (5.8%; peanut: 11, pistachio: 2, walnut: 6, almond: 1, pepper powder: 1, pepper paste: 1) out of 378 samples were screened as aflatoxin B1 positive by ELISA, but, 4 (1.1%; peanut: 2, pistachio:1, pepper powder: 1) out of the 22 samples screened were confirmed as aflatoxins positive at levels of 1.02~52.79 ng/g by LC/MS/MS. ELISA-LC/MS/MS system provides a more rapid, accurate and cost-effective method for the detection of aflatoxins in large number of samples.