Won-Bo Shim

Detection of ochratoxin A (OTA) in coffee using chemiluminescence resonance energy transfer (CRET) aptasensor

We report a chemiluminescence resonance energy transfer (CRET) aptasensor for the detection of ochratoxin A (OTA) in roasted coffee beans. The aptamer sequences used in this study are 5-DNAzyme-Linker-OTA aptamer-3-dabcyl. Dabcyl at the end of the OTA aptamer region plays as a quencher in CRET aptasensor. When hemin and OTA are added, the dabcyl-labeled OTA aptamer approaches to the G-quadruplex-hemin complex by formation of the G-quadruplex-OTA complex. The G-quadruplex-hemin complexes possess horseradish peroxidase (HRP)-like activity, and therefore, the HRP-mimicking DNAzyme (HRPzyme) catalyzes peroxidation in the presence of luminol and H2O2. Resonance energy transfer between luminol (donor) and dabcyl (acceptor) enables quenching of chemiluminescence signals. The signal decreases with increasing the concentration of OTA within the range of 0.1-100ngmL(-1) (limit of detection 0.22ngmL(-1)), and the level of recovery of the respective 1ngmL(-1) and 10ngmL(-1) spiked coffee samples was 71.5% and 93.3%. These results demonstrated the potential of the proposed method for OTA analysis in diverse foods.

Screening of a specific monoclonal antibody against and detection ofListeria monocytogenes whole cells using a surface plasmon resonance biosensor

In this study, a specific monoclonal antibody againstListeria monocytogenes was screened using an SPR biosensor Monoclonal antibodies were bound to protein L, after which theL. monocytogenes cells were subjected to an affinity assay. Protein L was immobilized on a carboxymethyl dextran (CM-Dex) surface via an amine coupling method and utilized repeatedly by regeneration. The monoclonal antibody, ‘A18’, was selected and employed for the high-sensitivity detection ofL. monocytogenes. Under optimized conditions, 103 cells/ml or 50 cells were detected by the SPR biosensor.

Determination of ochratoxin A in grains by immuno-ultrafiltration and HPLC-fluorescence detection after postcolumn derivatisation in an electrochemical cell

The paper presents a new sample clean-up method based on immuno-ultrafiltration for the analysis of ochratoxin A in cereals. In contrast to immunoaffinity chromatography, in immuno-ultrafiltration, the antibodies are used in non-immobilised form. Ochratoxin A was extracted with ACN/water (60/40, v/v), and the extract was loaded onto the ultrafiltration device. After a washing step with phosphate-buffered saline, containing 0.05% Tween 20, ochratoxin A was eluted with MeOH/acetic acid (99/1, v/v). The detection of ochratoxin A was carried out with high-performance liquid chromatography and a fluorescence detector coupled to an electrochemical cell (Coring cell). The electrochemical cell was used to eliminate matrix interferences by oxidising matrix compounds. The method was validated by repeatedly analysing spiked barley and rye samples as well as a certified wheat reference material. Recoveries and standard deviations (1xa0SD) were found to be 71u2009±u20099%, 77u2009±u200912% and 77u2009±u20098% in wheat, barley and rye, respectively. The limit of detection (S/Nu2009=u20093) and limit of quantitation (S/Nu2009=u200910) were determined to be 0.4xa0μgxa0kg-1 and 1xa0μgxa0kg-1. The analysis of the certified reference material resulted in ochratoxin A concentrations which were in the range assigned by the producer. Additionally, the effect of the electrochemical cell on other widely used clean-up techniques, namely the immunoaffinity clean-up and multifunctional columns (Mycosep #229), was evaluated. In all clean-up methods, an improvement of the chromatogram quality was registered.

Occurrence of ochratoxin A in Korean red paprika and factors to be considered in prevention strategy

A large amount—260,000 tons—of red paprika is consumed annually in Korea, where the people prefer hot and pungent to sweet foods. Concern has recently grown among consumers over contamination of paprika powder by mycotoxins; contamination can occur at any stage from pre-harvest to drying, storage, grinding, and eventually transport to the retail market. This study had dual aims: to investigate the current level of contamination of hot peppers by ochratoxin A and to identify the critical control points in the food chain. We measured the ochratoxin A (OTA) content of 200 samples from various sources including supermarkets, an online shopping mall, small stakeholder mills, Hazard Analysis and Critical Control Points (HACCP)-implemented factories, and an import company. Mycotoxin was determined using immunoaffinity column cleanup/HPLC quantification as well as strong anion exchange cleanup/stable isotope dilution assay. Monitoring revealed that approximately 30% of red paprika samples were OTA-positive, indicating a need to establish a maximum level for regulation. We selected two model factories that had adopted HACCP in different ways, and compared data in order to develop guidelines for alleviation of mitigation of the mycotoxin contamination.

Microbiological Hazard Analysis of Ginseng Farms at the Cultivation Stage to Develop a Good Agricultural Practices (GAP) Model

This study validated microbiological hazards of ginseng farms at the cultivation stage and sug- gested recommendations to develop a good agricultural practices (GAP) model. A total of 96 samples were collected from cultivation environments (soil, irrigation water, and atmosphere), plants (ginseng and its leaf), personnel hygiene (glove, cloth, and hand) of 3 ginseng farms (A, B, and C) and were tested to analyze sanitary indicator bacteria (aero- bic plate count, coliforms and Escherichia coli), major foodborne pathogens (E. coli O157:H7, Listeria monocytoge- nes, Salmonella spp., Staphylococcus aureus, and Bacillus cereus), and fungi. Total bacteria, coliform, and fungi in the 3 ginseng farms were detected at the level of 1.3~6.0, 0.1~5.0, and 0.4~4.9 v/g (or mL, hand, and 100 cm 2 ), respec- tively. Only irrigation water collected from one ginseng farm was confirmed to be E. coli positive. In case of patho- genic bacteria, B. cereus was detected at levels of 0.1~5.0 log CFU/g (or mL, hand, and 100 cm 2 ) in all samples, but other pathogen bacterias were not detected in any samples from all farms. Although E. coli were detected in irrigation water, the level of microbial for the three farms was lower than the regulation limit. According to the results, the gin- sengs produced from the 3 farms were comparatively safe with respect to microbiological hazard. However, cross-con- tamination of bacteria from environments and workers to ginseng has been considered as potential risks. Therefore, to minimize microbial contamination in ginseng, GAP model should be applied for ensuring the safety of ginsengs.

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.

Sol–gel immunoaffinity chromatography for the clean up of ochratoxin A contaminated grains

This paper describes the application of sol-gel immunoaffinity columns for clean up of ochratoxin A contaminated cereal crops. Monoclonal antibodies selective for OTA have been entrapped into the pores of a sol-gel matrix in order to prepare immunoaffinity columns. Different parameters such as amount of entrapped antibodies and loading conditions were optimized to obtain highest possible recoveries of OTA. The method has been found to be a suitable tool in sample preparation prior to HPLC-FLD determination and as selective as conventional commercially available immunoaffinity columns. In the clean up of different cereals mean recoveries of 82±5%, 90±6% and 91±3%, were obtained for wheat, barley and rye, respectively, with sol-gel columns containing 1mg of anti-OTA antibodies. The detection limit (signal-to-noise ratio, 3) was 0.5 μg/kg and the limit of quantification (signal-to-noise ratio, 10) determined to be 1 μg/kg. Sol-gel columns can be reused 7 times without significant loss of recovery. After 10 applications the recovery decreased to approx. 50%.

Investigation of Microbiological Hazard from Korean Leeks and Cultivation Area to Establish the GAP Model

ABSTRACT - This study is to investigate microbiological hazards which can be used as fundamental data to ade-quately control leeks hazards and develop leeks GAP model for those who want to get GAP system. The microbiolog-ical investigations on cultivation environments (soil and water), crops (leeks), personal hygiene (workers’ hands,clothes and gloves) and working tools (boxes) have been conducted for one year, so the period was classified undernon-cultivation, cultivation, and post harvest. Total bacteria was detected from soil (4.0~6.7 log CFU/g), leeks(4.6~5.1 log CFU/g), hands (ND~3.3 log CFU/hand) and gloves (ND~5.4 log CFU/cm 2 ) while nothing was detectedfrom the other samples. The coliform contamination of leeks (4.8~5.0 log CFU/g) was more high than that of soil(3.9~4.2 log CFU/g). In case of foodborne pathogens, only B. cereus was detected at the level of 0.5~4.6 log CFU/g(or hand, 100 cm 2 ). Fungi was observed at the level of 2.1~3.8 log CFU/g (or hand, 100 cm 2 ) excepting water andsome working tools. These results demonstrate that the contamination of leeks is comparatively higher than that of soilsample. The reason may be the cross-contamination by biological hazards presenting on soil. Therefore, it is necessaryto properly control soil and fertilizer for safety against biological hazards.Key words : good agricultural practices (GAP), leek, microbial contamination, microbial hazard

Quantitative analysis of lard in animal fat mixture using visible Raman spectroscopy

Food adulteration is a serious issue that requires verification and strict management due to healthcare, morality, and social value problems. In the context of fat, food manufacturers blend lard with vegetable oils or animal fats for convenience and gaining economic benefits. Thus, we herein report the classification of 4 animal fats, e.g., beef tallow, pork lard, chicken fat, duck oil, using Raman spectroscopy combined with simple calculation of intensity ratios of Raman signal at vibrational modes corresponding to unsaturated fatty acids and total fatty acids. Various calculated values of the species were compared to find a feature that is able to classify each fats using Raman peak ratio. As a result, we suggested Oil gauge (OG) as a standard feature for classification of the fats in Raman analysis field. Furthermore, a quantification of the lard in other fat was accomplished with good linear correlation using the OG values.

Profiles of Toxin genes and Antibiotic Susceptibility of Staphylococcus aureus Isolated from Perilla Leaf Cultivation Area

Thirty one of Staphylococcus aureus isolated from perilla leaf cultivation areas in Miryang were investigated on the characteristics, such as enterotoxin genes and antibiotic susceptibility. Five toxin genes (sea, seb, sec, sed, and see) were examined by PCR method. Disc diffusion method was used to examine the antibiotic suscep- tibility of S. aureus by using 18 types of antibiotic discs with different concentrations. Among enterotoxin-encoding genes, sea and sed genes were co-detected from 4 isolates (12.9%), sed gene was founded in 9 isolates (29.0%), and see gene was founded in 1 isolate (3.2%). However seb and sec and tsst were not detected in any isolates. As a result of antibiotic susceptibility test, 7 isolates (22.6%) were resistant to 12 antibiotics (penicillin, ampicillin, oxacillin, amoxicillin-clavulanic acid, cefazolin, cephalothin, imipenem, gentamicin, tetracycline, ofloxacin, norfloxacin, and erythromycin). 2 isolates (6.5%) were resistant to 5 antibiotics (penicillin, ampicillin, amoxicillin-clavulanic acid, gentamycin, and telithromycin). MRSA (Methicilline Resistant Staphylococcus aureus) was founded in packing vinyl, hands, and perilla leaves.