Jae-Hwang Lee

Specific PCR assays to determine bovine, porcine, fish and plant origin of gelatin capsules of dietary supplements.

Gelatin, a purified protein derived mostly from pig skin and bovine tissue, is used widely in both food and pharmaceutical industries. Here, to determine the species of origin of capsule gelatin, we developed a sensitive and reliable test using the polymerase chain reaction (PCR) method, which included 1) species-specific or universal primer sets, designed to detect short 16S ribosomal RNA (rRNA) gene sequences from cow, pig, and fish (tilapia) as well as genes encoding the large subunit of plant ribulose-1,5-bisphosphate carboxylase oxygenase and 2) species-specific PCR coupled with whole-genome amplification. This method was used to verify manufacturing label claims of 28 gelatin capsule samples sold as dietary supplements. The results from 27 samples were consistent with gelatin-related information on the manufacturer label, while one sample that mentioned tilapia gelatin was found to contain only bovine DNA. This rapid method can therefore be used to verify the authenticity of gelatin capsules.

Application for Identification of Food Raw Materials by PCR using Universal Primer

ABSTRACT - In order to determine an authenticity of food ingredient, we used DNA barcode method by univer-sal primers. For identification of animal food ingredients, LCO1490/HCO2198 and VF2/FISH R2 designed for ampli-fying cytochrome c oxidase subunit1 (CO1) region and L14724/H15915 for cytochrome b (cyt b) region onmitochondrial DNA were used. Livestock (cow, pig, goat, sheep, a horse and deer) was amplified by LCO1490/HCO2198, VF2/FISH R2 and L14724/H15915 primers. Poultry (chicken, duck, turkey and ostrich) was amplified byLCO1490/HCO 2198 and VF2/FISH R2 primers. But, Fishes (walleye pollack, herring, codfish, blue codfish, trout,tuna and rockfish) were only amplified by VF2/FISH R2 primers. For plant food ingredients, 3 types of primers (trnH/psbA, rpoB 1F/4R and rbcL 1F/724R) have been used an intergenic spacer, a RNA polymerase beta subunit and aribulose bisphosphate carboxylase region on plastid, respectively. Garlic, onion, radish, green tea and spinach wereamplified by trnH/psbA, rpoB 1F/4R and rbcL 1F/724R. The PCR product sizes were same by rpoB 1F/4R and rbcL1F/724R but, the PCR product size using trnH/psbA primer was different with others for plants each. We establishedPCR condition and universal primer selection for 17 items raw materials for foods and determine base sequences aimto PCR products in this study. This study can apply to determine an authenticity of foods through making an compar-ison between databases and base sequences in gene bank. Therefore, DNA barcode method using universal primerscan be a useful for species identification techniques not only raw materials but also processed foods that are difficultto analyze by chemical analysis.Key words: Universal primer, PCR (Polymerase Chain Reaction), DNA barcode, raw material

Development of PCR Method for Rapid Detection of Allergic Materials in Foods

The method for detection foods containing allergenic materials by PCR was developed in this study. To detect allergenic raw material from processed food, species specific primer which up to 200bp for PCR prod- uct were designed or selected from advanced research. As target materials, 14 items were selected (12 target materials for allergen in Korea, 2 target materials for allergen in foreign countries). The amplicon size for eggs, milk, buck- wheat, peanuts, beans, wheat, mackerel, crab, shrimp, pork, peach, tomato, almond, and sesame were confirmed 281, 131, 138, 120, 118, 127, 211, 174, 231, 138, 174, 132, 103, and 220bp, respectively. And any non-specific bands were not detected among each others. Detection method for allergenic material developed in this study could be used to investigate inaccurate goods for allergen labeling or non-intentional contaminant during processed foods manufactur- ing. In addition, the system will be usefully to detection accurate allergenic raw materials of export for other countries.

Identification of Faulty Red Pepper Powder Containing Seasoned Red-pepper Sauce

In this study, the experimental method has been investigated using molecular biological way to identify raw materials from seasoned red-pepper sauce which is one of the most popular spices in Korea. 6 kinds of seasoned red-pepper sauces were chosen as a sample containing chilli pepper, garlic, onion as a major ingredient and species specific primers were used for the identification of the raw material of processed food. Selected samples were pre-treated to remove salt (samples were washed with distilled water 3~4 times for desalting), after that, to amplify the extracted genes, whole genome amplification (WGA) kit was performed. Afterwards, PCR products were con- firmed through the electrophoresis. As a result, 102, 180, 280 bp of specific PCR products were confirmed for each major ingredients such as chilli pepper, garlic, onion. From this study, the gene extraction method was validated for the identification of ingredients from the spices and it would be applied to distinction of low quality chilli pepper pow- der including seasoned red-pepper sauce illegally.

A Comparison of Gene Extraction Methods for the Identification of Raw Materials from Processed Meat Products

ABSTRACT - In this study, effective gene extraction methods were compared to identify raw materials of pro-cessed meat products through molecular biological methods. Species specific primers were used to identify ingredi-ents of processed foods and, as a sample, 13 kinds of processed meat products including beef, pork and chicken.According to the type of sample, 13 kinds of samples were classified into liquid type, source type and powder type.The samples were pre-treated (centrifugation) and (or) performed Whole Gene Amplification (WGA) kit for amplifi-cation of the extracted DNA. As a result, it was possible to identify the raw material of products through the centrifu-gation of sample 1 ml for liquid type of processed meat products. For source type of products after gene extraction, itwas required to perform WGA for the identification of ingredients. For powder type products did not required anyfurther pre-treatment and WGA. In this study, it was an opportunity to confirm the possibility of identification of rawmaterial from the gene extraction of processed meat products and this method could be used to examine the authen-ticity of raw material of products. Key words: Gene extraction method, PCR (Polymerase Chain Reaction), Processed meat product, Species-specific primer

Development of Detection Method for Niphon spinosus, Epinephelus bruneus, and Epinephelus septemfasciatus using 16S rRNA Gene

Food Standardization Division, Food & Drug AdministrationAbstract Niphon spinosus, Epinephelus bruneus, and Epinephelus septemfasciatus are involved in the Perciformes Orderand Serranidae Family. When E. bruneus and E. septemfasciatus are fully grown, the striped pattern on the body graduallydisappears. Therefore, morphological classification of adult fishes is quite difficult to identify the differences to N. spinosus.In this study, we investigate the method to differentiate those using PCR. To design the primers, 16S rRNA region of N.spinosus, E. bruneus, and E. septemfasciatus registered in the GeneBank (www.ncbi.nlm.nih.gov) have been used and forthe analysis, Bio Edit ver. 7.0.9.0 was used. As a result, it was design NS-003-F/NS-005-R (136 bp), EB-001-F/EB-002-R (181 bp), and ES-001-F/ES-001-R (123 bp) primers for the differentiation of each 3 different fishes. Therefore, thespecies-specific primer sets would be a useful tool for scientific and speedy differentiation against the illegal distributionfor consumer protection.Keywords: Niphon spinosus, Epinephelus bruneus, Epinephelus septemfasciatus, PCR (Polymerase Chain Reaction), species-specific primer, 16S rRNA

Comparison of Irradiated Food with Electron Beam and Gamma-ray by PSL and TL Methods

This study was conducted to determine the PSL and TL properties of foods irradiated with electron beam and gamma-ray. 5 kinds of food including cereal, pulse, fish powder, dried vegetable and tea were irradiated at 0 to 10 kGy by electron beam accelerator or gamma-ray irradiator. The PSL analysis showed negative results for most of the non-irradiated samples. Non-irradiated shrimp powder showed intermediate result. Irradiated samples gave negative or intermediate or positive value which presented the limitation of PSL technique. In TL analysis, there were TL glow curves at around with low intensity on non-irradiated samples. Maximum peak in the range of was appeared on irradiated samples. TL ratio obtained by re-irradiation with 1 kGy was less than 0.1 on non-irradiated samples and higher than 0.1 on irradiated samples. Therefore, in PSL measurement, electron-beam irradiated samples could obtain more clear results. TL analysis showed obvious difference between non-irradiated and irradiated samples. But the identification was impossible for the sample of rice and lemon tea. Because of it`s low contents of mineral.

Detection Method for Identification of Pueraria mirifica (Thai kudzu) in Processed Foods

ABSTRACT - In this study, ribulose bisphosphate carboxylase (rbcL), RNApolymeraseC (rpoC1), intergenicspacer (psbA-trnH), and second internal transcribed spacer (ITS2) as identification markers for discrimination of P.mirifica in foods were selected. To be primer design, we obtained 719 bp, 520 bp, 348 bp, and 507 bp amplicon usinguniversal primers from selected regions of P. mirifica. The regions of rbcL, rpoC1 , and psbA-trnH were not proper fordesign primers because of high homology about P. mirifica, P. lobata, and B. superba. But, we had designed 4 pairsof oligonucleotide primers from ITS2 gene. Predicted amplicon from P. mirifica were obtained 137 bp and 216 bpusing finally designed primers SFI12-miri-6F/SFI12-miri-7R and SFI12-miri-6F/SFI12-miri-8R, respectively. Thespecies-specific primers distinguished P. mirifica from related species were able to apply food materials and processedfoods. The developed PCR method would be applicable to food safety management for illegally distributed productsin markets and internet shopping malls.Key words: PCR (Polymerase Chain Reaction), Pueraria mirifica, species-specific primer

Development of Detection Method for Oilfish (Ruvettus pretiosus and Lepidocybirium flavobrunneum) as a Food Materials not Usable in Foods

Since 1 June 2012, it is prohibited to sell oilfish as a food material but there are still many illegal cases of selling oilfish as if it is tuna or grilled Patagonian toothfish. So it is absolutely crucial to construct the system to distinguish the real food material from oilfish. There are two sorts of oil fish called Ruvettus pretiosus and Lepi- docybirium flavobrunneum involved in Percifomes order and Gempylidae class. 16S DNA gene region in mitochon- dria was selected to design the specific primers. For design species-specific primer, the theoretical experiment were performed for the sequences of R. pretiosus, L. flavobrunneum, Thunnus thynnus, Thunnus albacores, Makaira mit- sukurii and Xiphias gladius, registered at the Gene bank from the National Centre for Biotechnology Information, using BioEdit 7.0.9.0. program. Through the analysis of the result from experiments, it was possible to design the 4 kinds of primers to distinguish R. pretiosus and L. flavobrunneum. As a comparison group, 3 kinds of tuna and 4 kinds of billfishes were selected and experimental verification was performed. As a result, for R. pretiosus and L. flavobrun- neum, R.P-16S-006-F/R.P-16S-008-R and L.F-16S-004-F/L.F-16S-006-R primers were selected eventually and PCR condition was established. In addition, 178bp and 238bp of PCR products were confirmed from the established con- dition and non-specific band was not amplified among similar species. Therefore, the species-specific primers devel- oped in this study would be very useful and used in various ways such as internet shopping mall and illegal distributions with fast and scientific results.

Detection Characteristics of Gamma-Irradiated Seeds by using PSL, TL, ESR and GC/MS

In this study, we investigated the applicability of the photostimulated luminescence (PSL), ther- moluminescence (TL), electron spin resonance (ESR) and gas chromatography/mass spectrometry (GC/MS) methods for 5 seeds which are not allowed to be irradiated in Korea. All 5 seeds including evening primrose seed, safflower seed, rape seed, sunflower seed and flax seed were analyzed. Samples were irradiated at 1~10 kGy using a 60 Co gamma-ray irradiator. In PSL study, the photon counts of all the unirradiated samples showed negative (lower than 700). The photon counts of irradiated (1, 5, 10 kGy) samples showed positive (higher than 5,000). In TL analysis, results showed that it is possible to apply TL method to all foods containing minerals. In ESR measurements, the ESR signal (single-line) intensity of irradiated foods was higher than non-irradiated foods. The hydrocarbons 1,7-hexadec- adiene (C 16:2 ) and 8-heptadecene (C 17:1 ) from oleic acid were detected only in the irradiated samples before and after the treatment at doses ≥ 1 kGy, but they were not detected in non-irradiated samples before and after treatment. These two hydrocarbons could be used as markers to identify irradiated safflower seed, rape seed, Sunflower seed and flax seed. And then, the hydrocarbons 1,7,10-hexadecatriene (C 16:3 ) and 6,9-heptadecadiene (C 17:2 ) from linoleic acid were detected in the evening primrose seed, safflower seed and sunflower seed. According to the results, PSL, TL and GC/ MS methods were successfully applied to detect the irradiated foods. It is concluded that PSL, TL and GC/MS meth- ods are suitable for detection of irradiated samples and a combined method is recommendable for enhancing the reli- ability of detection results.