Development of certified reference materials for the determination of 5-methyltetrahydrofolate in spinach and kimchi cabbage

형석원,이준희 한국분석과학회 2021 학술대회논문집 Vol.2021 No.11

Certified reference materials (CRMs; KRISS CRM 108-05-010, 108-05-011) for the determination of 5-methyltetrahydrofolate (5-Me-THF) in spinach and kimchi cabbage were developed. The vegetables were freeze-dried, pulverized, sieved, V-mixed, and bottled. Determination of 5-Me-THF was conducted by isotope dilution ultra-performance liquid chromatography/mass spectrometry (ID –UPLC/MS) using randomly selected 10 bottles. A trienzyme method was adopted to release 5-Me-THF monoglutamate from the matrix bound polyglutamated one by treatments with protease for 1 h at 37 ℃ and the mixture of α-amylase and rat sera (conjugase) for 16 h at 37 ℃. The CRM candidates were monitored on 5-Me-THF at 25 ℃ for 7 days, -20 ℃ for 1 month, and -70 ℃ for 5 and 13 months to observe short-term and long-term stabilities. Both CRM candidates were stable under most monitoring conditions. The certified values of the CRMs were (16.93 ± 0.73) mg/kg and (11.66 ± 0.25) mg/kg for spinach and kimchi cabbage, respectively. The CRMs developed in this study are expected to be used in fields that require accurate measurement of related nutrients.

Quantification of Folic Acid in Human Serum Using Isotope Dilution Ultra-High-Pressure Liquid Chromatography/Mass Spectrometry

형석원,이준희,서혜영,김병주 대한화학회 2018 Bulletin of the Korean Chemical Society Vol.39 No.1

Analytical method for an accurate determination of folic acid in human serum has been established using isotope dilution ultra-high-pressure liquid chromatography/mass spectrometry (ID-UPLC/MS). Sample extract was prepared from 0.5 mL of human serum spiked with 13C5-folic acid solution and then LC–MS/MS experiment was performed on multiple reaction monitoring mode for 20 min. The method was tested for repeatability using homogeneous human serum and it showed good repeatability with relative standard deviation of 1.8%. Further evaluation was carried out through NIST SRM 1955 samples (level I, II, and III) and the measurement results were in good agreement with the certified values with good reproducibility over two periods. It was also revealed that the obtained relative expanded uncertainty was 12% which is much smaller than that of previously reported methods, indicating that this method is very reliable and accurate for determining the content of folic acid in human serum.

Bacillus sp. MS202에 의한 Dinitroaniline계 제초제인 Pendimethalin의 부분환원

이영근,장화형,장유신,형석원,정혜영 한국환경농학회 2004 한국환경농학회지 Vol.23 No.4

토양과 지하수에서 pendimethalin의 지속성은 환경에 해로운 영향을 미친다. 경남 마산에서 분리한 pendimethalin분해 균주는 API CHB50 kit 시험, FAME 분석, 그리고 165 rDNA 염기서열분석 결과로 Bacillus sp. MS202로 잠정적으로 동정하였다. TLC, GC, 그리고 GC-MS 분석에 의해 Bacillus sp. MS202가 pendimethalin의 -NO₂를 -NH₂로 환원시킨다는 것을 알 수 있었다. 이는 일반적으로 알려진 호기성 미생물에 의한 pendimethalin 분해가 탈알킬화가 우선한다는 보고와 상반되는 새로운 결과이다. The persistence of pendimethalin in soil and ground water has an injurious effect on ecosystem. Pendimethalin-degrading bacterium was isolated from Masan, Gyeongnam province and temporarily identified as Bacillus sp. MS202 by the analysis of API CHB50, kit, FAME, and 16S rDNA sequence. From the analysis of pendimethalin metabolite using TLC, GC, and GC-MS, we found that the degradation of pendimethalin by Bacillus sp. MS202 did not result in the dealkylated form, but the formation of the reduced compound, 6-amino-2-nitro-N(1-ethylpropyl)-3,4-xylidine or 2-amino-6-nitro-N(1-ethylpropyl)-3,4-xylidine.

Bacillus lentimorbus WJ5의 감마선유도 돌연변이체들에서 공통으로 발현되는 방사선 관련 유전자의 microarray 분석

이영근,장화형,장유신,허재호,형석원,정혜영 한국환경생물학회 2004 환경생물 : 환경생물학회지 Vol.22 No.3

본 연구에서는 감마선으로 유도된 돌연변이체들에서 공통으로 발현되는 방사선 관련 유전자들의 발현을 연구하기 위하여, B. lentimorbus WJ5의 방사선 유도 돌연변이체에서 발현되는 유전자를 DNA microarray로 동시에 탐색하였다. DNA microarray는 B. lentimorbus WJ5 genome을 무작위로 절단하여 2,000단편으로 구성하였으며, 감마선 (^(60)Co)으로 유도된 7돌연변이체의 발현을 정량적으로 관찰하였다. 클러스터 분석결과 발현된 408 유전자 중 27개가 감마선 유도 돌연변이체 모두에서 유의하게 발현이 증가되었다. 특히, 복구(mutL, mutM)에너지 대사 (acsA, sdhB, p하, yhjB, citB), protease(npr), 산화자극에 대한 환원 (HMM)관련 유전자들이 동시에 증가되었다. 이는 감마선 유도 돌연변이체들에서 자발적인 직/간접 복구 관련 유전자의 발현 증가는 방사선 노출 직후 보이는 stress response와는 다른 현상임을 나타내는 것으로 생각된다. To study the radiation related gene expression in mutants of Bacillus lentimorbus WJ5 induced by gamm radiation, the simultaneous gene expression was analyzed by DNA microarray. We constructed DNA chips including two thousand randomly digested genome spots of B. lentimorbus WJ5 and compared its quantitative aspect with seven mutants induced by gamma radiation (^(60)Co). From the cluster analysis of gene expression pattern, totally 408 genes were expressed and 27 genes were significantly upregulated by the gamma radiation in all mutants. Especially, genes involved in repair (mutL, mutM), energy metabolism (acsA, sdhB, pgk, yhjB, citB), protease (npr), and reduction response to oxidative stress (HMM) were simultaneously upregulated. It seems that the induction of the direct and/or indirect repair related genes in mutants induced by gamma radiation could be remarkably different from the adaptive responses against acute exposure to radiation.

뽕나무 첨가 배지에서 배양한 버섯균사체 배양물의 자동산화 억제 효과

김석종(Seck-Jong Kim),임동길(Dong-Kil Lim),형석원(Seok-Won Hyung),김미숙(Mee-Sook Kim),김정옥(Jeong-Ok Kim),김무남(Mu-Nam Kim),이강권(Kang-Kweon Lee),하영래(Yeong-Lae Ha) 한국식품영양과학회 2004 한국식품영양과학회지 Vol.33 No.2

BMM에 여러 가지 버섯균을 7일 동안 액체배양하고 균사를 제거한 배양물에 대한 항산화성에 관한 연구를 수행하였다. 시료(1 ㎎)를 275 μmol linoleic acid가 함유된 배양액에 첨가하고 shaking incubator(200 rpm, 40℃)에 16일 동안 배양하면서 생성된 POV와 MA의 함량의 조사하였다. 이 결과를 BM에서 배양한 버섯균사체배양물과 BMM 자체의 POV 및 MA 생성 결과와 비교하였다. AB-BMM과 HE-BMM의 항산화능이 다른 버섯균사체배양물이나 BMM보다 우수하였다. 따라서 이 연구결과는 뽕나무는 버섯균사체액체배양물의 항산화성을 증가시켰고, AB-BMM 및 HE-BMM은 항산화물로 활용할 수 있음을 의미한다. Effect of mulberry tree powders on the antioxidant activity of submerged-liquid culture of mushrooms was investigated. Agaricus blazei (AB), Hericicum erinacium (HE), Pleurotus ostreatus (PO), Phellinus linteus (PL) and Paecilomyces japonicus (PJ) were cultured in a shaking incubator (200 rpm, 25℃) for 7 days in culture media: 1) basal medium (BM) and 2) BM+1% mulberry tree powders (BMM). Hot water extracts from the submerged-liquid cultures of mushrooms and BMM were freeze-dried for the measurement of antioxidant activity, of which reaction mixture (25 mL: 10 mL of 0.2 M sodium phosphate buffer, pH 8.0; 4.5 mL distilled water; and 10.5 mL ethanol) contained 275 μmol linoleic acid and one mg test sample. The reaction mixture was incubated in a shaking incubator (200 rpm, 40℃) for 16 days. Peroxide value (POV) was measured for a period of over 16 days, and malonaldehyde (MA) was determined only for samples from the day 16 of incubation. Mycelial weight of mushroom strains cultured in BMM was greater than BM. The antioxidant activities of AB-cultured in BMM (AB-BMM) and HE-cultured in BMM (HE-BMM) were superior to those of other mushroom strains-cultured in BMM or BM and of BMM. These results suggest that mulberry tree powders enhance the antioxidant activity of submerged-liquid culture of mushroom strains. The AB-BMM and HE-BMM were the most active cultures.

뽕나무가루 첨가 배지에서 배양한 버섯균사체 배양물의 자유라디칼 유도 산화 억제

김석종(Seck-Jong Kim),임동길(Dong-Kil Lim),박철우(Cherl-Woo Park),세르보로다메(Rhoda Mae Cerbo),형석원(Seok-Won Hyung),이강권(Kang-Kweon Lee),김정옥(Jeong-Ok Kim),하영래(Yeong-Lae Ha) 한국식품영양과학회 2004 한국식품영양과학회지 Vol.33 No.2

뽕나무가루 첨가 배지에서 배양한 느타리버섯균사체 배양물의 조추출물이 자유라다칼로 유도한 linloeic acid의 산화를 대조구에 비해 75.9% 감소시켰고, mouse liver microsome 산화에서도 NADPH/Fe^(++) system에서 64.3% 감소시켰다. 이와 같은 효과는 조추출물로부터 용매분획한 분획물의 단독 효과보다 우수하였다. 상황 및 동충하초버섯균사체 배양물의 조추출물도 느타리버섯균사체 배양물의 조추출물과 효과가 유사하였지만 다소 낮았다. Antioxidant activity of extracts from the submerged-liquid culture of mushrooms was measured using two systems: linoleic acid and mouse liver microsomes induced by various free radical sources. Mushrooms of Pleurotus ostreatus (Neutari), Phellinus linteus (Sanghwang), Paecilomyces japonicus (Dongchunghacho), Hericicum erinacium (Norugungdengyee) and Agaricus blazei (Shinryeong) in 1% mulberry tree powder-supplemented medium were incubated in a shaking incubator (200 rpm, 25℃) for 3 days. Hot water extracts of mycelial cultures were freeze-dried, followed by fractioning with hexane, chloroform, ethylacetate, and butanol in the order. Antioxidant activity of each sample was examined in free radical-induced linoleic acid oxidation in phosphate-buffered saline (PBS) solution by measuring the amount of malonaldehyde (MA), and mouse liver microsomal systems by measuring the amount of thiobarbituric acid reactive substances (TBARS). In linoleic acid oxidation system, hot water extracts from the cultures of Pleurotus ostreatus, Phellinus linteus, and Paecilomyces japonicus exhibited stronger antioxidant activity than aqueous or butanol fraction and the combined fraction of hexane, chloroform and ethylacetate, but the hot water extract from Pleurotus ostreatus culture was the strongest activity. The antioxidant activity of the hot water extract from Pleurotus ostreatus culture was stronger than any other fractions in mouse microsomal system. These results suggest that hot water extract of Pleurotus ostreatus culture, and the cultures of Phellinus linteus and Paecilomyces japonicus could be useful for functional materials to reduce the oxidation of lipids in food systems induced by free radicals.