• Title/Summary/Keyword: LC/MS analysis

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Bacillus subtilis MJP1이 생산하는 항세균 물질의 분리.정제 및 특성규명 (Purification and Characterization of Antibacterial Compound Produced by Bacillus subtilis MJP1)

  • 임은정;양은주;장해춘
    • 한국미생물·생명공학회지
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    • 제38권1호
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    • pp.84-92
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    • 2010
  • B. subtilis MJP1이 생산하는 항균물질을 분리 정제하기 위하여 SPE, IEC, GFC를 통한 정제를 수행하였다. 정제된 항세균 물질은 tricine SDS-PAGE에서 하나의 band임을 확인 한 후 N-말단 아미노산 서열분석을 하였으나 N-말단이 blocking 되어 그 서열을 분석할 수 없였다. 이에 그 내부서열을 알아보기 위한 LC를 이용한 ESI-MS/MS를 시행하였으나 내부서열도 확인할 수 없었고, UPLC를 이용한 ESI-MS/MS를 시행한 결과 항세균 활성 물질은 분자량이 매우 유사한 2개의 peptide(3356.54 Da, 3400.5244 Da)가 존재함을 확인하였다. 정제된 항세균 물질의 항균 spectrum을 조사한 결과, L. monocytogenes에 가장 강한 항균활성을 나타내며 이외에 B. subtilis, S. aureus subsp. aureus, E. faecalis 등의 Gram 양성균에서 항균활성을 나타내였다. 정제된 B. subtilis 항세균 물질은 pH 3.0부터 pH 9.0 범위에서는 안정하였으며 $4^{\circ}C$, $30^{\circ}C$, $50^{\circ}C$에서 24시간, $100^{\circ}C$에서 5분 동안 매우 안정하였고, $70^{\circ}C$에서 24 시간, $100^{\circ}C$에서 30분 동안 처리한 구간부터 역가가 감소하여 $121^{\circ}C$에서 15분 동안 처리한 구간에서는 역가가 완전히 소실되었다. 효소 안정성 실험에서 정제된 항세균 물질은 일부 단백분해효소에 의해 분해되어 역가를 상실하였으며, lipase나 $\alpha$-amylase에서는 안정함을 나타냈다. 이로부터 정제된 항세균 물질이 넓은 pH 범위에서 안정하고, 비교적 높은 온도에서도 활성을 유지한다는 점을 관찰할 수 있었으며, 단백분해효소에 의해 분해되므로 bacteriocin임을 확인하였다. 본 연구를 통하여 B. subtilis MJP1으로부터 분리 정제된 bacteriocin은 class IIa군의 특성과 유사하며, B. subtilis MJP1은 본 분리 bacteriocin 이외에도 다른 항세균 물질과 항진균 물질을 생산하는 균주임을 알 수 있었다.

HPLC-UVD를 이용한 농산물 중 살균제 pyriofenone 분석법 확립 (Development of an Analytical Method for the Determination of Pyriofenone residue in Agricultural Products using HPLC-UVD)

  • 박혜진;김희정;도정아;권지은;윤지영;이지영;장문익;이규식
    • 농약과학회지
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    • 제18권2호
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    • pp.79-87
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    • 2014
  • 피리오페논은 아릴페닐케톤계 농약으로, 식물 병원성 곰팡이의 부착 및 침투를 억제하여 방제 효과를 나타내는 살균제이다. 미국에서는 2012년에 피리오페논을 사용등록 하였으며, 우리나라에서는 2013년에 참외와 고추에 대해 잔류허용기준 설정을 추진하고 있다. 이에 따라, 본 연구에서는 식품 중 피리오페논의 안전관리를 위한 시험법을 개발하고자 하였다. 피리오페논은 분자량(365.8)이 크고 증기압이 $1.9{\times}10^{-6}Pa$ ($25^{\circ}C$)로 휘발성이 낮은 편으로, 질소, 산소원자의 비공유전자쌍과 이중결합이 UVD에 높은 감도를 보여 HPLC-UVD를 분석기기로 선택하였다. 추출용매는 물질의 용해도와 추출효율을 고려하여 acetonitrile로 선정하였으며, 피리오페논이 옥탄올/물 분배계수(logPow, $25^{\circ}C$)가 3.2인 비극성 물질이므로 증류수, 포화식염수와 dichloromethane을 사용하여 극성 불순물을 제거하도록 액-액 분배하였다. 분배 추출액을 silica 카트리지를 이용하여 정제하는데, hexane으로 세척한 후 hexane/acetone(90/10, v/v)로 용출하면 간섭 물질로부터 피리오페논을 효과적으로 정제할 수 있었다. 본 연구에서 확립한 시험법을 통한 대표작물에 대한 회수율은 72.8~99.5%이었고 분석오차는 10% 미만으로 코덱스 가이드라인((CAC/GL 40)에 적합하였다. LC-MS로 재확인 시험을 하여 시험법의 신뢰성과 선택성을 확보하였다. 본 연구에서 개발한 시험법은 식품 중 피리오페논의 안전관리를 위한 시험법으로 활용될 것이다.

Liquid chromatography-mass spectrometry를 이용한 가열 조리된 어패류에서의 heterocyclic amines 함량 분석 (Determination of Heterocyclic Amines in Roasted Fish and Shellfish by Liquid Chromatography-Electrospray Ionization/Mass Spectrometry)

  • 이재환;백유미;이광근;신한승
    • 한국식품과학회지
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    • 제41권3호
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    • pp.326-333
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    • 2009
  • 국내에서 주로 섭취되는 어패류(고등어, 삼치, 조기, 대합, 바지락, 꼬막, 백합)를 가열조리 하였을 때 형성되는 HCAs를 분리, 동정하여 그 함량을 모니터링 하였다. 추출 및 정제는 고체상 추출 방법을 사용하였고 정성, 정량을 위한 분석장비는 LC/MS를 사용하였다. 모니터링 결과 생선류에서는 고등어(2.2-1,1117.7 ng/g), 삼치(9.1-443.6 ng/g), 조기(17.5-179.5 ng/g)순으로 HCAs가 많이 검출되었으며 특히 고등어 muscle에서 많은 양(1,1117.7 ng/g, Harman)이 검출되었다. 조개류 중에서는 대합(12.2-196.4 ng/g), 꼬막(2.2-76.2 ng/g), 바지락(1.7-25.8 ng/g), 백합(1.5-2.7 ng/g)순으로 많이 검출되었으며 대합의 Norharman(196.4 ng/g)이 가장 많이 검출되었다. 15종의 HCAs 중 ${beta}$-carbolines에 속하는 Norharman과 Harman이주로 검출되었으며 그 밖에 PhIP, Trp-P-1. Trp-P-2 등의 발암가능성이 있는 물질들이 검출되었다. 조리방법 측면에서 보면 간장을 이용한 조림방법보다는 직접적인 가열을 통한 구이방법이 많은 양의 HCAs를 형성시켰으며 구이 중에서도 muscle만 있는 부위에서의 검출함량보다는 muscle과 skin이 함께 있는 부위에서 검출함량이 더 많았다. 가열 조리한 어패류 분석방법에 대한 유효성을 확인해 본 결과 정량한계는 0.8-23.9 ng/mL, 검출한계는 0.2-7.2 ng/mL이었으며 회수율은 15.7-74.7%이었다.

Simultaneous Determination of Synthetic Phosphodiesterase-5 Inhibitors in Dietary Supplements by Liquid Chromatography-High Resolution/Mass Spectrometry

  • Kim, So-Hee;Kim, Ho-Jun;Son, Jung-Hyun;Jeon, Byoung-Wook;Jeong, Eun-Sook;Cha, Eun-Ju;Lee, Jae-Ick
    • Mass Spectrometry Letters
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    • 제3권2호
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    • pp.50-53
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    • 2012
  • After success of sildenafil for the treatment of erectile dysfunction, a large number of its analogues have been approved from FDA. Recently, the illegal dietary supplements which include sildenafil, vardenafil, tadalafil, or analogues of these drugs as ingredient have been widely distributed. Therefore, the determination of the residue of synthetic phosphodiesterase- 5 (PDE-5) inhibitors in dietary supplements is highly required due to indiscriminate and unintentional overdose caused nausea, chest pains, fainting and irregular heartbeat. In this paper, we report a rapid and sensitive analytical method for the simultaneous determination of nine phosphodiesterase-5 inhibitors by liquid chromatography-high resolution mass spectrometry. The present method was found to be accurate and reproducible with 40 ${\mu}g$/g of the limit of quantification for the nine PDE-5 inhibitors. The developed method can be successfully applied to the analysis of the seven illegal dietary supplements.

미생물 violacein 색소의 다섬교직포에서의 염색성 (Dyeing Properties of Microbial Violacein on Mutifiber Fabrics)

  • 최종명;김용숙
    • 한국의류산업학회지
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    • 제11권5호
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    • pp.818-826
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    • 2009
  • Dyeability of microbial violacein produced from Chromobacterum violaceum CV107 on to multifiber fabrics has been studied. The bluish-purple colourants were produced by cultivation of Chromobacterum violaceum using LB liquid medium for 2 days. The colourant was extracted with 80% acetone and identified as violacein by LC/MS analysis. The violacein could be dyed on not only natural fibers such as Cotton, Silk and Wool but also synthetic fibers such as Diacetate, Triacetate, Creslan 61 and Nylon 66. Maximum K/S values were shown at 540-580 nm according to different fiber with color appearance of purple or blue. An optimum pH and temperature under dyeing condition were 10 and $70^{\circ}C$, respectively. Any mordants were not improved colour density and quality on various fabrics. From this studies, pigments produced microbe have a high potentials for natural dyeing on fabrics. Finally, development of new colourants from microbe has made a possible change for new dyeing field in respects of eco-friend and repeatability of natural dyeing for apparels.

Isolation of Hyaluronidase Inhibitory Component from the Roots of Astraglus membranaceus Bunge (Astragali Radix)

  • Lee, Yun-Mi;Choi, Soo-Im;Lee, Jae-Won;Jung, Sun-Mi;Park, Sang-Min;Heo, Tae-Ryeon
    • Food Science and Biotechnology
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    • 제14권2호
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    • pp.263-267
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    • 2005
  • In order to isolate hyaluronidase (HAase) inhibitor from Astragali radix (AR), dried roots were extracted with ethanol, prior to sequential fractionations with n-hexane, chloroform, ethyl acetate, n-butanol, and aqueous fractions. The n-butanol soluble fraction was found to exhibit the most pronounced inhibitory effect (68%) on HAase, and the active components were separated using various chromatographic methods, including column chromatography and preparative HPLC. The active component was isolated from the n-butanol soluble fraction of AR and was structurally identified as calycosin-7-O-${\beta}$-D-glucopyranoside by LC-MS, IR, $^1H$ NMR, and $^{13}C$ NMR analysis. The $IC_{50}$ of calycosin-7-O-${\beta}$-D-glucopyranoside's HAase activity was found to be 3.7 mg/mL.

Comparison of linear and non-linear equation for the calibration of roxithromycin analysis using liquid chromatography/mass spectrometry

  • Lim, Jong-Hwan;Yun, Hyo-In
    • 대한수의학회지
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    • 제50권1호
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    • pp.11-17
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    • 2010
  • Linear and non-linear regressions were used to derive the calibration function for the measurement of roxithromycin plasma concentration. Their results were compared with weighted least squares regression by usual weight factors. In this paper the performance of a non-linear calibration equation with the capacity to account empirically for the curvature, y = ax$^{b}$ + c (b $\neq$ 1) is compared with the commonly used linear equation, y = ax + b, as well as the quadratic equation, y = ax$^{2}$+ bx + c. In the calibration curve (range of 0.01 to 10 ${\mu}g/mL$) of roxithromycin, both heteroscedasticity and nonlinearity were present therefore linear least squares regression methods could result in large errors in the determination of roxithromycin concentration. By the non-linear and weighted least squares regression, the accuracy of the analytical method was improved at the lower end of the calibration curve. This study suggests that the non-linear calibration equation should be considered when a curve is required to be fitted to low dose calibration data which exhibit slight curvature.

Residual determination of Ceftiofur in Raw Bovine Milk by Liquid Chromatography-Electrospray Mass Spectrometry

  • Lim, Jong-hwan;Park, Byung-kwon;Kim, Myoung-seok;Jang, Beom-Su;Kim, Doo;Yun, Hyo-in
    • 대한수의학회지
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    • 제44권3호
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    • pp.367-371
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    • 2004
  • This report describes the determination of ceftiofur residues in milk from treatment of lactating dairy cattle by intramuscular injection of three consecutive daily doses of about 1 mg /kg BW, the recommended label dosing. The separation of ceftiofur was achieved on $C_1_8$ reverse phase column. The mobile phase consisted of 0.1% trifluoracetic acid in water (A) and 0.05% acetic acid in acetonitrile (B) and grediently flowed at the flow rate of 0.4 mL/min. As a result of analysis of blank raw bovine milk samples, matrix interference was not shown. Limit of detection and limit of quantitaion was 0.5 ng/mL and 1 ng/mL, respectively. The values of precision and recovery satisfied the guideline of National Veterinary Research and Quarantine Service (NVRQS, Korea). The mean residual concentration of ceftiofur in milk did not exceed 3.71 ng/mL when ceftiofur was administered intramuscularly to lactating dairy cattle for 3 consecutive days at 1 mg/kg of BW per day. It is much lower than the proposed MRL (100 ng/mL) of ceftiofur in milk.

Features and Functions of Purple Pigment Compound in Halophytic Plant Suaeda japonica : Antioxidant/Anticancer Activities and Osmolyte Function in Halotolerance

  • Chung, Sang Ho
    • 한국자원식물학회지
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    • 제31권4호
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    • pp.342-354
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    • 2018
  • Suaeda japonica is a halophytic plant that lives in mudflat at intertidal zone of western and southern coastal areas of Korea. The seawater-living plants showed a purple color during their whole life. In contrast, freshwater-living plants displayed a green color in leaves. When seawater-living plants were transferred to potting soil, the purple color was gradually changed to green in the leaves. The extracted purple pigment compound exhibited typical characteristics of betacyanin that were represented by water solubility, pH- and temperature-dependent color changes, sensitivity to light, UV-Vis spectra, and gel electrophoretic migration pattern. The LC-MS analysis of the extracted pigment compound showed the presence of two major protonated molecular ions ($[M+H]^+$) at m/z 651.1 and m/z 827.1. Antioxidant activity of the pigment compound was determined using stable free radical DPPH assay. It was found to have an antioxidant activity that is linearly increased in proportion to the reaction time for up to 30 min, and the activity was comparable to that of control BHA at 9.0 mg/ml. The anticancer activity against several tumor cell lines was also examined following the MTT assay. The significant growth inhibitory effect was observed on two tumor cell lines, SW-156 (human kidney carcinoma) and HEC-1B (human endometrial adenocarcinoma). Probably, the pigment compound may function as an osmolyte to uphold halotolerant physiological processes in saline environment.

산업체에서의 식품 알레르기 관리 (Food Allergen Management in Industry)

  • 성동은;곽호석
    • 산업식품공학
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    • 제22권4호
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    • pp.283-294
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    • 2018
  • Food allergy is a chronic disease that is increasing all over the world, and it can even lead to a loss of life. To prevent any incidents resulting from food allergies, most countries keep strengthening their food allergen labeling requirements domestically and internationally, with a constant monitoring system against undeclared allergens and recall of offending products. In order to avoid economic losses to industry and damages to international relations from undeclared allergens, it is necessary to confirm each country's regulatory policy on food allergen labeling prior to exportation. Another required action is to try for a reduction of the cross-contamination risk of the allergens during manufacturing and storage, which should be verified by using an accurate and reliable analysis of food allergens. This paper is intended to provide an introduction to the regulation of food allergen labeling by country, allergen management methods to avoid cross-contamination, and allergen detection methods using ELISA, PCR, and LC/MS. Changes of allergenicity during thermal or nonthermal processing also will be investigated in our review. This review will be helpful for the food industry to better understand patients suffering from food allergies and to manage food allergens in food manufacturing.