Quantitative Analysis of Ergosterol as a Biomarker of Mold-contaminated Foods Using the Enzyme Biosensor

효소 바이오센서를 이용한 식품의 곰팡이 오염 지표물질인 Ergosterol 정량분석

  • Kim, Mi-Kyeong (Department of Food Science and Nutrition, Kyungpook National University) ;
  • Kim, Jong-Won (Department of Food Science and Nutrition, Kyungpook National University) ;
  • Kim, Mee-Ra (Department of Food Science and Nutrition, Kyungpook National University)
  • 김미경 (경북대학교 식품영양학과) ;
  • 김종원 (경북대학교 식품영양학과) ;
  • 김미라 (경북대학교 식품영양학과)
  • Published : 2009.04.30

Abstract

Ergosterol is the significant component of the cell wall of fungi. Its presence is regarded as evidence of fungi contamination in grain and other foods. Many studies on ergosterol detection have been carried out using chemical methods, but those methods required complicated pre-treatments and long analysis times. In this study, an amperometric biosensor was developed for fast and precise ergosterol detection. The biosensor system used the electron transfer of hydrogen peroxide produced from the reaction of ergosterol with cholesterol oxidase. The biosensor system consisted of a peristaltic pump, a syringe loading sample injector, an enzyme reactor, a fabricated flow-through cell containing a working electrode, a reference electrode and a counter electrode, and a potentiostat/recorder. The working electrode was prepared by coating modified multi-wall carbon nanotube (MWNT) on glassy carbon electrode. The $MWNT-NH_2$ coated glassy carbon electrode linearly responded to hydrogen peroxide in the range of $1{\times}10^{-5}{\sim}8{\times}10^{-5}$ M with a detection limit of $10^{-7}$ M in the basic performance test. The currents produced from the ergosterol biosensor showed the linearity in a range from $1.0{\times}10^{-6}$ M to $1.0{\times}10^{-5}$ M ergosterol.

본 연구에서는 식품의 곰팡이 초기오염 검출에 사용할 수 있는 ergosterol 분석용 바이오센서를 개발하고자 $MWNT-NH_2$ 유리탄소전극을 제작하고, cholesterol oxidase의 고정화를 통해 효소반응기를 제작하여, 바이오센서의 전기 화학적 ergosterol 검출장치를 구성하였다. FT-IR을 통해 MWNT에 기능기가 잘 도입되었음을 확인하였고, cholesterol oxidase 효소 고정화 시 coupling efficiency는 99% 이었다. 제작한 $MWNT-NH_2$ 유리탄소전극의 $H_2O_2$ 용액에 대한 농도별 전류를 분석한 결과 $1{\times}10^{-5}{\sim}8{\times}10^{-5}$ M 농도에서 우수한 선형관계를 나타내었고 검출한계는 $10^{-7}$ M이었다. 본 연구에서 제작한 ergosterol 센서를 이용하여 ergosterol에 대한 반응을 측정한 결과 $1.0{\times}10^{-6}{\sim}1.0{\times}10^{-5}$ M의 농도에서 좋은 선형관계를 나타내어 고감도로 ergosterol을 정량할 수 있는 것으로 확인되어 식품의 곰팡이 오염을 확인하는데 유용하게 사용될 수 있을 것으로 보인다.

Keywords

References

  1. Abramson D, Gan Z, Clear RM, Gilbert J, Marquardt RR. 1998. Relationships among deoxynivalenol, ergosterol and Fusarium exoantigens in Canadian hard and soft wheat. Int J Food Microbiol 45(3):217-224 https://doi.org/10.1016/S0168-1605(98)00164-0
  2. Battilani P, Chiusa G, Cervi C, Trevisan M, Ghebbioni C. 1996. Fungal growth and ergosterol content tomato fruits infected by fungi. Ital J Food Sci 8(4):283-289
  3. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities protein utilizing the principle of protein dye binding. Anal Biochem 72:248-256 https://doi.org/10.1016/0003-2697(76)90527-3
  4. de Sio F, Laratta B, Giovane A, Quagliuolo L, Castaldo D, Servillo L. 2000. Analysis of free and esterified ergosterol in tomato products. J Agric Food Chem 48(3):780-784 https://doi.org/10.1021/jf990475d
  5. Duesberg GS, Muster K, Krstic V, Burghard M, Roth S. 1998. Choromatographic size separation of single-wall carbon nanotubes. Appl Phys A 67(1):117-119 https://doi.org/10.1007/s003390050747
  6. Gourama H, Bullerman LB. 1995a. Detection of molds in foods and feeds: potential rapid and selective methods. J Food Prot 58(12):1389-1394 https://doi.org/10.4315/0362-028X-58.12.1389
  7. Gourama H, Bullerman LB. 1995b. Relationship between aflatoxin production and mold growth as measured by ergosterol and plate count. Lebensm Wiss Technol 28(2):185-189 https://doi.org/10.1016/S0023-6438(95)91372-6
  8. Hwang JH, Chun HS, Lee KG. 2004. Aflatoxins in foods-Analytical methods and reduction on toxicity by physicochemical process-. J Korean Soc Appl Biol Chem 47(1):1-16
  9. Kadakal C, Nas S, Ekinci R. 2005. Ergosterol as a new quality parameter together with patulin in raw apple juice produced from decayed apples. Food Chem 90(1-2):95-100 https://doi.org/10.1016/j.foodchem.2004.03.030
  10. Kim KS. 2006. Preparation and characterization of novel functionalized multiwall carbon nanotubes-conducting polymer nanocomposite. Master thesis. Kyungpook National University of Korea. pp 36-38
  11. Kim MR. 2004. Understanding food safety. Shinjeong. Seoul, Korea. pp 88-99
  12. Kim MR, Kim MJ. 2003. Isocitrate analysis using a potentiometric biosensor with immobilized enzyme in a FIA system. Food Res Int 36(3):223-230 https://doi.org/10.1016/S0963-9969(02)00140-0
  13. Lamper C, Teren J, Bartok T, Komoroczy R, Mesterhazy A, Sagi F. 2000. Predicting DON contamination in Fusarium-infected wheat grains via determination of the ergosterol content. Cereal Res Commun 28(3):337-344
  14. Masoom M, Townsend A. 1985. Determination of cholesterol by flow injection analysis with immobilized cholesterol oxidase. Anal Chim Acta 174:293-297 https://doi.org/10.1016/S0003-2670(00)84388-3
  15. Morgan MRA, Smith CJ, Williams PA. 1992. Food safety and quality assurance. In Application of immunoassay systems. Elsevier Applied Science. pp 207-214
  16. Parra A, Casero E, Pariente F, Vazquez L, Lorenzo E. 2007. Cholesterol oxidase modified gold electrodes as bioanalytical devices. Sens Actuators B 124(1):30-37 https://doi.org/10.1016/j.snb.2006.11.051
  17. Ravelet C, Grosset C, Alary J. 2001. Quantitation of ergosterol in river sediment by liquid chromatography. J Chromatogr Sci 39(6):239-242 https://doi.org/10.1093/chromsci/39.6.239
  18. Ryu D, Bullerman LB. 1999. Effect of cycling temperatures on the production of deoxynivalenol and zearalenone by Fusarium graminearum NRRL 5883. J Food Prot 62(12):1451-1455 https://doi.org/10.4315/0362-028X-62.12.1451
  19. Salinas E, Rivero V, Torriero AJ, Benuzzi D. 2006. Multienzymatic-rotating biosensor for total cholesterol determination in a FIA system. Talanta 70(2):244-250 https://doi.org/10.1016/j.talanta.2006.02.043
  20. Santhosh P, Manesh KM, Gopalan A, Lee KP. 2006a. Fabrication of a new polyaniline grafted multi-wall carbon nanotube modified electrode and its application for electrodchemical detection of hydrogen peroxide. Anal Chim Acta 575(1):32-38 https://doi.org/10.1016/j.aca.2006.05.075
  21. Santhosh P, Gopalan A, Lee KP. 2006b. Gold nanoparticles dispersed polyaniline grafted multiwall carbon nanotubes as newer electrocatalysts: Preparation and performances for methanol oxidation. J Catal 238(1):177-185 https://doi.org/10.1016/j.jcat.2005.12.014
  22. Sethi RS. 1994. Transducer aspects of biosensors. Biosens Bioelectron 9:243-264 https://doi.org/10.1016/0956-5663(94)80127-4
  23. Shi QC, Peng TZ. 2005. A Novel cholesterol oxidase biosensor based on Pt-nanoparticle/carbon nanotube modified electrode. Chinese Chem Lett 16(8):1081-1084
  24. Shin MC, Kim HS. 1993. Development of flow injection analysis system for amperometric determination of cholesterol using immobilized enzyme columns. Korean J Biotechnol Bioeng 8(4):328-335
  25. Srzednicki G, Craske J, Nimmuntavin C, Mantais LG, Wattananon S. 2004. Determination of ergosterol in paddy rice using solid phase extraction. J Sci Food Agric 84(15):2041-2046 https://doi.org/10.1002/jsfa.1909
  26. Toh TH, Prior BA, van der Merve MJ. 2001. Quantification of plasma membrane ergosterol of Saccharomyces cerevisiae by direct-injection atmospheric pressure chemical ionization/tandem mass spectrometry. Anal Biochem 288(1):44-51 https://doi.org/10.1006/abio.2000.4877
  27. Woo JH. 2005. Synthesis and characterization of multi-walled carbon nanotubes and their dispersion with copolymer in water and ethanol. Master thesis. Kyungpook National University of Korea. pp 17-28
  28. Weber N, Weitkamp P, Mukherjee KD. 2001. Fatty acid steryl, stanyl, and steroid esters by esterification and transesterification in vacuo using Candida rugosa lipase as catalyst. J Agric Food Chem 49(1):67-71 https://doi.org/10.1021/jf000830w
  29. Yoon MH, Kim KJ, Kim JH, Cho KH, Kim SJ. 1998. A study on the Aflatoxin B1 contents in domestic and import foods. Korean J Sanitation 13(2):115-1201
  30. Zhao C, Wan L, Jiang L, Wang Q, Jiao K. 2008. Highly sensitive and selective cholesterol biosensor based on direct electron transfer of hemoglobin. Anal Biochem 383(1):25-30 https://doi.org/10.1016/j.ab.2008.08.022