다양한 식품에서의 곰팡이독소 분석 개선 연구

Abstract

곰팡이독소는 농작물의 수확전후 저장 환경에 따라 곰팡이에 의해 생성될 수 있는 자연독소이다. 특히, 아플라톡신은 국제암연구소에서 1급발암물질로 지정하고 있고 데옥시니발레놀은 위장장애 등 인체에 위해한 영향을 끼쳐 세계 많은 국가들이 기준을 설정하여 관리하고 있으나 국가별로 식품의 종류와 그 기준범위가 매우 상이하다. 본 연구는 다양한 식품에서 아플라톡신과 데옥시니발레놀의 법적 규제기준을 충족하면서 식품 특성을 고려한 최적의 분석법을 개발하고자 수행되었다. 영유아식품 및 식용유지 중 아플라톡신 4종과 곡류 중 트리코테센류 type B를 대상으로 고성능 액체크로마토그래피를 이용한 정량 분석법을 각각 확립하였다. 필수적인 검증항목(정량한계, 검량선의 직선성, 분석의 재현성, 매트릭스 이펙트, 회수율 시험)을 검토하였다. 영유아용식품에 대한 아플라톡신 시험법은 식품공전의 방법이 적합하였으나, 정량한계를 낮추기위해 기기분석 조건을 변경하여 시험법을 검증하였다. 아플라톡신(B1, B2, G1, G2)에 대한 회수율은 89.5～92.3%로 양호한 결과를 얻었으며, 검출한계는 아플라톡신 B1 0.019, B2 0.015, G1 0.053 및 G2 0.012 ug/kg 이었다. 식용유지는 MSPD(matrix solid phase dispersion)법으로 아플라톡신을 추출해 내고 면역친화성 컬럼을 사용해 정제하여 형광검출기가 장착된 고성능액체크로마토그래피(HPLC/FLD)를 이용해 분석한 결과 직선성은 0.999 이상을 나타냈고 회수율은 85.9～93.0%의 양호한 결과를 얻었으며 상대표준편차는 5.7% 이하였다. 곡류 중 데옥시니발레놀, 3-아세틸데옥시니발레놀, 15-아세틸데옥시니발레놀의 분석은 84% ACN으로 추출한 후 Mycosep column으로 정제하여 HPLC/UV로 측정하였으며 이때의 이동상용매는 gradient 조건으로 동시분석 하였다. 시험법 검증결과 데옥시니발레놀, 3-아세틸데옥시니발레놀 및 15-아세틸데옥시니발레놀의 검출한계는 각각 0.2, 0.1 및 0.1 mg/kg, 직선성은 0.999 이상을 나타냈으며, 회수율은 85.0～107.3%, 상대표준편차는 4.6% 이하였다. 또한 확립된 시험방법으로 FAPAS QC sample을 분석하여 측정불확도를 추정한 결과 0.306 ± 0.006 mg/kg으로 산출되었다. 본 연구에서 개발된 모든 시험법의 신뢰성을 확보하고자 AOAC 분석법 가이드라인에 준하여 검증한 결과 모든 항목이 AOAC 가이드라인에 만족하여 시험법의 정확도와 재현성이 확보되었음을 확인하였다. 또한, 국제정도관리 프로그램(FAPAS)에 참여하여 만족할 만한 결과를 얻어 시험법의 정확도를 제고하였다. 확립한 시험법으로 국내 유통 중인 식용유지류, 영유아식품 및 곡류에서 아플라톡신, 트리코테센류 type B의 오염실태를 확인한 결과 안전한 수준임을 확인하였다. 본 연구에서 확립한 시험법은 곰팡이독소의 다양한 국제기준을 충족할 수 있어 공인시험법으로 활용가능 할 것으로 판단된다. |Mycotoxins are known as natural food toxins produced by certain fungi grown on various cropsduring pre-harvest and post-harvest conditions. Aflatoxins have been classified as carcino-genic to humans, and deoxynivalenol has a harmful effect on the human health such as vomiting and gastrointestinal disorders. Although mycotoxin, which can be dangerous to human health, is legally regulated worldwide, regulatory standards vary from country to country. The purpose of this study is to develop a pretreatment method suitable for characteristics because the composition of food is complex and diverse, and to prepare an analytical method that meets legal regulatory standards. Using high-performance liquid chromatography, analysis methods of aflatoxin of infant food and food oil and tricotesene type B of grain were established, respectively. We were determined the occurrence of aflatoxins(B1, B2, G1 and G2) in edible oils and infant-children foods in Korea. Therefore, this study developed condition of extract for aflatoxins(B1, B2, G1 and G2) in edible oils using a high performance liquid chromatography with florescence detector(HPLC/FLD). Aflatoxins were extracted from edible oils sample by means of MSPD(matrix solid phased dispersion), utilizing C18 as dispersing material and purified using immunoaffinity column. There regression line coefficients of determination were above 0.999. The recoveries for aflatoxins ranged from 85.9 to 93.0%, and relative standard deviations was below 5.7%. The newly developed method of aflatoxins effectively enhanced recoveries by using MSPD compare with Korea food code analytical method. Reviewing the current method, the recoveries of aflatoxins(B1, B2, G1 and G2)were 89.5～92.3% in order and their limits of quantitation (LOQ) were 0.019, 0.015, 0.053 and 0.012 ug/kg in order. A survery of aflatoxins was conducted on 110 edible oils and 106 infant-children foods. Samples were collected from market in Korea. Analytes were extracted from cereals by homogenization with acetonitrile:D.W.(84:16) and crude extracts was cleaned-up Myco-sep column. The analytical method using gradient solvent system was capable of separating a mixture of the DON, 3-ADON and 15-ADON. The overall method showed high precision and good linearity of calibration curves(above r2=0.999). The mean of recoveries for tested compounds ranged from 85.0 to 107.3%, and relative standard deviations was below 4.6% and their limits of quantitation (LOQ) were 0.2, 0.1 and 0.1 mg/kg in order. For quality control, Fapas QC material was digested same protocol as analyzed cereal sample. The evaluation parameters were selectivity, matrix-matched standards, linearity of calibration, accuracy, precision, the limit of quantitation (LOQ), and matrix effects. Most target compounds were found to be in acceptable quantities under the requirements of the validation guidelines. The optimized method was successfully applied to the analysis of mycotoxins in real samples. According to the result of the monitoring, aflatoxin B1 was detected five samples representing 4.7% and its level in infant-children food ranged from 0.06～0.14 ug/kg. No afltoxins contamination were detected in edible oils. A survey of deoxynivalenol and its derivatives was conducted on 458 cereals. Samples were collected from market in Korea. According to the result of the monitoring, deoxynivalenol and 15-ADON was detected 38 samples(8.3%) 2 sample(0.4%) rspectively. The analysis method established in this study can meet various regulation limit levels of mycotoxins and can be used as a certified analysis method