赭曲霉毒素A.B的提取、分析与鉴定

EXTRACTION PURIFICATION ASSAY AND IDENTIFICATION OF OCHRATOXIN A AND B

用赭曲霉菌(也称棕曲霉菌)生产菌株,接种于无菌的混合饲料中培养。培养物用乙腈—石油醚—氯仿提取,用碳酸氢钠水溶液进行液液分配,水相酸化后再用氯仿萃取,经硅胶柱层析净化,用苯:冰醋酸(9:1)洗脱。紫外光下分别收集绿色萤光区带和兰色萤光区带,采用紫外分光光度计检测。选用波长333nm的赭曲霉毒素A(Ochratoxin A.简称OA)和波长318nm的赭曲霉毒素B(Ochratoxin B.简称OB)收集液,分别将收集液(OA和OB)浓缩,然后结晶。OA产品为无色针状晶体;OB为淡黄色颗粒状晶体。OA和OB的UV最大吸收峰分别为333nm和318nm。萤光最大激发波长分别为335nm和320nm,最大发射波长分别为465nm和456nm,通过UV、HPLC、NMR、IR谱图及TLC的R_f值证明产品是赭曲霉毒素A和B。

The Aspergillus ochraceus strain was inoculated on mixed-feed of extinct bacteria and incubated. The toxin was extracted from the moldy materials with acetonitrile-petroleum etherchloroform. The extract was liquid-liquid partitioned with NaHCO_3solution. The supernatant was acidified with HCl or H_2SO_4and then reextacted with fresh chloroform. The extract was poured into a silica gel column and eluate with C_6H_6-CH_3COOH(9: 1). and the green was collected under UV light. Using ultraviolet-spectrophotometer, OA and OB in the eluate was determined with 333nm and 318nm, respectively. The eluate of OA and OB was concentrated and crystallized from benzent, respectively. The product of OA was colorless crystalline and OB was light yellow grains. The maximal UV absorptance peaks of OA and OB were 333nm and 318nm, respectively. Their fluorescence excitation wavelengths were 335nm and 320nm, and emission wavelengths 465nm and 456nm, respectively. It is identified as Ochratoxin A and B by UV, HPLC, NMR, IR, spectra and R_fofTLC.