饲料中真菌毒素分析方法及在体内代谢转化的研究
发布时间：2018-08-23 08:23
【摘要】：真菌毒素是真菌在适宜的环境条件下产生的次级有毒代谢产物,具有致癌、致畸及致突变等毒性作用。真菌毒素的摄入还可造成毒素及其代谢产物在畜禽体内残留,进而通过肉、奶等动物源性产品进入人食物链中,给消费者造成安全风险。目前,已经发现的具有毒性的真菌毒素有30余种,其中广受关注的真菌毒素主要为有黄曲霉毒素(AFB1)、赭曲霉毒素A(OTA)和脱氧雪腐镰刀菌烯醇(DON)等。许多国家针对不同产品已制定真菌毒素的限量标准。近年来,奶牛饲料和鲜奶中真菌毒素的污染受到广泛关注。本论文首先对饲料中常见的30种真菌毒素进行检测技术研究,进而针对目前污染较为严重但是容易被忽略的隐蔽型真菌毒素,建立灵敏、快捷、可靠的检测技术。采用建立的检测技术,对不同的饲料样本进行筛查,探明饲料中真菌毒素的暴露水平和暴露种类。然后,对典型真菌毒素AFB1和OTA进入奶牛体内后的代谢动力学情况进行研究,揭示典型真菌毒素的吸收、分布、代谢和排泄规律,对今后保障鲜奶安全、制定饲料中限量标准具有重要意义。本文主要研究内容和结论如下:(1)采用QuEChERS(Quick,Easy,Cheap,Effective,Rugged,Safe)前处理技术结合超高效液相色谱-串联质谱检测不同饲料样品(预混料、浓缩料和配合料)中30种真菌毒素的含量。饲料样品经过5 mL水和5 m L 1%甲酸乙腈溶液提取后取上清液氮吹至近干后,残渣经1 mL 5 mmol/L醋酸铵水溶液/乙腈溶液(80/20,v/v)复溶上机测定,采用基质加标结合同位素内标法进行定量分析。本方法在不同的线性范围内均线性良好,相关系数R2?0.99,检出限(limit of detection,LOD)在0.7~20?g/L之间,定量限(limit of quantitation,LOQ)在2~50?g/L之间。低、中、高浓度添加水平下30种真菌毒素平均回收率为72.0%~118.4%。该方法具有简单、快速、实用性强等优点,能适用于预混料、浓缩料和配合料中30种真菌毒素的定量分析。将建立的分析方法应用到38个饲料样品中39种真菌毒素的测定,其中,31份饲料样品被DON污染,污染率最高,含量为59.3~576.8μg/kg;有21份饲料样品被15-ADON污染,含量为62.3~984.3μg/kg;有7份饲料样品被D3G污染,含量为27.8~127.1μg/kg;另外,在不同的饲料中还发现了伏马毒素B1(FB1)、伏马毒素B2(FB2)、玉米赤霉烯酮(ZEN)、?-玉米赤霉烯酮(?-ZOL)、展青霉素(PAT)和AFB1等真菌毒素。(2)通过优化提取溶剂,HLB小柱的上样液,淋洗和洗脱溶剂等,首次建立了一种用于DON,3-乙酰基-脱氧雪腐镰刀菌烯醇(3-ADON)、15-乙酰基-脱氧雪腐镰刀菌烯醇(15-ADON)、镰刀菌烯酮(FUS-X)和3-葡萄苷化脱氧雪腐镰刀菌烯醇(D3G)富集、净化的前处理方法。净化样品液通过超高效液相色谱串联质谱分析。建立的分析方法通过方法学包括线性(R2≥0.99)、灵敏度(LOQ范围为0.08~4.85μg/L)、回收率(79.3%~108.1%)验证后表明,该分析方法快速、灵敏、准确。进而将建立的分析方法应用于31个饲料样品中的4种B型单端孢霉烯族类毒素和D3G的测定。其中,26个样品被不同真菌毒素污染,含量范围为2.1~864.5?g/kg,而D3G也在17个样本检测到,其浓度范围在2.1~34.8μg/kg。(3)建立了鲜奶、尿液、血浆、心、肝、脾、肺和肾中AFB1、AFM1、OTA和赭曲霉毒素?(OT?)四种真菌毒素的分析方法,样品经1400?L丙酮提取后取上清液氮气吹干,200?L 5 mmol/L醋酸铵水溶液/乙腈溶液(80/20,v/v)复溶上机测定。方法学结果表明四种真菌毒素在定量限、低、中和高添加水平下,回收率范围为为82.8%~114.1%。在0.1~200μg/L范围内线性关系良好(R2?0.990),LOD值在0.03~0.2μg/L之间,LOQ值在0.1~0.5μg/L之间。血浆和组织器官内4种真菌毒素的短期(8 h)、长期(20 d)和冷冻解冻三循环稳定性范围为80.9%~113.6%。证明建立的分析方法具有简单、快速、实用性强等优点,能适用于奶、尿、血、心、肝、脾、肺和肾中四种真菌毒素的定量分析。(4)将天然培养的富含AFB1或OTA的基体标准物质与饲料样本混合后,获得一定含量真菌毒素的奶牛饲料(AFB1的含量为1000?g/kg,OTA的含量为750?g/kg),饲喂奶牛,在10、35、45、60、120、180、240、360、540、720、1440、2160、2880 min处分别取血,在6 h屠宰奶牛取心、肝、脾、肺、肾、脑器官。采用(3)建立的分析方法对不同组织器官中真菌毒素及其典型代谢产物进行分析后。结果表明:AFB1在体内吸收速度较快,10 min血液中就可检测到AFB1,在35 min处达到峰值,48 h检测不到。AFB1向乳中的转化率为1.1%~1.6%,鲜奶中的AFM1清除期为2天。在心、脾、肺和肾检测到AFB1毒素(含量分别为1.6、4.1、3.3和5.6?g/kg),在脾和肾中检测到AFM1毒素(含量分别为0.7和0.8?g/kg)。OTA在奶牛中基本难以吸收,鲜奶、血液及各组织器官中均未检测到OTA和OT?。但是在尿液中检测到少量OTA和大量代谢物OT?毒素,最大值分别为1.8μg/L和324.6μg/L,表明OTA在体内不吸收,在体内代谢转化成OT?后排出体外。
[Abstract]:Mycotoxins are secondary toxic metabolites produced by fungi under suitable environmental conditions, which have carcinogenic, teratogenic and mutagenic effects. Intake of mycotoxins can also cause toxins and their metabolites to reside in livestock and poultry, and then enter the human food chain through animal-derived products such as meat and milk, resulting in a safe wind for consumers. At present, more than 30 kinds of toxic mycotoxins have been found, among which aflatoxin (AFB1), ochratoxin A (OTA) and deoxynivalenol (DON) are the main mycotoxins of concern. Many countries have formulated limits for mycotoxins in different products. In recent years, fungi in dairy feed and fresh milk have been widely used. Toxin contamination has attracted wide attention. In this paper, 30 kinds of common mycotoxins in feed were detected firstly, and then a sensitive, rapid and reliable detection technology was established for the concealed mycotoxins which are seriously contaminated but easily ignored. Different feed samples were screened by the established detection technology. Then, the metabolic kinetics of typical mycotoxins AFB1 and OTA in dairy cows was studied to reveal the absorption, distribution, metabolism and excretion of typical mycotoxins. The main research contents and conclusions are as follows: (1) QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) pretreatment combined with ultra-high performance liquid chromatography-tandem mass spectrometry were used to determine the content of 30 mycotoxins in different feed samples (premixes, concentrates and batches). Feed samples were extracted by 5 mL water and 5 mL 1% acetonitrile formate solution. After the supernatant was blown to nearly dry, the residue was determined by 1 mL 5 mmol/L ammonium acetate solution/acetonitrile solution (80/20, v/v) resolving on machine. The method was quantitatively analyzed by matrix addition and isotope internal standard method. The average recoveries of 30 mycotoxins were 72.0%~118.4% at low, medium and high concentration levels. The method is simple, rapid and practical. It can be applied to the quantitative analysis of 30 mycotoxins in premix, concentrate and batch. Thirty-nine mycotoxins in eight feed samples were determined, of which 31 feed samples were contaminated by DON with the highest contamination rate of 59.3-576.8 ug/kg, 21 feed samples were contaminated by 15-ADON with the content of 62.3-984.3 ug/kg, 7 feed samples were contaminated by D3G with the content of 27.8-127.1 ug/kg, and fumonisin B was also found in different feeds. 1 (FB1), fumonisin B2 (FB2), zearalenone (ZEN), zearalenone (?-ZOL), patulin (PAT) and AFB1 mycotoxins. (2) By optimizing the extraction solvent, HLB column sample solution, elution and elution solvent, a new kind of DON, 3-acetyl-deoxynivalenol (3-ADON), 15-acetyl-deoxynivalenol (3-ADON), deoxynivalenol (15-acetyl-deoxynivalenol) was established for the first time. Preconcentration and purification of 15-ADON, FUS-X and 3-glucosidated deoxynivalenol (D3G) were carried out. The purified sample solution was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry. The established analytical method included linearity (R2 < 0.99), sensitivity (LOQ range 0.08-4.85 ug/L), recovery (79 ug/L). 3%~108.1%. The method was proved to be rapid, sensitive and accurate. The established method was then applied to the determination of 4 B-type otoxins and D3G in 31 feed samples. Among them, 26 samples were contaminated by different mycotoxins in the range of 2.1~864.5 g/kg, and D3G was detected in 17 samples with a concentration range of 2.1~864.5 g/kg. A method for the determination of AFB1, AFM1, OTA and ochratoxin?(OT?) in milk, urine, plasma, heart, liver, spleen, lung and kidney was established. The samples were extracted with 1400? L acetone and then dried with nitrogen gas. The results of the method were determined by resolving ammonium acetate aqueous solution/acetonitrile solution (80/20, v/v). The recoveries of the four mycotoxins ranged from 82.8% to 114.1% at quantitative limits, low, moderate and high levels. The linear relationship was good in the range of 0.1-200 ug/L (R2?0.990), the LOD values were between 0.03 and 0.2 ug/L, and the LOQ values were between 0.1 and 0.5 ug/L. The short-term (8 h), long-term (20 d) and freeze-thaw cycles of the four mycotoxins in plasma and tissues were observed. The stability ranged from 80.9% to 113.6%. The method was proved to be simple, rapid and practical. It can be used for quantitative analysis of four mycotoxins in milk, urine, blood, heart, liver, spleen, lung and kidney. (4) After mixing naturally cultured matrix standard substances rich in AFB1 or OTA with feed samples, a certain amount of mycotoxin was obtained. Dairy cows were fed with diets containing 1 000?G/kg of AFB1 and 750?G/kg of OTA. Blood samples were taken at 10,35,45,60,120,180,240,360,540,720,1440,2160,2880 min. The heart, liver, spleen, lung, kidney and brain organs of dairy cows were slaughtered for 6 hours. Mycotoxins and their typical metabolites in different tissues and organs were analyzed by (3) method. The results showed that AFB1 could be detected in the blood within 10 minutes and reached its peak value at 35 minutes. The transformation rate of AFB1 to milk was 1.1%~1.6% at 48 hours. The elimination period of AFM1 in fresh milk was 2 days. AFB1 toxin was detected in heart, spleen, lung and kidney (the contents were 1.6, 4.1, 3.3 and 5.6 g/kg respectively), and in spleen and kidney. Atomic force microscope (AFM1) toxin was detected (0.7 and 0.8?G/kg respectively). OTA was hardly absorbed in dairy cows. OTA and OT were not detected in fresh milk, blood and tissues. However, a small amount of OTA and a large number of metabolite OT? Toxins were detected in urine, the maximum values were 1.8 and 324.6 ug/L respectively, indicating that OTA was not absorbed in the body and metabolized in the body. OT? Back out of the body.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S816
[Abstract]:Mycotoxins are secondary toxic metabolites produced by fungi under suitable environmental conditions, which have carcinogenic, teratogenic and mutagenic effects. Intake of mycotoxins can also cause toxins and their metabolites to reside in livestock and poultry, and then enter the human food chain through animal-derived products such as meat and milk, resulting in a safe wind for consumers. At present, more than 30 kinds of toxic mycotoxins have been found, among which aflatoxin (AFB1), ochratoxin A (OTA) and deoxynivalenol (DON) are the main mycotoxins of concern. Many countries have formulated limits for mycotoxins in different products. In recent years, fungi in dairy feed and fresh milk have been widely used. Toxin contamination has attracted wide attention. In this paper, 30 kinds of common mycotoxins in feed were detected firstly, and then a sensitive, rapid and reliable detection technology was established for the concealed mycotoxins which are seriously contaminated but easily ignored. Different feed samples were screened by the established detection technology. Then, the metabolic kinetics of typical mycotoxins AFB1 and OTA in dairy cows was studied to reveal the absorption, distribution, metabolism and excretion of typical mycotoxins. The main research contents and conclusions are as follows: (1) QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) pretreatment combined with ultra-high performance liquid chromatography-tandem mass spectrometry were used to determine the content of 30 mycotoxins in different feed samples (premixes, concentrates and batches). Feed samples were extracted by 5 mL water and 5 mL 1% acetonitrile formate solution. After the supernatant was blown to nearly dry, the residue was determined by 1 mL 5 mmol/L ammonium acetate solution/acetonitrile solution (80/20, v/v) resolving on machine. The method was quantitatively analyzed by matrix addition and isotope internal standard method. The average recoveries of 30 mycotoxins were 72.0%~118.4% at low, medium and high concentration levels. The method is simple, rapid and practical. It can be applied to the quantitative analysis of 30 mycotoxins in premix, concentrate and batch. Thirty-nine mycotoxins in eight feed samples were determined, of which 31 feed samples were contaminated by DON with the highest contamination rate of 59.3-576.8 ug/kg, 21 feed samples were contaminated by 15-ADON with the content of 62.3-984.3 ug/kg, 7 feed samples were contaminated by D3G with the content of 27.8-127.1 ug/kg, and fumonisin B was also found in different feeds. 1 (FB1), fumonisin B2 (FB2), zearalenone (ZEN), zearalenone (?-ZOL), patulin (PAT) and AFB1 mycotoxins. (2) By optimizing the extraction solvent, HLB column sample solution, elution and elution solvent, a new kind of DON, 3-acetyl-deoxynivalenol (3-ADON), 15-acetyl-deoxynivalenol (3-ADON), deoxynivalenol (15-acetyl-deoxynivalenol) was established for the first time. Preconcentration and purification of 15-ADON, FUS-X and 3-glucosidated deoxynivalenol (D3G) were carried out. The purified sample solution was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry. The established analytical method included linearity (R2 < 0.99), sensitivity (LOQ range 0.08-4.85 ug/L), recovery (79 ug/L). 3%~108.1%. The method was proved to be rapid, sensitive and accurate. The established method was then applied to the determination of 4 B-type otoxins and D3G in 31 feed samples. Among them, 26 samples were contaminated by different mycotoxins in the range of 2.1~864.5 g/kg, and D3G was detected in 17 samples with a concentration range of 2.1~864.5 g/kg. A method for the determination of AFB1, AFM1, OTA and ochratoxin?(OT?) in milk, urine, plasma, heart, liver, spleen, lung and kidney was established. The samples were extracted with 1400? L acetone and then dried with nitrogen gas. The results of the method were determined by resolving ammonium acetate aqueous solution/acetonitrile solution (80/20, v/v). The recoveries of the four mycotoxins ranged from 82.8% to 114.1% at quantitative limits, low, moderate and high levels. The linear relationship was good in the range of 0.1-200 ug/L (R2?0.990), the LOD values were between 0.03 and 0.2 ug/L, and the LOQ values were between 0.1 and 0.5 ug/L. The short-term (8 h), long-term (20 d) and freeze-thaw cycles of the four mycotoxins in plasma and tissues were observed. The stability ranged from 80.9% to 113.6%. The method was proved to be simple, rapid and practical. It can be used for quantitative analysis of four mycotoxins in milk, urine, blood, heart, liver, spleen, lung and kidney. (4) After mixing naturally cultured matrix standard substances rich in AFB1 or OTA with feed samples, a certain amount of mycotoxin was obtained. Dairy cows were fed with diets containing 1 000?G/kg of AFB1 and 750?G/kg of OTA. Blood samples were taken at 10,35,45,60,120,180,240,360,540,720,1440,2160,2880 min. The heart, liver, spleen, lung, kidney and brain organs of dairy cows were slaughtered for 6 hours. Mycotoxins and their typical metabolites in different tissues and organs were analyzed by (3) method. The results showed that AFB1 could be detected in the blood within 10 minutes and reached its peak value at 35 minutes. The transformation rate of AFB1 to milk was 1.1%~1.6% at 48 hours. The elimination period of AFM1 in fresh milk was 2 days. AFB1 toxin was detected in heart, spleen, lung and kidney (the contents were 1.6, 4.1, 3.3 and 5.6 g/kg respectively), and in spleen and kidney. Atomic force microscope (AFM1) toxin was detected (0.7 and 0.8?G/kg respectively). OTA was hardly absorbed in dairy cows. OTA and OT were not detected in fresh milk, blood and tissues. However, a small amount of OTA and a large number of metabolite OT? Toxins were detected in urine, the maximum values were 1.8 and 324.6 ug/L respectively, indicating that OTA was not absorbed in the body and metabolized in the body. OT? Back out of the body.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S816
【参考文献】
相关期刊论文 前10条
1 张琼;冉丛聪;陈丹;李s鷏，
本文编号：2198476
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