飼料中真菌毒素分析方法及在體內(nèi)代謝轉(zhuǎn)化的研究
發(fā)布時(shí)間：2018-08-23 08:23
【摘要】：真菌毒素是真菌在適宜的環(huán)境條件下產(chǎn)生的次級(jí)有毒代謝產(chǎn)物,具有致癌、致畸及致突變等毒性作用。真菌毒素的攝入還可造成毒素及其代謝產(chǎn)物在畜禽體內(nèi)殘留,進(jìn)而通過(guò)肉、奶等動(dòng)物源性產(chǎn)品進(jìn)入人食物鏈中,給消費(fèi)者造成安全風(fēng)險(xiǎn)。目前,已經(jīng)發(fā)現(xiàn)的具有毒性的真菌毒素有30余種,其中廣受關(guān)注的真菌毒素主要為有黃曲霉毒素(AFB1)、赭曲霉毒素A(OTA)和脫氧雪腐鐮刀菌烯醇(DON)等。許多國(guó)家針對(duì)不同產(chǎn)品已制定真菌毒素的限量標(biāo)準(zhǔn)。近年來(lái),奶牛飼料和鮮奶中真菌毒素的污染受到廣泛關(guān)注。本論文首先對(duì)飼料中常見(jiàn)的30種真菌毒素進(jìn)行檢測(cè)技術(shù)研究,進(jìn)而針對(duì)目前污染較為嚴(yán)重但是容易被忽略的隱蔽型真菌毒素,建立靈敏、快捷、可靠的檢測(cè)技術(shù)。采用建立的檢測(cè)技術(shù),對(duì)不同的飼料樣本進(jìn)行篩查,探明飼料中真菌毒素的暴露水平和暴露種類。然后,對(duì)典型真菌毒素AFB1和OTA進(jìn)入奶牛體內(nèi)后的代謝動(dòng)力學(xué)情況進(jìn)行研究,揭示典型真菌毒素的吸收、分布、代謝和排泄規(guī)律,對(duì)今后保障鮮奶安全、制定飼料中限量標(biāo)準(zhǔn)具有重要意義。本文主要研究?jī)?nèi)容和結(jié)論如下:(1)采用QuEChERS(Quick,Easy,Cheap,Effective,Rugged,Safe)前處理技術(shù)結(jié)合超高效液相色譜-串聯(lián)質(zhì)譜檢測(cè)不同飼料樣品(預(yù)混料、濃縮料和配合料)中30種真菌毒素的含量。飼料樣品經(jīng)過(guò)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,殘?jiān)?jīng)1 mL 5 mmol/L醋酸銨水溶液/乙腈溶液(80/20,v/v)復(fù)溶上機(jī)測(cè)定,采用基質(zhì)加標(biāo)結(jié)合同位素內(nèi)標(biāo)法進(jìn)行定量分析。本方法在不同的線性范圍內(nèi)均線性良好,相關(guān)系數(shù)R2?0.99,檢出限(limit of detection,LOD)在0.7~20?g/L之間,定量限(limit of quantitation,LOQ)在2~50?g/L之間。低、中、高濃度添加水平下30種真菌毒素平均回收率為72.0%~118.4%。該方法具有簡(jiǎn)單、快速、實(shí)用性強(qiáng)等優(yōu)點(diǎn),能適用于預(yù)混料、濃縮料和配合料中30種真菌毒素的定量分析。將建立的分析方法應(yīng)用到38個(gè)飼料樣品中39種真菌毒素的測(cè)定,其中,31份飼料樣品被DON污染,污染率最高,含量為59.3~576.8μg/kg;有21份飼料樣品被15-ADON污染,含量為62.3~984.3μg/kg;有7份飼料樣品被D3G污染,含量為27.8~127.1μg/kg;另外,在不同的飼料中還發(fā)現(xiàn)了伏馬毒素B1(FB1)、伏馬毒素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)通過(guò)優(yōu)化提取溶劑,HLB小柱的上樣液,淋洗和洗脫溶劑等,首次建立了一種用于DON,3-乙酰基-脫氧雪腐鐮刀菌烯醇(3-ADON)、15-乙酰基-脫氧雪腐鐮刀菌烯醇(15-ADON)、鐮刀菌烯酮(FUS-X)和3-葡萄苷化脫氧雪腐鐮刀菌烯醇(D3G)富集、凈化的前處理方法。凈化樣品液通過(guò)超高效液相色譜串聯(lián)質(zhì)譜分析。建立的分析方法通過(guò)方法學(xué)包括線性(R2≥0.99)、靈敏度(LOQ范圍為0.08~4.85μg/L)、回收率(79.3%~108.1%)驗(yàn)證后表明,該分析方法快速、靈敏、準(zhǔn)確。進(jìn)而將建立的分析方法應(yīng)用于31個(gè)飼料樣品中的4種B型單端孢霉烯族類毒素和D3G的測(cè)定。其中,26個(gè)樣品被不同真菌毒素污染,含量范圍為2.1~864.5?g/kg,而D3G也在17個(gè)樣本檢測(cè)到,其濃度范圍在2.1~34.8μg/kg。(3)建立了鮮奶、尿液、血漿、心、肝、脾、肺和腎中AFB1、AFM1、OTA和赭曲霉毒素?(OT?)四種真菌毒素的分析方法,樣品經(jīng)1400?L丙酮提取后取上清液氮?dú)獯蹈?200?L 5 mmol/L醋酸銨水溶液/乙腈溶液(80/20,v/v)復(fù)溶上機(jī)測(cè)定。方法學(xué)結(jié)果表明四種真菌毒素在定量限、低、中和高添加水平下,回收率范圍為為82.8%~114.1%。在0.1~200μg/L范圍內(nèi)線性關(guān)系良好(R2?0.990),LOD值在0.03~0.2μg/L之間,LOQ值在0.1~0.5μg/L之間。血漿和組織器官內(nèi)4種真菌毒素的短期(8 h)、長(zhǎng)期(20 d)和冷凍解凍三循環(huán)穩(wěn)定性范圍為80.9%~113.6%。證明建立的分析方法具有簡(jiǎn)單、快速、實(shí)用性強(qiáng)等優(yōu)點(diǎn),能適用于奶、尿、血、心、肝、脾、肺和腎中四種真菌毒素的定量分析。(4)將天然培養(yǎng)的富含AFB1或OTA的基體標(biāo)準(zhǔn)物質(zhì)與飼料樣本混合后,獲得一定含量真菌毒素的奶牛飼料(AFB1的含量為1000?g/kg,OTA的含量為750?g/kg),飼喂奶牛,在10、35、45、60、120、180、240、360、540、720、1440、2160、2880 min處分別取血,在6 h屠宰奶牛取心、肝、脾、肺、腎、腦器官。采用(3)建立的分析方法對(duì)不同組織器官中真菌毒素及其典型代謝產(chǎn)物進(jìn)行分析后。結(jié)果表明:AFB1在體內(nèi)吸收速度較快,10 min血液中就可檢測(cè)到AFB1,在35 min處達(dá)到峰值,48 h檢測(cè)不到。AFB1向乳中的轉(zhuǎn)化率為1.1%~1.6%,鮮奶中的AFM1清除期為2天。在心、脾、肺和腎檢測(cè)到AFB1毒素(含量分別為1.6、4.1、3.3和5.6?g/kg),在脾和腎中檢測(cè)到AFM1毒素(含量分別為0.7和0.8?g/kg)。OTA在奶牛中基本難以吸收,鮮奶、血液及各組織器官中均未檢測(cè)到OTA和OT?。但是在尿液中檢測(cè)到少量OTA和大量代謝物OT?毒素,最大值分別為1.8μg/L和324.6μg/L,表明OTA在體內(nèi)不吸收,在體內(nèi)代謝轉(zhuǎn)化成OT?后排出體外。
[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.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：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.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S816
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 張瓊;冉叢聰;陳丹;李s鷏，
本文編號(hào)：2198476
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