本文選題：赭曲霉毒素A + 分散式液液微萃取�。� 參考：《山東大學》2016年碩士論文

【摘要】：目的赭曲霉毒素A(Ochratoxin A,OTA)是一種由青霉屬和曲霉屬的真菌所產(chǎn)生的次級代謝產(chǎn)物。其毒性大,分布廣,對農(nóng)產(chǎn)品污染重。飼料發(fā)生霉變會嚴重影響飼料業(yè)以及畜牧業(yè)的正常生產(chǎn),霉菌毒素的含量檢測對保障牲畜的健康以及肉制品的安全至關(guān)重要。分散式液液微萃取(DLLME)技術(shù)作為一種新型的樣品前處理技術(shù),具有操作簡單、快速、有機溶劑用量少、成本低等優(yōu)點且兼具富集和凈化的作用。本文旨在建立基于DLLME-高效液相色譜法檢測飼料及豬肉中赭曲霉毒素A的方法；并應用該方法,對飼料生產(chǎn)企業(yè)生產(chǎn)的飼料中赭曲霉毒素A的污染程度及市售豬肉中OTA的蓄積水平進行調(diào)查與分析。方法1實驗條件1.1 OTA提取飼料樣品中OTA以甲醇-水的混合溶液超聲提取；豬肉樣品中OTA用甲醇、磷酸、氯化鈉溶液配成的混合溶液提取。1.2 OTA凈化及濃縮提取液經(jīng)離心、過濾后,取出1.00 mL與萃取劑混合,用注射器將混合液迅速注入乙酸溶液中,離心分離后移除上層水溶液,將沉積相氮吹至近干,再用甲醇復溶,進行高效液相色譜分析。1.3色譜條件使用的流動相為乙腈和2%的乙酸溶液,流動相流速為1.0mL/min;熒光檢測器檢測激發(fā)波長為333 nm,發(fā)射波長477 nm；柱溫30℃。依據(jù)保留時間定性,用峰面積外標法定量。1.4方法優(yōu)化多種因素會影響DLLME的萃取效率,本文對萃取劑的種類與體積、水相的pH與體積等因素進行了優(yōu)化；并對流動相的組成及比例進行優(yōu)化。1.5質(zhì)量控制為保證分析質(zhì)量,本實驗對方法的線性、檢出限、加標回收率、日內(nèi)精密度和日間精密度等指標進行實驗研究。2飼料及豬肉中OTA含量調(diào)查本實驗對來自全國部分省市自治區(qū)的飼料生產(chǎn)企業(yè)的飼料樣品麥麩、豆粕、棉粕、酒糟蛋白飼料(Distillers Dried Grains with Solubles,DDGS)中OTA含量進行了調(diào)查。并對濟南市的市售豬肉中OTA蓄積水平進行了調(diào)查。結(jié)果1.優(yōu)化的DLLME條件為：提取液經(jīng)離心、過濾后,取出1.00mL與200μL三氯甲烷混合,用注射器將混合液迅速注入5 mL pH為3的乙酸溶液中,離心分離后移除上層水溶液,將沉積相氮吹至近干,再用甲醇復溶,進行高效液相色譜分析。高效液相色譜流動相：檢測飼料中OTA時乙腈與乙酸溶液的比例采用梯度洗脫程序,檢測豬肉中OTA時乙腈與乙酸溶液的比例為50：50。2.本實驗建立的檢測飼料及豬肉中赭曲霉毒素A的方法對飼料及豬肉樣品中OTA的平均加標回收率均高于85%,且相對標準偏差小于5%。對于豬肉樣品,其檢出限為0.21 μg/kg;對飼料樣品,檢出限為0.25 μg/kg。3.所有飼料樣品中OTA總檢出率為46.7%,陽性樣品中OTA平均檢出量1.8μg/kg,最高檢出量18μg/kg。均未超出國家規(guī)定的100 μg/kg限量標準。不同種類飼料的檢測結(jié)果有差異,豆粕及棉粕中OTA檢出率及檢出量相對較高。將飼料按照產(chǎn)地進行分析,南方地區(qū)OTA檢出率及陽性樣品平均檢出量高于北方地區(qū)和西北地區(qū)。4.所采集樣品中100%的品牌豬肉及88.9%的非品牌豬肉檢出有OTA蓄積。品牌豬肉中OTA的最高含量為2.2 μg/kg,平均含量為0.37μg/kg。非品牌豬肉中OTA的最高含量為3.0 μg/kg,平均含量為0.36μg/kg。品牌豬肉中OTA濃度分布較非品牌豬肉集中。所有樣品中OTA的含量均低于糧食中OTA的最高限量5μg/kg。結(jié)論1.本實驗建立的DLLME與HPLC-FLD聯(lián)用測定飼料及豬肉中OTA含量的方法簡便、靈敏。使用DLLME對樣品提取液進行凈化及濃縮,大大減少了前處理過程中含氯有機溶劑的使用,減輕了對環(huán)境的危害,并保護了實驗操作者的健康；同時簡化了前處理過程,降低了分析成本。2.檢測發(fā)現(xiàn),飼料樣品中OTA的污染水平較低,低于國家限量標準。相比麥麩和DDGS,豆粕和棉粕較容易受到OTA污染。南方地區(qū)較北方地區(qū)和西北地區(qū)受霉菌毒素危害更重。3.豬肉中普遍檢出含有OTA蓄積,但檢出量較低,低于糧食中的OTA限量。品牌豬肉數(shù)據(jù)分布比非品牌豬肉更為集中。
[Abstract]:Objective ochratoxin A (Ochratoxin A, OTA) is a secondary metabolite produced by fungi of Penicillium and Aspergillus. It has large toxicity, wide distribution and heavy pollution to agricultural products. The mildew of feed will seriously affect the normal production of feed and animal husbandry. The detection of mycotoxin to protect the health of livestock and meat products The dispersive liquid liquid microextraction (DLLME) technology, as a new sample pretreatment technology, has the advantages of simple operation, rapid, low organic solvent consumption, low cost and low cost, and has the advantages of enrichment and purification. This paper aims to establish a method for the detection of ochratoxin A in feed and pork based on DLLME- high performance liquid chromatography. The method was used to investigate and analyze the pollution degree of ochratoxin A in feed produced by feed production enterprises and the accumulation level of OTA in the market pork. Method 1 experimental conditions 1.1 OTA extracted feed samples were extracted by ultrasonic extraction of methanol water mixed solution, and OTA with methanol, phosphoric acid and Sodium Chloride Solution in pork samples. The mixed solution of.1.2 OTA was extracted and extracted by centrifugation. After filtration, the mixture was extracted 1 mL from the extractant, and the mixture was quickly injected into the acetic acid solution by a syringe. After centrifugation, the upper water solution was removed and the sedimentary phase nitrogen was blown to the dry, then the methanol was reused, and the high performance liquid chromatography was used for the analysis of the.1.3 chromatographic conditions. The flow phase is acetonitrile and 2% acetic acid solution, the flow velocity is 1.0mL/min, the fluorescence detector detects the excitation wavelength of 333 nm, the emission wavelength is 477 nm, the column temperature is 30 C. According to the retention time, the extraction efficiency of DLLME can be affected by the optimization of various factors by the quantitative.1.4 method with the peak area external standard method. In this paper, the type and volume of the extractant and the water phase are discussed in this paper. The factors such as pH and volume are optimized, and the composition and proportion of the convective phase are optimized and the quality control of.1.5 is optimized to ensure the analysis quality. The experimental study on the linear, detection limit, the rate of recovery, the intraday precision and the day precision in the experiment study the OTA content in.2 feed and pork from the national Department The content of OTA in feed samples, wheat bran, soybean meal, cottonseed meal, Distillers Dried Grains with Solubles, DDGS, was investigated in the feed production enterprises in the provinces and autonomous regions. The accumulation level of OTA in the city sold in Ji'nan was investigated. The results of the 1. optimized DLLME conditions were: the extraction solution was centrifuged and filtered, and 1 was taken out. .00mL is mixed with 200 L trichloromethane, and the mixture is quickly injected into the acetic acid solution of 3 of 5 mL pH with syringe. After centrifugation, the upper water solution is removed and the sedimentary phase nitrogen is blown to the dry. The high performance liquid chromatography is used to analyze the liquid chromatography. The liquid chromatography of high performance liquid chromatography (HPLC) is used to detect the proportion of acetonitrile and acetic acid in the feed OTA. The gradient elution program was used to detect the ratio of acetonitrile and acetic acid in pork OTA when the ratio of acetonitrile and acetic acid was 50:50.2.. The method for detecting ochratoxin A in the feed and pork was higher than 85% in the feed and pork samples, and the relative standard deviation was less than 5%. to the pork samples, and the detection limit was 0.21 mu g/kg; The total detection rate of OTA in all feed samples was 0.25 mu g/kg.3.. The average detection rate of OTA in the positive samples was 46.7%, the average detection amount of OTA in the positive samples was 1.8 mu g/kg, and the highest detection amount 18 mu g/kg. was not beyond the national standard of 100 micron g/kg. The detection results of different kinds of feed were different, and the detection rate and detection rate of OTA in soybean meal and cottonseed meal were relatively high. The average detection rate of OTA detection rate and positive samples in South China is higher than that of 100% of the brand pork and 88.9% of the non branded pork in the north and Northwest China. The highest content of OTA in brand pork is 2.2 mu g/kg, and the average content is 0.37 mu g/kg. in non brand pork OT. The highest content of A was 3 mu g/kg and the concentration of OTA in the brand pork with the average content was more than the non branded pork. The content of OTA in all the samples was lower than the maximum limit of OTA in the grain 5 mu g/kg. conclusion. The method for the determination of OTA content in the feed and pork by the combined use of DLLME and HPLC-FLD in the experiment was simple and sensitive. The purification and concentration of the sample extraction liquid greatly reduced the use of chlorinated organic solvents in the pretreatment process, alleviated the harm to the environment and protected the health of the experimental operators. At the same time, the pretreatment process was reduced and the analysis cost was reduced by.2. detection. The pollution level of OTA in the feed samples was lower than the national limit standard. Compared with wheat bran and DDGS, soybean meal and cottonseed meal were more vulnerable to OTA pollution. In southern regions and northwestern regions, OTA accumulation was generally found in.3. pork, but the detection amount was lower than that of OTA in grain. The data distribution of brand pork was more concentrated than non brand pork.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S816.17;O657.72;TS251.7

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