饲料、猪肉中赭曲霉毒素A的液液分散微萃

发布时间：2018-06-10 20:18

本文选题：赭曲霉毒素A + 分散式液液微萃取　；参考：《山东大学》2016年硕士论文

【摘要】：目的赭曲霉毒素A(Ochratoxin A,OTA)是一种由青霉属和曲霉属的真菌所产生的次级代谢产物。其毒性大,分布广,对农产品污染重。饲料发生霉变会严重影响饲料业以及畜牧业的正常生产,霉菌毒素的含量检测对保障牲畜的健康以及肉制品的安全至关重要。分散式液液微萃取(DLLME)技术作为一种新型的样品前处理技术,具有操作简单、快速、有机溶剂用量少、成本低等优点且兼具富集和净化的作用。本文旨在建立基于DLLME-高效液相色谱法检测饲料及猪肉中赭曲霉毒素A的方法；并应用该方法,对饲料生产企业生产的饲料中赭曲霉毒素A的污染程度及市售猪肉中OTA的蓄积水平进行调查与分析。方法1实验条件1.1 OTA提取饲料样品中OTA以甲醇-水的混合溶液超声提取；猪肉样品中OTA用甲醇、磷酸、氯化钠溶液配成的混合溶液提取。1.2 OTA净化及浓缩提取液经离心、过滤后,取出1.00 mL与萃取剂混合,用注射器将混合液迅速注入乙酸溶液中,离心分离后移除上层水溶液,将沉积相氮吹至近干,再用甲醇复溶,进行高效液相色谱分析。1.3色谱条件使用的流动相为乙腈和2%的乙酸溶液,流动相流速为1.0mL/min;荧光检测器检测激发波长为333 nm,发射波长477 nm；柱温30℃。依据保留时间定性,用峰面积外标法定量。1.4方法优化多种因素会影响DLLME的萃取效率,本文对萃取剂的种类与体积、水相的pH与体积等因素进行了优化；并对流动相的组成及比例进行优化。1.5质量控制为保证分析质量,本实验对方法的线性、检出限、加标回收率、日内精密度和日间精密度等指标进行实验研究。2饲料及猪肉中OTA含量调查本实验对来自全国部分省市自治区的饲料生产企业的饲料样品麦麸、豆粕、棉粕、酒糟蛋白饲料(Distillers Dried Grains with Solubles,DDGS)中OTA含量进行了调查。并对济南市的市售猪肉中OTA蓄积水平进行了调查。结果1.优化的DLLME条件为：提取液经离心、过滤后,取出1.00mL与200μL三氯甲烷混合,用注射器将混合液迅速注入5 mL pH为3的乙酸溶液中,离心分离后移除上层水溶液,将沉积相氮吹至近干,再用甲醇复溶,进行高效液相色谱分析。高效液相色谱流动相：检测饲料中OTA时乙腈与乙酸溶液的比例采用梯度洗脱程序,检测猪肉中OTA时乙腈与乙酸溶液的比例为50：50。2.本实验建立的检测饲料及猪肉中赭曲霉毒素A的方法对饲料及猪肉样品中OTA的平均加标回收率均高于85%,且相对标准偏差小于5%。对于猪肉样品,其检出限为0.21 μg/kg;对饲料样品,检出限为0.25 μg/kg。3.所有饲料样品中OTA总检出率为46.7%,阳性样品中OTA平均检出量1.8μg/kg,最高检出量18μg/kg。均未超出国家规定的100 μg/kg限量标准。不同种类饲料的检测结果有差异,豆粕及棉粕中OTA检出率及检出量相对较高。将饲料按照产地进行分析,南方地区OTA检出率及阳性样品平均检出量高于北方地区和西北地区。4.所采集样品中100%的品牌猪肉及88.9%的非品牌猪肉检出有OTA蓄积。品牌猪肉中OTA的最高含量为2.2 μg/kg,平均含量为0.37μg/kg。非品牌猪肉中OTA的最高含量为3.0 μg/kg,平均含量为0.36μg/kg。品牌猪肉中OTA浓度分布较非品牌猪肉集中。所有样品中OTA的含量均低于粮食中OTA的最高限量5μg/kg。结论1.本实验建立的DLLME与HPLC-FLD联用测定饲料及猪肉中OTA含量的方法简便、灵敏。使用DLLME对样品提取液进行净化及浓缩,大大减少了前处理过程中含氯有机溶剂的使用,减轻了对环境的危害,并保护了实验操作者的健康；同时简化了前处理过程,降低了分析成本。2.检测发现,饲料样品中OTA的污染水平较低,低于国家限量标准。相比麦麸和DDGS,豆粕和棉粕较容易受到OTA污染。南方地区较北方地区和西北地区受霉菌毒素危害更重。3.猪肉中普遍检出含有OTA蓄积,但检出量较低,低于粮食中的OTA限量。品牌猪肉数据分布比非品牌猪肉更为集中。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S816.17;O657.72;TS251.7

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