食品中丙烯酰胺等有毒有害物质检测方法建立及其应用研究
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摘要
目的：通过实验建立和完善食品中赭曲霉毒素A、玉米赤霉烯酮、脱氧雪腐镰刀菌烯醇、T-2毒素4种真菌毒素(mycotoxin)、食品添加剂脱氢乙酸的灵敏、准-确、快速的国家标准分析方法,建立食品加工过程中产生的有害物质丙烯酰胺的简便、准确的分析方法,分析市售食品中丙烯酰胺的含量,加工条件对丙烯酰胺生成量的影响,并通过动物实验观察丙烯酰胺对SD大鼠生长发育情况及抗氧化功能的影响。
方法：毒物在食品中含量的分析方法的建立。(1)丙烯酰胺分析方法的建立：食品中丙烯酰胺经提取液提取后,用固相萃取小柱净化,用液相色谱测定,或者提取液衍生化为α,β-二溴丙酰胺后用气相色谱μ-ECD测定。(2)赭曲霉毒素A、玉米赤霉烯酮、脱氧雪腐镰刀菌烯醇、T-2毒素分析方法的建立：用不同提取液分别提取试样中的4种真菌毒素,经过滤、稀释后,稀释液通过键合有真菌毒素特异抗体的免疫亲和柱,真菌毒素键合在免疫亲和柱的抗体上,将免疫亲和柱上的杂质清洗后,用洗脱液将免疫亲和柱上的真菌毒素洗脱,用高效液相色谱的紫外检测器或荧光检测器定量测定。(3)食品中脱氢乙酸分析方法的建立：用氢氧化钠溶液提取试样中的脱氢乙酸,经脱脂、去蛋白处理后,用高效液相色谱紫外检测器测定,外标法定量。
丙烯酰胺在加工食品中的含量、毒性及形成机制研究：(1)分析测定了青岛地区市售的可能含有丙烯酰胺的212种食品中丙烯酰胺的含量,测定了7种加工条件及原料条件对丙烯酰胺含量的影响。(2)建立了组织中丙烯酰胺及其代谢产物的分析方法,建立了大鼠尿中8-羟基脱氧鸟苷(8-OHdG)的分析方法。(3)丙烯酰胺对大鼠抗氧化功能的影响：采用不同剂量丙烯酰胺对大鼠进行灌胃,2周后检测血液和肝组织匀浆中超氧化物歧化酶(SOD)活力、丙二醛(MDA)含量、谷胱甘肽过氧化物酶(GSH-Px)活性。并用LC-MS/MS分析测定大鼠尿中8-OHdG的含量。
1、通过实验建立的4种真菌毒素分析方法操作简便、准确,回收率高,精密度良好,其中赭曲霉毒素A的检出限为0.5μg/kg,样品的加标回收率为90.3%-106.0%,RSD1.75%;玉米赤霉烯酮的检出限为20μg/kg,加标回收率在98.2%-100.5%,相对标准偏差RSD1.0%；脱氧雪腐镰刀菌烯醇的检出限为0.1mg/kg,加标回收率在72.0%-85.9%之间,最大相对标准偏4.9%;T-2毒素的检出限为方法的定量限为2.0μg/kg,加标回收率在93.9%-103.1%,相对标准偏差RSD3.4%；脱氢乙酸的检出限0.5mg/kg,加标回收率在96.5%-99.5%,相对标准偏差RSD为1.09%。；所建立的丙烯酰胺分析方法中,气相色谱(GC-μECD)法的加标回收率为84.6%-98.5%,检出限(LOQ)为0.02mg/kg,液相色谱法的加标回收率为86.3%-96.9%,检出限(LOQ)为0.05mg/kg,两种分析方法一致性良好。
2、测定了青岛市场市售的212种食品中丙烯酰胺含量,除馒头未检出丙烯酰胺外,其余11类高温热加工的食品均含有丙烯酰胺,含量范围在39-1240μg/kg。
3、实验表明,加工条件及原料条件对丙烯酰胺的生成量有不同程度的影响,其中温度、加热时间、pH值、氨基酸种类、还原糖种类以及添加剂的种类对丙烯酰胺的生成有显著影响。
4、丙烯酰胺染毒组与空白组比较,肝匀浆和血浆SOD、血清GSH-Px的活力降低(P<0.05或P<0.01),MDA含量升高(P<0.01),但肝匀浆GSH-Px活力升高(P<0.05)。尿中8-OHdG含量升高(P<0.05)。
1、所建立的赭曲霉毒素A、玉米赤霉烯酮、脱氧雪腐镰刀菌烯醇、T-2毒素4种真菌毒素检测方法以及脱氢乙酸的分析方法,灵敏度高,准确性良好,加标回收率稳定,已被批准为推荐性国家标准。
2、市售的经高温加工食品中含有不等量的丙烯酰胺,温度、加热时间、pH值、氨基酸种类、还原糖种类等加工条件和原料成分对丙烯酰胺的形成有一定的相关性。
3、丙烯酰胺降低大鼠体内抗氧化酶的活性,增加脂质过氧化产物,并可引起DNA氧化损伤。
Abstract Objectives To establish and perfect national standard methods to detect 4 kinds of mycotoxins(ochratoxin A,zearalenone,deoxynivalenol and T-2 toxin)and dehydroacetic acid,to establish acrylamide in food products, to analyze the content of acrylamide in commercial food products and the impact of processing conditions on the amount of acrlamide, and to observe the effects of acrylamide on growth and anti-oxidant fuction of rats. Methods 1. Establishment of methods to analyze toxic materials in food products. (1) Analysis of acrylamide in food products. Acrylamide was detected by liquid chromatography after extracted by solid-phase extaction column purification, or detceted by gas chromatographyμ-ECD determination after derivatived intoα,β-dibromo-propanamide. (2) Methods to detect ochratoxin A, zearalenone, deoxynivalenol and T-2 toxin. After extracting the 4 kinds of mycotoxins from the samples, filtrating and diluting, the dilution passed an immunoaffinity column bonding with mycotoxin-specific antibody. Then the mycotoxins were bonded by the antibodies. The mycotoxins were washed off after washing the impurities, and were finally detected by UV detector or fluorescence detector of a high-performance liquid chromatography quantitatively. (3) Methods to detect dehydroacetic acid in food products. Dehydroacetic acid was extracted from samples by using sodium hydroxide, and then detected by UV detector of a high-performance liquid chromatography quantitatively.2. Analysis of acrylamide in food products, its toxicity and formation mechanism. (1) 212 kinds of commercial food products from 12 categories were detected for the acryamide content. Affection of 7 kinds of processing conditions on the cocentration of acrylamide. (2)The method to detcet acrylamide and its metabolites in tissues was established, as well as the method to detect 8-hydroxy-deoxyguanosine (8-OHdG) in rat urine. (3) Affection of acrylamide on the anti-oxidant activity of rats. Rats were fed with different dosage of acrymalide for 2 weeks, and then the activity of superoxide dismutase (SOD) and glutathione peroxydase (GSH-Px), and the content of malondialdephyde (MDA) in blood and liver tusse were detceted by using kits. And the 8-hydroxy-deoxyguanosine (8-OHdG) content in urine were analyzed by LC-MS/MS. Results 1.4 kinds of mycotoxins detection methods were established and all were easy-operating and accurate with high recovery rate, and the reproducibility was very good with good precision. The limited quantity (LOQ) of ochratoxin A was 0.5μg/kg, the recovery was 90.3%-106.0%, The relative standard deviation(RSD) was 1.75%; The LOQ of zearalenone was 20μg/kg, the recovery was 98.2%-100.5%, RSD was 1.0%; The LOQ of deoxynivalenol was 0.1mg/kg, recovery was 72.0%-85.9%, the maximum RSD was 4.9%; The LOQ of T-2 toxin was 2.0μg/kg, recovery was 93.9%-103.1%, RSD was 3.4%; The LOQ of dehydroacetic acid was 0.5mg/kg, recovery was 96.5%-99.5%, the RSD was 1.09%; The two kinds of analytical methods of acrylamide had a good consistency,The LOQ by GC-μ-ECD was 0.02mg/kg, recovery was 84.6%-98.5%, The LOQ by HPLC was 0.05mg/kg, recovery was 86.3%-96.9%.
2. The acrylamide contents of 212 kinds of commercial food products from 12 categories of Qingdao were 0-1240μg/kg.3. All the 7 kinds of processing conditions and raw materials affected the formation of acrylamide with different degrees. Temperature, heating time, pH, amino acid and reducing sugar species, as well as the types of additives have a significant effect on acrylamide formation.4. Compared with the control group, SOD activity of the liver homogenate and serum in groups of acrylamide exposure decreased (P<0.05,or P<0.01). GSH-Px activity of serum also increased, but increased in liver homogenate. MDA content increased(P<0.05),8-OhdG content increased (P＜0.05).
Conclusion 1 The five methods of detecting ochratoxin A, zearalenone, deoxynivalenol, T-2 toxin and dehydroacetic acid have been accepted as national standards.
2 The acrylamide contents of 212 kinds of commercial food products were different, Temperature, heating time, pH, amino acid and reducing sugar species, as well as the types of additives have a significant effect on acrylamide formation.
3.Acrylamide reduces the activity of antioxidant enzymes in rats, increasing lipid peroxidation, and can lead to oxidative damage of DNA.
方法：毒物在食品中含量的分析方法的建立。(1)丙烯酰胺分析方法的建立：食品中丙烯酰胺经提取液提取后,用固相萃取小柱净化,用液相色谱测定,或者提取液衍生化为α,β-二溴丙酰胺后用气相色谱μ-ECD测定。(2)赭曲霉毒素A、玉米赤霉烯酮、脱氧雪腐镰刀菌烯醇、T-2毒素分析方法的建立：用不同提取液分别提取试样中的4种真菌毒素,经过滤、稀释后,稀释液通过键合有真菌毒素特异抗体的免疫亲和柱,真菌毒素键合在免疫亲和柱的抗体上,将免疫亲和柱上的杂质清洗后,用洗脱液将免疫亲和柱上的真菌毒素洗脱,用高效液相色谱的紫外检测器或荧光检测器定量测定。(3)食品中脱氢乙酸分析方法的建立：用氢氧化钠溶液提取试样中的脱氢乙酸,经脱脂、去蛋白处理后,用高效液相色谱紫外检测器测定,外标法定量。
丙烯酰胺在加工食品中的含量、毒性及形成机制研究：(1)分析测定了青岛地区市售的可能含有丙烯酰胺的212种食品中丙烯酰胺的含量,测定了7种加工条件及原料条件对丙烯酰胺含量的影响。(2)建立了组织中丙烯酰胺及其代谢产物的分析方法,建立了大鼠尿中8-羟基脱氧鸟苷(8-OHdG)的分析方法。(3)丙烯酰胺对大鼠抗氧化功能的影响：采用不同剂量丙烯酰胺对大鼠进行灌胃,2周后检测血液和肝组织匀浆中超氧化物歧化酶(SOD)活力、丙二醛(MDA)含量、谷胱甘肽过氧化物酶(GSH-Px)活性。并用LC-MS/MS分析测定大鼠尿中8-OHdG的含量。
1、通过实验建立的4种真菌毒素分析方法操作简便、准确,回收率高,精密度良好,其中赭曲霉毒素A的检出限为0.5μg/kg,样品的加标回收率为90.3%-106.0%,RSD1.75%;玉米赤霉烯酮的检出限为20μg/kg,加标回收率在98.2%-100.5%,相对标准偏差RSD1.0%；脱氧雪腐镰刀菌烯醇的检出限为0.1mg/kg,加标回收率在72.0%-85.9%之间,最大相对标准偏4.9%;T-2毒素的检出限为方法的定量限为2.0μg/kg,加标回收率在93.9%-103.1%,相对标准偏差RSD3.4%；脱氢乙酸的检出限0.5mg/kg,加标回收率在96.5%-99.5%,相对标准偏差RSD为1.09%。；所建立的丙烯酰胺分析方法中,气相色谱(GC-μECD)法的加标回收率为84.6%-98.5%,检出限(LOQ)为0.02mg/kg,液相色谱法的加标回收率为86.3%-96.9%,检出限(LOQ)为0.05mg/kg,两种分析方法一致性良好。
2、测定了青岛市场市售的212种食品中丙烯酰胺含量,除馒头未检出丙烯酰胺外,其余11类高温热加工的食品均含有丙烯酰胺,含量范围在39-1240μg/kg。
3、实验表明,加工条件及原料条件对丙烯酰胺的生成量有不同程度的影响,其中温度、加热时间、pH值、氨基酸种类、还原糖种类以及添加剂的种类对丙烯酰胺的生成有显著影响。
4、丙烯酰胺染毒组与空白组比较,肝匀浆和血浆SOD、血清GSH-Px的活力降低(P<0.05或P<0.01),MDA含量升高(P<0.01),但肝匀浆GSH-Px活力升高(P<0.05)。尿中8-OHdG含量升高(P<0.05)。
1、所建立的赭曲霉毒素A、玉米赤霉烯酮、脱氧雪腐镰刀菌烯醇、T-2毒素4种真菌毒素检测方法以及脱氢乙酸的分析方法,灵敏度高,准确性良好,加标回收率稳定,已被批准为推荐性国家标准。
2、市售的经高温加工食品中含有不等量的丙烯酰胺,温度、加热时间、pH值、氨基酸种类、还原糖种类等加工条件和原料成分对丙烯酰胺的形成有一定的相关性。
3、丙烯酰胺降低大鼠体内抗氧化酶的活性,增加脂质过氧化产物,并可引起DNA氧化损伤。
Abstract Objectives To establish and perfect national standard methods to detect 4 kinds of mycotoxins(ochratoxin A,zearalenone,deoxynivalenol and T-2 toxin)and dehydroacetic acid,to establish acrylamide in food products, to analyze the content of acrylamide in commercial food products and the impact of processing conditions on the amount of acrlamide, and to observe the effects of acrylamide on growth and anti-oxidant fuction of rats. Methods 1. Establishment of methods to analyze toxic materials in food products. (1) Analysis of acrylamide in food products. Acrylamide was detected by liquid chromatography after extracted by solid-phase extaction column purification, or detceted by gas chromatographyμ-ECD determination after derivatived intoα,β-dibromo-propanamide. (2) Methods to detect ochratoxin A, zearalenone, deoxynivalenol and T-2 toxin. After extracting the 4 kinds of mycotoxins from the samples, filtrating and diluting, the dilution passed an immunoaffinity column bonding with mycotoxin-specific antibody. Then the mycotoxins were bonded by the antibodies. The mycotoxins were washed off after washing the impurities, and were finally detected by UV detector or fluorescence detector of a high-performance liquid chromatography quantitatively. (3) Methods to detect dehydroacetic acid in food products. Dehydroacetic acid was extracted from samples by using sodium hydroxide, and then detected by UV detector of a high-performance liquid chromatography quantitatively.2. Analysis of acrylamide in food products, its toxicity and formation mechanism. (1) 212 kinds of commercial food products from 12 categories were detected for the acryamide content. Affection of 7 kinds of processing conditions on the cocentration of acrylamide. (2)The method to detcet acrylamide and its metabolites in tissues was established, as well as the method to detect 8-hydroxy-deoxyguanosine (8-OHdG) in rat urine. (3) Affection of acrylamide on the anti-oxidant activity of rats. Rats were fed with different dosage of acrymalide for 2 weeks, and then the activity of superoxide dismutase (SOD) and glutathione peroxydase (GSH-Px), and the content of malondialdephyde (MDA) in blood and liver tusse were detceted by using kits. And the 8-hydroxy-deoxyguanosine (8-OHdG) content in urine were analyzed by LC-MS/MS. Results 1.4 kinds of mycotoxins detection methods were established and all were easy-operating and accurate with high recovery rate, and the reproducibility was very good with good precision. The limited quantity (LOQ) of ochratoxin A was 0.5μg/kg, the recovery was 90.3%-106.0%, The relative standard deviation(RSD) was 1.75%; The LOQ of zearalenone was 20μg/kg, the recovery was 98.2%-100.5%, RSD was 1.0%; The LOQ of deoxynivalenol was 0.1mg/kg, recovery was 72.0%-85.9%, the maximum RSD was 4.9%; The LOQ of T-2 toxin was 2.0μg/kg, recovery was 93.9%-103.1%, RSD was 3.4%; The LOQ of dehydroacetic acid was 0.5mg/kg, recovery was 96.5%-99.5%, the RSD was 1.09%; The two kinds of analytical methods of acrylamide had a good consistency,The LOQ by GC-μ-ECD was 0.02mg/kg, recovery was 84.6%-98.5%, The LOQ by HPLC was 0.05mg/kg, recovery was 86.3%-96.9%.
2. The acrylamide contents of 212 kinds of commercial food products from 12 categories of Qingdao were 0-1240μg/kg.3. All the 7 kinds of processing conditions and raw materials affected the formation of acrylamide with different degrees. Temperature, heating time, pH, amino acid and reducing sugar species, as well as the types of additives have a significant effect on acrylamide formation.4. Compared with the control group, SOD activity of the liver homogenate and serum in groups of acrylamide exposure decreased (P<0.05,or P<0.01). GSH-Px activity of serum also increased, but increased in liver homogenate. MDA content increased(P<0.05),8-OhdG content increased (P＜0.05).
Conclusion 1 The five methods of detecting ochratoxin A, zearalenone, deoxynivalenol, T-2 toxin and dehydroacetic acid have been accepted as national standards.
2 The acrylamide contents of 212 kinds of commercial food products were different, Temperature, heating time, pH, amino acid and reducing sugar species, as well as the types of additives have a significant effect on acrylamide formation.
3.Acrylamide reduces the activity of antioxidant enzymes in rats, increasing lipid peroxidation, and can lead to oxidative damage of DNA.
引文
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