CN109477822A - 用于从谷物、其它食物及饲料中萃取霉菌毒素的方法 - Google Patents
用于从谷物、其它食物及饲料中萃取霉菌毒素的方法 Download PDFInfo
- Publication number
- CN109477822A CN109477822A CN201780044926.2A CN201780044926A CN109477822A CN 109477822 A CN109477822 A CN 109477822A CN 201780044926 A CN201780044926 A CN 201780044926A CN 109477822 A CN109477822 A CN 109477822A
- Authority
- CN
- China
- Prior art keywords
- mycotoxin
- aflatoxin
- compound
- naphthalene
- acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 44
- 231100000678 Mycotoxin Toxicity 0.000 title claims abstract description 35
- 239000002636 mycotoxin Substances 0.000 title claims abstract description 35
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- UHOVQNZJYSORNB-UHFFFAOYSA-N monobenzene Natural products C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims abstract description 26
- -1 naphthalene compound Chemical class 0.000 claims abstract description 25
- UFWIBTONFRDIAS-UHFFFAOYSA-N naphthalene-acid Natural products C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 claims abstract description 18
- 125000000623 heterocyclic group Chemical group 0.000 claims abstract description 13
- 239000000243 solution Substances 0.000 claims abstract description 8
- 239000007864 aqueous solution Substances 0.000 claims abstract description 7
- 125000002843 carboxylic acid group Chemical group 0.000 claims abstract description 4
- 125000000542 sulfonic acid group Chemical group 0.000 claims abstract description 4
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- 238000002965 ELISA Methods 0.000 claims description 30
- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 claims description 29
- 239000000126 substance Substances 0.000 claims description 25
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- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 10
- RWQKHEORZBHNRI-BMIGLBTASA-N ochratoxin A Chemical compound C([C@H](NC(=O)C1=CC(Cl)=C2C[C@H](OC(=O)C2=C1O)C)C(O)=O)C1=CC=CC=C1 RWQKHEORZBHNRI-BMIGLBTASA-N 0.000 claims description 9
- VYLQGYLYRQKMFU-UHFFFAOYSA-N Ochratoxin A Natural products CC1Cc2c(Cl)cc(CNC(Cc3ccccc3)C(=O)O)cc2C(=O)O1 VYLQGYLYRQKMFU-UHFFFAOYSA-N 0.000 claims description 8
- LINOMUASTDIRTM-QGRHZQQGSA-N deoxynivalenol Chemical compound C([C@@]12[C@@]3(C[C@@H](O)[C@H]1O[C@@H]1C=C(C([C@@H](O)[C@@]13CO)=O)C)C)O2 LINOMUASTDIRTM-QGRHZQQGSA-N 0.000 claims description 8
- 229930002954 deoxynivalenol Natural products 0.000 claims description 8
- XTEGVFVZDVNBPF-UHFFFAOYSA-N naphthalene-1,5-disulfonic acid Chemical compound C1=CC=C2C(S(=O)(=O)O)=CC=CC2=C1S(O)(=O)=O XTEGVFVZDVNBPF-UHFFFAOYSA-N 0.000 claims description 8
- DAEYIVCTQUFNTM-UHFFFAOYSA-N ochratoxin B Natural products OC1=C2C(=O)OC(C)CC2=CC=C1C(=O)NC(C(O)=O)CC1=CC=CC=C1 DAEYIVCTQUFNTM-UHFFFAOYSA-N 0.000 claims description 8
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- MVIOINXPSFUJEN-UHFFFAOYSA-N benzenesulfonic acid;hydrate Chemical compound O.OS(=O)(=O)C1=CC=CC=C1 MVIOINXPSFUJEN-UHFFFAOYSA-N 0.000 claims 1
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- FEPBITJSIHRMRT-UHFFFAOYSA-N 4-hydroxybenzenesulfonic acid Chemical compound OC1=CC=C(S(O)(=O)=O)C=C1 FEPBITJSIHRMRT-UHFFFAOYSA-N 0.000 description 10
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/23—Removal of unwanted matter, e.g. deodorisation or detoxification by extraction with solvents
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
- A23L7/10—Cereal-derived products
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Abstract
本发明涉及一种用于从谷物和其它食物或者从饲料中萃取霉菌毒素的方法以及其随后的量化。应用领域为食品工业,饲料工业或者生物技术。本发明的目的在于开发一种萃取方法,利用该方法可以将具有不同溶解特性的霉菌毒素统一萃取出。已经显示出,借助于水性的、经缓冲的萘化合物和/或苯化合物或其杂环类似物可以将疏水的以及亲水的霉菌毒素萃取出。根据本发明的方法的特征在于,经过缓冲的、具有至少一个磺酸基团或至少一个羧酸基团的萘化合物和/或苯化合物和/或其杂环类似物的溶液与谷物或者其它食物或者饲料相接触,随后将水性溶液分离并且确定所萃取出的霉菌毒素在水性溶液中的含量。
Description
技术领域
本发明涉及一种用于从谷物和其它食物或者从饲料中萃取霉菌毒素的方法以及其随后的量化。应用领域为食品工业,饲料工业或者生物技术。
背景技术
霉菌毒素是由霉菌产生的次级物质交换产物,其在脊椎动物中在极少量的情况下就有毒性。目前已知约200种不同的霉菌毒素,其由超过300种菌种产生。霉菌毒素这一概念包括一系列具有不同结构和作用的化学化合物,其可总结为以下的物质类别:黄曲霉毒素,赭曲霉毒素,单端孢霉烯,例如脱氧雪腐镰刀菌烯醇,伏马菌素,交链孢菌毒素,镰刀菌毒素,麦角生物碱。
在食品工业和饲料工业中,黄曲霉毒素类,伏马菌素类,赭曲霉毒素A,脱氧雪腐镰刀菌烯醇(DON),HT-2毒素(简称HT2),T-2毒素(简称T2)和玉米赤霉烯酮这些霉菌毒素是有特别意义的。由于其明显较高的危险潜力和宽广的播散,食物和饲料中非常微小的量已经会对人和动物的健康产生急性或慢性的损伤。因此,在世界范围内,立法机构在不同的真值表中针对不同霉菌毒素颁布了国家适用的阈值。在此,实例为2006年的欧盟法规1881。此外,在此方面,欧盟内部对检测系统(例如ELISA)的最低要求通过2014年的法规519确定。因此,存在强烈需求研究食物或者饲料可能存在的霉菌毒素污染。这里,先前存在的问题在于,上面提到的微菌毒素的分子结构十分不同并且由此具有非常不同的特性,例如不同的溶解性。由此,疏水的黄曲霉素在水中是不溶的,而与此相对,伏马菌素和脱氧雪腐镰刀菌烯醇是溶于水的(图1)。因此,黄曲霉素必须使用水-甲醇或者水-乙醇混合物从试样中萃取出来。其它的霉菌毒素,如T2和HT2(图1)在其极性和可萃取性方面处于中间位置,但也需要溶剂,例如甲醇进行萃取。
目前为止,霉菌毒素的萃取使用有机溶剂,如乙醇,乙腈或者甲醇进行。然而这里会出现大量随后需清理的有机溶剂。作为其替代方案,开发出了这样的方法,在这些方法中,通过添加特定的物质,如环状糊精,含蛋白质的组分(如牛血清蛋白)或助溶剂(如非离子表面活性剂,如氚核(Triton)或者玻雷吉(Brij))可萃取出特定的霉菌毒素。这些不使用有机溶剂的方法通常在最近几年获得了专利(US2014/0356978;WO2015/188205;WO2016/057044)。然而在不用有机溶剂进行的萃取中,对赭曲霉毒素的萃取要么没有说明,要么由于在分子中所含的羧基官能团而要求使用特殊的缓冲剂(Mishra等,2016,FoodAdd.Contam.33:500-508)。所有相关霉菌毒素的萃取方法还没有得以说明。
由于所提及的缺点,这些方法并不是用于迅速、简便、环保并且经济地分析食物,例如谷物试样中不同霉菌毒素的存在的选择手段。
发明内容
本发明的目的在于,利用唯一一种萃取剂将非常不同的霉菌毒素尽可能统一地从食物和饲料中萃取出来。
由此引导出本发明的任务,即开发出一种萃取方法,利用该萃取方法可以将具有不同溶解性的霉菌毒素统一萃取出。
尤其引导出这样的目的,即,将黄曲霉毒素,脱氧雪腐镰刀菌烯醇,赭曲霉毒素A,玉米赤霉烯酮,伏马菌素和T2/HT2等霉菌毒素从谷物(玉米,小麦,黑麦,燕麦,大麦及其来自小麦族的亲属),饲料,坚果,大豆,玉米麸质和大米,还有无花果,枣,葡萄干和开心果中萃取出然后量化。
该目的通过根据权利要求1的方法解决。其它可能的实施形式从从属权利要求、说明书和实例中给出。
令人惊奇地发现,借助于水性的、经缓冲的萘化合物和/或苯化合物或其杂环类似物可以将疏水的以及亲水的霉菌毒素萃取出。
根据本发明的方法的特征在于,经缓冲处理的、具有至少一个磺酸基团或至少一个羧酸基团的萘化合物和/或苯化合物和/或其杂环类似物的溶液与谷物或者其它食物或者饲料相接触,随后将水性溶液分离并且确定所萃取出的霉菌毒素在水性溶液中的含量。
根据本发明,单独或者混合使用通式I的萘化合物或苯化合物或其杂环类似物,从而特别有效地萃取非常不同种类的霉菌毒素。
式I:
其中,X、R1和R2的意义如下:
X=萘基或苯基或其杂环类似物
R1=至少一个磺酸基团或者至少一个羧酸基团
R2=未取代或者选自下列的功能基团:羟基醚,烷基醚,烷氧基醚,氨基醚,巯基醚,卤素醚和硫醚,其在分子中处于相对于酸基的邻位、间位或者对位,其中排除α氨基酸。
下面列出可以萃取种类非常不同的霉菌毒素的实例:
1,5-萘二磺酸
2,6-萘二磺酸
4-羟基苯磺酸
苯磺酸盐,
4-甲基苯磺酸,
苯-1,3-二磺酸,
1-萘酚-3,6-二磺酸,
3-磺基苯甲酸,
4-磺基苯甲酸,
2-羟基苯甲酸,
2,6-二羟基苯甲酸,
2,5-二羟基苯甲酸,
2,4-二羟基苯甲酸,
3,4-二羟基苯甲酸,
3,5-二羟基苯甲酸,
2-羟基-5-磺基苯甲酸。
1,5-萘二磺酸,2,6-萘二磺酸和/或对羟基苯磺酸的单独以及组合使用证明是特别有利。
随后将所产生的含有所萃取的霉菌毒素的水性上清液分离并用于分析。由此可以例如利用酶催化、酶免疫、色谱支持的和/或免疫亲和色谱方法进行对霉菌毒素的确定或者进一步清洁。
对于所有接下来进行说明的试验,使用经认证的参照材料(表1)。对于每种霉菌毒素测量一个空白试样和一个污染的参照试样。
表1用于检测所要求的方法的萃取功效的、经认证的霉菌毒素参照材料
*借助于HPLC未检测到;参见Trilogy公司的认证以获取更详细的解释,如所使用方法的检测极限
**使用Trilogy公司(美国,密苏里,华盛顿)的材料
接下来以玉米中黄曲霉毒素的确定进一步说明本发明。参照萃取方法规定1g的试样称重,以5ml 70%的甲醇水溶液在震荡下萃取10min(表2)。在离心或者过滤后将上清液以蒸馏水进行1:7的稀释(例如100μl萃取物+600μl水)并且以市面上常见的商用ELISA定量黄曲霉毒素的含量。使用 Aflatoxin Total(商品号4701,R-Biopharm股份公司,达姆施塔特,参见表2)。基础依据是抗原抗体反应。微量滴定盘的凹陷以针对抗黄曲霉毒素抗体的捕捉抗体涂层。添加校准剂或萃取的试样溶液、酶标记黄曲霉毒素(酶共轭物)和抗黄曲霉毒素抗体。空白的和酶标记的黄曲霉毒素竞争黄曲霉毒素抗体连接位置。同时,抗黄曲霉毒素抗体也由固定捕捉抗体结合。未被结合的酶标记黄曲霉毒素随后重新在清洗步骤中去除。检定通过添加底物溶液/色原溶液来进行。所结合的酶共轭物将色原转化为蓝色的最终产物。终止剂的添加引起由蓝至黄的色变。测量在450nm下以光度测定进行;所测得的溶液光密度(OD)又与试样中的黄曲霉毒素的浓度成比例。
表2所使用的参照萃取和为与所要求保护的方法的萃取效果相比较所使用的ELISA系统
接下来示出用于从玉米中萃取黄曲霉毒素的优选方法作为实例。为此将1g均质化的玉米试样以5ml由250mM1,5-萘二磺酸组成的溶液缓冲处理至pH为8,与100mM三-羟基氨基甲烷(TRIS)混合,震荡10min并离心或过滤。将澄清的上清液用蒸馏水进行1:7的稀释并如上所述在ELISA中测量。
结果在表3中示出。这里需注意,出于可对比性原因,结果作为“%为单位的信号降低”给出。信号降低由作为测试系统的ELISA的竞争格式给出。对新的、所要求保护的方法和现存的参照萃取之间对比的萃取产率进行评估。萃取物的高分析浓度(由高萃取产率所致)导致测量信号(OD450nm)的降低。%为单位的信号降低的计算如下:
信号降低[%]=100–(OD阳性试样/OD阴性试样×100)
表3:使用所要求保护的物质1,5-萘二磺酸(在TRIS缓冲液中,pH 8.0)时玉米中黄曲霉素相比于参照萃取的萃取产率。OD值在市场可得的ELISA AflatoxinTotal中测得。
如果将参照萃取的值与所要求保护的萃取的值相比较,则会发现其非常相似。由此通过所要求保护的方法成功地定量地从基质玉米中萃取黄曲霉毒素。尤其是空白试样OD值微小的不同可以通过测量系统ELISA中所要求保护的物质的次级效应来解释。通过相互作用轻微干扰了萃取物(试样)中的黄曲霉毒素和黄曲霉毒素-酶-共轭物之间对于抗体连接位置的竞争。这也是为什么不探测浓度值,而是将结果以“信号降低[%]”给出的原因。这些浓度值会产生错误的图像,因为ELISA的影响并不是所要求保护的萃取的一部分。ELISA系统可以相应地设定为这些物质,由此给出了测量系统的正确性。
无关于上述实例,研究来自食物领域或饲料领域的其它试样也是可能的,例如小麦、黑麦、燕麦、大麦和小麦族的其它亲属、饲料、坚果、大豆、玉米麸质和大米。还有无花果,枣,葡萄干和开心果。
同样地,萃取不限制在上述黄曲霉素。由此,利用根据本发明的方法萃取脱氧雪腐镰刀菌烯醇,赭曲霉毒素A,玉米赤霉烯酮,伏马菌素和T2/HT2也是可能的。同样可以利用根据本发明的方法萃取其它毒素,例如麦角生物碱,桔霉素和杂色曲霉素。
研究表明,该方法的有效性受到合适的缓冲系统的正面影响。因此,萘基或苯基化合物或其杂环类似物的水溶液应在pH 5-10的范围内经缓冲处理。在pH7.5-8.5范围内的三-羟甲基-氨基甲烷(Tris)和含“咪唑”的缓冲液被认为是优选的(参见表3中的实施例)。通过以适当的比例混合异丙基咪唑和咪唑盐酸盐来制备“咪唑”缓冲液。其他缓冲系统,如磷酸盐和EPPS(N-(2-羟乙基)-哌嗪-N'-(3-丙磺酸)也是可能的,但根据浓度和霉菌毒素可能具有不同的萃取结果。
使用具有至少一个氮取代位的芳香类苯化合物或者萘化合物的萃取方法也是可能的。
相对于目前已知的萃取方法,该方法具有这样的优点,即,可以以环保的、水性萃取剂高效并尽可能完全地从相关的食品类别中萃取出所有重要的霉菌毒素。多种不同霉菌毒素的分析不再像传统的方法那样需要单独的试样称量和单独的萃取剂,而是仅需要一次试样称量和利用所要求保护的方法进行的通用萃取。由此为该新方法的使用者提供了显著的时间和材料节省,这会降低成本并且避免了暴露于传统方法的萃取剂中有害健康的溶剂。
接下来通过实施例进一步说明本发明。
具体实施方式
实例1
表4:与参照萃取(参照,参见表2)相比,在使用不同缓冲剂(100mM)和pH值的情况下,使用所要求保护的物质1,5-萘二磺酸(1,5-NDS,250mM),由玉米中萃取黄曲霉毒素的萃取产率。OD值在市场可得的ELISA Aflatoxin Total中测得。
实例2
表5:与参照萃取(参照,参见表2)相比,在使用不同缓冲剂(100mM)和pH值的情况下,使用所要求保护的物质2,6-萘二磺酸(2,6-NDS,125mM),由玉米中萃取黄曲霉毒素的萃取产率。OD值在市场可得的ELISA Aflatoxin Total中测得。
实例3
表6:与参照萃取(参照,参见表2)相比,在使用不同缓冲剂(100mM)和pH值的情况下,使用所要求保护的物质1,5-萘二磺酸(1,5-NDS,250mM),由小麦中萃取脱氧雪腐镰刀菌烯醇DON的萃取产率。OD值在市场可得的ELISA DON中测得。
实例4
表7:与参照萃取(参照,参见表2)相比,在使用不同缓冲剂(100mM)和pH值的情况下,使用所要求保护的物质2,6-萘二磺酸(2,6-NDS,125mM),由小麦中萃取脱氧雪腐镰刀菌烯醇DON的萃取产率。OD值在市场可得的ELISA DON中测得。
实例5
表8:
与参照萃取(参照,参见表2)相比,在使用不同缓冲剂(100mM)和pH值的情况下,使用所要求保护的物质1,5-萘二磺酸(1,5-NDS,250mM),由玉米中萃取赭曲霉毒素A的萃取产率。OD值在市场可得的ELISA Ochratoxin A 30/15中测得。
实例6
表9:与参照萃取(参照,参见表2)相比,在使用不同缓冲剂(100mM)和pH值的情况下,使用所要求保护的物质2,6-萘二磺酸(2,6-NDS,125mM),由玉米中萃取赭曲霉毒素A的萃取产率。OD值在市场可得的ELISA Ochratoxin A 30/15中测得。
实例7
表10:与参照萃取(参照,参见表2)相比,在使用不同缓冲剂(2,6-NDS,100mM)和pH值的情况下,使用所要求保护的物质1,5-萘二磺酸(1,5-NDS,250mM),由玉米中萃取玉米赤霉毒素的萃取产率。OD值在市场可得的ELISA Zearalenon中测得。
实例8
表11:与参照萃取(参照,参见表2)相比,在使用不同缓冲剂(2,6-NDS,100mM)和pH值的情况下,使用所要求保护的物质1,5-萘二磺酸(1,5-NDS,50mM)或者2,6-萘二磺酸,由玉米中萃取伏马菌素的萃取产率。OD值在市场可得的ELISA Fumonisin中测得。
实例9
表12:与参照萃取(参照,参见表2)相比,在使用不同缓冲剂(2,6-NDS,100mM)和pH值的情况下,使用所要求保护的物质1,5-萘二磺酸(1,5-NDS,250mM)或者2,6-萘二磺酸,由玉米中萃取T2和HT2的萃取产率。OD值在市场可得的ELISA T2/HT2中测得。
实例10
表13:与参照萃取(参照,参见表2)相比,在使用不同缓冲强度和pH值的情况下,使用所要求保护的物质1,5-萘二磺酸(1,5-NDS,250mM)或者2,6-萘二磺酸(2,6-NDS,100mM),由玉米中萃取黄曲霉毒素的萃取产率。OD值在市场可得的ELISA Aflatoxin Total中测得。
*N-(2-羟乙基)哌嗪-N‘-(3-丙烷磺酸)
实例11
表14:与参照萃取(参照,参见表2)相比,在pH为8.0(5mM磷酸盐)的情况下,使用不同浓度的所要求保护的物质1,5-萘二磺酸,由玉米中萃取黄曲霉毒素的萃取产率。OD值在市场可得的ELISA Aflatoxin Total中测得。
参照 | 300mM | 200mM | 100mM | 50mM | 10mM | |
OD<sub>空白</sub> | 1.320 | 1.516 | 1.611 | 1.601 | 1.596 | 1.601 |
OD<sub>阳性</sub> | 0.325 | 0.309 | 0.373 | 0.452 | 0.752 | 0.920 |
信号降低[%] | 75 | 80 | 77 | 72 | 53 | 43 |
实例12
表15:与参照萃取(参照,参见表2)相比,在pH为8.0(5mM磷酸盐)的情况下,使用不同浓度的所要求保护的物质2,6-萘二磺酸,由玉米中萃取黄曲霉毒素的萃取产率。OD值在市场可得的ELISA Aflatoxin Total中测得。
实例13
表16:与参照萃取(参照,参见表2)相比,在添加50mM的磷酸盐(pH8.0)的情况下,使用所要求保护的物质4-羟基苯磺酸(375mM;4-OH-PSN),对于脱氧雪腐镰刀菌烯醇、伏马菌素和玉米赤霉烯酮(真值表参见表1)的萃取产率。OD值在市场可得的ELISA系列中测得。
*DON,脱氧雪腐镰刀菌烯醇;Fumo,伏马菌素;Zea,玉米赤霉烯酮
实例14
表17:与参照萃取(参照,参见表2)相比,在添加50mM的磷酸盐(pH8.0)的情况下,使用所要求保护的物质4-羟基苯磺酸(375mM;4-OH-PSN),对于黄曲霉素、赭曲霉毒素和T2/HT2(真值表参见表1)的萃取产率。OD值在市场可得的ELISA 系列中测得。
*Afla,黄曲霉毒素;OTA,赭曲霉毒素
实例15
表18:与参照萃取(参照,参见表2)相比,在pH为8.0(5mM磷酸盐)的情况下,使用不同浓度的所要求保护的物质4-羟基苯磺酸,由玉米中萃取黄曲霉毒素的萃取产率。OD值在市场可得的ELISA Aflatoxin Total中测得。
实例16
表19:与参照萃取(参照,参见表2)相比,在pH为8.0(5mM磷酸盐)的情况下,使用不同浓度的所要求保护的物质4-羟基苯磺酸,由玉米中萃取赭曲霉毒素的萃取产率。OD值在市场可得的ELISA Ochratoxin A30/15中测得。
实例17
表20:与参照萃取(参照,参见表2)相比,在pH为8.0(5mM磷酸盐)的情况下,组合使用所要求保护的物质1,5-萘二磺酸(1,5-NDS,250mM)和4-羟基苯磺酸(从10mM至150mM的不同浓度),由玉米中萃取赭曲霉毒素的萃取产率。OD值在市场可得的ELISA Ochratoxin A30/15中测得。
实例18
表21:与参照萃取(参照,参见表2)相比,在7.5至9.0的不同pH情况下,组合使用所要求保护的物质1,5-萘二磺酸(1,5-NDS,250mM)和4-羟基苯磺酸(4-OH-PSN,50mM和75mM),由玉米中萃取赭曲霉毒素的萃取产率。OD值在市场可得的ELISA Ochratoxin A30/15中测得。
实例19
表22:与参照萃取(参照,参见表2)相比,在pH为8.0(5mM磷酸盐)的情况下,组合使用所要求保护的物质2,6-萘二磺酸(125mM)和4-羟基苯磺酸(从20mM和150mM的不同浓度),由玉米中萃取赭曲霉毒素的萃取产率。OD值在市场可得的ELISA Ochratoxin A30/15中测得。
参照 | 70mM | 60mM | 50mM | 40mM | 30mM | 20mM | |
OD<sub>空白</sub> | 1.952 | 1.213 | 1.421 | 1.457 | 1.474 | 1.555 | 1.647 |
OD<sub>阳性</sub> | 0.441 | 0.327 | 0.430 | 0.395 | 0.494 | 0.452 | 0.409 |
信号降低[%] | 77 | 73 | 70 | 73 | 67 | 71 | 75 |
实例20
表23:
与参照萃取(参照,参见表2)相比,在7.5至9.0的不同pH情况下,组合使用所要求保护的物质2,6-萘二磺酸(2,6-NDS,125mM)和4-羟基苯磺酸(4-OH-PSN,50mM和75mM),由玉米中萃取赭曲霉毒素的萃取产率。OD值在市场可得的ELISA OchratoxinA30/15中测得。
附图说明
图1:在食品工业和饲料工业中重要的霉菌毒素列表。在列表之外,也示出了其化学结构式和在水性环境中的溶解性。
Claims (11)
1.用于从谷物和其它食物或者从饲料中萃取霉菌毒素的方法,其特征在于,使用通式I的萘化合物和/或苯化合物和/或其杂环类似物的水性、经缓冲处理的溶液,
其中,X、R1和R2的意义如下
X=萘基或苯基或其杂环类似物
R1=至少一个磺酸基团或者至少一个羧酸基团
R2=未取代或者选自下列的功能基团:羟基醚,烷基醚,烷氧基醚,氨基醚,巯基醚,卤素醚和硫醚,其在分子中处于相对于酸基的邻位、间位或者对位,其中排除α氨基酸。
2.根据权利要求1所述的方法,其特征在于,使用一种或者多种萘化合物和/或苯化合物和/或其杂环类似物。
3.根据权利要求1至2中任意一项所述的方法,其特征在于,使用具有X=萘,R1=二磺酸或者X=苯,R1=磺酸和R2=羟基的化合物。
4.根据权利要求1至3中任意一项所述的方法,其特征在于,使用1,5-萘二磺酸,2,6-萘二磺酸和/或羟基苯磺酸。
5.根据权利要求1至4中任意一项所述的方法,其特征在于,所使用的萘化合物和/或苯化合物和/或其杂环类似物根据其最大溶解度以5至600mM的浓度来使用。
6.根据权利要求1至5中任意一项所述的方法,其特征在于,萘化合物和/或苯化合物和/或其杂环类似物以溶液、粉末形式或者片剂形式施加。
7.根据权利要求1至6中任意一项所述的方法,其特征在于,萘化合物和/或苯化合物和/或其杂环类似物的水性溶液在pH 5-10的范围内经缓冲处理。
8.根据权利要求1至7中任意一项所述的方法,其特征在于,所萃取出的霉菌毒素的含量在抗体支持的系统,如ELISA或者免疫层析系统中确定。
9.根据权利要求1至8中任意一项所述的方法,其特征在于,所萃取出的霉菌毒素的含量在色谱支持的系统中确定。
10.根据权利要求1至9中任意一项所述的方法,其特征在于,利用免疫亲和色谱方法对所萃取出的霉菌毒素进一步清洁。
11.根据权利要求1至10中任意一项所述的方法用于移除黄曲霉毒素B1,黄曲霉毒素B2,黄曲霉毒素G1,黄曲霉毒素G2,黄曲霉毒素M1,黄曲霉毒素M2,伏马菌素B1,伏马菌素B2,伏马菌素B3,脱氧雪腐镰刀菌烯醇,赭曲霉毒素A,玉米赤霉烯酮,T-2,HT-2,桔霉素,柄曲霉素和麦角生物碱的应用。
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