Patent Grant
11627750
This application is a national stage of international application no. PCT/EP2017/000721, filed on Jun. 21, 2017, and claims the benefit of priority under 35 USC 119 of EP application no. 16001588.9, filed on Jul. 18, 2016.
The invention relates to a method for extracting mycotoxins from grain and other food products or from animal feed and its subsequent quantification. Fields of application are the food industry, the animal feed industry or biotechnology.
Mycotoxins are secondary metabolic products formed by mould fungi that can already have a toxic effect among vertebrae even in tiny quantities. Currently, around 200 different mycotoxins are known, which are formed by over 300 types of fungus. The term “mycotoxins” comprises a series of chemical compounds with different structures and effects, which can be classified as the following substance groups: Aflatoxins, ochratoxins, trichothecenes such as deoxynivalenol, fumonisins, alternaria toxins, fusarium toxins, ergot alkaloids.
Of particular importance in the food and animal feed industries are mycotoxins of the aflatoxin group, the fumonisin group, ochratoxin A, deoxynivalenol (DON), HT-2-toxin (or HT2), T-2-toxin (or T2) and zearalenone. Due to their extremely high hazard potential and broad dissemination, the tiniest quantities in food products or animal feed can already acutely or chronically impair the health of humans and animals. For this reason, legislators worldwide have set limit values for the different mycotoxins in different matrices, which are valid at national level. As an example, reference is made to EU directive 1881 from 2006. Further, in this context, minimal standards for test systems (e.g. ELISA) were determined in the EU by directive 519 from 2014. For this reason, there is an extraordinarily high level of interest in examining food products or animal feed for any mycotoxin contamination. Here, the fact has proven to be problematic in the past that the aforementioned mycotoxins are extremely heterogeneous in terms of their molecular structure, and therefore have a wide range of properties such as different dissolving properties. Thus, the hydrophobic aflatoxins are indissoluble in water, while by contrast, the fumonisins and deoxynivalenol are soluble in water (
To date, mycotoxins have been extracted using organic solvents such as ethanol, acetonitrile or methanol. However, large quantities of organic solvents are created during the process, which must then be disposed of. As an alternative, methods have been developed in which certain mycotoxins can be extracted through the addition of specific substances such as cyclodextrins, components containing proteins (e.g. cattle serum albumin) or solubilising agents (e.g. non-ionic tensides such as triton or Brij). Such methods without organic solvents have frequently been patented in recent years (US 2014/0356978; WO 2015/188205; WO 2016/057044). However, with regard to extractions without organic solvents, the extraction of ochratoxin is either not described or requires the use of a special buffer due to the carboxyl function contained in the molecule (Mishra et al., 2016, Food Add.Contam. 33: 500-508). An extraction method for all relevant mycotoxins has not yet been described.
Due to the aforementioned disadvantages, these methods are not the agent of choice in order to analyse food products such as grain samples for the presence of a wide range of mycotoxins quickly, simply, in an environmentally friendly manner and at low cost.
The goal of the invention is to extract a wide range of mycotoxins (
From this, the objective of the present invention is derived of developing an extraction method with which it is possible to uniformly extract mycotoxins with different dissolving properties.
In particular, this results in the objective of extracting the mycotoxins aflatoxin, deoxynivalenol, ochratoxin A, zearalenone, fumonisin and T2/HT2 from grain (maize, wheat, rye, oats, barley and their relatives from the triticeae tribe), animal feed, nuts, soya, maize gluten and rice, as well as figs, dates, raisins and pistachios, and then to quantify them.
This objective is attained by means of a method according to claim 1. Further possible embodiments are presented in the subclaims, the description and the examples.
Surprisingly, it has been found that with the aid of aqueous, buffered naphthyl and/or phenyl compounds or their heterocyclical analogues, both hydrophobic and hydrophilic mycotoxins can be extracted.
The method according to the invention is characterized in that the buffered solutions of naphthyl and/or phenyl compounds and/or their heterocyclical analogues, which carry at least one sulphonic acid or at least one carbonate acid group, are brought into contact with the grain or other food products or animal feed, the aqueous solution is then separated and the content of the extracted mycotoxins in the aqueous solution is determined.
According to the invention, naphthyl or phenyl compounds or their heterocyclical analogues of the general formula I are used individually or as a mixture in order to particularly effectively extract mycotoxins of a wide range of different groups.
in which X, R1 and R2 have the following meaning:
Examples are listed below that are capable of extracting mycotoxins of a wide range of different groups:
The sole but also combined use of 1,5-naphtyldisulphonic acid, 2,6-naphthyldisulphonic acid and/or p-hydroxyphenyl sulphonic acid emerged as being particularly advantageous.
The result aqueous supernatant, which contains the extracted mycotoxins, is then separated and used for analysis. In this way, the determination or further purification of the mycotoxins can be achieved, for example with the aid of enzymatic, enzyme immunological, chromatographically supported and/or immuno-affinity chromatographic methods.
Certified reference materials (Table 1) are used for all tests described below. For each mycotoxin, a blank sample and a contaminated reference sample are measured.
The invention will now be explained with reference to the determination of aflatoxin in maize. The reference extraction method provides for a sample weigh-in of 1 g, which is extracted with 5 ml 70% methanol in water for 10 min while shaking (Table 2). After centrifugation or filtration, the supernatant is diluted 1:7 with distilled water (e.g. 100 μL extract+600 μL water) and the aflatoxin content is quantified in a standard commercial ELISA. The RIDASCREEN® Aflatoxin Total (art. no. 4701, R-Biopharm AG, Darmstadt, see also Table 2) is used. The basis is the antigen-antibody reaction. The recesses in the microtitre plates are coated against anti-aflatoxin antibodies with capture antibodies. Calibrators or extracted sample solution, enzyme-marked aflatoxin (enzyme conjugate) and anti-aflatoxin antibodies are added. Free and enzyme-marked aflatoxin compete for the aflatoxin antibody binding sites. At the same time, the anti-aflatoxin antibodies are also bound by the immobilised capture antibodies. Non-bound enzyme-marked aflatoxin is then removed again in a washing step. Verification is conducted by adding substrate/chromogenic solution. Bound enzyme conjugate converts the chromogen into a blue end product. The addition of the stop reagent leads to a colour change from blue to yellow. The measurement is conducted photometrically at 450 nm; the measured optical density (OD) of the solution is conversely proportional to the aflatoxin concentration in the sample.
As an example, a preferred method for the extraction of aflatoxin from maize is described below. For this purpose, 1 g of homogenised maize sample with 5 ml of a solution consisting of 250 mM 1,5-naphthyl disulphonic acid buffered to pH 8 is mixed with 100 mM tris-(hydroxymethyl)-aminomethane (tris), shaken for 10 min and centrifuged or filtered. The clear supernatant is diluted 1:7 with distilled water and measured in the ELISA as already described above.
The results are shown in Table 3. Here, it should be noted that for comparability reasons, the results are given as a “signal reduction in %”. The signal reduction results from the competitive format of the ELISA as a test system. The extraction yield should be evaluated as a comparison between the new, claimed method and the established reference extraction. A high analyte concentration in the extract (caused by a high extraction yield) leads to a reduction of the measurement signal (OD450 nm). The signal reduction in [%] is calculated as follows:
Signal reduction[%]=100−(ODpositive sample/ODnegative sample×100)
If one compares the values of the reference extraction with those of the claimed extraction, it becomes clear that these are very similar. Aflatoxin was thus successfully quantitatively extracted from the matrix maize using the claimed method. Slight differences in the OD values, above all of the blank samples, can be explained by secondary effects of the claimed substances in the ELISA measurement system. Through interactions, the competition for antibody binding sites between the aflatoxin from the extract (sample) and aflatoxin-enzyme conjugate is slightly disrupted. This is also the reason why no concentration values have been determined, but the results are instead given as “signal reduction [%]”. This would create a false picture, since the influencing of the ELISA is not a part of the claimed extraction. The ELISA systems can accordingly be set to such substances, so that the accuracy of the measurement system is provided.
Independently of the example given above, it is however also possible to study other samples from the food or animal feed area such as wheat, rye, oats, barley and their relatives from the triticeae tribe, animal feed, nuts, soya, maize gluten and rice. Furthermore, figs, dates, raisins and pistachios are also of interest.
Equally, the extraction is not limited to the above aflatoxins. Thus, it is also possible to extract deoxynivalenol, ochratoxin A, zearalenone, fumonisin and T2/HT2 with the method according to the invention. Equally, other toxins such as ergot alkaloids, citrinin and sterigmatocystin can be extracted with the method according to the invention.
Studies have shown that the effectiveness of the method is positively influenced by a suitable buffer system. Thus, the aqueous solution of naphthyl and/or phenyl compounds and/or their heterocyclical analogues should be buffered in the range of pH 5-10. Here, buffers containing tris-(hydroxy methyl)-aminomethane (tris) and “Imidazol” are preferred in the region of pH 7.5-8.5 (see example in Table 3). The “Imidazol” buffer was prepared by mixing the isopropylimidazol and Imidazol hydrochloride in the corresponding proportions. Other buffer systems such as phosphate and EPPS N-(2-hydroxyethyl)-piperazine-N′-(3-propane sulphonic acid) are also possible, but can have different extraction results depending on the concentration and mycotoxin.
An extraction method is also possible in which aromatic phenyl or naphthyl compounds are used with at least on position substituted with nitrogen.
The method has the advantage over the extraction methods known to date that all important mycotoxins can be extracted efficiently and almost entirely from relevant food product matrices with an environmentally friendly, aqueous extraction agent. Unlike the standard methods, the analysis of several different mycotoxins no longer requires separate sample weigh-ins and individual extraction agents, but simply a single sample weigh-in and universal extraction with the claimed method. This results in significant time and material savings for the user of this new method, which leads to a cost reduction and the avoidance of exposure to solvents from the extraction agents of standard methods, which are harmful to health.
The invention will now be explained in greater detail with reference to illustrative embodiments.
Illustrative Embodiments
| Number | Date | Country | Kind |
|---|---|---|---|
| 16001588 | Jul 2016 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/000721 | 6/21/2017 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/014988 | 1/25/2018 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20050165076 | Ammermann | Jul 2005 | A1 |
| 20090110563 | Takita | Apr 2009 | A1 |
| 20090124678 | Semar | May 2009 | A1 |
| 20090269859 | Liu | Oct 2009 | A1 |
| 20130203613 | Burmeister | Aug 2013 | A1 |
| 20140356978 | Jabour et al. | Dec 2014 | A1 |
| 20160278405 | Sarver, Jr. | Sep 2016 | A1 |
| 20170099862 | Binder et al. | Apr 2017 | A1 |
| Number | Date | Country |
|---|---|---|
| 2962710 | Apr 2016 | CA |
| M1931079 | Nov 1994 | IT |
| 2014114982 | Jul 2014 | WO |
| 2015188205 | Dec 2015 | WO |
| 2016057044 | Apr 2016 | WO |
| Entry |
|---|
| Wacoo et al, “Methods for Detection of Aflatoxins in Agricultural Food Crops”, Hindawi Publishing Corporation, Journal of Applied Chemistry, vol. 2014, Article ID 706291, Published Aug. 25, 2014. (Year: 2014). |
| English Translated Abstract of Fiorenzo et al Patent Publication ITM1931079, Published Nov. 26, 1994. (Year: 1994). |
| International Search Report (PCT/ISA/210) dated Aug. 28, 2017, by the European Patent Office as the International Searching Authority for International Application No. PCT/EP2017/000721. |
| Written Opinion (PCT/ISA/237) dated Aug. 28, 2017, by the European Patent Office as the International Searching Authority for International Application No. PCT/EP2017/000721. |
| Number | Date | Country | |
|---|---|---|---|
| 20190281870 A1 | Sep 2019 | US |
