Method for extracting mycotoxins from grain, other food products and animal feed

Abstract

The invention relates to a method for extracting mycotoxins from grain and other food products or from feed and its subsequent quantification. Fields of application are the food industry, the animal feed industry or biotechnology. The objective of the present invention is to develop an extraction method with which it is possible to uniformly extract mycotoxins with different dissolving properties. It was 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.

Claims

1. A method for extracting one or more mycotoxins from grain, a food product or animal feed, wherein an aqueous, buffered solution comprising one or more naphthyl and/or phenyl compounds and/or their heterocyclical analogues of formula I is brought into contact with the grain, food product or animal feed, and the aqueous solution is then separated, ##STR00002## wherein: X comprises one or more napthyl and/or phenyl compounds and/or their heterocyclical analouges, R1 is at least one sulphonic acid group or at least one carbonic acid group, and R2 is H or selected from a functional group of hydroxy, alkyl, alkoxy, amino, sulfhydryl, halogen and thioether, which are arranged in the o, m or p position in relation to the acid group in the molecule, wherein α-amino acids are excluded.

2. The method of claim 1, wherein X is naphthyl, R1 is disulphonic acid and R2 is hydroxy, or X is phenyl, R1 is sulphonic acid and R2 is hydroxy.

3. The method according to claim 1, wherein the one or more naphthyl and/or phenyl compounds and/or their heterocyclical analogues are selected from the group consisting of 1,5-naphthyl disulphonic acid, 2,6-naphthyl disulphonic acid and hydroxyphenyl sulphonic acid.

4. The method according to claim 1, wherein the one or more naphthyl and/or phenyl compounds and/or their heterocyclical analogues are present in a concentration of 5 to 600 mM of each compound.

5. The method according to claim 1, wherein the one or more naphthyl and/or phenyl compounds and/or their heterocyclical analogues are in a solution, in powder or in tablet form.

6. The method according to claim 1, wherein the buffered, aqueous solution of the one or more naphthyl and/or phenyl compounds and/or their heterocyclical analogues are in the range of pH 5-10.

7. The method according to claim 1, wherein the one or more extracted mycotoxins are subjected to further purification using immunoaffinity chromatography columns.

8. The method according to claim 1, wherein the one or more mycotoxins being extracted are selected from the group consisting of aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1, aflatoxin M2, fumonisin B1, fumonisin B2, fumonisin B3, deoxynivalenol, ochratoxin A, zearalenone, T-2, HT-2, citrinin, sterigmatocystin ergot alkaloids and mixtures thereof.

9. The method according to claim 1, wherein the separated aqueous solution is analyzed by determining a concentration of the one or more extracted mycotoxins in the aqueous solution.

10. The method according to claim 9, wherein the concentration of the one or more extracted mycotoxins is determined in an antibody-supported system.

11. The method according to claim 10, wherein the antibody-supported system is an ELISA or a lateral flow systems.

12. The method according to claim 9, wherein the concentration of the one or more extracted mycotoxins is determined in a chromatograph-supported system.

Description

EXAMPLE 1

(1) TABLE-US-00004 TABLE 4 Extraction yield of aflatoxin from maize using the claimed substances 1,5-naphthyl disulphonic acid (1,5-NDS, 250 mM) using different buffers (100 mM) and pH values compared to reference extraction (ref., see also Table 2). The OD value was calculated in the commercially available ELISA RIDASCREEN ® Aflatoxin Total. 1,5-NDS 1,5-NDS 1,5-NDS 1,5-NDS Tris Tris Imidazol Imidazol Ref. pH 8.0 pH 8.5 pH 8.0 pH 8.5 OD.sub.Blank 1.591 1.966 1.917 1.684 1.578 OD.sub.Positive 0.474 0.525 0.491 0.401 0.326 Signal 71 73 74 76 79 reduction [%]

EXAMPLE 2

(2) TABLE-US-00005 TABLE 5 Extraction yield of aflatoxin from maize using the claimed substances 2,6-naphthyl disulphonic acid (2,6-NDS, 125 mM) using different buffers (100 mM) and pH values compared to reference extraction (ref., see also Table 2). The OD value was calculated in the commercially available ELISA RIDASCREEN ® Aflatoxin Total. 2,6-NDS 2,6-NDS 2,6-NDS 2,6-NDS Tris Tris Imidazol Imidazol Ref. pH 8.0 pH 8.5 pH 8.0 pH 8.5 OD.sub.Blank 1.505 1.686 1.711 1.654 1.645 OD.sub.Positive 0.377 0.435 0.356 0.403 0.393 Signal 75 74 79 76 76 reduction [%]

EXAMPLE 3

(3) TABLE-US-00006 TABLE 6 Extraction yield of aflatoxin from maize using the claimed substances 1,5-naphthyl disulphonic acid (1,5-NDS, 250 mM) using different buffers (100 mM) and pH values compared to reference extraction (ref., see also Table 2). The OD value was calculated in the commercially available ELISA RIDASCREEN ® DON. 1,5-NDS 1,5-NDS 1,5-NDS 1,5-NDS Tris Tris Imidazol Imidazol Ref. pH 8.0 pH 8.5 pH 8.0 pH 8.5 OD.sub.Blank 2.440 2.682 2.722 2.649 2.638 OD.sub.Positive 0.738 0.795 0.842 0.744 0.811 Signal 70 70 69 72 69 reduction [%]

EXAMPLE 4

(4) TABLE-US-00007 TABLE 7 Extraction yield of aflatoxin from maize using the claimed substances 2,6-naphthyl disulphonic acid (2,6-NDS, 125 mM) using different buffers (100 mM) and pH values compared to reference extraction (ref., see also Table 2). The OD value was calculated in the commercially available ELISA RIDASCREEN ® DON. 2,6-NDS 2,6-NDS 2,6-NDS 2,6-NDS Tris Tris Imidazol Imidazol Ref. pH 8.0 pH 8.5 pH 8.0 pH 8.5 OD.sub.Blank 2.463 2.694 2.667 2.610 2.657 OD.sub.Positive 0.886 0.923 0.923 0.871 0.906 Signal 64 66 65 67 66 reduction [%]

EXAMPLE 5

(5) TABLE-US-00008 TABLE 8 Extraction yield of aflatoxin from maize using the claimed substances 1,5-naphthyl disulphonic acid (1,5-NDS, 250 mM) using different buffers (100 mM) and pH values compared to reference extraction (ref., see also Table 2). The OD value was calculated in the commercially available ELISA RIDASCREEN ® Ochratoxin A 30/15. 1,5-NDS 1,5-NDS 1,5-NDS 1,5-NDS Tris Tris Imidazol Imidazol Ref. pH 8.0 pH 8.5 pH 8.0 pH 8.5 OD.sub.Blank 1.551 1.447 1.374 1.479 1.277 OD.sub.Positive 0.305 0.338 0.306 0.366 0.296 Signal 80 77 78 75 77 reduction [%]

EXAMPLE 6

(6) TABLE-US-00009 TABLE 9 Extraction yield of ochratoxin A from maize using the claimed substances 2,6-naphthyl disulphonic acid (2,6- NDS, 125 mM) using different buffers (100 mM) and pH values compared to reference extraction (ref., see also Table 2). The OD value was calculated in the commercially available ELISA RIDASCREEN ® Ochratoxin A 30/15. 2,6-NDS 2,6-NDS 2,6-NDS 2,6-NDS Tris Tris Imidazol Imidazol Ref. pH 8.0 pH 8.5 pH 8.0 pH 8.5 OD.sub.Blank 1.551 1.602 1.462 1.425 1.374 OD.sub.Positive 0.305 0.407 0.309 0.324 0.263 Signal 80 75 79 77 81 reduction [%]

EXAMPLE 7

(7) TABLE-US-00010 TABLE 10 Extraction yield of zearalenone from maize using the claimed substances 1,5-naphthyl disulphonic acid (1,5-NDS, 250 mM) or 2,6-naphthyl disulphonic acid using different buffers (2,6- NDS, 100 mM) and pH values compared to reference extraction (ref., see also Table 2). The OD value was calculated in the commercially available ELISA RIDASCREEN ® zearalenone. 1,5-NDS 1,5-NDS 2,6-NDS 2,6-NDS Tris Imidazol Tris Imidazol Ref. pH 8.0 pH 8.5 pH 8.0 pH 8.0 OD.sub.Blank 3.093 2.594 2.540 2.403 2.214 OD.sub.Positive 0.227 0.355 0.268 0.245 0.301 Signal 93 86 89 90 86 reduction [%]

EXAMPLE 8

(8) TABLE-US-00011 TABLE 11 Extraction yield of fumonisin from maize using the claimed substances 1,5-naphthyl disulphonic acid (1,5-NDS, 50 mM) or 2,6-naphthyl disulphonic acid using different buffers (2,6- NDS, 100 mM) and pH values compared to reference extraction (ref., see also Table 2). The OD value was calculated in the commercially available ELISA RIDASCREEN ® fumonisin. 1,5-NDS 1,5-NDS 2,6-NDS 2,6-NDS Tris Imidazol Tris Imidazol Ref. pH 8.0 pH 8.0 pH 8.0 pH 8.0 OD.sub.Blank 1.193 0.719 0.762 0.874 0.881 OD.sub.Positive 0.339 0.174 0.151 0.202 0.188 Signal 72 76 80 77 79 reduction [%]

EXAMPLE 9

(9) TABLE-US-00012 TABLE 12 Extraction yield of T2 and HT2 from maize using the claimed substances 1,5-naphthyl disulphonic acid (1,5-NDS, 250 mM) or 2,6-naphthyl disulphonic acid using different buffers (2,6- NDS, 100 mM) and pH values compared to reference extraction (ref., see also Table 2). The OD value was calculated in the commercially available ELISA RIDASCREEN ® T2/HT2. 1,5-NDS 1,5-NDS 2,6-NDS 2,6-NDS Tris Imidazol Tris Imidazol Ref. pH 8.0 pH 8.0 pH 8.0 pH 8.0 OD.sub.Blank 1.676 0.947 0.921 0.868 0.799 OD.sub.Positive 0.467 0.289 0.282 0.270 0.265 Signal 72 69 69 69 67 reduction [%]

EXAMPLE 10

(10) TABLE-US-00013 TABLE 13 Extraction yield of aflatoxin from maize using the claimed substances 1,5-naphthyl disulphonic acid (1,5-NDS, 250 mM) or 2,6-naphthyl disulphonic acid (2,6-NDS, 100 mM) and pH values compared to reference extraction (ref., see also Table 2). The OD value was calculated in the commercially available ELISA RIDASCREEN ® Aflatoxin Total. 1,5-NDS 1,5-NDS 2,6-NDS 2,6-NDS Phosphate Epps* Phosphate Phosphate 200 mM 100 mM 25 mM 75 mM Ref. pH 8.0 pH 8.5 pH 8.0 pH 8.5 OD.sub.Blank 1.487 1.542 1.607 1.474 1.445 OD.sub.Positive 0.326 0.303 0.350 0.339 0.350 Signal 78 80 78 77 76 reduction [%] *(N-(2-hydroxyethyl)-piperazine-N′-(3-propane sulphonic acid)

EXAMPLE 11

(11) TABLE-US-00014 TABLE 14 Extraction yield of aflatoxin from maize using the claimed substance 1,5-naphthyl disulphonic acid in different concentrations at pH 8.0 (5 mM phosphate) compared to reference extraction (ref., see also Table 2). The OD value was calculated in the commercially available ELISA RIDASCREEN ® Aflatoxin Total. 300 Ref. mM 200 mM 100 mM 50 mM 10 mM OD.sub.Blank 1.320 1.516 1.611 1.601 1.596 1.601 OD.sub.Positive 0.325 0.309 0.373 0.452 0.752 0.920 Signal 75 80 77 72 53 43 reduction [%]

EXAMPLE 12

(12) TABLE-US-00015 TABLE 15 Extraction yield of aflatoxin from maize using the claimed substance 2,6-naphthyl disulphonic acid in different concentrations at pH 8.0 (5 mM phosphate) compared to reference extraction (ref., see also Table 2). The OD value was calculated in the commercially available ELISA RIDASCREEN ® Aflatoxin Total. Ref. 100 mM 75 mM 50 mM 10 mM 5 mM OD.sub.Blank 1.544 1.860 1.854 1.881 1.962 1.973 OD.sub.Positive 0.438 0.489 0.521 0.620 1.101 1.051 Signal 72 74 72 67 44 47 reduction [%]

EXAMPLE 13

(13) TABLE-US-00016 TABLE 16 Extraction yield of deoxynivalenol, fumonisin and zearalenone (for matrices see Table 1) using the claimed substance 4-hydroxyphenyl sulphonic acid (375 mM; 4-OH-PSN) with the addition of 50 mM phosphate (pH 8.0) compared to the reference extraction (ref., see also Table 2). The OD value was calculated in the commercially available ELISA RIDASCREEN ® series (see Table 2). DON DON Fumo* Fumo* Zea* Zea* ref. 4-OH-PSN ref. 4-OH-PSN ref. 4-OH-PSN OD.sub.Blank 2.337 2.577 1.261 0.814 3.012 2.338 OD.sub.Positive 0.786 0.824 0.398 0.195 0.195 0.294 Signal 66 68 68 76 94 87 reduction [%] *DON, deoxynivalenol; fumo, fumonisin; zea, zearalenone

EXAMPLE 14

(14) TABLE-US-00017 TABLE 17 Extraction yield of aflatoxin, ochratoxin and T2/HT2 (for matrices see Table 1) using the claimed substance 4-hydroxyphenyl sulphonic acid (375 mM; 4-OH- PSN) with the addition of 50 mM phosphate (pH 8.0) compared to the reference extraction (ref., see also Table 2). The OD value was calculated in the commercially available ELISA RIDASCREEN ® series (see Table 2). Afla* Afla* OTA* OTA* T2/HT2 T2/HT2 ref. 4-OH-PSN ref. 4-OH-PSN ref. 4-OH-PSN OD.sub.Blank 1.701 1.821 1.551 1.557 1.889 0.965 OD.sub.Positive 0.451 0.673 0.305 0.308 0.525 0.342 Signal 73 63 80 80 72 65 reduction [%] *Afla, aflatoxin; OTA, ochratoxin

EXAMPLE 15

(15) TABLE-US-00018 TABLE 18 Extraction yield of aflatoxin from maize using the claimed substance 4- naphthyl disulphonic acid in different concentrations at pH 8.0 (5 mM phosphate) compared to reference extraction (ref., see also Table 2). The OD value was calculated in the commercially available ELISA RIDASCREEN ® Aflatoxin Total. Ref. 600 mM 500 mM 400 mM 300 mM 200 mM 100 mM OD.sub.Blank 1.485 1.692 1.707 1.712 1.722 1.692 1.649 OD.sub.Positive 0.440 0.512 0.533 0.547 0.568 0.676 0.855 Signal 70 70 69 68 67 60 48 reduction [%]

EXAMPLE 16

(16) TABLE-US-00019 TABLE 19 Extraction yield of aflatoxin from maize using the claimed substance 4- naphthyl disulphonic acid in different concentrations at pH 8.0 (5 mM phosphate) compared to reference extraction (ref., see also Table 2). The OD value was calculated in the commercially available ELISA RIDASCREEN ® Ochratoxin A 30/15. Ref. 600 mM 500 mM 400 mM 300 mM 200 mM 50 mM OD.sub.Blank 1.620 1.308 1.365 1.345 1.383 1.475 1.692 OD.sub.Positive 0.239 0.264 0.229 0.264 0.241 0.267 0.309 Signal 85 80 83 80 83 82 82 reduction [%]

EXAMPLE 17

(17) TABLE-US-00020 TABLE 20 Extraction yield of ochratoxin from maize with combined use of the claimed substances 1,5-naphthyl disulphonic acid (1,5-NDS, 250 mM) and 4-hydroxy phenyl sulphonic acid (different concentrations of 10 mM to 150 mM) at pH 8.0 (5 mM phosphate) compared to reference extraction (ref., see also Table 2). The OD value was calculated in the commercially available ELISA RIDASCREEN ® Ochratoxin A 30/15. 250 mM 1,5-NDS Ref. 150 mM 100 mM 75 mM 50 mM 20 mM 10 mM OD.sub.Blank 1.551 1.120 1.231 1.360 1.352 1.494 1.495 OD.sub.Positive 0.305 0.330 0.375 0.321 0.355 0.422 0.485 Signal 80 71 70 76 74 72 68 reduction [%]

EXAMPLE 18

(18) TABLE-US-00021 TABLE 21 Extraction yield of ochratoxin from maize with combined use of the claimed substances 1,5-naphthyl disulphonic acid (1,5-NDS, 250 mM) and 4-hydroxy phenyl sulphonic acid (4-OH-PSN, 50 mM and 75 mM) at different pH values of 7.5 to 9.0 compared to reference extraction (ref., see also Table 2). The OD value was calculated in the commercially available ELISA RIDASCREEN ® Ochratoxin A 30/15. 250 mM 1,5-NDS 50 mM 4-OH-PSN 75 mM 4-OH-PSN pH pH pH pH pH pH pH pH Ref. 7.5 8.0 8.5 9.0 7.5 8.0 8.5 9.0 OD.sub.Blank 1.551 1.342 1.249 1.210 1.265 1.244 1.223 1.139 1.108 OD.sub.Positive 0.305 0.348 0.292 0.205 0.240 0.379 0.358 0.278 0.257 Signal 80 74 77 83 81 70 71 76 77 reduction [%]

EXAMPLE 19

(19) TABLE-US-00022 TABLE 22 Extraction yield of ochratoxin from maize with combined use of the claimed substances 2,6-naphthyl disulphonic acid (125 mM) and 4-hydroxy phenyl sulphonic acid (different concentrations of 20 mM to 150 mM) at pH 8.0 (5 mM phosphate) compared to reference extraction (ref., see also Table 2). The OD value was calculated in the commercially available ELISA RIDASCREEN ® Ochratoxin A 30/15. Ref. 70 mM 60 mM 50 mM 40 mM 30 mM 20 mM OD.sub.Blank 1.952 1.213 1.421 1.457 1.474 1.555 1.647 OD.sub.Positive 0.441 0.327 0.430 0.395 0.494 0.452 0.409 Signal 77 73 70 73 67 71 75 reduction [%]

EXAMPLE 20

(20) TABLE-US-00023 TABLE 23 Extraction yield of ochratoxin from maize with combined use of the claimed substances 2,6-naphthyl disulphonic acid (2,6-NDS, 125 mM) and 4-hydroxy phenyl sulphonic acid (4-OH-PSN, 50 mM and 75 mM) at different pH values of 7.5 to 9.0 compared to reference extraction (ref., see also Table 2). The OD value was calculated in the commercially available ELISA RIDASCREEN ® Ochratoxin A 30/15. 125 mM 2,6-NDS 50 mM 4-OH-PSN 75 mM 4-OH-PSN pH pH pH pH pH pH pH pH Ref. 7.5 8.0 8.5 9.0 7.5 8.0 8.5 9.0 OD.sub.Blank 1.986 1.698 1.608 1.558 1.477 1.562 1.479 1.405 1.416 OD.sub.Positive 0.484 0.408 0.292 0.358 0.266 0.424 0.342 0.281 0.329 Signal 76 76 82 77 82 73 77 80 77 reduction [%]

LEGEND FOR THE FIGURES

(21) FIG. 1: A list of mycotoxins relevant to the food and animal feed industries are shown. Next to the list, their chemical structural formulae and their dissolving properties in an aqueous environment are shown.