Antimycotoxin composition

Abstract

The invention relates to the use of a composition comprising at least one strain of Bacillus subtilis, for the degradation of mycotoxins.

Claims

1. A method for the treatment of animals suffering from a pathology related to the ingestion of at least one mycotoxin, or the prevention of said pathologies, the method comprising a step of administering to an animal in need thereof, a composition comprising or consisting essentially of strain NOL03, said strain being filed with the CNCM under number CNCM I-4607.

2. The method according to claim 1, wherein said composition comprises said strain NOL03, and at least one of the following strains, Strain NOL01, filed with the CNCM under number CNCM I-4606, Strain NOL02, filed with the CNCM under number CNCM I-5043, and Strain NOL04, filed with the CNCM under number CNCM I-4608.

3. The method according to claim 1, wherein said pathologies are related to the ingestion of at least two mycotoxins with different structures.

4. The method according to claim 1, where wherein said composition comprises Strain NOL03 deposited with the CNCM under number CNCM I-4607, Strain NOL01 deposited with the CNCM under number CNCM I-4606, and Strain NOL02 deposited with the CNCM under number CNCM I-5043.

5. The method according to claim 1, wherein said at least one mycotoxin is chosen from aflatoxins B1, B2, M1, G1 and G2, toxin T-2, toxin HT-2, deoxynivalenol (or nivalenol), zearalenone, a fumonisin B family mycotoxin, and ochratoxin A.

6. The method according to claim 5, wherein said fumonisin B family mycotoxin is fumonisin B1.

7. The method according to claim 3, wherein said at least two mycotoxins comprise deoxynivalenol (DON) and a fumonisin B family mycotoxin.

8. The method according to claim 4, wherein said at least one mycotoxin is selected from deoxynivalenol (DON) and a fumonisin B family mycotoxin.

9. The method according to claim 8, wherein said fumonisin B family mycotoxin is fumonisin B1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a graph of the growth of NOL01 as a function of different concentrations of ZEA in the medium (0, 0.1, 0.5, 1 and 10 ppm). The x-axis shows the time in hours and the y-axis shows the absorbance of the solution correlated with the growth of the bacteria.

(2) FIG. 2 shows a graph of the growth of NOL02 as a function of different concentrations of ZEA in the medium (0, 0.1, 0.5, 1, 5 and 10 ppm). The x-axis shows the time in hours and the y-axis shows the absorbance of the solution correlated with the growth of the bacteria.

(3) FIG. 3 shows a graph of the growth of NOL03 as a function of different concentrations of ZEA in the medium (0, 0.1, 0.5, 1, 5 and 10 ppm). The x-axis shows the time in hours and the y-axis shows the absorbance of the solution correlated with the growth of the bacteria.

(4) FIG. 4 shows the percentage of breakdown of the ZEA by strains NOL01 (black), NOL02 (white) and NOL03 (gray) as a function of the temperature in degrees Celsius.

(5) FIG. 5 shows the percentage of breakdown of the ZEA by strains NOL01 (black), NOL02 (white) and NOL03 (gray) as a function of pH of the medium.

(6) FIG. 6 shows a histogram showing the number of bacteria (log CFU/mL) as a function of time (0, 16 and 72 h) of bacteria NOL01 (black bar), NOL02 (dark gray bar), NOL03 (light gray bar) and NOL04 (white bar) cultivated without ZEA toxins, and bacteria NOL01 (bar with diagonal crosshatching), NOL02 (bar with horizontal crosshatching), NOL03 (bar with vertical crosshatching) and NOL04 (bar with dots) cultivated with 1 μg/ML of ZEA toxin.

(7) FIG. 7 is a graph showing the percentage of reduction of ZEA toxin over time (in hours) for strains NOL01 (diamonds), NOL02 (squares), NOL03 (triangles) and NOL04 (crosses).

(8) FIG. 8 shows a graph of the growth of the bacteria according to the invention NOL01 (dark gray), NOL02 (light gray) and NOL03 (black) in the presence of ZEA and as a function of the medium: TSB-rich medium (square dot), minimal medium MM (round dot). The x-axis shows the time in hours, and the y-axis shows the absorbance of the solution correlated with the growth of the bacteria (OD.sub.600nm).

EXAMPLES

Example 1—In Vitro Tests

(9) The inventors tested the properties of reducing the mycotoxin concentration of bacterial strains NOL01, NOL02, NOL03 and NOL04 on ZEA (zearalenone).

(10) 1—Effect of the Presence of Toxins on Cell Growth

(11) The ZEA toxins used for this test are provided by Sigma-Aldrich (Milan, Italy), the purity of which is greater than 99%. The toxins are dissolved in acetonitrile at 1 mg/mL, then filtered on a filtration tube with 0.2 μm cellulose membrane (RC/G) (LABOCHEM Science S.r.l., ITALY). An appropriate volume of standard ZEA solution is added to the TSB medium to obtain the desired final concentration (1 μg/mL). The different bacterial layers were cultivated in a TSB culture medium in the presence or absence of 1 μg/mL of ZEA at 30° C., with agitation at 150 rpm, under aerobic conditions.

(12) The culture data are shown in FIG. 6. These results show that strains NOL01, NOL02, NOL03 and NOL04 develop as well, or even more quickly in the presence of ZEA at a concentration of 1 μg/mL of medium.

(13) Next, different concentrations of ZEA in the medium were tested, and the results showed that strains NOL01, NOL02 and NOL03 developed equivalently under all tested ZEA concentrations, as shown by FIGS. 1 to 3.

(14) Lastly, different pH and temperature conditions were also tested. The obtained results show that NOL01, NOL02 and NOL03 have a reduced activity of the ZEA at 10, 20 and 30° C. (FIG. 4) as well as at pH 5, 7 and 8 (FIG. 5).

(15) These results show that the strains of Bacillus subtilis of the invention have the ability to develop in the presence of high concentrations of mycotoxins, and that the property of reducing the mycotoxin concentration is preserved under many conditions, in particular pH and temperature.

(16) 2—Measurement of the Quantity of Toxins after Culture

(17) After the various aforementioned cultures, the inventors tested the presence of toxins.

(18) In order to measure the quantity of toxin, the inventors used DonTest™ and ZearalaTest™ absorption kits (VICAM/Waters Corporation, Milford, Mass., USA).

(19) 0.5 mL of supernatant of each culture is mixed with 1.5 mL of phosphate buffer (PBS) and applied on the corresponding columns for desorption of the toxin, at the speed of one drop per second.

(20) The columns are next washed with 5 mL of distilled water at the same speed.

(21) The toxins are next eluted with 2 mL of methanol, and the eluates are dried at 50° C. The dry products are next transferred into a mixture of 250 μl H.sub.2O:MeOH 85:15, before LC mass spectrometry analysis.

(22) For the detection of the ZEA toxin, the inventors used the Agilent 1100 HPLC system device. The conditions are as follows: analytical column: Phenomenex® PFP (100 mm×4.6 mm, 2.6 μm), 40° C.; mobile phase: Water+Methanol+Acetonitrile 50+25+25 (v/v/v) ISOCRATIC; flow rate: 0.800 mL/min; retention time: 6.7 min; Fluorescence 274 nm λ(ex) and 440 nm λ(em).

(23) The results obtained for the ZEA toxin are shown in the following table, and in FIG. 3:

(24) TABLE-US-00003 TABLE 3 Percentage ZEA reduction* time (h) NOL01 NOL02 NOL03 NOL04  6  8.7 ± 4.6  6.0 ± 0.0 33.5 ± 1.9  2.2 ± 0.1 16 95.5 ± 1.5 57.0 ± 3.1 96.1 ± 4.2 93.2 ± 1.7 24 97.7 ± 0.6 53.6 ± 3.0 96.6 ± 1.9 94.4 ± 4.9 72 96.9 ± 0.6 64.8 ± 2.1 99.4 ± 1.1 95.5 ± 1.0 *average of the % obtained over 3 independent studies and standard deviation

(25) These results show an almost total reduction of the ZEA (more than 94%) after 24 h with NOL01, NOL03 and NOL04 and more than 50% for NOL02.

(26) To confirm these results, the inventors repeated the same experiments for the ZEA toxin. The results are indicated in the following table:

(27) TABLE-US-00004 TABLE 4 Percentage ZEA reduction* time (h) NOL01 NOL02 NOL03 NOL04 24* 97.7 ± 0.6 53.6 ± 3.0 96.6 ± 1.9 94.4 ± 4.9 24** 95.7 ± 2.5 56.5 ± 2.5 91.3 ± 1.4 91.8 ± 0.8 Value P 0.238 0.264 0.415 0.090 *average of the % obtained over 3 independent studies and standard deviation

(28) Since the value P is greater than 0.05, the results are considered to be repeatable. *: first series of tests, **: second series of tests.

(29) 3—Adsorption Test on Bacteria

(30) The inventors next tested whether the toxins are adsorbed on the bacteria.

(31) To that end, the bacteria cultures after 24 h in the presence of ZEA were treated as follows:

(32) The bacterial pellets are transferred into 1 mL of an acetonitrile:water mixture (90:10; v/v), and the mixtures are agitated vigorously using a vortex, and lastly centrifuged for 20 min at 14,000 G. The obtained supernatants are next dried at 50° C. and the dry residues are transferred into 1 mL of a water:methanol 85:15 v/v mixture for analysis by spectrometry as described in detail above.

(33) The ZEA desorption results are indicated in the following table:

(34) TABLE-US-00005 TABLE 5 Strain Percentage of desorbed ZEA* (NOL01) 3.2 (NOL02) 11.9 (NOL03) 4.0 (NOL04) 4.4 *average of the % obtained over 3 independent studies and standard deviation

(35) These results suggest that, in light of the small quantity of desorbed ZEA, the toxin is either internalized in the bacteria, or broken down by said bacteria.

(36) 4—Test for Breakdown by the Bacteria

(37) In order to test the hypothesis of breakdown by the bacteria, the inventors tested the presence of toxin directly in the bacteria.

(38) To that end, from bacteria cultivated for 24 h with 1 μg/mL of ZEA, the bacterial pellets were transferred into 1 mL of an acetonitrile:water mixture (90:10; v/v) and lysed by sonication. The sonication conditions are as follows: probe diameter: 3.175 mm, frequency: 20 kHz, amplitude: 50%, sonication: 15 sec, then stop 15 sec (6 times), sonication duration: 90 seconds total (without pause times), temperature: 4° C.

(39) After sonication, the specimens are centrifuged for 20 min at 14,000 G. The obtained supernatants are next dried at 50° C. and the dry residues are transferred into 1 mL of a water:methanol 85:15 v/v mixture for analysis by spectrometry as described in detail hereinafter. Device: Aquity UPLC® BEH C18 (100 mm×2.1 mm, 1.7 μm)—50° C.; UV absorbance at 220 nm; Fluorescence: 274 nm λ(ex) and 440 nm λ(em). The retention time of the ZEA was 11.5 min.

(40) The ZEA results extracted from the different pellets are as follows:

(41) TABLE-US-00006 TABLE 6 Strain Percentage of desorbed ZEA* (NOL01) 2.7 (NOL02) 0.1 (NOL03) 0.0 (NOL04) 1.4 *average of the % obtained over 3 independent studies and standard deviation

(42) These results show that the ZEA is not detectable, even when the bacteria are lysed, meaning that the mycotoxin is broken down by the bacteria.

(43) 5—Culture in Anaerobic Conditions

(44) The Inventors also tested bacterial growth conditions according to 1—, but in the absence of oxygen.

(45) They next measured the breakdown of the ZEA mycotoxin under these conditions, according to the protocols described above.

(46) The results are provided in the following table:

(47) TABLE-US-00007 TABLE 7 Type of Percentage of ZEA reduction** incubation NOL01 NOL02 NOL03 NOL04 aerobic* 97.7 ± 0.06 53.6 ± 3.0 96.6 ± 1.9 94.4 ± 4.9 anaerobic* 92.9 ± 1.4  44.5 ± 5.0 92.6 ± 2.8 90.3 ± 0.5 Value P 0.259 0.080 0.530 0.121 *the tests were done after 24 h of culture in the presence of 1 μg/mL of mycotoxin **average of the % obtained over 3 samples of standard deviation

(48) These results show that, irrespective of the culture condition (presence or absence of oxygen), the bacteria are capable of breaking down the mycotoxins.

(49) 6—Culture in Minimal Mediums

(50) The inventors also tested the bacterial growth conditions according to 1—, but in a minimal medium, that is to say, a depleted, non-enriched medium (MM), without adding carbon other than that provided by the mycotoxins, the composition of which is as follows for 1 L of culture medium:

(51) TABLE-US-00008 TABLE 8 Compound Quantity K.sub.2HPO.sub.4 2.72 g KH.sub.2PO.sub.4 1 g Na.sub.2SO.sub.4 0.284 g NaNO.sub.3 0.17 g KCl 0.15 g CaCl2 × 6H.sub.2O 22 mg MnCl.sub.2 × 4H.sub.2O 15 mg FeCl.sub.3 × 6H.sub.2O 2.16 mg MgCl.sub.2 × 6H.sub.2O 25 mg NH.sub.4Cl 1 g Yeast extract 0.05% Water Qs

(52) These results show that the strains of Bacillus subtilis of the invention have the ability to develop in a minimal medium MM, even if the growth level is less than in a rich medium TSB.

(53) Following the different aforementioned cultures and additional cultures done according to 1—but in the presence of 20 μg/mL of ZEA in MM medium, in the presence of 1 μg/mL of ZEA under anaerobic conditions in a MM medium, in a minimal medium re-enriched with NO.sub.3 (MMN) and in a minimal medium re-enriched with NO.sub.3 and glucose (MMNG), the inventors tested the presence of toxins according to 2—at t=72 h.

(54) The results obtained for the ZEA toxin are shown in the following table:

(55) TABLE-US-00009 TABLE 9 Percentage of ZEA reduction* Condition Medium ZEA NOL01 NOL02 NOL03 Aerobic TSB 1 μg/mL 96.9 64.8 99.4 MM 1 μg/mL 100 100 100 20 μg/mL  99.7 48.9 99 Anaerobic TSB 1 μg/mL 92.9 44.5 92.3 MM 1 μg/mL 32.5 24.1 32.5 MMN 1 μg/mL 23 20.4 80.1 MMNG 1 μg/mL 98.4 80.3 96.4 *average of the % obtained over 3 samples

(56) These results show, under aerobic conditions, a reduction of the ZEA obtained in minimal medium MM comparable to that observed in rich medium TSB, irrespective of the initial ZEA concentration.

(57) Under anaerobic conditions, a same breakdown level of the mycotoxins as in the rich medium TSB is achieved by the three strains according to the invention, tested in minimal medium re-enriched with NO.sub.3 and glucose (MMNG). The same finding is reached for NOL03 in minimal medium re-enriched with NO.sub.3 (MMN).

(58) These results show that irrespective of the culture medium (rich, minimal or re-enriched minimal), the bacteria according to the invention are capable of breaking down the mycotoxins, with greater efficacy in rich and re-enriched minimal mediums.

(59) 7—Reduction of Mycotoxins Other than ZEA

(60) The inventors also conducted bacterial cultures according to the invention according to 1—but in the presence of mycotoxins other than ZEA, then measured the quantity of toxin after culture according to 2—with appropriate absorption kits for each of the tested mycotoxins, at t=72 h.

(61) TABLE-US-00010 TABLE 10 Percentage of reduction* NOL01 NOL02 NOL03 ZEA 96.9 64.8 99.4 FBI 4.7 18.7 8.8 OTA 7.3 10.0 15.9 T-2 7.2 1.0 4.2 *average of the % obtained over 3 samples

(62) These results show, under aerobic conditions, a reducing capacity of NOL01 on ZEA, OTA and T-2, the effect observed on the last two mycotoxins nevertheless being lower. Likewise, they show a reducing capacity of NOL02 and NOL03 on ZEA, FB.sub.1 and OTA, the observed effect on these last two mycotoxins nevertheless being lower.

(63) 8—Efficacy Test of the NOL01, NOL02 and NOL03 Bacteria in Combination

(64) The inventors also conducted bacterial cultures according to the invention according to 1—in the presence of ZEA, AFB.sub.1 and FB.sub.1, in rich medium (TSB) or minimal medium re-enriched with NO.sub.3 and glucose (MMNG), but with an inoculum containing 1 μg/mL of the three bacteria according to the invention NOL01, NOL02, NOL03 implemented in equal proportions.

(65) TABLE-US-00011 TABLE 11 Percentage of reduction* at t = 72 h NOL01 NOL02 NOL03 POOL Aerobic TSB ZEA 96.9 64.8 99.4 100 Anaerobic MMNG ZEA 98.4 80.3 96.4 Not tested AFB.sub.1 1.6 0.5 0.7 11.7 FB.sub.1 3.0 4.4 2.0 18.1 *average of the % obtained over 3 samples

(66) These results show a capacity to reduce the presence of ZEA mycotoxins by the NOL01, NOL02 and NOL03 bacteria in combination, whether in aerobic conditions in rich medium (TSB), or in anaerobic conditions in minimal medium re-enriched with NO.sub.3 and glucose (MMNG). An ability has also been noted to reduce the presence of AFB.sub.1 and FB.sub.1 of this pool under anaerobic conditions and MMNG medium, although it is more reduced.

(67) 9—Measure of Efficacy Against Metabolites of ZEA

(68) The inventors verified the ability of strains NOL01, NOL02 and NOL03 according to the invention to reduce mycotoxins, according to 1—but on the metabolites of ZEA, under aerobic conditions in minimal medium (MM) and under anaerobic conditions in minimal medium re-enriched with NO.sub.3 and glucose (MMNG).

(69) TABLE-US-00012 TABLE 12 Percentage of reduction* at t = 72 h Aerobic Anaerobic MM MMNG NOL01 NOL02 NOL03 NOL01 NOL02 NOL03 ZEA 100 98.9 99.0 100 96.3 96.3 α-ZOL 100 98.4 99.0 100 56.6 100 β-ZOL 100 96.6 99.0 100 47.0 100 α-ZAL 99.0 85.3 96.0 100 32.7 79.4 β-ZAL 98.9 80.0 93.0 100 44.5 87 ZAL 99.0 90.8 85.7 100 20.2 68.6 *average of the % obtained over 3 independent samples and standard deviation

(70) These results confirm that irrespective of the culture conditions (presence or absence of oxygen, rich, re-enriched minimal or minimal medium), the bacteria according to the invention are capable of breaking down the ZEA as well as the metabolites thereof.

Example 2—In Vivo Test in Pigs

(71) The inventors also tested the bacteria according to the invention under in vivo conditions in pigs in order to demonstrate their efficacy in animals.

(72) 1—Protocol

(73) The inventors conducted a 21-day feeding test in an experimental station on 64 weaned piglets, divided into 2 tests, one on ZEA and the other on DON (or 32 piglets per test).

(74) The following diets were applied for each of these tests (or 8 piglets per diet): (1) uncontaminated food without additive (2) uncontaminated food+additive (3) contaminated food without additive (4) contaminated food+additive

(75) For diets (3) and (4), the food distributed to the animal was artificially contaminated in an amount of 0.1 mg ZEA/kg of complete food at 12% moisture, or the maximum content recommended by European regulations according to Recommendation no. 2006/576/EC by the European Commission.

(76) For the test on DON, the same 4 diets were applied, with an artificial contamination of the food distributed in an amount of 0.9 mg DON/kg of complete food at 12% moisture for diets (3) and (4).

(77) The quantity of mycotoxins was analyzed at the beginning of the test and good homogeneity of the contamination of the distributed food was confirmed.

(78) The additive as added corresponded to the combination of the 3 strains NOL01, NOL2 and NOL3 according to the invention implemented in equal proportions. This additive, in powdered form, was added to the food distributed after artificial contamination with mycotoxins and before distribution to the animals, and in sufficient quantity to reach 10.sup.9 bacterial colonies per kg of food.

(79) About 1.5 kg of food is distributed per day per piglet.

(80) The animals were distributed at 1 animal per metabolic cage for the test on ZEA, and 1 animal per pen for the test on DON.

(81) An adaptation period of 3 weeks before launching the test diets was respected in order to avoid stressing the animals during the transition to post-weaning and to acclimate those who were in metabolic cages for the test on ZEA.

(82) At the end of this 3-week adaptation period, the tests began and lasted 3 weeks (21 days).

(83) 2—Analyses and Zootechnical Parameters

(84) The following specimens were collected throughout the test: Blood samples at 0, 7, 14 and 21 days on all animals, Collection of feces and urine between the 17.sup.th and 21.sup.st days for the test on ZEA, Collection of feces at 21 days, at slaughter for the test on DON, Collection of tissue samples at 21 days: liver, kidneys, muscles, reproductive organs, intestinal tracts.

(85) On the collected samples, as a priority the samples collected at the end of the test, the following analyses were done: Detection of ZEA and its metabolites α-zearalenol and β-zearalenol in the plasma, urine and feces collected on the animals from the ZEA test, Detection of DON and its metabolites in the serum collected from the animals from the test on DON.

(86) For both tests, the following zootechnical parameters were noted: body weight, average daily food consumption, mortality, morbidity. For the tests on ZEA, the following parameters were also noted: size of the vulva and/or degree of inflammation of the vulva, weight of the reproductive organs.

(87) The invention is not limited to the described embodiments, and other embodiments will appear clearly to one skilled in the art.