ADSORBENT OF MICOTOXINS BASED ON A BETAINE DERIVATIVE FOR BALANCED ANIMAL FOODS

Abstract

The present invention provides a mycotoxin adsorbent based on a highly specific betaine derivative for trichothecenes A and B, and especially for vomitoxin (or deoxynivalenol) and T-2 toxin; as well as a process for preparing said mycotoxin adsorbent. The mycotoxin adsorbent of the invention is obtained by modifying the surface of an aluminosilicate by means of an organic amphoteric compound with a carboxyl group that provides it with high polarity properties. The mycotoxin adsorbent is useful for preparing balanced feed from animals that avoid the toxic effects of mycotoxins.

Claims

1. A mycotoxin adsorbent characterized in that it comprises an organically modified aluminosilicate with a betaine derivative of formula (I) or with a salt of said derivative: ##STR00007## wherein R represents alkyl having 1 to 12 carbon atoms and n is 1 to 12.

2. The mycotoxin adsorbent according to claim 1, wherein the aluminosilicate is a tectosilicate, a phyllosilicate or a mixture of both.

3. The mycotoxin adsorbent according to claim 1, wherein the betaine derivative has the formula (Ia): ##STR00008##

4. The mycotoxin adsorbent according to claim 1, wherein the salt of the betaine derivative has the formula (Ib): ##STR00009##

5. The mycotoxin adsorbent according to claim 1, wherein the aluminosilicate has a cation exchange capacity of at least 20 milliequivalents per 100 grams of material.

6. The mycotoxin adsorbent according to claim 1, wherein the aluminosilicate has a cation exchange capacity of 55 milliequivalents per 100 grams of material.

7. The mycotoxin adsorbent according to claim 1, wherein the betaine derivative or the salt of said derivative is used in a proportion of 25% to 120% of the cation exchange capacity of the aluminosilicate used.

8. An animal feed additive characterized in that it comprises the mycotoxin adsorbent according to claim 1.

9. A premix for preparing the animal feed additive according to claim 8.

10. A balanced feed formulation for animals characterized in that it comprises the mycotoxin adsorbent according to claim 1.

11. The mycotoxin adsorbent according to claim 2, wherein the aluminosilicate has a cation exchange capacity of at least 20 milliequivalents per 100 grams of material.

12. The mycotoxin adsorbent according to claim 3, wherein the aluminosilicate has a cation exchange capacity of at least 20 milliequivalents per 100 grams of material.

13. The mycotoxin adsorbent according to claim 4, wherein the aluminosilicate has a cation exchange capacity of at least 20 milliequivalents per 100 grams of material.

14. An animal feed additive characterized in that it comprises the mycotoxin adsorbent according to claim 2.

15. An animal feed additive characterized in that it comprises the mycotoxin adsorbent according to claim 3.

16. An animal feed additive characterized in that it comprises the mycotoxin adsorbent according to claim 4.

17. A balanced feed formulation for animals characterized in that it comprises the mycotoxin adsorbent according to claim 2.

18. A balanced feed formulation for animals characterized in that it comprises the mycotoxin adsorbent according to claim 3.

19. A balanced feed formulation for animals characterized in that it comprises the mycotoxin adsorbent according to claim 4.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0034] The inventors of the present application experimentally explored the hypothesis of increasing the polarity of the surface of a mycotoxin adsorbent of the organoaluminosilicate type by using a carboxyl functional group in one of the aliphatic chains of a quaternary ammonium compound in order to efficiently adsorb the mycotoxins.

[0035] In this way, the present invention is based on the fact that a modification of the surface of the aluminosilicates by a treatment as described in the present application, can be used to increase its capacity for mycotoxin adsorption. The treatment to be followed depends on what is intended to be achieved, but generally two main characteristics of the surface are manipulated, which are the hydrophilic character and the hydrophobic or organophilic character (Lara et al 1998).

[0036] Particularly, in the present invention the modification of the surface is carried out with an organic amphoteric compound with a carboxyl functionality, in such a way that the surface achieves a highly polar character. The organic compound that is used for the modification of the surface may occupy part or all the active sites on the surface of the aluminosilicate.

[0037] The aluminosilicate used can be a tectosilicate or a phyllosilicate or a mixture of both, with the proviso that the material that is used has a cation exchange capacity of at least 20 milliequivalents per 100 grams of material, and preferably 55 milliequivalents per 100 grams of material.

[0038] The selection of the organic compound depends on the specificity and efficiency that is desired in the adsorption of mycotoxins. In the case of the present invention, preferably the organic compound used is a betaine (trimethylglycine or TMG). This organic compound is used in a proportion of 25% to 120% of the cation exchange capacity of the aluminosilicate used. The reaction is carried out in aqueous medium with stirring at a temperature between 15 and 85 C. and with a time of 0.25 to 3 hours. The product is separated by filtration, dried at a temperature between 40 and 150 C. and granulated or ground to meshes between 100 and 325.

[0039] The additive object of the present invention is a low inclusion adsorbent that is added to foods contaminated with trichothecenes, at a ratio of 0.025% to 0.2% of the weight of the food.

[0040] According to the first aspect, the invention relates to a mycotoxin adsorbent characterized in that it comprises an organically modified aluminosilicate with a betaine derivative of general formula (I) or a salt of said derivative:

##STR00002##

[0041] wherein R represents an alkyl group with 1 to 12 carbon atoms and n is 1 to 12.

[0042] In the case that R is equal to 1 and n is equal to 1, the betaine derivative of formula (I) is trimethylglycine, known simply as Betaine, according to figure (Ia):

##STR00003##

[0043] In one embodiment, the invention relates to a mycotoxin adsorbent characterized in that it comprises an organically modified aluminosilicate with a betaine derivative of formula (I) or a salt of said derivative, wherein the aluminosilicate can be a tectosilicate, a phyllosilicate or a mix of both.

[0044] In still another embodiment, the invention relates to a mycotoxin adsorbent characterized in that it comprises an organically modified aluminosilicate with a betaine derivative of formula (I), wherein R represents a methyl group and n is 1 to 12, more preferably n is 1 to 6, and more preferably n is equal to 1.

[0045] The invention also relates to a mycotoxin adsorbent characterized in that it comprises an organically modified aluminosilicate with a betaine derivative of formula (I), wherein n is equal to 1 and R represents alkyl with 1 to 6 carbon atoms, preferably R represents alkyl with 1 to 3 carbon atoms and more preferably R represents alkyl with 1 carbon atom.

[0046] Another additional particular embodiment, the invention relates to a mycotoxin adsorbent characterized in that it comprises an aluminosilicate organically modified with a salt of the betaine derivative of formula (I), preferably said salt is Betaine Hydrochloride according to formula (Ib):

##STR00004##

[0047] In another embodiment, the mycotoxin adsorbent according to the invention comprises an aluminosilicate organically modified with the betaine derivative of formula (I) or a salt of said derivative, wherein the aluminosilicate has a cation exchange capacity of at least 20 milliequivalents per 100 grams of material and more preferably 55 milliequivalents per 100 grams of material.

[0048] The mycotoxin adsorbent according to the present invention may further comprise an aluminosilicate organically modified with the betaine derivative of formula (I) or a salt of said derivative, which is used in a proportion of 25% to 120% of the capacity of cation exchange (CEC) of the aluminosilicate used, preferably used in a proportion of 60% to 120% CEC.

[0049] In a further embodiment, the mycotoxin adsorbent according to the invention may comprise an aluminosilicate organically modified with the betaine derivative of formula (I) or a salt of said derivative, having a cation exchange capacity of at least 55 milliequivalents per 100 grams of material.

[0050] The invention also relates to a mycotoxin adsorbent comprising an aluminosilicate organically modified with the betaine derivative of formula (I), (Ia), or a salt of said derivative, particularly betaine hydrochloride having the formula (Ib), wherein said betaine derivative of formula (I) or the salt of said derivative, is used in a proportion of 25% to 120% of the cation exchange capacity (CEC) of the aluminosilicate used and more preferably is used in a proportion from 60% to 120% CIC.

[0051] According to the second aspect, the invention relates to a premix for preparing the mycotoxin adsorbent of the present invention, an additive for animal feed and a balanced feed formulation for animals including the mycotoxin adsorbent of the invention.

[0052] According to the third aspect, the invention relates to the use of the mycotoxin adsorbent of the present invention, in the preparation of an additive for balanced feed of animals and in the preparation of a formulation of a balanced feed, to treat or prevent one or more harmful effects or symptoms in the digestive tract associated with trichothecene poisoning, particularly trichothecenes A and B, and especially vomitoxin (or deoxynivalenol) and T-2 toxin.

[0053] The invention involves the use of a mycotoxin adsorbent characterized in that it comprises an organically modified aluminosilicate with a betaine derivative according to formula (I) or a salt of said derivative, in the preparation of an additive for animal feed to treat or prevent one or more deleterious effects or symptoms in the digestive tract associated with trichothecene A and/or B poisoning, such as vomiting, diarrhea, irritation, hemorrhages or necrosis in the digestive tract associated with trichothecene intoxication.

[0054] In the present invention, one or more deleterious effects or one or more symptoms in the digestive tract associated with trichothecene poisoning can be selected from the group consisting of: Diacetoxyscirpenol (DAS), HT-2 toxin (HT-2), T-2 toxin (T-2), neosolaniol (NEO), deoxynivalenol (DON) or vomitoxin, 3-acetyldeoxynivalenol (3-AcDON), nivalenol (NIV), fusarenon-X (Fus-X), trichotecolone (TRI) and a combination of the above.

[0055] Also, another embodiment of the invention relates to the use of an additive for animal feed in accordance with the invention in the preparation of a balanced feed formulation to reduce or eliminate the deleterious effects or symptoms in the digestive tract associated with the trichothecene intoxication in animals, specifically the harmful effects of trichothecenes A and/or B, and more specifically of deoxynivalenol (or vomitoxin) and T-2 toxin.

[0056] The invention also consists of a mycotoxin adsorbent according to the invention for use as an animal feed additive to treat or prevent one or more deleterious effects or one or more symptoms in the digestive tract associated with trichothecene poisoning, wherein the additive for animal fodder is to treat or prevent vomiting, diarrhea, irritation, hemorrhages, necrosis or oral lesions.

[0057] The present invention further relates to a process for preparing the mycotoxin adsorbent of the invention comprising the following steps:

[0058] a) contacting an aluminosilicate with cation exchange capacity of at least 20 milliequivalents per 100 grams of material in an aqueous medium with stirring at a temperature between 15 and 85 C. and with a time of 0.25 to 3 hours with a derivative of betaine of formula (I) or (Ia), or with a salt of said derivative, particularly hydrochloride of a betaine derivative of formula (Ib) and more particularly betaine hydrochloride, in a proportion of 25% to 120% of the cation exchange capacity of the aluminosilicate used;

[0059] b) Separate by filtration;

[0060] c) Dry at a temperature between 40 and 150 C.; and

[0061] d) Granulate or grind to meshes between 100 and 325.

[0062] The following examples show that the objective value of protection against the effects of vomitoxin and T-2 toxin by the mycotoxin adsorbent of the present invention turns out to be particularly high. Said examples, which include the preparation of the mycotoxin adsorbent of the present invention and its in vivo evaluation, all of which is provided for the purpose of illustration and not limitation.

Example 1

Preparation of the Micotoxin Adsorbent of the Invention

Method with Betaine

[0063] Raw Materials to be Used. [0064] The characteristics of the organic compound used for the surface treatment are shown in Table 2. [0065] The base aluminosilicate used is an aluminosilicate of the Bentonite type, with cation exchange capacity of 55 meg/100 g.

TABLE-US-00002 TABLE 2 Characteristics of the betaine used. MOLECULAR QUATERNARY TYPE STRUCTURE WEIGHT LOAD Betaine Amphoteric Quaternary [00005] 117 +1

[0066] Formulation.

[0067] The initial experimental design was developed based on the substitution percentage of the cation exchange capacity (CEC) of the base aluminosilicate. A substitution percentage of 60% to 120% of CEC was taken as a base.

[0068] Experimental Development of Formulations.

[0069] Materials and Equipment.

[0070] 1. Laboratory glassware

[0071] 2. Magnetic stirrer.

[0072] 3. Drying stove.

[0073] 4. Mortar or laboratory mill.

[0074] 5. 200 mesh screen.

[0075] Process. [0076] The exchange procedure is carried out according to the state of the art (S L Lemke, P G Grant and T D Phillips Adsortion of Zearalenone by Organophilic Montmorillonite Clay J Agric. Food Chem. (1998), pp. 3789-3796). In this particular case, hydrochloric acid is added to the water in order to obtain a positive charge in the nitrogen. [0077] The final phase of the reaction is carried out with a time of 2 hours. [0078] Filter the mixture. [0079] Dry the sample in the oven at a temperature of around 105 C. [0080] Grind the sample. [0081] Sieve in 200 mesh. [0082] Carry out corresponding analyses according to the sample.

Example 2

Preparation of the Micotoxin Adsorbent of the Invention

Method with Betaine Hydrochloride

[0083] Raw Materials to be Used. [0084] The characteristics of the organic compound used for the surface treatment are shown in Table 3. [0085] The base aluminosilicate used is an aluminosilicate of the Bentonite type, with cation exchange capacity of 55 meq/100 g.

TABLE-US-00003 TABLE 3 Characteristics of the betaine used. MOLECULAR QUATERNARY TYPE STRUCTURE WEIGHT LOAD Betaine Quaternary Amphoteric [00006] 153.6 +1

[0086] Formulation.

[0087] The initial experimental design was developed based on the substitution percentage of the cation exchange capacity (CEC) of the base aluminosilicate. A substitution percentage of 60% to 120% of CEC was taken as a base.

[0088] Experimental Development of Formulations.

[0089] Materials and Equipment.

[0090] 6. Laboratory glassware

[0091] 7. Magnetic stirrer.

[0092] 8. Drying oven.

[0093] 9. Mortar or laboratory mill.

[0094] 10. 200 mesh screen.

[0095] Process. [0096] The exchange procedure is carried out according to the state of the art (S L Lemke, P G Grant and T D Phillips Adsortion of Zearalenone by Organophilic Montmorillonite Clay J Agric. Food Chem. (1998), pp. 3789-3796). In this case, due to the hydrochloride in the betaine used, no hydrochloric acid is added. [0097] The final phase of the reaction is carried out with a time of 2 hours. [0098] Filter the mixture. [0099] Dry the sample in the oven at a temperature of around 105 C. [0100] Grind the sample. [0101] Sieve in 200 mesh. [0102] Carry out corresponding analyses according to the sample.

Example 3

In Vivo Evaluation in Rats with a Mycotoxin Adsorbent of the Invention Designed to Adsorb Vomitoxin (or Deoxynivalenol)

[0103] In this example it is used as an adsorbent of mycotoxins, an aluminosilicate organically modified with a betaine hydrochloride, which we call Zeotri B2 or ZB2.

[0104] In the experiment, 30 freshly weaned male Sprague Dawley rats distributed in 3 groups and with one week of adaptation were used. A single rat was used as a repetition. The negative and positive control groups as well as the challenge group had 10 rats per treatment. The rats were received from the bioterium of the Benemerita Autonomous University of Puebla (BUAP). The experimental time was 35 days. The level of contamination with Vomitoxin (or DON) was 12 mg/kg (12 ppm or 12,000 ppb) in the food. The Zeotri B2 adsorbent dose was 1.5 kg/Ton of feed.

[0105] The DON present in the feed affected the growth rate of the animal; this is observed in the weight gain due to the decrease in the consumption of the food.

[0106] At the used concentration of 12,000 ppb of DON, there were statistically significant differences in the productive parameters among the three treatments: weight gain, food consumption and feed conversion. An important observation is that the group treated with Zeotri B2 had less variation in the final weight and this is confirmed because there is less standard error.

[0107] The European Union states that a product is considered effective, based on a biomarker when it protects a 40%. In this case, the effectiveness was estimated based on the weight gain with respect to the positive control group, which is the weight recovered by the group that consumed the adsorbent. The effectiveness of the mycotoxin adsorbent of the present invention, based on the weight gain, was surprisingly ZB2=64.1%.

[0108] Tables 4 to 6 below show the productive results in initial and final weight, as well as weight gains, feed consumption and feed conversion.

TABLE-US-00004 TABLE NO. 4 Initial and Final Weights, Weight gain, Alimentary conversion and food consumption. Weight in g Initial Final (56 Weight Food Food (21 days old) das de edad) gain conversion consumption Means Means Means Means Means Treatments standard error standard error standard error standard error standard error Negative control 108 7.84 .sup.a 256 9.05 .sup.a 148 7.22 .sup.a 4.51 0.058 .sup.a 666 8.55 .sup.a Positive 100 5.27 .sup.a 209 10.4 .sup.b 109 6.61 .sup.b 5.22 0.195 .sup.b 569 19.1 .sup.b Control DON Challenge 97 2.42 .sup.a 231 4.09 .sup.ab 134 2.94 .sup.a 4.68 0.045 .sup.a 627 5.99 .sup.a Zeotri B2 + DON Averages with different letters are statistically significant for p < 0.05.

TABLE-US-00005 TABLE NO. 5 Weight gain during the experimental period Gain of weight Gain of weight Gain of weight Gain of weight Gain of weight from from from from from 0 to 7 days 7 to 14 days 14 to 21 days 21 to 28 days 28 to 35 days Means Means Means Means Means Treatments standard error standard error standard error standard error standard error Negative Control 36 2.84 .sup.a 31 0.84 .sup.a 27 3.02 .sup.a 27 1.73 .sup.a 28 1.87 .sup.a Positive 24 2.45 .sup.b 24 3.04 .sup.a 21 2.92 .sup.a 13 3.89 .sup.b 27 3.63 .sup.a control DON Challenge 28 1.54 .sup.ab 29 1.21 .sup.a 26 2.75 .sup.a 26 3.66 .sup.a 25 2.30 .sup.a Zeotri B2 + DON Averages with different letters are statistically significant for p < 0.05.

TABLE-US-00006 TABLE NO. 6 Food consumption during the experimental period. Food Food Food Food Food consumption consumption consumption consumption consumption from from from from from 0 to 7 days 7 to 14 days 14 to 21 days 21 to 28 days 28 to 35 days Means Means Means Means Means Treatments standard error standard error standard error standard error standard error Negative Control 107 1.01 .sup.a 120 1.27 .sup.a 134 5.72 .sup.a 141 3.44 .sup.a 165 3.19 .sup.a Positive 98 4.75 .sup.a 109 4.84 .sup.a 109 5.41 .sup.b 121 4.83 .sup.b 132 3.10 .sup.b control DON Desafo 103 2.39 .sup.a 119 1.75 .sup.a 126 1.71 .sup.a 137 2.12 .sup.a 142 4.10 .sup.b Zeotri B2 + DON Averages with different letters are statistically significant for p < 0.05.

[0109] The experimental results obtained show that in the DON present in the diet of the positive control group, it affected the consumption of food. The effect of DON was manifested from the third week of food consumption. The same happened in the challenge group ZB2, but to a lesser degree.

[0110] The levels of inclusion of DON in this experiment (12,000 ppb) affected the consumption of food and therefore the weight gain. These results showed statistically significant differences of these parameters between the groups.

[0111] The ZB2 mycotoxin adsorbent at a dose of 1.5 kg/T was able to decrease the effects of 12,000 ppb of DON with statistical differences against the positive control group, in the parameters evaluated.

Example 4

In Vivo Evaluation in Rats with a Mycotoxin Adsorbent of the Invention Designed to Adsorb Vomitoxin (or Deoxynivalenol) at a Higher Dose of Inclusion

[0112] A second experiment was carried out with rats to evaluate the effectiveness of the adsorbent at a dose slightly higher than 2 kg/T, with a DON contamination of 12.5 ppm.

[0113] In the experiment, we used 60 freshly weaned male Sprague Dawley rats distributed in 3 treatments. A week of adaptation of the rats was also considered and the experiment lasted 35 days. A rat was used as a repetition.

[0114] The dose of the mycotoxin adsorbent of the present invention (ZB2) as mentioned, was 2 kg/T of feed and the contamination level of DON was 12.5 ppm, but in addition there was a level of contamination of Zearalenone of 10.2 ppm. The results are presented in table 7.

TABLE-US-00007 TABLE 7 Initial and final weights of the rats after 35 days of experimentation, as well as data on weight gain, feed consumption and feed conversion. Weight in g Gain of Conversion Consumption Stocks Final weight food food Means Means Means Means Means Treatments Standard error Standard error Standard error Standard error Standard error Negative Control 85 2.67 .sup.a 210 9.67 .sup.a 125 9.24 .sup.a 4.61 0.172 .sup.a 576 25.1 .sup.a Positive 86 1.78 .sup.a 169 3.69 .sup.b .sup.83 3.45 .sup.b 6.15 0.287 .sup.b 511 5.71 .sup.b Control DON Challenge 88 1.91 .sup.a 196 7.40 .sup.a 108 6.40 .sup.ab 5.62 0.406 .sup.b 607 5.73 .sup.a Zeotri B2 + DON Averages with different letters are statistically significant for p < 0.05.

[0115] It is observed that the mycotoxin adsorbent of the invention, Zeotri B2, allowed a protection based on the surprisingly 59.5% weight gain, with statistical difference.

Example 5

In Vivo Evaluation of the Mycotoxin Adsorbent of the Invention Against the Adverse Effects of 1.8 ppm of T-2 Toxin from a Culture of Fusarium Sporotrichioides, in Broilers in the Growth Phase (Days 1 to 28 of Age)

[0116] In order to have information on the efficacy of the mycotoxin adsorbent described here, in poultry farming, an experiment was carried out with broiler chicken, using food contaminated with T2 toxin, another trichothecene.

[0117] We used 112 1-day-old chickens, distributed in 4 treatments of 4 repetitions with 7 chickens per repetition. Table 7 below shows the distribution of the treatments and the level of contamination with Toxin T2, which was of the order of 1900 ppb. The mycotoxin adsorbent of the invention was used at 2 kg/T. The experiment lasted 28 days and the base food was commercial.

TABLE-US-00008 TABLE 8 Design of the experiment with Toxin T2 and Zeotri B2. Treatment Zeotri B2 (g/kg) Toxin T-2 (g/kg) Negative Control 0 0 innocuity 2 0 Positive Control 0 1900 Challenge Zeotri 2 1825

[0118] At the end of the experimental period, the chickens were weighed to calculate the weight gain as well as the feed conversion. Table 9 presents the final results.

TABLE-US-00009 TABLE 9 Initial and final weights of chickens after 28 days of experimentation, as well as data on weight gain, feed consumption and feed conversion. Weight in g Gain of Conversion Consumption Initial Final weight food of food Means Means Means Means Means Treatments standard error standard error standard error standard error standard error Negative control 47 0.52 .sup.a 1525 21.11 .sup.a 1478 21.03 .sup.a 1.44 0.022 .sup.a 2126 19.1 .sup.a Safety 47 0.51 .sup.a 1541 20.36 .sup.a 1494 20.19 .sup.a 1.47 0.025 .sup.a 2197 50.4 .sup.a Positive Control 47 0.52 .sup.a 1418 28.44 .sup.b 1371 28.44 .sup.b 1.51 0.045 .sup.a 2076 100 .sup.a Toxin T-2 Challenge Zeotri 47 0.51 .sup.a .sup.1488 19.49 .sup.ab .sup.1441 19.38 .sup.ab 1.45 0.019 .sup.a 2092 20.6 .sup.a Averages with different letters are statistically significant for p < 0.05.

[0119] From this experiment it is observed that the mycotoxin adsorbent of the invention offered a good protection with statistical difference against the negative effect of Toxin T2.

[0120] It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.