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    • 专利摘要:
    Procedure for evaluating the capacity of mycotoxin adsorption in monogastric and polygastric animals. The present invention relates to a method for evaluating the capacity of adsorption of mycotoxins in monogastric and polygastric animals, comprising the steps of: (a) arranging a sample with mycotoxins on membranes composed of dextran and polyethylene gels; (b) adding pepsin to the sample; (c) adding the mycotoxin collector to be analyzed in a part of the sample; (d) subjecting this part of the sample with mycotoxin scavenger successively to different pH conditions and quantifying the concentration of mycotoxins adsorbed by the mycotoxin scavenger after each pH condition; (e) repeating step (d) with the other part of the sample without the mycotoxin trap; (f) comparing the adsorption results obtained with the part of the sample that contains the mycotoxin trap and the part of the sample that does not contain the mycotoxin trap. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2668215A1
    申请号:ES201631464
    申请日:2016-11-16
    公开日:2018-05-17
    发明作者:Jaime Borrell Valls
    申请人:Biovet S A；BIOVET SA；
    IPC主号:
    专利说明:

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    DESCRIPTION
    EVALUATION PROCEDURE FOR MICOTOXIN ADSORTION CAPACITY IN MONOGASTRIC AND POLYGASTRIC ANIMALS
    Field of the Invention
    The present invention relates to the field of mycotoxin analytics. In particular, the present invention relates to a method of evaluating the adsorption capacity of mycotoxins in monogastric and polygastric animals in which it is intended to simulate the conditions in the digestive tract in order to precisely determine said adsorption capacity.
    Background of the invention
    Mycotoxins are fungal metabolites that affect all crops and therefore livestock production, since cereals used in the manufacture of feed are contaminated by them.
    In the industry there are various systems used for the control or elimination of mycotoxins so that they are not absorbed by animals and therefore their pathogenic effects and their incidence on production parameters are minimized. In order to advise or decide when to use one system or another for the control of mycotoxins, a series of analyzes on the ability of each of these systems to absorb mycotoxins must be performed.
    In 1977, field studies on the control of mycotoxin binding or adsorption were initiated by Dr. Hernández (finally unpublished results), in which an approach to the digestive structure of monogastric animals is performed, studying a sample contaminated with aflatoxin in a stomach-like system at pH = 3.
    Subsequently, in "Newer methods which simulate the GIT (and estimate absorption)" (Doll et al., 2004, Doll and Danicke, 2004 and Avantaggiato et al., 2004) the study is extended considering also adsorption at intestinal level, performing a larger study at pH = 3 and pH = 6, corresponding to the pH of the duodenum.This technique, although more exact, does not take into account the last section of the intestine, that is the colon.
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    This last section affects the fixation of mycotoxins, so that a desorption of mycotoxins occurs and they have access to the bloodstream through the venous system of the upper hemorrhoids, which are a branching of the portal vein, whereby some mycotoxins pass directly to the hepatic portal system and other mycotoxins would pass into the bloodstream through the system of middle and lower hemorrhoids, which are a branch of the vena cava, transporting the mycotoxins adsorbed to the general systemic circulation.
    Therefore, there is a need for a more precise procedure that allows the evaluation of mycotoxin adsorption capacity in both monogastric and polygastric animals in order to avoid their presence.
    The present invention aims to disclose a method by which this need is met.
    Summary Description of the Invention
    The present invention relates to a method of evaluating the adsorption capacity of mycotoxins in monogastric and polygastric animals.
    Description of the invention
    The present invention relates to a method of evaluating the adsorption capacity of mycotoxins in monogastric and polygastric animals, comprising the steps of:
    (a) arrange a sample with mycotoxins on membranes composed of dextran and polyethylene gels;
    (b) add pepsin to the sample;
    (c) add the mycotoxin collector to be analyzed in a part of the sample;
    (d) subject this part of the sample with mycotoxin scavenger successively to different pH conditions and quantify the concentration of mycotoxins adsorbed by the mycotoxin scavenger after each pH condition;
    (e) repeat step (d) with the other part of the sample without the mycotoxin scavenger;
    (f) compare the adsorption results obtained with the part of the sample that contains the mycotoxin sensor and the part of the sample that does not contain the mycotoxin sensor.
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    By way of example, said mycotoxin scavenger is glucomannan, enzymes, clinoptilolite, silicoglycidol or a combination thereof.
    This procedure is intended to mimic the route of the food bolus through the digestive system of animals, both in relation to the adsorption system at the level of the epithelium of the enterocytes of the different intestinal sections that will be simulated through a filtration system, such as of the different pH values that can be found in the digestive system.
    The intestinal epithelium that lines the inside of the colon and rectum is formed by various layers that allow the permeability of molecules of various molecular weights.
    As shown in step (a) of the procedure, the filter that would mimic this section of the epithelium is formed by membranes composed of various dextran gels (Purath, J. and Flodin, P. Nature 183, 1657; 1959) and polyethylene (Moore, J; J. Polym. Sci, 2,835; 1964) that adsorb amino acids, fatty acids and hydrolyzed carbohydrates during digestion.
    In the first section of the intestinal epithelium, molecular weight substances (in g / mol) from 83 to 291 are adsorbed, for example amino acids (lysine 146.19, methionine 149.21, leucine 131.17), fatty acids ( butyric acid 88.11, stearic acid 284.48, oleic acid 288.3) and sugars (glucose 180.1; fructose 180.16). All of them come from the digestion of nutritional principles of food. The above numbers all refer to the molecular weight of the mentioned compound.
    In the second section of the intestinal epithelium, molecular weight substances from 291 to 833 are adsorbed, for example free mycotoxins not fixed by the scavenger (deoxinivalenol 296, zearalenone 318, aflatoxins 312-330, except aflatoxin G2 having a molecular weight of 346, ochratoxin 403 and fumonisin 721) and enzymes, such as sucrose 342.29. The above numbers all refer to the molecular weight of the mentioned compound.
    Complex molecules of molecular weight from 833 to 12,500 resistant to digestive juices, such as subunits of digestive enzymes (peptidases) and simple components of starches (amylose), are adsorbed in the third section of the intestinal epithelium.
    In a preferred embodiment of the invention, said animals are monogastric. By "monogastric" are understood those animals that have a simple stomach, with a
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    Average storage capacity like that of the human being. Monogastric animals do not do astral fermentation, however, some, such as rabbits and horses, do a postgastric fermentation, because they have a functional blind, which inside has microorganisms capable of efficiently digesting portions of fiber (cellulose and hemicellulose). Examples of monogastric animals include omnivores, such as humans, rats and pigs; carnivores, such as dogs and cats; and herbivores, such as horses and rabbits.
    In a more preferred embodiment, in the case of monogastric animals, the pH conditions in step d) are pH 3, subsequently pH 5 and finally pH 9.
    In another preferred embodiment of the invention, said animals are polygastric. By "polygastric" are meant animals, also called ruminants, in which the anatomical structure of their stomachs is complex because it consists of 4 compartments. The four compartments are: the reticulum, rumen, omasum and abomasum. The first three are jointly called pre-stomachs and have an aglandular mucosa (epithelium without the ability to produce juices with digestive function).
    In a more preferred embodiment, in the case of polygastric animals, the pH conditions in step d) are pH 6.1, subsequently pH 2.5, subsequently pH 7.5, subsequently pH 4.2 and finally pH 7 .
    Preferably, the quantification of the concentration of mycotoxins that is carried out in the comparison stage f) is carried out by ELISA.
    In a more preferred example embodiment of step (d) in the case of monogastric animals:
    (d1) Adsorption at pH 3 is carried out with a toxic aqueous solution, a pH 3 buffer of, for example, 1M disodium phosphate (Na2H2PO4) and 0.1M phosphoric acid (H3PO4) (which simulates the stomach pH) and a mycotoxin collector of concentration 0.05% w / w. They are stirred for 2 hours at 38 ° C or 42 ° C. A centrifugation (10 min, 4,500 r.p.m.) is then performed at 15 ml of sample aliquot.
    (d2) Next, the pH of the sample is changed to pH 5 by a suitable base, such as sodium bicarbonate. The sample is stirred for 2 hours at 38 ° C and then centrifugation (10 min, 4,500 rpm) is performed at 15 ml of sample aliquot.
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    (d3) Next, the pH of the sample is changed to pH 9 by a suitable base, such as sodium bicarbonate. The sample is stirred for 2 hours at 38 ° C and then centrifugation (10 min, 4,500 rpm) is performed at 15 ml of sample aliquot.
    In a more preferred example embodiment of step d) in the case of polygastric animals:
    (d1) Adsorption at pH 6.1 is performed with a toxic aqueous solution, a buffer of pH 6.1 of, for example, Na2HPO4 (dibasic sodium phosphate): NaHPO4 (monobasic sodium phosphate) in volume ratio 15:85 , and a mycotoxin collector of concentration 0.05% w / w. They are stirred for 2 hours at 42 ° C. A centrifugation (10 min, 4,500 r.p.m.) is then performed at 15 ml of sample aliquot.
    (d2) Next, the pH of the sample is changed to pH 2.5 by a suitable acid, for example HCl. The sample is stirred for 2 hours at 38 ° C and then centrifugation (10 min, 4,500 rpm) is performed at 15 ml of sample aliquot.
    (d3) Next, the pH of the sample is changed to pH 7.5 by a suitable base, such as sodium bicarbonate. The sample is stirred for 2 hours at 38 ° C and then centrifugation (10 min, 4,500 rpm) is performed at 15 ml of sample aliquot.
    (d4) Next, the pH of the sample is changed to pH 4.2 by a suitable acid, for example HCl. The sample is stirred for 2 hours at 38 ° C and then centrifugation (10 min, 4,500 rpm) is performed at 15 ml of sample aliquot.
    (d5) Next, the pH of the sample is changed to pH 7 by a suitable base, such as sodium bicarbonate. The sample is stirred for 2 hours at 38 ° C and then centrifugation (10 min, 4,500 rpm) is performed at 15 ml of sample aliquot.
    In the following, the invention will be illustrated by examples that are not intended to limit the scope of the present invention.
    EXAMPLES
    1. Simulation of a procedure of an adsorption study of mycotoxins of collectors for use in monogastric animals.
    A solution of distilled water is prepared with the amount of mycotoxins that is considered appropriate (for example, 300 ppb of aflatoxins, 1 ppm of zearalenone can be used
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    or fumonisin B1 in alcoholic solution). Pepsin is added to this solution containing mycotoxins at the rate of 17,331 international units (IU) per liter.
    The sample is divided into two parts, one that will be analyzed as is (acts white) and another to which 0.5 g / liter of the mycotoxin collector is added.
    Since in the intestinal tract of the monogastrics the bolus passes through a pH of 3, after 5 and finally 9, we proceed to reproduce these conditions by adjusting the pH.
    (one) . First, HCl (hydrochloric acid) is used to lower the pH to 3. It is stirred at 38-42 ° C for 2 hours. A 15 ml sample of the blank and another of the test solution are taken and centrifuged 10 minutes at 4,500 rpm. The supernatant containing the amount of free mycotoxins (therefore those not adsorbed by the mycotoxin collector) is collected and quantified by ELISA.
    The adsorption result is calculated as: control free mycotoxin concentration - free mycotoxin concentration of the sample with collector.
    (2) . Samples are taken and brought to pH 5 using 0.5 M NaHCÜ3 (monosodium carbonate). A 15 ml sample of the blank and another of the test solution are taken and centrifuged 10 minutes at 4,500 rpm. The supernatant containing the amount of free mycotoxins is collected (therefore those not adsorbed by the mycotoxin collector) and quantified by ELISA. The adsorption result is calculated as in step (1).
    (3) . Samples are taken and brought to pH 9 using 0.5 M NaHCÜ3 (monosodium carbonate). A 15 ml sample of the blank and another of the test solution are taken and centrifuged 10 minutes at 4,500 rpm. The supernatant containing the amount of free mycotoxins is collected (therefore those not adsorbed by the mycotoxin collector) and quantified by ELISA. The adsorption result is calculated as in step (1).
    The results obtained in a mycotoxin adsorption test for an initial concentration of Fumonisin 2 ppm and mycotoxin collector dose 0.5 g / kg, are:
     Adsorption (pg / l)% adsorption Desorption (pg / l)% desorption
     pH 3 (stomach)  1999.2 99.96 0.8 0.04
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     pH 5 (duodenum)  1801.28 90.10 197.92 9.86
     pH 9 (rectum)  1668.89 83.43 132.39 6.62
    % adsorption of the entire digestive system: 83.48%
    2. Simulation of a procedure of an adsorption study of mycotoxins of collectors for use in polygastric animals.
    A solution of distilled water is prepared with the amount of mycotoxins that is considered appropriate (for example 300 ppb of aflatoxins, 1 ppm of zearalenone or fumonisin B1 can be used in alcoholic solution). Pepsin is added to this solution containing mycotoxins at the rate of 17,331 international units (IU) per liter.
    The sample is divided into two parts, one that will be analyzed as is (acts white) and another to which 0.5 g / liter of the mycotoxin collector is added.
    Since in the intestinal tract of the polygastrics the food bolus will go through a pH of 6.1, after 2.5, then 7.5, to go back down to 4.2 and finally 7, these conditions will be reproduced by adjusting the pH
    (1) First, 0.5 M NaHCO3 is used to buffer the pH to 6.1. Stir at 38-42 ° C for 2 hours. A 15 ml sample of the blank and another of the test solution are taken and centrifuged 10 minutes at 4,500 rpm. The supernatant containing the amount of free mycotoxins (therefore those not adsorbed by the mycotoxin collector) is collected and quantified by ELISA.
    The adsorption result is calculated as: control free mycotoxin concentration - free mycotoxin concentration of the sample with collector.
    (2) . Samples are taken and brought to pH 2.5 using HCl (hydrochloric acid). A 15 ml sample of the blank and another of the test solution are taken and centrifuged 10 minutes at 4,500 rpm. The supernatant containing the amount of free mycotoxins is collected (therefore those not adsorbed by the mycotoxin collector) and quantified by ELISA. The adsorption result is calculated as in step (1).
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    (3) Samples are taken and brought to pH 7.5 by using 0.5 M NaHCO3. A 15 ml sample of the blank and another of the test solution are taken and centrifuged 10 minutes at
    4,500 rpm The supernatant containing the amount of free mycotoxins is collected (therefore those not adsorbed by the mycotoxin collector) and quantified by ELISA. The adsorption result is calculated as in step (1).
    (4) . Samples are taken and brought to pH 4.2 through the use of HCl (hydrochloric acid). A 15 ml sample of the blank and another of the test solution are taken and centrifuged 10 minutes at 4,500 rpm. The supernatant containing the amount of free mycotoxins is collected (therefore those not adsorbed by the mycotoxin collector) and quantified by ELISA. The adsorption result is calculated as in step (1).
    (5) Samples are taken and brought to pH 7 through the use of 0.5 M NaHCO3. A 15 ml sample of the blank and another of the test solution are taken and centrifuged 10 minutes at
    4,500 rpm The supernatant containing the amount of free mycotoxins is collected (therefore those not adsorbed by the mycotoxin collector) and quantified by ELISA. The adsorption result is calculated as in step (1).
    The comparative results obtained in a mycotoxin adsorption test for initial concentration of Aflatoxin B1 300ppb and various mycotoxin-capturing substances at doses of all 0.5 g / kg, are:
     Mycotoxin Sensors  pH 6, 1 pH 2.5 pH 7.5 pH 4.2 pH 7 25
     Glucomannan  21.66% 20.23% 13.73% 17.73% 42.12%
     Enzymes  99.91% 100% 99.06% 100% 100%
     Clinoptilolite  25.81% 67% 61% 63.70% 60.4%
     Silicoglycidol  95.67% 100% 100% 63.70% 100%
    According to the table, it can be seen that with the process of the present invention it has been elucidated that both enzymes, such as silicoglycidol, have a similar mycotoxin uptake capacity, while that of glucomannan and clinoptilolite is much lower.
    权利要求:
    Claims (8)
    [1]
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    1. Procedure for evaluating the ability of mycotoxin adsorption in monogastric and polygastric animals, comprising the steps of:
    (a) arrange a sample with mycotoxins on membranes composed of dextran and polyethylene gels;
    (b) add pepsin to the sample;
    (c) add the mycotoxin collector to be analyzed in a part of the sample;
    (d) subject this part of the sample with mycotoxin scavenger successively to different pH conditions and quantify the concentration of mycotoxins adsorbed by the mycotoxin scavenger after each pH condition;
    (e) repeat step (d) with the other part of the sample without the mycotoxin scavenger;
    (f) compare the adsorption results obtained with the part of the sample that contains mycotoxin scavenger and the part of the sample that does not contain the mycotoxin scavenger.
    [2]
    2. - Evaluation procedure according to claim 1, wherein said animals are monogastric.
    [3]
    3. - Evaluation procedure according to claim 1 or 2, wherein the pH conditions in step d) for monogastric animals is pH 3, subsequently pH 5 and finally pH 9.
    [4]
    4. - Evaluation procedure according to claim 1, wherein said animals are polygastric.
    [5]
    5. - Evaluation procedure according to claim 1 or 4, wherein the pH conditions in step d) for polygastric animals is pH 6.1, subsequently pH 2.5, subsequently pH 7.5, subsequently pH 4.2 and finally pH 7.
    [6]
    6. - Evaluation procedure according to any of the preceding claims, wherein the quantification of the concentration of mycotoxins is carried out by ELISA.
    [7]
    7. - Evaluation procedure according to any of the preceding claims, wherein the comparison in step f) is carried out by subtracting between the concentration of control mycotoxins and the concentration of free mycotoxins in the sample with collector.
    [8]
    8. Evaluation procedure according to any of the preceding claims, wherein the mycotoxin collector added in step (c) is glucomannan, enzymes, clinoptilolite, silicoglycidol or a combination thereof.
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