Application of beta zeolite as multi-toxin binder in animal feed and related methods

Abstract

The present invention relates to the application of beta zeolite in animal feed as a toxin binder. The beta zeolites, which contain 12 membered ring systems with Bronsted and Lewis acidic sites, have high binding efficacy against common toxins present in animal feed. This study aimed to evaluate the binding efficacy of the disclosed H beta zeolite (HBZ) has high binding efficiecy against major mycotoxins such as aflatoxin B1, ochratoxin A (OTA), zearalenone, mycophenolic acid, cyclopiazonic acid, Fumonisin B1, T-2 and patulin.

Claims

1. A method for binding or sequestering mycotoxins present in animal feed comprising administering an effective amount of a mycotoxin binder, wherein the mycotoxin binder comprises H beta zeolite (HBZ), said HBZ having a pore size of from about 1 to about 15 ?.

2. The method of claim 1, wherein the mycotoxin is selected from the group consisting of aflatoxin (afla B1), ochratoxin (OTA), zearalenone (zea), mycophenolic acid (MPA), cyclopiazonic acid (CPA), fumonisin (fun B1), tricothecenes (T-2) and patulin (pat).

3. The method of claim 1, wherein the mycotoxin is a toxic secondary metabolite that is produced by fungus, bacterial toxins or ergot alkaloids.

4. The method of claim 1, wherein the mycotoxin binder is a multi-toxin binder.

5. The method of claim 1 whereby the beta zeolite has a pore size of about 5 ?.

6. The method of claim 1 whereby the beta zeolite has a Si/Al ratio of between about 10 to about 50.

7. The method of claim 6 whereby the beta zeolite has a Si/Al ratio of about 25.

8. A method for binding or sequestering mycotoxins present in animal feed comprising administering an effective amount of a mycotoxin binder, wherein the mycotoxin binder comprises H beta zeolite (HBZ), said HBZ having a Si/Al ratio in the range between about 10 to about 50, whereby the HBZ has a pore size of about 1 to about 15 ?.

9. The method of claim 8 whereby the beta zeolite has a Si/Al ratio of about 25.

10. A method for binding or sequestering mycotoxins present in animal feed comprising administering an effective amount of a mycotoxin binder, wherein the mycotoxin binder comprises H beta zeolite (HBZ), said HBZ having a pore size of about 5 ?.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a chart of isotherm linear plot of HBZ.

(2) FIG. 2 is a chart of pore width analysis of HBZ by BJH method.

(3) FIG. 3 is a chart of powder X-Ray Diffraction (pXRD) analysis of HBZ.

(4) FIG. 4 is a chart of the binding efficacy of HBZ at three different concentration of the binder; each experimental data point represents mean net binding (mean+/?standard deviation (n=3)). Significant differences between the concentrations of HBZ were denoted with different letters in superscripts (ANOVA, p<0.05).

(5) FIG. 5 is a chart on effect of acidic and near neutral pH on binding of HBZ against OTA. Each experimental data represents mean+/?standard error, n=3. Significant differences between the pH of HBZ were denoted with different letters in superscripts (ANOVA, p<0.05).

(6) FIG. 6 is a chart on effect of different pH on adsorbed OTA in HBZ. Each experimental data represents mean+/?standard error, n=3. Significant differences between the pH were denoted with different letters in superscripts (ANOVA, p<0.05).

(7) FIG. 7 is a chart on influence of methanol on adsorbed OTA in HBZ Each experimental data represents mean+/?standard error, n=3.

(8) FIG. 8 is a chart on effect of contact time on adsorption rate of OTA by HBZ. Each experimental data represents mean, n=3.

(9) FIG. 9 is a chart of in vivo OTA binding assessment of HBZ. Each experimental data represents mean+/?standard error (n=6 replicates, 2 birds/replicate). Significant difference between the treatment groups were denoted with different letters (p<0.05).

(10) FIG. 10 is a chart of in vitro net binding efficacy of HBZ against mycotoxins. Each experimental data represents mean net binding+/?standard error (n=3).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(11) Disclosed herein is a toxin binder comprising microporous zeolite materials having 12 membered ring systems. According to certain embodiments, the disclosed toxin binder comprises either a NH.sub.4+ or H+ form of beta zeolite. In according to further embodiments, the disclosed beta zeolites have pore sizes in the range of about 1 to about 15 ?, but preferably about 5 ?. According to still further embodiments, disclosed beta zeolites have a Si/Al ratio in the range between about 10 to about 50, preferably around about 25. In yet further embodiments, disclosed beta zeolites have Lewis and Bronsted acidic sites at the surface as well as inside the pores. According to certain embodiments, the disclosed toxin binder is adapted for use as multitoxin binders in animal feed supplements. In further embodiments, the disclosed toxin binder is adapted for binding a mycotoxin, wherein the mycotoxin is selected from the group consisting of aflatoxin B1, ochratoxin A, zearalenone, mycophenolic acid, cyclopiazonic acid, fumonisin B1, T-2 and patulin and also extends to toxic secondary metabolites produced by fungus, bacterial exotoxins, bacterial endotoxins, ergot alkaloids and pesticides.

(12) The term toxin means, any substances including metabolites produced by microbial origin which are toxic in nature (e.g., Mycotoxins, bacterial endotoxins, Ergot alkaloids).

(13) The term pesticides mean substances present in the animal feed capable of exerting cidal action against pests.

(14) The term binders means, non-absorbable material which is capable of binding target molecules (e.g., Mycotoxins, bacterial endotoxins, ergot alkaloids).

(15) The term porous materials refers to materials having pores in the framework.

(16) The term microporous materials means, materials having a pore size of less than about 20 ?.

(17) The term pore size means the measure of internal diameter of the channels present in zeolites.

(18) The term multitoxin binder means, binder capable of binding more than one toxins.

(19) The term Lewis acidic sites means, the presence of positive Al.sup.3+ ion in the materials which has the tendency to accept the electrons.

(20) The term Bronsted acidic sites means, the presence of positive NH.sub.4+ ion in the materials which has the tendency to donate the electrons.

EXAMPLE 1

(21) Materials and Methods

(22) The mycotoxins analysed in the current invention were done through high performance liquid chromatography (HPLC). The mycotoxins used were obtained from Sigma-Aldrich, India. The separations were performed with a reverse phase C18 analytical column, Phenomenex, Luna C18 (250 mm?4.6 mm i.d., 5 ?m particle size) at 1 ml/min flow rate. The mobile phase composition and detection wavelength used for quantification are set out in Table 1. The chromatographic system consisted of Shimadzu LC-20AD, equipped with fluorescence detector and diode array detector interfaced with LC solutions software (version 1.25).

(23) TABLE-US-00001 TABLE 1 Mobile Phase composition and detector/wavelength used for quantification of toxins Serial Name of the No. mycotoxin Mobile Phase Composition Detector/wavelength 1 Afla B1 Water:Acetonitrile = 70:30 Fluorescent/Excitation wavelength: 365 Emission wavelength: 440 nm 2 OTA 2% Aqueous glacial acetic acid:Acetonitrile = Fluorescent/Excitation 40:60 wavelength: 333 Emission wavelength: 443 nm 3 Zea 2% Aqueous glacial acetic acid:Acetonitrile = Fluorescent/Excitation 40:60 wavelength: 274 Emission wavelength: 450 nm 4 MPA Acetonitrile:Water adjusted to pH UV/284 nm 3.0 with ortho-phosphoric acid = 60:40 5 CPA Water:Methanol in ratio of 30:70 UV/284 nm v/v containing 300 mg/l zinc sulphate 6 PAT Water:Acetonitrile = 90:10 UV/284 nm 7 Fum B1 0.1M phosphate buffer pH adjusted Excitation wavelength: 335 to 3.35 with orthophosphoric acid:Methanol = Emission wavelength: 440 nm 30:70

(24) Statistical analysis. All the analyses were carried out in multiple replicates. The statistical analysis was performed using ANOVA with STATGRAPHICS plus 5.1. The differences at p<0.05 were denoted with different superscripts and were considered significant.

(25) In vitro binding method. The biphasic in vitro binding method involved adsorption at pH 3.2 (0.1 M citrate buffer) followed by desorption at pH 6.8 (0.1 M phosphate buffer). A known amount of binder (10 mg) was taken and was suspended in 1 ml of mycotoxin solution prepared in 0.1 M citrate buffer pH 3.2. The suspension was vortexed for 1 minute and was then kept in a shaking water bath at 40? C. for 45 minutes. After incubation the mixture was centrifuged (Eppendorf, 5810R) at 10000 rpm for 10 minutes at room temperature. The supernatant was removed and transferred into micro centrifuge tubes. The pellet was suspended with 1 ml of 0.1 M phosphate buffer pH 6.8 and vortexed for 1 minute. The mixture was kept in shaking water bath at 40? C. for 45 minutes. After incubation the mixture was centrifuged at 10000 rpm for 10 minutes at room temperature. The supernatants were removed and transferred into micro centrifuge tubes. The supernatants were analyzed by HPLC as per the method described above and the net binding percentage (net binding=adsorption binding percentage?desorption binding percentage) was calculated.

EXAMPLE 2

(26) Beta zeolite was synthesized using a hydrothermal method having a molar composition of 30 TEAOH (tetra ethyl ammonium hydroxide)-50SiO.sub.2-Al.sub.20.sub.3-750H.sub.20 and Si/Al ratio of 25. The material was synthesized based on the reported procedure (Ding L, Zheng Y, Zhang Z, Ring Z, Chen J. 2006. Effect of agitation on the synthesis of zeolite beta and its synthesis mechanism in the absence of alkali. Microporous and Mesoporous Materials. 94 1-8) of porous material synthesis which is widely used as catalyst in petrochemical industries. The synthesised material exchanged with 0.1 M ammonium nitrate for 24 hours (NH.sub.4.sup.+ form). After Ammonium exchange, the product was filtered and calcinated at 550? C. for 12 hours (H.sup.+ form). The final product so obtained is called H beta zeolite (HBZ) which was subsequently used for the further studies in this invention.

EXAMPLE 3

(27) The analyses were carried out to characterize the HBZ. The HBZ was characterized for BET surface area, BJH pore size using N.sub.2 sorption analyzer (Quantachrome Autosorb) and powder X-ray diffraction studies (pXRD, Rigaku diffractometer using CuK? (?=0.154 nm) radiation).

(28) The nitrogen adsorption desorption isotherms and BJH pore size distribution HBZ are shown in FIGS. 1 and 2 and the results of surface area analysis by BET method are given in Table 2. The pore width (FIG. 2) was found to be 5.5 ?. These findings suggest that the smaller pore width of HBZ would have played a role in entrapping smaller molecules such as mycotoxins which could have led to a confinement effect (Li, C. 2004. Chiral synthesis on catalysts immobilized in microporous and mesoporous materials. Catal. Rev. 46: 419-492).

(29) A narrow loop of type II isotherm was observed, which confirms the formation of a well-ordered microporous structure with uniform pore size distribution. The significant reduction in the amount of nitrogen adsorbed in the case of HBZ in both monolayer and the multilayer region confirms the formation of small pore size. It is also observed that the capillary condensation step which gives the direct measure of the pore diameter of the materials is shifted towards the low relative pressure for HBZ revealing a reduction in the pore diameter of the HBZ.

(30) TABLE-US-00002 TABLE 2 BET surface area analysis of H-beta zeolite (HBZ). Surface area Materials (m.sup.2/g) HBZ 333

(31) The material was further characterized by pXRD and the reflections are shown in FIG. 3. The reflections of HBZ at higher 20 values between 21? and 23? confirms the formation of beta zeolite (Kang, Z., Zhang, X., Liu, H., Qiu, J., and Yeung, K. L. 2013. A rapid synthesis route for Sn-Beta zeolites by steam-assisted conversion and their catalytic performance in Baeyer-Villiger oxidation. Chem. Eng. J. 218: 425-432).

EXAMPLE 4

(32) In vitro OTA binding was evaluated for HBZ by biphasic binding studies to find the least concentration necessary to adsorb/sequester the entire OTA used in the experiment. The procedure involves the suspension of HBZ: 0.75% (0.75 mg) in OTA solution (1 ?m/mL prepared in citrate buffer pH 3.2) for 1 hour at 40? C. in shaking water bath. Subsequently the net binding of OTA was calculated as briefed in Example 1. Similarly, the evaluation at 0.25% and 0.5% were also done. FIG. 4 shows the influence of different concentrations of HBZ against OTA binding. High binding was observed at 0.5% and 0.75% HBZ. A dose related increase in binding was observed at 0.25% and 0.5% which was statistically significant (p<0.05, n=3). This indicates 0.5% adsorbent concentration was sufficient to bind the OTA concentration used in the experiment.

EXAMPLE 5

(33) An experiment was conducted to evaluate the influence of pH on binding of OTA against HBZ. The OTA adsorption was evaluated individually at pH 3.2 and pH 6.8 as described in Example 1 with 1 ?g/mL OTA and the results were shown in FIG. 5. HBZ exhibited maximum binding at pH 3.2 as well as pH 6.8 conditions. Statistically no difference in binding efficacy was observed for HBZ at pH 6.8 with respect to pH 3.2 (p>0.05, n=3). In most of the adsorption process, the effect of binding of mycotoxins from aqueous medium is highly dependent on pH, as pH affects the surface charge of adsorbents as well as the degree of ionization of toxins. This confirms HBZ could bind OTA irrespective of the ionization state (either ionized or unionized).

(34) During the digestion process in monogastric animals, the pH of the food bolus changes to a large extent depending upon the gastro intestinal (GI) compartments i.e., from pH 6.5 (crop) to pH 3.0 (gizzard) and then to pH 7.5 (distal part of intestine, Fengying, G., Jie, G., Hui, R., and Guoqing, H. (2011) In Vitro Evaluating the Activities and Stabilities of the Multihydratases Produced by Aspergillus Niger Zju-Y1 in Simulated Poultry Digestive Tract pH Levels. Procedia Eng. 18, 405). Hence, the effect of pH simulating the pH of the GI tract was evaluated. OTA are adsorbed to adsorbents (HBZ) initially at pH 6.8 as per Example 1 and the pellets were sequentially evaluated for the desorption with 1 ml of pH 3.0, 5.0, 6.8 and 7.5 and the supernatants at each step were analyzed for OTA content. The residual OTA content adsorbed were calculated and the results were shown in FIG. 6. HBZ was observed with minimal (<5%) desorption at pHs (3.2, 5, 6.8) and around 16% desorption at pH 7.5 (FIG. 6). This study suggests that the bound OTA in HBZ remains almost intact as it travels through GI tract. These results and the findings suggests that the HBZ adsorbed OTA are almost stable on the entire pH range relevant to the GI tract of monogastrics.

EXAMPLE 6

(35) The Chemisorption Index (CI), strength of adsorption of OTA with HBZ was assessed using the method described previously with some modifications (Dwyer M. R., Kubena L., Harvey R. B., Mayura K., Sarr A. B., Buckley S., Bailey R. H. and Phillips T. D. 1997. Effects of Inorganic Adsorbents and Cyclopiazonic Acid in Broiler Chickens. Poultry Science. 76: 1141-1149). Briefly, 10 mg of adsorbents were added to 1 ml of water containing 1 ?g/m of OTA (C.sub.initial and incubated at 40? C. for 1 hour. After 1 hour, the tubes were centrifuged and the supernatants were analyzed for OTA and the amount of OTA bound was estimated (C.sub.bound). To the pellet, 1 ml of 20% methanol was added and incubated for 1 hour in shaking water bath, centrifuged and the supernatants were analyzed for OTA (C.sub.unbound). The percentage of OTA bound to HBZ at each step is shown in FIG. 7. The CI was calculated by using the following Equation 1,
CI=(C.sub.Bound?C.sub.Unbound)/C.sub.initial. Equation 1. Chemisorption Index.

(36) The CI results are tabulated in Table 3. CI of HBZ was found to be 0.78. The results suggest that HBZ has the highest propensity and tightest binding for HBZ at the analyzed methanol concentration.

(37) TABLE-US-00003 TABLE 3 Assessment of Chemisorption Index (CI) for H-beta zeolite (HBZ). CI 20% Adsorbents methanol HBZ 0.78

EXAMPLE 7

(38) Further the experiments were carried out to identify the interactions associated with HBZ and OTA. The interactions associated with HBZ and OTA were evaluated through thermodynamic studies (Avantaggiato, G., Greco, D., Damascelli, A., Solfrizzo, M., and Visconti, A. (2014) Assessment of Multi-mycotoxin Adsorption Efficacy of Grape Pomace. J. Agric. Food Chem. 62, 497-507; Ringot, D., Lerzy, B., Bonhoure, J. P., Auclair, E., Oriol, E., and Larondelle, Y. (2005) Effect of temperature on in vitro ochratoxin A biosorption onto yeast cell wall derivatives. Process. Biochem.: 3008-3016). The parameters arrived through the thermodynamic studies are: Gibbs free energy change, ?G? (kJ/mol), is the fundamental criterion of spontaneity and is given by the Gibbs-Helmholtz equation (Eq.2),
?G?=?RT ln K.sub.0 Equation 2. Gibbs-Helmholtz equation.
Where, K.sub.0 is the equilibrium constant, R is the universal gas constant and T is the absolute temperature (K).

(39) The equilibrium constant K.sub.0 for the adsorption reaction is determined by the Equation 3 where, Q.sub.eq (mol/kg) is the molar OTA concentration in adsorbed phase and C.sub.eq is the residual OTA concentration in equilibrium (mol/L).
K.sub.0=Q.sub.eq/C.sub.eq Equation 3. Equilibrium constant.

(40) The constant of equilibrium constant (K.sub.0) is expressed in terms of enthalpy and entropy changes is given by Van't Hoff equation (Eq. 4), where R is the universal gas constant, T is the absolute temperature (K), ?H? is the enthalpy change (KJ/mol) and ?S? is the entropy change (KJ/mol.K). The slope and intercept of the plot of 1/T vs ln K.sub.0 were used to calculate ?H? and ?S? values.
Ln K.sub.0=(??H?/RT)+(?S?/R) Equation 4. Van't Hoff equation

(41) The thermodynamic parameters (?G?, ?H? and ?S?) for OTA with HBZ was calculated at different temperature (278 K, 288 K, 298 K, 308 K and 318 K) and results are tabulated in Table 4. The negative values of ?G? (Table 4) were observed with HBZ which indicates a spontaneous adsorption process. The slope and intercept from the plots of ln K.sup.0 versus 1/T were used to determine the thermodynamic parameters (?H? and ?S?) according to the Van't Hoff equation (Table 4). The plots obtained for OTA at experimental temperature conditions gave correlation of R.sup.2=0.9612 for HBZ (data not shown). The values of ?H? (standard enthalpy) and ?S? (standard entropy) are shown in Table 4. The negative value of ?H? for OTA confirms the exothermic nature of the phenomenon.

(42) The enthalpy was found to be less than 20 KJ/mol, indicating a physisorption phenomenon allowing attaining of the equilibrium rapidly. The magnitude of ?S? values also indicates the nature of interactions between adsorbate and adsorbent. HBZ exhibited positive ?S? value, suggesting the hydrophobic interaction primarily between adsorbent and adsorbate. In general, enthalpy cost was associated with hydrophobic interactions but reverse in observed with HBZ which suggests the binding also involves polar non-covalent interactions. Negative enthalpy and positive entropy value were also observed with other sorbents(Lin, F.-Y., and Chen, W. Y. (2001) Microcalorimetric Studies on the Interaction Mechanism between Proteins and Hydrophobic Solid Surfaces in Hydrophobic Interaction Chromatography: Effects of Salts, Hydrophobicity of the Sorbent, and Structure of the Protein. Anal. Chem. 73: 3875-3883. Ringot, D., Lerzy, B., Bonhoure, J. P., Auclair, E., Oriol, E., and Larondelle, Y. (2005) Effect of temperature on in vitro ochratoxin A biosorption onto yeast cell wall derivatives. Process. Biochem. 40: 3008-3016).

(43) TABLE-US-00004 TABLE 4 Thermodynamic parameters for OTA adsorption by H-beta zeolite (HBZ). Temper- ?S? ature ?G? ?H? (KJ/ Material (K) K.sub.0 ln K.sub.0 (KJ/mol) (KJ/mol) mol .Math. K) HBZ 278 7592.30 8.93 ?20.65 ?14.22 22.76 288 5424.86 8.59 ?20.58 298 4754.36 8.46 ?20.97 308 4305.28 8.36 ?21.42 318 3244.48 8.08 ?21.37

EXAMPLE 8

(44) The rate of adsorption of OTA with HBZ was evaluated at various time intervals at pH 6.8 and pH 3.2 individually at 1% w/v (10 mg/ml) as well as 0.2% (2 mg/ml) dosage (triplicate independent experiments) at 1 ?g/ml of OTA (Avantaggiato, G., Greco, D., Damascelli, A., Solfrizzo, M., and Visconti, A. (2014) Assessment of Multi-mycotoxin Adsorption Efficacy of Grape Pomace. J. Agric. Food Chem. 62: 497-507). Samples were withdrawn at appropriate time intervals (1-60 min). Supernatant liquid portions were analyzed for residual OTA content and the binding percentages were calculated as described in Example 1. The results are shown in FIG. 8.

(45) HBZ was observed with very rapid adsorption of OTA and establishment of equilibrium in a short time. The effect of contact time is of significant importance in mycotoxin reduction by adsorption as most of the major toxins were absorbed very rapidly in the GI tract (Avantaggiato, G., Greco, D., Damascelli, A., Solfrizzo, M., and Visconti, A. (2014) Assessment of Multi-mycotoxin Adsorption Efficacy of Grape Pomace. J. Agric. Food Chem. 62: 497-507). OTA is reported to absorb rapidly from the GI tract by passive absorption (Ringot, D., Lerzy, B., Bonhoure, J. P., Auclair, E., Oriol, E., and Larondelle, Y. (2005) Effect of temperature on in vitro ochratoxin A biosorption onto yeast cell wall derivatives. Process. Biochem. 40: 3008-3016). The maximum adsorption of >80% was reached within 5 min for HBZ. No rapid change in adsorption percentage was seen after 5 minutes in HBZ. This shows establishment of equilibrium was achieved in a shorter time with HBZ. Such rapid uptake of toxins and establishment of equilibrium by HBZ in short period implies the greater efficacy of material.

EXAMPLE 9

(46) One of the approaches to estimate the in vivo binding potential of the adsorbents is the analysis of mycotoxin content in excreta receiving adsorbents and comparing it with the control group receiving no adsorbents. The in vivo binding potential of the material was evaluated in broiler birds.

(47) The in vivo excretion trial was done with the six week old Vencobb-400 breed birds. The each study group had six replicates with two birds per replicate. The birds were adapted for five days with ad libitum feed (corn soya based mash feed) and water. Then the birds were starved for 24 hours to empty the gut contents. After the starvation period, each bird was fed with 50 g of feed (contaminated with 200 ppb OTA) of the respective treatment group as shown in Table 5. Water was given ad libitum throughout the trial. The excreta samples were collected for 72 hrs. The collected excreta samples were dried at 50? C. for 48 hours. The OTA in excreta samples were extracted as follows: 5 g of dried excreta samples were extracted with 20 ml of solvent (Acetonirile and 10% glacial acetic acid=1:1) using shaking incubator (Orbitrek, LT) for 60 minutes at 250 revolutions per minute (rpm). After stirring, the mixture was filtered. The filtrate was transferred into a separating funnel and 20 ml hexane) was added, shaken vigorously for 10 minutes and allowed to stand for 10 minutes for the layer separation. The upper hexane layer was discarded to remove fats and oil. The same procedure was repeated twice. The lower layer was further extracted with 50 ml of chloroform thrice. All the chloroform layers were pooled and passed through sodium sulphate bed kept in a funnel. The chloroform was removed using rotary evaporator (Heidolph, Hei-VAP advantage) and the residue was reconstituted in 5 ml of 50% aqueous acetonitrile. The reconstituted layer was centrifuged at 10000 rpm for 10 minute (Eppendorf, 5810R) and the supernatants were quantified by HPLC for OTA as described in Example 1.

(48) TABLE-US-00005 TABLE 5 Treatment groups used for the in vivo OTA excretion study. All treatment groups were given OTA# contaminated feed. HBZ dose Group Number of replicates* (per ton of feed) Control 6 Treatment 1 (T1) 6 0.5 kg Treatment 2 (T2) 6 1 kg *2 birds/replicate; #200 ppb OTA fortified in mash feed; HBZH-beta zeolite.

(49) The results were presented in FIG. 9. All the treatment groups showed significantly higher amount of OTA in excreta in comparison with control. In this study, the chicken fed on feed spiked with OTA showed significantly higher OTA excreted in all the treatment groups compared to control (p<0.05, FIG. 9) which confirms the in vivo binding potential of the material supplemented.

EXAMPLE 10

(50) HBZ was further evaluated for its multiple mycotoxins binding property. The stock solutions of mycotoxins were prepared in acetonitrile and stored at 4? C. The working stock solutions (afla B1 (0.5 ?g/ml), OTA (1 ?g/ml), fum B1 (2 ?g/ml), MPA (5 ?g/ml), CPA (5 ?g/ml), zea (1 ?g/ml), PAT (5 ?g/ml) and T-2 (2 ?g/ml) of individual mycotoxins were prepared in pH 3.2 0.1 M citrate buffer as described in Example 1. The biphasic in vitro studies were carried out for the all the mentioned toxins individually as per the procedure briefed in Example 1 and the net binding was calculated. FIG. 10 shows the net toxin binding results of HBZ and the results shows HBZ has multi toxin binding property.

(51) The foregoing description and drawings comprise illustrative embodiments of the present inventions. The foregoing embodiments and the methods described herein may vary based on the ability, experience, and preference of those skilled in the art. Merely listing the steps of the method in a certain order does not constitute any limitation on the order of the steps of the method. The foregoing description and drawings merely explain and illustrate the invention, and the invention is not limited thereto, except insofar as the claims are so limited. Those skilled in the art who have the disclosure before them will be able to make modifications and variations therein without departing from the scope of the invention.