Patent Publication Number: US-9901108-B2

Title: Mycotoxin-reducing composition

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     The present application is a divisional of U.S. application Ser. No. 12/447,837, filed Sep. 15, 2009, which is the National Stage of International Application Number PCT/GB2007/004191, filed Nov. 1, 2007, each of which is hereby incorporated by reference herein in its entirety, including any figures, tables, nucleic acid sequences, amino acid sequences, or drawings. 
    
    
     FIELD OF THE INVENTION 
     This invention relates to reducing the mycotoxin content of a foodstuff. 
     BACKGROUND OF THE INVENTION 
     Mycotoxins are toxins produced by funghi that are known to affect adversely the nutrition and health of humans and animals. The production of mycotoxins is the result of the natural biological process of funghi and has occurred over thousands of years. However, mycotoxin production has been influenced by climate change and changes in agricultural practise. 
     Mycotoxins are produced by a wide range of funghi including  Aspergillus  (Aflatoxin and Ochratoxin),  Fusarium , (Zearalenone, Deoxynivalenol, Fumonisin) and  Penicillium  (PR toxin and Roquefortin). These toxins have a considerable pharmacological effect, even at very low concentrations (parts per billion), while toxicity may be further enhanced by metabolism in vivo, particularly by the liver. Detoxification of most toxins occurs in the liver, while in the gastro-intestinal tract (GIT), under certain conditions, detoxification may also be achieved by micro-organisms. 
     A large number of mycotoxins have been identified. Currently, there are five main groups of particular agricultural interest: the Aflatoxins, the trichothecene (for example Deoxynivalenol), the Zearalenone group, the Fumonisins and the endophyte toxins. 
     Aflatoxins can cause growth reduction, suppressed immunity, reduced feed efficiency and increased mortality in cattle, among other symptoms. In pigs, reduced feed efficiency, increased mortality, and lower growth rates can be observed. In poultry, there are similar symptoms and a decreased ability to metabolize fat, protein and starch. 
     Zearalenone in cattle and pigs mimics oestrogen and produces a considerable reduction in reproductive performance, reduced growth, reduced milk production and reduced feed efficiency. In poultry, increased mortality is observed. 
     Deoxynivalenol (DON), an example of a trichothecene, causes severe symptoms in cattle, pigs and poultry, including gastric effects such as vomiting, reduced growth rates, reduced egg production, scours and reduced feed efficiency. 
     Fumonisin produces negative effects via a reduction in blood circulation and cardiac output, at least in part by agonising sphingosine receptors. In this way they reduce growth and cause pulmonary oedema in swine and poultry. This reduction of circulation affects all major organs including the liver and can exacerbate and enhance the effects of other toxins that may also be present. 
     Ochratoxin can be carcinogenic in man and produces immuno-supression in farm animals. 
     Lolitrem B ( Acremonium  lolii in Ryegrass) is an example of an endophyte toxin that produces a form of grass staggers often confused with hypomagnesaemia. 
     Sporidesmin ( Pithomyces  spp. in Ryegrass) is an endophyte that causes facial eczema and liver damage in sheep. 
     Ergovaline ( Acremonium coenophialum ) is an endophyte toxin found in tall fescue, which reduces prolactin release and reduces blood flow. 
     It is in the interests of the health of both humans and animals that mycotoxins are reduced, or preferably removed altogether, from the food chain. 
     Current techniques used to reduce mycotoxin content in a foodstuff involve the use of mycotoxin-binding agents, such as bentonite clay, to which the toxins bind and can therefore be removed with the clay. However, binding and removal of mycotoxins are only partly successful. Some mycotoxins remain toxic even when attached to a binding agent, while some toxins do not bind efficiently at normal in vivo concentrations. Higher contamination levels are also an issue as the current recommended levels of binders may not be sufficient to remove all toxins present. 
     An alternative technique is to add to foodstuffs enzymes, or microorganisms, that break down mycotoxins, to reduce toxicity. However, this is often not effective at reducing mycotoxin content sufficiently, which may be due to a dynamic equilibrium in the gasto-intestinal tract, which prevents excretion of the toxins. 
     An effective technique for reducing mycotoxin toxicity is therefore still required. 
     SUMMARY OF THE INVENTION 
     The present invention is based on the surprising realisation that a composition containing an enzyme, a mycotoxin-binding agent and a microorganism capable of taking up a mycotoxin is unexpectedly effective at reducing mycotoxin toxicity. 
     According to a first aspect of the invention, a composition comprises an enzyme, a mycotoxin-binding agent and a microorganism capable of taking up a mycotoxin. 
     According to a second aspect of the invention, a method of reducing the toxicity of a mycotoxin in a foodstuff comprises the step of contacting the foodstuff with a composition as defined above. 
     According to a third aspect of the invention, a foodstuff comprises a composition as defined above. 
     According to a fourth aspect of the invention, a composition as defined above is useful in therapy, in particular the treatment of a disease caused by a mycotoxin. 
     According to a fifth aspect of the invention, a composition as defined above is used in the manufacture of a medicament for the treatment of a disease caused by a mycotoxin. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
       The invention is described with reference to the following figures, wherein: 
         FIG. 1  is a graph indicating the binding percentage of various mycotoxins and mycotoxin-binding agents; 
         FIG. 2  illustrates the effect of Saccharomyces cerevisiae uptake of DON, which is then modified by the yeast, followed by release and binding of the modified toxin to bentonite; 
         FIG. 3  illustrates the effect of Saccharomyces cerevisiae uptake of Zearalenone; 
         FIG. 4  illustrates the reduction of fumonisin using Saccharomyces cerevisiae; 
         FIG. 5  shows the increase in milk production seen in a dairy herd, contaminated with Deoxynivalenol and Zearalenone, after application of a composition according to the invention; and 
         FIG. 6  shows the increase in milk production seen in a dairy herd, contaminated with Vomitoxin, after application of a composition according to the invention. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The invention is based on the finding that combining an enzyme, a mycotoxin-binding agent and a microorganism that is capable of taking up a mycotoxin is surprisingly effective at reducing the toxicity of mycotoxins present in a foodstuff. 
     The present inventor has realised that, when an enzyme alone is used, a dynamic equilibrium exists that prevents toxins from being defecated. Therefore, the sequestration by a microorganism and binding by a binding agent act as an aid to the ultimate degradation of the toxin by the enzymes and, optionally, microorganisms in the composition. 
     As used herein, the term “enzyme” is to be given its usual meaning in the art, i.e. a biological catalyst. For the avoidance of doubt, an enzyme is a protein. The enzyme is preferably at least partially purified, i.e. is provided as a cell extract, isolate or purified preparation. The enzyme is present in the composition in addition to any enzymes present in the microorganism, i.e. the enzymes, microorganism and mycotoxin-binding agent are present as three separate components of the composition. The purpose of the enzyme in the composition of the invention is to modify one or more mycotoxins, to reduce the toxicity of the toxin. Preferably, the enzyme breaks down the toxin, partially or completely, thereby removing the toxicity of the toxin. 
     Therefore, the one or more enzymes of the composition preferably break down the toxin. The enzyme may further induce a modification of a toxin so that binding to the binding agent is increased, pending further degradation. Without wishing to be bound by theory, the inventor believes that epoxidation and/or dehydrogenation of the toxin, in particular, improves binding. An increase in binding of the toxin to the binding agent may be detected by a toxin binding assay, as detailed in Example B, experiment 4 . Other enzymes, that do not necessarily act directly upon a mycotoxin, may be used to produce radicals that modify the toxin. 
     This may be achieved with or without binding of the toxin to the enzyme. An example of a suitable enzyme of this type is lignin peroxidase. In this embodiment, where radicals are used, the enzyme may be replaced with a biomimetic catalyst (such as the haem prosthetic group), an organo-metallic catalyst or a metallic catalyst. 
     Any enzyme, or combination of enzymes, with the ability to reduce the toxicity of a mycotoxin is within the scope of the invention. Preferred enzymes include an esterase, lipase, protease, oxidase, amino acid oxidase, lactonohydrolase, peroxidase, lactoperoxidase, manganese peroxidase, epoxidase, polysaccharase and dehydrogenase. Any combination of preferred enzymes can be used and the skilled person will realise that the specific combination of enzymes required may depend on the toxin or toxins to be treated. Particularly preferred enzymes include cellulases, epoxidases, dehydrogenases, hemicellulases (also known as xylanases) and lipases, preferably a triacylglycerol lipase (E.C. number 3.1.1.3), such as lipase AE 02 (obtainable from  Candida  Rugosa and commercially available from Mann Associates, Cambridge, UK) or “Lipase OF” (commercially available from Meito Sangyo, Japan). The combination preferably includes a lipase. A preferred combination of enzymes is the combination of a cellulase, a hemicellulase (xylanase) and a lipase. A preferred combination of enzymes suitable for use in the invention is shown in Table 2, below. 
     The second component of the composition according to the invention is a mycotoxin-binding agent. One or more binding agents can be included. Preferably, 2, 3, 4 or more different binding agents are included. The term “binding” refers both to ionic or covalent chemical bonding and weaker physical adsorption involving van der waals interactions and/or hydrogen bonding. In a preferred embodiment, the binding agent has a charged surface. The charge may be positive or negative. Binding refers to the non-transient association of a mycotoxin to the binding agent. In a preferred embodiment, the binding has a minimum affinity between the mycotoxin and binding agent such that 10 mg of binding agent will bind 50% of a 200 μg/l (i.e. physiological) concentration of toxin. Binding may be determined using the method provided in Example B, experiment 4 . Mycotoxin-binding agents are known in the art; examples of suitable binding agents are zeolites, montmorillonite, bentonite clays, diatomaceous earth, activated charcoals, fibrous plant materials such as wheat bran and alfalfa fibre, yeast cell wall extract and polysaccharides. Any agent that can bind a mycotoxin or a mycotoxin breakdown product is within the scope of the invention. Preferred mycotoxin-binding agents include a bentonite clay, preferably sodium bentonite or more preferably calcium bentonite, and a polysaccharide, preferably glucomannan, more preferably an esterified glucomannan. The glucomannan is preferably obtained as a yeast cell wall extract, as is recognised in the art. Glucomannan-containing mycotoxin adsorbents are commercially available, for example Mycosorb®, obtainable from Alltech UK, Stamford, UK. 
     Preferably, the binding agent has at least one ionic, i.e. charged, surface, which will allow (strong) ionic bonding between the toxin and binding agent to occur. The charge may be positive or negative. Examples of ionic binding agents are bentonites such as sodium bentonite and calcium bentonite. 
     The third component of the composition according to the invention is a microorganism. As used herein, the term “microorganism” refers to bacteria and yeasts. The skilled person will recognise that the microorganism is preferably not harmful to the health of the human or animal which will ingest the foodstuff to which the composition will be applied, i.e. the microorganism is preferably non-pathogenic. The purpose of the microorganism is to uptake mycotoxins. Any microorganism that can uptake mycotoxins can be used according to the invention. Once taken up, the microorganism sequesters the toxin, which cannot then be taken up by the host animal. The microorganism&#39;s endogenous enzymes may then breakdown the toxins, although this is not an absolute requirement. Preferably, the microorganism is a yeast, more preferably a saccharomyces, most preferably  Saccharomyces cerevisiae.    
     The microorganism has the function of physically taking up the toxin to sequester it within the bacterial or yeast cell. In the case of one toxin at least (DON), the toxin is modified in the cell and analogues are released, which are then able to bind to the binders where further enzyme degradation can take place. Therefore, in a preferred embodiment, the microorganism additionally performs a second function, that of expressing one or more enzymes capable of breaking down a mycotoxin. 
     One or more different microorganisms can be included in the composition of the invention. In one embodiment, at least two microorganisms are included; the first microorganism is primarily responsible for mycotoxin uptake and the second is primarily responsible for mycotoxin degrading enzyme expression. For the avoidance of doubt, in the embodiments where a microorganism expresses one or more enzymes capable of breaking down a mycotoxin, the composition must still contain the enzyme component as a separate component of the composition. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Example Composition according to the Invention. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Approx 
               
               
                 Name 
                 Source/Company 
                 Specification 
                 kg 
                 % wt 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 FerMos 
                 JACKLYN IND 
                 FerMos, 25 kg Bags 
                 7.445 
                 35%  
               
               
                 Mannan 
                   
                   
                   
                   
               
               
                 oligosaccharide 
                   
                   
                   
                   
               
               
                 (Binding Agent) 
                   
                   
                   
                   
               
               
                 Terra-Green 
                 OIL DRI 
                 Terra-Green, 24/48 
                 6.68742 
                 30%  
               
               
                 24/48 LVM 
                   
                 LVM, 50 lb bags 
                   
                   
               
               
                 (Binding Agent) 
                   
                   
                   
                   
               
               
                 Sodium 
                 Eastern Mineral 
                 Sodium Bentonite 
                 2.06116 
                 10%  
               
               
                 bentonite 
                   
                 Granular 
                   
                   
               
               
                 (Binding Agent) 
                   
                   
                   
                   
               
               
                 Diabond 
                 Eastern Mineral 
                 200 Bags of Diabond 
                 2.06116 
                 10%  
               
               
                 Granular 
                   
                 granular, 50 lb bags 
                   
                   
               
               
                 (Binding Agent) 
                   
                   
                   
                   
               
               
                 Dried molasses 
                 Bartlett Milling 
                 Dried molasses, 50 lb 
                 1.03058 
                 5% 
               
               
                   
                   
                 bags. 
                   
                   
               
               
                 
                   S. 
                   cerevisiae 
                 
                 JACKLYN IND 
                 Bulk Yeast 10 B/g, 20 kg 
                 1 
                 5% 
               
               
                 Yeast 
                   
                 box 
                   
                   
               
               
                 Vitamin A, A-30 
                 RC BWATER 
                 Vitamin A, A-30, 50 lb 
                 0.51529 
                 2% 
               
               
                   
                   
                 bag 
                   
                   
               
               
                 Vitamin E, E-20 
                 RC BWATER 
                 Vitamin E, E-20, 50 lb 
                 0.51529 
                 2% 
               
               
                   
                   
                 bag 
                   
                   
               
               
                 Enzyme Premix 
                 Micron 
                 Mycotex SW enzymes 
                 0.03405 
                 &lt;1%   
               
               
                 IV (See Table 
                   
                   
                   
                 (0.16%)   
               
               
                 2) 
                   
                   
                   
                   
               
               
                 Light mineral oil  
                 AKEY 
                 Light mineral oil, 387 lb 
                 0.22809 
                 1% 
               
               
                   
                   
                 drum 
                   
                   
               
               
                   
                 TOTAL 
                   
                 21.53204 
                 100.00%    
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Enzyme Formulation suitable for use  
               
               
                 in the Composition of the Invention 
               
            
           
           
               
               
               
               
               
            
               
                 Name 
                 Source 
                 Specification 
                 kg 
                 % wt 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Cellulase 300 
                 BIOCAT 
                 Cellulase 300 
                 0.49000 
                 32.67% 
               
               
                 TR 
                   
                 TR, 75,000/g, 
                   
                   
               
               
                 Hemicellulase 
                 BIOCAT 
                 Hemicellulase 
                 0.49000 
                 32.67% 
               
               
                 1500 
                   
                 1500 
                   
                   
               
               
                 Lipase OF 
                 Meito 
                 400,000 U/g 
                 0.49000 
                 32.67% 
               
               
                   
                 Sangyo 
                   
                   
                   
               
               
                 Flow Agent 
                 DUNLEARY 
                 Hubersorb 600, 
                 0.03000 
                 2.00% 
               
               
                   
                   
                 30 lb bag 
                   
                   
               
               
                   
                   
                 TOTAL 
                 1.50000 
                 100.00% 
               
               
                   
               
            
           
         
       
     
     In addition to these essential components, additional components can be included in a composition of the invention, if necessary. Examples of suitable additional components are food supplements such as vitamins and minerals, mineral oil, a flow agent such as a fine, precipitated calcium silicate powder (e.g. hubersorb), and a sugar source such as dried molasses. An example of a formulation according to the invention is provided in Table 1. 
     The three essential components are preferably prepared as a mixture, optionally with a flow agent (as in Table 2), and subsequently combined with the additional components when needed for application to a foodstuff. 
     Any amount of each of the three essential components of the composition can be used that achieves the desired result of reducing mycotoxin levels. Preferably, between 0.1 kg and 50 kg of the composition is applied to each tonne of feed, more preferably between 0.2 kg and 10 kg per tonne, e.g. 0.5, 1, 1.2, 1.5, 2.5, 5, 7.5 kg or more per tonne. Suitable levels will be apparent to the skilled person. 
     In a preferred embodiment, the composition is applied to the feed such that the microorganism is present at approximately 1×10 9  cells per tonne of feed or greater. More preferably, the microorganism is present at 2×10 9 ′ 3×10 9 ′ 4×10 9 ′ or 5×10 9  cells per tonne of feed or greater. Yet more preferably, 1×10 10  cells per tonne of feed is applied, more preferably 1×10 11  cells per tonne of feed or greater is applied, for example greater than 2×10 11  cells per tonne of feed, more preferably greater than 1×10 12 , 2×10 12  cells per tonne of feed or more. The skilled person will realise that the precise amount of composition that is required per tonne of feed will depend on the feed to be treated, the toxins present and the intended recipient of the feed. For example, an animal that consumes a small amount of feed per day will generally require a higher concentration of microorganism applied to the feed than an animal that consumes a large amount of feed per day. For cattle, an example of a suitable amount is 2.2×10 11  cells per tonne of feed, while for swine and poultry a preferred amount is 1.1×10 12  cells per tonne of feed. 
     In the composition, the microorganism is present at any suitable level that allows detoxification to occur when applied to a foodstuff. Preferably, the microorganism is present at approximately 1×10 9  cells or greater per kilogram of the composition, more preferably 1×10 1 ° cells per kilogram of composition, or greater, for example 1×10 11  or 1×10 12  cells per kilogram, or greater. The skilled person will appreciate that the amount of microorganism in the composition will affect the amount of composition required to achieve the desired effect, in particular the cells per tonne of feed recited above. In the example composition of Table 1, yeast is included at approx 5% (by weight). 1 Kg of the composition defined by Table 1 contains approximately 5×10 11  yeast cells. Therefore, applying 0.5 Kg of this composition to a tonne of feed will provide approximately 2.5×10 11  cells per tonne of feed, while applying 2.5 Kg of this composition to a tonne of feed will provide approximately 1.25×10 12  cells per tonne of feed. 
     The one or more enzyme is present in the composition at an effective level. Less than 5% by weight of enzyme is preferred, more preferably less than 1%, more preferably between 0.01% and 0.5% by weight, yet more preferably between 0.15% and 0.25% by weight. The composition of Table 1 contains an enzyme mixture totalling 0.16% by weight, including the flow agent. 
     The binder is present in the largest proportion, at an effective level. Preferably, at least 50% by weight of the composition should be binding agent, more preferably 60 to 90%. The composition of Table 1 contains a mixture of binding agents totalling approx 85%. 
     For the avoidance of doubt, a suitable range for each of the essential ingredients in the composition of the invention is: microorganism—0.1% to 10% by weight, preferably 2% to 8%, i.e. 5%; enzyme—less than 5% by weight of enzyme is preferred, more preferably less than 1%, more preferably between 0.01% and 0.5% by weight, yet more preferably between 0.15% and 0.25% by weight; binder—preferably at least 50% by weight of the composition is binding agent, more preferably 60 to 90%. The balance (if any) can be made up with the additional agents recited above, if required. 
     The composition of the invention can be in any suitable formulation, for example a solid such as a powder or granulate, a gel or a liquid. Preferably, the formulation is a solid, more preferably a powder. 
     The composition of the invention is effective at reducing mycotoxin levels. Preferably, the composition is used to reduce mycotoxins in a foodstuff. As used herein, the term “foodstuff” is to be given its usual meaning in the art, referring to any material that is eaten by a human or animal for nutrition. Foodstuffs comprise carbohydrates, fats and/or proteins among other components. The foodstuff can be of animal or vegetable origin. Human food and animal feeds are within the scope of the term. In a preferred embodiment, the foodstuff is an animal feed of plant origin, commonly known as fodder, more preferably a cereal foodstuff. The term “foodstuff” includes materials that must be processed before safe consumption by a human or animal. A preferred example of such a material are “distiller&#39;s grains”. These grains, also known as “distiller&#39;s dried grains”, are a cereal by-product of the distillation process. Distiller&#39;s grains are known in the art. Briefly, they are produced in distilleries by drying mash and are used commonly as a fodder for livestock, especially ruminants. Detoxifying distiller&#39;s grains, by reduction of mycotoxin content, is therefore within the scope of the invention. 
     The composition can be added to the foodstuff any way that is effective at reducing the mycotoxin content. Contact between the composition and foodstuff is required. The preferred method is to treat the foodstuff before ingestion to remove, preferably completely, the toxicity. This is “detoxification” of the foodstuff before consumption. An alternative method is to treat the feed immediately before ingestion, or simultaneous with ingestion, and to rely on the invention to complete the detoxification during digestion. This post-ingestion detoxification is particularly suitable for use in animals, preferably ruminants such as cattle and sheep. 
     A method of reducing the toxicity of a mycotoxin or mycotoxins in a foodstuff comprises the step of contacting the foodstuff with a composition as defined herein. The mycotoxin can be any mycotoxin. Preferred mycotoxins include Zearalenone, a trichothecene such as Deoxynivalenol, Aflatoxin, Fumonisin, Roquefortin, ochratoxin or an endophyte toxin, such as sporidesmin, ergovaline or Lolitrem B. 
     A foodstuff of any human or non-human animal, preferably non-human mammal, can be detoxified according to the invention. Preferably, the foodstuff is for a farm animal such as cattle, horses, pigs or poultry. 
     Without wishing to be bound by theory, detoxification is thought to be achieved in a number of ways, acting in synergy. The enzyme component breaks down toxins and/or improves the toxin binding to the mycotoxin-binding agent. The binding agent prevents the mycotoxins from being absorbed by the animal and allows their excretion. The microorganism uptakes and sequesters the toxins and may also break down the toxins. Any toxin that is released by the microorganism is thought to bind more strongly to the binding agent, where further degradation (by the enzymes) may occur. Therefore, the actions of the microorganism and/or enzyme are thought to potentiate the binding of the toxin to the binding agent. These three components therefore work in an advantageous complex synergy that could not be predicted. 
     The toxin may therefore be removed from the foodstuff by enzyme breakdown, by binding to the binding agent and/or sequestration in the micro-organism, thus removing it from the foodstuff; if detoxification occurs in an animal, the removal is ultimately from the gastrointestinal tract in faecal material. These sequestrations have the further effects of preventing absorption into the animal and facilitating the action of the enzymes of the invention in the gastrointestinal tract. 
     The skilled person will realise that the health of animals and humans can be adversely affected by the presence of a mycotoxin in a foodstuff. The compositions of the invention can therefore be used in therapy, to treat a disease caused by a mycotoxin. 
     The invention is further described with reference to the following non-limiting examples. 
     EXAMPLES 
     Example A - Experimental Degradation of Toxins and Identification of Catalysts 
     The modification of various toxins by enzymes was examined by screening the activity of a number of a selection of enzymes against various toxins. The change in the toxin was measured by a monoclonal antibody in an Elisa test. Since the monoclonal antibody retains, in many cases, the ability to cross react with the fragments produced by the enzyme catalysis, this provides a definitive screening tool but not a quantitative measure of the enzymes activity. 
     Suitable enzymes which may be used as part of the invention are shown in Table 3 and examples of their action are shown in Tables 4-6. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Examples of theEnzymes suitable for use in the  
               
               
                 Composition of the Invention. 
               
            
           
           
               
               
               
            
               
                 ENZYME 
                 EC NUMBER 
                 ACTION 
               
               
                   
               
               
                 Esterase 
                 3.1.1.x 
                 Ester hydrolysis 
               
               
                 Lipase 
                 3.1.1.3 
                 Ester hydrolysis 
               
               
                 Protease 
                 3.4.x.x 
                 Hydrolysis of peptides 
               
               
                 Oxidase 
                 1.4.3 
                 Oxidations 
               
               
                 Aminoacid oxidase 
                 1.4.3.2 &amp; 3 
                 Oxidation of amino acid NH 2   
               
               
                 Lacotonohydrolase 
                 3.1.1.25 
                 Hydrolysis of lactones 
               
               
                 Peroxidase 
                 1.11.1.7 
                 Peroxidation reactions 
               
               
                 Lactoperoxidase 
                 1.11.1.7 
                 Peroxidation reactions 
               
               
                 Manganese peroxidase 
                 1.11.1.13 
                 Production of Mn +++   
               
               
                 Epoxidase 
                 3.3.2.3 
                 Hydrolysis of peroxides 
               
               
                 Polysaccharase 
                 3.2.1 
                 Hydrolysis of sugar cross links 
               
               
                 Dehydrogenases 
                 1.1 
                 Oxidation by removal of hydrogen 
               
               
                   
               
            
           
         
       
     
     Experiment 1: Fumonisin Modification by Lipase 
     Lipase AE 02 (Mann Associates, Cambridge, UK) was incubated with Fumonisin 200 μg/liter and measured after 1 hour at 37° C. The remaining fumonisin was measured by ELISA assay. Boiled enzyme controls were also used in each case. It can be seen that there was a considerable reduction in the Fumonisin reacting with the antibody indicating degradation of the toxin. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 4 
               
               
                   
                   
               
               
                   
                   
                 pH 6.4 
                 pH 4.5 
                 No Buffer 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Std Norm 
                 0.06 
                 0.045 
                 0.028 
               
               
                   
                 Experimental  
                 0.039 
                 0.046 
                 0.01 
               
               
                   
                 % degradation 
                 30 
                 0 
                 50 
               
               
                   
                 Std boiled 
                 0.049 
                 0.054 
                 0.046 
               
               
                   
                 Experimental  
                 0.039 
                 0.046 
                 0.014 
               
               
                   
                 % degradation 
                 22 
                 15 
                 70 
               
               
                   
                   
               
            
           
         
       
     
     Experiment 2: Fumonisin Modification by Lipase and Amine Oxidase 
     Fumonisin was further examined by incubating it as in Table 3, but with an aminoxidase to remove the amine groups. The lipase 2 again achieved degradation while the oxidase also degraded the toxin. There was no apparent synergy of using both enzymes. This demonstrates that a combination of enzymes, alone, is not effective at improving mycotoxin degradation. 
     
       
         
           
               
               
               
               
               
               
             
               
                   
                 TABLE 5 
               
               
                   
                   
               
               
                   
                   
                   
                   
                   
                 Lipase 
               
               
                   
                   
                   
                 Lipase 
                   
                 AE 02 + 
               
               
                   
                   
                 Blank 
                 AEO 2 
                 Oxidase 
                 Oxidase 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Results 
                 0.38 
                 0.45 
                 0.41 
                 0.46 
               
               
                   
                 % max abs Ave 
                 20.89 
                 24.91 
                 22.6 
                 23.23 
               
               
                   
                 ppm 
                 0.26 
                 0.18 
                 0.2 
                 0.19 
               
               
                   
                 % degradation 
                   
                 31.90% 
                 22.10% 
                 26.00% 
               
               
                   
                   
               
            
           
         
       
     
     Experiment 3: Enzyme modification of Zearalenone 
     Enzyme degradation of Zearalenone was examined using a variety of preparations. Of these preparations, some achieved degradation but the composition according to the invention (as defined in Table 1) produced a synergistic effect. Repeats of the experiment followed by analysis by HPLC showed that the formulation was capable of reducing the toxin levels to zero. 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                   
                 Formulation 
                 Cellulase 
                   
                   
                   
               
               
                   
                 defined in 
                 From 
                 Xylanase 
                 Nitrilase 
                 Lipase 
               
               
                 Enzyme 
                 Table 1 
                 Table 2 
                 of Table 2 
                 (Control) 
                 AE 02 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Experimental 
                 0.0010 
                 0.014 
                 0.022 
                 0.024 
                 0.006 
               
               
                 Control 
                 0.019 
                 0.015 
                 0.026 
                 0.017 
                 0.012 
               
               
                 % reduction in 
                 47 
                 7 
                 17 
                 0 
                 12 
               
               
                 apparent toxin 
                   
                   
                   
                   
                   
               
               
                 concentration 
                   
                   
                   
                   
                   
               
               
                   
               
            
           
         
       
     
     Example B - Experimental Binding of Toxins and Identification of the Properties of Binders 
     Experiment 4 
     Toxin at physiological levels of approx. 200 μg/L was mixed with a constant quantity (50 mg) of binder, agitated for 30 min, centrifuged and the toxin remaining in solution measured using a RIDASCREEN® FAST mycotoxin test kit (available from R-Biopharm GmbH, Darmstadt, Germany). The results are determined by reference to a standard binding curve for the toxin and binder. 
     The results are shown in  FIG. 1 , which shows the binding percentage at a typical (physiological) rumen concentration. The binding abilities of various components are demonstrated at a physiological concentration of toxin. It can be seen from the variability in the results that a mixture of binders is preferable to a single entity. (Volclay=Na bentonite; Teragreen=Ca bentonite, FerMOS=polysaccharide binding complex). 
     Example C - Sequestration of Toxins and Subsequent Enzyme Degradation of the Toxin 
     In the following experiments (5, 6, 7) active yeast cultures ( Saccharomyces Cerevisiae  NYCC R404) were charged with the appropriate toxin at 200μg/L and the disappearance from the supernatant followed by ELISA assays. 
       FIGS. 2, 3, and 4  illustrate the effects of the microorganism and its endogenous microbial enzymes on the mycotoxins DON, Zearalenone and Fumonisin. In each case, the yeast take up the toxin, rapidly reducing the toxicity of the culture. The toxin is then metabolised and released, in an altered form, where it is able to bind the binding agent, for subsequent degradation. These examples illustrate the synergy of the composition according to the invention. 
     Experiment 5: DON Uptake and Degradation by Yeast Enzymes 
     Yeast ( Saccharomyces Cerevisiae  NYCC R404) rapidly take up DON from solution, preventing uptake into the animal. The yeast metabolise the DON and release break-down products (metabolites) which are able to bind to two different bentonite binders. Further degradation can then occur on the binders. See  FIG. 2 . 
     Experiment 6: Zearalenone Degradation by Yeast Enzymes 
     Rapid uptake by yeast ( Saccharomyces Cerevisiae  NYCC R404) is demonstrated with the toxin Zearalenone. The enzyme modification occurs in the cell and metabolite peak is then produced, as with DON, before it is subsequently degraded. See  FIG. 3 . 
     Experiment 7: Fumonisin Degradation by Yeast Enzymes 
     Yeast ( Saccharomyces Cerevisiae  NYCC R404) can remove fumonisin from the gastro-intestinal tract by sequestration, as shown in  FIG. 4 . Again, a peak of metabolites is produced early on before further degradation over time. 
     Example D - DON (Deoxynivalenol and Zearalenone) in a Dairy Herd 
     Treatment of a small herd receiving contaminated feed. Prior to treatment the 49 head herd exhibited rough hair coat, abortions, scours, erratic appetites, a lower and erratic milk production, feed in manure and stressed demeanour in the animals. 
       FIG. 5  shows that as the desired formulation (as defined in Table 1) was added, at a rate of 0.5 kilos /1000 kilos of feed for 2 days, and 0.25 kilos 1000 kilos of feed thereafter, milk production increased. Milk production is shown in lbs. 
     Example E - Deoxynivalenol (DON) in a Dairy Herd 
     Feed containing corn silage known to be contaminated with Vomitoxin (DON), and determined to be at 16.6 ppm, was fed to a 160 head dairy herd which was used in a one month experiment. Enzymes and binders (as part of the formulation defined in Table 1) were fed at 0.5 kilo per 1000 kilo of total mixed ration. Milk production is shown in  FIG. 6 , adjusted for 150 DIM. 
     Example F - Porcine Deoxynnivalenol 
     In a group of pigs where reproduction and growth performance was poor and the conception rate was less than 50%, the number born alive was at 5 pigs per litter. Grower finisher days were over 200 days and animals were very variable. A toxin challenge was identified. Feed analysis showed 4.1 and 4.8 ppm of DON. All breeding pigs were immediately placed on feed containing 2.5 kg/tonne of the formulation (as defined in Table 1). 
     After 21 days first service, conception was back to normal at over 90% and pigs born alive returned to Greater than 10 . Pig feed intake in grower finisher was back to normal. Subsequently, pig variation in grower finisher also returned to normal. 
     Boars also benefited. Exposed to DON, their semen volume and sperm concentration decreased by 50% within 7 days. On treatment they returned to normal also in approximately 7 days.