Patent Publication Number: US-2009226570-A1

Title: Detoxification of feed products

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority or the benefit under 35 U.S.C. 119 of European application no. 08152315.1 filed Mar. 5, 2008 and U.S. provisional application No. 61/034,176 filed Mar. 6, 2008, the contents of which are fully incorporated herein by reference. 
    
    
     CROSS-REFERENCE TO SEQUENCE LISTING 
     This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference. 
     FIELD OF THE INVENTION 
     The present invention relates to a method for detoxification of feed products contaminated by the mycotoxin aflatoxin. 
     BACKGROUND OF THE INVENTION 
     Aflatoxins are naturally occurring mycotoxins that are produced by many species of  Aspergillus , most notably  Aspergillus flavus  and  Aspergillus parasiticus . Aflatoxins are toxic and carcinogenic. 
     Aflatoxin producing members of  Aspergillus  are common and widespread in nature. They can colonize and contaminate grain before harvest or during storage. Host crops are particularly susceptible to infection by  Aspergillus  following prolonged exposure to a high humidity environment or damage from stressful conditions such as drought, a condition which lowers the barrier to entry. 
     Crops which are frequently affected include cereals, such as maize, sorghum, millet, rice and wheat, and oilseeds, such as rape, peanut, soybean, sunflower and cotton. 
     When cereal grain is used in ethanol production and the starch is consumed the aflatoxin is concentrated in the fermentation by-products, e.g., in the distillers&#39; dried grain which is used as a feed product, and aflatoxin in the fermentation by-products may be increased three-fold relative to the cereal grain. Thus, distillers&#39; grains contaminated with aflatoxins can pose risks to the safety of animals consuming these products and with the widespread use of distiller&#39;s grains in dairy cattle feed there is also a potential human safety concern due to aflatoxin residues in the milk. 
     Inactivation of aflatoxin by the use of microorganisms is disclosed in WO 2006/053357. Enzymatic inactivation of other mycotoxins is disclosed in WO 96/12414. There is a need for further methods of detoxification of animal feed products, e.g., such as fermentation by-products, including distillers&#39; wet and dried grain, contaminated by the mycotoxin aflatoxin. 
     SUMMARY OF THE INVENTION 
     In a first aspect the invention provides a process for producing a feed product from a vegetable material, said process comprising treating said vegetable material with an enzyme that degrades aflatoxin, to produce a feed product having a reduced level of aflatoxin. 
     In a second aspect the invention provides a process for degrading aflatoxin in a vegetable material which process comprises treating said vegetable material with an enzyme. 
     In a third aspect the invention provides a use of an enzyme for degrading aflatoxin. 
     The enzyme is preferably selected from the group consisting of laccase, cutinase, and carboxypeptidase. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Aflatoxin 
     In the context of this invention the term “aflatoxin” comprises any type of aflatoxin. The term “aflatoxin” also comprises any derivative of aflatoxin which is susceptible for modification by an enzyme, e.g., a laccase, a cutinase or a carboxypeptidase. 
     At least 13 different types of aflatoxin are produced in nature. Aflatoxin B1, which is considered the most toxic, and B2 are produced by both  Aspergillus flavus  and  Aspergillus parasiticus . Aflatoxin G1 and G2 are produced exclusively by  A. parasiticus.    
     Aflatoxins M1 and M2 were originally discovered in the milk of cows which fed on moldy grain. These compounds are products of a conversion process in the animal&#39;s liver. However, aflatoxin M1 is also present in the fermentation broth of  Aspergillus parasiticus.    
     While the presence of  Aspergillus  in feed products does not always indicate harmful levels of aflatoxin are also present, it does imply a significant risk in consumption of that product. 
     Vegetable Material 
     The vegetable material may comprise cereal(s), e.g., one or more of corn, wheat, barley, rye, rice, sorghum and millet, legumes, e.g., one or more of soybean, pea, and peanut, oilseeds, e.g., rape, soybean, sunflower and cotton. The vegetable material may be milled, e.g., wet or dry milled grain, including milling fractions comprising gluten, protein, starch, bran and/or oil. 
     The vegetable material may be a vegetable material which apart from an unwanted level of aflatoxin is suitable for production of an animal feed product. The vegetable material can also be a vegetable material suspected of comprising an unwanted level of aflatoxin, and/or a vegetable material having an unknown level of aflatoxin, including vegetable material not comprising a detectable level of aflatoxin. 
     The process of the invention may be combined with any process in which a product suitable as an animal feed product is produced, either as the main product or as a byproduct. Thus the vegetable material of the invention may be the mash of a process for producing a fermentation product. Preferably said fermentation product is an ethanol product, e.g., beer, potable ethanol, fuel ethanol and/or industrial ethanol. The process of the invention may be performed prior to, during or after the fermentation step with the purpose of degrading aflatoxin present in the vegetable material comprised in the mash to produce a product, e.g., the spend grains or the destillers&#39; vet or dried grain with a reduced amount of aflatoxin. Similarly, the vegetable material of the invention may be the grain in a steeping step in a wet milling process, in which process also a product suitable as an animal feed product is produced. 
     The vegetable material may be a material which apart from an unwanted or unknown level of aflatoxin is suitable for consumption by an animal, i.e., an animal feed product according to the definition below. 
     Animal Feed Products 
     The term “animal” includes all animals, including human beings. Examples of animals are cattle, (including but not limited to cows and calves); mono-gastric animals, e.g., pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys and chicken (including but not limited to broiler chicks, layers); and fish (including but not limited to salmon). 
     The term “feed” or “feed product” means any compound, preparation, mixture, or composition suitable for, or intended for intake by an animal. 
     The feed product may be a product which apart from an unwanted level of aflatoxin is suitable for consumption by an animal. The feed product can also be a product suspected of comprising an unwanted level of aflatoxin, and/or a product having an unknown level of aflatoxin, including products not comprising a detectable level of aflatoxin. 
     Preferably the feed product comprises cereal(s), e.g., one or more of corn, wheat, barley, rye, rice, sorghum and millet, legume(s), e.g., one or more of soybean, pea, and peanut, oilseed(s), e.g., rape, soybean, sunflower and cotton. The feed product may be milled, e.g., wet or dry milled grain, including milling fractions comprising gluten, protein, starch, bran and/or oil. 
     Laccases 
     In the context of this invention the term “laccases” include enzymes comprised by the enzyme classification E.C. 1.10.3.2. Preferred are the below mentioned enzymes as well as enzymes with homologous sequence, especially recombinant and/or substantially purified enzymes. 
     The laccases may be derived from any sources, preferably from a microorganism, such as a fungus or a bacterium. Preferably, the laccase employed is derived from a strain of  Polyporus  sp., in particular a strain of  Polyporus pinisitus  or  Polyporus versicolor , or a strain of  Myceliophthera  sp., e.g.,  M. thermophila  or a strain of  Rhizoctonia  sp., in particular a strain of  Rhizoctonia praticola  or  Rhizoctonia solani , or a strain of a  Rhus  sp., in particular  Rhus vernicifera.    
     In specific embodiments of the invention the oxidoreductase is the  Polyporus pinisitus  laccase (also called  Trametes villosa  laccase) described in WO 96/00290, the  Myceliophthora thermophila  laccase described in WO 95/33836, or a laccase having an amino acid sequence homologous to any of these sequences. 
     Further, the laccase may be a  Scytalidium  sp. laccase, such as the  S. thermophilium  laccase described in WO 95/33837 or a  Pyricularia  sp. laccase, such as the  Pyricularia oryzae  laccase which can be purchased from SIGMA under the trade name SIGMA no. L5510, or a  Coprinus  sp. laccase, such as a  C. cinereus  laccase, especially a  C. cinereus  IFO 30116 laccase, or a  Rhizoctonia  sp. laccase, such as a  R. solani  laccase, especially the neutral  R. solani  laccase described WO 95/07988. 
     In preferred embodiments the laccase is a laccase from  Myceliophthora thermophila  (MtL) having the amino acid sequence deposited as GENESEQP: AAR88500 and shown herein as SEQ ID NO: 3, a laccase from  Polyporus pinsitus  (PpL) having the amino acid sequence deposited as UNIPROT: Q99044 and shown herein as SEQ ID NO: 4, a laccase from  Streptomyces coelicolor  ScL having the amino acid sequence deposited as SWISSPROT: Q9XAL8 and shown herein as SEQ ID NO: 5, or a laccase having an amino acid sequence homologous to any of these sequences. 
     The laccase must be present in the medium to be detoxified in effective amounts. Preferably the laccase is present in concentrations of 0.01-100 mg enzyme protein per kg dry matter, preferably 0.1-10 mg enzyme protein per kg dry matter, or more preferably 1-5 mg enzyme protein per kg dry matter. 
     The Mediator 
     In an embodiment wherein a laccase is applied a mediator acting as electron may be used together with the laccase. The mediator should be present in the medium to be detoxified in effective amounts. 
     Various mediators are known; see, e.g., WO 94/12620, WO 94/12621, WO 95/01626, WO 96/00179 and WO 99/23887. Mediators therein are hereby incorporated by reference. 
     Preferred for the invention is a mediator selected from methylsyringate (MES), phenothiazine-10-propionicacid (PPT), n-(4-cyanophenyl)acetohydroxamic acid (NCPA), acetosyringone, syringaldehyde, p-coumaric acid, ‘2,2-azinobis(3-ethylbenzthiazoline-6-sulfonate), 1-hydroxybenzotriazole, 2,4-pentanedione, and phenothiazine. 
     Said mediators are commercially available or can be made by methods known to the art. 
     Cutinases 
     In the context of this invention the term “cutinases” include enzymes comprised by the enzyme classification E.C.3.1.1.74. Preferred are the below mentioned enzymes as well as enzymes with homologous sequence, especially recombinant and/or substantially purified enzymes. 
     The cutinase may be derived from a microorganism, preferably from a fungus or a bacterium. Particularly, the cutinase may be derived from a strain of  Humicola , particularly  H. insolens , more particularly  H. insolens  strain DSM1800 (U.S. Pat. No. 5,827,719) or from a strain of  Fusarium , e.g.,  F. roseum culmorum , or particularly  F. solani  f.sp. pisi (WO 90/09446; WO 94/14964, WO 94/03578). The fungal cutinase may also be derived from a strain of  Rhizoctonia , e.g.,  R. solani , or a strain of  Alternaria , e.g.,  A. brassicicola  (WO 94/03578). 
     Preferred are the cutinases shown in SEQ ID NO: 1; the  Humicola insolens  cutinase (corresponding to the mature part of SEQ ID NO: 2 of U.S. Pat. No. 5,827,719, and of SEQ ID NO:1 of WO 01/92502), and in SEQ ID NO: 2; the  Fusarium solani  f.sp. pisi according to FIG. 1D of WO 94/14964, as well as a laccase having an amino acid sequence homologous to any of these sequences. 
     The cutinase may also be a variant of a parent cutinase such as those described in WO 00/34450, or WO 01/92502, all of which are hereby incorporated by reference. The cutinase may be the variant of the  Humicola insolens  cutinase comprising the substitutions E6Q, G8D, A14P, N15D, E47K, S48E, R51P, A88H, A91H, A130V, E179Q and R189V, which is disclosed at p. 24, line 11 of WO 2001/092502 and used in example 1 herein. 
     The cutinase must be present in the medium to be detoxified in effective amounts. Preferably the cutinase is present in concentrations of 0.01-100 mg enzyme protein per kg dry matter, preferably 0.1-10 mg enzyme protein per kg dry matter, or more preferably 1-5 mg enzyme protein per kg dry matter. 
     Carboxypeptidases 
     In the context of this invention the term the term “carboxypeptidase” refers to an enzyme that cleaves the C-terminal peptide bond of a peptide or polypeptide chain. The group comprises but is not limited to the enzymes assigned to enzyme subclass EC 3.4.16, Serine-type carboxypeptidases. 
     Preferred are the below mentioned enzymes, especially recombinant and/or substantially purified enzymes. The carboxypeptidase may be derived from any sources, preferably from a microorganism, such as a fungus or a bacterium. In preferred embodiments the carboxypeptidase is derived from  Aspergillus oryzae , preferably such as the carboxypeptidases shown in SEQ ID NO: 5 and in SEQ ID NO: 6. 
     The carboxypeptidase must be present in the medium to be detoxified in effective amounts. Preferably the carboxypeptidase is present in concentrations of 0.01-100 mg enzyme protein per kg dry matter, preferably 0.1-10 mg enzyme protein per kg dry matter, or more preferably 1-5 mg enzyme protein per kg dry matter. 
     The Medium 
     In an embodiment the enzyme is degrading the aflatoxin in a medium comprising the feed product. The medium is preferably aqueous and may be a liquid, a paste or a slurry. To form a suitable medium water may be added to the feed product. The enzyme and if relevant the mediator, may be comprised, either separately or together, in solid or liquid formulations suitable for application to said medium. 
     The detoxifixation efficiency of the invention depends on, e.g., availability of oxygen, pH, temperature and buffer of the medium. For example, the treatment may take place at a pH-value at which the relative activity of the actual enzyme is at least 50%, at least 60%, at least 70%, at least 80%, or even at least 90%. Likewise, for example, the treatment may take place at a temperature at which the relative activity of the actual enzyme is at least 50%, at least 60%, at least 70%, at least 80%, or even at least 90%. The relative activity is calculated relative to the activity at the pH value where the highest activity is observed. 
     Oxygen in the Medium 
     When a laccase is applied the source of oxygen required may be oxygen from the atmosphere or an oxygen precursor for in situ production of oxygen. Oxygen from the atmosphere will usually be present in sufficient quantity. If more O 2  is needed, additional oxygen may be added, e.g., as pressurized atmospheric air or as pure pressurized oxygen. 
     pH in the Medium 
     Depending, inter alia, on the characteristics of the enzyme employed, the pH in the medium employed should normally be in the range of 5-11, preferably in the range 6-10, e.g., 6.5-8.5. 
     Temperature in the Medium 
     Preferably a reaction temperature is applied which is close to the optimum temperature for the enzyme. In numerous embodiments of the invention, temperatures in the range of 10-65° C., more preferably 30-50° C. should be employed. 
     Treatment Duration 
     The duration of treatment depends, inter alia, on the treatment type, the type of item to be treated, the properties of the medium, e.g., temperature and pH and the type and amounts of enzyme employed. 
     The enzymatic reaction is continued until the desired result is achieved, following which it may or may not be stopped by inactivating the enzyme, e.g., by a heat-treatment step. 
     For detoxification purposes treatment times in the range of 1 minute to 1 week may be employed. In many cases a treatment time in the range of 6 to 48 hours will be suitable. 
     By the process of the invention the content of aflatoxin in the feed product is preferably reduced to less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, or even less than 5%, such as less than 4, 3, 2 or even 1% relative to the level prior to the process. 
     Identity 
     The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “identity”. 
     For purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970 , J. Mol. Biol.  48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000 , Trends in Genetics  16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” 
     (obtained using the—nobrief option) is used as the percent identity and is calculated as follows: 
       (Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment) 
     For purposes of the present invention, the degree of identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the—nobrief option) is used as the percent identity and is calculated as follows: 
       (Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment) 
     Homologous Sequence 
     The term “homologous sequence” is defined as a predicted protein that gives an E value (or expectancy score) of less than 0.001 in a tfasty search (Pearson, W. R., 1999, in  Bioinformatics Methods and Protocols , S. Misener and S. A. Krawetz, ed., pp. 185-219) with a specified sequence. 
     The term “homologous sequence” may also be defined as a sequence that has a degree of identity at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or even 100%, to a specified sequence. 
     EXAMPLES 
     Materials and Methods 
     Enzymes 
     A cutinase which is a variant of the  Humicola insolens  cutinase shown in SEQ ID NO: 1 with the substitutions E6Q, G8D, A14P, N15D, E47K, S48E, R51P, A88H, A91H, A130V, E179Q and R189V. 
     A laccase from  Myceliophthora thermophila  (MtL) having the amino acid sequence shown herein as SEQ ID NO: 3. 
     A laccase from  Streptomyces coelicolor  (ScL) having the amino acid sequence shown herein as SEQ ID NO: 4. 
     A laccase from  Polyporus pinsitus  (PpL) having the amino acid sequence shown herein as SEQ ID NO: 5. 
     A carboxypeptidase from  Aspergillus oryzae  (CPY) having the amino acid sequence shown herein as SEQ ID NO: 7. 
     Mediators 
     Methylsyringate (MeS) 
     Phenothiazine-10-propionicacid (PPT)
 
Assay: Reactions were performed in 600 microL volumes in eppendorf tubes comprising aflatoxin 30 microM, sodium acetate 100 mM and enzyme 0.1 mg EP/mL. In reactions involving laccase 0.2 mM mediator was included. In control reactions the enzyme volume was substituted with an equivalent amount of H 2 O. The reactions were incubate 24 hours at 37° C. before being terminated by adding 600 microL of a 100 microM acetonitrile stop solution. Reactions were stored at −20° C. until chromatographic analysis.
 
Chromatographic analysis: Samples were centrifugated and the supernatant analysed for aflatoxin by HPLC-DAD as described by Smedsgaard ( J. Chromatogr . A, 1997, 760: 264-270). The DAD scanned from 200-600 nm. Separation was done on a Phenomenex (Torrance, Calif.) Luna C18(2) 10×2 mm ID, 3 micrometer, column 2, using a linear gradient moving form 5% to 100% acetonitrile in 20 min. Residual aflatoxin was calculated relative to the control.
 
     Example 1 
       
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Residual aflatoxin after 24 hours incubation with 3 laccases 
               
               
                 (MtL, PpL or ScL) and MeS as mediator at pH 4.5 or pH 7.0 
               
            
           
           
               
               
               
               
            
               
                   
                 Enzyme 
                 pH 
                 Residual aflatoxin (%) 
               
               
                   
                   
               
               
                   
                 Control 
                 4.5 
                 100  
               
               
                   
                 MtL + MeS 
                 4.5 
                 48 
               
               
                   
                 ScL + MeS 
                 4.5 
                 39 
               
               
                   
                 MtL 
                 4.5 
                 63 
               
               
                   
                 Control 
                 7.0 
                 100  
               
               
                   
                 MtL + MeS 
                 7.0 
                  8 
               
               
                   
                 PbL + MeS 
                 7.0 
                 45 
               
               
                   
                 ScL + MeS 
                 7.0 
                  0 
               
               
                   
                 MtL 
                 7.0 
                 70 
               
               
                   
                   
               
            
           
         
       
     
     Example 2 
       
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Residual aflatoxin after 24 hours incubation 
               
               
                 with a cutinase at pH 4.5 or pH 7.0 
               
            
           
           
               
               
               
               
            
               
                   
                 Enzyme 
                 pH 
                 Residual aflatoxin (%) 
               
               
                   
                   
               
               
                   
                 Control 
                 4.5 
                 100  
               
               
                   
                 Cutinase 
                 4.5 
                 49 
               
               
                   
                 Control 
                 7.0 
                 100  
               
               
                   
                 Cutinase 
                 7.0 
                 62 
               
               
                   
                   
               
            
           
         
       
     
     Example 3 
       
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Residual aflatoxin after 24 hours incubation 
               
               
                 with a carboxypeptidase at pH 4.5. 
               
            
           
           
               
               
               
            
               
                   
                 Enzyme 
                 Residual aflatoxin (%) 
               
               
                   
                   
               
               
                   
                 Control 
                 100  
               
               
                   
                 carboxypeptidase 
                 66