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    • 专利摘要:
    FOOD ADDITIVE COMPOSITION. A feed additive composition comprising a direct-fed live microorganism (DFM) in combination with xylanase (eg, endo-1,4-β-d-xylanase) and β-glucanase (and optionally a fiber degradation enzyme additional), in which DFM is selected from the group consisting of an enzyme-producing strain; a strain that ferments C5-sugar; a strain that produces short-chain fatty acids; a strain that promotes endogenous fibrolytic microflora; or combinations thereof. The DFM can be selected from the group consisting of: Bacillus subtilis AGTP BS3BP5, Bacillus subtilis AGTP BS442, B. subtilis AGTP BS521, B. subtilis AGTP BS918, Bacillus subtilis AGTP BS1013, B. subtilis AGTP BS1069, B. subtilis AGTP 944, B. pumilus AGTP BS 1068 or B. pumilus KX11-1, Enterococcus faecium ID7, Propionibacterium acidipropionici P169, Lactobacillus rhamnosus CNCM-I-3698, Lactobacillus farciminis CNCM-I-3699, a strain that has all the characteristics thereof, any derivative or variant thereof and combinations thereof and the additional fiber degradation enzyme may be selected from the group consisting of cellobiohydrolase (EC 3.2.1.91 and EC 3.2.1.176), β-glucosidase (EC 3.2.1.21), β -xylosidase (EC 3.2.1.37), feruloyl esterase (EC 3.1.1.73), a- arabinofuranosidase (...).
    公开号:BR112015002449B1
    申请号:R112015002449-1
    申请日:2013-08-02
    公开日:2021-07-06
    发明作者:Elijah Gituanjah Kiarie；Susan Lund Arent；Mai Faurschou Isaksen；Marion Bernardeau；Luis Fernando Romero Millán；Pãivi Helena Nurminen；Sofia Forssten；Mari Ellen Davis；Daniel Petri；Elizabeth Ann Galbraith
    申请人:Dupont Nutrition Biosciences Aps；
    IPC主号:
    专利说明:

    FIELD OF THE INVENTION
    [001] The present invention relates to methods for improving feed compositions using specific direct fed live microorganisms in combination with a xylanase and a β-glucanase and a feed additive composition comprising live directly fed microorganisms in combination with a xylanase and a β-glucanase. The present invention also relates to uses and kits. BACKGROUND OF THE INVENTION
    [002] Supplemental enzymes are used as feed additives for animals, in particular poultry and swine, as a means to improve nutrient utilization and production performance characteristics. Enzyme blends are available to enhance the nutritional value of diets containing cereal grains, soybean meal, animal protein meals or foods with high fiber content and industrial by-products.
    [003] The concept of Direct Fed Microbials (DFM) involves the feeding of beneficial live microbes to animals such as chickens or pigs so that, when administered in adequate amounts, they confer health benefits on the host . Probiotics is another term for this category of feed additives. Probiotics or DFM have been shown to improve animal performance in controlled studies. DFM includes bacteria and or directly fed yeast-based products.
    [004] Although combinations of DFMs with some enzymes have been considered, the interaction between DFMs and enzymes has never been fully understood. The present invention relates to new specific combinations which, surprisingly, significantly improve the production performance characteristics of animals.
    [005] Continued pressure on global stop grain markets has resulted in trends for the swine and poultry industries to seek alternative cost-effective ingredient options such as co-products (by-products) from the biofuels and milling industries. However, a characteristic of alternative ingredients is the high content of non-starch polysaccharides (Non-Starch Polysaccharide- NSP, fiber) which, for non-ruminants, are of low nutritional value, as they are not digestible, limit the nutrient consumption of an animal and negatively influence energy and nutrient use. It follows that the successful application of alternative fibrous ingredients in diets for monogastrics will be dependent on the availability of technologies to efficiently utilize the energy contained in dietary fiber, reduce the risks associated with its anti-nutritional properties and potential economic benefits when formulated correctly in diets. SUMMARY OF THE INVENTION
    [006] A seminal finding of the present invention is that degradation of dietary material derived from plant cell wall particles, which has a high content of non-starch polysaccharides (NSP), by xylanases can be optimized to improve animal performance when combining xylanase and a β-glucanase with one or more specific live directly fed microorganisms (DFMs) selected for their ability to digest structural plant cell wall carbohydrates and/or their ability to produce short chain fatty acids (Short Chain Fatty Acid - SCFA) from pentoses (eg arabinoxylans) contained in the NSP fraction of ingredients under anaerobic conditions.
    [007] The reason this combination improves performance is that the solubilization of fibers, specifically hemicellulose, from the diet is maximized in the gastrointestinal tract (Gastro Intestinal Tract - GIT) of animals. This solubilization of hemicellulose would not always be sufficient to increase performance because released C5-sugars are not an efficient energy source for animals when they are absorbed (Savory C., J.Br. J. Nut. 1992, 67: 103-114 ), but they are a more efficient energy source when converted to short chain fatty acids (SCFA) either through microorganisms in the GIT or by DFMs.
    [008] Therefore, the energy value from plant products (eg, wheat, corn, oats, barley and co-products (by-products) of cereals or readily accessible mixed grain diet for monogastrics) can be optimized by combining xylanase and a β -glucanase and specific DFMs that can produce SCFAs from pentoses in the NSP fraction under anaerobic conditions or that can modulate microbial populations in the GIT to increase SCFA production from released sugars. DFMs can adapt their metabolism to synergistically increase fiber hydrolysis in combination with xylanase and β-glucanase. Use of DFMs with fibrolytic enzymes can confer additional benefits and maximize the benefits of carbohydrates.
    [009] Specific DFMs selected for their enzymatic activities can be considered as a glycan-driven bacterial food chain. The specifically selected DFMs taught here may preferentially utilize dietary fiber, a feature that allows them to perform the initial glycan digestion steps to release shorter, more soluble polysaccharides for other bacteria, eg, other endogenous GIT microflora. The specific DFMs were selected for their metabolism, which adjusts according to the glycans released by enzymes (eg, xylanase and β-glucanase) to improve the effectiveness of the enzymes taught here and the combination of DFM(s) compared to the use of a combination of enzymes individually or the use of DFM(s) individually.
    [0010] Without intending to be bound by theory, in the present invention, dietary material derived from plant cell wall particles, which is rich in glycans from specific sources, such as cellulose, hemicellulose and pectin (plant material), or glycosaminoglycans enter the Distal intestine in the form of particles are attacked by specific glycan degrading DFMs which are capable of binding directly to these insoluble particles and digesting their glycan components. After this initial degradation of glycan-containing particles, more soluble glycan fragments can be digested by secondary glycan degraders present in the cecum, which contribute to the released reservoir of short-chain fatty acid (SCFA) fermentation products that is derived from both types of degraders. Since SCFAs arise from carbohydrate fermentation and/or protein fermentation and deamination by the native anaerobic microflora in the GIT, the SCFA concentration may be an index of the population of anaerobic organisms. SCFA can actually confer a number of advantages to the host animal, acting as a metabolic fuel for gut, muscle, kidney, heart, liver and brain tissues and also conferring bacteriostatic and bactericidal properties against organisms such as Salmonella and E. coli.
    [0011] The nutritional value of fibers in non-ruminants can be derived primarily through the production of short-chain fatty acids (SCFA) via fermentation of solubilized or degraded fibers by effective fiber degradation (for example, a xylanase and a β- glucanase, suitably in combination with other fiber degradation). Individual feed xylanase is not sufficient to use fibrous ingredients in animal (especially non-ruminant) diets. There is a wide variety of chemical characteristics among plant ingredients in the feed. An enzymatic application depends on the characteristics of the plant material (feed). As an example only, in wheat grains, arabinoxylans predominate, however, in wheat bran (a co-product or by-product of wheat milling), the β-glycan content increases from 8 g-1 DM (in grains) to more than 26 g kg-1 DM.
    [0012] SCFAs have different energy values and some can serve as glucose precursors and some can contribute to the maintenance of intestinal integrity and health. The inventors have found that the specific combinations described here preferentially shift the fermentation process in the animal's GIT towards the production of more valuable/useful SCFAs such as butyric acid and/or propionic acids.
    [0013] In one aspect, the present invention provides a feed additive composition comprising a live direct-fed micro-organism (DFM) in combination with a xylanase and a β-glucanase, wherein the DFM is selected from the group consisting of a enzyme-producing strain; a strain that ferments C5-sugar; a strain that produces short-chain fatty acids; a strain that promotes endogenous fibrolytic microflora; or combinations thereof.
    [0014] The present invention further provides a method to:i) improve the performance of an individual, or ii) improve the digestibility of a raw material in a feed (e.g., nutrient digestibility, such as amino acid digestibility), oriii) improve nitrogen retention, oriv) improve feed conversion ratio (FCR), orv) improve weight gain in an individual, hear) improve feed efficiency in an individual, heari) shift the fermentation process in the gastrointestinal tract of the individual for the production of butyric acid and/or propionic acid,
    [0015] method which comprises administering, to an individual, a live directly fed microorganism (DFM) in combination with a xylanase and a β-glucanase, in which the DFM is selected from the group consisting of a strain that produces enzyme; a strain that ferments C5-sugar; a strain that produces short-chain fatty acids; a strain that promotes endogenous fibrolytic microflora; or combinations thereof.
    [0016] The present invention further provides a premix comprising an additive composition according to the present invention or a live directly fed micro-organism (DFM), a xylanase and a β-glucanase, in which the DFM is selected from the group which consists of an enzyme-producing strain; a strain that ferments C5-sugar; a strain that produces short-chain fatty acids; a strain that promotes endogenous fibrolytic microflora; or combinations thereof and at least one vitamin and/or at least one mineral.
    [0017] In yet another aspect, the present invention provides a feed comprising an additive composition according to the present invention or a premix according to the present invention.
    [0018] The present invention further provides a feed comprising a live directly fed microorganism (DFM) in combination with a xylanase and a β-glucanase, wherein the DFM is selected from the group consisting of a strain that produces enzyme; a strain that ferments C5-sugar; a strain that produces short-chain fatty acids; a strain that promotes endogenous fibrolytic microflora; or combinations thereof.
    In another aspect, there is provided a method of preparing a food comprising mixing a feed component with a feed additive composition according to the present invention or a premix according to the present invention.
    [0020] Another aspect of the present invention is a method of preparing a food comprising mixing a feed component with a live directly fed micro-organism (DFM) in combination with a xylanase and a β-glucanase, wherein the DFM is selected from the group consisting of an enzyme-producing strain; a strain that ferments C5-sugar; a strain that produces short-chain fatty acids; a strain that promotes endogenous fibrolytic microflora; or combinations thereof.
    [0021] The present invention further provides the use of a live directly fed microorganism (DFM) in combination with a xylanase and a β-glucanase, in which the DFM is selected from the group consisting of a strain that produces enzyme; a strain that ferments C5-sugar; a strain that produces short-chain fatty acids; a strain that promotes endogenous fibrolytic microflora; or combinations thereof: i) improve the performance of an individual, orii) improve the digestibility of a raw material in a feed (eg nutrient digestibility such as amino acid digestibility), oriii) improve nitrogen retention, ouiv) improve the feed conversion ratio (FCR), or v) improve the weight gain in an individual, hear) improve the feed efficiency in an individual, or vii) shift the fermentation process in the gastrointestinal tract of the individual for the production of butyric acid and/or propionic acid.
    [0022] Another aspect relates to a kit comprising a live directly fed microorganism (DFM), a xylanase and a β-glucanase, in which the DFM is selected from the group consisting of a strain that produces enzyme; a strain that ferments C5-sugar; a strain that produces short-chain fatty acids; a strain that promotes endogenous fibrolytic microflora; or combinations thereof (and optionally at least one vitamin and/or optionally at least one mineral) and instructions for administration. BRIEF DESCRIPTION OF THE DRAWINGS
    [0023] Figure 1 shows the effects of xylanase and β-glucanase sem or with live direct-fed microorganism (DFM) of Bacillus on the fecal counts of lactobacilli and E. coli (colony forming unit/gram of log transformed feces , Log10 cfu/g). DETAILED DESCRIPTION OF THE INVENTION
    Preferably, the enzyme(s) used in the present invention is/are exogenous to DFM. In other words, the enzyme(s) is/are preferably added to or mixed with the DFM.
    [0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20th ED., John Wiley and Sons, New York (1994) and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991)) provide those experts in the art a general dictionary of many of the terms used in this description.
    [0026] The present description is not limited by the exemplary material methods described herein and any methods and materials similar or equivalent to those described herein may be used in the practice or testing of embodiments of the present description. Numeric ranges are inclusive of the numbers that define the range. Unless otherwise noted, all nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written from left to right in amino to carboxy orientation, respectively.
    [0027] The headings provided here are not limitations on the various aspects or modalities of this description, which may be obtained by reference to the descriptive report as a whole. Consequently, the terms defined immediately below are more fully defined by reference to the descriptive report as a whole.
    [0028] Amino acids are cited here using the amino acid name, the three letter abbreviation or the single letter abbreviation.
    [0029] The term "protein", as used herein, includes proteins, polypeptides and peptides.
    As used herein, the term "sequence of amino acids" is synonymous with the term "polypeptide" and/or the term "protein". In some cases, the term "sequence of amino acids" is synonymous with the term "peptide". In some cases, the term "sequence of amino acids" is synonymous with the term "enzyme".
    The terms "protein" and "polypeptide" are used interchangeably herein. In the present description and in the claims, conventional one-letter and three-letter codes for amino acid residues can be used. The 3-letter code for amino acids is as defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It should also be understood that a polypeptide may be encoded by more than one nucleotide sequence by virtue of the degeneracy of the genetic code.
    [0032] All E.C. enzyme classifications cited herein refer to the classifications provided in Enzyme Nomenclature - Recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology - ISBN 0-12226164-3.
    [0033] Other definitions of terms may appear throughout the descriptive report. Before exemplary embodiments are described in greater detail, it should be understood that the present description is not limited to the particular embodiments described, as these may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.
    [0034] When a range of values is provided, it should be understood that each intervening value, up to the tenth of the unit of the minimum limit, unless the context clearly guides otherwise, between the maximum and minimum limits of said range, is also specifically described. Each smaller range between any specified value or intervening value in a specified range and any other specified or intervening value in this specified range is covered within the present description. The maximum and minimum limits of these minor ranges may be independently included or excluded from the range and each range where either, none or both limits are included in the minor ranges is also covered within the present description, subject to any specifically excluded limit in the specified range . When the specified range includes one or both of these limits, ranges excluding either or both of these included limits are also included in the present description.
    [0035] It should be noted that, as used herein and in the appended claims, the singular forms "a", "an", "the" and "a" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an enzyme" includes a plurality of such candidate agents and reference to "feed" includes reference to one or more feeds and their equivalents known to those of skill in the art, and so on.
    [0036] The publications discussed herein are provided solely for their description prior to the filing date of this Order. Nothing herein should be construed as an admission that these publications constitute the state of the art to the appended claims.
    [0037] Enzymes for use in the present invention can be produced by means of solid or submerged culture, including continuous, batch and continuous flow processes. The culture is carried out in a growth medium comprising an aqueous medium of mineral salts, organic growth factors, the carbon and energy source material, molecular oxygen and, of course, an initial inoculum of one or more species of microorganisms individuals to be employed.
    [0038] The DFM for use in the present invention may be an enzyme-producing strain.
    [0039] The DFM for use in the present invention may be a C5-sugar fermenting cepaque.
    [0040] The DFM for use in the present invention can be a strain that produces short chain fatty acid.
    [0041] The DFM for use in the present invention can be a strain that promotes endogenous fibrolytic microflora.
    [0042] The strain that produces enzyme and/or the strain that ferments C5-sugar and/or the strain that produces short-chain fatty acid and/or the strain that promotes the endogenous fibrolytic microflora according to the present invention can be selected from the group consisting of the following genera: Bacillus, Enterococcus, Lactobacillus, Propionibacterium and combinations thereof.
    [0043] The strain that produces enzyme and/or the strain that ferments C5-sugar and/or the strain that produces short-chain fatty acid and/or the strain that promotes the endogenous fibrolytic microflora according to the present invention may be by the minus one selected strain of the Bacillus genus, particularly Bacillus subtilis, B. licheniformis, B. amyloliquefaciens or B. pumilus.
    [0044] The strain that produces enzyme and/or the strain that ferments C5-sugar and/or the strain that produces short-chain fatty acid and/or the strain that promotes the endogenous fibrolytic microflora according to the present invention may be by the minus one selected strain of the Enterococcus genus, particularly Enterococcus faecium.
    [0045] The enzyme-producing strain and/or the C5-sugar fermenting strain and/or the short-chain fatty acid-producing strain and/or the strain that promotes the endogenous fibrolytic microflora according to the present invention can be selected from the group consisting of: Bacillus subtilis AGTP BS3BP5, Bacillus subtilis AGTP BS442, B. subtilis AGTP BS521, B. subtilis AGTP BS918, Bacillus subtilis AGTP BS1013, B. subtilis AGTP BS1069, B. subtilis AGTP 944, Bacillus subtilis BS 2084 NRRL B-50013), Bacillus subtilis LSSAO1 (NRRL B-50104), Bacillus subtilis 3A-P4 (PTA-6506), Bacillus subtilis 22C-P1 (PTA-6508), Bacillus licheniformis BL21 (NRRL B-50134), Bacillus subtilis BS-27 (NRRL B-50105), Bacillus subtilis BS18 (NRRL B-50633), Bacillus subtilis 15A-P4 (PTA-6507), Bacillus subtilis BS278 (NRRL B-50634), Bacillus licheniformis BL842 (NRRL B-50516) , B. pumilus AGTP BS 1068, B. pumilus KX11-1, Enterococcus faecium ID7, Propionibacterium acidipropionici P169, Lactobacillus rhamnosus CNCM-I-3698, Lactoba cillus farciminis CNCM-I-3699 or a strain having all the characteristics thereof, any derivative or variant and combinations thereof.
    [0046] The enzyme producing strain and/or the C5-sugar fermenting strain and/or the short chain fatty acid producing strain and/or the fibrolytic endogenous microflora promoting strain for use in the present invention is preferably , a viable bacteria.
    [0047] The enzyme producing strain and/or the C5-sugar fermenting strain and/or the short chain fatty acid producing strain and/or the fibrolytic endogenous microflora promoting strain for use in the present invention may be in the form of a endospore.
    The xylanase for use in the present invention is preferably an endo-1,4-β-d-xylanase (E.C. 3.2.1.8).
    [0049] In some embodiments, preferably, xylanase and β-glucanase are used in combination with at least one other degrading fiber. The (other) fiber degradation can be selected from the group consisting of a cellobiohydrolase (EC 3.2.1.91 and EC 3.2.1.176), a β-glycosidase (EC 3.2.1.21), a β-xylosidase (EC 3.2 .1.37), a feruloyl esterase (EC 3.1.1.73), an α-arabinofuranosidase (EC 3.2.1.55), a pectinase (eg, an endopolygalacturonase (EC 3.2.1.15), an exopolygalacturonase (EC 3.2.1.67), or a lyase of pectate (EC 4.2 .2.2)) or combinations thereof.
    [0050] Suitably, there may be more than one other fiber degradation, suitably more than two, suitably more than three, suitably more than four, suitably more than five.
    Suitably, the feed additive composition according to the present invention or the composition comprising a DFM in combination with a xylanase, a β-glucanase and at least one other degradation shifts the fermentation process in the gastrointestinal tract of the individual to the production of butyric acid and/or propionic acid. DIRECT FED LIVE MICRO-ORGANISM (DFM)
    [0052] The term "living microorganism" is used interchangeably with "microorganism".
    [0053] The DFM for use in the present invention may be any suitable DFM which is an "enzyme producing strain" - such as an enzyme producing strain of Bacillus. To determine whether a DFM is an "enzyme-producing strain", the DFM assay defined herein as "DFM-producing enzyme assay" can be used. A DFM is considered an enzyme-producing DFM if it is classified as an enzyme-producing DFM using the "enzyme-producing DFM assay" taught here.
    [0054] The DFM for use in the present invention may be any suitable DFM which is a "C5-sugar fermenting strain". To determine whether a DFM is a "C5-sugar fermenting strain", the DFM assay defined herein as "C5-sugar fermenting DFM assay" can be used. A DFM is considered to be a C5-sugar fermenting DFM if it is classified as a C5-sugar fermenting DFM using the "C5-sugar fermenting DFM assay" taught here.
    [0055] The DFM for use in the present invention may be any suitable DFM which is a "strain producing short chain fatty acid (SCFA)". To determine whether a DFM is a "strain producing SCFA", the DFM assay defined herein as "DFM assay producing SCFA" can be used. A DFM is considered to be a DFM that produces SCFA if it is classified as a DFM that produces SCFA using the "DFM that produces SCFA" taught here.
    [0056] The DFM for use in the present invention may be any suitable DFM which is a "strain that promotes endogenous fibrolytic microflora". To determine whether a DFM is a "strain that promotes endogenous fibrolytic microflora", the DFM assay defined herein as the "fibrolytic DFM assay that promotes endogenous microflora" can be used. A DFM is considered to be a fibrolytic DFM that promotes endogenous microflora if it promotes or stimulates endogenous microflora using the assay taught here.
    [0057] The DFM for use in the present invention may be any suitable DFM which is an "enzyme producing strain", a "C5-sugar fermenting strain", a "SCFA producing strain", a "microflora promoting strain endogenous fibrolytic" or combinations thereof.
    [0058] Suitably, the DFM for use in the present invention may be a DFM which is a strain that would be classified as being an "enzyme producing strain" and/or a "C5-sugar fermenting strain" and/or a "strain that produces SCFA" and/or a "strain that promotes endogenous fibrolytic microflora". Suitably, DFM can be a strain that is classified as having more than one type of activity, eg at least 2, suitably at least 3, suitably all 4 activities, eg enzyme production activity, fermentation activity of C5-sugar, SCFA production activity and/or activity to promote endogenous fibrolytic microflora.
    [0059] The DFMs according to the present invention confer benefits to animals fed with high levels of by-products rich in vegetable fibers, such as dry distillers grains with solubles (DDGS). ENZYME-PRODUCING DFM TEST:
    [0060] High-throughput screening of these test strains was performed by plating one dot per replicate of 2 microliters of liquid culture onto 15.0 mL of various substrate media types of interest on plates with 100 x 100 x 15 mm grids. Cellulase, α-amylase, zeinase, soy protease, esterase, lipase and xylanase activities were determined based on specific substrate utilization by the individual strains. Components of the media used to test substrate utilization properties from the enzymatic activity of environmentally derived strains are described in Table 1. Assay plates were allowed to dry for 30 minutes after application of the culture, and then incubated at 32° C for 24 hours. Enzyme activities for each strain were determined by measuring the substrate degradation zone in millimeters, as indicated by clearing the surrounding edge of colony growth. Average values of repeated plates were recorded. Table 1 - Media components used to assay enzymatic activities illustrated by use properties of environmentally derived Bacillus substrate.


    [0061] In one embodiment, the enzyme-producing strain produces one or more of the following enzymatic activities: cellulase activity, α-amylase activity, xylanase activity, esterase activity, lipase activity, β-mannanase activity, protease (eg zeinase activity or soy protease) and combinations thereof.
    [0062] In one embodiment, the enzyme-producing strain preferably produces one or more of the following enzymatic activities: cellulase activity, xylanase activity, β-mannanase activity, or combinations thereof.
    [0063] In one modality, the DFM that produces enzyme is a strain selected from the group consisting of the species Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Bacillus amyloliquefaciens or mixtures thereof.
    [0064] In one modality, preferably, the DMF strain that produces enzyme is selected from the group consisting of:
    [0065] Bacillus subtilis AGTP BS3BP5 (NRRL B-50510)
    [0066] Bacillus subtilis AGTP BS442 (NRRL B-50542)
    [0067] Bacillus subtilis AGTP BS521 (NRRL B-50545)
    [0068] Bacillus subtilis AGTP BS918 (NRRL B-50508)
    [0069] Bacillus subtilis AGTP BS1013 (NRRL B-50509)
    [0070] Bacillus pumilus AGTP BS 1068 (NRRL B-50543)
    [0071] Bacillus subtilis AGTP BS1069 (NRRL B-50544)
    [0072] Bacillus subtilis AGTP 944 (NRRL B-50548)
    [0073] Bacillus pumilus AGTP KXII-1 (NRRL B-50546)
    [0074] Bacillus subtilis 15A-P4 (PTA-6507)
    [0075] Bacillus subtilis BS 2084 (NRRL B-50013)
    [0076] Bacillus subtilis LSSAO1 (NRRL B-50104)
    [0077] Bacillus subtilis 3A-P4 (PTA-6506)
    [0078] Bacillus subtilis 22C-P1 (PTA-6508)
    [0079] Bacillus licheniformis BL21 (NRRL B-50134)
    [0080] Bacillus subtilis BS-27 (NRRL B-50105)
    [0081] Bacillus subtilis BS18 (NRRL B-50633)
    [0082] Bacillus subtilis BS278 (NRRL B-50634)
    [0083] Bacillus licheniformis BL842 (NRRL B-50516)
    [0084] or any derivative or variant thereof,
    [0085] and combinations thereof.
    [0086] The DFM enzyme producing strain may be one or more of the strains taught in US 61/527,371 and US 61/526,881, both of which are incorporated herein by reference. DFM TEST THAT FERMENTS C5-SUGAR:
    Bacillus strains are grown overnight on tryptic soy agar (Difco) plates at 32°C and lactic acid bacteria are grown overnight in MRS medium (Difco) under anaerobic conditions at 37°C. API 50 CHB and API 50 CHL media (bioMerieux, Marcy l'Etoile, France) are inoculated with DFM in pure culture (Bacillus or lactic acid bacteria, respectively) and applied to API 50CH® strips according to the manufacturer's instructions. The strips are incubated at 32°C (Bacillus) or 37°C (lactic acid bacteria) under anaerobic conditions and monitored at 24 and 48 hours for colorimetric changes.
    [0088] The term "C5-sugar", as used herein, means any type of sugar with 5 carbons. C5-sugars can be said here as pentoses.
    [0089] C5-sugars include D-arabinose, L-arabinose, D-ribose, D-xylose and L-xylose.
    [0090] In one modality, the DFM strain that ferments C5-sugar is selected from the group consisting of:
    [0091] Bacillus subtilis 15A-P4 (PTA-6507)
    [0092] Bacillus subtilis AGTP BS918 (NRRL B-50508)
    [0093] Bacillus subtilis BS 2084 (NRRL B-50013)
    [0094] Bacillus subtilis LSSAO1 (NRRL B-50104)
    [0095] Enterococcus faecium ID7
    [0096] Lactobacillus lactis DJ6 (PTA 6102)
    [0097] Lactococcus lactis ID7 (PTA 6103)
    [0098] or combinations thereof. DFM ASSAY THAT PRODUCES SHORT CHAIN FATTY ACID (SCFA):
    A 1% vol/vol inoculum of a 48 hour culture of a DFM is used to inoculate 10 ml tubes of modified Sodium Lactate Calc (NLB) (1% sodium lactate; Sigma-Aldrich, St Louis, MO; 1% tryptone; Oxoid Ltd., Hampshire, England, 0.5% yeast extract; Oxoid Ltd.; and 0.5% KH2PO4) devoid of sodium lactate and supplemented with a proportional amount ( 1% w/vol) of one of nine different carbohydrates (lactate, glucose, galactose, arabinose, sucrose, starch, xylose, cellobiose, fructose, Sigma-Aldrich, St. Louis, MO). Cultures are grown under anaerobic conditions at 32 °C and, after 0, 24, 48 and 72 hours of incubation, duplicate tubes are centrifuged at 5000 xg for 10 min and spent broth collected from each culture. The production of short-chain fatty acids in the consumed broth was measured using high-performance liquid chromatography (High Pressure Liquid Chromatography - HPLC). Duplicate samples of 1 ml of consumed culture broth are taken from each sampling tube and mixed with 10 ml of 0.005 M H2SO4. Three ml of each diluted sample are filtered through a 0.2 micron filter into vials HPLC and capped. Samples are analyzed for acetate, lactate, propionic acid and butyric acid with a Waters 2695 Separation Module (Milford, MA) using a 300 x 7.8 mm Aminex HPX-87H column from Bio-Rad (Hercules, CA) . All analytes are detected with a Waters 2410 RI detector.
    [00100] In one embodiment, the strain that produces short decay fatty acid (SCFA) may be Propionibacterium acidipropionici P169.
    [00101] In another modality, the strain that produces short decay fatty acid (SCFA) may be Enterococcus faecium ID7.
    [00102] The term "short chain fatty acid", as used herein, includes volatile fatty acids as well as lactic acid.
    [00103] In one embodiment, SCFA can be selected from the group consisting of: acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, 2-methylbutyric acid and lactic acid.
    [00104] In one embodiment, the SCFA can be butyric acid. DFM ASSAY THAT PROMOTES ENDOGENOUS FIBROLITIC MICROFLORA:
    [00105] A pen experiment is carried out to determine the effects of a DFM on broilers compared to a control without DFM. Samples are collected on days 11 and 42 of the experiment. In each collection, one bird is collected from each pen for a total of eight birds per treatment. Birds are sacrificed and the total gastrointestinal tract (GIT) below the gizzard to the ileal-cecal junction is collected from each bird. Samples from the cecum of each bird are cut into pieces and cecum and digestive tissue are collected in a Whirlpak bag and kneaded in 99 ml of 0.1% peptone at 7.0 hold/s for 60 seconds to release bacterial cells associated with the mucosa of the cecal tissue. Aliquots of the mixed solution containing bacteria from the cecal and digestive mucosa are frozen in liquid nitrogen and stored at -20 °C until further analysis. Genomic DNA is isolated from 250 µl of each sample by phenol/chloroform extraction and purified using the Roche Applied analysis kit (Roche Diagnostics Corp., Indianapolis, IN). DNA from two birds per treatment is pooled in equal amounts and sent for pyrosequencing as a single sample, resulting in four samples per treatment of each age. Pyrosequencing by bacterial marker encoded FLX amplicon is performed as previously described (Dowd, et al BMC Microbiol. July 24, 2008; 8:125). The V1-V3 region of the 16S rRNA gene is amplified in each pooled sample using the 28 F (5'-GAGTTTGATCNTGGCTCAG) and 519R (5'-GTNTACNGCGGCKGCTG) primers. Pyrosequencing data is processed and analyzed using Qiime v.1.4.0 software. Briefly, raw sequence data is tracked and refined based on quality. All sequences are refined to 350 bp. The sequences are finally decomposed into individual samples based on barcode sequences. Barcode markers and primers are removed from the sequences and non-bacterial ribosomal sequences are removed. The sequences are grouped into Operational Taxonomic Units (OTUs) at 97% similarity using uclust. Representative sequences from each OTU are then aligned using PyNAST and the taxonomy is assigned by sequence comparison with known bacterial 16S rRNA gene sequences in the SILVA database using the RDP classifier. A random subsampling of sequences is performed to normalize each sample so that the same number of sequences are analyzed. Analysis of Variance (ANOVA) is used to determine if any fibrolytic microflora (rate) is significantly affected by the treatment.
    [00106] The term "fibrolytic microflora", as used herein, means a group of microorganisms which are capable of processing complex plant polysaccharides by virtue of their ability to synthesize cellulolytic and hemicellulolytic enzymes.
    [00107] The term "endogenous", as used herein, means present in (or originating from) the GIT of an individual (eg, an animal). In other words, the endogenous fibrolytic microflora is not a DFM. Endogenous fibrolytic microflora is not added to the individual's feed.
    [00108] Preferably, the enzyme producing strain and/or the C5-sugar fermenting strain and/or the short chain fatty acid producing strain and/or the fibrolytic endogenous microflora promoting strain for use in the present invention comprises a viable microorganism. Preferably, the enzyme producing strain and/or the C5-sugar fermenting strain and/or the short chain fatty acid producing strain and/or the fibrolytic endogenous microflora promoting strain comprises a viable bacteria or a viable yeast or a viable fungus.
    [00109] In a preferred embodiment, the enzyme-producing strain and/or the C5-sugar fermenting strain and/or the short-chain fatty acid-producing strain and/or the strain that promotes endogenous fibrolytic microflora comprises a viable bacteria .
    [00110] The term "viable microorganism" means a microorganism that is metabolically active or capable of differentiating.
    [00111] In one embodiment, the strain that produces enzyme and/or the strain that ferments C5-sugar and/or the strain that produces short-chain fatty acid and/or the strain that promotes endogenous fibrolytic microflora may be a bacterium that it forms spores and therefore the term DFM can be understood from or contain spores, for example bacterial spores. Therefore, in one embodiment, the term "viable microorganism" as used herein can include microbial spores such as endospores or conidia.
    [00112] In another modality, the strain that produces enzyme and/or the strain that ferments C5-sugar and/or the strain that produces short-chain fatty acid and/or the strain that promotes the endogenous fibrolytic microflora in the additive composition according to with the present invention is not comprised of or does not contain microbial spores, for example, endospores or conidia.
    [00113] The microorganism may be a naturally occurring microorganism or it may be a transformed microorganism. The microorganism can also be a combination of suitable microorganisms.
    [00114] In some respects, the strain that produces enzyme and/or the strain that ferments C5-sugar and/or the strain that produces short-chain fatty acid and/or the strain that promotes endogenous fibrolytic microflora according to the present invention can be one or more of the following: a bacterium, a yeast or a fungus.
    [00115] Preferably, the strain that produces enzyme and/or the strain that ferments C5-sugar and/or the strain that produces short-chain fatty acid and/or the strain that promotes the endogenous fibrolytic microflora according to the present invention it is a probiotic microorganism.
    [00116] In the present invention, the term live directly fed micro-organism (DFM) encompasses directly fed bacteria, directly fed yeast, directly fed fungi and combinations thereof.
    [00117] Preferably, the strain that produces enzyme and/or the strain that ferments C5-sugar and/or the strain that produces short-chain fatty acid and/or the strain that promotes endogenous fibrolytic microflora is a directly fed bacteria.
    [00118] Suitably, the enzyme-producing strain and/or the C5-sugar fermenting strain and/or the short-chain fatty acid-producing strain and/or the strain promoting endogenous fibrolytic microflora may comprise a bacterium of one or plus of the following genera: Bacillus, Lactobacillus, Propionibacterium and combinations thereof.
    [00119] In one embodiment, the strain that produces enzyme and/or the strain that ferments C5-sugar and/or the strain that produces short-chain fatty acid and/or the strain that promotes endogenous fibrolytic microflora can be selected from a strain of the genus Bacillus.
    [00120] In one modality, the strain that produces enzyme and/or the strain that ferments C5-sugar and/or the strain that produces short-chain fatty acid and/or the strain that promotes endogenous fibrolytic microflora can be selected from the following Bacillus spp.: strains of Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, B. pumilus, B. coagulans, B. amyloliquefaciens, B. stearothermophilus, B. brevis, B. alkalophilus, B. clausii, B. halodurans, B. megaterium , B. circulans, B. lautus, B. thuringiensis and B. lentus.
    In at least some embodiments, the strain(s) of B. subtilis is/are Bacillus subtilis AGTP BS3BP5, Bacillus subtilis AGTP BS442, B. subtilis AGTP BS521, B. subtilis AGTP BS918, Bacillus subtilis AGTP BS1013 , B. subtilis AGTP BS1069, B. subtilis AGTP 944.
    [00122] In at least some embodiments, the strain(s) of B. subtilis is/are Bacillus subtilis 15A-P4 (PTA-6507), LSSAO1 (NRRL B-50104).
    [00123] In at least some embodiments, the B. pumilus strain is B. pumilus AGTP BS 1068 or B. pumilus KX11-1.
    Strains 3A-P4 (PTA-6506), 15A-P4 (PTA-6507) and 22C-P1 (PTA-6508) are publicly available from the American Type Culture Collection (ATCC). Strains 2084 (NRRL B-500130); LSSA01 (NRRL-B-50104); BS27 (NRRL B-50105) are publicly available from the Agricultural Research Service Culture Collection (NRRL). The strain of Bacillus subtilis LSSA01 is sometimes referred to as B. subtilis 8. These strains are taught in US 7,754,469 B2.
    [00125] Danisco USA, Inc., of Waukesha, Wisconsin, USA, has deposited, under the Budapest Treaty, the following biological deposits with the Agricultural Research Service Culture Collection (NRRL) with original deposit dates and detailed accession numbers below:

    Danisco USA, Inc., of Waukesha, Wisconsin, USA, has authorized DuPont Nutrition Biosciences ApS of Langebrogade 1, PO Box 17, DK-1001, Copenhagen K, Denmark, to refer to these biological materials deposited with the this Patent Application and gave unconditional and irrevocable consent for the deposited material to be made available to the public.
    [00127] Agtech Products, Inc., of W227 N752 Westmound Drive, Waukesha, WI 53186, USA, has deposited, under the Budapest Treaty, the following biological deposit with the Agricultural Research Service Culture Collection (NRRL) with the date of deposit original and accession number detailed below:

    [00128] Agtech Products, Inc. has authorized DuPont Nutrition Biosciences ApS de Langebrogade 1, PO Box 17, DK-1001, Copenhagen K, Denmark to refer to this biological material deposited in this Application and has given unconditional consent and irrevocable for the deposited material to be made available to the public.
    [00129] The table below summarizes the enzyme production capabilities of the selected strains using the "DFM Producing Enzyme Assay" above:
    [00130] Summary of enzymatic activity of candidate strains directly fed live microorganism1.Table 2. Cellulase, xylanase and β-mannanase activities of Bacillus strains.


    [00131] 1Mannanase (eg β-mannanase) is the name given to a class of enzymes that can hydrolyze 1,4- β-D- glycosidic bonds of β-mannan, galactomannan and glucomannan into mannan and mannose oligosaccharides, thereby breaking down hemicellulose containing mannan, one of the main components of plant cell walls. β-mannanase is endo-1,4-β-D-mannanase (E.C. 3.2.1.78).
    [00132] Suitably, the enzyme-producing strain and/or the C5-sugar fermenting strain and/or the short-chain fatty acid-producing strain and/or the strain promoting endogenous fibrolytic microflora for use in the present invention may be selected a strain of the Propionibacterium genus. In one embodiment, the DFM for use in the present invention can be selected from the species Propionibacterium acidipropionici.
    [00133] In one embodiment, the DFM for use in the present invention is Propionibacterium acidipropionici P169.
    Agtech Products, Inc., of W227 N752 Westmound Drive, Waukesha, WI 53186, USA, deposited on July 28, 2003, under the Budapest Treaty, Propionibacterium acidipropionici P169 with the American Type Culture Collection (ATCC), Manassas , VA 20110-2209, USA, as Accession No. PTA-5271. Propionibacterium acidipropionici P169 has been cited in assigned US Patent 6,951,643B2 and is publicly available from the ATCC.
    [00135] In one embodiment, the enzyme-producing strain and/or the C5-sugar fermenting strain and/or the short-chain fatty acid-producing strain and/or the strain promoting endogenous fibrolytic microflora for use in the present invention may be a strain of the Enterococcus genus.
    [00136] In one embodiment the DFM for use in the present invention can be selected from species of Enterococcus faecium.
    [00137] In one embodiment, the DFM for use in the present invention may be Enterococcus faecium ID7.
    [00138] Lactococcus lactis ID7 (which was later reclassified as Enterococcus faecium ID7) was deposited on June 22, 2004 under the Budapest Treaty as Lactococcus lactis ID7 in the American Type Culture Collection (ATCC), Manassas, VA 201102209, USA, as Accession No. PTA-6103. Lactococcus lactis ID7 (which was later reclassified as Enterococcus faecium ID7) has been cited in assigned US Patent 7,384,628 and is publicly available from the ATCC. When "Enterococcus faecium ID7" is used herein, it should be understood that the name of this organism is interchangeable with "Lactococcus lactis ID7", which has been deposited as Accession No. PTA-6103. Enterococcus faecium ID7 is also publicly available from Danisco Animal Nutrition, Denmark.
    [00139] In one embodiment, the enzyme-producing strain and/or the C5-sugar fermenting strain and/or the short-chain fatty acid-producing strain and/or the strain promoting endogenous fibrolytic microflora for use in the present invention may be a strain of the Lactobacillus genus.
    [00140] In one embodiment, the strain that produces enzyme and/or the strain that ferments C5-sugar and/or the strain that produces short-chain fatty acid and/or the strain that promotes endogenous fibrolytic microflora can be selected from the following Lactobacillus spp: Lactobacillus buchneri, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus kefiri, Lactobacillus bifidus, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Labacillus fercirius, Lactobacillus rectos Lactobacillus lactis, Lactobacillus delbreuckii, Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus farciminis, Lactobacillus rhamnosus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus johnsonii and Lactobacillus jenseniie combinations of any of the same.
    [00141] In one embodiment, DFM can be selected from one or more of the following strains: Lactobacillus rhamnosus CNCM-I-3698 and Lactobacillus farciminis CNCM-I-3699. These strains have been deposited with the Collection Nationale de Cultures de Microorganims (CNCM) 25, Rue due Docteur Roux, F75724 Paris Cedex 15, France, on December 8, 2006 by Sorbial, Route de Spay 72,700 Allonnes, France and all rights, titles and interests in the warehouses were later transferred to Danisco France SAS 20, Rue de Brunel, 75017 Paris, France.
    [00142] Danisco France SAS has authorized DuPont Nutrition Biosciences ApS de Langebrogade 1, PO Box 17, DK-1001,Copenhagen K, Denmark to refer to this biological material deposited in this Patent Application and has given unconditional and irrevocable consent for the deposited material to be made available to the public.
    In at least some embodiments, DFM can be selected from Lactobacillus lactis DJ6 (PTA 6102) and/or Lactococcus lactis ID7 (PTA 6103).
    [00144] Agtech Products, Inc., of W227 N752 Westmound Drive, Waukesha, WI 53186, USA, has deposited, under the Budapest Treaty, the following biological deposits with the American Type Culture Collection (ATCC), Manassas, VA 20110-2209 , USA, with the dates of the original deposits and the access numbers specified below:

    [00145] Agtech Products, Inc. has authorized DuPont Nutrition Biosciences ApS de Langebrogade 1, PO Box 17, DK-1001, Copenhagen K, Denmark to refer to this biological material deposited in this Application and has given unconditional consent and irrevocable for the deposited material to be made available to the public.
    [00146] In at least one embodiment, more than one strain of those described here are combined.
    [00147] Therefore, the enzyme producing strain and/or the C5-sugar fermenting strain and/or the short chain fatty acid producing strain and/or the fibrolytic endogenous microflora promoting strain used in the present invention may be a combination of at least two, conveniently at least three, suitably at least four DFM strains described herein, for example, DFM strains selected from the group consisting of Bacillus subtilis AGTP BS3BP5, Bacillus subtilis AGTP BS442, B. subtilis AGTP BS521, B. subtilis AGTP BS521, B. subtilis AGTP BS3BP BS918, Bacillus subtilis AGTP BS1013, B. subtilis AGTP BS1069, B. subtilis AGTP 944, B. pumilus AGTP BS 1068, B. pumilus KX11-1, Propionibacterium P169, Lactobacillus rhamnosus CNCM-I-3698 or Lactobacillus farciminis CNCM 3699.
    In one embodiment, preferably the DFM may be one or more of the group consisting of Bacillus subtilis AGTP BS3BP5, Bacillus subtilis AGTP BS442, B. subtilis AGTP BS521, B. subtilis AGTP BS918, Bacillus subtilis AGTP BS1013, B subtilis AGTP BS1069, B. subtilis AGTP 944, B. pumilus AGTP BS 1068, B. pumilus KX11-1 and a combination thereof.
    Any derivative or variant of Bacillus, Lactobacillus or Propionibacterium is also included and is useful in the methods described and claimed herein.
    In some embodiments, variant Bacillus strains having all the characteristics of Bacillus subtilis AGTP BS3BP5, Bacillus subtilis AGTP BS442, B. subtilis AGTP BS521, B. subtilis AGTP BS918, Bacillus subtilis AGTP BS1013, B. subtilis AGTP BS1069, B. subtilis AGTP 944, B. pumilus AGTP BS 1068 or B. pumilus KX11-1 are also included and are useful in the methods described and claimed herein.
    [00151] As used herein, a "variant" has at least 80% genetic sequence identity with the strains described using amplified polymorphic DNA polymerase chain reaction analysis (RAPD-PCR) . The degree of identity of genetic sequences can vary. In some embodiments, the variant has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence genetic identity with the strains described using RAPD-PCR analysis.
    [00152] Six primers that can be used for RAPD-PCR analysis include the following:
    [00153] Primer 1 (5'-GGTGCGGGAA-3'), Primer 2 (5'-GTTTCGCTCC-3'), Primer 3 (5'-GTAGACCCGT-3'), Primer 4 (5'-AAGSCFACGT-3') , Primer 5 (5'-AACGCGCAAC-3'), Primer 6 (5'-CCCGTCAGCA-3'). RAPD analysis can be performed using Ready-to-Go™ RAPD Analysis Beads (Amersham Biosciences, Sweden), which are designed as premixed reactions, pre-dispensed to perform RAPD analysis
    [00154] The directly fed bacteria used in the present invention may be of the same type (genus, species and strain) or may comprise a mixture of genera, species and/or strains.
    [00155] Preferably, the DFM to be used in accordance with the present invention is a microorganism that is generally recognized as safe and which is preferably GRAS approved.
    [00156] Those skilled in the art will be readily aware as to the species and strains of microorganisms of which specific genera described herein that are used in the food and/or agricultural industries and which are generally considered suitable for animal consumption.
    [00157] Preferably, the DFM used in accordance with the present invention is one which is suitable for animal consumption.
    [00158] Advantageously, where the product is a feed or feed additive composition, the viable DFM should remain effective throughout the "shelf life" or "shelf life" of the product during which the feed or feed additive composition is put to sale by the retailer. Desired time periods and normal shelf life will vary from feed to feed and those skilled in the art will recognize that shelf lives will vary with feed type, feed size, storage temperature, processing conditions, material of packaging and packaging equipment.
    [00159] In some modalities, it is important that the DFM is heat tolerant, that is, it is thermotolerant. This is particularly the case when the feed is pelleted. Therefore, in one embodiment, the DFM can be a thermotolerant microorganism, such as a thermotolerant bacteria including, for example, Bacillus spp.
    [00160] In some embodiments, it may be preferable for the DFM to be a bacterium that produces spores, such as Bacilli, eg Bacillus spp. Bacilli are able to form stable endospores when growing conditions are unfavorable and are very resistant to heat, pH, moisture and disinfectants.
    [00161] Suitably, DFM is not an inactivated microorganism.
    [00162] In one embodiment, DFM can be a viable or non-viable microorganism that is used in isolated or semi-isolated form. DFM can be used in combination with or without the growth medium in which it was grown.
    [00163] In one modality, DFM is capable of producing colony-forming units when cultivated on an appropriate medium. Appropriate means may comprise (or consist of) a feed or a feed constituent.
    [00164] In one embodiment, DFM is unable to produce colony-forming units when cultivated on an appropriate medium. Appropriate means may comprise (or consist of) a feed or a feed constituent.
    Regardless of whether DFM is capable or unable to produce colony-forming units when cultured on an appropriate medium - cells may still be metabolically active (eg, even if they are unable to divide).
    [00166] In one embodiment, DFM can be administered as non-viable cells.
    [00167] In one embodiment, DFM can be administered as a viable microorganism.
    [00168] DFM can be properly dosed.
    Suitably, dosages of DFM in the feed can be between about 1x103 CFU/g feed to about 1x109 CFU/g feed, suitably between about 1x104 CFU/g feed to about 1x108 CFU/g feed feed, suitably between about 7.5x104 CFU/g feed and about 1x107 CFU/g feed.
    [00170] In one modality, DFM is dosed into the feed at more than about 1x103 CFU/g of feed, suitably more than about 1x104 CFU/g of feed, suitably more than about 7.5x104 CFU/ g of feed.
    Suitably, dosages of DFM in the feed additive composition may be between about 1x105 CFU/g composition to about 1x1013 CFU/g composition, suitably between about 1x106 CFU/g composition to about 1x1012 CFU /g composition, suitably between about 3.75x107 CFU/g composition to about 1x1011 CFU/g composition.
    [00172] In one embodiment, DFM is dosed into the feed additive composition at more than about 1x105 CFU/g composition, suitably more than about 1x106 CFU/g composition, suitably more than about 3, 75x107 CFU/g composition.
    In a preferred embodiment, the DFM can be dosed in the additive composition between about 5x107 to about 1x109 CFU/g, suitably between about 1x108 to about 5x108 CFU/g of composition.
    [00174] In another preferred embodiment, the DFM may be dosed in the additive composition between about 5x103 to about 5x105 CFU/g, suitably between about 1x104 to about 1x105 CFU/g of composition.FIBER DEGRADATION ENZYMES
    [00175] DFM as taught herein can be used in combination with at least one xylanase and at least one β-glucanase (and optionally at least one additional fiber degradation).
    [00176] β-glucanase or endo-glucanase is the name given to a class of enzymes that can hydrolyze (1,3)-β-D-glycosidic and/or (1,4)-eD-glycosidic bonds of (1, 4)-β-glucan, (1,3; 1.4)-β-glucan and cellulose in glucose and glucose oligosaccharides, thus breaking down cellulose and hemicellulose, the main components of plant cell walls.
    The β-glucanase for use in the present invention can be any commercially available β-glucanase.
    [00178] In one embodiment, the β-glucanase is an endoglucanase, eg an endo-1,4-β-D-glucanase (classified as E.C. 3.2.1.4).
    Suitably, the β-glucanase for use in the present invention may be a β-glucanase from Bacillus, Trichoderma, Aspergillus, Thermomyces, Fusarium and Penicillium.
    [00180] In one embodiment, the fiber degradation may be a β-glucanase produced from one or more of the expression hosts selected from the group consisting of: Bacillus lentus, Aspergillus niger, Trichoderma reesei, Penicillium funiculosum, Trichoderma longibrachiatum , Humicola insolens, Bacillus amyloliquefaciens, Aspergillus aculeatus, Aspergillus aculeatus.
    [00181] In one embodiment, fiber degradation may be one or more of the following commercial products, which comprise at least one of β-glucanase fiber degradation: Econase® GT or Econase® BG (available from AB Vista), Rovabio Excel® (available from Adisseo), Endofeed® DC and Amylofeed® (available from Andres Pintaluba SA), AveMix® XG10 (from Aveve), Natugrain®, Natugrain®TS, or Natugrain® TS/L (available from BASF), Avizyme® 1210, Avizyme® SX, Grindazym® GP, Grindazym® GV, Porzyme® 8100, Porzyme® 9102, Porzyme® tp100, AXTRA® XB, Avizyme® 1100, Avizyme® 1110, Avizyme® 1202, Porzyme® sf or Porzyme® SP (available from Danisco Animal Nutrition), Bio-Feed Plus®, Ronozyme A®, Ronozyme VP® or Roxazyme G2® (available from DSM), Hostazym C® (available from Huvepharma), Kemzyme W dry or Kemzyme W liquid (available from from Kemin), Biogalactosidase BL (available from Kerry Ingredients), Safizyme G (available from Le Saffre) or Feedlyve AGL (available from Lyven).
    [00182] In one embodiment, β-glucanase can be obtained from Axtra®XB.
    [00183] β-glucanase can be administered in any suitable amount.
    [00184] In one embodiment, the β-glucanase for use in the present invention may be present in the feed in a range from about 50 BGU/kg feed to about 50000 BGU/kg feed, suitably about 100 BGU/kg feed of feed at about 1000 BGU/kg of feed.
    [00185] The β-glucanase for use in the present invention may be present in the feed in a range from about 75 BGU/kg feed to about 400 BGU/kg feed, suitably about 150 BGU/kg feed to about 200 BGU/kg of feed.
    [00186] In one embodiment, β-glucanase is present in the feed at less than 1000 BGU/kg feed, suitably less than about 500 BGU/kg feed, suitably less than 250 BGU/kg feed.
    [00187] In one embodiment, β-glucanase is present in the feed at more than 75 BGU/kg of feed, suitably more than 100 BGU/kg of feed.
    Suitably, β-glucanase is present in the additive composition in the range from about 150 BGU/g composition to about 3000 BGU/g composition, suitably in the range from about 300 BGU/g composition to about 1500 BGU/g of composition.
    [00189] In one embodiment, β-glucanase is present in the feed additive composition at less than 5000 BGU/g composition, suitably at least 4000 BGU/g decomposition, suitably at least 3000 BGU/g decomposition, suitably at less than 2000 BGU/g of composition.
    [00190] In one embodiment, β-glucanase is present in the feed additive composition at more than 50 BGU/g composition, suitably at more than 100 BGU/g decomposition, suitably at more than 125 BGU/g decomposition .
    [00191] In some embodiments, β-glucanase activity can be calculated using the "β-Glucanase Activity Assay (BGU)" as taught here.
    [00192] In one embodiment, the β-glucanase for use in the present invention may have β-glucanase activity as determined using the "β-glucanase Activity Assay (BUG)" taught herein.
    [00193] The term "degrading fibers", as used herein, may include one or more of the following degrading fibers: a xylanase (eg an endo-1,4-β-D-xylanase (EC 3.2. 1.8) or a 1,4 β-xylosidase (EC 3.2.1.37)), a β-glucanase (EC 3.2.1.4), a cellobiohydrolase (EC 3.2.1.91 and EC 3.2.1.176), a β-glucosidase ( EC 3.2.1.21), a feruloyl esterase (EC 3.1.1.73), an α-arabinofuranosidase (EC 3.2.1.55), a pectinase (eg an endopolygalacturonase (EC 3.2.1.15), an exopolygalacturonases (EC 3.2.1.67) ) or a pectate lyase (EC 4.2.2.2)) or combinations thereof.
    [00194] The term "fiber degrading other", as used herein, may include one or more of the following degrading fibers: a cellobiohydrolase (EC 3.2.1.176 and EC 3.2.1.91), a β-glucosidase ( EC 3.2.1.21), a β-xylosidase (EC 3.2.1.37), a feruloyl esterase (EC 3.1.1.73), an α-arabinofuranosidase (EC 3.2.1.55), a pectinase (eg an endopolygalacturonase (EC 3.2). .1.15), an exopolygalacturonase (EC 3.2.1.67) or a pectate lyase (EC 4.2.2.2)) or combinations thereof.
    [00195] It will also be understood by those skilled in the art that "another fiber degradation" may encompass various fiber degradation options.
    [00196] In one embodiment, DFM as taught herein can be used in combination with at least one xylanase, at least one β-glucanase, and at least one other fiber degradation.
    [00197] In another embodiment, DFM as taught herein can be used in combination with at least one xylanase, at least one β-glucanase and two (or at least two) of other degrading fibers.
    [00198] In another embodiment, DFM as taught herein can be used in combination with at least one xylanase, at least one β-glucanase and three (or at least three) other degrading fibers.
    [00199] In another embodiment, DFM as taught herein can be used in combination with at least one xylanase, at least one β-glucanase and four (or at least four) other degrading fibers.
    [00200] In one embodiment, DFM as taught herein can be used in combination with a broth or solid state fermentation product containing measurable enzymatic activity or activities of the present invention.
    [00201] In one embodiment, DFM as taught herein can be used in combination with the enzymes of the present invention, enzymes which are in isolated or purified form.
    [00202] In one embodiment, DFM as taught herein can be used in combination with the enzymes of the present invention, enzymes which are exogenous to DFM in the composition (for example, if DFM is an enzyme-producing strain).
    [00203] Preferably, the fiber degradation enzyme(s) is(are) present in the feed in the range of about 0.05 to 5 g of enzyme protein per metric ton (Metric Ton - MT) of feed (or mg/kg).
    Suitably, each of fiber degradation may be present in the feed in the range of about 0.05 to 5 g of enzyme protein per metric ton (MT) of feed (or mg/kg).
    Suitably, total fiber degradations are present in the feed in the range of about 0.05 to 5 g of enzyme protein per metric ton (MT) of feed (or mg/kg).
    [00206] Preferably, the fiber degradation enzyme(s) is(are) present in the additive composition (or premix) in the range of from about 0.05 to 100 mg of protein/g composition (eg at a total dietary inclusion of 50 to 1000 g/MT).
    [00207] Suitably, each of fiber degradation is present in the additive composition (or premix) in the range of about 0.05 to 100 mg protein/g composition (for example, at a total dietary inclusion of 50 at 1000 g/MT).
    [00208] Suitably, those of total fiber degradation are present in the additive composition (or premix) in the range of about 0.05 to 100 mg protein/g of composition (for example, in a total inclusion in the diet of 50 to 1000 g/MT).
    [00209] In a preferred embodiment, the fiber degradation (e.g., each fiber degradation or the total fiber degradation) may be in the additive composition (or premix) in the range of about 50 to about 700 g/MT of feed. Suitably, fiber degradation (e.g. each fiber degradation or total fiber degradation) may be in the additive composition (or premix) at about 100 to about 500 g/MT of feed.
    [00210] In one embodiment, the other fiber degradation enzyme(s) for use in the present invention may comprise (or consist essentially of or consist of) a cellobiohydrolase ( EC 3.2.1.91 and EC 3.2.1.176).
    [00211] In another embodiment, the other fiber degradation enzyme(s) for use in the present invention may comprise (or consist essentially of or consist of) a β-glycosidase ( EC 3.2.1.21).
    Suitably, the fiber degradation other may comprise (or consist essentially of or consist of) a cellobiohydrolase (EC 3.2.1.91 and EC 3.2.1.176), a β-glycosidase (EC 3.2.1.21) or combinations the same.
    [00213] In another embodiment, the other fiber degradation enzyme(s) for use in the present invention may comprise (or consist essentially of or consist of) a β-xylosidase ( EC 3.2.1.37).
    In one embodiment, the fiber degradation enzyme(s) for use in the present invention may comprise (or consist essentially of or consist of) a feruloyl esterase (EC 3.1.1.73 ).
    [00215] In another embodiment, the fiber degradation other for use in the present invention may comprise (or consist essentially of or consist of) an α-arabinofuranosidase (E.C. 3.2.1.55).
    [00216] In yet another embodiment, the other fiber degradation enzyme(s) for use in the present invention may comprise (or consist essentially of or consist of) a pectinase (by example, an endopolygalacturonase (EC 3.2.1.15), an exopolygalacturonases (EC 3.2.1.67) or a pectate lyase (EC 4.2.2.2)).
    [00217] In a preferred embodiment, the other fiber degradation enzyme(s) for use in the present invention may comprise (or consist essentially of or consist of) one or more ( suitably two or more, suitably three) pectinases selected from the group consisting of: an endopolygalacturonase (EC 3.2.1.15), an exopolygalacturonase (EC 3.2.1.67) and a pectate lyase (EC 4.2.2.2).
    [00218] In one embodiment, the other fiber degradation enzyme(s) for use in the present invention may comprise (or consist essentially of or consist of) a cellobiohydrolase ( EC 3.2.1.91 and EC 3.2.1.176), a β-glycosidase (EC 3.2.1.21), a β-xylosidase (EC 3.2.1.37), a feruloyl esterase (EC 3.1.1.73), an α-arabinofuranosidase (EC 3.2.1.55) and/or a pectinase (eg, an endopolygalacturonase (EC 3.2.1.15), an exopolygalacturonase (EC 3.2.1.67) or a pectate lyase (EC 4.2.2.2).
    [00219] The present invention relates to the combination of at least one xylanase with at least one β-glucanase and at least one specific DFM, as taught herein.
    [00220] In a preferred embodiment, the at least one xylanase, the at least one β-glucanase and the at least one specific DFM as taught herein may be combined with another degrading fiber as taught herein.
    [00221] The present invention further relates to the combination of at least one xylanase and at least one β-glucanase with at least two, such as at least three or at least four or at least five, of other fiber degradation and at least one specific DFM as taught here.
    [00222] Xylanase is the name given to a class of enzymes that degrade the linear polysaccharide beta-1,4-xylan to xylose, thus breaking down hemicelluloses, one of the main components of plant cell walls.
    [00223] The xylanase for use in the present invention can be any commercially available xylanase.
    Suitably, the xylanase may be an endo-1,4-β-d-xylanase (classified as EC 3.2.1.8).
    [00225] In one embodiment, preferably the xylanase is an endoxylanase, for example an endo-1,4-β-xylanase. The classification of an endo-1,4-β-d-xylanase is E.C. 3.2.1.8.
    In one embodiment, the present invention relates to a DFM in combination with an endoxylanase, for example an endo-1,4-β-d-xylanase and another enzyme.
    [00227] All E.C. classifications of enzymes cited herein refer to the classifications given in Enzyme Nomenclature - Recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology - ISBN 0-12226164-3
    Suitably, the xylanase for use in the present invention may be a xylanase from Bacillus or Trichoderma.
    [00229] In one embodiment, the xylanase may be a xylanase that comprises (or consists of) an amino acid sequence shown herein as SEQ ID NO. 1, a xylanase that comprises (or consists of) an amino acid sequence shown herein as SEQ ID NO. 2 or a xylanase which comprises (or consists of) an amino acid sequence shown herein as SEQ ID NO. 3 (FveXyn4), a xylanase from Trichoderma reesei, Econase XT™ or Rovabio Excel™.
    [00230] In one embodiment, the xylanase can be the xylanase Axtra XAP® or Avizyme 1502® or AxtraXB™, all commercially available products from Danisco S/A.
    [00231] In a preferred embodiment, the xylanase for use in the present invention may be one or more of the xylanases in one or more of the commercial products below:




    [00232] In one embodiment, the xylanase may be a xylanase that comprises (or consists of) a polypeptide sequence shown herein as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12; or a variant, homologue, fragment or derivative thereof having at least 75% identity (such as at least 80%, 85%, 90%, 95%, 98% or 99% identity) to SEQ ID NO. 1 or SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12; or a polypeptide sequence comprising SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12 with a conservative substitution of at least one of the amino acids.
    [00233] In one embodiment, the xylanase may comprise a polypeptide sequence shown herein as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3 or a variant, homologue, fragment or derivative thereof having at least 98.5% (e.g. at least 98.8 or 99 or 99.1 or 99.5%) identity with SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3.
    [00234] SEQ ID NO. 1: mklssflytasIvaa / PTA / EPPQAADSINKLIKNKGKLYYGTITDPNLLGVAKDTAIIKADFGAVTPEN SGKWDATEPSQGKFNFGSFDQWNFAQQNGLKVRGHTLVWHSQLPQWVKNINDKATLTK VIENHVTQWGRYKGKIYAWDWNEIFEWDGTLRKDSHFNNVFGNDDYVGIAFRAARKADP NAKLYINDYSLDSGSASKVTKGMVPSVKKWLSQGVPVDGIGSQTHLDPGAAGQIQGALTAL ANSGVKEVAITELDIRTAPANDYATVTKACLNVPKCIGITVWGVSDKNSWRKEHDSLLFDAN YNPKPAYTAWNALR
    [00235] SEQ ID NO. 2: / PTA / EPRQAADSINKLIKNKGKLYYGTITDPNLLGVAKDTAIIKADFGAVTPENSGKWDATEP SQGKFNFGSFDQWNFAQQNGLKVRGHTLVWHSQLPQVWKNINDKATLTKVIENHVTQW GRYKGKIYAWDVVNEIFEWDGTLRKDSHFNNVFGNDDYVGIAFRAARKADPNAKLYINDYS LDSGSASKVTKGMVPSVKKWLSQGVPVDGIGSQTHLDPGAAGQIQGALTALANSGVKEVAI TELDIRTAPANDYATVTKACLNVPKCIGITVWGVSDKNSWRKEHDSLLFDANYNPKPAYTAVVNALR
    [00236] SEQ ID NO. 3: QAADSINKLIKNKGKLYYGTITDPNLLGVAKDTAIIKADFGAVTPENSGKWDATEPSQGKFNF GSFDQWNFAQQNGLKVRGHTLVWHSQLPQWVKNINDKATLTKVIENHVTQWGRYKGKIYAWDWNEIFEWDGTLRKDSHFNNVFGNDDYVGIAFRAARKADPNAKLYINDYSLDSGSASK VTKGMVPSVKKWLSQGVPVDGIGSQTHLDPGAAGQIQGALTALANSGVKEVAITELDIRTAP ANDYATVTKACLNVPKCIGITVWGVSDKNSWRKEHDSLLFDANYNPKPAYTAWNALR
    [00237] SEQ ID NO. 4: mklssflytasIvaa / To / EPRQASDSINKLIKNKGKLYYGTITDPNLLGVAKDTAIIKADFGAVTPEN SGKWDATEPSQGKFNFGSFDQWNFAQQNGLKVRGHTLVWHSQLPQVWKNINDKATLTK VIENHVTNVVGRYKGKIYAWDWNEIFDWDGTLRKDSHFNNVFGNDDYVGIAFRAARKADP NAKLYINDYSLDSGSASKVTKGMVPSVKKWLSQGVPVDGIGSQTHLDPGAAGQIQGALTAL ANSGVKEVAITELDIRTAPANDYATVTKACLNVPKCIGITVWGVSDKNSWRKEHDSLLFDAN YNPKAAYTAWNALR
    [00238] SEQ ID NO. 5: ZPTA / EPRQASDSINKLIKNKGKLYYGTITDPNLLGVAKDTAIIKADFGAVTPENSGKWDATEP SQGKFNFGSFDQWNFAQQNGLKVRGHTLVWHSQLPQWVKNINDKATLTKVIENHVTNW GRYKGKIYAWDWNEIFDWDGTLRKDSHFNNVFGNDDYVGIAFRAARKADPNAKLYINDYS LDSGSASKVTKGMVPSVKKWLSQGVPVDGIGSQTHLDPGAAGQIQGALTALANSGVKEVAI TELDIRTAPANDYATX / TKACLNVPKCIGITVWGVSDKNSWRKEHDSLLFDANYNPKAAYTAV VNALR
    [00239] SEQ ID NO. 6: QASDSINKLIKNKGKLYYGTITDPNLLGVAKDTAIIKADFGAVTPENSGKWDATEPSQGKFNF GSFDQWNFAQQNGLKVRGHTLVWHSQLPQWVKNINDKATLTKVIENHVTNWGRYKGKIY AWDWNEIFDWDGTLRKDSHFNNVFGNDDYVGIAFRAARKADPNAKLYINDYSLDSGSASK VTKGMVPSVKKWLSQGVPVDGIGSQTHLDPGAAGQIQGALTALANSGVKEVAITELDIRTAP ANDYATVTKACLNVPKCIGITVWGVSDKNSWRKEHDSLLFDANYNPKAAYTAWNALR
    [00240] SEQ ID NO. 7:mvsfkylflaasalgalaAPVEVEESSWFNETHEFAERAGTPSSTGWNNGYYYSFWTDNGGTV NYQNGNGGSYSVQWKDTGNFVGGKGWNPGSARTINYSGSFNPSGNAYLTVYGWTTNPLV EYYIVENYGTYNNGYYYSFWTDNGGTV NYQNGNGGSYSVQWKDTGNFVGGKGWNPGSARTINYSGSFNPSGNAYLTVYGWTTNPLV EYYIVENYGTYNNGYYYSFWTDNGGTV NYQNGNGGSYSVQWKDTGNFVGGKGWNPGSARTINYSGSFNPSGNAYLTVYGWTTNPLV EYYIVENYGTYNNGYYYSFWTDNGGTV
    [00241] SEQ ID NO. 8:APVEVEESSWFNETALHEFAERAGTPSSTGWNNGYYYSFWTDNGGTVNYQNGNGGSYSV QWKDTGNFVGGKGWNPGSARTINYSGSFNPSGNAYLTVYGWTTNPLVEYYIVENYGTYNP GNGGTYRGSVYSDGANYNIYTATRYNAPSIEGDNYGSTEKTNTGTVQYWS
    [00242] SEQ ID NO. 9:AGTPSSTGWNNGYYYSFWTDNGGTVNYQNGNGGSYSVQWKDTGNFVGGKGWNPGSAR TINYSGSFNPSGNAYLTVYGWTTNPLVEYYiVENYGTYNPGNGGTYRGSVYSDGANYNIYT ATRYNAPSIEGDKTFTQYWSVRQSKRTGGQIVTTANG
    [00243] SEQ ID NO. 10:MVSFTSLLAAVSAVTGVMALPSAQPVDGMSWERDPPTNVLDKRTQPTTGTS GGYYFSFWTDTPNSVTYTNGNGGQFSMQWSGNHVGGKGWMPGTSRTIKY SGSYNPNGNSYLAVYGWTRNPLIEYYIVENFGTYNPSSGGQKKVRNLKNVQSDVNTGYVNTGGYNTGYVNTG
    [00244] SEQ ID NO. 11:/.PSAQPVDGMSVyERDPPTNVLDKRTQPTTGTSGGYYFSFWTDTPNSVTYTNGNGGQFS MQWSGNGNHVGGKGWMPGTSRTIKYSGSYNPNGNSYLAVYGWTRNPLIEYYIVENFGTY NPSSGGQKKGEVNVDGSVYDYNTYVKSTGLQRNQGSGG
    [00245] SEQ ID NO. 12:TQPTTGTSGGYYFSFWTDTPNSVTYTNGNGGQFSMQWSGNGNHVGGKGWMPGTSRTIK YSGSYNPNGNSYLAVYGWTRNPLIEYYIVENFGTYNPSSGGQKKGEVNVDGSVYDIYVSTR VNAPSIDGNKTFQQYWSVRNVNGNSYLAVYGWTRNPLIEYYIVENFGTYNPSSGGQKKGEVNVDGSVYDIYVSTR VNAPSIDGNKTFQQYWSVRAVENGLQRSSVG
    Preferably, the xylanase is present in the feed in the range of about 500 XU/kg to about 16,000 XU/kg feed, more preferably about 750 XU/kg feed to about 8000 XU/kg feed and even more preferably about 1000 XU/kg feed to about 4000 XU/kg feed.
    [00247] In one embodiment, xylanase is present in the feed at more than about 500 XU/kg of feed, suitably more than about 600 XU/kg of feed, suitably more than about 700 XU/kg of feed feed, suitably more than about 800 XU/kg feed, suitably more than about 900 XU/kg feed, suitably more than about 1000 XU/kg feed.
    [00248] In one embodiment, xylanase is present in the feed at less than about 16,000 XU/kg of feed, suitably less than about 8000 XU/kg of feed, suitably less than about 7000 XU/kg of feed , suitably less than about 6000 XU/kg feed, suitably less than about 5000 XU/kg feed, suitably less than about 4000 XU/kg feed.
    Preferably, the xylanase is present in the additive composition in the range of from about 100 XU/g to about 320,000 XU/g of composition, more preferably about 300 XU/g of composition to about 160,000 XU/g of composition and even more preferably about 500 XU/g composition to about 50,000 XU/g composition and even more preferably about 500 XU/g composition to about 40,000 XU/g composition.
    [00250] In one embodiment, the xylanase is present in the feed additive composition at more than about 100 XU/g decomposition, suitably more than about 200 XU/g decomposition, suitably more than about 300 XU/g decomposition, suitably more than about 400 XU/g decomposition, more suitably about 500 XU/g composition.
    [00251] In one embodiment, the xylanase is present in the feed additive composition at less than about 320,000 XU/g of composition, suitably less than about 160,000 XU/g of composition, suitably less than about 50,000 XU/ g of composition, suitably less than about 40,000 XU/g of composition, suitably less than about 30,000 XU/g of composition.
    [00252] Xylanase activity can be expressed in xylanase units (Xylanase Units - XU) measured as taught in the "Xylanase Activity Assay (XU)" taught here. See also Bailey, M.J. Biely, P. and Poutanen, K., Journal of Biotechnology, Volume 23, (3), May 1992, 257-270, the teaching of which is incorporated herein by reference.
    [00253] In one embodiment, suitably, the enzyme is classified using the E.C. classification above and the E.C. classification designates an enzyme having that activity when tested in the "Xylanase Activity Assay (XU)" taught here for determination of 1 XU.
    In one embodiment, the xylanase for use in the present invention may possess xylanase activity as determined using the "Xylanase Activity Assay (ABX U/g)" taught herein. ENZYMATIC ACTIVITIES AND ASSAYS
    In one embodiment, the additive composition may comprise a DFM in combination with a xylanase and a β-glucanase.
    [00256] In modality, xylanase activity can be calculated using the "Xylanase Activity Assay (XU)" taught here.
    [00257] In another embodiment, β-glucanase activity can be calculated using the "β-Glucanase Activity Assay (BGU)" taught here.
    [00258] Suitably, DFM in combination with a xylanase and a β-glucanase can be dosed as defined in the table below:

    [00259] Enzyme activity given in units can be calculated for each enzyme as taught in the preceding sections.
    [00260] In some embodiments, the additive composition may comprise a DFM in combination with a xylanase, a β-glucanase and a fiber degrading other as taught herein.
    [00261] Appropriately, DFM, xylanase, β-glucanase and other fiber degradation can be dosed as defined in the table below:


    [00262] In one modality, preferably, the ration comprises the following:
    [00263] a xylanase in at least 1000 XU/kg to 5000 kg/XU (suitably in at least 2000 XU/kg to 4500 XU/kg) of feed;
    a β-glucanase at at least 100 BGU/kg to 4000BGU/kg (suitably at least 150 BGU/kg to 3000 BGU/kg); and
    [00265] a DFM as taught herein in at least 50,000CFU/g to 200,000 CFU/g (suitably at least 70,000 CFU/g to 175,000 CFU/g) of feed.
    [00266] In another modality, preferably, the ration comprises the following:
    [00267] a xylanase in at least 1000 XU/kg to 5000 kg/XU (suitably in at least 2000 XU/kg to 4500 XU/kg) of feed;
    [00268] a β-glucanase at at least 100 BGU/kg to 4000BGU/kg (suitably at least 150 BGU/kg to 3000 BGU/kg); and
    [00269] a DFM as taught herein in at least 37,500CFU/g to 100,000 CFU/g (suitably at least 37,500 CFU/g to 75000 CFU/g) of feed.
    [00270] In another modality, preferably, the ration comprises the following:
    [00271] a xylanase in at least 1000 XU/kg to 5000 kg/XU (suitably in at least 2000 XU/kg to 4500 XU/kg) of feed;
    [00272] a β-glucanase in at least 200 to 2000 CMC U/kg (suitably at least 500 to 1500 CMC U/kg) of feed;
    [00273] a DFM as taught herein in at least 50,000 CFU/g to 200,000 CFU/g (suitably at least 70,000 CFU/g to 175,000 CFU/g) of feed; and
    [00274] a fiber degradation mixture further comprising at least 800 to 3500 ABX L/kg (suitably at least ABX 1000 to 2750 U/g) of feed and pNPG 500 to 3000 U/kg (suitably at least 600 to 2000 pNPG L/kg) of feed.
    [00275] In another modality, preferably, the ration comprises the following:
    [00276] a xylanase in at least 1000 XU/kg to 5000 kg/XU (suitably in at least 2000 XU/kg to 4500 XU/kg) of feed;
    [00277] a β-glucanase in at least 200 to 2000 CMC U/kg (suitably at least 500 to 1500 CMC U/kg) of feed;
    [00278] a DFM as taught herein in at least 37,500 CFU/g to 100,000 CFU/g (suitably at least 37,500 CFU/g to 75000 CFU/g) of feed; and
    [00279] a fiber degradation mixture further comprising at least 800 to 3500 ABX L/kg (suitably at least ABX 1000 to 2750 U/g) of feed and pNPG 500 to 3000 U/kg (suitably at least 600 to 2000 pNPG L/kg) of feed.
    [00280] In one modality, the DFM can be dosed according to the number of xylanase units present in the composition. In one embodiment, DFM can be dosed in the range of 6.25x101 CFU DFM: 1 XU enzyme to 2x109 CFU DFM: 1 XU enzyme; preferably in the range of 1.88x104 CFU DFM : 1 XU enzyme to 1.0x107 CFU DFM : 1 XU enzyme. The DFM taught here can be used in combination with a xylanase and a β-glucanase.
    [00281] In another embodiment, the DFM taught here can be used in combination with a xylanase, a β-glucanase and a fiber degrading one. In a further preferred embodiment, the fiber degrading one can be a β-glycosidase.
    [00282] In one embodiment, the xylanase for use in the present invention may have xylanase activity as determined using the "Xylanase Activity Assay (ABX U/g)" taught herein.
    [00283] In another embodiment, the β-glucanase for use in the present invention may have β-glucanase activity as determined using the "β-glucanase Activity Assay (CMC U/g)" taught herein.
    [00284] In yet another embodiment, the β-glycosidase for use in the present invention may have β-glycosidase activity as determined using the "β-glycosidase Activity Assay (pNPG U/g)" taught herein.
    [00285] In one modality, the DFM taught here can be used in combination with a xylanase and a β-glucanase, wherein the xylanase and β-glucanase have the activities shown in the tables below:

    [00286] 1A unit ABX is defined as the amount of enzyme required to produce 1 μmol of xylose reducing sugar equivalents per minute at 50 °C and pH 5.3.
    [00287] 2A unit of CMC activity releases 1 μmol of reducing sugars (expressed as glucose equivalents) in one minute at 50 °C and pH 4.8.
    [00288] In a preferred embodiment, the DFM taught here can be used in combination with a xylanase, a β-glucanase and a β-glucosidase, wherein the xylanase, β-glucanase and β-glucosidase have the activities shown in the tables below :

    [00289] 1A unit ABX is defined as the amount of enzyme required to produce 1 μmol of xylose reducing sugar equivalents per minute at 50 °C and pH 5.3.
    [00290] 2A unit of CMC activity releases 1 μmol of reducing sugars (expressed as glucose equivalents) in one minute at 50 °C and pH 4.8.
    [00291] 3A unit of pNPG denotes 1 mole of nitro-phenol released from para-nitrophenyl-BD-glucopyranoside per minute at 50 °C and pH 4.8.
    [00292] In one embodiment, the xylanase and β-glucanase for use in the present invention may comprise (or consist essentially of or consist of) more than about 3000 ABX u/g of xylanase activity and about 2000 to 2600 CMC u/g of β-glucanase activity, respectively.
    Suitably, the xylanase, β-glucanase and β-glycosidase for use in the present invention may comprise (or consist essentially of or consist of) more than about 3000 ABX u/g of xylanase activity, about 2000 to 2600 CMC u/g of β-glucanase activity more than about 2000 pNPG u/g of β-glucosidase activity, respectively.
    [00294] In one embodiment, the xylanase for use in the present invention may comprise (or consist essentially of or consist of) at least 2000 ABX u/g of xylanase activity (suitably at least 2500 ABX u/g of activity, suitably by the minus 3000 ABX u/g of activity), as determined using the "Xylanase Activity Assay (ABX U/g)".
    Suitably, the xylanase for use in the present invention may comprise (or consist essentially of or consist of) about 2000 to about 5000 ABX u/g of xylanase activity (suitably at least about 2500 to about 4000 ABX u/g activity, suitably at least about 3000 to about 4000 ABX u/g activity), as determined using the "Xylanase Activity Assay (ABX U/g)".
    [00296] In another embodiment, the β-glucanase for use in the present invention may comprise (or consist essentially of or consist of) at least 1000 CMC u/g of β-glucanase activity (suitably at least 1500 CMC u/g of activity, suitably at least 2000 CMC u/g activity), as determined using the "β-Glucanase Activity Assay (CMC U/g)".
    Suitably, the β-glucanase for use in the present invention may comprise (or consist essentially of or consist of) about 600 to about 4000 CMC u/g of β-glucanase activity (suitably at least about 1000 to about 3000 CMC u/g activity, suitably at least about 1500 to about 2600 CMC u/g activity), as determined using the "β-Glucanase Activity Assay (CMC U/g)".
    [00298] In another embodiment, the β-glycosidase for use in the present invention may comprise (or consist essentially of or consist of) at least 300 pNPG u/g of β-glycosidase activity (suitably at least 500 pNPG u/g of activity, suitably at least 1,000 pNPG u/g activity, or suitably at least 2000 pNPG u/g activity), as determined using the "β-Glycosidase Activity Assay (pNPG U/g)".
    Suitably, the β-glycosidase for use in the present invention may comprise (or consist essentially of or consist of) about 200 to about 4000 pNPG u/g of β-glycosidase activity (suitably at least about 300 to about 3000 pNPG u/g activity, suitably at least about 1000 to about 3000 pNPG u/g activity, suitably at least about 2000 to about 3000 pNPG u/g activity), as determined using the " β-Glycosidase Activity Assay (pNPG U/g)".
    Suitably, the DFM taught here can be used in combination with a xylanase and a β-glucanase which comprises (or consists essentially of or consists of) at least 2000 ABX u/g of xylanase activity (suitably at least 2500 ABX u/g activity, suitably at least 3000 ABX u/g activity), as determined using the "Xylanase Activity Assay (ABX U/g)"; and at least 1,000 CMC u/g β-glucanase activity (suitably at least 1500 CMC u/g activity, suitably at least 2000 CMC u/g activity), as determined using the "β-Glucanase Activity Assay (CMC U/g)".
    Suitably, the DFM taught here can be used in combination with a xylanase, a β-glucanase and a β-glycosidase that comprises (or consists essentially of or consists of) at least 2000 ABX u/g of xylanase activity ( suitably at least 2500 ABX u/g activity, suitably at least 3000 ABX u/g activity), as determined using the "Xylanase Activity Assay (ABX U/g)"; and at least 1,000 CMC u/g β-glucanase activity (suitably at least 1500 CMC u/g activity, suitably at least 2000 CMC u/g activity), as determined using the "β-Glucanase Activity Assay (CMC U/g)"; and at least 300 pNPG u/g β-glycosidase activity (suitably at least 500 pNPG u/g activity, suitably at least 1000 pNPG u/g activity or suitably at least 2000 pNPG u/g activity), as determined using the "β-Glycosidase Activity Assay (pNPG U/g)".
    [00302] In one embodiment, the DFM taught here can be used in combination with a xylanase and a β-glucanase that comprises (or consists essentially of or consists of) about 2000 to about 5000 ABX u/g of xylanase activity (suitably at least about 2500 to about 4000 ABX u/g activity, suitably at least about 3000 to about 4000 ABX u/g activity), as determined using the "Xylanase Activity Assay (ABX U/ g)"; and about 600 to about 4000 CMC u/g β-glucanase activity (suitably at least about 1000 to about 3000 CMC u/g activity, suitably at least about 1500 to about 2600 CMC u/g of activity) as determined using the "β-Glucanase Activity Assay (CMC U/g)".
    Suitably, the DFM taught herein can be used in combination with a xylanase, a β-glucanase and a β-glycosidase which comprises (or consists essentially of or consists of) about 2000 to about 5000 ABX u/g of xylanase activity (suitably at least about 2500 to about 4000 ABX u/g activity, suitably at least about 3000 to about 4000 ABX u/g activity), as determined using the "Xylanase Activity Assay ( ABX U/g)"; about 600 to about 4000 CMC u/g of β-glucanase activity (suitably at least about 1000 to about 3000 CMC u/g of activity, suitably at least about 1500 to about 2600 CMC u/g of activity), as determined using the "β-Glucanase Activity Assay (CMC U/g)"; and about 200 to about 4000 pNPG u/g of β-glycosidase activity (suitably at least about 300 to about 3000 pNPG u/g activity, suitably at least about 1000 to about 3000 pNPG u/g of activity or suitably at least about 2000 to about 3000 pNPG u/g activity), as determined using the "β-Glycosidase Activity Assay (pNPG U/g)"."XYLANASE ASSAY (XU)"
    Xylanase activity can be expressed in xylanase units (XU) measured at a pH of 5.0 with AZCL-arabinoxylan (azurine cross-linked wheat arabinoxylan, 100 mg Xylazyme tablets, Megazyme) as substrate. Hydrolysis by endo-(1-4) β-D-xylanase (xylanase) produces water-soluble colored fragments and their release rate (increase in absorbance at 590 nm) can be directly related to the enzymatic activity. Xylanase units (XU) are determined relative to a standard enzyme (Danisco Xylanase, available from Danisco Animal Nutrition) under conventional reaction conditions, which are 40 °C, reaction time of 10 min in McIlvaine's buffer, pH of 5.0.
    [00305] The xylanase activity of the standard enzyme is determined as the amount of reducing sugar end groups released from a spelled xylan substrate per min at a pH of 5.3 and 50 °C. The reducing sugar end groups react with 3,5-dinitrosalicylic acid and the formation of the reaction product can be measured as the increase in absorbance at 540 nm. Enzyme activity is quantified against a standard curve for xylose (reducing sugar equivalents). One unit of xylanase (XU) is the amount of standard enzyme that releases 0.5 μmol of reducing sugar equivalents per min at pH 5.3 and 50 °C."XYLANASE ACTIVITY ASSAY (ABX U/G)"
    [00306] The xylanase activity can be expressed in acidic birch xylanase units (ABX U) measured at a pH of 5.3 with birch 4-O-methyl glucuronoxylan as substrate. Pipette 1.8 ml of 1% Birch 4-O-Methyl Glucuronoxylan Substrate Solution into each sample tube. Incubate for 10 to 15 minutes, allowing to equilibrate to 50 °C. Pipette 0.2 ml of enzyme dilution using positive displacement pipettes or equivalent. Centrifuge to mix. Incubate each sample at 50 °C for exactly 5 minutes. Add 3 ml of 3.5-nitrosalicylic acid (DNS) sodium salt solution at 1% and mix. Cover the tops of the sample tubes with lids to prevent evaporation. Place the sample tubes in a boiling bath for exactly 5 minutes. Cool sample tubes for 10 minutes in an ice/water bath. Incubate the sample tube for 10 minutes at room temperature. Transfer the contents of the sample tube to cuvettes and measure at 540 nm against deionized water. Correct the absorption to the base color by subtracting the corresponding blank enzyme. Enzyme activity is quantified against a standard curve for xylose (reducing sugar equivalents).
    [00307] A unit of ABX is defined as the amount of enzyme required to produce 1 μmol of xylose reducing sugar equivalents per minute at 50 °C and pH 5.3."B-GLUCANASE ACTIVITY ASSAY (CMC U/ G)"
    [00308] The β-glucanase activity can be expressed in CMC units measured at a pH of 4.8 with carboxy methyl cellulose (CMC) sodium salt as substrate. Pipette 1 ml of 1% carboxy methyl cellulose (CMC) sodium salt solution (prepared with 0.05 M sodium acetate buffer) into blank sample tubes. Incubate tubes in a 50°C water bath for 10 minutes. Pipette 1 ml of the enzyme dilution at 15 second intervals into the sample tubes. Mix tubes after each addition. After 10 minutes, add 3 ml of 1% 3,5-dinitrosalicylic acid (DNS) sodium salt in the same order and time as the enzyme was added to the sample tubes. Add 3 ml DNS to blank sample tubes. After adding the DNS, remove the sample tubes to another rack outside the 50°C water bath. Add 1 ml of diluted enzyme to the corresponding blank sample. Cap the tubes and boil for exactly 5 minutes. Remove from the 100°C water bath and place in an ice bath for 10 minutes. Leave at room temperature for 10 to 15 minutes. Transfer to 3 ml cuvettes. Using the reagent blank to zero the spectrophotometer, each sample is read at 540 nm against deionized water. Enzyme activity is quantified against a standard curve of glucose (reducing sugar equivalents).
    [00309] One CMC unit of activity releases 1 μmol of reducing sugars (expressed as glucose equivalents) in one minute at 50 °C and pH 4.8."B-GLUCANASE (BGU) ACTIVITY ASSAY"
    [00310] Beta-glucanase activity can be expressed in beta-glucanase units (BGU) measured at a pH of 5.0 with AZCL-glucan (barley β-glucan cross-linked with azurine, Glucazyme 100 mg tablets, Megazyme ) as a substrate. Hydrolysis by beta-glucanase produces soluble colored fragments and their release rate (increase in absorbance at 590 nm) can be directly related to enzymatic activity. Beta-glucanase units (BGU) are determined relative to a standard enzyme (Multifect BGL, available from Danisco Animal Nutrition) under standard reaction conditions, which are 50°C, reaction time of 10 min in acetate buffer at 0.1 M, pH 5.0.
    [00311] The beta-glucanase activity of the standard enzyme is determined as the amount of reducing sugar end groups released from a barley glucan substrate per min at a pH of 5.0 and 50 °C. The reducing sugar end groups react with 3,5-dinitrosalicylic acid and the formation of the reaction product can be measured as an increase in absorbance at 540 nm. Enzyme activity is quantified against a standard curve of glucose (reducing sugar equivalents). A unit of beta-glucanase (BGU) is the amount of standard enzyme that releases 2.4 μmol of reducing sugar equivalents per min at a pH of 5.0 and 50 °C."B-GLYCOSIDASE ACTIVITY ASSAY (PNPG U/G)"
    [00312] The β-glucosidase activity can be expressed in pNPG units measured at a pH of 4.8 with para-nitrophenyl-BD-glucopyranoside (pNPG) as substrate. Pipette 1 ml of 3% nitrophenyl-beta-D-glucopyranoside (pNPG) solution (prepared with 0.05M sodium acetate buffer) into duplicate sample tubes for each of sample and control. Place in a 50°C water bath for 5 minutes. Add 200 μl of the sample or control to their respective tubes in duplicate at 15-30 second intervals. To the reagent blank tube, add 200 μl of sodium acetate buffer. Centrifuge each tube after sample addition. Allow the tubes to incubate for exactly 10 minutes. After 10 minutes of incubation, add 500 μl of a 1M sodium carbonate solution to stop the reaction. Centrifuge each tube after addition and place the tube in a rack outside the water bath. Add 10 ml of milli-Q water to each tube and centrifuge to mix. Using the reagent blank to zero the spectrophotometer, the concentration of 4-nitrophenol is measured by reading each sample at 400 nm.
    [00313] One unit of pNPG denotes 1 μmol of nitro-phenol released from para-nitrophenyl-BD-glucopyranoside per minute at 50 °C and pH 4.8. BENEFITS
    [00314] The interaction of DFMs with xylanase and β-glucanase (and optionally at least one other fiber-degrading enzyme) is complicated and, not wanting to be limited by theory, it is very surprising that an increase in the production of short-chain fatty acids in the GIT of animals.
    [00315] It has been found that combining the specific DFMs taught herein with at least one xylanase and at least one β-glucanase (and optionally at least one additional fiber-degrading enzyme) is particularly advantageous in feeds and/or in an individual who is fed a feed that is rich in fibrous by-products (eg from the biofuel and milling industries).
    [00316] Surprisingly, it has been found that the nutritional value and digestibility of feeds comprising substantial amounts (sometimes 30-60%) of fibrous by-products (having a high content of non-starch polysaccharides eg fiber) can be significantly improved , as well as the performance and weight gain of an individual fed such feeds.
    [00317] An advantage of the present invention is the improved feed conversion ratio (FCR) observed using the combination of the present invention.
    [00318] Without wishing to be bound by theory, the degradation of dietary material derived from plant cell wall particles, which is rich in non-starch polysaccharides (Non-Starch Polysaccharide- NSP), by xylanases can be optimized to improve the performance of animals when combining xylanase (eg, endo-1,4-β-d-xylanase) with one or more β-glucanases (and optionally in combination with one or more other degrading fibers (eg, a cellobio). -hydrolase (EC 3.2.1.91 and EC 3.2.1.176), a β-glycosidase (EC 3.2.1.21), a β-xylosidase (EC 3.2.1.37), a feruloyl esterase (EC 3.1.1.73), an α- arabinofuranosidase (EC 3.2.1.55), a pectinase (eg, an endopolygalacturonase (EC 3.2.1.15), an exopolygalacturonase (EC 3.2.1.67) or a pectate lyase (EC 4.2.2.2)) or combinations thereof)) and one or more specific Direct Fed Microbials (DFMs) live microorganisms selected for their ability to produce enzymes and/or its ability to produce short chain fatty acids (Short Chain Fatty Acid - SCFA) from pentoses of the NSP fraction under anaerobic conditions and/or its ability to promote endogenous populations of fibrolytic microflora in the GIT of an individual and /or its ability to degrade C5-sugars.
    [00319] The reason this combination improves performance is that the solubilization of fibers, specifically hemicellulose, from the diet is maximized in the gastrointestinal tract (Gastro Intestinal Tract - GIT) of animals. This solubilization of hemicellulose would not always be sufficient to increase performance because released C5-sugars are not an efficient source of energy for animals when they are absorbed (Savory CJ Br. J. Nut. 1992, 67: 103-114), but they are a more efficient energy source when converted to short chain fatty acids (SCFA) either by microorganisms in the GIT or by DFMs.
    [00320] Therefore, the energy value from plant products (for example, wheat, corn, oats, barley and cereal co-products (by-products) or readily accessible mixed grain diet for monogastrics) can be optimized by combining xylanase (by example, endo-1,4-β-d-xylanase) and β-glucanase (and optionally at least one additional fiber-degrading enzyme (including, but not limited to, a cellobiohydrolase (EC 3.2.1.91 and EC 3.2.). 1.176), a β-glycosidase (EC 3.2.1.21), a β-xylosidase (EC 3.2.1.37), a feruloyl esterase (EC 3.1.1.73), an α-arabinofuranosidase (EC 3.2.1.55), a pectinase ( for example, an endopolygalacturonase (EC 3.2.1.15), an exopolygalacturonase (EC 3.2.1.67) or a pectate lyase (EC 4.2.2.2)) or combinations thereof)) and specific DFMs that can produce SCFA from pentoses in the fraction NPS under anaerobic conditions and/or that are able to modulate microbial populations in the GIT to increase production. tion of SCFA from the released sugars and/or that can use C-5 sugars. DFMs can adapt their metabolism to synergistically increase fiber hydrolysis in combination with xylanase and β-glucanase (and optionally at least one additional fiber-degrading enzyme). Use of DFMs that can produce (fibrolytic) enzymes can confer additional benefits and maximize the benefits of added enzymes.
    [00321] Specific DFMs selected for their enzymatic activities can be considered as a glycan-driven bacterial food chain. The specifically selected DFMs taught here can preferentially use dietary fiber, a feature that allows them to perform the initial glycan digestion steps to release shorter, more soluble polysaccharides for other bacteria, eg, other endogenous microflora in the GIT. The specific DFMs were selected for their metabolism, which adapts according to the glycans released by enzymes (eg, xylanase and β-glucanase (and, optionally, at least one additional fiber-degrading enzyme)) to improve efficiency of the enzymes described here and the combination of DFM(s) compared to using a combination of enzymes individually or using DFM(s) individually.
    [00322] Without intending to be bound by theory, in the present invention, dietary material derived from plant cell wall particles, which is rich in glycans from specific sources, such as cellulose, hemicellulose and pectin (plant material) or glycosaminoglycans, enter in the distal intestine in the form of particles that are attacked by specific glycan degrading DFMs, which are capable of binding directly to these insoluble particles and digesting their glycan components. After this initial degradation of glycan-containing particles, more soluble glycan fragments can be digested by secondary glycan degraders present in the cecum, which contribute to the released reservoir of short-chain fatty acid (SCFA) fermentation products that are derived from both the types of degraders. Since SCFAs arise from carbohydrate fermentation and/or protein fermentation and deamination by native anaerobic microflora in the GIT, the SCFA concentration may be an index of the population of anaerobic organisms. SCFA can actually provide a number of advantages to the host animal, acting as a metabolic fuel for gut, muscle, kidney, heart, liver and brain tissues and also conferring bacteriostatic and bactericidal properties against organisms such as Salmonella and E. coli.
    [00323] The nutritional value of fibers in non-ruminants can be derived primarily through the production of short-chain fatty acids (SCFA), through fermentation of solubilized fibers or degraded by effective fiber-degrading enzymes (e.g., xylanases and β -glucanase and/or an additional fiber-degrading enzyme as taught herein). Xylanase in individual feed is not sufficient to use fibrous ingredients in animal diets (especially non-ruminants). There are a wide variety of chemical characteristics among plant-based feed ingredients. Enzyme application depends on the characteristics of the plant material (feed). As an example only, in wheat grain, arabinoxylans predominate, however, in wheat bran (a co-product (by-product) of wheat milling), the β-glucan content increases from 8 g-1 DM (in grains) to more than 26 g-1 DM. An enzyme matrix containing a complex of xylanase and β-glucanase (and optionally at least one other fiber-degrading enzyme) can improve the nutritional value of diets rich in co-product(s) (by-product(s)).
    [00324] SCFA have different energy values and some can serve as glucose precursors and some can contribute to the maintenance of intestinal integrity and health. The inventors have found that the specific combinations taught here preferentially move the fermentation process in the animal's GIT towards the production of more valuable/useful SCFAs.
    [00325] Without wishing to be bound by theory, the present inventors have found that NPSs can be effectively degraded by a combination of a DFM according to the present invention and a xylanase and a β-glucanase (and optionally at least one enzyme of additional fiber degradation). Furthermore, this particular combination was found to release C-5 sugars that normally have only marginal nutritional value to the animal. However, using combinations as claimed herein, it is possible to have microorganisms in the GIT (or the DFM of the present invention) or endogenous fibrolytic microflora (which is stimulated by the (DFM) combinations of the present invention) that convert such C-5 sugars into nutritionally valuable and useful components, ie short-chain fatty acids. These short-chain fatty acids can be used by the animal. Thus, the system allows to improve the nutritional value of a feed for an animal.
    [00326] Advantageously, the combination of a live direct-fed micro-organism, a xylanase and a β-glucanase (and optionally at least one additional fiber-degrading enzyme), as taught here, surprisingly increases the fiber degradation of a composition. feed additive, premix, feed or feed component, which leads to improved performance of an individual. In particular, the combination of the present invention improves the digestibility of a raw material in a feed, resulting in an increase in the bioavailability of nutrients (eg digestibility) and metabolizable energy therein. FORMULATION OF DFM WITH ENZYMES
    [00327] The DFM of the present invention and enzymes may be formulated in any suitable form to ensure that the formulation comprises viable DFMs and active enzymes.
    [00328] In one embodiment, the DFM and enzymes can be formulated as a dry powder or a granule.
    [00329] The dry powder or granule can be prepared by means known to those skilled in the art, such as in a microingredient mixer.
    [00330] For some embodiments, the DFM and/or the enzyme(s) can be coated, for example, encapsulated. Suitably, DFM and enzymes can be formulated within the same coating or encapsulated within the same capsule. Alternatively, one or two or three or four of the enzymes can be formulated within the same coating or encapsulated within the same capsule and the DFM can be formulated in a coating to separate one or more or all of the enzymes. In some embodiments, such as where DFM is capable of producing endospores, DFM can be supplied without any coating. In such circumstances, DFM endospores can simply be mixed with one or two or three or four enzymes. In the latter case, the enzymes can be coated, for example, encapsulated, for example, one or more or all of the enzymes can be coated, for example, encapsulated. Enzymes may be encapsulated in the form of mixtures (i.e. comprising one or more, two or more, three or more or all) of enzymes or may be encapsulated separately, for example, as individual enzymes. In a preferred embodiment, all four enzymes can be coated, eg encapsulated, together.
    [00331] In one embodiment, the coating protects the enzymes against heat and can be considered a thermoprotector.
    In one embodiment, the additive composition is formulated as a dry powder or granule as described in WO2007/044968 (referred to as TPT granules), which is incorporated herein by reference.
    [00333] In some embodiments, DFM (eg, DFM endospores, for example) can be diluted with a diluent such as powdered starch, limestone or the like.
    [00334] In another embodiment, the feed additive composition can be formulated by applying, for example, by spraying the enzyme(s) on a carrier substrate, such as ground wheat, for example.
    [00335] In one embodiment, the feed additive composition according to the present invention may be formulated as a premix. By way of example, the premix alone may comprise one or more feed components, such as one or more minerals and/or one or more vitamins.
    [00336] In one embodiment, the DFM and/or enzymes for use in the present invention are formulated with at least one physiologically acceptable vehicle selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or a wheat component , sucrose, starch, Na2SO4, talc, PVA, sorbitol, benzoate, sorbitate, glycerol, sucrose, propylene glycol, 1,3-propane diol, glucose, parabens, sodium chloride, citrate, acetate, phosphate, calcium, metabisulfite, formate and mixtures thereof. PACKAGING
    [00337] In one embodiment, the feed additive composition and/or premix and/or feed or feed component according to the present invention is packaged.
    [00338] In a preferred embodiment, the feed additive composition and/or premix and/or feed or feed component is packaged in a bag, such as a paper bag.
    [00339] In an alternative embodiment, the feed additive composition and/or premix and/or feed or feed component can be sealed in a container. Any suitable container can be used. BY-PRODUCTS
    [00340] The animal feed industry has seen an increase in the feeding of by-products such as, for example, the processing of biofuels, for animals (raising this form of animal feed from 0 to 10% to the current extremes of 30 to 60% ). These dietary cost savings have been a great opportunity for the industry to save on feed intake costs, but come with a number of challenges. By-products are often high fiber products (eg at least about 40% fiber). Consequently, the inclusion of fiber-rich by-product (eg DDGS) can have a negative impact on the animal's growth performance and carcass characteristics. In addition to negative effects on animal growth and carcass quality, changes in nutrient digestibility have implications for manure handling, storage and decomposition (eg, swine manure).
    The term "by-product", as used herein, means any fibrous plant material, e.g. one comprising at least approximately 20% or 30% fiber).
    [00342] In one embodiment, the term by-product means any by-product of a high fiber feed material.
    [00343] In one modality, the by-product as mentioned here may be selected from one or more of the following products: corn germ meal, corn meal, hominy ration, corn gluten ration, dry distillery grains with solubles ( Dried Distillers Grains With Solubles - DDGS), dry distillers grains (DDG), gluten bran, wheat trimmings, wheat bran or combinations thereof.
    [00344] In one embodiment, the feed component of the present invention comprises a fibrous by-product, such as corn germ bran, corn bran, corn bran, hominy feed, corn gluten feed, dry distillery grains with solubles, dry distillery grains, gluten bran, wheat trimmings, wheat bran or combinations thereof.
    [00345] In one embodiment, the individual to whom the combination of DFM, xylanase and β-glucanase (and optionally at least one additional fiber-degrading enzyme) of the present invention or feed additive composition of the present invention is administered is also fed a feed comprising a fibrous by-product, such as corn germ bran, corn bran, corn bran, hominy ration, corn gluten feed, dry distillery grains with solubles, dry distillery grains, bran of gluten, wheat trimmings, wheat bran or combinations thereof. BREAKDOWN OR DEGRADATION
    The enzyme (or composition comprising the enzyme) of the present invention or as described herein can be used to break down (degrade) insoluble arabinoxylan (AXinsol) or soluble arabinoxylan (AXsol) or combinations thereof or degradation products of AXinsol.
    [00347] The term "break" or "degrade" are synonymous with hydrolysis. NON-AMYLACE POLYSACCHARIDES (NSPs)
    [00348] Most of the common plant ingredients in a pet food are made up of carbohydrates, making carbohydrates a crucial factor in animal production. In addition to well-digestible nutrients such as starch and sugars, the carbohydrate fraction of plant origin includes non-digestible (fibrous) components such as cellulose, hemicellulose, pectins, beta-glucans and lignin.
    [00349] All these difficult-to-digest components, excluding lignin, are classified as a group referred to here as non-starch polysaccharides (Non-Starch Polysaccharides - NSPs). The NSP fraction is well known for the anti-nutritional effects it can exert.
    [00350] In one embodiment, the term fiber may be used interchangeably with the term NSP.
    [00351] Within the group of NSPs, hemicellulose itself is a heterogeneous subgroup predominantly composed of xylans, arabinans, galatans, glucans and mannans. Arabinoxylan is the major fraction of NSP in several of the most important feed materials, including wheat and corn. ARABINOXILANA (AX)
    The term "arabinoxylans" (AX), as used herein, means a polysaccharide consisting of a xylan backbone (1,4-linked xylose units) with L-arabinofuranose (L-arabinose in its ring form with 5 atoms) randomly linked by 1α^2 and/or 1α^3 bonds to the xylose units in the entire chain. Arabinoxylan is a hemicellulose found in both primary and secondary cell walls of plants. Arabinoxylan can be found in grain bran such as wheat, corn (green corn), rye and barley.
    [00353] Arabinoxylan (AX) is found in close association with the plant cell wall, where it acts as a glue that connects various building blocks of the plant cell wall and tissue, giving it both structural strength and rigidity.
    [00354] Since xylose and arabinose (the constituents of arabinoxylans) are two pentoses, arabinoxylans are generally classified as pentosans.
    [00355] AX is the major non-starch polysaccharide (NSP) fraction in several of the most important feed materials, including wheat and corn.
    [00356] Its abundance, location within plant material and molecular structure cause AX to have a major negative impact on feed digestibility, effectively reducing the nutritional value of the raw materials in which it is present. This makes AX an important anti-nutritional factor, reducing the efficiency of animal production.
    [00357] AXs can also contain substantial amounts of water (which can be referred to as their water holding capacity) - this can cause soluble arabinoxylans to result in (high) viscosity - which is a disadvantage in many applications. WATER-INSOLUBLE ARABINOXILANA (AXINSOL)
    [00358] Water-insoluble arabinoxylan (AXinsol), also known as water-insoluble arabinoxylan (WU-AX), constitutes a significant part of the dry matter of plant material.
    [00359] In wheat, AXinsol can represent 6.3% of dry matter. In wheat bran and wheat DDGS, AXinsol can represent about 20.8% or 13.4% of dry matter (weight/weight).
    [00360] In rye, AXinsol can represent 5.5% of dry matter.
    [00361] In corn, AXinsol can represent 5.1% of dry matter. In corn DDGS, AXinsol can represent 12.6% of dry matter.
    [00362] AXinsol causes retention of nutrients in the feed. Large amounts of well-digestible nutrients, such as starch and proteins, remain locked in clumps of cell wall material or attached to AX side chains. These locked-in nutrients will not be available for digestion and subsequent absorption in the small intestine. WATER SOLUBLE ARABINOXILANA (AXSOL)
    [00363] Water-soluble arabinoxylan (AXsol), also known as water extractable arabinoxylan (WE-AX), can cause problems in the production of biofuels and/or malt and/or brewing and/or feed, as it can cause an increase viscosity due to the water binding capacity of AXsol.
    [00364] In feeds, AXsol may have an anti-nutritional effect, particularly in monogastrics, as they cause a considerable increase in the viscosity of the intestinal contents, caused by the extraordinary water binding capacity of AXsol. Increased viscosity can affect feed digestion and nutrient use as it can prevent proper mixing of food with digestive enzymes and bile salts and/or delay nutrient availability and absorption and/or stimulate fermentation in the intestine. thick.
    [00365] In wheat, AXsol can represent 1.8% of dry matter. In wheat bran and wheat DDGS, AXsol can represent around 1.1% or 4.9% of dry matter (weight/weight).
    [00366] In rye, AXsol can represent 3.4% of dry matter.
    [00367] In barley, AXsol may represent 0.4 to 0.8% of dry matter.
    [00368] In corn, AXsol can represent 0.1% of the dry matter. In corn DDGS, AXinsol can represent 0.4% of dry matter.
    [00369] Furthermore, however, in addition to the amount of AXsol present in plant material, when a xylanase solubilizes AXinsol in plant material, it can release pentosans and/or oligomers that contribute to the AXsol content of plant material.
    [00370] A significant advantage of some of the xylanases described here is that they have the ability to solubilize AXinsol as well as quickly and efficiently disaggregate solubilized oligomers and/or pentosans, thus enzymes are able to solubilize AXinsol without increasing viscosity and /or decrease the viscosity.
    [00371] The breakdown of AXsol can decrease viscosity.
    [00372] The breakdown of AXsol can release nutrients. VISCOSITY
    [00373] The present invention can be used to reduce viscosity in any process where the water binding capacity of AXsol causes an undesirable increase in viscosity.
    [00374] The present invention relates to the reduction of viscosity by the breakdown (degradation) of AXsol or by the breakdown (degradation) of polymers and/or oligomers produced when solubilizing AXinsol.
    In the present invention, a reduction in viscosity can be calculated by comparing a sample comprising the xylanase of the present invention (or taught herein) to another comparable sample without the xylanase of the present invention (or taught herein).
    [00376] Comparison of the viscosity reduction profiles of the xylanase of the present invention with that of the reference xylanases on the market shows the performance of the enzyme. The aim is to improve enzyme performance compared to the market benchmark. Reference enzymes for individual applications are provided in the examples below.
    [00377] In one embodiment of the present invention, the xylanases taught herein are viscosity reducers.
    [00378] The enzyme or feed additive composition of the present invention can be used as - or in the preparation of - a feed.
    [00379] The term "feed" is used here as a synonym for "food".
    [00380] In one embodiment, the feed of the present invention comprises a fiber-rich feed material and/or at least one by-product of the at least one fiber-rich feed material, such as corn germ meal, corn meal, feed of hominy, corn gluten, dry distillers grains with solubles (DDGS), dry distillers grains (DDG), wheat gluten bran, wheat trimmings, wheat bran or combinations thereof.
    [00381] In one embodiment, the individual to whom the combination of DFM, xylanase and β-glucanase (optionally in combination an additional fiber-degrading enzyme) of the present invention or feed additive composition of the present invention is administered is also fed with a food comprising a high fiber feed material and/or at least one by-product of the at least one high fiber feed material such as corn germ bran, corn bran, hominy ration, corn gluten, grains dry distillers with solubles (DDGS), dry distillers grains (DDG), wheat gluten bran, wheat trimmings, wheat bran or combinations thereof.
    [00382] Suitably, in one embodiment, the cereal component of a bird's diet may be wheat or barley with rye, wheat bran, wheat bran, oats, oat husks, although the vegetable components may be soy flour with or without other protein foods such as canola, rapeseed meal, etc., as long as the diet contains wheat-barley as the main foods and is formulated to meet the nutritional requirements of the birds being fed.
    [00383] The feed according to the present invention can be in the form of a solution or as a solid - depending on the use and/or mode of application and/or mode of administration.
    [00384] When used as - or in the preparation of - a feed - such as functional feed - the enzyme or composition of the present invention may be used in conjunction with one or more of: a nutritionally acceptable carrier, a nutritionally acceptable diluent, an excipient nutritionally acceptable, a nutritionally acceptable adjuvant, a nutritionally active substance.
    [00385] In a preferred embodiment, the enzyme or feed additive composition of the present invention is mixed with a feed component to form a feed.
    [00386] The term "feed component", as used herein, means all or part of the feed. Feed part can mean one feed constituent or more than one feed constituent, for example 2 or 3 or 4. In one embodiment, the term "feed component" encompasses a premix or premix constituents.
    [00387] Preferably, the feed may be a forage, or a premix thereof, a compound feed, or a premix thereof. In one embodiment, the feed additive composition according to the present invention may be mixed with a compound feed, a compound feed component or a premix of a compound feed or a forage, a forage component or a premix. mixture of a forage.
    [00388] The term fodder, as used herein, means any food which is supplied to an animal (rather than the animal obtaining fodder on its own). Forage covers plants that have been cut down.
    [00389] The term forage includes silage, compacted and pelleted feed, oils and mixed feed and also germinated grains and legumes.
    [00390] Forage can be obtained from one or more of the plants selected from: corn (green corn), alfalfa (lucerna), barley, gherkin, cabbage, Chau moellier, kale, rapeseed (canola), kale (turnip), turnip , clover, hybrid clover, red clover, subterranean clover, white clover, fescue, brome, millet, oats, sorghum, soybean, trees (pruned tree shoots), wheat and vegetables.
    [00391] The term "compound feed" means a commercial feed in the form of a meal, a pellet, nuts, cake or agglomerate. Compound feeds can be blended from various raw materials and additives. These mixtures are formulated according to the specific requirements of the target animal.
    Composite rations can be complete rations that provide all the required daily nutrients, concentrates that provide a portion of the ration (protein, energy) or supplements that provide only additional micronutrients such as minerals and vitamins.
    [00393] The main foods used in compound feeds are feed grains, which include corn, wheat, wheat bran, soybeans, sorghum, oats and barley.
    Suitably, a premix, as said herein, can be a composition comprised of microfoods, such as vitamins, minerals, chemical preservatives, antibiotics, fermentation products and other essential foods. Premixes are generally compositions suitable for blending into commercial feeds.
    [00395] Any food of the present invention may comprise one or more feed materials selected from the group comprising a) cereals, such as small grains (e.g. wheat, barley, rye, oats, triticale and combinations thereof) and/or large grains such as corn or sorghum; b) products derived from cereals, such as corn germ meal, corn meal, hominy ration, corn gluten meal, dry distillery grains with solubles (DDGS), dry distillery grains (DDG), gluten meal , wheat trimmings, wheat bran or combinations thereof; c) protein obtained from sources such as soy, sunflower, peanut, lupine, peas, broad beans, cotton, canola, fish meal, dry plasma protein, meat and bone meal, potato protein, whey, copra , Sesame; d) oils and fats obtained from vegetable and animal sources; e) minerals and vitamins.
    [00396] In one embodiment, the food comprises or consists of corn, DDGS (such as cDDGS), wheat, wheat bran or a combination thereof.
    [00397] In one embodiment, the feed component may be corn, DDGS (eg cDDGS), wheat, wheat bran or a combination thereof.
    [00398] In one embodiment, the food comprises or consists of corn, DDGS (such as cDDGS) or a combination thereof.
    [00399] In one embodiment, a feed component may be corn, DDGS (such as corn DDGS (cDDGS)) or a combination thereof.
    [00400] A food of the present invention may contain at least 30%, at least 40%, at least 50% or at least 60% by weight of corn and soybean bran or total corn and soybean fat or wheat bran or bran of sunflower.
    [00401] A feed of the present invention may contain between about 5 to about 40% of corn DDGS. For poultry - the feed can, on average, contain between about 7 to 12% of corn DDGS. For swine (pigs) - the feed can contain, on average, 5 to 40% of corn DDGS.
    [00402] A food of the present invention may contain corn as a single grain, in which case the food may comprise between about 35% to about 85% corn.
    [00403] In foods comprising mixed grains, for example comprising corn and wheat, for example, the food may comprise at least 10% corn.
    [00404] Additionally or alternatively, a feed of the present invention may comprise at least one fiber-rich feed material and/or at least one by-product of the at least one fiber-rich feed material to provide a fiber-rich feed. Examples of high fiber feed materials include: wheat, barley, rye, oats, cereal by-products such as corn gluten bran, wet cake, dry distillers grains (DDG), dry distillers grains with solubles (DDGS ), wheat bran, wheat semolina, wheat chips, rice bran, rice husk, oat husk, palm seed and citrus pulp. Some protein sources can also be considered as high in fiber: protein obtained from sources such as sunflower, lupine, broad bean and cotton.
    [00405] In one embodiment, the food of the present invention comprises at least one fiber-rich material and/or at least one by-product of the at least one fiber-rich feed material selected from the group consisting of dry distillery grains with solubles ( DDGS) - particularly corn DDGS (cDDGS), wet cake, dry distillery grains (DDG) - particularly corn DDG (cDDG), wheat and wheat bran, for example.
    [00406] In one embodiment, the food of the present invention comprises at least one fiber-rich material and/or at least one by-product of the at least one fiber-rich feed material selected from the group consisting of dry distillery grains with solubles ( DDGS) - particularly cDDGS, wheat and wheat bran, for example.
    [00407] In the present invention, the feed can be one or more of the following: a compound feed and premix, including pellets, nuts or cake (cattle); a crop or crop residues: corn, soybeans, sorghum, oats, barley, copra, straw, beet residues; Fish's flour; flesh and bone; molasses; bagasse and cake press; oligosaccharides; conserved forage plants: silage; seaweed; seeds and grains, whether whole or prepared by crushing, grinding, etc.; germinated cereals and legumes; Yeast extract.
    [00408] The term feed, in the present invention, also encompasses, in some embodiments, food for pets. A pet food is a plant or animal material intended for consumption by pets, such as dog food or cat food. Pet foods, such as dog and cat foods, can be either in a dry form, such as dog food, or in a wet canned form. Cat food may contain the amino acid taurine.
    [00409] The term feed, in the present invention, also encompasses, in some embodiments, food for fish. Fish food usually contains the macronutrients, trace elements and vitamins needed to keep captive fish in good health. Fish food can be in the form of a flake, pellet or tablet. Pellet shapes, some of which sink quickly, are often used for larger fish or bottom-feeding species. Some fish foods also contain additives, such as beta-carotene or sex hormones, to artificially increase the color of the ornamental fish.
    [00410] The term feed, in the present invention, also encompasses, in some embodiments, food for birds. Bird food includes food that is used both in aviaries and to feed pet birds. Typically, bird food comprises a variety of seeds, but it can also include tallow (mutton or mutton fat).
    [00411] As used herein, the term "contacted" refers to the direct or indirect application of the enzyme (or composition comprising the enzyme) of the present invention with the product (eg, feed). Examples of application methods that can be used include, but are not limited to, treating the product with a material comprising the additive composition, mixing by direct application of the feed additive composition with the product, spraying the feed additive composition onto the surface of the product. or soaking the product in a preparation of the feed additive composition.
    [00412] In one embodiment, the feed additive composition of the present invention is preferably mixed with the product (e.g., food). Alternatively, the additive composition can be included in the emulsion or raw material of a food.
    [00413] For some applications, it is important that the composition is available above or on the surface of a product to be affected/treated. This allows the composition to confer one or more of the following favorable characteristics: performance benefits.
    [00414] The enzyme (or composition comprising the enzyme) of the present invention can be applied to intersperse, coat and/or impregnate a product (e.g., food or feed material) with a controlled amount of said enzyme.
    Suitably, the additive composition may simply be administered to the individual at the same time as feeding the animal as a food.
    Preferably, the enzyme (or composition comprising the enzyme) of the present invention will be thermally stable to heat treatment up to about 70°C; up to about 85°C; or up to about 95 °C. The heat treatment can be carried out for up to about 1 minute; up to about 5 minutes; up to about 10 minutes; up to about 30 minutes; up to about 60 minutes. The term thermally stable means that at least about 75% of the enzyme that was present/active in the additive before heating to the specified temperature is still present/active after it cools to room temperature. Preferably, at least about 80% of the enzyme that is present and active in the additive prior to heating to the specified temperature is still present and active after cooling to room temperature.
    [00417] In a particularly preferred embodiment, the enzyme (or composition comprising the enzyme) of the present invention is homogenized to produce a powder.
    [00418] In an alternative preferred embodiment, the enzyme (or composition comprising the enzyme) of the present invention is formulated into granules as described in WO2007/044968 (referred to as TPT granules), incorporated herein by reference.
    [00419] In another preferred embodiment, when the feed additive composition is formulated into granules, the granules comprise a hydrated salt barrier coated over the protein core. The advantage of such a salt coating is to improve thermotolerance, improve storage stability and protect against other feed additives which would otherwise have an adverse effect on the enzyme.
    [00420] Preferably, the salt used for salt coating has an activity in water greater than 0.25 or constant humidity greater than 60% at 20 °C.
    [00421] Preferably, the salt coating comprises Na2SO4.
    [00422] The method of preparing an enzyme (or composition comprising the enzyme) of the present invention may also comprise the additional step of granulating the powder. The powder can be mixed with other components known in the art. The powder or a mixture comprising the powder can be forced through a die and the resulting ribbons cut into suitable pellets of varying length.
    [00423] Optionally, the granulation step can include a steam treatment, or conditioning phase, before forming the pellets. The mixture comprising the powder can be placed in a conditioner, for example a steam injection mixer. The mixture is heated in the conditioner to a specified temperature, such as 60 to 100 °C, typical temperatures being 70 °C, 80 °C, 85 °C, 90 °C or 95 °C. Residence time can range from seconds to minutes and even hours, such as 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minute 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes and 1 hour.
    [00424] It should be understood that the enzyme (or composition comprising the enzyme) of the present invention is suitable for addition to any suitable feed material.
    [00425] It will be understood by those skilled in the art that different animals require different foods and the same animal may need different foods, depending on the purpose for which the animal is raised.
    [00426] Optionally, the feed can also contain additional mineral salts such as, for example, additional calcium and/or vitamins.
    [00427] Preferably, the food is a mixture of soy flour and corn.
    [00428] In one modality, preferably, the kibble is not pet food.
    [00429] In another aspect, a method for producing a food is provided. Food is typically produced in feed mills where raw materials are first ground to a suitable particle size and then mixed with appropriate additives. The food can then be produced as a puree or pellets; the later step typically involves a method whereby the temperature is raised to a target level and then the feed is passed through a die to produce pellets of a particular size. Pellets are allowed to cool. Subsequently, liquid additives such as fat and enzymes can be added. Food production may also involve an additional step which includes extrusion or expansion before pelletizing - in particular by means of appropriate techniques which may include at least the use of steam.
    [00430] The food can be a food for a monogastric animal, such as poultry (for example, broilers, layers, broiler breeders, turkeys, ducks, geese, waterfowl), pigs (all age categories), a pet (eg dogs, cats) or fish; preferably the food is for birds.
    [00431] In one modality, the feed is not for a layer.
    [00432] By way of example only, a feed for chickens, for example, broilers, may comprise one or more of the ingredients listed in the table below, for example, in the % by age indicated in the table below:

    [00433] By way of example only, the diet specification for chickens, such as broilers, may be as shown in the table below:


    [00434] By way of example only, a feed for laying hens may comprise one or more of the ingredients listed in the table below, for example, in the % by age indicated in the table below:

    [00435] As an example only, the diet specification for laying hens can be as shown in the table below:


    [00436] By way of example only, a turkey food may comprise one or more of the ingredients listed in the table below, for example, in the % by age indicated in the table below:

    [00437] By way of example only, the diet specification for turkeys may be as shown in the table below:


    [00438] As an example only, a feed for piglets may comprise one or more of the ingredients listed in the table below, for example, in the % by age indicated in the table below:

    [00439] As an example only, the diet specification for piglets can be as shown in the table below:


    [00440] By way of example only, a growth/slaughter feed for pigs may comprise one or more of the ingredients listed in the table below, for example, in the % by age indicated in the table below:

    [00441] By way of example only, the specification of swine diet/slaughter for pigs can be as shown in the table below:

    MOIST CAKE, DRY DISTILLERY GRAINS (DDG) AND DRY DISTILLERY GRAINS WITH SOLUBLES (DDGS)
    [00442] Moist cake, dry distillery grains and dry distillery grains with solubles are products obtained after the removal of ethyl alcohol by means of distillation, from fermentation yeasts, of a grain or a mixture of grains through the methods employed in the grain distillation industry.
    [00443] The stillage from the distillation (eg comprising water, grain remnants, yeast cells, etc.) is separated into a "solid" part and a liquid part.
    [00444] The solid part is called "wet cake" and can be used as animal feed as such.
    [00445] The liquid part is (partially) evaporated into a syrup (soluble).
    [00446] When the wet mass is dry, it is dry distilleries grains (DDG).
    [00447] When the wet mass is dried together with the syrup(solubles) it is distillery grains dried with solubles (DDGS).
    [00448] The wet cake can be used in dairy operations and with confined beef cattle.
    [00449] Dry DDGS can be used in feed for farm animals (eg dairy, beef and pork) and feed for poultry.
    [00450] DDGS from corn is a very good source of protein for dairy cows. CORN GLUTEN BRAN
    [00451] In one aspect, the corn by-product may be corn gluten meal (CGM).
    [00452] CGM is a powdered by-product of the corn milling industry. CGM is useful, for example, in animal feed. It can be used as a low-cost protein source for feed such as pet food, livestock feed and poultry feed. It is an especially good source of the amino acid cysteine, but it must be balanced with other proteins such as lysine. FOOD ADDITIVE COMPOSITION
    The feed additive composition of the present invention and/or the food comprising the same may be used in any suitable form.
    [00454] The additive composition of the present invention can be used in the form of solid or liquid preparations or alternatives thereof. Examples of solid preparations include powders, pastes, boluses, capsules, lozenges, tablets, powders and granules which can be wettable, lyophilized or spray dried. Examples of liquid preparations include, but are not limited to, aqueous, organic or aqueous-organic solutions, suspensions and emulsions.
    [00455] In some applications, the feed additive compositions of the present invention may be mixed with food or administered in drinking water.
    [00456] In one aspect, the present invention relates to a method of preparing a feed additive composition comprising mixing a xylanase, a β-glucanase (and optionally at least one additional fiber degradation enzyme) and a DFM as taught herein with a vehicle, diluent or excipient for feed and (optionally) packaged. PREMIX
    [00457] The feed and/or food additive composition can be combined with at least one mineral and/or at least one vitamin. Compositions so derived may be referred to herein as a premix. FORMS
    [00458] The feed additive composition of the present invention and other components and/or the food comprising the same may be used in any suitable form.
    [00459] The additive composition of the present invention can be used in the form of solid or liquid preparations thereof. Examples of solid or liquid preparations or alternatives thereof. Examples of solid preparations include powders, pastes, boluses, capsules, lozenges, tablets, powders and granules which can be wettable, lyophilized or spray dried. Examples of liquid preparations include, but are not limited to, aqueous, organic or aqueous-organic solutions, suspensions and emulsions.
    In some applications, the DFM or feed additive compositions of the present invention may be mixed with food or administered in drinking water. In one embodiment, the dosage range for inclusion in water is from about 1x103 CFU/animal/day to about 1x1010 CFU/animal/day, and more preferably about 1x107 CFU/animal/day.
    [00461] Suitable examples of forms include one or more of: powders, pastes, boluses, pellets, tablets, pills, capsules, eggs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate, delayed release applications, modified, sustained, pulsed or controlled.
    [00462] By way of example, if the composition of the present invention is used in the form of a solid, for example, in pellet form, it may also contain one or more of the following: excipients such as microcrystalline cellulose, lactose, citrate sodium, calcium carbonate, dibasic calcium phosphate and glycine; disintegrants such as starch (preferably corn starch, potato starch or tapioca starch), sodium starch glycolate, sodium croscarmellose and certain complex silicates; granulation binders such as polyvinylpyrrolidone, hydroxy propyl methyl cellulose (HPMC), hydroxy propyl cellulose (HPC), sucrose, gelatin and gum acacia; lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
    [00463] Examples of nutritionally acceptable carriers for use in preparing the forms include, for example, water, saline solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose , magnesium stearate, talc, silicic acid, surfactants, viscous paraffin, aromatic oil, fatty acid monoglycerides and diglycerides, fatty acid esters, hydroxy methyl cellulose, polyvinylpyrrolidone and the like.
    Preferred excipients for the forms include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.
    [00465] For aqueous suspensions and/or elixirs, the composition of the present invention can be combined with various sweetening or flavoring agents, coloring matter or colorants, with emulsifying agents and/or suspending agents and with diluents such as water, propylene glycol and glycerin and combinations thereof.
    [00466] Non-hygroscopic whey is often used as a vehicle for DFMs (particularly bacterial DFMs) and is a good medium to initiate growth.
    [00467] Pastes containing bacterial DFMs can be formulated with a vegetable oil and inert gelling ingredients.
    [00468] Fungal products can be formulated with grain by-products as vehicles.
    [00469] In one embodiment, preferably, the feed additive composition according to the present invention is not in the form of a microparticle system, such as the microparticle system taught in WO2005/123034. DOSAGE
    [00470] The DFM and/or feed additive composition according to the present invention can be designed for a single dosage or it can be designed for feeding on a daily basis.
    [00471] The optimal amount of the composition (and each component therein) to be used in the combination of the present invention will depend on the product to be treated and/or the method of contact of the product with the composition and/or intended use for it.
    [00472] The amount of DFM and enzymes used in the compositions must be an amount sufficient to be effective and to remain sufficiently effective in improving the performance of animals fed with feed products containing said composition. This period of time for effectiveness should extend to at least the time of use of the product (for example, feed additive composition or feed containing the same). COMBINATION WITH OTHER COMPONENTS
    [00473] The DFM and enzyme(s) for use in the present invention can be used in combination with other components. Thus, the present invention also relates to combinations. DFM in combination with the xylanase and a β-glucanase (and optionally at least one additional fiber-degrading enzyme) can be referred to herein as "the feed additive composition of the present invention".
    [00474] In a preferred embodiment, "the feed additive composition of the present invention" may comprise (or consist essentially of or consist of) the DFM in combination with the xylanase and a β-glucanase and an additional fiber-degrading enzyme, as taught herein (e.g. suitably at least two, suitably at least three additional fiber-degrading enzymes).
    [00475] In another preferred embodiment, "the feed additive composition of the present invention" may comprise (or consist essentially of or consist of) the DFM in combination with the xylanase and a β-glucanase and an additional fiber-degrading enzyme, as taught herein (e.g. suitably at least four, suitably at least five additional fiber-degrading enzymes).
    [00476] The combination of the present invention comprises the feed additive composition of the present invention (or one or more of its constituents) and another component which is suitable for animal consumption and capable of conferring a medical or physiological benefit to the consumer.
    [00477] In one embodiment, preferably, the "other component" is not another enzyme or another DFM.
    [00478] The components can be prebiotics. Prebiotics are typically non-digestible carbohydrates (oligo- or polysaccharides) or a sugar alcohol that is not degraded or absorbed in the upper digestive tract. Known prebiotics used in commercial products and useful in accordance with the present invention include inulin (fructooligosaccharides or FOS) and transgalactooligosaccharides (GOS or TOS). Suitable prebiotics include palatinose oligosaccharides, soy oligosaccharides, alginate, xanthan, pectin, locust bean gum (LBG), inulin, guar gum, galactooligosaccharide (GOS), fructooligosaccharide (FOS), non-degradable starch, lactosucrose, lactulose, lactitol, maltitol, maltodextrin, polydextrose (i.e. Litesse®), lactitol, lactosucrose, soy oligosaccharides, palatinose, isomalto-oligosaccharides, gluco-oligosaccharides and xylo-oligosaccharides.
    [00479] In one embodiment, the present invention relates to the combination of the feed additive composition according to the present invention (or one or more of the constituents thereof) with a prebiotic. In another embodiment, the present invention relates to an additive composition comprising (or consisting essentially of or consisting of) a DFM in combination with a xylanase, a β-glucanase, an amylase, a phytase, a protease and a prebiotic.
    [00480] The prebiotic can be administered simultaneously with (for example, mixed together with or distributed simultaneously by the same or different routes) or sequentially with (for example, by the same or different routes) the additive composition (or constituents thereof) according to the present invention.
    [00481] Other components of the combinations of the present invention include polydextrose, such as Litesse®, and/or a maltodextrin and/or lactitol. These other components can optionally be added to the additive composition to aid the drying process and aid in the survival of the DFM.
    [00482] Other examples of other suitable components include one or more of the following: thickening agents, gelling agents, emulsifiers, binders, crystal modifiers, sweeteners (including artificial sweeteners), rheology modifiers, stabilizers, antioxidants, dyes, enzymes, carriers, vehicles, excipients, diluents, lubricating agents, flavoring agents, colorants, suspending agents, disintegrating agents, granulation binders, etc. These other components can be natural. These other components can be prepared using chemical and/or enzymatic techniques.
    [00483] In one embodiment, DFM and/or enzymes can be encapsulated. In one embodiment, the feed additive composition and/or DFM and/or enzymes is/are formulated as a dry powder or granules, as described in WO2007/044968 (referred to as TPT granules) - which is incorporated herein by reference.
    [00484] In a preferred embodiment, the DFM and/or enzymes for use in the present invention can be used in combination with one or more lipids.
    [00485] For example, the DFM and/or enzymes for use in the present invention can be used in combination with one or more lipid micelles. The lipid micelle can be a simple lipid micelle or a complex lipid micelle.
    [00486] The lipid micelle can be an aggregate of oriented molecules of amphipathic substances, such as a lipid and/or an oil.
    [00487] As used herein, the term "thickener or gelling agent" refers to a product that prevents separation by slowing or preventing the circulation of particles, whether droplets of immiscible liquids, air, or insoluble solids. Thickening occurs when hydrated individual molecules cause an increase in viscosity, delaying separation. Gelation occurs when hydrated molecules bind to form a three-dimensional network that holds the particles, thereby immobilizing them.
    [00488] The term "stabilizer", as used herein, is defined as an ingredient or combination of ingredients that prevents a product (eg, a feed product) from changing over time.
    [00489] The term "emulsifier", as used herein, refers to an ingredient (eg, a feed ingredient) that prevents the separation of emulsions. Emulsions are two immiscible substances, one present in the form of droplets, contained within the other. Emulsions can consist of water-in-oil, where the droplet or dispersed phase is oil and the continuous phase is water; or water-in-oil, where water becomes the dispersed phase and the continuous phase is oil. Foams, which are gas-in-liquid, and suspensions, which are solid-in-liquid, can also be stabilized through the use of emulsifiers.
    [00490] As used herein, the term "binder" refers to an ingredient (eg, a feed ingredient) that binds the product together through a physical or chemical reaction. During "gelling", for example, water is absorbed, providing a binding effect. However, binders can absorb other liquids, such as oils, keeping them inside the product. In the context of the present invention, binders would typically be used in solid state or low moisture products, for example for bakery products: cakes, donuts, bread and others.
    [00491] "Carriers" or "vehicles" means materials suitable for administering DFM and/or enzymes and include any such materials known in the art such as, for example, any liquid, gel, solvent, liquid diluent, solubilizer or the like that it is non-toxic and does not interact with any components of the composition in a deleterious way.
    [00492] In one embodiment, the feed, premix, feed or feed additive composition of the present invention may be mixed with at least one physiologically acceptable carrier selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or a wheat component, sucrose, starch, Na2SO4, talc, PVA, sorbitol, benzoate, sorbitate, glycerol, sucrose, propylene glycol, 1,3-propane diol, glucose, parabens, sodium chloride, citrate, acetate, phosphate , calcium, metabisulfite, formate and mixtures thereof.
    [00493] Examples of excipients include one or more of the following: microcrystalline cellulose and other celluloses, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine, starch, milk sugar and high molecular weight polyethylene glycols.
    [00494] Examples of disintegrants include one or more of the following: starch (preferably corn starch, potato starch or tapioca starch), sodium starch glycolate, sodium croscarmellose and certain complex silicates.
    [00495] Examples of granulation binders include one or more of the following: polyvinylpyrrolidone, hydroxy propyl methyl cellulose (HPMC), hydroxy propyl cellulose (HPC), sucrose, maltose, gelatin and acacia.
    [00496] Examples of lubricating agents include one or more of the following: magnesium stearate, stearic acid, glyceryl behenate and talc.
    [00497] Examples of suitable diluents include one or more of the following: water, ethanol, propylene glycol and glycerin and combinations thereof.
    [00498] The other components can be used simultaneously (eg when they are mixed together or even when they are distributed by different routes) or sequentially (eg they may be administered by different routes).
    [00499] Preferably, when the feed additive composition of the present invention is mixed with other component(s), DFM remains viable.
    [00500] In one embodiment, preferably, the feed additive composition according to the present invention does not comprise chromium or organic chromium.
    [00501] In one embodiment, preferably, the additive according to the present invention does not contain sorbic acid. CONCENTRATES
    [00502] DFMs for use in the present invention may be in the form of concentrates. Typically, these concentrates comprise a substantially high concentration of a DFM.
    [00503] Feed additive compositions according to the present invention may have a viable cell content (colony forming units, CFU) that is in the range of at least 104 CFU/g (suitably including at least 105 CFU/g, such as at least 106 CFU/g, for example at least 107 CFU/g, at least 108 CFU/g, such as at least 109 CFU/g) to about 1010 CFU/g (or even about 1011 CFU/g). g or about 1012 CFU/g).
    [00504] When DFM is in the form of a concentrate, the feed additive compositions according to the present invention may have a viable cell content in the range of at least 109 CFU/g to about 1012 CFU/g, preferably by minus 1010 CFU/g to about 1012 CFU/g.
    [00505] Powders, granules and liquid compositions in the form of concentrates may be diluted with water or resuspended in water or other suitable diluents, for example a suitable growth medium such as milk or mineral or vegetable oils, to obtain compositions ready to use.
    The DFM or feed additive composition of the present invention or the combinations of the present invention in the form of concentrates can be prepared according to methods known in the art.
    [00507] In one aspect of the present invention, the enzyme or feed is contacted by a composition in a concentrated form.
    The compositions of the present invention can be lyophilized or spray dried by methods known in the art.
    [00509] Typical processes for producing particles using a spray drying process involve a solid material that is dissolved in an appropriate solvent (eg a culture of a DFM in a fermentation medium). Alternatively, the material can be suspended or emulsified in a non-solvent to form a suspension or emulsion. Other ingredients (as discussed above) or components, such as antimicrobial agents, stabilizing agents, dyes and drying process aids, may optionally be added at this stage.
    [00510] The solution is then atomized to form a fine mist of droplets. The droplets immediately enter a drying chamber where they contact a drying gas. Solvent is evaporated from the droplets in the drying gas to solidify the droplets, thereby forming particles. The particles are then separated from the drying gas and collected. INDIVIDUAL
    The term "subject", as used herein, means an animal to which an additive composition according to the present invention is being or has been administered or a food comprising said additive composition according to the present invention.
    [00512] The term "individual", as used herein, means an animal. Preferably, the individual is a mammal, bird, fish or crustacean including, for example, cattle or a domesticated animal (for example, a pet).
    [00513] In one modality, the "individual" is a farm animal.
    [00514] The term "farmed animal", as used herein, refers to any farm animal. Preferably, the farm animal is one or more of cows or bulls (including calves), poultry, pigs (including piglets), birds (including chickens, chickens and turkeys), birds, fish (including freshwater fish such as salmon, cod, trout and carp, for example koi carp and marine fish such as sea bass), crustaceans (such as shrimp, mussels and scallops), horses (including racehorses), sheep (including lambs).
    [00515] In one modality, the term farm animal and/or birds and/or chickens does not include laying hens.
    [00516] In another embodiment, the "individual" is a domesticated animal or a pet or kept in a zoo environment.
    [00517] The term "pet or domesticated animals or animals kept in a zoo environment", as used herein, refers to any animal, including relevant canines (eg dogs), felines (eg cats), rodents (eg, guinea pigs, rats, mice), birds, fish (including freshwater fish and marine fish) and horses. PRODUCTION OF SHORT CHAIN FATTY ACIDS (SCFA)
    [00518] The term "short chain fatty acid", as used herein, includes volatile fatty acids as well as lactic acid. In one embodiment, SCFA can be selected from the group consisting of: acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, 2-methylbutyric acid and lactic acid, preferably propionic acid and/or butyric acid .
    [00519] In one embodiment, the SCFA can be butyric acid and/or propionic acid.
    [00520] Short chain fatty acids (particularly volatile fatty acids, eg propionic acid and butyric acid and lactic acid) can be analyzed using the following method.
    [00521] Chromatographic analysis of volatile fatty acids and lactic acid, eg SCFA, to be performed from simulation samples with pivalic acid as an internal standard, as previously described (Ouwehand et al., February 2009; 101 (3 ): 367-75). Concentrations of acetic, propionic, butyric, isobutyric, valeric, isovaleric, 2-methylbutyric acid and lactic acid are determined. PERFORMANCE
    [00522] As used herein, "animal performance" can be determined by feed efficiency and/or animal weight gain and/or feed conversion ratio and/or nutrient digestibility in a feed (e.g., digestibility of amino acids) and/or digestible energy and metabolizable energy in a feed and/or by nitrogen retention.
    [00523] Preferably, "animal performance" is determined by feed efficiency and/or animal weight gain and/or feed conversion.
    [00524] By "improved animal performance" is meant that there is an increase in feed efficiency and/or increased weight gain and/or reduced feed conversion ratio and/or an improvement in the digestibility of nutrients or energy in a feed and/or an improvement in nitrogen retention arising from the use of the additive composition of the present invention in feed compared to a feed which does not comprise said feed additive composition.
    [00525] Preferably, by "improved animal performance" is meant that there is an increase in feed efficiency and/or increased weight gain and/or reduced feed conversion ratio.
    [00526] As used herein, the term "feed efficiency" refers to the amount of weight gain in an animal that occurs when the animal is fed ad libitum or a specified amount of feed over a period of time.
    [00527] By "increase in feed efficiency" is meant that the use of an additive composition according to the present invention in the feed results in an increase in weight gain per unit of food consumption compared to an animal without the said feed additive composition is present. FOOD CONVERSION RATE (FCR)
    [00528] As used herein, the term "feed conversion ratio" refers to the amount of feed fed to an animal to increase the animal's weight by a specified amount.
    [00529] An improved feed conversion ratio means a reduced feed conversion ratio.
    [00530] By "reduced feed conversion rate" or "improved feed conversion rate" is meant that the use of a feed additive composition results in a smaller amount of feed needed by an animal to increase the animal's weight by a specified amount compared to the amount of feed required to increase the animal's weight by the same amount when the feed does not include said feed additive composition. NUTRIENT DIGESTIBILITY
    Nutrient digestibility, as used herein, means the fraction of a nutrient that disappears from the gastrointestinal tract or a specified segment of the gastrointestinal tract, eg, the small intestine. Nutrient digestibility can be measured as the difference between what is given to the individual and what comes out in the individual's feces or between what is given to the individual and what is left in the digesta in a specified segment of the gastrointestinal tract, for example, the ileum.
    [00532] Nutrient digestibility, as used here, can be measured by the difference between the consumption of a nutrient and the nutrient excreted through total excretion collection over a period of time; or using an inert marker that is not absorbed by the animal and allows the researcher to calculate the amount of nutrients that have disappeared from the entire gastrointestinal tract or a segment of the gastrointestinal tract. Such an inert marker can be titanium dioxide, chromium oxide or acid-insoluble ash. Digestibility can be expressed as a percentage of nutrient in the ration or as mass units of digestible nutrient per mass of nutrient in the ration.
    [00533] Nutrient digestibility, as used herein, encompasses starch digestibility, fat digestibility, protein digestibility, and amino acid digestibility.
    [00534] Energy digestibility, as used herein, means the gross feed energy consumed minus the gross stool energy or the total feed energy consumed minus the gross energy of the digesta remaining in a specified segment of the animal's gastrointestinal tract, for example , Hi Leo. Metabolizable energy, as used here, refers to apparent metabolizable energy and means the gross energy of the feed consumed minus the gross energy contained in faeces, urine, and gaseous products of digestion. Energy digestibility and metabolizable energy can be measured as the difference between gross energy intake and gross energy excreted in feces or the digesta present in the specified segment of the gastrointestinal tract using the same methods as for measuring nutrient digestibility, with appropriate corrections for nitrogen excretion to calculate the metabolizable energy of feed. NITROGEN RETENTION
    [00535] Nitrogen retention, as used herein, means the individual's ability to retain nitrogen from the diet as body mass. A negative nitrogen balance occurs when nitrogen excretion exceeds daily consumption and is often observed when muscle mass is being lost. A positive nitrogen balance is often associated with muscle growth, particularly in growing animals.
    [00536] Nitrogen retention can be measured as the difference between nitrogen input and nitrogen excreted through total collection of excreta and urine over a period of time. It is to be understood here that nitrogen excreted includes undigested protein from the feed, endogenous proteinaceous secretions, microbial protein and urinary nitrogen. CARCASS INCOME AND MEAT INCOME
    [00537] The term carcass yield, as used herein, means the amount of carcass as a proportion of live body weight after a commercial or experimental slaughter process. The term carcass means the body of an animal that has been slaughtered for food, such as the head, viscera, part of the limbs and the feathers or skin removed. The term meat yield, as used herein, means the amount of edible meat as a percentage of live body weight or the amount of a beef meat specified as a proportion of live body weight. WEIGHT GAIN
    [00538] The present invention further provides a method for increasing weight gain in an individual, for example poultry or swine, comprising feeding to said individual a feed comprising an additive composition according to the present invention.
    [00539] An "increase in weight" refers to an animal having increasing body weight which is being fed a feed comprising a feed additive composition compared to an animal being fed a feed without said additive composition be present. OTHER PROPERTIES
    [00540] In one embodiment, the additive composition for action, feed, food or method according to the present invention may not modulate (e.g. enhance) the individual's immune response.
    [00541] In another embodiment, the feed, feed, food or method additive composition according to the present invention may not improve the survival (e.g., reduce mortality) of the individual.
    [00542] In a preferred embodiment, the feed, feed, feed or method additive composition according to the present invention may not modulate (e.g., enhance) the immune response or enhance the survival (e.g., reduce mortality) of the individual. PROBIOTIC
    [00543] For some applications, it is believed that the DFM in the composition of the present invention may exert a probiotic culture effect. It is also within the scope of the present invention to add to the composition of the present invention another probiotic and/or prebiotics.
    [00544] Here, a prebiotic is:
    [00545] "a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of beneficial bacteria".
    [00546] The term "probiotic culture", as used herein, defines live microorganisms (including bacteria or yeast, for example) which, for example, when ingested or applied locally in sufficient numbers, beneficially affect the host organism, or that is, they confer one or more demonstrable health benefits on the host organism. Probiotics can improve microbial balance on one or more mucosal surfaces. For example, the mucosal surface can be the intestine, urinary tract, respiratory tract, or skin. Although there are no minimum and maximum limits for consumption of probiotics, it has been suggested that at least 106-1012, preferably at least 106-1010, preferably 108-109 CFU as a daily dose will be effective to achieve beneficial health effects in an individual. ISOLATED
    [00547] In one aspect, preferably, the enzyme used in the present invention is in isolated form. The term "isolated" means that the enzyme is at least substantially free of at least one other component with which the enzyme is naturally associated in nature and as found in nature. The enzyme of the present invention can be provided in a form that is substantially free of one or more contaminants with which the substance might otherwise be associated. Thus, for example, it can be substantially free of one or more potentially contaminating polypeptides and/or nucleic acid molecules. PURIFIED
    [00548] In one aspect, preferably the enzyme and/or the DFM according to the present invention is in a purified form. The term "purified" means that the enzyme and/or DFM is present at an elevated level. The enzyme and/or DFM is desirably the predominant component present in a composition. Preferably, it is present at a level of at least about 90% or at least about 95% or at least about 98%, said level being determined on a dry weight/weight basis with respect to the total composition in question. .
    It is considered, within the scope of the present invention, that embodiments of the invention may be combined in such a way that combinations of any of the features described herein are included within the scope of the present invention. In particular, it is considered within the scope of the present invention that any of the therapeutic effects of the bacteria can be exhibited concomitantly. AMINO ACID SEQUENCES
    The scope of the present invention also encompasses enzyme amino acid sequences that have the specific properties as defined herein.
    As used herein, the term "sequence of amino acids" is synonymous with the term "polypeptide" and/or the term "protein". In some cases, the term "sequence of amino acids" is synonymous with the term "peptide". In some cases, the term "sequence of amino acids" is synonymous with the term "enzyme".
    The amino acid sequence can be prepared/isolated from a suitable source or it can be produced synthetically or it can be prepared using recombinant DNA techniques.
    Preferably, the amino acid sequence, when referring to and when falling within the scope of the present invention per se, is not a native enzyme. In this regard, the term "native enzyme" means an entire enzyme that is in its native environment and when it has been expressed by its native nucleotide sequence.
    [00554] SEQUENCE IDENTITY OR SEQUENCE HOMOLOGY
    [00555] The present invention also encompasses the use of sequences that have a degree of sequence identity or sequence homology with the amino acid sequence of a polypeptide having the specific properties defined herein or any nucleotide sequence encoding such a polypeptide (hereinafter referred to as "(a" homologous sequence(s)"). Here, the term "homologous" means an entity that has a certain homology to the amino acid sequences in question and the nucleotide sequences in question. term "homology" can be equated with "identity".
    [00556] The homologous amino acid sequence and/or nucleotide sequence must provide and/or encode a polypeptide which retains the functional activity and/or increases the activity of the enzyme.
    [00557] The term "nucleotide sequence", in relation to the present invention, includes genomic DNA, cDNA, synthetic DNA and RNA. Preferably, it means DNA, more preferably the cDNA sequence which encodes the sequence of the present invention.
    [00558] In the present context, in some embodiments, a homologous sequence is taken to include an amino acid or nucleotide sequence which may be at least 97% identical, preferably at least 98 or 99% identical to the sequence in question.
    [00559] In some embodiments, a homologous sequence is said to include an amino acid or nucleotide sequence that may be at least 85% identical, preferably at least 90 or 95% identical to the sequence in question.
    [00560] Typically, homologues will comprise the same active sites, etc. than the amino acid sequence in question, for example. While homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention, it is preferred to express homology in terms of sequence identity.
    In one embodiment, a homologous sequence is made to include an amino acid sequence or a nucleotide sequence that has one or more additions, deletions and/or substitutions compared to the sequence in question.
    [00562] In one embodiment, the present invention relates to a protein whose amino acid sequence is represented herein or a protein derived from this (parent) protein by substitution, deletion or addition of one or more amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9 amino acids or more amino acids, such as 10 or more than 10 amino acids, in the parent protein amino acid sequence and having the activity of the parent protein.
    Typically, homologs will comprise the same sequences encoding active sites, etc., as the target sequence. While homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention, it is preferred to express homology in terms of sequence identity.
    [00564] Homology comparisons can be performed with the naked eye or, more generally, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
    [00565] The % homology can be calculated over contiguous sequences, that is, one sequence is aligned with another sequence and each of the amino acids in one sequence is directly compared to the corresponding amino acid in the other sequence, one residue of each turn. This is called a "no gap" alignment. Typically, such gap-free alignments are performed only over a relatively short number of residues.
    [00566] Although this is a very simple and consistent method, it fails to take into account that, for example, in an otherwise identical sequence pair, an insertion or deletion will cause the following amino acid residues to be placed out of alignment , thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take possible insertions and deletions into account, without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximize local homology.
    [00567] However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting superior relatedness between the two sequences compared - you will get a higher score than one with many gaps. "Affine gap costs" are commonly used, which charge a relatively high cost for the existence of a gap and a smaller penalty for each residue after the gap. This is the most commonly used gap scoring system. High gap penalties will, of course, produce optimized alignments with fewer gaps. Most alignment programs allow gap penalties to be modified. However, it is preferred to use the predefined values when using this software for sequence comparisons.
    [00568] Calculating the maximum % homology, therefore, first requires the production of an optimal alignment taking into account gap penalties. A suitable computer program for performing such an alignment is the Vector NTI (Invitrogen Corp.). Examples of software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. 1999 Short Protocols in Molecular Biology, 4th Ed - Chapter 18), BLAST 2 (see FEMS Microbiol Lett 1999, 174 (2 ): 247-50; FEMS Microbiol Lett 1999, 177 (1): 187-8 and tatiana@ncbi.nlm.nih.gov), FASTA (Altschul et al. 1990 J. Mol Biol: 403-410) and AlignX, for example. At least BLAST, BLAST 2 and FASTA are available for offline and online search (see Ausubel et al. 1999, pages 7-58 to 7-60).
    [00569] Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used, which assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is the BLOSUM62 matrix - the default matrix for the BLAST program package. Vector NTI programs generally use public default values or a custom symbol comparison table, if provided (see the user manual for details). For some applications, it is preferable to use default values for the Vector NTI package.
    [00570] Alternatively, the percent homologies can be calculated using the multiple alignment function of the Vector NTI (Invitrogen Corp.), based on an algorithm, in a manner analogous to CLUSTAL (Higgins DG & a Sharp PM (1988), Gene 73 (1), 237-244).
    [00571] Once the software has produced an optimal alignment, it is possible to calculate the % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
    [00572] Gap penalties should be used to determine sequence identity, so preferably, the following parameters are used for pairwise alignment:

    [00573] In a modality, CLUSTAL may be used with the gap penalty and gap extension defined as defined above.
    Suitably, the degree of identity with respect to an amino acid sequence is determined over at least 20 contiguous amino acid residues, preferably over at least 30 contiguous residues, preferably over at least 40 contiguous residues preferably over at least 50 contiguous residues, preferably over at least 60 contiguous residues, preferably over at least 100 contiguous residues.
    Suitably, the degree of identity with respect to the amino acid sequence can be determined across the entire sequence taught herein.
    [00576] The sequences may also have deletions, insertions or substitutions of amino acid residues that produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphiphatic nature of the residues, as long as the secondary binding activity of the substance is maintained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine and tyrosine.
    [00577] Conservative substitutions can be made, for example, in accordance with the Table below. Amino acids in the same block in the second column, preferably in the same row in the third column, can be substituted for each other:

    [00578] The present invention also encompasses homologous substitution (substitution and alteration are both used herein to signify the exchange of an existing amino acid residue for an alternative residue) which can occur, for example, equivalent substitution, such as basic with basic, acid by acid, polar by polar, etc. Non-homologous substitution can also occur, ie, from one residue class to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as as B), ornithine norleucine (hereinafter referred to as O), pyridylalanine, thienylalanine, naphthylalanine and phenylglycine.
    [00579] Substitutions can also be made for unnatural amino acids, including; *alpha and alpha-disubstituted amino acids*, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*, pI-phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-amino butyric acid*, Ly-amino butyric acid*, L-α-amino isobutyric acid*, L-ε-amino caproic acid#, 7-amino acid heptanoic*, L-methionine sulfone#*, L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L-hydroxyproline#, L-thioproline*, methyl derivatives of phenylalanine (Phe), such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino)#, L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic (1,2,3 acid ,4-tetrahydroisoquinoline-3-carboxylic acid)*, L-diaminopropionic acid # and L-Phe (4-benzyl)*. The notation * was used for the purposes of the above discussion (relating to homologous or non-homologous substitution), to indicate the hydrophobic nature of the derivative, while # was used to indicate the hydrophilic nature of the derivative, #* indicates amphiphatic characteristics.
    [00580] Variant amino acid sequences can include suitable spacer groups that can be inserted between any two amino acid residues in the sequence, including alkyl groups such as methyl, ethyl or propyl groups, in addition to amino acid spacers such as glycine or β residues -alanine. Another form of variation, which involves the presence of one or more amino acid residues in the peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, "peptoid form" is used to refer to variant amino acid residues where the α-carbon substituent group is on the residue's nitrogen atom instead of the α-carbon. Processes for preparing peptides in peptoid form are known in the art, for example, Simon R.J. et al., PNAS (1992) 89 (20), 9367-9371 and Horwell D.C., Trends Biotechnol. (1995) 13 (4), 132-134.
    [00581] In one embodiment, the xylanase for use in the present invention may comprise a polypeptide sequence herein with a conservative substitution of at least one of the amino acids.
    Suitably, there may be at least 2 conservative substitutions, such as at least 3 or at least 4 or at least 5.
    [00583] Suitably there may be less than 15 conservative substitutions, for example less than 12, less than 10 or less than 8 or less than 5. EXAMPLES Example 1: Responses of broilers fed wheat-based diets containing xylanase, β-glucanase and live microorganisms fed directly MATERIAL AND METHODS
    [00584] The use of animals and experimental protocol has been approved by the Institutional Animal Experiment Committee. The diet was formulated to be energy and nutrient balanced for broilers (0-21 days old) (Table 1, Diet I). The cereal component of the diet was wheat, barley, rye, wheat bran, wheat bran or combinations thereof, while the protein component was soy bran and the fat source was rapeseed oil. No synthetic antimicrobials or anticoccidial drugs were included and the diet was provided as a puree. The base diet was divided into portions and the respective enzymes and DFMs added to constitute experimental diets identified in Table 2.
    [00585] Each supplement was supplied in a premix and added to the blender during diet preparation. Diets containing the DFM were mixed first and the blender was discharged between each diet to avoid cross contamination. Samples were collected from each treatment diet from the beginning, middle and end of each batch and mixed together to confirm enzyme activities and the presence of DFM in the feed before starting the animal experiment. Additional samples from each treatment diet were retained and stored until needed at a temperature of -20 °C ± 2 °C for analysis.
    [00586] Male Ross broilers (308) were obtained as day-old chicks from a commercial hatchery. The chicks were individually weighed and placed in 32 brooder cages (8 birds per cage) so that the average weight of birds per cage was similar. The four treatments (Table 2) were then randomly divided into eight cages each. On day 12, birds were transferred to growth cages. The space allocation per bird in the brooder and growth cages was 530 and 640 cm2, respectively. The brooder and growth cages were installed in acclimatized chambers. The temperature was maintained at 31 °C in the first week and then gradually reduced to 22 °C until the end of the third week. The birds received 20 hours of fluorescent lighting and free access to diet and water. Diets were fed from d 0 to 21. Body weights were recorded at weekly intervals throughout the 21 d experimental period. Mortality was recorded daily. Data were analyzed using the SAS GLM procedure.


    [00587] 1The enzymes (xylanase (Danisco Xylanase, an endo-1,4-β-D-xylanase (E.C. 3.2.1.8)) and β-glucanase Axtra®XB) are commercial products supplied by Danisco Animal Nutrition.
    [00588] 2A live microorganism fed directly based on three Bacillus strains selected for the ability to secrete enzymes supplied by Danisco Animal Nutrition in equal proportions of strains AGTP BS918 (NRRL B-50508), AGTP BS3BP5 (NRRL B-50510) and AGTP BS1013 (NRRL B-50509). RESULTS Table 3: Effects of xylanase, β-glucanase and a live micro-organism fed directly based on bacilli on the productive performance of a young broiler


    [00589] N.B. Different letters after the values show statistical differences (P<0.10) between values in this column.
    [00590] Birds fed a combination of xylanase, the fiber-degrading enzyme (β-glucanase) and a bacillus-based DFM grew faster than the control and numerically better than birds fed diets with enzymes only. Body weight at 21 days and body weight gain were numerically better in animals fed three combinations of xylanase, β-glucanase and DFM compared to control.II. NUTRIENTS AND ENERGY RETENTION/MATERIAL DIGESTIBILITY AND METHODS
    [00591] The use of animals and experimental protocol has been approved by the Institutional Animal Experiment Committee. A wheat-barley based diet was formulated to be energy and nutrient balanced for broilers (0-21 days of age) (Table 1, Diet II). Titanium dioxide was included at 0.30% to allow determination of dietary component retention. No synthetic antimicrobials or anticoccidial drugs were included and the diet was provided as a puree. The base diet was divided into portions and the respective enzymes and DFMs added to constitute experimental diets identified in Table 4. Each supplement was pre-mixed and the mixer was washed to avoid cross-contamination of the treated diets. Samples were collected from each treatment diet from the beginning, middle and end of each batch and mixed together to confirm enzyme activities and the presence of DFM in the feed before starting the animal experiment. Additional samples from each treatment diet are retained and stored until needed at -20 °C ± 2 °C for analysis. Table 4: Identification of treatments

    [00592] 1The enzymes (xylanase (Danisco Xylanase, an endo-1,4-β-D-xylanase (E.C. 3.2.1.8)) and β-glucanase Axtra®XB) are commercial products supplied by Danisco Animal Nutrition.
    [00593] 2A live microorganism fed directly based on three Bacillus strains selected for the ability to secrete enzymes supplied by Danisco Animal Nutrition in equal proportions of strains AGTP BS918 (NRRL B-50508), AGTP BS3BP5 (NRRL B-50510) and AGTP BS1013 (NRRL B-50509).
    [00594] The study involved a cage experiment, which was performed to obtain fecal samples for measurements of energy and nutrient digestibility. Day-old male broiler chicks (Ross 308) were obtained from a commercial hatchery. The chicks were individually weighed on arrival and stratified by body weight and distributed into 30 cages (five birds per cage) so that the average weight of birds per cage was similar. The four dietary treatments were then randomly divided into six identical cages. The study was conducted from day 0 to 21, during which the birds had free access to their assigned dietary treatments and water. The brooder and ambient temperatures were fixed at 32 and 29 ◦C, respectively, during the first week. After that, the heat supply in the brooder was turned off and room temperature was maintained at 29 ◦C throughout the experiment. Light was provided for 24 h throughout the experiment. On days 17, 18, 19 and 20, excreta samples were collected and stored frozen at -20 ◦C for determination of energy and nutrient retention/digestibility. Care was taken during collection of faecal samples to avoid contamination from feathers and other foreign materials. Faecal samples were pooled within a well-mixed cage using a blender and two representative samples per cage were taken. Samples were lyophilized. The dried samples were ground to pass through a 0.5 mm sieve and stored in airtight plastic containers at -4 °C until chemical analysis. Diet and faecal samples were analyzed for dry matter, crude protein (as nitrogen), gross energy, fat (as hexane extracts) and neutral detergent fiber according to official AOAC analysis methods. Titanium (digestibility marker) was analyzed according to the procedures described by Lomer et al. (2000, Analyst 125: 23392343). Retention/digestibility was calculated using standard procedures (Adeola, O. 2001. Digestion and Balance Techniques in Pigs. Pages 903-916 in Swine Nutrition, 2nd ed. AJ Lewis and LL Southern, ed. CRC Press, Washington, DC). Data were analyzed using the SAS (2004) General Linear Models procedure. RESULTS Table 5: Effects of xylanase, fiber-degrading enzyme and a live microorganism fed directly on bacilli on nutrient retention/digestibility and metabolizable energy in young broiler chickens.


    [00595] N.B. Different letters after the values show statistical differences (P<0.10) between values in this column.
    [00596] A combination of xylanase, β-glucanase and live microorganism fed directly based on bacilli improved the dietary energy utilization of young broilers when compared to bacilli, control or xylanase alone or a combination of xylanase and β-glucanase (Table 5). This could be related to increased retention of energy nutrients such as fiber, fat and nitrogen (Table 5). The increased fat retention due to the three-component combinations is notable and could be related to better digestion and absorption of dietary fat as well as the production and absorption of short-chain fatty acids from fermentation. The observed benefits of the three-component combination of xylanase, β-glucanase, bacillus/propionic DFM that improve energy and nutrient use can also be speculatively associated with improved intestinal health and function through positive modulation of microbiota and absorption function /digestive of the intestine.III. PRODUCTION OF LACTIC ACID IN THE CECUM MATERIALS AND METHODS In vitro simulation of chicken cecum
    [00597] A chicken cecum model was developed from a previously described in vitro human colon system (Makivuokko et al. 2006; Nutrition and Cancer 52:94-104, Makelainen et al. 2009; International Dairy Journal 19: 675-683). This in vitro cecum model is comprised of four connected vessels inoculated with fresh cecal microbes. A wheat-whole wheat bran-based diet was formulated to be energy and nutrient balanced for young broilers (Table 1, Diet III). No synthetic antimicrobials or anticoccidials were included in the baseline diet. The basic diet was divided into portions and the respective enzymes and DFMs added to constitute experimental diets identified in Table 6. The different rations were subjected to a simulated digestion of the upper gastrointestinal tract before being fed to the cecum system in vitro during a simulation 5 hours. The pots mimic chicken cecum compartments, each having the same pH (6.25). Chromatographic analysis of lactic acid from the cecal sham samples was performed with pivalic acid as an internal standard in a similar manner as described previously (Ouwehand et al. 2009; The British Journal of Nutrition 101: 367-375).Table 6: Identification of treatments
    1Danisco Xylanase, Danisco Animal Nutrition.
    [00598] 2ACCELLERASE® TRIO™ Enzyme Complex contains a potent combination of multiple enzymatic activities, including β-glucanases (CMC 200 U/kg), xylanases (eg endoxylanases - endo-1,4-β-D-xylanase (EC 3.2) .1.8)) (> 1200 ABX L/kg) and β-glucosidases (> 800 pNPG L/kg) supplied by DuPont Industrial Bioscences.
    [00599] 3A live microorganism fed directly based on three Bacillus strains selected for the ability to secrete enzymes supplied by Danisco Animal Nutrition in equal proportions of strains AGTP BS918 (NRRL B-50508), AGTP BS3BP5 (NRRL B-50510) and AGTP BS1013 (NRRL B- 50509).RESULTSTable 7: Effects of xylanase, a mixture of fiber-degrading enzymes and a live micro-organism fed directly on lactic acid production in the chicken cecum

    [00600] N.B. Different letters after the values show statistical differences (P<0.10) between values in this column.
    [00601] The combination of xylanase + a mixture of other fiber degradation enzymes + live micro-organism fed directly based on bacilli increased the production of cecal lactic acid compared to a single enzyme or combinations of individual enzymes. Lactic acid is produced by lactic acid bacteria, in which lactobacilli and streptococci predominate; these bacteria are known to have health-promoting properties in the gut (Walter, 2008; Applied and Environmental Microbiology 74: 4985-4996). Lactic acid has antibacterial effects on pathogens such as E. coli and Salmonella (Nout et al. 1989; International Journal of Food Microbiology 8, 351-361) and lactobacilli can inhibit the adhesion of E. coli to the intestine (Hillman et al. al., 1994; Journal of Applied Microbiology 76: 294-300). High concentrations of lactic acid, due to a combination of three components of xylanase, fiber-degrading enzymes and directly fed live micro-organism should therefore reflect the increased population and activity of these microbes related to gut health. IV. CECAL MICROBIAL POPULATION MATERIALS AND METHODS
    Broiler chickens are assigned to pens based on initial body weight and randomly provided experimental diets using a recognized experimental design. Birds have free access to experimental diets for a period between days 0 to 21.
    [00603] Fecal samples are collected daily from d18 to d20 and stored at -20 °C. On d 21, birds are sacrificed by cervical dislocation and the contents of the cecum obtained and stored frozen at -20°C for determination of cecal VFA.
    [00604] DNA extraction: 0.2 g of cecal digesta was suspended in PBS and then further isolated via a bead-beating step and then automatically with MagMax using a commercial kit, MagMAX™ Total Nucleic Acid Isolation Kit (Applied Biosystems). The amount of isolated DNA was determined using a full spectrum UV/Vis Nanodrop ND-1000 (Wilmington, DE, USA). Flow cytometry used as described above (Apajalahti et al. 2002, Appl Environ Microbiol 68 (10): 4986-4995) for enumeration of total or specific bacteria in samples.
    [00605] PCR Procedures: Isolated DNA is analyzed by qPCR (Quantitative Polymerase Chain Reaction) using an Applied Biosystem. Specific primers are used to detect the microbial genus specifically of interest as described in 3.Table 8: References where genus specific primers can be found for qPCR quantification of the microbial population in digesta


    [00606] The combination of xylanase + (mannanase or β-Glucanase) + DFMs induces a change in the cecal microbial population in favor of Lactobacillus and/or other specific groups known as fibrolytic bacteria: Ruminococcus, Bacteroides, Roseburia.Example 2: Effect of 2 xylanases and other fiber degradation enzymes (FDE-blend) and DFM (live microorganism fed directly based on Bacillus; live microorganisms fed directly based on Lactobacillus) when fed individually or in combination on the performance of growth and cecal volatile fatty acids in young broilers fed corn-based diets EXPERIMENT 1 MATERIAL AND METHODS
    [00607] The use of animals and experimental protocol was approved by the Institutional Animal Experiment Committee. The staple diet, as fed, is formulated to be balanced for energy and protein and to meet requirements for growing birds of this age and genotype (Table 9). The cereal component of the diet is corn and the protein component can be soy bran with or without other protein foods such as canola, rapeseed seed meal, etc. Corn co-products, such as DDGS or corn germ or corn gluten meal, can be included, either individually or in combination, as long as the diet is formulated to meet the nutrient needs of the birds to be fed. No synthetic antimicrobials or anticoccidial drugs are included and the diet is provided as a puree. A common digestibility marker (titanium dioxide, como oxide or celite) is included at 3 g/kg to allow determination of the digestibility of dietary components. The staple diet is divided into portions and the respective enzymes and DFMs added to constitute experimental diets identified in Table 10. Each supplement is pre-mixed and the blender is washed to avoid cross-contamination of the treated diets. Samples are taken from each treatment diet from the beginning, middle and end of each batch and mixed together to confirm enzyme activities and the presence of DFM in the feed before starting the animal experiment. Additional samples from each treatment diet are retained and stored until needed at -20°C ± 2°C for analysis.Table 9: Diet composition based on corn (% as fed) for d 0-21 broilers


    [00608] 1Xylanases (eg endo-1,4-β-D-xylanase (E.C.3.2.1.8) from two organisms of different origin
    [00609] Bacillus 2DFM selected as an enzyme-producing strain
    [00610] 3DFM of Lactobacillus, known to be a strain that ferments C5-sugar; a strain that produces short-chain fatty acids; a strain that promotes endogenous fibrolytic microflora; or combinations thereof
    [00611] 4FDE-blend: Combination of fiber-degrading enzyme activities, including beta-glucanase, beta-glucosidase, beta-xylosidase and/or alpha-arabinofuranosidase
    [00612] Chickens are assigned to pens based on body weight and initial experimental diets randomly allocated using a recognized experimental design. Birds have free access to the experimental diets for a period from day 0 to 21. Body Weight (BW), feed intake (FI) and mortality are recorded to calculate body weight gain (Body Weight Gain - BWG), Feed Conversion Ratio - FCR and Feed Conversion Efficiency - FCE.
    [00613] Fecal samples are collected daily on d18 to d20 and stored at -20 °C for determination of nutrients and fiber retention and AME and AMEn contents. At 21 d, birds are euthanized by cervical dislocation and the cecal contents (from Meckel's diverticulum about 1 cm above the ileocecal junction) obtained and stored frozen at -20 °C for determination of ileal digestibility of components and VFA caecal.
    [00614] Daily excreta fecal samples are pooled for each cage and dried in an oven at 60°C, while ileal samples were pooled on the basis of cage/pen and lyophilized. Diet, faecal and ileal samples are finely ground and well mixed for analysis. All samples are analyzed for dry matter, nitrogen, fat and gross energy according to AOAC (2005) procedures. Soluble and insoluble non-starch polysaccharides are tested in diets and faeces according to Englyst et al. (1988), whereas neutral detergent fiber, neutral detergent insoluble nitrogen are tested according to the methods of Tilley and Terry (1962). Digestibility marker is analyzed according to the standard selected marker procedure.
    [00615] Chromatographic analysis of volatile fatty acids and lactic acid, eg SCFA, will be performed from simulation samples with pivalic acid as an internal standard in a manner similar to that described above (Ouwehand et al., February 2009; 101 ( 3): 367-75). The concentrations of acetic, propionic, butyric, isobutyric, valeric, isovaleric, 2-methylbutyric acid and lactic acid are determined.
    [00616] Ileal digestibility coefficient and apparent component retention coefficient are calculated according to Adeola et al., 2010 (Poult Sci September 2010; 89(9): 1947-1954).
    [00617] The cage (fenced) is the experimental unit. ANOVA is performed using SAS General Linear Models (SAS Inst. Inc., Cary, NC). When F-proportions indicate significance, treatment means are separated. RESULTS
    [00618] Treated groups fed the entire combination: xylanase plus a secondary fiber degradation enzyme and a DFM (Bacillus or LB), have higher BWG (g/bird/day) and/or a lower FCR (g BW gain/ g of feed intake) and/or better digestibility of nutrients, energy and fiber/or retention than the control or these additives fed alone or in combination with two components.
    [00619] The combination of xylanases (xylanase 1 and/or 2) + a mixture of FDE + DFMs significantly increases total VFA in the ileum and/or cecum and the concentration of butyric acid or propionic acid in the ileum and/or cecal digesta of chickens .II. GROWTH PERFORMANCE Experiment I MATERIALS AND METHODS
    [00620] The use of animals and experimental protocol has been approved by the Institutional Animal Experiment Committee. The corn/soybean based diet was formulated to be energy and nutrient balanced for broilers (0-21 days old) (Table 9, Diet I). No synthetic antimicrobials or anticoccidial drugs were included and the diet was provided as a puree. The basic diet was divided into portions and the respective enzymes and DFMs added to constitute experimental diets identified in Table 11.Table 11: Identification of treatments used in experiment I

    [00621] aEnzymes (xylanase (Danisco Xylanase, an endo-1,4-β-D-xylanase (E.C. 3.2.1.8)) and β-glucanase (Axtra ® XB)) are commercial products supplied by Danisco Animal Nutrition
    [00622] bDFM based on three Bacillus strains (equal proportions of strains AGTP BS918 (NRRL B-50508), AGTP BS3BP5 (NRRL B-50510) and AGTP BS1013 (NRRL B-50509)), selected for their ability to secrete enzymes
    [00623] cXylanase FveXyn4 (an endo-1,4-β-D-xylanase (E.C.3.2.1.8)) shown as SEQ ID NO. 3 shown herein (also described in PCT/CN2012/079650, which is incorporated herein by reference), Danisco Animal Nutrition.
    [00624] All supplements were provided in a premix that was added to the blender during diet preparation. Diets containing the DFM were mixed first and the blender was discharged between each diet to avoid cross contamination. Samples were collected from each treatment diet from the beginning, middle and end of each batch and mixed together to confirm enzyme activities and the presence of DFM in the feed before starting the animal experiment. Additional samples from each treatment diet were retained and stored until needed at a temperature of -20 °C ± 2 °C for analysis. Male broilers (Hubbard-Cobb) were obtained as day-old chicks from a commercial hatchery. On day 0, the chicks were weighed and placed in 72 cages (8 birds per cage) so that the average weight of birds per cage was similar individually. The nine treatments (Table 11) were then randomly divided into eight cages each. The cages were housed in air-conditioned chambers. The temperature was maintained at 31 °C in the first week and then gradually reduced to 22 °C until the end of the third week. Birds received 20 hours of fluorescent lighting and free access to diets and water for the duration of the study. Body weight and feed intake were recorded at the beginning and end of the 21 d experimental period. Mortality was recorded daily. Feed conversion ratios were calculated by dividing total feed intake by live plus dead bird weight gain. Data were analyzed using the SAS General Linear Models (SAS Inst. Inc., Cary, NC). When F-proportions indicate significance, the treatment means are separated.RESULTS, Experiment ITable 12: Effects of xylanase, β-glucanase and a live microorganism fed directly based on bacilli on the productive performance of young broilers

    [00625] N.B. Different letters after the values show statistical differences (P<0.10) between values in this column.
    [00626] Treated groups fed the entire combination: xylanase + β-glucanase + Bacillus DFM combination had higher BWG (g/bird/day) and lower FCR (g BW gain/g feed intake) than the control or these additives powered individually or in combination of two components (Table 12). This was the case when xylanase 1 and xylanase 2 were administered. EXPERIMENT IMATERIALS AND METHODS
    [00627] The use of animals and experimental protocol was approved by the Institutional Animal Experiment Committee. The corn/soybean based diet was formulated to be energy and nutrient balanced for broilers (0-21 days old) (Table 9, Diet I). No synthetic antimicrobials or anticoccidial drugs were included and the diet was provided as a puree. The base diet was divided into portions and the respective enzymes and DFMs added to constitute experimental diets identified in Table 13.Table 13: Identification treatments for Experiment II

    [00628] aEnzymes (xylanase (Danisco Xylanase, an endo-1,4-β-D-xylanase (E.C. 3.2.1.8)) and β-glucanase (Axtra ® XB)) are commercial products supplied by Danisco Animal Nutrition
    [00629] bDFM based on Enterococcus (Enterococcus faeciumID7 (referred to as Lactococcus lactis ID7 in United States Patent Assigned No. 7,384,628 and filed with the ATCC as deposit PTA-6103 and later reclassified as Enterococcus faecium ID7)),
    [00630] cXylanase FveXyn4 (an endo-1,4-β-D-xylanase (E.C. 3.2.1.8)) shown as SEQ ID NO. 3 shown here (also described in PCT/CN2012/079650, which is incorporated herein by reference), Danisco Animal Nutrition
    [00631] All supplements were provided in a premix that was added to the blender during diet preparation. Diets containing the DFM were mixed first and the blender was discharged between each diet to avoid cross contamination. Samples were collected from each treatment diet from the beginning, middle and end of each batch and mixed together to confirm enzyme activities and the presence of DFM in the feed before starting the animal experiment. Additional samples from each treatment diet were retained and stored until needed at a temperature of -20 °C ± 2 °C for analysis. Male Broiler (Hubbard-Cobb) chicks were obtained as day-olds from a commercial hatchery. On day 0 the chicks were weighed and allocated to 72 cages (8 birds per cage) so that the average weight of birds per cage was similar individually. The nine treatments (Table 13) were then randomly divided into eight cages each. The cages were housed in air-conditioned chambers. The temperature was maintained at 31°C in the first week and then gradually reduced to 22°C until the end of the third week. Birds received 20 hours of fluorescent lighting and free access to diets and water for the duration of the study. Body weights were recorded at the beginning and end of the 21 d experimental period. Mortality was recorded daily. Data were analyzed using the SAS GLM procedure.RESULTS, EXPERIMENT IITable 14: Effects of xylanase, β-glucanase and a live microorganism fed directly on Enterococcus on the productive performance of young broilers


    [00632] N.B. Different letters after the values show statistical differences (P<0.10) between values in this column.
    [00633] There was a numerical improvement in the body weight gain of broilers when the combination of xylanase + β-glucanase + Enterococcus DFM was supplemented with xylanase + β -glucanase or xylanase + Enterococcus DMF (Table 14).III. PRODUCTION OF VOLATILE FATTY ACID IN THE CECO MATERIALS AND METHODS
    A basal diet based on corn-soybean meal-rapeseed meal was formulated to be balanced for energy and nutrients for young broilers (Table 9, Diet II). No synthetic antimicrobials or anticoccidials were included in the baseline diet. The base diet was divided into portions and the respective enzymes and DFMs added to constitute experimental diets identified in table 15. Subsequent procedures were similar to those described for Example 1, part III. Chromatographic analysis of volatile fatty acids from simulation samples (see Example 1, part III) was performed with pivalic acid as an internal standard in a similar manner as described previously (Ouwehand et al. 2009; The British Journal of Nutrition 101: 367- 375 the teaching of which is incorporated herein by reference). The concentrations of acetic, propionic, butyric, isobutyric, valeric, isovaleric and 2-methylbutyric acid were evaluated. Table 15: Identification of treatments

    [00635] 1A live microorganism fed directly based on three Bacillus (equal proportions of strains AGTP BS918 (NRRL B-50508), AGTP BS3BP5 (NRRL B-50510) and AGTP BS1013 (NRRL B-50509)), selected by their ability to secrete enzymes provided by Danisco Animal Nutrition.
    [00636] The xylanase enzymes ((Danisco Xylanase, an endo-1,4-β-D-xylanase (EC 3.2.1.8)) and β-glucanase (Axtra® XB)) are commercial products supplied by Danisco animal nutrition.RESULTSTable 16: Effects of xylanase, β-glucanase and a live micro-organism fed directly on the production of acetic and butyric acids and total volatile fatty acids (VFA) in chicken cecum

    [00637] N.B. Different letters after the values show statistical differences (P<0.10) between values in this column.
    [00638] The combination of xylanase + β-glucanase + directly fed live micro-organism increased the production of acetic acid, butyric acid and volatile fatty acids (VFA) in the cecum compared with DFM individually, enzymes or combinations of enzymes individually (Table 16). Volatile fatty acids can provide a significant amount of energy for chickens. Butyric acid is also known to improve gastrointestinal health and reduce the incidence of colon cancer in humans (Brons et al., 2002, Trends Food Science and Technology. 13: 251-261, which is incorporated herein by reference). 3: Effect of xylanase and other fibrolytic enzymes (β-Glucanase or mixture of fiber degradation enzymes (FDE-mix)) and DFM (live microorganism fed directly based on Bacillus) when fed individually or in combination on performance and nutrient digestibility in pigs (25 60 kg) fed mixed grain-based diets MATERIAL AND METHODS
    [00639] The use of animals and experimental protocol was approved by the Animal Experiment Committee. The staple diet, as fed, is formulated to be balanced for energy and protein and to meet the requirements for raising pigs of this age and genotype (Table 17). The main composition of main ingredients (type and inclusion levels) in the base diet may vary as per table 17, as the diet is formulated to meet the nutritional requirements of the fed pigs. A common digestibility marker (titanium dioxide, chromium oxide or celite) is included at 3 g/kg to allow determination of the digestibility of dietary components. No synthetic antimicrobials or anticoccidial drugs are included and the diet is provided as a puree. The staple diet is divided into parts which are then treated with the enzymes and DFMs identified in Table 18. During feed feeding, the blender is washed to avoid cross-contamination of the diets. Samples are taken from each treatment diet from the beginning, middle and end of each batch and mixed together to confirm enzyme activities and the presence of DFM in the feed. Samples from each treatment diet are retained during mixing and stored at -20°C until needed.


    [00640] 1FDE Blend: Combination of fiber-degrading enzyme activities, including beta-glucanase, beta-glucosidase, beta-xylosidase and/or alpha-arabinofuranosidase
    [00641] 2DFM based on Bacillus selected as a strain that produces enzymes
    [00642] The experiment is designed and conducted to match the growth phase (<25 to ~60 kg body weight). Experimental diets are fed for 42 days, 6 weeks. A group of female and male pigs close to the initial target body weight are acquired from the same herd (genetics). Upon arrival, pigs are weighed and distributed to treatment diets using a recognized experimental design so that each treatment has a minimum of 8 repeat pens. Body weight and feed intake are monitored weekly to calculate feed conversion efficiency for mortality corrected weight gain efficiency. Fresh faecal samples are collected at weeks 3 and 6 to allow for calculation of dietary component digestibility.
    [00643] Growing pigs (initial body weight 30 kg) are fitted with a T-cannula in the distal ileum for the purposes of the experiment. Pigs are housed in individual pens (1.2 x 1.5 m) in a controlled environment. Each pen was equipped with a feeder and a drinking fountain and had concrete floors. The experiment was designed and performed to provide a minimum of 6 replicates per treatment. All pigs are fed at a level of 3 times their maintenance energy requirement (106 kcal ME per kg0.75; NRC, 1998) and in two equal portions at 08:00 and 17:00 h. Animals are allowed free access to water through a drinking fountain. Pig weights are recorded at the beginning and end of each period and the amount of feed provided per day is recorded. The trial period lasts 15 d. The first 10 days of each period are considered a period of adaptation to the diet. Fresh stool samples are collected on d 11 to 13 and ileal samples are collected for 8 h on d 14 and 15 using conventional procedures. For ileal collection, a plastic bag is connected to the tubing of a cannula and the digesta flows into the collection bag. Bags are removed whenever they are full of digesta - or at least once every 30 minutes and immediately frozen at -20°C.
    [00644] Stool and ileal samples are thawed, mixed by animals and diet and a subsample collected for chemical analysis. A staple diet sample is also collected and analyzed. Digest samples were lyophilized and finely ground prior to chemical analysis. Stool samples are dried in an oven and finely ground for analysis. All samples were analyzed for dry matter, digestibility marker, gross energy, crude protein, fat and neutral detergent fiber according to conventional procedures (AOAC, 2005).
    [00645] Values for apparent and total ileal digestibility of energy and nutrients are calculated as previously described (Stein et al., 2007. J. Anim Sci 85: 172-180). The enclosure is the experimental unit. Data is submitted to SAS MIXED procedures. RESULTS
    [00646] Treated groups fed the entire combination: xylanase plus a secondary fiber degradation enzyme (β-Glucanase or FDE-mix) and a DFM (live microorganism fed directly based on Bacillus) have higher BWG and/or a lower FCR (gain BW g/g feed intake) and/or high digestibility of nutrients and/or energy and/or dry matter and/or fiber.Example 4: Effects of xylanase, β-glucanase and a micro-organism live feed directly based on bacterial strains that produce propionic acid on nutrient retention/digestibility and metabolizable energy in young broilers Composition of experimental wheat-based diets used in Example 4Table 19: Composition of wheat-based diet for broiler chickens cut (% as fed)

    MATERIAL AND METHODS
    [00647] The use of animals and experimental protocol has been approved by the Institutional Animal Experiment Committee. The wheat and barley based diet was formulated to be energy and nutrient balanced for broilers (0-21 days old) (Table 19). Titanium dioxide was included at 0.30% to allow determination of dietary component retention. No synthetic antimicrobials or anticoccidial drugs were included and the diet was provided as a puree. The base diet was divided into portions and the respective enzymes and DFMs added to constitute experimental diets identified in Table 20. Each supplement was pre-mixed and the blender was washed to avoid cross-contamination of treated feed. Samples were collected from each treatment diet from the beginning, middle and end of each batch and mixed together to confirm enzyme activities and the presence of DFM in the feed before starting the animal experiment. Additional samples from each treatment diet are retained and stored until needed at -20°C ± 2°C for analysis. Table 20: Identification of treatments

    [00648] 1 Live microorganism fed directly on the basis of strains that produce propionic acid (Propionibacterium acidipropionici P169 PTA-5271, Omni-Bos® P169).
    The enzymes (xylanase (Danisco Xylanase, an endo-1,4-β-D-xylanase (E.C. 3.2.1.8)) and β-glucanase (Axtra® XB)) are commercial products supplied by Danisco Animal Nutrition.
    [00650] The study involved a cage experiment, which was carried out to obtain faecal samples for measurements of energy and nutrient digestibility. Day-old male broiler chicks (Ross 308) were obtained from a commercial hatchery. The chicks were individually weighed on arrival and stratified by body weight and distributed into 30 cages (five birds per cage) so that the average weight of birds per cage was similar. The four dietary treatments were then randomly divided into six identical cages. The study was conducted from day 0 to 21, during which the birds had free access to their assigned dietary treatments and water. The brooder and ambient temperatures were fixed at 32 and 29 ◦C, respectively, during the first week. After that, the heat supply in the brooder was turned off and room temperature was maintained at 29 ◦C throughout the experiment. Light was provided for 24 h throughout the experiment. On days 17, 18, 19 and 20, excreta samples were collected and stored frozen at -20 ◦C for determination of energy and nutrient retention/digestibility. Care was taken during collection of faecal samples to avoid contamination from feathers and other foreign materials. Faecal samples were pooled within a well-mixed cage using a blender and two representative samples per cage were taken. Samples were lyophilized. The dried samples were ground to pass through a 0.5 mm sieve and stored in airtight plastic containers at -4 °C until chemical analysis. Diet and faecal samples were analyzed for dry matter, crude protein (as nitrogen), gross energy, fat (as hexane extracts) and neutral detergent fiber according to official AOAC analysis methods. Titanium (digestibility marker) was analyzed according to the procedures described by Lomer et al. (2000, Analyst 125: 23392343), which is incorporated herein by reference. Retention/digestibility was calculated using standard procedures (Adeola, O. 2001. Digestion and Balance Techniques in Pigs. Pages 903-916 in Swine Nutrition, 2nd ed. AJ Lewis and LL Southern, ed. CRC Press, Washington, DC, both of which are hereby incorporated by reference). Data were analyzed using the SAS (2004) General Linear Models procedure.RESULTSTable 21: Effects of xylanase, fiber-degrading enzyme and a live microorganism fed directly on bacterial strains that produce propionic acid on nutrient retention /digestibility and metabolizable energy in young broilers


    [00651] N.B. Different letters after the values show statistical differences (P<0.10) between values in this column.
    [00652] A combination of xylanase, β-glucanase and a live microorganism fed directly on bacilli strains improved dietary energy utilization compared to control or xylanase alone or a combination of xylanase and β-glucanase (Table 20). This could be related to increased retention of energy nutrients relative to dry matter such as fat (Table 20). The increased fat retention due to the three-component combinations is notable and could be related to increased digestion and absorption of dietary fat as well as the production and absorption of short-chain fatty acids from fermentation. The observed benefits of the three-component combination of xylanase, β-glucanase, bacillus/propionic DMF on energy and better nutrient utilization could also be speculatively associated with better intestinal health and function through positive modulation of microbiota and absorption function/ digestive tract.Example 5: Responses of broilers when fed corn-based diets containing xylanase, other fiber-degrading enzymes, and direct-fed live microorganism that produces propionic acid
    [00653] Composition of experimental diets used in Example 5Table 22: Composition of corn-based diets for broilers (% as fed)
    MATERIALS AND METHODS
    [00654] The use of animals and experimental protocol has been approved by the Institutional Animal Experiment Committee. The corn-based diet was formulated to be energy and nutrient balanced for broilers (0-21 days old) (Table 22). No synthetic antimicrobials or anticoccidial drugs were included and the diet was provided as a puree. The base diet was divided into portions and the respective enzymes and DFMs added to constitute experimental diets identified in Table 23. Each supplement was pre-mixed and the blender was washed to avoid cross-contamination of the treated diets. Samples were collected from each treatment diet from the beginning, middle and end of each batch and mixed together to confirm enzyme activities and the presence of DFM in the feed before starting the animal experiment. Additional samples from each treatment diet are retained and stored until needed at -20 °C ± 2 °C for analysis. Table 23: Identification of Treatments

    [00655] 1Danisco Xylanase, Danisco Animal Nutrition
    [00656] 2ACCELLERASE® TRIO ™ Enzyme Complex contains a potent combination of multiple enzymatic activities, including β-glucanases (CMC 200 U/kg), xylanases (eg, endoxylanases, endo-1,4-β-D-xylanase (EC 3.2) .1.8)) (> 1200 ABX L/kg) and β-glucosidases (> 800 pNPG L/kg) (DuPont Industrial Bioscences).
    [00657] 3 Live microorganism fed directly on the basis of strains that produce propionic acid (Propionibacteriumacidipropionici P169 PTA-5271, Omni-Bos® P169)
    [00658] Day-old chicks were purchased from a commercial hatchery and, on arrival, birds were weighed and marked for identification and distributed into six blocks by body weight and randomly assigned to 4 treatments (Table 23) within a block with ten birds per unit in a randomized complete block design. From d 1 onwards, ad libitum access to drinking water was allowed. Birds were weighed on days 0 and 21 and their weights were recorded; feed intake was also monitored and documented in daily bird weight. Birds were monitored daily and variations in their appearance or behavior were recorded. At the end of each feeding period, parameters such as weight gain, feed intake, feed conversion, feed efficiency and mortality were determined. Data were analyzed as a randomized complete block design using the SAS software GLM procedure (SAS Institute, Inc., 2006).RESULTSTable 24: Effects of xylanase, a mixture of other fiber-degrading enzymes, and a live microorganism directly fed with propionic acid on the productive performance of young broilers

    [00659] N.B. Different letters after the values show statistical differences (P<0.10) between values in this column.
    [00660] Birds fed a combination of xylanase, a mixture of other fiber-degrading enzymes, and a propionic acid-based DFM had better FCR than the control and were numerically better than birds fed enzyme-only diets (Table 24).Example 6: Effects of xylanase and β-glucanase without or with live microorganism fed directly based on bacilli strains on growth performance, microbial counts and nutrient digestibility in growing pigs Composition of experimental diets used in Example 6 Table 25: Feed diet composition for growing pigs (20-60 kg body weight) (% as fed)
    MATERIALS AND METHODS
    [00661] Two experiments were conducted to evaluate the growth performance, faecal microbial counts and digestibility effects of a mixture of xylanase and β-glucanase enzymes fed without or with live microorganism fed directly based on bacilli strains on growth growing pigs. The Institutional Animal Care and Use Committee approved the use of pigs and country-relevant welfare guidelines were used. A total of 42 pigs ([^Yorkshire x Landrace] x ^Duroc) housed in groups of two were used in experiment 1 and 72 pigs of the same breed housed in groups of three were used in experiment 2. Each pen had clear plastic sides sheets and plastic covered expanded sheet metal liners in a temperature-controlled room (22 ± 2 °C).
    [00662] Respective staple diets were formulated to meet the nutritional recommendations of the NRC for pigs (NRC, 1998; Table 25, diet I for experiment 1 and diet II for experiment 2). In each experiment, a batch of the staple diet is manufactured and divided into two servings and each serving subsequently mixed with additives identified in Table 26. Table 26: Identification of Treatments

    [00663] 1 Live micro-organism fed directly based on three Bacillus strains (equal proportions of strains AGTP BS918 (NRRL B-50508), AGTP BS3BP5 (NRRL B-50510) and AGTP BS1013 (NRRL B-50509)), selected by its ability to secrete enzymes provided by Danisco Animal Nutrition.
    The xylanase ((Danisco Xylanase, an endo-1,4-β-D-xylanase (E.C. 3.2.1.8)) and β-glucanase (Axtra® XB) enzymes) are commercial products supplied by Danisco Animal Nutrition.
    [00665] The treatments identified in table 26 were assigned to 7 and 8 enclosures repeated in experiments 1 and 2, respectively. Assignment of pens to treatments was random based on the pig's body weight at the start of the experiment. Body weight and feed intake were recorded on a weekly basis and used to calculate the feed conversion ratio. Pigs were offered experimental diets for 42 days in both experiments. Food and water were freely available at all times during experimentation. In experiment 2, fresh stool samples were collected on days 38, 39 and 40 to determine nutrients, energy and fiber digestibility, as well as fecal microbial counts. One gram of the composite fecal sample from each pen was diluted with 9 mL of 1% peptone broth (Becton, Dickinson and Co., Franklin Lakes, NJ) and then homogenized. Viable counts of bacteria in the fecal samples were then performed by placing 10-fold dilutions (in 1% peptone solution) on MacConkey agar plates (Difco Laboratories, Detroit, MI) and medium III agar plates for lactobacilli (Medium 638, DSMZ, Braunschweig, Germany) to isolate E. coli and Lactobacillus, respectively. Medium III agar plates for lactobacilli were then incubated for 48 h at 39 °C under anaerobic conditions. MacConkey agar plates were incubated for 24 h at 37°C. Colonies of E. coli and Lactobacillus were counted immediately after removal from the incubator. Prior to chemical analysis, faecal samples were thawed and dried at 60°C for 72 h, after which they were finely ground to a size such that they could pass through a 1 mm sieve. All feed and faecal samples were then analyzed for dry matter, gross energy and acid detergent fiber following the procedures described by the AOAC (Official Methods of Analysis). Chromium (digestibility marker) was analyzed following the method described by Williams et al. 1962, J. Anim. Sci. 59:381-389, which is incorporated herein by reference. Digestibility was calculated using conventional procedures (Adeola, O. 2001. Digestion and Balance Techniques in Pigs. Pages 903-916 in Swine Nutrition, 2nd ed. AJ Lewis and LL Southern, ed. CRC Press, Washington, DC - the teaching of the which are incorporated herein by reference). Growth performance data (BW, ADFI, ADG, and FCR) were subjected to the SAS GLM procedures with treatments, experiments, and interactions as effects in the model. Initial analysis revealed that the interactions were not significant and, as such, required further analysis; subsequently main effects of treatments are presented. Microbial count data were log transformed along with digestibility and subjected to one-way analysis of variance using SAS GLM procedures.RESULTSTable 27: Effects of xylanase and β-glucanase without or with live microorganism fed directly based on strains of bacilli on growth performance in growing pigs

    [00666] NB Different letters after the values show statistical differences (P<0.10) between values in this column.Table 28: Effects of xylanase and β-glucanase without or with live microorganisms fed directly based on bacilli strains on dry matter, nitrogen, fiber and energy digestibility (%) in growing pigs

    [00667] N.B. Different letters after the values show statistical differences (P<0.10) between values in this column.
    [00668] A combination of xylanase, β-glucanase and a live microorganism fed directly based on bacillus improved pig growth performance and utilization of dietary nutrients and energy compared to control or individual enzymes (Tables 27 and 28). It was also observed that combinations with three components result in more fiber degradation and promote the proliferation of lactobacilli bacteria in the intestine (Figure 1).Example 7: Effects of xylanase, other fiber degradation enzymes and live directly fed microorganisms on the production of short chain fatty acids in the large intestine of pigs Composition of the experimental diets used in Example 7Table 29: Feed diet composition for growing pigs (20-60 kg body weight) (% as fed)

    MATERIALS AND METHODS
    [00669] In order to establish a porcine large intestine model, a method has been adapted from (Boisen and Fernandez 1997, Animal Feed Science and Technology 68: 277-286), the teaching of which is incorporated herein by reference) to generate porcine iliac effluent in vitro. In summary, 1.35 kg of complete crushed feed (based on corn and wheat, details see table 29) were combined with 3.00 L of phosphate buffer (0.1 M, pH 6) and 1.20 L of 0.2M HCl in a 3-liter bucket with a resealable lid. The pH was adjusted to 2 using 10M HCl or NaOH. Then, 120 mL of a pre-prepared pepsin solution was added (250 mg of pepsin (Sigma-Aldrich, Inc., St. Louis, MO) per mL of water). The bucket was sealed and incubated at 39°C for 2 hours with shaking in order to simulate digestion in the stomach. To simulate digestion in the small intestine, 1.20 L of phosphate buffer (0.2 M, pH 6.8) and 600 mL of 0.6 M NaOH were added to the solution and the pH was adjusted to 6. 8 using 10M NaOH or HCl as above. After neutralization, 120 ml of pre-prepared pancreatin solution (1000 mg of pancreatin (Sigma-Aldrich) per ml of water) was added, the bucket sealed and incubated at 39°C for 4 hours with shaking. After incubation, the liquid was separated by filtration using a double-layer infusion bag and twice folded in half (Jumbo Nylon Coarse, LD Carlson Company, Kent, OH). The remaining pellet was homogenized and divided into 128 g portions, each weighed into separate 250 mL Pyrex vials. Vials were subsequently stored at -20°C. As an inoculant for a large bowl of microbiota, the cecal contents were collected from 12 growing pigs. The contents were homogenized, mixed with 10% glycerol and 14 g aliquots weighed in 15 mL conical flasks. The conical vials were then sealed and stored at -80°C.
    [00670] Simulation experiments in the large intestine of pigs were performed in duplicate operations, each with a control and 3 treatments (Table 30). Each treatment was tested in triplicate. For each in vitro swine large intestine fermentation experiment, a total of 24 Pyrex flasks were used with simulated ileal effluent and a 15 mL conical flask with cecal content. The vials were thawed overnight and 240 mL of a sterile 0.1 M phosphate buffer solution (pH 6) with 4 g/L mucin (Sigma-Aldrich) was added to each vial, similar to the methods described in (Christensen et al., 1999, Journal of the Science of Food and Agriculture 79, 755-762) and Aristoteli and Willcox, 2003, Infection and Immunity 71: 5565-5575), the teaching of which is incorporated herein by reference. The inoculant was thawed for 30 minutes at room temperature, while the Pyrex flasks were pre-warmed at 39°C for 30 minutes in a shaking water bath, then treatments in 1 mL of a 1% peptone solution and 450 μL of 0.1 M phosphate buffer (see table 30) was added. Table 30: Treatments tested for porcine large intestine fermentation in vitro*

    [00671] *Enzyme and directly fed live microorganisms were included at a rate similar to 500 g per metric ton when including in the feed; each experiment was performed in duplicate operations; treatments were measured in triplicate in each series; # Baseline diet is corn control diet (CC) or wheat control diet (WC), as described in Table 29; f Xylanase Y5 is (Danisco Xylanase, an endo-1,4-β-D-xylanase (EC 3.2.1.8)) or NGX (FveXyn4 (an endo-1,4-β-D-xylanase (EC 3.2.1.8)) ) shown as SEQ ID NO.3 herein (also described in PCT/CN2012/079650, which is hereby incorporated by reference), Danisco Animal Nutrition) with a guaranteed activity of 4000 XU/kg of feed; Φ Fiber degradation enzyme is the mixture of Accel enzymes. (Accelerase Trio, ACCELLERASE® TRIO™ Enzyme Complex contains a combination of various enzymatic activities, including β-glucanases (CMC 200 U/kg), xylanases (eg, endoxylanases, endo-1,4-β-D-xylanase (EC) 3.2.1.8)) (> 1200 ABX L/kg) and β-glucosidases (> 800 pNPG L/kg) (DuPont Industrial Bioscences) or Axtra® XB β-glucanase with a guaranteed activity of 360 BGU β-glucanase/kg + Directly fed live microorganism is based on Bacillus (equal proportions of strains AGTP BS918 NRRL B-50508, BS1013 AGTP NRRL B-50509 and AGTP BS3BP5 NRRL B-50510), with a guaranteed activity of 3.0 x 108 cfu per gram of product, Propionibacterium acidipropionici P169 or PTA-5271 OmniBos® P169, with a guaranteed activity of 2.1 x 109 cfu per gram of product.
    Vials were flushed with CO2 gas for 30 seconds while 250 µL of cecal inoculant was added (based on Coles et al. 2005, Animal Feed Science and Technology 123: 421-444, the teaching of which is incorporated herein for reference) and a 10 mL baseline sample was collected, the baseline pH determined and the sample stored at -20°C. The vials were capped, gently mixed and placed in a shaking water bath at 39°C and 160 rpm. After 12 h, another 10 mL sample was collected, the pH determined and the sample stored at -20 °C. For quantification of volatile fatty acid (Volatile Fatty Acid - VFA) by means of high performance liquid chromatography (High Pressure Liquid Chromatography - HPLC), samples were thawed and centrifuged at 16.1 rad for 20 minutes and the supernatant was filtered through a 0.22 µm mixed cellulose ester membrane (milex-GS, Merck Millipore Corp., Billerica, MA). Of the filtrate, 20 µL was injected into a Waters Alliance 2695 Separations Module (Waters Corp., Milford, MA) equipped with a Shodex SH-G protection column (Waters) and a 300 x 7.8 mm Aminex HPX-87H column (Biorad Laboratories, Inc., Hercules, CA). An isocratic method was applied with a mobile phase consisting of 16.8 mM phosphoric acid in water/acetonitrile (98:2, v/v) at a flow rate of 0.525 mL/min and column temperature of 35 °C . Volatile fatty acids were detected using a photodiode array detector (Photo Diode Array - PDA) Waters 2996 (Waters) in absorption at 211 nm. Instrument control, data acquisition, data processing were achieved with Waters Empower 3 (Waters) software. Volatile fatty acids were quantified using standard curves generated from high-grade (>99.9%) reagents (Sigma Aldrich, St. Louis, MO). Linear dilutions of the standards in 16.8 mM phosphoric acid in water/acetonitrile (98:2, v/v) were prepared at 6 concentrations, ranging from 0.05% to 2.0%. The concentration of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid (the sum of which is presented as total VFA) and lactic acid was determined. Statistical analysis for each experiment was performed as one-way operation-blocked ANOVA using the SPSS GLM procedure (version 17, SPSS Inc., Chicago, IL). Significance was stated for P < 0.10; means of treatment were separated using Duncan's multiple band test.
    [00673] In wheat-based diets, a significant increase in total VFA and lactic acid production after 12 hours of simulation in the large intestine of pigs was observed when xylanase NGX, Accelerase Trio fiber-degrading enzyme mixture and a DFM were added and compared to the control without supplementation (Table 31, experiments 1 and 2). Use of DFM based on Propionibacterium acidipropionici P169 significantly increased propionate levels and had greater simulated colonic content acidification in the combined treatment compared to the control (Table 31, Experiment 1). In corn-based diets, the combined treatment of xylanase Y5, Accelerase Trio fiber-degrading enzyme mixture and DFM significantly increased butyrate levels compared to control after 12 h of simulated fermentation in the large intestine of pigs, with an increase additional total VFA when DFM based on Propionibacterium acidipropionici P169 was used (Table 31, experiments 3 and 5). Replacement of the Accelerase Trio enzyme mixture with Axtra® XB β-glucanase and use of Bacillus-based DFM in the corn diet with Y5 resulted in a significant increase in total VFA and lactate compared to the control treatment (Table 31, experiment 4) .Table 31: Mean abundance of propionate, butyrate, total volatile fatty acids (VFA) and lactate (% as is), as well as pH differences compared to baseline samples after 12 h of large intestine fermentation of pigs in vitro


    [00674] a, bValues with different superscripts within a column are significantly different at P <0.10; NS, not significant; SEM, standard error of the mean; details of treatment see Table 26.Example 8. Effects of xylanase, other fibrolytic enzymes and directly fed live microorganisms on fiber degradation in the large intestine of pigs Composition of experimental diet used in Example 8Table 32: Composition of growth feed diet for pigs (20-60 kg body weight) (% as fed)

    [00675] To demonstrate the disappearance of dry matter (Dried Matter - DM) and fiber degradation, iliac effluents were generated and fermentation in the large intestine set up as described in Example 7. In summary, the wheat-based diet (CW, see Table 32) was used as a control without any treatment, as well as CW together with xylanase Y5 (Treatment 1), CW with Y5 and fiber degradation enzyme mixture Accelerase Trio (Treatment 2), CW with Y5, Accelerase in combination with a live microorganism fed directly based on three Bacillus strains (Treatment 3); details on enzyme and DFM treatments see Table 33.Table 33: Identification of Treatments*

    [00676] * Enzyme and live directly fed microorganisms were included at a rate similar to 500 g per metric ton when including in the feed; each experiment was performed in duplicate operations; treatments were measured in triplicate in each series; # The staple diet is a wheat control (WC) diet as described in table 32; f Xylanase Y5 is (Danisco Xylanase, an endo-1,4-β-D-xylanase (E.C. 3.2.1.8)) with a guaranteed activity of 4000 XU/kg of feed; * Fiber degradation enzyme is Accel. (Accelerase Trio, ACCELLERASE® TRIO™ Enzyme Complex contains a potent combination of multiple enzymatic activities, including β-glucanases (CMC 200 U/kg), xylanases (eg, endoxylanases, endo-1,4-β-D-xylanase ( EC 3.2.1.8)) (> 1200 ABX L/kg) and β-glucosidases (> 800 pNPG L/kg) (DuPont Industrial Bioscences); the enzyme mixture was dosed to ensure a guaranteed 360 BGU β-glucanase activity /kg of feed. + Directly fed live microorganism is based on Bacillus (equal proportions of strains AGTP BS918 NRRL B-50508, BS1013 AGTP NRRL B-50509 and AGTP BS3BP5 NRRL B-50510), with a guaranteed activity of 3 .0 x 108 cfu per gram of product or Propionibacterium acidipropionici P169 PTA-5271 Omni-Bos® P169, with a guaranteed activity of 2.1 x 109 cfu per gram of product.
    [00677] Effects of Treatment on Disappearance of DM and Fiber. At 0 and 48 hours after the experiment, liquid was filtered and the remaining solids were collected and sent for analysis of approximate nutrients of dry matter (Dry Matter - DM), acid and neutral detergent fiber (ADF and NDF, respectively), the latter were generated based on MD according to the methods described in (Association of Analytical Chemists (AOAC) 2007, 18th edition. AOAC, Washington, DC). Data were calculated as percentage disappearance, statistical analysis was performed as one-way ANOVA blocked by operation using the SPSS GLM procedure (version 17, SPSS Inc., Chicago, IL). Significance was stated for P < 0.10, means of treatment were separated using Duncan's multiple band test.
    [00678] In the wheat-based diet tested, the combined treatment with the mixture of fiber-degrading enzymes Y5 Xylanase, Accelerase Trio and DFM based on three Bacillus had the greatest disappearance of DM, ADF and NDF compared with CW without any enzyme and DFM supplementation (Table 34).Table 34: Percentage of disappearance of dry matter, acid and neutral detergent fiber during 48 h of fermentation in the large intestine of pigs in vitro


    [00679] a, b Values with different superscripts within a column are significantly different at P < 0.10; SEM, standard error of the mean; details of the treatment see Table 7.2.
    [00680] All publications mentioned in the above descriptive report are hereby incorporated by reference. Various modifications and variations of the described methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. While the present invention has been described in relation to specific preferred embodiments, it is to be understood that the invention as claimed will not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention, which are obvious to those skilled in the art of biochemistry and biotechnology or related fields, are intended to be within the scope of the following claims.
    权利要求:
    Claims (6)
    [0001]
    1. Feed additive composition, characterized by the fact that it comprises two or more strains of directly fed live microorganisms (DFM), a xylanase and a β-glucanase, wherein the DFM strains are two or more DFM strains selected from the group which consists of AGTP BS918 (NRRL B-50508), AGTP BS3BP5 (NRRL B50510) and AGTP BS1013 (NRRL B-50509), wherein one or more of the DFM strains is an enzyme-producing strain; DFM is a strain that ferments C5 sugar; ewhere xylanase and β-glucanase are exogenous to DFM strains.
    [0002]
    2. Feed additive composition, according to claim 1, characterized in that (a) one or more of the DFM strains is a strain that produces short-chain fatty acid; and/or(b) one or more of the DFM strains is a strain that promotes endogenous fibrolytic microflora; and/or(c) DFM is a viable bacterium; and/or(d) the directly fed live microorganism is in the form of an endospore.
    [0003]
    3. Additive composition according to claim 1 or 2, characterized in that the additive composition comprises an additional fiber degradation enzyme.
    [0004]
    4. Additive composition according to claim 3, characterized in that the additional fiber degradation enzyme is selected from the group consisting of a cellobiohydrolase (EC 3.2.1.91 and EC 3.2.1.176), a β- glycosidase (EC 3.2.1.21), a β-xylosidase (EC 3.2.1.37), a feruloyl esterase (EC 3.1.1.73), an α-arabinofuranosidase (EC 3.2.1.55), a pectinase (eg, an endopolygalacturonase (EC) 3.2.1.15), an exopolygalacturonase (EC 3.2.1.67) or a pectate lyase (EC 4.2.2.2)) or combinations thereof, preferably the additional fiber degradation enzyme is selected from the group consisting of a cellobiohydrolase (EC 3.2.1.176 and EC 3.2.1.91), a β-glycosidase (EC 3.2.1.21) or combinations thereof.
    [0005]
    5. Additive composition, according to any one of claims 1 to 4, characterized in that it is formulated as a premix comprising one or more vitamins and/or one or more minerals.
    [0006]
    6. Method of improving the performance of an individual or improving the digestibility of a feedstock in a feed (eg nutrient digestibility such as amino acid digestibility) or improving nitrogen retention, or improving the feed conversion ratio (FCR) or improve weight gain in an individual or increase feed efficiency in an individual or move the fermentation process in the individual's gastrointestinal tract to the production of butyric acid and/or propionic acid, characterized by the fact that it comprises administering to an individual a feed additive composition as defined in any one of claims 1 to 5.
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    KR20150038587A|2015-04-08|
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    PH12015500137A1|2015-03-02|
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    CA2880485C|2021-03-30|
    JP2015528705A|2015-10-01|
    CA2880485A1|2014-02-06|
    BR112015002449A2|2017-08-01|
    MX2015001260A|2015-05-08|
    WO2014020141A1|2014-02-06|
    CN112806471A|2021-05-18|
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    法律状态:
    2019-07-23| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2020-06-09| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|
    2020-12-08| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|
    2021-04-27| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-07-06| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 02/08/2013, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
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    US201261679084P| true| 2012-08-03|2012-08-03|
    US61/679,084|2012-08-03|
    PCT/EP2013/066254|WO2014020141A1|2012-08-03|2013-08-02|Feed additive composition|
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