Use of bacterial amylases in poultry feed

Abstract

The present invention relates to a method and use of at least one bacterial amylase in poultry feed to improve the nutritional value of the feed. The invention also relates to poultry feed and poultry feed additives for improving the nutritional value of the feed, which poultry feed additives comprise at least one bacterial amylase and at least one vitamin and / or mineral.

Description

[0001] This application is a divisional application of a Chinese invention patent application with an application date of December 1, 2014, application number 201480064636.0, and invention name “Use of bacterial amylase in poultry feed”.

[0002] Reference to a sequence listing

[0003] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. Background of the Invention Field of the Invention

[0005] The present invention relates to a method and use of at least one bacterial amylase in poultry feed for improving the nutritional value of the feed. The present invention also relates to poultry feed and poultry feed additives for improving the nutritional value of the feed, which poultry feed additives include at least one bacterial amylase and at least one vitamin and / or mineral.

[0006] Related technical notes

[0007] WO 03 / 068256A1 describes an amylase feed supplement for improving ruminant nutrition. The amylase used is a fungal amylase produced by Aspergillus oryzae. Tricarico et al., in Animal Science, 2005, 81:365-374 describe the effect of an Aspergillus oryzae extract containing alpha-amylase activity on ruminal fermentation and milk production in Holstein dairy cows.

[0008] US Patent No. 3,250,622 discloses the use of specific additives comprising proteolytic and amylolytic enzymes and gumases in close association with a ground malt carrier for stimulating milk production in dairy cows. The source of the enzymes is not specified.

[0009] Rojo et al., (Animal Feed Science and Technology, 123-124 (2005), 655-665) studied the effects of exogenous amylases from Bacillus licheniformis and Aspergillus niger on rumen starch digestion and lamb performance.

[0010] WO 01 / 41795 A1 relates to the use of a combination of a protease and a quaternary aminocarboxylic acid inner salt in the treatment and / or prevention of coccidiosis and bacterial infections. Alpha-amylase is an optional enzyme that may be added.

[0011] Gracia et al., (Animal Feed Sci Tech, 150:3-4, (2009), 303-315) showed that a multi-enzyme complex with xylanase, protease and alpha-amylase activities improved weight gain and FCR in broiler chickens aged 1-4 days.

[0012] It is an object of the present invention to provide alternative, preferably improved amylases which improve feed utilization and / or weight gain in poultry. SUMMARY OF THE INVENTION

[0014] The present invention relates to the use of at least one bacterial amylase in poultry feed for improving the nutritional value of the feed, wherein the bacterial amylase is a polypeptide having at least 80% identity to amino acids 1-481 of SEQ ID NO:2.

[0015] The present invention also relates to a method for improving the nutritional value of poultry feed, the method comprising the step of adding to the feed at least one bacterial amylase, wherein the bacterial amylase is a polypeptide having at least 80% identity to amino acids 1-481 of SEQ ID NO:2.

[0016] The present invention further relates to a poultry feed additive comprising at least one bacterial amylase, together with (i) at least one vitamin, (ii) at least one mineral, or (iii) at least one vitamin and at least one mineral, wherein the bacterial amylase is a polypeptide having at least 80% identity to amino acids 1-481 of SEQ ID NO: 2, and further to a poultry feed comprising the poultry feed additive and 10w%-40w% soy meal.

[0017] definition

[0018] Amylase: In the present context, amylases are enzymes that catalyze the endo-hydrolysis of starch and other linear and branched oligo- and polysaccharides. In a particular embodiment, the amylase for use according to the invention has alpha-amylase activity, i.e., catalyzes the endo-hydrolysis of 1,4-alpha-glycosidic bonds in oligo- and polysaccharides. Alpha-amylases act in a random manner on, for example, starch, glycogen and related polysaccharides and oligosaccharides, releasing reducing groups in the alpha-configuration.

[0019] Amylases belong to the EC 3.2.1.-group, such as EC 3.2.1.1 (α-amylase, 1,4-α-D-glucan glucanohydrolase), EC 3.2.1.2 (β-amylase), EC 3.2.1.3 (glucan 1,4-α-glucosidase, amyloglucosidase, or glucoamylase), EC 3.2.1.20 (α-glucosidase), EC 3.2.1.60 (glucan 1,4-α-maltotetrasoamylase), EC 3.2.1.68 (isoamylase), EC 3.2.1.98 (glucan 1,4-α-maltohexasoylase), or EC 3.2.1.133 (glucan 1,4-α-maltohydrolase). The amylase of the present invention is an α-amylase belonging to the group EC 3.2.1.1. EC numbers refer to the 1992 Enzyme Nomenclature published by NC-IUBMB Academic Press, San Diego, California, as published in Eur. J. Biochem. 1994, 223, 1-5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur. J. Biochem. 1997, 250, 1-6; and Eur. J. Biochem. 1999, 264, Supplements 1-5, 610-650. The nomenclature is periodically supplemented and updated; see, for example, http: / / www.chem.qmw.ac.uk / iubmb / enzyme / index.html .

[0020] Amylase activity can be determined by any suitable method. In general, the assay-pH and assay-temperature can be adapted to the enzyme under investigation. Examples of pH assays are pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Examples of assay-temperatures are 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 80°C, 90°C, or 95°C. Preferred pH values ​​and temperatures are within the physiological range, such as a pH of 3, 4, 5, 6, 7, or 8, and a temperature of 30°C, 35°C, 37°C, or 40°C. A preferred assay is the reducing sugar assay of Example 4 herein. Alternatively, the following amylase assay can be used: Substrate: Phadebas tablets (Pharmacia Diagnostic; a cross-linked, insoluble, blue starch polymer that is mixed with poultry serum albumin and a buffer substance and made into tablets). Assay temperature: 37°C. Assay pH: 4.3 (or 7.0 if desired). Reaction time: 20 min. After suspension in water, the starch is hydrolyzed by alpha-amylase, giving soluble blue fragments. The absorbance of the resulting blue solution measured at 620 nm is a function of the alpha-amylase activity. One fungal alpha-amylase unit (1 FAU) is the amount of enzyme that decomposes 5.26 g of starch per hour under standard assay conditions. The preferred starch is Merck, soluble starch Erg. B. 6, batch 9947275. More detailed assay instructions, APTSMYQI-3207, are available from Novozymes A / S (Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark).

[0021] Concentrates: The term "concentrates" means feeds with a high protein and energy concentration, such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (e.g., from corn, oats, rye, barley, wheat, whole or prepared by cracking, milling, etc.), oilseed cakes (e.g., from cottonseed, safflower, sunflower, soybean, rapeseed / canola, peanut or groundnut), palm kernel cake, yeast-derived materials and distillers grains (e.g., wet distillers grains (WDS) and distillers dried grains with solubles (DDGS)).

[0022] Feed conversion ratio: The term "feed conversion ratio" (FCR) describes how efficiently feed is utilized. The lower the FCR, the better the feed is utilized. The FCR can be determined on the basis of an animal experiment comprising a first treatment in which the amylase for use according to the invention is added to the animal feed at a desired concentration (e.g., 100 to 400 mg enzyme protein / kg feed) and a second treatment (control) in which no amylase is added to the animal feed. In a specific embodiment, the FCR is improved (i.e., reduced) by at least 1.0%, preferably at least 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, or at least 2.5%, compared to the control. In a further specific embodiment, the FCR is improved (i.e., reduced) by at least 2.6%, 2.7%, 2.8%, 2.9%, or at least 3.0%, compared to the control. In still further specific embodiments, the FCR is improved (ie, decreased) by at least 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, or at least 3.8% compared to a control.

[0023] Poultry: The term "poultry" means domesticated birds raised by humans for the eggs they produce and / or their meat and / or their feathers. Poultry includes broiler chickens and laying hens. Poultry includes members of the order Galliformes (birds), especially the order Galliformes (which includes chickens, guinea fowl, quail and turkeys) and the family Anatidae, in which they are commonly called "waterfowl" and include ducks and geese. Poultry also includes other birds killed for their meat, such as squabs. Examples of poultry include chickens (including laying hens, broiler chickens and chicks), ducks, geese, pigeons (including but not limited to pigeons), guinea fowl, turkeys and quail.

[0024] Poultry feed: The term "poultry feed" means feed given to poultry, such as chickens (including laying hens, broilers and chicks), ducks, geese, pigeons (including but not limited to pigeons), guinea fowl, turkeys and quail. Poultry feed typically includes a premix that is added to, for example, a concentrate and then fed to the poultry.

[0025] Poultry feed additive: The term "poultry feed additive" means a premix including, for example, vitamins and / or minerals. Premix is ​​a term recognized in the art for certain feed additives. They can be solid or liquid. For example, a trace mineral premix is ​​a composition that is intended to be added to animal feed and includes trace minerals of the desired type and amount. A vitamin premix is ​​a composition that is intended to be added to animal feed and includes vitamins of the desired type and amount. Some premixes include vitamins and trace minerals and optional formulation agents, preservatives, antibiotics, other feed ingredients, or any combination thereof, which are mixed with high protein and / or energy feeds (e.g., concentrates) before being given to poultry.

[0026] Sequence identity: The relatedness between two amino acid sequences is described by the parameter "sequence identity".

[0027] For purposes of the present invention, use as in EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Genetics Trend (Trends Genet.) 16:276-277) (preferred 3.0.0 version or updated version) Ni Deer (Needle) program implemented in Ni Deer (Needle) algorithm (Ni Deer (Needleman) and Weng Shi (Wunsch), 1970, Journal of Molecular Biology (J.Mol.Biol.) 48:443-453) determine the sequence identity degree between two amino acid sequences.Use version 6.1.0.The optional parameters used are gap opening penalty 10, gap extension penalty 0.5, and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.The output (using-non-simplified option to obtain) of " the longest consistency " of Ni Deer annotation is used as percentage consistency, and is calculated as follows:

[0028] (number of identical residues x 100) / (length of alignment - total number of gaps in the alignment)

[0029] Weight gain: The term "weight gain" means how much weight an animal gains over a certain period of time. Improved weight gain means an improved daily, weekly, biweekly or monthly weight gain (expressed in g or kg / relative period) relative to a control to which amylase is not added. This is preferably determined in an assay as described in the FCR paragraph above.

[0030] This application involves the following projects:

[0031] 1. Use of at least one bacterial amylase in poultry feed to improve the nutritional value of the feed, wherein the bacterial amylase is a polypeptide having at least 80% identity to amino acids 1-481 of SEQ ID NO: 2.

[0032] 2. The use as described in item 1, wherein the poultry feed has reduced apparent metabolizable energy compared to a standard poultry feed.

[0033] 3. The use according to any one of items 1 to 2, wherein the bacterial amylase is added to the poultry feed in a dosage of between 0.01 and 200 mg enzyme protein / kg diet.

[0034] 4. The use according to any one of items 1 to 3, for improving weight gain and / or feed conversion rate of poultry.

[0035] 5. A method for improving the nutritional value of poultry feed, the method comprising the step of adding to the feed at least one bacterial amylase, wherein the bacterial amylase is a polypeptide having at least 80% identity to amino acids 1-481 of SEQ ID NO:2.

[0036] 6. The method of claim 5, wherein the poultry feed has reduced apparent metabolizable energy compared to a standard poultry feed.

[0037] 7. The method of any one of items 5 to 6, wherein the bacterial amylase is added to the poultry feed in a dosage of between 0.01 and 200 mg enzyme protein / kg diet.

[0038] 8. The method according to any one of items 5 to 7, for improving weight gain and / or feed conversion rate of poultry.

[0039] 9. A poultry feed additive comprising at least one bacterial amylase, together with (i) at least one vitamin, (ii) at least one mineral, or (iii) at least one vitamin and at least one mineral, wherein the bacterial amylase is a polypeptide having at least 80% identity to amino acids 1-481 of SEQ ID NO:2.

[0040] 10. The poultry feed additive according to item 9, wherein the poultry feed additive improves the nutritional value of poultry feed.

[0041] 11. The poultry feed additive according to any one of items 9 to 10, wherein the poultry feed additive improves the weight gain or feed conversion rate of poultry.

[0042] 12. A poultry feed comprising the poultry feed additive according to any one of items 9 to 11 and 10w%-40w% soybean meal.

[0043] 13. The poultry feed of item 12, wherein the poultry feed has reduced apparent metabolizable energy compared to a standard poultry feed.

[0044] 14. The poultry feed of any one of items 12 to 13, wherein the bacterial amylase is added to the poultry feed in a dosage of between 0.01 and 200 mg enzyme protein / kg diet.

[0045] 15. The poultry feed according to any one of items 12 to 14, wherein the poultry feed improves weight gain and / or feed conversion rate of poultry. DETAILED DESCRIPTION OF THE INVENTION

[0047] It was surprisingly found that adding amylase to poultry feed improves the nutritional value of the feed. If the apparent metabolizable energy (AME) of the poultry feed is reduced by 100 kcal / kg, the weight gain and FCR of the poultry become worse compared to poultry on a standard diet. However, the inventors surprisingly found that including an amylase of the present invention in this reduced AME diet improves the weight gain and FCR of poultry.

[0048] Thus, in a first aspect, the present invention relates to a method for improving the nutritional value of poultry feed, the method comprising the step of adding to the feed at least one bacterial amylase, wherein the bacterial amylase is a polypeptide having at least 80% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 85% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 90% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 91% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 92% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 93% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 94% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 95% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 96% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 97% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 98% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 99% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the polypeptide comprises, or consists of, amino acids 1-481 of SEQ ID NO:2.

[0049] In a further aspect of the first aspect of the invention, the present invention relates to a method for improving the nutritional value of poultry feed, the method comprising the step of adding at least one bacterial amylase to the feed, wherein the bacterial amylase is a polypeptide having at least 80% identity to amino acids 1-481 of SEQ ID NO:2, and wherein the bacterial amylase improves the weight gain and / or feed conversion rate of poultry. In one embodiment, the bacterial amylase is a polypeptide having at least 85% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 90% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 91% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 92% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 93% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 94% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 95% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 96% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 97% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 98% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 99% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the polypeptide comprises, or consists of, amino acids 1-481 of SEQ ID NO:2.

[0050] In an embodiment of the first aspect, the bacterial amylase improves feed conversion rate. In one embodiment, the bacterial amylase improves weight gain. In a further embodiment, the bacterial amylase improves feed conversion rate and improves weight gain. In a preferred embodiment, in a diet, the bacterial amylase improves the feed conversion rate of poultry, wherein the poultry feed has a reduced apparent metabolizable energy compared to a standard poultry feed. In a preferred embodiment, in a diet, the bacterial amylase improves the weight gain of poultry, wherein the poultry feed has a reduced apparent metabolizable energy compared to a standard poultry feed. In a preferred embodiment, in a diet, the bacterial amylase improves feed conversion rate and weight gain of poultry, wherein the poultry feed has a reduced apparent metabolizable energy compared to a standard poultry feed.

[0051] In a further embodiment of the first aspect of the invention, the poultry feed has reduced apparent metabolizable energy compared to a standard poultry feed. In one embodiment, the apparent metabolizable energy is between 85% and 99.9% of a standard poultry feed, such as between 90% and 99.5%, between 91% and 99%, between 92% and 99%, between 93% and 98.5%, or between 94% and 98% of a standard poultry feed, wherein the energy of the standard poultry feed is 3050 kcal / kg for days 1-21 and 3170 kcal / kg for days 22-40.

[0052] In a further embodiment of the first aspect of the invention, the bacterial amylase is added to the poultry feed at a dose of between 0.01 and 200 mg enzyme protein / kg diet, such as 0.05-100, 0.1-50, 0.2-35, 0.4-20, 0.5-25, 0.6-15, 0.8-8, 1-10 or 1-6. mg enzyme protein / kg. In a further embodiment of the first aspect of the invention, the bacterial amylase is added to the poultry feed at a dose of between 1 and 5000 KNU / kg feed, such as 5-2500, 10-1000, 15-600, 20-400, 25-300, 30-200, 40-160 KNU / kg feed.

[0053] In a second aspect, the present invention relates to a method for improving the nutritional value of poultry feed, the method comprising the step of adding at least one bacterial amylase to the feed, wherein the bacterial amylase is a polypeptide having at least 80% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 85% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 90% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 91% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 92% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 93% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 94% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 95% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 96% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 97% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 98% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 99% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the polypeptide comprises, or consists of, amino acids 1-481 of SEQ ID NO:2.

[0054] In a further aspect of the second aspect of the invention, the present invention relates to a method for improving the nutritional value of poultry feed, the method comprising the step of adding at least one bacterial amylase to the feed, wherein the bacterial amylase is a polypeptide having at least 80% identity to amino acids 1-481 of SEQ ID NO:2, and wherein the bacterial amylase improves weight gain and / or feed conversion of poultry. In one embodiment, the bacterial amylase is a polypeptide having at least 85% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 90% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 91% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 92% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 93% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 94% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 95% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 96% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 97% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 98% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 99% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the polypeptide comprises, or consists of, amino acids 1-481 of SEQ ID NO:2.

[0055] In an embodiment of the second aspect, the bacterial amylase improves feed conversion efficiency. In one embodiment, the bacterial amylase improves weight gain. In a further embodiment, the bacterial amylase improves feed conversion efficiency and improves weight gain. In a preferred embodiment, in a diet, the bacterial amylase improves the feed conversion efficiency of poultry, wherein the poultry feed has a reduced apparent metabolizable energy compared to a standard poultry feed. In a preferred embodiment, in a diet, the bacterial amylase improves the weight gain of poultry, wherein the poultry feed has a reduced apparent metabolizable energy compared to a standard poultry feed. In a preferred embodiment, in a diet, the bacterial amylase improves feed conversion efficiency and weight gain of poultry, wherein the poultry feed has a reduced apparent metabolizable energy compared to a standard poultry feed.

[0056] In a further embodiment of the second aspect of the invention, the poultry feed has reduced apparent metabolizable energy compared to a standard poultry feed. In one embodiment, the apparent metabolizable energy is between 85% and 99.9% of a standard poultry feed, such as between 90% and 99.5%, between 91% and 99%, between 92% and 99%, between 93% and 98.5%, or between 94% and 98% of a standard poultry feed, wherein the energy of the standard poultry feed is 3050 kcal / kg for days 1-21 and 3170 kcal / kg for days 22-40.

[0057] In a further embodiment of the second aspect of the invention, the bacterial amylase is added to the poultry feed at a dosage between 0.01 and 200 mg enzyme protein / kg diet, such as 0.05-100, 0.1-50, 0.2-35, 0.4-20, 0.5-25, 0.6-15, 0.8-8, 1-10 or 1-6.mg enzyme protein / kg. In a further embodiment of the first aspect of the invention, the bacterial amylase is added to the poultry feed at a dosage between 1 and 5000 KNU / kg feed, such as 5-2500, 10-1000, 15-600, 20-400, 25-300, 30-200, 40-160 KNU / kg feed.

[0058] In a second aspect, the present invention relates to a poultry feed additive comprising at least one bacterial amylase, together with (i) at least one vitamin, (ii) at least one mineral, or (iii) at least one vitamin and at least one mineral, wherein the bacterial amylase is a polypeptide having at least 80% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 85% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 90% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 91% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 92% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 93% identity to amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 94% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 95% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 96% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 97% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 98% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 99% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the polypeptide comprises amino acids 1-481 of SEQ ID NO:2, or consists of it. In a preferred embodiment, the poultry feed additive improves the nutritional value of poultry feed.

[0059] In one embodiment of the third aspect of the invention, the invention relates to a poultry feed additive comprising at least one bacterial amylase, together with (i) at least one vitamin, (ii) at least one mineral, or (iii) at least one vitamin and at least one mineral, wherein the bacterial amylase is a polypeptide having at least 80% identity to amino acids 1-481 of SEQ ID NO: 2, and wherein the poultry feed additive improves weight gain or feed conversion of poultry. In one embodiment, the bacterial amylase is a polypeptide having at least 85% identity to amino acids 1-481 of SEQ ID NO: 2. In one embodiment, the bacterial amylase is a polypeptide having at least 90% identity to amino acids 1-481 of SEQ ID NO: 2. In one embodiment, the bacterial amylase is a polypeptide having at least 91% identity to amino acids 1-481 of SEQ ID NO: 2. In one embodiment, the bacterial amylase is a polypeptide having at least 92% identity to amino acids 1-481 of SEQ ID NO: 2. In one embodiment, the bacterial amylase is a polypeptide having at least 93% identity to amino acids 1-481 of SEQ ID NO: 2. In one embodiment, the bacterial amylase is a polypeptide having at least 94% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 95% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 96% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 97% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 98% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the bacterial amylase is a polypeptide having at least 99% identity with amino acids 1-481 of SEQ ID NO:2. In one embodiment, the polypeptide comprises amino acids 1-481 of SEQ ID NO:2, or consists of it. In a preferred embodiment, the poultry feed additive improves the nutritional value of poultry feed.

[0060] In a fourth aspect, the present invention relates to a poultry feed comprising the poultry feed additive of the third aspect and 10w%-40w% of soy flour. In a preferred embodiment, the poultry feed comprising the poultry feed additive of the third aspect and 10w%-40w% of soy flour improves the feed conversion rate of poultry. In a preferred embodiment, the poultry feed comprising the poultry feed additive of the third aspect and 10w%-40w% of soy flour improves the weight gain of poultry. In a preferred embodiment, the poultry feed comprising the poultry feed additive of the third aspect and 10w%-40w% of soy flour improves the weight gain and / or feed conversion rate of poultry.

[0061] In an embodiment of the fourth aspect, the bacterial amylase improves feed conversion rate. In one embodiment, the bacterial amylase improves weight gain. In a further embodiment, the bacterial amylase improves feed conversion rate and improves weight gain. In a preferred embodiment, in a diet, the bacterial amylase improves the feed conversion rate of poultry, wherein the poultry feed has a reduced apparent metabolizable energy compared to a standard poultry feed. In a preferred embodiment, in a diet, the bacterial amylase improves the weight gain of poultry, wherein the poultry feed has a reduced apparent metabolizable energy compared to a standard poultry feed. In a preferred embodiment, in a diet, the bacterial amylase improves feed conversion rate and weight gain of poultry, wherein the poultry feed has a reduced apparent metabolizable energy compared to a standard poultry feed.

[0062] In a further embodiment of the fourth aspect of the invention, the poultry feed has reduced apparent metabolizable energy compared to a standard poultry feed. In one embodiment, the apparent metabolizable energy is between 85% and 99.9% of a standard poultry feed, such as between 90% and 99.5%, between 91% and 99%, between 92% and 99%, between 93% and 98.5%, or between 94% and 98% of a standard poultry feed, wherein the energy of the standard poultry feed is 3050 kcal / kg for days 1-21 and 3170 kcal / kg for days 22-40.

[0063] In a further embodiment of the fourth aspect of the invention, the bacterial amylase is added to the poultry feed at a dose of between 0.01 and 200 mg enzyme protein / kg diet, such as 0.05-100, 0.1-50, 0.2-35, 0.4-20, 0.5-25, 0.6-15, 0.8-8, 1-10 or 1-6. mg enzyme protein / kg. In a further embodiment of the first aspect of the invention, the bacterial amylase is added to the poultry feed at a dose of between 1 and 5000 KNU / kg feed, such as 5-2500, 10-1000, 15-600, 20-400, 25-300, 30-200, 40-160 KNU / kg feed.

[0064] In a specific embodiment, the amylase in the form of being added to the feed or when contained in the feed additive is clearly defined. It is clearly defined that the amylase preparation is at least 50% pure on a protein basis. In other specific embodiments, the amylase preparation is at least 60%, 70%, 80%, 85%, 88%, 90%, 92%, 94%, or at least 95% pure. Purity can be determined by any method known in the art, such as by SDS-PAGE, or by size exclusion chromatography (see Example 12 of WO01 / 58275).

[0065] Well-defined amylase preparations are advantageous. For example, it is much easier to dose correctly an amylase that is substantially free of interfering or contaminating other enzymes into the feed. The term dosed correctly specifically refers to the goal of obtaining consistent and constant results, and the ability to optimize the dose based on the desired effect.

[0066] Specifically, amylase preparations with the above-mentioned purity levels can be obtained using recombinant production methods, whereas these amylase preparations are not easy to obtain when produced by traditional fermentation methods and there are high variability between batches.

[0067] The bacterial amylase for purposes according to the invention is included in a poultry diet or poultry feed additive in an effective amount. It is now desired that the effective amount is less than 400 mg enzyme protein / kg diet dry matter, preferably less than 350, 300, 250, 200, 150, 100 or less than 50 mg enzyme protein / kg diet dry matter (ppm). On the other hand, the effective amount can be higher than 0.01 mg enzyme protein / kg diet dry matter, preferably higher than 0.5, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.75 or higher than 1 mg enzyme protein / kg diet dry matter (ppm). Therefore, non-limiting examples of preferred dosage ranges are: 0.01-200 mg enzyme protein / kg, preferably 0.05-100, 0.1-50, 0.5-25 or 1-10 mg enzyme protein / kg. Additional examples of preferred dosage ranges, all in mg enzyme protein / kg, are: 0.2-35, 0.4-20, 0.6-15, 0.8-8, and 1-6. In other non-limiting examples, preferred dosage ranges for bacterial amylases of the invention are between 1 and 5000 KNU / kg feed, such as 5-2500, 10-1000, 15-600, 20-400, 25-300, 30-200, 40-160 KNU / kg feed.

[0068] To determine the mg of amylase protein per kg of feed, amylase is purified from the feed composition and the specific activity of the purified amylase is determined using the desired amylase assay. The amylase activity of the feed composition may also be similarly determined using the same assay and based on these two determinations, the dosage expressed as mg amylase protein / kg of feed is calculated.

[0069] The same rules apply to determining the mg of amylase protein in the feed additive. Of course, if the sample uses an amylase used in the preparation of a feed additive or feed, the specific activity can be determined from this sample (without the need to purify the amylase from the feed composition or additive).

[0070] In order to classify and identify bacteria taxonomically, reference is made to Bergey's Manual of Systematic Bacteriology (1986), Vol. 2, ISBN 0-683-0783. Alternatively, well-known 16S rRNA sequence analysis can be used (see, for example, Johansen et al., International Journal of Systematic Bacteriology (Int. J. Syst. Bacteriol), 1999, 49, 1231-1240, particularly the methods section of the second column of page 1233); or a taxonomic expert can be consulted, for example, a taxonomic expert from DSMZ or other recognized depository institutions.

[0071] As used herein, the term bacterial indicates that the amylase is derived from bacteria. The term "derived from" includes enzymes that can be obtained from or obtained from wild-type bacterial strains, and variants thereof. Variants may have at least one substitution, insertion and / or deletion of at least one amino acid residue. The term variant also includes shufflant, hybrid, chimeric enzyme and consensus enzyme. Variants can be produced by any method known in the art, such as site-directed mutagenesis, random mutagenesis, consensus derivation process (EP 897985) and gene rearrangement (WO 95 / 22625, WO 96 / 00343), etc. For the present purpose, when at least one bacterial amylase is used to design, derive or prepare a variant, an amylase variant may be referred to as bacterial. The term bacterial does not refer to a possible recombinant production host, but only to the host origin of the amylase encoding gene.

[0072] Amylases for use according to the invention are preferably derived from strains of the genus Bacillus, such as strains of Bacillus amyloliquefaciens, Bacillus circulans, Bacillus halmapalus, Bacillus licheniformis, Bacillus megaterium, Bacillus sp., Bacillus stearothermophilus, and Bacillus subtilis; preferably strains from Bacillus amyloliquefaciens, Bacillus halmapalus, Bacillus licheniformis, Bacillus sp., Bacillus subtilis, and Bacillus stearothermophilus.

[0073] Examples of wild-type amylases are those derived from Bacillus licheniformis, such as the Swissprot entry name AMY_BACLI, the primary accession number P06278 (SEQ ID NO: 8), and the commercial amylase sold under the trade name DURAMYL by Novozymes; those derived from Bacillus amyloliquefaciens, such as the Swissprot entry name AMY_BACAM, the primary accession number P00692 (SEQ ID NO: 7), and the commercial amylase sold under the trade name BAN by Novozymes; those derived from Bacillus megaterium, such as the Swissprot entry name AMY_BACME, the primary accession number P20845; those derived from Bacillus circulans, such as the Swissprot entry name AMY_BACCI, the primary accession number P08137; those derived from Bacillus stearothermophilus, such as the Swissprot entry name AMY_BACST, the primary accession number P06279 (SEQ ID NO:9), and commercial amylases sold under the trade name TERMAMYL SC by Novozymes. Another example is derived from Bacillus subtilis, as shown in the Swissprot entry name AMY_BACSU, primary accession number P00691.

[0074] Examples of amylases contained in commercial products are: BAN, Stainzyme, Termamyl SC, Natalase, and Duramyl (all from Novozymes), and in the products Validase BAA and Validase HT (from Valley Research). Further specific examples of amylases for use according to the invention are the amylases contained in the following commercial products: Clarase, DexLo, GC 262SP, G-Zyme G990, G-Zyme G995, G-Zyme G997, G-Zyme G998, HTAA, Optimax 7525, Purastar OxAm, Purastar ST, Spezyme AA, Spezyme α, Spezyme BBA, Spezyme δAA, Spezyme DBA, Spezyme Ethyl, Spezyme Fred (GC521), Spezyme HPA, and Ultraphlow (all from Genencor); Validase HT340L, Valley Thin 340L (all from Valley Research); Avizyme 1500, Dextro 300L, Kleistase, Maltazyme, Maxamyl, Thermozyme, Thermatex, Starzyme HT 120L, Starzyme Super Conc, and Ultraphlo.

[0075] Additional non-limiting examples of amylases are:

[0076] An amylase having, comprising or consisting of amino acids 1-481, 1-484, 1-486, or 1-513 of SEQ ID NO: 2 (wherein "1" refers to the starting amino acid of the mature peptide, Ala, with reference to the sequence listing);

[0077] An amylase having, comprising or consisting of amino acids 1-483 of SEQ ID NO: 4;

[0078] An amylase having, comprising or consisting of amino acids 1-483 of SEQ ID NO:5;

[0079] An amylase having, comprising or consisting of amino acids 1-481 of SEQ ID NO: 6 (wherein "1" refers to the starting amino acid of the mature peptide, Val, with reference to the sequence list);

[0080] An amylase having, comprising or consisting of amino acids 1-483 of SEQ ID NO: 7 (wherein "1" refers to the starting amino acid of the mature peptide, Val, with reference to the sequence list);

[0081] An amylase having, comprising or consisting of amino acids 1-483 of SEQ ID NO: 8 (wherein "1" refers to the starting amino acid of the mature peptide, Ala, with reference to the sequence listing); and

[0082] An amylase having, comprising or consisting of amino acids 1-515 of SEQ ID NO: 9 (wherein "1" refers to the starting amino acid of the mature peptide, Ala, with reference to the sequence listing);

[0083] Together with any fragments or variants of the above-specified amylases that retain amylase activity.

[0084] Fragments are polypeptides that have one or more amino acids missing at the amino and / or carboxyl termini. Preferably, the fragment comprises at least 450 amino acid residues, more preferably at least 460 amino acid residues, even more preferably at least 470 amino acid residues, and most preferably at least 480 amino acid residues. Additionally preferred fragments comprise at least 481, 483, 484, or at least 513 amino acid residues. Examples of enzymatically active fragments of the amylase of SEQ ID NO: 2 are sequences having amino acids 1-481, 1-484, and 1-486 thereof.

[0085] In a particular embodiment, the amylase for use according to the invention is granulation stable and / or thermostable. The dissolution temperature (Tm) of an enzyme is a measure of its thermostability. The amylase of the invention can have a Tm of at least 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C or at least 95°C, as determined by differential scanning calorimetry (DSC). DSC is carried out in 10 mM sodium phosphate, 50 mM sodium chloride buffer (pH 7.0). The scan rate is constant, e.g., 1.5°C / min. The scan interval can be from 20°C to 100°C. Another buffer can be selected for scanning, e.g., a buffer of pH 5.0, 5.5, 6.0 or pH 6.5. In a further alternative embodiment, a higher or lower scan rate may be used, such as a slower scan rate of 1.4°C / min, 1.3°C / min, 1.2°C / min, 1.1°C / min, 1.0°C / min, or 0.9°C / min.

[0086] In another preferred embodiment, the amylase for use according to the invention has at least 35% activity at pH 7.0 and 37° C. relative to the activity at the optimum pH and 37° C. More preferably, the activity at pH 7.0 and 37° C. is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, or at least 75% of the activity at the optimum pH and 37° C. (see Table 1 of Example 2).

[0087] In another preferred embodiment, the amylase of the invention has at least 25% activity at pH 7.0 and 37° C. and in the presence of 5 mM bile salts relative to the activity at the optimum pH and 37° C. in the absence of bile salts. More preferably, the activity at pH 7.0 and 37° C. and in the presence of 5 mM bile salts is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, or at least 65% of the activity at the optimum pH and 37° C. and in the absence of bile salts (see Table 2 of Example 2).

[0088] In still further more preferred embodiments, the specific activity of the amylase of the invention at pH 7.0 and 37°C is at least 10%, more preferably at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or at least 70% relative to the specific activity of the amylase of TERMAMYL SC at pH 5.0 and 37°C (see Table 3 of Example 2).

[0089] In another preferred embodiment, the specific activity of the amylase of the present invention at pH 7.0 and 37°C and in the presence of 5 mM bile salts is at least 10%, more preferably at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or at least 75% relative to the specific activity of the amylase of TERMAMYL SC at pH 5.0 and 37°C and in the presence of 5 mM bile salts (see Table 4 of Example 2).

[0090] The activities mentioned in the preferred embodiments above can be suitably determined using a reducing sugar assay, for example, as described in Example 2, preferably using waxy corn as a substrate. The detailed process is described in Example 2.

[0091] In another specific embodiment, the amylase for purposes according to the present invention is stable in the presence of a protease. The example of a protease is a digestive protease and a feed protease, such as described in, for example, WO 01 / 58275, WO01 / 58276, WO 2004 / 111220 2004 / 111221, WO 2004 / 072221 and WO 2005 / 035747. The example of a digestive protease is pancreatin and pepsin. Can be in the presence of a protease (e.g., pepsin, 70mg / l), in a buffer, with a desired pH (e.g., pH 2,3,4 or 5), by hatching 0.5mg of the purified amylase protein / ml for a desired time (e.g., 30,45,60,90 or 120 minutes), and then increase pH to a desired pH (e.g., pH 4,5,6 or 7) and use, for example, the reducing sugar assay of Example 2 herein to measure residual activity to determine protease stability. Relative to a control (non-protease treated sample), the residual amylase activity is preferably at least 20%, preferably at least 30%, 40%, 50%, 60%, 70%, 80%, or at least 90%.

[0092] Poultry feed and poultry feed additives

[0093] The invention also relates to poultry feed and poultry feed additives comprising an amylase according to the invention. Preferably, the poultry feed or poultry feed additive is enriched with an amylase according to the invention. The term "enriched" indicates that the amylase activity of the composition has been increased, e.g., the enrichment factor is at least 1.1, such as at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 2.0, at least 3.0, at least 4.0, at least 5.0, at least 10.

[0094] In one embodiment, the poultry feed or poultry feed additive comprises polypeptide of the present invention and one or more formulations. In one embodiment, the poultry feed or poultry feed additive comprises polypeptide of the present invention, together with (i) at least one vitamin, (ii) at least one mineral or (iii) at least one vitamin and at least one mineral. In one embodiment, the poultry feed or poultry feed additive comprises polypeptide of the present invention, together with one or more formulations and (i) at least one vitamin, (ii) at least one mineral or (iii) at least one vitamin and at least one mineral. In one embodiment, the poultry feed or poultry feed additive further comprises one or more amino acids. In one embodiment, the poultry feed or poultry feed additive further comprises one or more other feed ingredients. In one embodiment, the poultry feed or poultry feed additive further comprises one or more amino acids and one or more other feed ingredients.

[0095] In one embodiment, this poultry feed additive is made up of polypeptide of the present invention and one or more components from the following group, and this group is made up of the following: vitamins, mineral substances, formulations and other feed ingredients. In one embodiment, this poultry feed additive is made up of polypeptide of the present invention, one or more vitamins and one or more formulations. In one embodiment, this poultry feed additive is made up of polypeptide of the present invention, one or more mineral substances and one or more formulations. In one embodiment, this poultry feed additive is made up of polypeptide of the present invention, one or more mineral substances, one or more vitamins and one or more formulations. In one embodiment, this poultry feed additive is made up of polypeptide of the present invention, one or more mineral substances, one or more vitamins, one or more other feed ingredients and one or more formulations.

[0096] In one embodiment, the poultry feed is composed of a polypeptide of the present invention, one or more concentrates and one or more components, and the one or more components are from the group consisting of the following: vitamins, minerals, formulations and other feed ingredients. In one embodiment, the poultry feed is composed of a polypeptide of the present invention, one or more concentrates, one or more vitamins and one or more formulations. In one embodiment, the poultry feed is composed of a polypeptide of the present invention, one or more concentrates, one or more minerals and one or more formulations. In one embodiment, the poultry feed is composed of a polypeptide of the present invention, one or more concentrates, one or more minerals, one or more vitamins and one or more formulations. In one embodiment, the poultry feed is composed of a polypeptide of the present invention, one or more concentrates, one or more minerals, one or more vitamins, one or more other feed ingredients and one or more formulations.

[0097] The poultry feed has a crude protein content of 50 to 800 g / kg and furthermore comprises at least one amylase as claimed herein.

[0098] In addition to the amylase for use according to the invention as described above, the final poultry feed composition according to the invention comprises at least soy flour and a trace mineral and / or vitamin premix as described above. For example, the feed composition comprises 10w%-40w% soy flour and 0.5w%-2w% trace minerals and vitamins.

[0099] The dietary content of calcium, available phosphorus and amino acids in the complete animal diet is calculated on the basis of feed tables (e.g. Feed Tables (Veevoedertabel) 1997, Chemical Composition Data (gegevens over chemische samenstelling), Digestibility and Nutritional Value of Feeds (verteerbaarheid en voederwaarde van voedermiddelen), Central Feed Bureau (Central Veevoederbureau), Runderweg 6, 8219pk Lelystad, ISBN 90-72839-13-7).

[0100] In a preferred embodiment, the poultry feed composition comprises, for each kg of feed: vitamin A, 7,000-9,000 UI; vitamin D3, 2,000-2,500 UI; vitamin E, 15-20 mg; vitamin K3, 1.5-2.5 mg; thiamine, 1-2 mg; riboflavin, 6-7 mg; pyridoxine, 2-3 mg; pantothenic acid, 10-12 mg; folic acid, 0.5-1.5 mg; biotin, 0.1-1 mg; iron, 100-150 mg; zinc, 100-150 mg; manganese, 100-150 mg; cobalt, 1-2 mg; copper, 10-20 mg.

[0101] Crude protein is calculated by multiplying nitrogen (N) by a factor of 6.25, i.e. crude protein (g / kg) = N (g / kg) X 6.25. Nitrogen content is determined by the Kjeldahl method (AOAC, 1984, Official Methods of Analysis 14th Edition, Association of Official Analytical Chemists, Washington, DC).

[0102] Metabolizable energy can be calculated on the basis of the European Table of Energy Values ​​for Poultry Feed-stuffs, Spelderholt Poultry Research and Extension Center, 7361DA Beekbergen, The Netherlands, Grafisch bedrijf Ponsen & looijen, Wageningen. ISBN 90-71463-12-5.

[0103] The poultry feed of the present invention may also contain animal protein, such as meat and bone meal, feather meal, and / or fish meal, typically in an amount of 0-25%. The poultry feed of the present invention may also contain distillers dried grains with solubles (DDGS), typically in an amount of 0-30%.

[0104] In yet further specific embodiments, the poultry feed of the present invention comprises 0-80% corn; and / or 0-80% sorghum; and / or 0-70% wheat; and / or 0-70% barley; and / or 0-30% oats; and / or 10%-40% soybean meal; and / or 0-25% fish meal; and / or 0-25% meat and bone meal; and / or 0-20% whey.

[0105] The animal feed may include vegetable protein. In specific embodiments, the protein content of the vegetable proteins is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (w / w). The vegetable protein may be derived from vegetable protein sources such as beans and cereals, for example, materials from plants of the Fabaceae (Leguminosae), Cruciferae, Chenopodiaceae, and Poaceae, such as soy meal, lupin meal, rapeseed meal, and combinations thereof.

[0106] In a specific embodiment, the vegetable protein source is a material from one or more plants of the Fabaceae family, such as soybeans, lupines, peas, or beans. In another specific embodiment, the vegetable protein source is a material from one or more plants of the Chenopodiaceae family, such as beets, sugar beets, spinach, or quinoa. Other examples of vegetable protein sources are rapeseed and cabbage. In another specific embodiment, soybean is a preferred vegetable protein source. Other examples of vegetable protein sources are cereals, such as barley, wheat, rye, oats, maize (corn), rice, and sorghum.

[0107] Preparation agent

[0108] The poultry feed additive of the present invention can be formulated as a liquid or solid. For liquid formulations, the formulation can include a polyol (such as, for example, glycerol, ethylene glycol or propylene glycol), a salt (such as, for example, sodium chloride, sodium benzoate, potassium sorbate) or a sugar or sugar derivative (such as, for example, dextrin, glucose, sucrose and sorbitol). Therefore, in one embodiment, the poultry feed additive is a liquid composition comprising a polypeptide of the present invention and one or more formulations selected from the following list, the list consisting of the following: glycerol, ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, dextrin, glucose, sucrose and sorbitol.

[0109] For solid formulations, the formulation can be, for example, as granules, spray-dried powders or agglomerates. Formulation agents can include salts (organic or inorganic zinc, sodium, potassium or calcium salts, for example, such as calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch or sugar or sugar derivatives (such as, for example, sucrose, dextrin, glucose, lactose, sorbitol).

[0110] In one embodiment, the solid poultry feed additive is in the form of particles. The particles can have a matrix structure in which the components are uniformly mixed. However, the particles typically include a core particle and one or more coatings, which are typically salt and / or waxy coatings. The core particle can be a uniform mixture of an amylase of the present invention and optionally one or more salts or inert particles (having an amylase of the present invention applied thereto).

[0111] In one embodiment, the material of the core particle is selected from the group consisting of inorganic salts (such as calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch or sugar or sugar derivatives (such as, for example, sucrose, dextrin, glucose, lactose, sorbitol), sugar or sugar derivatives (such as, for example, sucrose, dextrin, glucose, lactose, sorbitol), small organic molecules, starch, flour, cellulose and minerals.

[0112] The salt coating is typically at least 1 μm thick and may be a specific salt or a mixture of salts, for example Na2SO4, K2SO4, MgSO4 and / or sodium citrate. Other examples are those described in, for example, WO 2008 / 017659, WO 2006 / 034710, WO 1997 / 05245, WO 1998 / 54980, WO1998 / 55599, WO 2000 / 70034, or polymer coatings such as described in WO 2001 / 00042.

[0113] In another embodiment, the poultry feed additive is a solid composition comprising amylase of the present invention and one or more formulations selected from the following list, the list consisting of the following: sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch and cellulose. In a preferred embodiment, the formulation is selected from one or more of the following compounds: sodium sulfate, dextrin, cellulose, sodium thiosulfate and calcium carbonate. In a preferred embodiment, the solid composition is in granular form. In one embodiment, the solid composition is in granular form and comprises a core particle, an enzyme layer comprising amylase of the present invention and a salt coating.

[0114] In other embodiments, the compounding agent is selected from one or more of the following compounds: glycerol, ethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch and cellulose. In a preferred embodiment, the compounding agent is selected from one or more of the following compounds: 1,2-propylene glycol, 1,3-propylene glycol, sodium sulfate, dextrin, cellulose, sodium thiosulfate and calcium carbonate.

[0115] Vitamins and minerals

[0116] In another embodiment, the poultry feed or poultry feed additive may include one or more vitamins, such as one or more fat-soluble vitamins and / or one or more water-soluble vitamins. In another embodiment, the poultry feed or poultry feed additive may optionally include one or more minerals, such as one or more trace minerals and / or one or more macro minerals.

[0117] Typically fat-soluble and water-soluble vitamins and trace minerals form part of a so-called premix for addition to feed, whereas macrominerals are usually added to the feed separately.

[0118] Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, such as vitamin K3.

[0119] Non-limiting examples of water-soluble vitamins include vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid, and panthothenate, such as Ca-D-pantothenate.

[0120] Non-limiting examples of trace minerals include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, and zinc.

[0121] Non-limiting examples of macrominerals include calcium, magnesium, potassium, and sodium.

[0122] In Table A of WO 01 / 58275 the nutritional requirements of these components are listed (for poultry and piglets / pigs as examples). The nutritional requirements refer to the specified concentrations of these components that should be provided in the diet.

[0123] In an alternative embodiment, the animal feed additive of the present invention comprises at least one of the individual components specified in Table A of WO 01 / 58275. At least one refers to any one, one or more of one or two or three or four etc. up to all thirteen, or up to all fifteen individual components. More specifically, the at least one individual component is included in the additive of the present invention in an amount to provide an in-feed-concentration within the range specified in the fourth column or the fifth column or the sixth column of Table A.

[0124] Amino Acids

[0125] The composition of the invention may also include one or more amino acids. Examples of amino acids for animal feed are lysine, alanine, beta-alanine, threonine, methionine and tryptophan.

[0126] Other feed ingredients

[0127] Poultry feed or poultry feed additives comprising the amylase of the invention may further include colorants, stabilizers, growth improving additives and flavor compounds / flavorings, polyunsaturated fatty acids (PUFA); reactive oxygen generating substances, antimicrobial peptides and antifungal polypeptides.

[0128] Examples of colorants are carotenoids, such as beta-carotene, astaxanthin, and lutein.

[0129] Examples of flavouring compounds / flavours are cresols, anisole, deca-, undeca- and / or dodeca-lactones, ionones, irones, gingerol, piperidine, propylidene phatalide, butylidene phatalide, capsaicin and tannins.

[0130] Examples of antimicrobial peptides (AMPs) are CAP18, Leucocin A, Tritrpticin, Protegrin-1, Thanatin, Defensin, Lactoferrin, Lactoferrin peptides, and Ovispirin such as Novispirin (Robert Lehrer, 2000), Plectasins and Statins, including compounds and polypeptides disclosed in WO 03 / 044049 and WO 03 / 048148, and variants or fragments thereof that retain antimicrobial activity.

[0131] Examples of antifungal polypeptides (AFPs) are peptides of Aspergillus giganteus and Aspergillus niger, together with variants and fragments thereof which retain antifungal activity, as disclosed in WO 94 / 01459 and WO 02 / 090384.

[0132] Examples of polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids such as arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid.

[0133] Examples of reactive oxygen generating species are chemicals such as perborate, persulfate or percarbonate; and enzymes such as oxidases, oxygenases or synthetases.

[0134] The present invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

[0135] Examples

[0136] Example 1: α-Amylase activity

[0137] The α-amylase activity was determined using the AMYK-kit commercially available from Roche Diagnostics with catalog number 11876473. The substrate was 4,6-ethylidene(G7)-p-nitrophenyl(G1)-α,D-maltoheptaside (ethylidene-G7PNP). α-amylase splits off ethylidene-G n , and the resulting G is then cleaved by the enzyme α-glucosidase (part of the kit) with the formation of glucose and yellow p-nitrophenol n -p-Nitrophenyl.

[0138] The rate of formation of p-nitrophenol (which is a measure of the reaction rate and thus of the alpha-amylase activity) is observed at 405 nm, for example with a Konelab 30 analyser (commercially available from Thermo Electron Corporation), for example using a measurement time of 2 min.

[0139] The reaction conditions are: temperature 37°C, pH: 7.0, reaction time: 5 min. Calcium chloride 0.03 M with Brij 0.0025% (Sigma B 4184) is preferably used as a stabilizer. The alpha-amylase activity may be given relative to a standard, for example in units of KNU(S), which is determined relative to an alpha-amylase standard of known KNU(S) activity.

[0140] A more detailed assay description (EB-SM-0221.02) as well as the KNU(S) TERMAMYL SC standard are available from Novozymes (Kroger Huyol 36, DK-2880 Bagsveld) on request.

[0141] Example 2: Amylase pH curves with and without bile salts

[0142] This experiment was used to determine the pH profiles of three alpha-amylases, two bacterial amylases of the invention and a prior art fungal Aspergillus oryzae amylase, with and without added bile salts.

[0143] The amylases used were purified Bacillus stearothermophilus amylase variant (from TERMAMYL SC), purified Bacillus sp. amylase variant (from STAINZYME), and for comparison purified Aspergillus oryzae amylase (from FUNGAMYL). All of these enzyme preparations are commercially available from Novozymes (Kroger Hujol 36, DK-2880 Bagsveld, Denmark).

[0144] Reducing sugar determination

[0145] Enzyme buffer: 50 mM acetate, 50 mM imidazole, 50 mM malonic acid, 1 mM CaCl2, 0.01% Triton X-100. Adjust to pH 2.0, 3.0, 4.0, 5.0, 6.0 or 7.0 with HCl / NaOH.

[0146] Substrate buffer: 1.5 mg / ml pullulan (waxy corn, e.g. waxy corn 04201 from Cerestar, batch number WM5671), 50 mM acetate, 50 mM imidazole, 50 mM malonic acid, 1 mM CaCl2. Adjust to desired pH with HCl / NaOH (as above). Incubate at 100°C for 5 min. Prepare substrate buffer with or without 5 mM bile salts (i.e. sodium taurocholate, 500 g / mol, commercially available from, e.g., LGC promochem).

[0147] Amylase activity was detected with reducing sugar assay. In brief, 50 μl enzyme (diluted in enzyme buffer so that it falls within the linear range of the assay) was mixed with 100 μl substrate buffer in PCR-MTP (Thermo-Fast96, ABgene, catalog number AB-0600). MTP was incubated at 37 ° C for 15 min, after which 75 μl stop solution (100 mM p-hydroxybenzoic acid hydrazide, 180 mM K-Na-tartaric acid, 2% NaOH) was added, and the plates were incubated at 95 ° C for 10 min. 150 μl was then transferred from each well to 96-well MTP, and at 410 nm, absorbance was monitored as a measure of amylase activity.

[0148] The results (average of two determinations) are shown in Tables 1-4 below. Table 1 shows the activity of each enzyme at the indicated pH in the absence of bile salts. For each enzyme, the maximum activity is set to 100%. Table 2 shows the same as Table 1, but in the presence of 5 mM bile salts. Table 3 shows the activity of each enzyme for each mg of enzyme protein at the indicated pH in the absence of bile salts, relative to the maximum enzyme activity determined in this experiment, which is the activity of the TERMAMYL SC enzyme at pH 5.0 (100%). Therefore, the activity of each enzyme has been normalized relative to this activity. On the basis of the specific activity, the amount of enzyme protein of each enzyme is determined. Table 4 shows the same as Table 3, but in the presence of 5 mM bile salts. Here, the activity of the TERMAMYL SC enzyme at pH 5.0 in the presence of 5 mM bile salts is a reference value (100%).

[0149] Table 1: Relative activity in the absence of bile salts

[0150] Enzyme / pH 2 3 4 5 6 7 FUNGAMYL 0.0 0.0 77.4 93.4 100.0 25.6 STAINZYME 0.3 0.8 2.8 22.2 79.7 100.0 TERMAMYL SC 0.1 1.8 29.4 100.0 86.0 71.1

[0151] Table 2: Relative activity in the presence of bile salts

[0152] Enzyme / pH 2 3 4 5 6 7 FUNGAMYL 0.0 0.0 53.5 71.8 68.6 16.1 STAINZYME 0.0 0.0 0.8 2.5 61.4 78.1 TERMAMYL SC 0.0 0.0 10.4* 76.0 68.6 59.7

[0153] *One measurement discarded due to obvious error

[0154] Table 3: Absolute activity normalized to TERMAMYL SC in the absence of bile salts

[0155] Enzyme / pH 2 3 4 5 6 7 FUNGAMYL 0.0 0.0 10.9 13.2 14.1 3.6 STAINZYME 0.1 0.4 1.4 10.7 38.3 48.0 TERMAMYL SC 0.1 1.8 29.4 100.0 86.0 71.1

[0156] Table 4: Absolute activity normalized to TERMAMYL SC in the presence of bile salts

[0157] Enzyme / pH 2 3 4 5 6 7 FUNGAMYL 0.0 0.0 9.9 13.3 12.7 3.0 STAINZYME 0.0 0.0 0.5 1.6 38.8 49.3 TERMAMYL SC 0.0 0.0 13.7 100.0 90.2 78.6

[0158] These results show that although bile salts appear to slightly reduce the amylase activity, the activity is still satisfactory in the presence of 5 mM bile salts. These results also show that bile salts do not lead to a shift in the optimal pH.

[0159] Furthermore, these results show that at pH 7, each of the Bacillus amylases of the invention had a relative activity of more than 50%, which was not the case for the comparative fungal amylases.

[0160] Finally, Tables 3 and 4 demonstrate that, at least under these conditions, the amylase from TERMAMYL SC has significantly higher activity / mg enzyme than the other two amylases tested.

[0161] Example 3: Broiler chicken feed (“starter”)

[0162] A broiler feed ("early stage") comprising a mixture of compounds according to the invention can be prepared by mixing the following ingredients together at room temperature using conventional mixing equipment.

[0163]

[0164] Example 4: Broiler Feed ("Grower")

[0165] Broiler feed ("growing period") containing a mixture of compounds according to the invention can be prepared by mixing the following ingredients together at room temperature using conventional mixing equipment.

[0166]

[0167] Example 5: Apparent Metabolizable Energy of a Diet

[0168] In vivo trials were conducted on the vital performance of broiler chickens that had been fed a basal diet as specified in Examples 3 and 4, based on soya flour and supplemented with TERMAMYL SC.

[0169] The study was conducted with a basal diet and with a corn / soy diet comprising corn replacing 40% of the soy. In the trial, TERMAMYL SC was used in graded inclusions of 40 KNU / kg feed, 80 KNU / kg feed, 120 KNU / kg feed and 160 KNU / kg feed.

[0170] Table 5: Summary of experimental treatments (7 birds / cage treated with 9 replicates)

[0171] deal with describe T1 Basic diet T2 60% basal diet + 40% corn diet (soybean meal replaced by corn) T3 T1+40KNU / kg feed RZ TERMAMYL SC T4 T1+80KNU / kg feed RZ TERMAMYL SC T5 T1+120KNU / kg feed RZ TERMAMYL SC T6 T1+160KNU / kg feed RZ TERMAMYL SC T7 T2+40KNU / kg feed RZ TERMAMYL SC T8 T2+80KNU / kg feed RZ TERMAMYL SC T9 T2+120KNU / kg feed RZ TERMAMYL SC T10 T2+160KNU / kg feed RZ TERMAMYL SC

[0172] animal

[0173] A total of 630 birds were delivered as day-old Cobb 500 male chickens. For the trial, a completely randomized design consisting of 10 treatments with 9 replicates per treatment (7 birds per treatment) was used, with treatment groups assigned to wire cages.

[0174] nest

[0175] The experiments were carried out at the UFPR experimental unit. The treatment number was shown on the cage or pen, the number of replicates and the treatment group had to be visible at each pen. The maximum number of birds per pen and the maximum number of spaces per bird were in accordance with current legislation. Feed and water were provided for ad libitum access. The birds were housed in metal cages equipped with drinking troughs and feeders. The temperature and lighting regime were in accordance with the recommendations of broiler farms. On a daily basis, all pens were checked for sick and dead birds. The identification number, pen, age and weight of each dead bird were recorded on the pen record form. The general health status, weight, mortality and cause of death and fill grade of the dead birds were recorded together with morphological changes / symptoms.

[0176] Measurement

[0177] Animals consumed the same diet until 14 days of age, and thereafter the experimental diet was formulated by adding acid insoluble ash (AIA) as a marker, using both based on Rostano et al. (2011). After the experimental diet adaptation period, fecal sampling was maintained for four days (21 to 24 days) by partial collection. There were two collections per day, one in the morning and the other in the evening, at about 9:00 am and 17:30 pm, respectively, with a plastic scraper, avoiding the collection of feces near the adjacent cages, troughs and drinking troughs. These samples were then placed in a freezer, and just after sampling, the freezer was cleaned of remaining feces to prevent contamination of the next sampling. Subsequently, the fecal specimens were dried in a forced ventilation oven at 65°C until constant weight.

[0178] These chickens were fasted for two hours and fed for one and a half hours to fill the digestive tract for ileal digestibility measurement. The birds were slaughtered by cervical dislocation and the ileal contents were taken 4 cm below Meckel's diverticulum and 4 cm above the ileocecal region. The content samples were quickly frozen in liquid nitrogen and then stored in a freezer. Subsequently, the contents were freeze-dried.

[0179] Diet, feces and ileal contents were analyzed for dry matter (DM), crude protein (CP), gross energy (GE), fat (F) and acid insoluble ash (AIA). Before calculating metabolism and digestibility, dry matter data (DM 105°C) were collected, and then metabolizable and digestible and metabolizable energy were calculated by the fraction collection method using acid insoluble ash (AIA) as a marker. Digestibility was calculated according to the following equation: Digestibility = 100 - (100 * (diet marker % / labeled feces %) * (% feces nutrients / dietary nutrients %)).

[0180] After calculating the dietary coefficients for digestibility and metabolizability, we calculated the digestibility of corn according to Mattson et al. (1965) by the following formula: CD content = CD reference + ((CD inclusion 40–CD reference %)) / (corn MS * % inclusion)).

[0181] Statistical analysis

[0182] Data were analyzed by the study investigator and mortality was considered in the data analysis.Statistical analyses of apparent metabolizable energy and digestibility parameters were performed according to standard least squares procedures appropriate to the study design and characteristics of the data set and including checks for outliers.

[0183] result

[0184] The data that best fits the biological interpretation were evaluated. When the results did not satisfy linear regression, the data were analyzed by linear regression Plato's method for the best enzyme reaction. The results are summarized in Table 6.

[0185] Table 6: Appearance of diet, diet with corn and corn with different levels of TERMAMYL SC Metabolizable energy (AME).

[0186]

[0187] *l=3860r=158**l=4023r=183;l=maximumAMEr=optimal level

[0188] These results show that the addition of an alpha-amylase of the invention to poultry feed improves the apparent metabolizable energy of the diet, and especially of the diet where 40% of the soy meal was replaced with corn.

[0189] Example 6: Animal testing in chickens

[0190] A second in vivo study was conducted on the vital performance of broiler chickens that had been fed the basal diet as specified below.

[0191] This study investigated the supplemental effect of Termamyl SC (bacterial α-amylase, IUB No. 3.2.1.1.) on broiler chicken performance using a model with a corn / soybean diet.

[0192] Performance parameters were recorded weekly and from 1 to 7 days, 1 to 14 days, 1 to 21 days, 1 to 28 days, 1 to 35 days, 1 to 40 days, and 22 to 40 days.

[0193] Table 7: Overview of experimental treatments

[0194]

[0195] * Treatments T2, T3 and T4 were obtained by mixing diets T1 and T5.

[0196] Amylase: Termamyl SC (SEQ ID NO: 2), 80 KNU / kg feed

[0197] Table 8: Ingredients

[0198]

[0199]

[0200] 1 Determined using NIR.

[0201] Table 9: Estimated composition of the basic diet

[0202]

[0203]

[0204] 1 Composition / kg feed: vit.A, 9,000UI; vit.D3, 2,500UI; vit.E, 20mg; vit.K3, 2.5mg; thiamine, 2mg; riboflavin, 6mg; pyridoxine, 3mg; cyanocobalamin, 0.015mg, pantothenic acid, 12mg; niacin, 35mg; folic acid, 1.5mg; biotin, 0.1mg; iron, 100mg; zinc, 130mg; manganese, 130mg; cobalt, 2mg; copper, 20mg; iodine, 2mg; selenium, 0.25mg.

[0205] 2Apparent metabolizable energy.

[0206] Poultry and nests

[0207] A total of 1,800 one-day-old male Cobb x Cobb 500 slow-feathering chicks were used. Treatment groups were assigned to floor pens using a completely randomized arrangement consisting of 8 treatments with 9 replicates and 25 birds / replicate.

[0208] Feed and water were provided for ad libitum feeding. Ambient temperature was controlled using infrared heating lamps to maintain bird comfort. On the first day, ambient temperature was adjusted to 32°C and reduced by 1°C every two days until a comfortable temperature was reached. Birds were placed on rice husk litter.

[0209] Experimental diet

[0210] All diets were produced in a 400 kg capacity mixer. The mixer was cleaned and vacuumed between each batch. The diets were provided as a mash. The chemical composition of corn and soybean meal was analyzed before the start of the study.

[0211] Experimental design

[0212] The design was completely randomized with 8 treatments and 9 replications of 25 birds each.

[0213] Measured response

[0214] Body weight, weight gain and feed conversion were assessed at 7, 14, 21, 28, 35 and 40 days of age. Feed conversion was calculated as a correction for the weight of dead birds. At each box, responses were recorded on a separate available form.

[0215] Statistical analysis

[0216] The study investigators analyzed the data. Statistical analyses were performed following appropriate procedures (SAS Institute, 2009).

[0217] result

[0218] Table 10: Weight gain from 1 to 40 days of age of broiler chickens fed a diet with enzymes, g

[0219]

[0220] a-d Means with different superscript letters are significantly different (P < 0.05) - Tukey's test.

[0221] Table 11: Feed conversion ratio, g:g, from 1 to 40 days of age for broiler chickens fed a diet with enzymes

[0222]

[0223]

[0224] a-d Means with different superscript letters are significantly different (P < 0.05) - Tukey's test.

[0225] Table 12: Cumulative weight gain from 1 to 40 days of age, g

[0226]

[0227] a-c Means with different superscript letters are significantly different (P < 0.05) - Tukey's test.

[0228] Table 13: Cumulative feed conversion from 1 to 40 days of age, g:g

[0229]

[0230] a-c Means with different superscript letters are significantly different (P < 0.05) - Tukey's test.

[0231] Table 14: Regression equations of the estimated measurements relative to the energy level of the diets fed to broiler chickens from 1 to 40 days Procedure

[0232]

[0233] 1 Regression equations for energy levels from 1 to 21 days (2,950; 3,000; 3,050; 3,100 and 3,150 kcal / kg) and from 22 to 40 days (3,070; 3,120; 3,170; 3,220 and 3,270 kcal / kg). The coefficient of determination (r) was obtained using all data. 2 ).

[0234] 2 Linear effect (P<0.10).

[0235] 3 For each parameter, the response to graded addition of energy was determined based on the mean value. The difference between energy levels (0, 50, 100, 150 and 200 kcal / kg) was used to obtain the relative bioequivalence.

[0236] In summary, these trials performed well with the addition of the amylase of the invention, especially with regard to improved weight gain and FCR during the growing and later stages.

Claims

1. Use of at least one bacterial amylase in poultry feed to improve the nutritional value of the feed, wherein the bacterial amylase is a polypeptide having at least 80% identity to amino acids 1-481 of SEQ ID NO:

2.

2. The use according to claim 1, wherein the poultry feed has a reduced apparent metabolizable energy compared to a standard poultry feed.

3. Use according to any one of claims 1 to 2, wherein the bacterial amylase is added to the poultry feed in a dosage of between 0.01 and 200 mg enzyme protein / kg diet.

4. Use according to any one of claims 1 to 3, for improving weight gain and / or feed conversion rate of poultry.

5. A method for improving the nutritional value of poultry feed, the method comprising the step of adding to the feed at least one bacterial amylase, wherein the bacterial amylase is a polypeptide having at least 80% identity to amino acids 1-481 of SEQ ID NO:

2.

6. The method of claim 5, wherein the poultry feed has reduced apparent metabolizable energy compared to a standard poultry feed.

7. The method of any one of claims 5 to 6, wherein the bacterial amylase is added to the poultry feed at a dosage of between 0.01 and 200 mg enzyme protein / kg diet.

8. A method according to any one of claims 5 to 7 for improving weight gain and / or feed conversion efficiency in poultry.

9. A poultry feed additive comprising at least one bacterial amylase, together with (i) at least one vitamin, (ii) at least one mineral, or (iii) at least one vitamin and at least one mineral, wherein the bacterial amylase is a polypeptide having at least 80% identity to amino acids 1-481 of SEQ ID NO:

2.

10. A poultry feed comprising the poultry feed additive according to claim 9 and 10w%-40w% soybean meal.

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