Patent Application
20230180791
The present invention relates to uses of at least one carbohydrase in combination with an animal feed with a defined A/X fiber fraction, to an animal feed with a defined A/X fiber fraction and comprising at least one enzyme having carbohydrase activity in an amount determined suitable to the A/X fiber fraction of the feed and methods of making thereof.
Improving the growth performance of farm animals is needed in a world with a growing population eating more animal protein, and it is the object of the present invention to devise solutions which helps meet this challenge.
Xylans are hemicelluloses found in all land plants (Popper and Tuohy, Plant Physiology, 2010, 153:373-383). They are especially abundant in secondary cell walls and xylem cells. In grasses, with type II cell walls, glucurono arabinoxylans are the main hemicellulose and are present as soluble or insoluble dietary fiber in many grass based food and feed products.
Plant xylans have a β-1,4-linked xylopyranose backbone that can be substituted at the O2 or O3 position with arabinose, glucuronic acid and acetic acid in a species and tissue specific manner.
Soybean meal is used around the world in animal feed and polypeptides having xylanase activity that are capable of breaking down the highly branched xylan backbone in the cell wall in order to release more xylose and other nutrients which are trapped inside the cell wall as disclosed in WO 2003/106654 and WO 2017/103159 have been developed.
It has been recently discovered that some factors, such as high A/X soybean meal fiber fractions, the ratio of the mass of arabinoxylans in soybean meal to the total mass of xylose in the soybean meal, lead to different levels of activity of the added feed enzymes, such as carbohydrases, preferably xylanases. The present invention provides an animal feed with a defined A/X soybean meal ratio and comprising polypeptides having carbohydrase activity in an amount determined suitable based on the the A/X soybean meal ratio of the feed and uses and methods of making thereof.
The present invention relates to uses of at least one carbohydrase in combination with an animal feed with a defined A/X fiber fraction for improving the nutritional value of an animal feed.
The present invention further relates to uses of at least one carbohydrase in combination with an animal feed with a defined A/X fiber fraction for increasing digestibility of an animal feed.
The present invention further relates to uses of at least one carbohydrase in combination with an animal feed with a defined A/X fiber fraction for improving one or more performance parameters in an animal.
The present invention also relates to an animal feed with a defined A/X fiber fraction and comprising polypeptides having carbohydrase activity in an amount determined suitable to the A/X fiber fraction of the feed.
The present invention further relates to a method of composing an animal feed comprising at least one carbohydrase, and comprising the step of adjusting the amount of the one or more polypeptides in the animal feed depending on the A/X fiber fraction of the feed.
Animal: The term “animal” refers to any animal except humans. Examples of animals are monogastric animals, including but not limited to pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks, quail, guinea fowl, geese, pigeons (including squabs) and chicken (including but not limited to broiler chickens (referred to herein as broiles), chicks, layer hens (referred to herein as layers)); pets such as cats and dogs; horses.
Animal feed: The term “animal feed” refers to any compound, preparation, or mixture suitable for, or intended for intake by an animal. Animal feed for a monogastric animal typically comprises concentrates as well as vitamins, minerals, enzymes, direct fed microbial, amino acids and/or other feed ingredients (such as in a premix) whereas animal feed for ruminants generally comprises forage (including roughage and silage) and may further comprise concentrates as well as vitamins, minerals, enzymes direct fed microbial, amino acid and/or other feed ingredients (such as in a premix).
Apparent metabolizable energy (AME): The term “Apparent metabolizable energy (AME)” is the gross energy of the feed consumed minus the gross energy contained in the feces, urine, and gaseous products of digestion.
Arabinoxylan-containing material: The term “Arabinoxylan-containing material” means any material containing arabinoxylan. Arabinoxylan is a hemicellulose found in both the primary and secondary cell walls of plants, including woods and cereal grains, consisting of copolymers of two pentose sugars, arabinose and xylose. The arabinoxylan chain contains a large number of 1,4-linked xylose units. Many xylose units are substituted with 2-, 3—or 2,3-substituted arabinose residues.
Examples of arabinoxylan-containing material are forage, roughage, seeds and grains (either whole or prepared by crushing, milling, etc from, e.g., soybeans corn, oats, rye, barley, wheat), trees or hard woods (such as poplar, willow, eucalyptus, palm, maple, birch), bamboo, herbaceous and/or woody energy crops, agricultural food and feed crops, animal feed products, cassava peels, cocoa pods, sugar cane, sugar beet, locust bean pulp, vegetable or fruit pomaces, wood waste, bark, shavings, sawdust, wood pulp, pulping liquor, waste paper, cardboard, construction and demolition wood waste, industrial or municipal waste water solids or sludge, manure, by-product from brewing and/or fermentation processes, wet distillers grain, dried distillers grain, spent grain, vinasse and bagasse.
Forage as defined herein also includes roughage. Forage is fresh plant material such as hay and silage from forage plants, grass and other forage plants, grass and other forage plants, seaweed, sprouted grains and legumes, or any combination thereof. Examples of forage plants are Alfalfa (Lucerne), birdsfoot trefoil, brassica (e.g., kale, rapeseed (canola), rutabaga (swede), turnip), clover (e.g., alsike clover, red clover, subterranean clover, white clover), grass (e.g., Bermuda grass, brome, false oat grass, fescue, heath grass, meadow grasses, miscanthus, orchard grass, ryegrass, switchgrass, Timothy-grass), corn (maize), hemp, millet, barley, oats, rye, sorghum, soybeans and wheat and vegetables such as beets. Crops suitable for ensilage are the ordinary grasses, clovers, alfalfa, vetches, oats, rye and maize. Forage further includes crop residues from grain production (such as corn stover; straw from wheat, barley, oat, rye and other grains); residues from vegetables like beet tops; residues from oilseed production like stems and leaves form soybeans, rapeseed and other legumes; and fractions from the refining of grains for animal or human consumption or from fuel production or other industries.
Preferred sources of arabinoxylan-containing materials are forage, roughage, seeds and grains, sugar cane, sugar beet and wood pulp.
Body Weight Gain: The term “body weight gain” means an increase in live weight of an animal during a given period of time, e.g., the increase in weight from day 1 to day 21.
Carbohydrase: In the present context, a carbohydrase is an enzyme that catalyzes the breakdown of carbohydrates into simple sugars.
Examples of carbohydrases include, but are not limited to, glucanases, xylanase, pectinase, galactosidases, cellulose, mannanases, debranching enzymes and amylases.
Primary targets of carbohydrases are cellulose, arabinoxylans and mixed linked glucans from cereals and pectin polysaccharides and oligosaccharides from plant protein sources. For example, xylanase degrades the linear polysaccharide beta-1,4-xylan into xylose. It helps to breakdown cell wall and thus exposing starch and augmenting digestion.
Preferred carbohydrases according to the present invention are xylanses (as defined in more detail below) and α-galactosidases. α-galactosidase (α-GAL, also known as α-GAL A; E.C. 3.2.1.22) is a glycoside hydrolase enzyme that hydrolyses the terminal alpha-galactosyl moieties from glycolipids and glycoproteins.
Examples of carbohydrases useful in the present context are carbohydrases from Thermomyces lanuginosus or Trichderma reeseibeta-glucanases produced by fermentation of genetically modified micro-organisms as for example Aspergillus oryzae or Bacillus amyloliquefaciens.
The carbohydrase for use according to the invention is stable in the presence of protease. The protease stability may be determined by incubating 0.5 mg purified carbohydrase enzyme protein/ml in a buffer at a desired pH (e.g. pH 3, 4, or 5), for the desired time (e.g. 30, 45, 60, 90, or 120 minutes) in the presence of protease (e.g. pepsin, 70 mg/I), and then raising pH to the desired pH (e.g. pH 4, 5, 6, or 7) and measuring residual activity. The residual carbohydrase activity is preferably at least 20%, preferably at least 30, 40, 50, 60, 70, 80, or at least 90% relative to the control (a non-protease-treated sample).
In the use according to the invention the carbohydrases can be fed to the animal before, after, or simultaneously with the diet of the animal. The latter is preferred.
In a particular embodiment, the carbohydrases, in the form in which they are added to the feed, or when being included in a feed additive, are well-defined. Well-defined means, that the enzyme preparation is at least 50% pure on a protein-basis. In other particular embodiments the enzyme preparation is at least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95% pure. Purity may be determined by any method known in the art, e.g. by SDS-PAGE, or by Size-exclusion chromatography (see Example 12 of WO 01/58275).
Concentrates: The term “concentrates” means feed with high protein and energy concentrations, such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (either whole or prepared by crushing, milling, etc. from e.g. soybeans, corn, oats, rye, barley, wheat), oilseed press cake (e.g. from cottonseed, safflower, sunflower, soybean (such as soybean meal), rapeseed/canola, peanut or groundnut), palm kernel cake, yeast derived material and distillers grains (such as wet distillers grains (WDS) and dried distillers grains with solubles (DDGS).
Dietary Fiber: The term dietary fiber generally refers to the coarse, indigestible plant matter, composed primarily of polysaccharides such as cellulose, that when eaten by humans stimulates intestinal peristalsis. For example, dietary fiber can include cell wall materials such as cellulose, hemicelluloses, lignin, and pectins, along with gums and mucilages that are not digested by the body. Dietary fiber includes polysaccharides, oligosaccharides, lignin, and associated plant substances. Soluble and insoluble fibres make up the two basic categories of dietary fibre. Cellulose, hemicellulose and lignin—are not soluble in water whereas pectins, gums and mucilages—become gummy in water.
Sources of dietary fiber suitable for use in products and quantification in accordance with the disclosure include, but are not limited to, cereal brans, barley, psyllium, legumes, insulin, fructo-oligosaccharides, polydextrose, vegetable sources, fruit sources, grain sources, nuts, and flax seeds.
The amount of dietary fiber in a sample can be quantified by standard methods. These methods include, without being limited to, dissoluting the sample to produce a dietary fiber solution and then centrifuging the dietary fiber solution to produce a pellet and a supernatant liquid. After separating the supernatant liquid from the pellet, the pellet can be analyzed to determine a content of non-dietary fiber components in the pellet. The dietary fiber content in the pellet can be determined from the content of the non-dietary fiber components in the pellet. By using centrifugation to help isolate the dietary fiber in the sample, fiber loss may be minimized, leading to a more accurate determination of the content of dietary fiber in the sample.
Effect of carbohydrase on BWcFCR vs NC: The term “Effect of carbohydrase on BWcFCR vs NC” refers to the change in FCR when fed a diet with addition of carbohydrases vs. the same diet without carbohydrases. This effect is not expressed in units, but in a difference in units (points). For example the difference in FCR is one point from 1.57 to 1.56.
Feed Conversion Ratio: The term “feed conversion ratio” the amount of feed fed to an animal to increase the weight of the animal by a specified amount. An improved feed conversion ratio means a lower feed conversion ratio. By “lower feed conversion ratio” or “improved feed conversion ratio” it is meant that the use of a feed additive composition in feed results in a lower amount of feed being required to be fed to an animal to increase the weight of the animal by a specified amount compared to the amount of feed required to increase the weight of the animal by the same amount when the feed does not comprise said feed additive composition. To be able to compare different groups, flocks, houses or diets, the FCR is corrected for weight differences, the body weight corrected feed conversion ratio (BWcFCR) and mortality, the mortality corrected feed conversion ratio. For the purpose of the present invention, for birds, body weight correction is done by subtracting 1 point in FCR per each 30 g of extra body weight, e.g. from 1.57 to 1.56.
To be able to compare different groups, flocks or trials, the feed intake per pen is adjusted for the total number of days the birds in the pen are on the trial and called mortality corrected FCR (mFCR). The relationship is as follows:
Total bird days=(#birds per pen×days on trial)+day bird died.
mortality adjusted feed intake per bird=(pen intake/total bird days)×total trial days
Mortality corrected FCR=mortality adjusted feed intake per bird/body weight gain per bird
Feed efficiency: The term “feed efficiency” means the amount of weight gain per unit of feed when the animal is fed ad-libitum or a specified amount of food during a period of time. By “increased feed efficiency” it is meant that the use of a feed additive composition according the present invention in feed results in an increased weight gain per unit of feed intake compared with an animal fed without said feed additive composition being present.
Fiber Fraction: The term “fiber fraction” or “dietary fiber fraction” for the purpose of this invention refers to the mass fraction (weight fraction), the ratio of the mass of a fiber component of a feed or food to the mass of another fiber component of the feed or food.
Specifically, this invention relates to the A/X ratio, or A/X fiber fraction, which is the ratio of the mass of arabinose (A) in a feed or food to the the mass of xylose (X) in the feed or food.
A/X total feed ratios, the A/X ratio in the total diet, were calculated from the measured A/X corn ratio and the measured A/X soybean meal ratio by using the ((percent corn in the diet×measured mass of arabinose in corn)+(percent soybean meal in diet×measured mass of arabinose in soybean meal))/((percent corn in diet×measured mass of xylose in corn)+(percent soybean meal in diet×measured mass of xylose in soybean meal)).
For the purpose of illustrating the calculation of the A/X ratio in a total diet an example is given as follows: A broiler diet contains 57.43% corn and 37.6% soybean meal. The measured content of arabinose in corn was 1.72 g per 100 g corn and the measured content of xylose in corn was 2.41 g per 100 g corn. The measured content of arabinose in soybean meal was 2.42 g per 100 g soybean meal and the measured content of xylose in soybean meal was 1.25 g per 100 g soybean meal. Therefore, the calculated arabinose to xylose ratio in the total diet is 1.02=((57.43/100)*1.72)+((37.6/100)*2.42)/((57.43/100)*2.41)+((37.6/100)*1.25).
A/X corn ratio: The term “A/X corn fiber fraction” or “A/X corn ratio” for the purpose of this invention refers to the ratio of the mass of arabinoxylans in a sample of corn to the the mass of xylose in the same sample of corn.
Insoluble A/X ratio: The term “Insoluble A/X ratio” or “Corn insoluble A/X ratio” is determined as the water non-extractable arabinose (insoluble arabinose) over the water non-extractable xylose (insoluble xylose) content in corn, indicative of the structural features of corn arabinoxylan.
soluble A/X ratio: The term “soluble A/X ratio” or “Corn soluble A/X ratio” is determined as the water extractable arabinose (soluble arabinose) over the water extractable xylose (soluble xylose) content in corn. The water extractable arabinose (soluble Arabinose) is calculated by subtracting the insoluble Arabinose from the total arabinose. The water extractable xylose (soluble xylose) is calculated by subtracting the insoluble xylose from the total xylose.
A/X soybean meal ratio: The term “A/X soybean meal fiber fraction” or “A/X soybean meal ratio” or “SBM A/X ratio” for the purpose of this invention refers to the ratio of the mass of arabinoxylans in a sample of soybean meal to the the mass of xylose in the same sample of soybean meal. A/X ratios in soybean meal typically range from 1.53 to 2.60, with a distribution as shown in
Mature polypeptide: The term “mature polypeptide” means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.
In the present invention, the mature polypeptide may be amino acids 1 to 208 of SEQ ID NO: 1 as disclosed in EP 16178681.9.
Near-infrared Spectroscopy: As used herein, the term “near-infrared spectroscopy (NIRS)” refers to a spectroscopic method that uses the near-infrared region of the electromagnetic spectrum (from about 700 nm to 2500 nm).
Non-starch polysaccharide (NSP): The term “non-starch polysaccharide (NSP)” refers to those polysaccharides (complex carbohydrates), other than starches, found in foods. They are the major part of dietary fibre and can be measured more precisely than total dietary fibre; include cellulose, pectins, glucans, gums, mucilages, inulin, and chitin (and exclude lignin). NSP fractions, include soluble and insoluble NSPs and constituent sugars.
Nutrient Digestibility: The term “nutrient digestibility” means the fraction of a nutrient that disappears from the gastro-intestinal tract or a specified segment of the gastro-intestinal tract, e.g., the small intestine. Nutrient digestibility may be measured as the difference between what is administered to the subject and what. comes out in the faeces of the subject, or between what is administered to the subject and what remains in the digesta on a specified segment of the gastro intestinal tract, e.g., the ileum.
Nutrient digestibility as used herein may be measured by the difference between the intake of a nutrient and the excreted nutrient by means of the total collection of excreta during a period of time; or with the use of an inert marker that is not absorbed by the animal, and allows the researcher calculating the amount of nutrient that disappeared in the entire gastro-intestinal tract or a segment of the gastro-intestinal tract. Such an inert marker may be titanium dioxide, chromic oxide or acid insoluble ash. Digestibility may be expressed as a percentage of the nutrient in the feed, or as mass units of digestible nutrient per mass units of nutrient in the feed. Nutrient digestibility as used herein encompasses starch digestibility, fat digestibility, protein digestibility, and amino acid digestibility.
Energy digestibility as used herein means the gross energy of the feed consumed minus the gross energy of the faeces or the gross energy of the feed consumed minus the gross energy of the remaining digesta on a specified segment of the gastro-intestinal tract of the animal, e.g., the ileum. Metabolizable energy as used herein refers to apparent metabolizable energy and means the gross energy of the feed consumed minus the gross energy contained in the faeces, urine, and gaseous products of digestion. Energy digestibility and metabolizable energy may be measured as the difference between the intake of gross energy and the gross energy excreted in the faeces or the digesta present in specified segment of the gastro-intestinal tract using the same methods to measure the digestibility of nutrients, with appropriate corrections for nitrogen excretion to calculate metabolizable energy of feed.
Parent or parent xylanase: The term “parent” or “parent xylanase” means a xylanase to which a substitution is made to produce the xylanase variants used in the present invention. The parent may be a naturally occurring (wild-type) polypeptide or a variant or fragment thereof.
Roughage: The term “roughage” means dry plant material with high levels of fiber, such as fiber, bran, husks from seeds and grains and crop residues (such as stover, copra, straw, chaff, sugar beet waste).
Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.
For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), e.g., version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the—nobrief option) is used as the percent identity and is calculated as follows:
(Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment)
For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), e.g., version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the—nobrief option) is used as the percent identity and is calculated as follows:
(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment)
Rapidly digested starch: The term “Rapidly digested starch” refers to starch that is rapidly digested after 20 minutes and measured using an in vitro assay developed by Englyst et al. (1999).
Resistant starch: The term “Resistant starch” refers to starch that is resistant to digestion by endogenous or exogenous enzymes. Measured using an in vitro assay developed by Englyst et al., (1999) or calculated as the starch remaining after rapidly digested starch and slowly digested starch are subtracted from the total starch.
Salt-soluble protein: Solubility of proteins relates to surface hydrophobic (protein-protein) and hydrophilic (protein-solvent) interaction, in food case, such solvent is the water, and therefore the protein solubility is classified as a hydrophilic property. The term “Salt-soluble protein” or “protein solubility” provides an indication of the susceptibility of the protein and starch granules in corn to enzymatic attack. Protein solubility of corn can be influenced by moisture content at harvest and drying time and temperature (Odjo et al., 2012). Salt-soluble protein is measured using the procedure NF V03-741 recommended by AFNOR (2008) and presented as equivalent mg of albumine or mg proteins/100 ml. Brief methods are described by Janas et al., 2010
Slowly digested starch: The term “Slowly digested starch” refers to starch that is slowly digested after 120 minutes and measured using an in vitro assay developed by Englyst et al. (1999) which includes the measurement of total starch, rapidly digested starch and resistant starch.
Total starch: The term “total starch” refers to a natural vegetable polymer consisting of long linear unbranched chains of alpha-1,4-linked D-glucose units (amylose) and or long alpha-1,6-branched glucose units (amylopectin). The methods to evaluate total starch include the measurement of glucose released through the use of alpha-amylases and amyloglucosidases that are specifically active on the alpha(1-4) and alpha (1-6) linkages. Total starch can be measured by multiple methods, not limited to those described by Englyst et al. (1999), Hall (2015) or McCleary et al. (2018).
Variant: The term “variant” means a polypeptide having xylanase activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position. The variants of the present invention have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the xylanase activity of the polypeptide of SEQ ID NO: 1 as disclosed in EP 16178681.9.
Wild-type xylanase: The term “wild-type” xylanase means a xylanase expressed by a naturally occurring microorganism, such as a bacterium, yeast, or filamentous fungus found in nature.
Xylanase: In the present context, a xylanase is an enzyme that degrades the linear polysaccharide beta-1,4-xylan into xylose. It helps to breakdown cell wall and thus exposing starch and augmenting digestion.
Preferably, the term “xylanase” refers to a glucuronoarabinoxylan endo-1,4-beta-xylanase (E.C. 3.2.1.136) that catalyses the endohydrolysis of 1,4-beta-D-xylosyl links in some glucuronoarabinoxylans.
The known xylanases are classified into enzyme families based on sequence similarity (cazy.org). The enzymes with mainly endo-xylanase activity have previously been described in Glycoside hydrolase family (GH) 5, 8, 10, 11, 30 and 98. The enzymes within a family share some characteristics such as 3D fold and they usually share the same reaction mechanism. Some GH families have narrow or mono-specific substrate specificities while other families have broad substrate specificities.
Commercially available GH10 and GH11 xylanases are often used to break down the xylose backbone of arabinoxylan. In animal feed this results in a degradation of the cereal cell wall with a subsequent improvement in nutrient release (starch and protein) encapsulated within the cells. Degradation of xylan also results in the formation of xylose oligomers that may be utilised for hind gut fermentation and therefore can help an animal to obtain more digestible energy. However, such xylanases are sensitive to side chain steric hindrance and whilst they are effective at degrading arabinoxylan from wheat, they are not very effective on the xylan found in the seeds of Poaceae species, such as corn or sorghum.
WO 2003/106654 discloses numerous polypeptides with putative xylanase activity. Variants of the GH30 xylanase of SEQ ID NO 190 are described in WO 2003/106654 in order to overcome inherent pH and thermo-stability issues. A number of polypeptides of WO2003/106654 are of relevance to the present invention. WO 2017/103159 also discloses a GH30 subfamily 8 polypeptide having xylanase activity, wherein the GH30 subfamily 8 polypeptide have xylanase activity of relevance to the present invention.
In one embodiment of this invention the polypeptide having xylanase activity is a GH30 family xylanase, for example a xylanase derived from Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis or Paenibacillus pabuli.
Xylanase activity can be determined with 0.2% AZCL-glucuronoxylan as substrate in 0.01% TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37° C. One unit of xylanase activity is defined as 1.0 μmole of azurine produced per minute at 37° C., pH 6 from 0.2% AZCL-glucuronoxylan as substrate in 200 mM sodium phosphate pH 6.
Xylose-containing material: The term “Xylose-containing material” means any material containing xylose. Xylose is a pentose sugar and the main building block for the hemicellulose xylan. Xyloce may be extracted from wood, sugar cane or coconuts. It also naturally occurs in small amounts in berries, spinach, broccoli, and pears and as part of the dietary fiber arabinoxylan. The arabinoxylan chain contains a large number of 1,4-linked xylose units. Many xylose units are substituted with 2-, 3—or 2,3-substituted arabinose residues.
As mentioned above, the present invention relates to uses of at least one carbohydrase in combination with an animal feed with a defined A/X fiber fraction for improving the nutritional value of an animal feed.
The carbohydrase of the present invention may be added to the feed in the form of a single component carbohydrase or in the form of a multicomponent carbohydrase composition, such as commercially available from Rovabio® Advance (further referred to as “RVB”) comprising an Endo-1,4-β-xylanase, β-xylosidase, Endo-1,3 1,4-β-glucanase, laminarinase, α-arabinofuranosidase, α-glucuronidase, ferulic acid esterase, endo-1,4-β-glucanase, cellobiohydrolase, β-glucosidase, polygalacturonase, pectin esterase, endo-1,5-α-arabinanase, α-galactosidase, rhamnogalacturonase, aspartic protease, metallo protease, endo-1,4-β-mannanase, β-manosidase.
Preferred Catbohydrases are Xylanses as Defined Above, for Example:
Clostridium acetobutylicum:
Pseudoalteromonas tetraodonis:
Paenibacillus sp-19179:
Pectobacterium carotovorum subsp. Carotovorum:
Ruminococcus sp. CAG.330
Streptomyces sp-62627:
saccharobutylicum:
panacisoli.
rhizosphaerae:
Use of at Least One Carbohydrase in Combination with an Animal Feed
The present invention is also directed to uses of at least one carbohydrase in combination with an animal feed with an A/X soybean meal ratio between 1.8 and 2.6, preferably between 1.9 and 2.5, preferably between 2.0 and 2.3, preferably between 2.1 and 2.3, most preferably between 2.15 and 2.25.
The present invention further relates to the use of at least one carbohydrase in combination with an animal feed with an A/X soybean meal ratio between 1.8 and 2.6, preferably between 1.9 and 2.5, preferably between 2.0 and 2.3, preferably between 2.1 and 2.3, most preferably between 2.15 and 2.25 for increasing digestibility of said animal feed.
In the present invention, the digestibility may be improved by at least 1%, such as by at least 1.5%, at least 2.0%, at least 2.5%, at least 3%, at least 3.5%, at least 4% or at least 5%.
The present invention further relates to the use of at least one carbohydrase in combination with an animal feed with an A/X soybean meal ratio between 1.8 and 2.6, preferably between 1.9 and 2.5, preferably between 2.0 and 2.3, preferably between 2.1 and 2.3, most preferably between 2.15 and 2.25 for improving the nutritional value of said animal feed.
In the present invention, the nutritional value of the feed may be improved by at least 1%, such as by at least 1.5%, at least 2.0%, at least 2.5%, at least 3%, at least 3.5%, at least 4% or at least 5%.
The present invention further relates to the use of at least one carbohydrase in combination with an animal feed with an A/X soybean meal ratio between 1.8 and 2.6, preferably between 1.9 and 2.5, preferably between 2.0 and 2.3, preferably between 2.1 and 2.3, most preferably between 2.15 and 2.25 for improving one or more performance parameters in an animal.
In the present invention, the one or more performance parameters may be improved by at least 1%, such as by at least 1.5%, at least 2.0%, at least 2.5%, at least 3%, at least 3.5%, at least 4% or at least 5%.
In an embodiment, the improvement in the performance of an animal is an increase in body weight gain. In another embodiment, the improvement is an improved feed conversion ratio. In a further embodiment, the improvement is an increased feed efficiency. In a further embodiment, the improvement is an increase in body weight gain and/or an improved feed conversion ratio and/or an increased feed efficiency.
In the present invention, the improvements are compared to the same feed but excluding the carbohydrase or excluding adjusting the amount of carbohydrase to the A/X fiber fraction of the feed.
The term improving the nutritional value of an animal feed means improving the digestibility and availability of nutrients in the feed. The nutritional values refers in particular to improving the solubilization and degradation of the arabinoxylan-containing fraction (e.g., such as hemicellulose) of the feed, thereby leading to increased release of nutrients from cells in the endosperm that have cell walls composed of highly recalcitrant hemicellulose. Consequently, an increased release of arabinoxylan oligomers indicates a disruption of the cell walls and as a result the nutritional value of the feed is improved resulting in improved animal performance, such as increased growth rate and/or weight gain and/or feed conversion (i.e., the weight of ingested feed relative to weight gain). In addition the arabinoxylan oligomer release may result in improved utilization of these components per se either directly or by bacterial fermentation in the hind gut thereby resulting in a production of short chain fatty acids that may be readily absorbed in the hind and utilised in the energy metabolism.
In one aspect, the present invention relates to an animal feed with a defined A/X fiber fraction and comprising at least one carbohydrase as defined above.
Preferably, the A/X fiber fraction in the feed is in a range from 0.61 to 0.97.
More preferably, the A/X soybean meal fiber fraction in the feed is between 1.8 and 2.6, preferably between 1.9 and 2.5, preferably between 2.0 and 2.3, preferably between 2.1 and 2.3, most preferably between 2.15 and 2.25.
The polypeptide having carbohydrase activity may be dosed between 0.1 to 150 ppm enzyme protein per kg animal feed, such as 0.5 to 100 ppm, 1 to 75 ppm, 2 to 50 ppm, 3 to 25 ppm, 2 to 80 ppm, 5 to 60 ppm, 8 to 40 ppm, 10 to 30 ppm, 13 to 75 ppm, 15 to 50 ppm, 17.5 to 40 ppm, 25 to 75 ppm or 30 to 60 ppm enzyme protein per kg animal feed, or any combination of these intervals.
The final total carbohydrase concentration in the feed is within the range of 50-500 mg per kg animal feed, such as 60 to 450 mg, 70 to 400 mg, 80 to 350 mg, 90 to 300 mg, 100 to 300 mg, 110 to 250 mg, 120 to 200 mg per kg animal feed, or any combination of these intervals.
The final total carbohydrase concentration in the feed is within the range of 0.00001% to 0.1%, preferably 0.0001% to 0.1%, preferably 0.001% to 0.1%, preferably 0.01% to 0.1%, preferably 0.01 to 0.05%.
Animal feed compositions or diets have a relatively high content of protein. Poultry and pig diets can be characterised as indicated in Table B of WO 01/58275, columns 2-3. Fish diets can be characterised as indicated in column 4 of this Table B. Furthermore such fish diets usually have a crude fat content of 200-310 g/kg.
An animal feed composition according to the invention has a crude protein content of 50-800 g/kg. The protein source may be vegetable protein source and/or animal protein.
The vegetable proteins may be derived from vegetable protein sources, such as legumes and cereals, for example, materials from plants of the families Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, and Poaceae, such as soybean meal, lupin meal, rapeseed meal, and combinations thereof. The protein content of the vegetable proteins is at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% (w/w).
Preferably, the vegetable protein source may be material from one or more plants of the family Fabaceae, e.g., soybean, lupine, pea, or bean. The vegetable protein source may also be material from one or more plants of the family Chenopodiaceae, e.g. beet, sugar beet, spinach or quinoa. Other examples of vegetable protein sources are rapeseed, and cabbage. In the present invention, soybean is a preferred vegetable protein source. Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oat, maize (corn), rice, and sorghum.
Besides the vegetable protein as defined above, the animal feed of the 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 animal feed of the invention may also comprise Dried Distillers Grains with Solubles (DDGS), typically in amounts of 0-30%.
Preferably, the protein source is selected from the group consisting of soybean, wild soybean, beans, lupin, tepary bean, scarlet runner bean, slimjim bean, lima bean, French bean, Broad bean (fava bean), chickpea, lentil, peanut, Spanish peanut, canola, sunflower seed, cotton seed, rapeseed (oilseed rape) or pea or in a processed form such as soybean meal, full fat soybean meal, soy protein concentrate (SPC), fermented soybean meal (FSBM), sunflower meal, cotton seed meal, rapeseed meal, fish meal, bone meal, feather meal, whey or any combination thereof.
Furthermore, or in the alternative (to the crude protein content indicated above), the animal feed composition of the invention may have a content of metabolisable energy of 10-30 MJ/kg. In present invention, the energy source may be selected from the group consisting of maize, corn, soybeans, sorghum, barley, wheat, oats, rice, triticale, rye, beet, sugar beet, spinach, potato, cassava, quinoa, cabbage, switchgrass, millet, pearl millet, foxtail millet or in a processed form such as milled corn, milled maize, potato starch, cassava starch, milled sorghum, milled switchgrass, milled millet, milled foxtail millet, milled pearl millet, or any combination thereof.
Furthermore, or in the alternative (to the crude protein content indicated above), the animal feed composition of the invention may have a content of calcium of 0.1-200 g/kg; and/or a content of available phosphorus of 0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or a content of methionine plus cysteine of 0.1-150 g/kg; and/or a content of lysine of 0.5-50 g/kg.
In particular, the content of metabolisable energy, crude protein, calcium, phosphorus, methionine, methionine plus cysteine, and/or lysine may be within any one of ranges 2, 3, 4 or 5 in Table B of WO 01/58275 (R. 2-5).
Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25, i.e. Crude protein (g/kg)=N (g/kg)×6.25. The nitrogen content is determined by the Kjeldahl method (A.O.A.C., 1984, Official Methods of Analysis 14th ed., Association of Official Analytical Chemists, Washington D.C.).
Metabolisable energy can be calculated on the basis of the NRC publication Nutrient requirements in swine, ninth revised edition 1988, subcommittee on swine nutrition, committee on animal nutrition, board of agriculture, national research council. National Academy Press, Washington, D.C., pp. 2-6, and the European Table of Energy Values for Poultry Feed-stuffs, Spelderholt centre for poultry research and extension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen & looijen bv, Wageningen. ISBN 90-71463-12-5.
The dietary content of calcium, available phosphorus and amino acids in complete animal diets is calculated on the basis of feed tables such as Veevoedertabel 1997, gegevens over chemische samenstelling, verteerbaarheid en voederwaarde van voedermiddelen, Central Veevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.
Preferably, the animal feed of the invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70% wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybean meal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or 0-20% whey.
Animal feed can e.g. be manufactured as mash feed (non-pelleted) or pelleted feed. Typically, the milled feed-stuffs are mixed and sufficient amounts of essential vitamins and minerals are added according to the specifications for the species in question. Enzymes can be added as solid or liquid enzyme formulations. For example, for mash feed a solid or liquid enzyme formulation may be added before or during the ingredient mixing step. For pelleted feed the (liquid or solid) carbohydrase/enzyme preparation may also be added before or during the feed ingredient step. Typically a liquid enzyme preparation comprises the carbohydrase of the invention optionally with a polyol, such as glycerol, ethylene glycol or propylene glycol, and is added after the pelleting step, such as by spraying the liquid formulation onto the pellets. The carbohydrase may also be incorporated in a feed additive or premix that can then be added to the final feed.
Alternatively, the carbohydrase can be prepared by freezing a mixture of liquid enzyme solution with a bulking agent such as ground soybean meal, and then lyophilizing the mixture.
In present invention, the animal feed may further comprise one or more additional enzymes; one or more eubiotics; one or more vitamins; one or more minerals, and one or more amino acids, as described below.
The animal feed of the present invention may be produced by any known process.
For example, the animal feed of the present invention is prepared by a process comprising the steps of:
In one embodiment, the at least one carbohydrase is added in step (a). In one embodiment, the carbohydrase is added in step (c).
In the present process, the animal feed may be pelleted by steam treating the mixture of (a) to obtain a moisture content below 20% by weight of the mixture, and pressing the steam treated mixture to form pellets. Preferably, the animal feed is pelleted by steam treating the mixture of (a) to obtain a moisture content below 20% by weight of the mixture wherein the steam treatment is between 60° C. and 100° C. when measured at the outlet of the conditioner, and pressing the steam treated mixture to form pellets. In the present process, the total residence time in step b) may be between 1 second and 10 minutes. In the present process, the temperature of the pellets after pelleting of the steam treated mixture may be between 70° C. and 105° C.
In the present invention, the compositions and/or the animal feed described herein may optionally include one or more enzymes. Enzymes can be classified on the basis of the handbook Enzyme Nomenclature from NC-IUBMB, 1992), see also the ENZYME site at the internet: http://www.expasy.ch/enzyme/. ENZYME is a repository of information relative to the nomenclature of enzymes. It is primarily based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUB-MB), Academic Press, Inc., 1992, and it describes each type of characterized enzyme for which an EC (Enzyme Commission) number has been provided (Bairoch A. The ENZYME database, 2000, Nucleic Acids Res 28:304-305). This IUB-MB Enzyme nomenclature is based on their substrate specificity and occasionally on their molecular mechanism; such a classification does not reflect the structural features of these enzymes.
Another classification of certain glycoside hydrolase enzymes, such as endoglucanase, galactanase, mannanase, dextranase, and galactosidase is described in Henrissat et al, “The carbohydrate-active enzymes database (CAZy) in 2013”, Nucl. Acids Res. (1 Jan. 2014) 42 (D1): D490-D495; see also www.cazy.org.
Thus the composition, the animal feed or the animal feed additive of the present invention may also comprise at least one other enzyme selected from the group comprising of acetylxylan esterase (EC 3.1.1.23), acylglycerol lipase (EC 3.1.1.72), alpha-amylase (EC 3.2.1.1), beta-amylase (EC 3.2.1.2), arabinofuranosidase (EC 3.2.1.55), cellobiohydrolases (EC 3.2.1.91), cellulase (EC 3.2.1.4), feruloyl esterase (EC 3.1.1.73), galactanase (EC 3.2.1.89), alpha-galactosidase (EC 3.2.1.22), beta-galactosidase (EC 3.2.1.23), beta-glucanase (EC 3.2.1.6), beta-glucosidase (EC 3.2.1.21), triacylglycerol lipase (EC 3.1.1.3), lysophospholipase (EC 3.1.1.5), lysozyme (EC 3.2.1.17), alpha-mannosidase (EC 3.2.1.24), beta-mannosidase (mannanase) (EC 3.2.1.25), phytase (EC 3.1.3.8, EC 3.1.3.26, EC 3.1.3.72), phospholipase A1 (EC 3.1.1.32), phospholipase A2 (EC 3.1.1.4), phospholipase D (EC 3.1.4.4), protease (EC 3.4), pullulanase (EC 3.2.1.41), pectinesterase (EC 3.1.1.11), xylanase (EC 3.2.1.8, EC 3.2.1.136), beta-xylosidase (EC 3.2.1.37), or any combination thereof.
The composition, the animal feed or the animal feed additive of the invention may also comprise a galactanase (EC 3.2.1.89) and a beta-galactosidase (EC 3.2.1.23).
The composition, the animal feed or the animal feed additive of the present invention may also comprise a phytase (EC 3.1.3.8 or 3.1.3.26). Examples of commercially available phytases include Bio-Feed™ Phytase (Novozymes), Ronozyme® P, Ronozyme® NP and Ronozyme® HiPhos (DSM Nutritional Products), Natuphos™ (BASF), Natuphos™ E (BASF), Finase® and Quantum® Blue (AB Enzymes), OptiPhos® (Huvepharma), AveMix® Phytase (Aveve Biochem), Phyzyme® XP (Verenium/DuPont) and Axtra® PHY (DuPont). Other preferred phytases include those described in e.g. WO 98/28408, WO 00/43503, and WO 03/066847.
The composition, the animal feed or the animal feed additive of the present invention may also comprise a xylanase (EC 3.2.1.8). Examples of commercially available xylanases include Ronozyme® WX (DSM Nutritional Products), Econase® XT and Barley (AB Vista), Xylathin® (Verenium), Hostazym® X (Huvepharma), Axtra® XB (Xylanase/beta-glucanase, DuPont) and Axtra® XAP (Xylanase/amylase/protease, DuPont), AveMix® XG 10 (xylanase/glucanase) and AveMix® O2 CS (xylanase/glucanase/pectinase, Aveve Biochem), and Naturgrain (BASF).
The composition, the animal feed or the animal feed additive of the invention may also comprise a protease (EC 3.4). Examples of commercially available proteases include Ronozyme® ProAct (DSM Nutritional Products), Winzyme Pro Plus® (Suntaq International Limited) and Cibenza® DP100 (Novus International).
The composition, the animal feed or the animal feed additive of the invention may also comprise an alpha-amylase (EC 3.2.1.1). Examples of commercially available alpha-amylases include Ronozyme® A and RONOZYME® RumiStar™ (DSM Nutritional Products).
The composition, the animal feed or the animal feed additive of the invention may also comprise a multicomponent enzyme product, such as FRA® Octazyme (Framelco), Ronozyme® G2, Ronozyme® VP and Ronozyme® MultiGrain (DSM Nutritional Products), Rovabio® Excel or Rovabio® Advance (Adisseo).
The composition, the animal feed or the animal feed additive of the invention may additionally comprise eubiotics. Eubiotics are compounds which are designed to give a healthy balance of the micro-flora in the gastrointestinal tract. Eubiotics cover a number of different feed additives, such as probiotics, prebiotics, phytogenics (essential oils) and organic acids which are described in more detail below.
In the present invention, the composition, the animal feed or the animal feed additive may further comprise one or more additional probiotic. In particular, the animal feed composition may further comprise a bacterium from one or more of the following genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium and Megasphaera or any combination thereof.
Preferably, the composition, the animal feed or the animal feed additive further comprises a bacterium from one or more of the following strains: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Enterococcus faecium, Enterococcus spp, and Pediococcus spp, Lactobacillus spp, Bifidobacterium spp, Lactobacillus acidophilus, Pediococsus acidilactici, Lactococcus lactis, Bifidobacterium bifidum, Propionibacterium thoenii, Lactobacillus farciminus, Lactobacillus rhamnosus, Clostridium butyricum, Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, Lactobacillus salivarius ssp. salivarius, Megasphaera elsdenii, Propionibacteria sp.
More preferably, the composition or the animal feed of the present invention further comprises a bacterium from one or more of the following strains of Bacillus subtilis: 3A-P4 (PTA-6506), 15A-P4 (PTA-6507), 22C-P1 (PTA-6508), 2084 (NRRL B-500130), LSSA01 (NRRL-B-50104), BS27 (NRRL B-501 05), BS 18 (NRRL B-50633), BS 278 (NRRL B-50634), DSM 29870, DSM 29871, DSM 32315, NRRL B-50136, NRRL B-50605, NRRL B-50606, NRRL B-50622 and PTA-7547.
More preferably, the composition or the animal feed of the present invention further comprises a bacterium from one or more of the following strains of Bacillus pumilus: NRRL B-50016, ATCC 700385, NRRL B-50885 or NRRL B-50886.
More preferably, the composition or the animal feed of the present invention further comprises a bacterium from one or more of the following strains of Bacillus lichenformis: NRRL B 50015, NRRL B-50621 or NRRL B-50623.
More preferably, the composition or the animal feed of the present invention further comprises a bacterium from one or more of the following strains of Bacillus amyloliquefaciens: DSM 29869, DSM 29869, NRRL B 50607, PTA-7543, PTA-7549, NRRL B-50349, NRRL B-50606, NRRL B-50013, NRRL B-50151, NRRL B-50141, NRRL B-50147 or NRRL B-50888.
The bacterial count of each of the bacterial strains in the composition, the animal feed or the animal feed additive is between 1×104 and 1×1014 CFU/kg of dry matter, preferably between 1×106 and 1×1012 CFU/kg of dry matter, and more preferably between 1×107 and 1×1011 CFU/kg of dry matter. Preferably, the bacterial count of each of the bacterial strains in the composition, the animal feed or the animal feed additive is between 1×108 and 1×1010 CFU/kg of dry matter.
The bacterial count of each of the bacterial strains in the composition, the animal feed or the animal feed additive is between 1×105 and 1×1015 CFU/animal/day, preferably between 1×107 and 1×1013 CFU/animal/day, and more preferably between 1×108 and 1×1012 CFU/animal/day. Preferably, the bacterial count of each of the bacterial strains in the composition, the animal feed or the animal feed additive is between 1×109 and 1×1011 CFU/animal/day. More preferably, the amount of probiotics is 0.001% to 10% by weight of the composition or the animal feed or animal feed additive.
In the present invention, the one or more bacterial strains may be present in the form of a stable spore.
Examples of commercial products are Cylactin® (DSM Nutritional Products), Alterion (Adisseo), Enviva PRO (DuPont Animal Nutrition), Syncra® (mix enzyme+probiotic, DuPont Animal Nutrition), Ecobiol® and Fecinor® (Norel/Evonik) and GutCare® PY1 (Evonik).
Prebiotics are substances that induce the growth or activity of microorganisms (e.g., bacteria and fungi) that contribute to the well-being of their host. Prebiotics are typically non-digestible fiber compounds that pass undigested through the upper part of the gastrointestinal tract and stimulate the growth or activity of advantageous bacteria that colonize the large bowel by acting as substrate for them. Normally, prebiotics increase the number or activity of bifidobacteria and lactic acid bacteria in the GI tract.
Yeast derivatives (inactivated whole yeasts or yeast cell walls) can also be considered as prebiotics. They often comprise mannan-oligosaccharids, yeast beta-glucans or protein contents and are normally derived from the cell wall of the yeast, Saccharomyces cerevisiae.
In the present invention, the amount of prebiotics may be 0.001% to 10% by weight of the composition. Examples of yeast products are Yang® and Agrimos (Lallemand Animal Nutrition).
The composition or the animal feed of the invention may further comprise one or more amino acids. Examples of amino acids which are used are lysine, alanine, beta-alanine, threonine, methionine and tryptophan. In the present invention, the amount of amino acid may be 0.001% to 10% by weight of the composition or the animal feed.
In the present invention, the composition or the animal feed may include one or more vitamins, such as one or more fat-soluble vitamins and/or one or more water-soluble vitamins. In addition, the composition or the animal feed may optionally include one or more minerals, such as one or more trace minerals and/or one or more macro minerals.
Usually fat- and water-soluble vitamins, as well as trace minerals form part of a so-called premix intended for addition to the feed, whereas macro minerals are usually separately added to the feed.
Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, e.g., vitamin K3.
Non-limiting examples of water-soluble vitamins include vitamin C, vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g., Ca-D-panthothenate.
Non-limiting examples of trace minerals include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, iodine, selenium and zinc.
Non-limiting examples of macro minerals include calcium, magnesium, phosphorus, potassium and sodium.
In the present invention, the amount of vitamins may be 0.001% to 10% by weight of the composition or the animal feed. Preferably, the amount of minerals is 0.001% to 10% by weight of the composition or the animal feed.
The nutritional requirements of these components (exemplified with poultry and piglets/pigs) are listed in Table A of WO 01/58275. Nutritional requirement means that these components should be provided in the diet in the concentrations indicated.
In the alternative, the composition or the animal feed of the invention comprises at least one of the individual components specified in Table A of WO 01/58275. At least one means either of, one or more of, one, or two, or three, or four and so forth up to all thirteen, or up to all fifteen individual components. More specifically, this at least one individual component is included in the additive of the invention in such an amount as to provide an in-feed-concentration within the range indicated in column four, or column five, or column six of Table A.
Preferably, the composition or the animal feed of the invention comprises at least one of the below vitamins, preferably to provide an in-feed-concentration within the ranges specified in the below Table 1 (for piglet diets, and broiler diets, respectively).
The composition or the animal feed of the invention may further comprise colouring agents, stabilisers, growth improving additives and aroma compounds/flavourings, polyunsaturated fatty acids (PUFAs); reactive oxygen generating species, antioxidants, anti-microbial peptides, anti-fungal polypeptides and mycotoxin management compounds.
Examples of colouring agents are carotenoids such as beta-carotene, astaxanthin, and lutein.
Examples of aroma compounds/flavourings are creosol, anethol, deca-, undeca—and/or dodeca-lactones, ionones, irone, gingerol, piperidine, propylidene phatalide, butylidene phatalide, capsaicin and tannin.
Examples of antimicrobial peptides (AMP's) are CAP18, Leucocin A, Tritrpticin, Protegrin-1, Thanatin, Defensin, Lactoferrin, Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000), Plectasins, and Statins, including the compounds and polypeptides disclosed in WO 03/044049 and WO 03/048148, as well as variants or fragments of the above that retain antimicrobial activity.
Examples of antifungal polypeptides (AFP's) are the Aspergillus giganteus, and Aspergillus niger peptides, as well as variants and fragments thereof which retain antifungal activity, as disclosed in WO 94/01459 and WO 02/090384.
Examples of polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid.
Examples of reactive oxygen generating species are chemicals such as perborate, persulphate, or percarbonate; and enzymes such as an oxidase, an oxygenase or a syntethase.
Antioxidants can be used to limit the number of reactive oxygen species which can be generated such that the level of reactive oxygen species is in balance with antioxidants.
Mycotoxins, such as deoxynivalenol, aflatoxin, zearalenone and fumonisin can be found in animal feed and can result in nmegative animal performance or illness.
Compounds which can manage the levels of mycotoxin, such as via deactivation of the mycotoxin or via binding of the mycotoxin, can be added to the feed to ameliorate these negative effects. Examples of mycotoxin management compounds are Vitafix®, Vitafix Ultra (Nuscience), Mycofix®, Mycofix® Secure, FUMzyme®, Biomin® BBSH, Biomin® MTV (Biomin), Mold-Nil®, Toxy-Nil® and Unike® Plus (Nutriad).
In the present invention, the polypeptide having carbohydrase activity of the composition may be formulated as a solid formulation;
In the present invention, the polypeptide having carbohydrase activity of the composition may also be formulated as a liquid formulation;
In the present invention, the liquid formulation may further comprise 20%-80% polyol (i.e. total amount of polyol), preferably 25%-75% polyol, more preferably 30%-70% polyol, more preferably 35%-65% polyol or most preferably 40%-60% polyol. Preferably, the liquid formulation comprises 20%-80% polyol, more preferably 25%-75% polyol, more preferably 30%-70% polyol, more preferably 35%-65% polyol or most preferably 40%-60% polyol wherein the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600. More perferably, the liquid formulation comprises 20%-80% polyol (i.e. total amount of polyol), more preferably 25%-75% polyol, more preferably 30%-70% polyol, more preferably 35%-65% polyol or most preferably 40%-60% polyol wherein the polyol is selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG).
In the present invention, the liquid formulation may further comprise preservative, preferably selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof. Preferably, the liquid formulation comprises 0.02% to 1.5% w/w preservative, more preferably 0.05% to 1.0% w/w preservative or most preferably 0.1% to 0.5% w/w preservative. More preferably, the liquid formulation comprises 0.001% to 2.0% w/w preservative (i.e. total amount of preservative), preferably 0.02% to 1.5% w/w preservative, more preferably 0.05% to 1.0% w/w preservative or most preferably 0.1% to 0.5% w/w preservative wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof.
In the present invention, the liquid formulation may comprise one or more formulating agents (such as those described herein), preferably a formulating agent selected from the list consisting of 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, PVA, acetate and phosphate, preferably selected from the list consisting of 1, 2-propylene glycol, 1, 3-propylene glycol, sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate.
In the present invention, the solid formulation may be for example as a granule, spray dried powder or agglomerate (e.g. as disclosed in WO2000/70034). The formulating agent may comprise a salt (organic or inorganic zinc, sodium, potassium or calcium salts such as e.g. 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 a sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol).
Preferably, the formulating agents of the solid formulation are selected from the list consisting of 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. Preferably, the formulating agent is selected from one or more of the following compounds: sodium sulfate, dextrin, cellulose, sodium thiosulfate, magnesium sulfate and calcium carbonate.
Preferably, the composition of the present invention is an enzyme granule comprising the enzymes of the invention optionally combined with one or more additional enzymes. The granule is composed of a core, and optionally one or more coatings (outer layers) surrounding the core.
Typically, the granule size, measured as equivalent spherical diameter (volume based average particle size), of the granule is 20-2000 μm, particularly 50-1500 μm, 100-1500 μm or 250-1200 μm.
The core can be prepared by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation.
Methods for preparing the core can be found in Handbook of Powder Technology; Particle size enlargement by C. E. Capes; Volume 1; 1980; Elsevier. Preparation methods include known granule formulation technologies, e.g.:
The core may include additional materials such as fillers, fibre materials (cellulose or synthetic fibres), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances.
The core may include a binder, such as synthetic polymer, wax, fat, or carbohydrate.
The core may include a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposing catalyst and/or an acidic buffer component, typically as a homogenous blend.
In the present invention, the core may comprise a material selected from the group consisting of 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 a sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol), sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol), small organic molecules, starch, flour, cellulose and minerals and clay minerals (also known as hydrous aluminium phyllosilicates). Preferably, the core comprises a clay mineral such as kaolinite or kaolin.
The core may also include an inert particle with the enzyme absorbed into it, or applied onto the surface, e.g., by fluid bed coating.
The core may have a diameter of 20-2000 μm, particularly 50-1500 μm, 100-1500 μm or 250-1200 μm.
The core may be surrounded by at least one coating, e.g., to improve the storage stability, to reduce dust formation during handling, or for coloring the granule. The optional coating(s) may include a salt and/or wax and/or flour coating, or other suitable coating materials.
The coating may be applied in an amount of at least 0.1% by weight of the core, e.g., at least 0.5%, 1% or 5%. The amount may be at most 100%, 70%, 50%, 40% or 30% by weight of the core.
The coating is preferably at least 0.1 μm thick, particularly at least 0.5 μm, at least 1 μm or at least 5 μm. In some embodiments the thickness of the coating is below 100 μm, such as below 60 μm, or below 40 μm.
The coating should encapsulate the core unit by forming a substantially continuous layer. A substantially continuous layer is to be understood as a coating having few or no holes, so that the core unit is encapsulated or enclosed with few or no uncoated areas.
The layer or coating should in particular be homogeneous in thickness.
The coating can further contain other materials as known in the art, e.g., fillers, antisticking agents, pigments, dyes, plasticizers and/or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.
Preferably, the enzyme granules of the invention may comprise a core comprising the enzymes of the invention, one or more salt coatings and one or more wax coatings.
Examples of enzyme granules with multiple coatings are shown in WO1993/07263, WO1997/23606 and WO2016/149636.
The salt coating may comprise at least 60% by weight of a salt, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight. The salt coating may be as described in WO1997/05245, WO1998/54980, WO1998/55599, WO2000/70034, WO2006/034710, WO2008/017661, WO2008/017659, WO2000/020569, WO2001/004279, WO1997/05245, WO2000/01793, WO2003/059086, WO2003/059087, WO2007/031483, WO2007/031485, WO2007/044968, WO2013/192043, WO2014/014647 and WO2015/197719 or polymer coating such as described in WO 2001/00042.
The salt in the coating may have a constant humidity at 20° C. above 60%, particularly above 70%, above 80% or above 85%, or it may be another hydrate form of such a salt (e.g., anhydrate).
The salt may be an inorganic salt, e.g., salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids (less than 10 carbon atoms, e.g., 6 or less carbon atoms) such as citrate, malonate or acetate. Examples of cations in these salts are alkali or earth alkali metal ions, the ammonium ion or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminium. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, sorbate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate. In particular alkali- or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used.
Specific examples of suitable salts are NaCl (CH20° C.=76%), Na2CO3 (CH20° C.=92%), NaNO3 (CH20° C.=73%), Na2HPO4 (CH20° C.=95%), Na3PO4 (CH25° C.=92%), NH4Cl (CH20° C.=79.5%), (NH4)2HPO4 (CH20° C.=93,0%), NH4H2PO4 (CH20° C.=93.1%), (NH4)2SO4 (CH20° C.=81.1%), KCl (CH20° C.=85%), K2HPO4 (CH20° C.=92%), KH2PO4 (CH20° C.=96.5%), KNO3 (CH20° C.=93.5%), Na2SO4 (CH20° C.=93%), K2SO4 (CH20° C.=98%), KHSO4 (CH20° C.=86%), MgSO4 (CH20° C.=90%), ZnSO4 (CH20° C.=90%) and sodium citrate (CH25° C.=86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4, Mg(NO3)2, magnesium acetate, 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, sodium acetate, sodium benzoate, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate and zinc sorbate.
The salt may be in anhydrous form, or it may be a hydrated salt, i.e. a crystalline salt hydrate with bound water(s) of crystallization, such as described in WO 99/32595. Specific examples include anhydrous sodium sulfate (Na2SO4), anhydrous magnesium sulfate (MgSO4), magnesium sulfate heptahydrate (MgSO4.7H2O), zinc sulfate heptahydrate (ZnSO4.7H2O), sodium phosphate dibasic heptahydrate (Na2HPO4.7H2O), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate and magnesium acetate tetrahydrate.
The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water soluble, in particular having a solubility at least 0.1 g in 100 g of water at 20° C., preferably at least 0.5 g per 100 g water, e.g., at least 1 g per 100 g water, e.g., at least 5 g per 100 g water. The salt may be added from a salt solution where the salt is completely dissolved or from a salt suspension wherein the fine particles are less than 50 μm, such as less than 10 μm or less than 5 μm.
A wax coating may comprise at least 60% by weight of a wax, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight.
Specific examples of waxes are polyethylene glycols; polypropylenes; Carnauba wax; Candelilla wax; bees wax; hydrogenated plant oil or animal tallow such as polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC), polyvinyl alcohol (PVA), hydrogenated ox tallow, hydrogenated palm oil, hydrogenated cotton seeds and/or hydrogenated soybean oil; fatty acid alcohols; mono-glycerides and/or di-glycerides, such as glyceryl stearate, wherein stearate is a mixture of stearic and palmitic acid; micro-crystalline wax; paraffin's; and fatty acids, such as hydrogenated linear long chained fatty acids and derivatives thereof. A preferred wax is palm oil or hydrogenated palm oil.
The granulate of the present invention may also be produced as a non-dusting granulate, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art. The coating materials can be waxy coating materials and film-forming coating materials. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591.
The granulate may further comprise one or more additional enzymes. Each enzyme will then be present in more granules securing a more uniform distribution of the enzymes, and also reduces the physical segregation of different enzymes due to different particle sizes. Methods for producing multi-enzyme co-granulates is disclosed in the ip.com disclosure IPCOM000200739D.
Another example of formulation of enzymes by the use of co-granulates is disclosed in WO 2013/188331.
The present invention also relates to protected enzymes prepared according to the method disclosed in EP 238,216.
Thus, preferably, the present invention provides a granule, which comprises:
In the present invention, the coating comprises a salt coating as described herein.
Preferably, the coating comprises a wax coating as described herein. More preferably, the coating comprises a salt coating followed by a wax coating as described herein. The polypeptide having carbohydrase activity and other polypeptides may be co-granulated.
In the present invention, the composition may further comprise one or more components selected from the list consisting of one or more carriers. The carrier may be selected from the group consisting of water, 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, maltodextrin, glucose, sucrose, sorbitol, lactose, wheat flour, wheat bran, corn gluten meal, starch, kaolin and cellulose or any combination thereof.
In present invention, the composition may further comprise one or more additional enzymes; one or more eubiotics; one or more vitamins; one or more minerals, and one or more amino acids, as described below.
Methods of composing an Animal Feed
The present invention is also directed to methods of composing an animal feed comprising at least one enzyme and with an A/X soybean meal ratio between 1.8 and 2.6, preferably between 1.9 and 2.5, preferably between 2.0 and 2.3, preferably between 2.1 and 2.3, most preferably between 2.15 and 2.25.
An embodiment of the present invention therefore is a method of composing an animal feed
Another embodiment of the present invention is a method of composing an animal feed comprising at least one enzyme, and comprising the steps of
Quantitative analysis of the dietary fiber content in animal feed can be performed by standard methods, such as wet chemistry, HPLC, GLC or spectrophotometry, and enzyme assays.
Wet chemistry methods include the methods as used by Englyst et al. (Englyst H N, Quigley ME, Hudson GJ (1994). Determination of dietary fiber as non-starch polysaccharides with gas-liquid chromatographic, high-performance liquid chromatographic or spectrophotometric measurement of constituent sugars. Analyst 119, 1497-1509.) to determine the non-starch polysaccharides (NSP) in the form of soluble, insoluble and total NSP fractions from feed samples are separated. Briefly, 5 mL of sodium acetate buffer is added to the ground feed sample followed by serial enzymatic treatment for starch removal. The sample is then centrifuged to collect the soluble NSP fraction. The remaining pellet is the insoluble NSP. Both portions are acid hydrolysed and the monosaccharides are determined using gas chromatography, high performance liquid chromatography or spectrophotometry.
Enzyme assays can be purchased as kits, for example from Megazyme.
Additionally, dietary fiber feactions may be calculated from other fractions or as a percent of NDF as in the paper as disclosed by Balckom et al. (Mourtzinis S, Arriaga F J, Bransby D, Balcom KS (2014). A simplified method for monomeric carbohydrateanalysis of corn stover biomass. GCB Bioenergy 6, 300-304.).
The quantitative analysis of the steps al) and a2) of the method according to the present
invention is rather time and cost consuming. Near infrared measurements (NIR) of the respective animal feed would be a more time and cost efficient alternative for determining the A/X soybean meal ratio in an animal feed. However, near infrared spectroscopy does not give the results with the desired precision. Accordingly, neither quantitative analysis nor near infrared spec-troscopy alone are suitable for a cost and time efficient determination of the A/X soybean meal ratio in the total amount of the animal feed.
According to the present invention this problem is solved in that the near infrared absorptions obtained for a sample of an animal feed are correlated with the corresponding values of the quantitative analysis of the same. The thus obtained correlation of the values of the quantitative analysis with the absorptions of the NIR measurement is preferably depicted or plotted as a calibration graph, which facilitates the matching of the absorptions of the NIR measurements of other sample with the corresponding exact values for the parameters based on the quan-titative analysis.
Another object of the present invention is therefore a computer-implemented method of composing an animal feed comprising at least one enzyme, comprising the steps of
A1) nearinfrared (NIR) spectroscopy to obtain an NIR spectrum;
A2) matching the absorption intensities at the respective wavelengths or wavenumbers in the NIR spectrum obtained in A1) with a calibration graph or equation;
A3) a quantitative analysis of the dietary fiber in the sample
A4) a determination of the A/X soybean meal ratio in the total amount of processed feedstuff raw material and/or feedstuff; and
The present invention further comprises the step of generating a calibration graph or equation as in A2) by additionally following the steps
a1) a quantitative analysis of the A/X soybean meal ratio in the sample;
a2) a determination of the A/X soybean meal ratio in the total amount of the animal feed; and
Depending on the spectrometer used, the near-infrared (NIR) spectra of step A) can be recorded at wavelengths between 400 and 2,500 nm with any suitable infrared spectroscopes working either on the monochromator principle or on the Fourier transform principle. Preferably, the NIR spectra are recorded between 1,000 and 2,500 nm. Wavelengths are easily converted into the respective wavenumbers and therefore, the NIR spectra can of course also be recorded at the corresponding wavenumbers. Organic compounds rich in O—H bonds, C—H bonds and N—H bonds are suitable for the detection by means of near-infrared spectroscopy. However, a biological sample such as an animal feed contains a multitude of different organic compounds and thus represents a complex matrix. Notwithstanding every biological substance has a unique near-infrared spectrum, comparable to an individual finger print. Consequently, two biological substances having exactly the same spectrum can be assumed to have the same physical and chemical composition and thus to be identical. On the other hand, if two biological substances have different spectra, it can be assumed that they are different, either in terms of their physical or chemical characteristics or in both terms. Due to their individual and highly specific absorption bands the signals of organic compounds and their intensities in NIR spectra can be easily attributed and correlated to a specific organic compound and its concentration in a sample of known weight. Thus, the NIR spectroscopy allows a reliable prediction or assessment of for example the amount of dietary fiber in a sample. Since the same sample of a specific animal feed is subjected to the quantitative analysis in step a) and to the NIR spectroscopy in step A), it is also possible to attribute and correlate absorptions and their intensities in an NIR spectrum to parameters, such as the amount of arabinoxylans in the sample. Once, the absorption intensities at the respective wavelengths or wavenumbers have been successfully matched, i.e. attributed and correlated to the parameters of interest and their values, the NIR spectroscopy allows a reliable prediction or assessment of the dietary fiber in the animal feed. For this purpose a large number of NIR spectra, e.g. 100, 200, 300, 400, 500 or more, of an animal feed are recorded, and the absorption intensities at the respective wavelengths or wavenumbers are matched with the corresponding parameters and their values.
The relationship between the A/X fiber fraction in the feed and carbohydrase efficacy can be described by the following equation:
In general, the relationship shows that body weight corrected feed conversion ratio improves as total carbohydrase dose (as mg of enzyme protein/kg) and increases and A/X soybean meal ratio nears the defined range. This relationship is illustrated in
a. Comprising a an A/X soybean meal ratio between 1.8 and 2.6, preferably between 1.9 and 2.5, preferably between 2.0 and 2.3, preferably between 2.1 and 2.3, most preferably between 2.15 and 2.25; and
b. At least one carbohydrase.
one or more additional enzymes;
one or more eubiotics, such as probiotics, prebiotics and organic acids;
one or more vitamins;
of one or more carriers;
one or more minerals;
one or more amino acids; and
one or more other feed ingredients.
Effect of Carbohydrase on BWcFCR vs NC=1.629−1.533*SBM A/X ratio+0.352*SBM A/X ratio2−0.007*carbohydrase dose;
and the improvement in body weight corrected feed conversion ratio compared with a non-enzyme treatment control.
Effect of Carbohydraseon BWcFCR vs NC=1.629−1.533*SBM A/X ratio+0.352*SBM A/X ratio2−0.007*carbohydrase dose;
The present invention will be further illustrated by the following examples.
The study evaluated different levels of dietary xylanase (SEQ ID NO 2), alpha-galactosidease (SEQ ID NO 17) and multi-carbohydrase (RVB) in a corn and soybean meal based diet with a A/X soybean meal fiber fraction of 1.87 compared to control basal diet fed broilers.
Trial CS010-19; In Aug 2019, Cobb 500, male broilers (n=2100) were randomly assigned to one of seven experimental diets from day of hatch to day 42 post-hatch. There were 15 replicate pens per experimental diet and 20 birds per pen from day 0 to 7 and 18 birds per pen from day 7 to 42. All diets (see Tables 2 and 3) were fed in mash form and birds had ad libitum access to feed and fresh water. Water was provided via nipple drinkers and feed was provided via one hanging tube feeder per pen. A chick feeder tray was placed in each pen for approximately the first 4 days. Birds will be placed on their respective treatment diets upon receipt (day 0) according to the experimental design. The treatments included a positive control (PC; nutrient adequate) as well as a negative control (NC; reduction in energy levels) and the xylanase and alpha-galactosidase were included in the NC to create the experimental diets (Tables 2 and 3).
The chicks were observed daily for signs of unusual grow-out patterns or health problems. Body weights, feed consumption and feed conversion were measured weekly and feeding phases were: starter, d0 to 14; grower, day 15 to 28; and finisher, day 29 to 42. The enzymes were provided in powder form. The test material was mixed into the treatment feeds under directions provided by DSM. The treatment feeds were then placed into the pens according to the pen design for this study.
Temperature, humidity, lighting, feeder and water space was similar for all test groups. Clean wood shavings were used as bedding. Lighting was available via incandescent lights.
The xylanase of SEQ ID NO 2 was used in the concentrations indicated in table 2. The α-galactosidase of SEQ ID NO 17 was used in the concentrations indicated in table 2. A multicomponent carbohydrase composition, commercially available as Rovabio® Advance (further referred to as “RVB”), was used in the concentrations indicated in table 2. Diets were fed either as a non-supplemented, low energy diet (negative control), a non-supplemented, high energy diet (positive control) or as a low energy diet supplemented with SEQ ID NO 2, SEQ ID NO 2 and SEQ ID NO 17 or RVB. The treatments were as follows (Table 2):
On day 14, 21 and day 42, body weight gain (kg) was measured and feed conversion ratio (g/g) was calculated.
There was no effect of xylanase supplementation without or with alpha-galactosidase on growth performance when compared with birds fed the NC.
BWcFCR was only slightly (not significantly) improved compared to birds fed the NC.
Birds fed the NC and multi-carbohydrase gained similar to birds fed the PC but not different than birds fed the NC or all other enzyme doses and combinations.
The study evaluated different levels of dietary xylanase (SEQ ID NO 2), alpha-galactosidease (SEQ ID NO 17) and multi-carbohydrase (RVB) in a corn and soybean meal based diet with a A/X soybean meal fiber fraction of 1 0.98 compared to control basal diet fed broilers.
Trial (BR 190305) was performed from Aug. 30, 2019 to Apr. 6, 2020 at a Research Center in Brazil for DSM Nutritional Products. A total of 2,100 one-day-old male Cobb 500/Ross broilers were allocated in 84 experimental floor pens (2.0 m2). Birds were selected at placement and distributed by body weight. Each experimental box contains 3 nipple drinkers and one 18 kg tubular feeder. Birds were vaccinated for Marek's disease at the hatchery. Water and feed were provided ad libitum. Temperature and lighting program were in accordance with breeder recommendation (Cobb, 2016). Temperature was controlled to maintain bird comfort throughout the experimental period using infrared lamps, fans and foggers when needed. All pens were daily checked for sick and dead birds. Pen number, age and body weight of each dead bird was recorded in an excel sheet. General health status, weight, mortality and estimated cause of death were recorded. The experiment was consisting of 7 treatments, 12 replicates with 25 birds each in a completely randomized design.
Experimental diets were based on corn and soybean meal as main ingredients. The positive and negative control diets were formulated to contain the following nutrient levels (Table 8). A 3-phases feeding program was used: starter (1 to 14 d), grower (15 to 28 d) and finisher (29 to 42 d). Feeds were provided ad libitum as mash. The quantity of feed required for each dietary treatment was supplemented on top with the test enzyme. The products were applied in granulated form according to the instructions.
The xylanase of SEQ ID NO 2 was used in the concentrations indicated in table 7. The α-galactosidase of SEQ ID NO 17 was used in the concentrations indicated in table 7. A multicomponent carbohydrase composition, commercially available as Rovabio® Advance (further referred to as “RVB”), was used in the concentrations indicated in table 7. Diets were fed either as a non-supplemented, low energy diet (negative control), a non-supplemented, high energy diet (positive control) or as a low energy diet supplemented with SEQ ID NO 2, SEQ ID NO 2 and SEQ ID NO 17 or RVB. The treatments were as follows (Table 7):
Chicks were individually weighed into groups of 25 birds per pen before placement. Bird weights, averaged by pen were recorded on 1, 7, 14, 21, 28, 35 and 42 days of age. Body weight gain (BWG), feed intake (FI), feed conversion ratio (FOR) corrected for the weight of dead birds/day and feed consumption were evaluated for each phase and overall.
Supplementation with xylanase at 5 mg/kg without or with alpha-galactosidase or 2 mg/kg xylanase with alpha-galactosidase improved FOR and BWcFDR comparable to the P0.
Supplementation with Xylanase at 5 mg/kg with 2 mg/kg alpha-galactosidase improved BWcFCR compared to birds fed the NO.
There were no significant differences between birds fed xylanase without or with alpha-galactosidase and birds fed the NO and the mult-carbohydrase.
The study evaluated different levels of dietary xylanase (SEQ ID NO 2), alpha-galactosidease (SEQ ID NO 17) and multi-carbohydrase (RVB) in a corn and soybean meal based diet with a A/X soybean meal fiber fraction of 2.23 compared to control basal diet fed broilers.
Trial BR 190304; A total of 1,400 Cobb 500 one-d-old male broilers were placed in 56 experimental pens. The experiment consisted of 7 treatments, 8 replicates with 25 birds each in a completely randomized design.
A total of 1400 one-day-old male Cobb 500 slow feathering broilers were allocated in 56 experimental floor pens (2.80 m2). Birds were selected at placement and distributed by body weight. Each experimental box contained 5 nipple dinkers and one 18 kg tubular feeder. Birds were vaccinated for Marek's disease at the hatchery. Water and mash feeds will be provided ad libitum. The experiment was carried out at UFSM experimental facilities in a climate-controlled poultry house. Temperature and lighting program were in accordance with breeder recommendation (Cobb, 2016). Temperature was controlled to maintain bird comfort throughout the experimental period using infrared lamps, fans and foggers when needed. All pens were daily checked for sick and dead birds. Pen number, age and body weight of each dead bird were recorded in an excel sheet. General health status, weight, mortality and estimated cause of death were recorded.
All-vegetable corn and soybean meal mash diets were formulated. Experimental diets were based on corn and soybean meal as main ingredients. The positive and negative control diets were formulated to contain the following nutrient levels (Table 13. A 3-phases feeding program was used: starter (1 to 14 d), grower (14 to 28 d) and finisher (28 to 42 d). Feeds was be provided ad libitum as mash. The quantity of feed required for each dietary treatment was supplemented on top with the test enzyme. Products were applied in granulated form according to the instructions.
The xylanase of SEQ ID NO 2 was used in the concentrations indicated in table 12. The α-galactosidase of SEQ ID NO 17 was used in the concentrations indicated in table 12. A multicomponent carbohydrase composition, commercially available as Rovabio® Advance (further referred to as “RVB”), was used in the concentrations indicated in table 12. Diets were fed either as a non-supplemented, low energy diet (negative control), a non-supplemented, high energy diet (positive control) or as a low energy diet supplemented with SEQ ID NO 2, SEQ ID NO 2 and SEQ ID NO 17 or RVB.
The treatments were as follows (Table 12):
1Composition per kg of product: Vitamin A 11,000,000 UI; Vitamin D3 4,000,000 UI; Vitamin E 55,000 UI; Vitamin K3 3,000 mg; Vitamin B1 2,300 mg; Vitamin B2 7,000 mg; Pantothenic acid 12 g; Vitamin B6 4,000 mg; Vitamin B12 25,000 mcg; Nicotinic acid 60 g; Folic acid 2,000 mg, Biotin 250 mg; Selenium 300 mg.
2Composition per kg of product: Iron 100 g; Copper 20 g; Manganese 130 g; Zinc 130 g; Iodine 2,000 mg.
Chicks were individually weighed into groups of 25 birds per pen before placement. Bird weights, averaged by pen were recorded on 1, 7, 14, 21, 28, 35, and 42 days of age. Body weight gain (BWG), feed intake (FI), feed conversion ratio (FCR) corrected for the weight of dead birds were evaluated each phase and from 1 to 21 d, 22 to 42 d, and 1 to 42 d.
Supplementation with Xylanase at 2 or 5 mg/kg with alpha-galactosidase increased BWG compared with birds fed the NC.
Supplementation with Xylanase at 5 mg/kg with 2 mg/kg alpha-galactosidase improved BWcFCR compared to birds fed the NC and equivalent to birds fed the PC.
Birds fed 5 mg/kg xylanase with alpha-galactosidase gained body weight comparable to birds fed the NC with the multi-carbohydrase but had an improved BWcFCR.
The supplementation of the basal feed having a high A/X soybean meal ratio of 2.23 with xylanase leds to a significantly improved BWcFCR and body weight gain.
The supplementation of the basal feed having a medium A/X soybean meal ratio of 1.98 with xylanase leds to an improved BWcFCR and body weight gain.
The supplementation of the basal feed having a low A/X soybean meal ratio of 1.87 with xylanase leds to small to no improvement in BWcFCR and body weight gain.
The same trend can be observed for supplementation with the multi-carbohydrase RVB and alpha-galactosidase.
The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20175236.7 | May 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/063187 | 5/18/2021 | WO |
