EP4297583A1 - Verfahren zur verbesserung der kohlenhydratverdaulichkeit durch eine carbohydratase in einem tierfutter unter verwendung von serinprotease - Google Patents

Verfahren zur verbesserung der kohlenhydratverdaulichkeit durch eine carbohydratase in einem tierfutter unter verwendung von serinprotease

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Publication number
EP4297583A1
EP4297583A1 EP22714769.1A EP22714769A EP4297583A1 EP 4297583 A1 EP4297583 A1 EP 4297583A1 EP 22714769 A EP22714769 A EP 22714769A EP 4297583 A1 EP4297583 A1 EP 4297583A1
Authority
EP
European Patent Office
Prior art keywords
protease
animal feed
proteases
polypeptide
carbohydrase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22714769.1A
Other languages
English (en)
French (fr)
Inventor
Aaron COWIESON
Eduardo Antonio Della Pia
Jose-Otavio SORBARA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novozymes AS
DSM IP Assets BV
Original Assignee
Novozymes AS
DSM IP Assets BV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novozymes AS, DSM IP Assets BV filed Critical Novozymes AS
Priority claimed from PCT/EP2022/025071 external-priority patent/WO2022179757A1/en
Publication of EP4297583A1 publication Critical patent/EP4297583A1/de
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01007—Inulinase (3.2.1.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01025—Beta-mannosidase (3.2.1.25), i.e. mannanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01032—Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1-3-beta-xylanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)

Definitions

  • the present invention is related to a method for boosting the activity of a carbohydrase in an animal feed and/or improving digestibility of a carbohydrate in an animal.
  • Carbohydrates are energy-providing feed components composed of carbon, hydrogen and oxygen. They should make up about 75 percent of an animal's diet. The energy they provide powers muscular movements. Carbohydrates also produce the body heat that helps keep the animal warm. They aid in the use of proteins and fats by the body. Carbohydrates are not stored in the body. They must be provided in the animal's diet every day. Unused carbohydrates are converted into fats to be stored.
  • Carbohydrates can be classified into two categories: storage carbohydrates and structural carbohydrates.
  • Storage carbohydrates include starch and simple sugars, such as fructose and saccharose. These carbohydrates, along with lipids in the embryonic part of seeds, are the main energy sources for the new plants that will be emerging from cereal seeds.
  • Structural carbohydrates on the other hand, including the well-known non-starch polysaccharides, are responsible for cellular form and structure, and are located mostly in the outer cellular membrane. Structural carbohydrates, commonly referred to as "fiber,” are hardly digested by monogastric animals due to lack of suitable endogenous enzymes. Thus, the majority of structural carbohydrates are fermented in the hind gut, where they may release limited useful energy levels.
  • Carbohydrases are specific commercial enzyme preparations that attack carbohydrates releasing energy that would be otherwise lost for the animal. They work mainly by opening the cell wall structure of intact plant cells, release thus not only energy (starch), but also other nutrients, such as protein, minerals and lipids. In addition, plant cell wall fractions increase intestinal viscosity that leads to reduced nutrient absorption, accelerated proliferation of pathogens, such as Escherichia coli, and other problems, such as sticky droppings and dirty eggs. Today carbohydrases are becoming increasingly popular in animal feeds.
  • proteases can boost the activity of carbohydrases to hydrolyze carbohydrates in an animal feed and thus potentially improving digestibility of carbohydrates in an animal.
  • the present invention provides a method for boosting the activity of a carbohydrase or improving hydrolyzation of a carbohydrate by a carbohydrase in an animal feed, and a method for improving digestibility of a carbohydrate in an animal by using one or more proteolytic enzymes, i.e., proteases.
  • the present invention also provides a feed composition comprising one or more proteolytic enzymes, i.e., proteases and a carbohydrase for improving hydrolyzation of carbohydrates in an animal feed and/or for improving digestibility of a carbohydrate in an animal and use thereof.
  • proteolytic enzymes i.e., proteases and a carbohydrase for improving hydrolyzation of carbohydrates in an animal feed and/or for improving digestibility of a carbohydrate in an animal and use thereof.
  • animal refers to any animal except humans.
  • 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 broilers), chicks, layer hens (referred to herein as layers)); pets such as cats and dogs; horses (including but not limited to hotbloods, coldbloods and warm bloods), crustaceans (including but not limited to shrimps and prawns) and fish (including but not limited to amberjack, arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia,
  • animal feed refers to any compound, preparation, or mixture suitable for or intended for intake by an animal and capable of maintaining life and/or promoting production of the animal without any additional substance being consumed except water.
  • 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)
  • 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).
  • concentrates refers to 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. 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)).
  • 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. corn, oats, rye, barley, wheat), oilseed press cake (e.g. from cottonseed, safflower, sunflower, soybean (such as
  • forage is fresh plant material such as hay and silage from forage plants, grass and other forage plants, seaweed, sprouted grains and legumes, or any combination thereof.
  • 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.
  • 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 soy beans, rapeseed and other legumes; and fractions from the refining of grains for animal or human consumption or from fuel production or other industries.
  • the term “roughage” refers to 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).
  • the term “animal feed additive” refers to an ingredient or combination of ingredients added to the animal feed, usually used in micro quantities and requires careful handling and mixing.
  • Such ingredient includes but is not limited to vitamins, amino acids, minerals, enzymes, eubiotics, colouring agents, 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 etc..
  • PUFAs polyunsaturated fatty acids
  • hydrolyzation refers to the carbohydrates to be hydrolysed are broken down under acidic or alkaline conditions and/or in the presence of an enzyme into soluble sugar molecules.
  • a polysaccharide can be hydrolysed into monosaccharaides or disaccharides or oligosaccharides by treating with concentrated hydrochloric acid
  • starch can be broken into maltose by treating with concentrated hydrochloric acid or an amylase.
  • the hydrolyzation of carbohydrates is improved when more carbohydrates, for example, 1%, 2%, 3%, 4%, 5% or more carbohydrates, are broken down into soluble sugar molecules in the presence of the feed composition according to the present invention compared to a feed composition otherwise.
  • the present invention provides a method for boosting the activity of a carbohydrase in an animal feed comprising adding to the animal feed one or more proteolytic enzymes, i.e., proteases.
  • the present invention also provides a method for improving hydrolyzation of a carbohydrate in an animal feed comprising adding to the animal feed one or more proteolytic enzymes, i.e., proteases, and a carbohydrase.
  • the present invention further provides a method for improving digestibility of a carbohydrate in an animal comprising administering to the animal one or more proteolytic enzymes, i.e., proteases, and a carbohydrase in an animal feed.
  • proteolytic enzymes i.e., proteases
  • proteolytic enzymes or proteases catabolize peptide bonds in proteins breaking them down into fragments of amino acid chains, or peptides.
  • proteases are classified on the basis of their catalytic mechanism into the following groups: serine proteases (S), cysteine proteases (C), aspartic proteases (A), metalloproteases (M), and unknown, or as yet unclassified, proteases (U) (see Flandbook of Proteolytic Enzymes, A. J. Barrett, N. D. Rawlings, J. F. Woessner (eds), Academic Press (1998)).
  • the protease according to the present invention is a serine protease, preferably an acid stable serine protease, and more preferably a S8 protease.
  • protease includes not only natural or wild-type proteases, but also any mutants, variants, fragments etc. thereof exhibiting protease activity, as well as synthetic proteases, such as shuffled proteases, and consensus proteases.
  • Such genetically engineered proteases can be prepared as is generally known in the art, e. g. by site-directed mutagenesis, by PCR (using a PCR fragment containing the desired mutation as one of the primers in the PCR reactions), or by random mutagenesis. The preparation of consensus proteins is described in e. g. EP 0897985.
  • the protease according to the present invention is a microbial protease, the term microbial indicating that the protease is derived from, or originates from a microorganism, or is an analogue, a fragment, a variant, a mutant, or a synthetic protease derived from a microorganism. It may be produced or expressed in the original wild-type microbial strain, in another microbial strain, or in a plant; i.e. the term covers the expression of wild-type, naturally occurring proteases, as well as expression in any host of recombinant, genetically engineered or synthetic proteases.
  • microorganisms are bacteria, e. g. bacteria of the phylum Actinobacteria phy. nov., e. g. of class I: Actinobacteria, e. g. of the Subclass V: Actinobacteridae, e. g. of the Order I: Actinomycetales, e. g. of the Suborder XII: Streptosporangineae, e. g. of the Family II: Nocardiopsaceae, e. g. of the Genus I: Nocardiopsis, e. g. Nocardiopsis sp. NRRL 18262, and Nocardiopsis alba ; e.g.
  • microorganisms are fungi, such as yeast or filamentous fungi.
  • Preferred protease according to the present invention is an acid stable serine protease obtained or obtainable from the Genus: Nocardiopsis, such as those derived from Nocardiopsis dassonvillei DSM 43235 (A1918L1), Nocardiopsis prasina DSM 15649 (NN018335L1), Nocardiopsis prasina (previously alba) DSM 14010 (NN18140L1), Nocardiopsis sp. DSM 16424 (NN018704L2), Nocardiopsis alkaliphila DSM 44657 (NN019340L2) and Nocardiopsis lucentensis DSM 44048 (NN019002L2); or the Genus: Bacillus, e.g.
  • Bacillus horneckiae Bacillus sp TY145, Bacillus sp-13380, Bacillus idriensis, Bacillus sp-62451 and Bacillus oceanisediminis; as well as homologous proteases.
  • protease includes not only natural or wild-type proteases, but also any mutants, variants, fragments etc. thereof exhibiting protease activity, as well as synthetic proteases, such as shuffled proteases, and consensus proteases.
  • Such genetically engineered proteases can be prepared as is generally known in the art, e. g. by Site-directed Mutagenesis, by PCR (using a PCR fragment containing the desired mutation as one of the primers in the PCR reactions), or by Random Mutagenesis. The preparation of consensus proteins is described in e. g. EP 0897985.
  • acid-stable proteases for use according to the invention are a) the proteases derived from Nocardiopsis sp. NRRL 18262, and Nocardiopsis alba ; b) proteases of at least 60, 65, 70, 75, 80, 85, 90, or at least 95% amino acid identity to any of the proteases of (i) ; c) proteases of at least 60, 65, 70, 75, 80, 85, 90, or at least 95% identity to any of SEQ ID NO : 1, and/or SEQ ID NO : 2.
  • any computer program known in the art can be used. Examples of such computer programs are the Clustal V algorithm (Higgins, D. G., and Sharp, P. M. (1989), Gene (Amsterdam), 73, 237-244 ; and the GAP program provided in the GCG version 8 program package (Program Manual for the Wisconsin Package, Version 8, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-453.
  • Clustal V algorithm Higgins, D. G., and Sharp, P. M. (1989), Gene (Amsterdam), 73, 237-244
  • GAP program provided in the GCG version 8 program package (Program Manual for the Wisconsin Package, Version 8, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-453.
  • the protease for use according to the invention can be produced or expressed in the original wild-type microbial strain, in another microbial strain, or in a plant; i. e. the term covers the expression of wild-type, naturally occurring proteases, as well as expression in any host of recombinant, genetically engineered or synthetic proteases.
  • the protease activity is at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 97% of the reference activity.
  • the step b) buffer pH-value may be 1.0, 1.5, 2.0, 2.5, 3.0, 3.1, 3.2, 3.3, or 3.4.
  • the residual protease activity as compared to the reference is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 97%.
  • pH values of 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5 can be applied for the step d) buffer.
  • the term A280 1.0 means such concentration (dilution) of said pure protease which gives rise to an absorption of 1.0 at 280 nm in a 1 cm path length cuvette relative to a buffer blank.
  • pure protease refers to a sample with a A280/A260 ratio above or equal to 1.70.
  • the protease for use according to the invention besides being acid- stable, is also thermostable.
  • thermostable means one or more of the following: That the temperature optimum is at least 50 °C, 52 °C, 54 °C, 56 °C, 58 °C, 60 °C, 62 °C, 64 °C, 66 °C, °68 C, or at least °70 C.
  • protease defined by polypeptides having S8 protease activity, wherein the polypeptide is selected from the list consisting of: a) a polypeptide having a sequence identity of at least 70% to any one of SEQ ID NOs 3-6; b) a variant of any one of SEQ ID NOs: 3-6, wherein the variant has protease activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions; c) a polypeptide comprising the polypeptide of (a 1 ) or (b 1 ) and a N-terminal and/or C-terminal His-tag and/or HQ
  • proteases which are covered by definitions above are Ronozyme ® ProAct (DSM Nutritional Products AG, Switzerland), Ronozyme ® ProAct360 (DSM Nutritional Products Ltd., Switzerland), AxtraPro (subtilisin-type protease, Dupont, USA), Poultrygrow (mixture of different proteases, Jefo, USA), Cibenza DP100 (Novus, USA).
  • the intended dosage of the protease is 0.01-200 mg protease enzyme protein per kg final feed.
  • the protease may be provided in a dosage of between 1,000 units/kg animal feed and 1,000,000 units/kg animal feed, for example in one of the following amounts (dosage ranges): 1,000, 2,000, 4,000, 6,000, 8,000, 10,000, 15,000, 20,000, 30,000, 50,000, 80,000, 100,000, 150,000, 200,000, 250,000, 300,000, 500,000, 600,000, 800,000, 1,000,000 units/kg animal feed.
  • One protease unit is the amount of enzyme that releases 1 pmol of p-nitroaniline (pNA) from 1 mM substrate (such as N-succinyl-Ala-Ala-Pro-Phe-pNA) per minute at pH 9.0 and 37°C.
  • the carbohydrase may be any carbohydrase which can be added into an animal feed for feeding an animal.
  • carbohydrases include but are not limited to hemicellulases, pectinases, glucanases, amylases (such as a-amylase, b-amylase and y-amylase), xylanases, galactosidases maltases and mixtures thereof.
  • Preferred xylanases are l,4-R-xylanases, for example endo-l,4-R-xylanases produced by Trichoderma reesei.
  • carbohydrase mixtures are mixtures of different xylanases or carbohydrase blends comprising at least two enzymes selected from the group consisting of xylanases, glucanases, hemicellulases, pectinases, amylases, galactosidases, maltases.
  • Mixtures containing a combination of more than 10 active enzymes can be produced by only one non-genetically modified fungus, as for example Talaromyces versatilis.
  • carbohydrases which are covered by the definitions above and can be boosted by proteases according to the invention are Ronozyme ® VP (Glucanase, DSM Nutritional Products AG, Switzerland), Ronozyme ® WX (Xylanase, DSM Nutritional Products Ltd., Switzerland), RONOZYME ® HiStarch (Amylase from Bacillus licheniformis, DSM Nutritional Products AG, Switzerland), Rovabio (Carbohydrase blend, Adisseo, France), ECONASE ® (Xylanase, AB Enzymes, Germany), CIBENZA CSM (mixture of xylanase, b-glucanase and galactosidase, Novus International, USA) and AllzymeSSF (Carbohydrase blend, Alltech USA).
  • Ronozyme ® VP Glucanase, DSM Nutritional Products AG, Switzerland
  • Ronozyme ® WX Xylanase
  • the carbohydrase is provided in a dosage of between 10 units/kg animal feed and 5,000 units/kg animal feed, for example in one of the following: 10, 20, 40, 50, 60, 80, 100, 200, 500, 800, 1,000, 2,000, 3,000, 4,000 and 5,000 units/kg animal feed.
  • the activity of a carbohydrase can be represented by hydrolyzation of a carbohydrate by the carbohydrase, which can be easily measured by a process known in the art, for example, by testing the hydrolysate of the carbohydrate treated by the carbohydrase as shown in the examples of the present application.
  • the activity of a carbohydrase is boosted when the carbohydrate breaks more carbohydrate, such as 1%, 2%, 3%, 4%, 5% or more, into soluble sugar molecules in the presence of a protease compared to the same conditions without the protease.
  • the activity of a carbohydrase may be boosted or the hydrolyzation of carbohydrates may be improved by 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20% or more based on the amount of the hydrolysate obtained from the hydrolyzation with the carbohydrase.
  • carbohydrates are the basic source of energy for all animals and must be provided in the animal diets every day.
  • the carbohydrates suitable for the animal feed according to the present invention may be monosaccharaides such as galactose, glucose, fructose, ribose, arabinoses and xylose; disaccharides such as maltose, sucrose and lactose; oligosaccharides such as arabinoxylans; and/or polysaccharides such as starch including amylose, amylopectin and modified starches, non-starch polysaccharides (NSP) including pentosans, glycogen, fibre, cellulose, hemicellulose, chitin, pectin and/or hydrocolloids.
  • NSP non-starch polysaccharides
  • the carbohydrates to be hydrolysed by the carbohydrase may be oligosaccharides and/or polysaccharides.
  • the carbohydrates are arabinoxylans, starch or NSP such as fibre, cellulose, hemicellulose and pectin.
  • the carbohydrates may be in the forms of concentrates such as soy-bean meal, corn, wheat, wheat middlings, oats, rye, barley and sorghum; and/or roughages such as hay and pasture plants, or mixture thereof.
  • the carbohydrates in the animal feed are in the forms of soy-bean meal, corn, wheat, wheat middlings, oats, rye or barley, or mixture thereof.
  • the animal feed according to the present invention is an animal diet based on soy-bean meal, corn and/or wheat.
  • the animal feed according to the present may also include micro-ingredients.
  • micro-ingredients include but are not limited to aroma compounds; antimicrobial peptides; polyunsaturated fatty acids (PUFAs); reactive oxygen generating species; at least one enzyme, and fat- and water-soluble vitamins, as well as minerals.
  • aroma compounds include but are not limited to aroma compounds; antimicrobial peptides; polyunsaturated fatty acids (PUFAs); reactive oxygen generating species; at least one enzyme, and fat- and water-soluble vitamins, as well as minerals.
  • PUFAs polyunsaturated fatty acids
  • reactive oxygen generating species at least one enzyme
  • fat- and water-soluble vitamins as well as minerals.
  • antimicrobial peptides examples include CAP18, leucocin A, protegrin-1, thanatin, defensin, lactoferrin, lactoferricin, and ovispirin such as novispirin (Robert Lehrer, 2000), plectasins, and statins.
  • polyunsaturated fatty acids are Cig-, C20- and C22- polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid.
  • reactive oxygen generating species are chemicals such as perborate, persulphate, or percarbonate; and enzymes such as an oxidase, an oxygenase or a syntethase.
  • enzymes examples include phytase (EC 3.1.3.8 or 3.1.3.26), galactanase (EC 3.2.1.89), alpha-galactosidase (EC 3.2.1.22), phospholipase A 1 (EC 3.1.1.32), phospholipase A2 (EC 3.1.1.4), lysophospholipase (EC 3.1.1.5), phospholipase C (EC 3.1.4.3), and/or phospholipase D (EC 3.1.4.4).
  • phytase EC 3.1.3.8 or 3.1.3.26
  • galactanase EC 3.2.1.89
  • alpha-galactosidase EC 3.2.1.22
  • phospholipase A 1 EC 3.1.1.32
  • phospholipase A2 EC 3.1.1.4
  • lysophospholipase EC 3.1.1.5
  • phospholipase C EC 3.1.4.3
  • phospholipase D
  • fat-soluble vitamins include but are not limited to vitamin A, vitamin D3, and vitamin K, e.g. vitamin K3.
  • water-soluble vitamins include but are not limited to vitamin B12, biotin and choline, vitamin Bi, vitamin B2, vitamin Be, niacin, folic acid and panthothenate, e.g. Ca-D-panthothenate.
  • minerals include but are not limited to calcium, phosphorus, sodium, potassium, magnesium, chlorine, iodine, iron, manganese, copper, molybdenum, cobalt and zinc.
  • Common mineral supplements in feed are: limestone, Bone meal, oyster shell, sodium chloride, dicalcium phosphate, manganese sulphate, potassium iodide, and superphosphate.
  • Sources of minerals include meat scraps, fish meal, milk products, ground limestone (calcium), ground oyster shells (calcium), dicalcium phosphate (calcium, phosphorus), defluorinated rock phosphate (phosphorus, calcium), steamed bone meal (phosphorus, calcium), salt (sodium, chlorine, iodine), manganese sulfate (manganese), manganese oxide (manganese), zinc carbonate (zinc), zinc oxide (zinc).
  • the animal feed according to the present invention may further include any number of components typical for an animal feed, such as proteins, carbohydrates as defined above, fats and additional additives.
  • suitable types of proteins include, but are not limited to, meat scraps (lysine), fish meal (lysine, methionine), poultry by-product meal (tryptophan, lysine), blood meal, liver and glandular meal, feather meal (hydrolyzed), animal tankage, milk products, cottonseed meal, peanut meal, soybean meal, sesame meal, sunflower seed meal.
  • feed ingredients (maize, barley, safflower, milo, wheat, rice, bran, etc.) contain approximately 2-5% fat and linoleic acid.
  • Sources of fats include animal tallow (beef), lard, corn oil, and other vegetable oils.
  • Additional additives include but are not limited to minerals as defined above; antioxidants like BHT (Butylated hydroxytoluene), santoquin, ethoxyquin, butylated hydroxyanisode and diphenyl paraphenyl diamine; pellet binders such as sodium bentonite (clay), liquid or solid by-products of the wood pulp industry, molasses, and guarmeal; coloring agents such as xanthophylls, synthetic carotinoid, and canthaxanthin; probiotics such as strains of lactobacillus and streptococcus; and/or antibiotics such as penicillin, streptomycin, tetracyclines, and aureomycin.
  • antioxidants like BHT (Butylated hydroxytoluene), santoquin, ethoxyquin, butylated hydroxyanisode and diphenyl paraphenyl diamine
  • pellet binders such as sodium bentonite (cla
  • the present invention provides a method for boosting the activity of a carbohydrase in an animal feed comprising adding to the animal feed one or more proteolytic enzymes, i.e., proteases, wherein: a) the protease is an acid stable serine protease, and more preferably a S8 protease, in a dosage of between 10,000 units/kg feed and 30,000 units/kg feed; b) the carbohydrase is hemicellulase, pectinase, amylase, xylanase or mixture thereof; c) the animal feed is an animal diet based on soy-bean meal, corn and/or wheat; and d) optionally the activity of a carbohydrase is increased by 5% or more.
  • proteases i.e., proteases
  • the present invention provides a method for improving hydrolyzation of a carbohydrate in an animal feed comprising adding to the animal feed one or more proteolytic enzymes, i.e., proteases, and a carbohydrase, wherein: a) the protease is an acid stable serine protease, and more preferably a S8 protease, in a dosage of between 10,000 units/kg feed and 30,000 units/kg feed; b) the carbohydrase is hemicellulase, pectinase, amylase, xylanase or mixture thereof; c) the animal feed is an animal diet based on soy-bean meal, corn and/or wheat; and d) optionally the activity of a carbohydrase is increased by 5% or more.
  • the present invention provides a feed composition comprising one or more protease as defined below and a carbohydrase or carbohydrase mixture.
  • Proteases of such compositions are:
  • A Acid stable proteases selected from the group consisting of: a) the proteases derived from Nocardiopsis sp. NRRL 18262, and Nocardiopsis alba ; b) proteases of at least 60, 65, 70, 75, 80, 85, 90, or at least 95% amino acid identity to any of the proteases of (i) ; c) proteases of at least 60, 65, 70, 75, 80, 85, 90, or at least 95% identity to any of SEQ ID NO : 1, and/or SEQ ID NO : 2. or
  • Proteases defined by polypeptides having S8 protease activity wherein the polypeptide is selected from the list consisting of: a) (a 1 ) a polypeptide having a sequence identity of at least 70% to any one of SEQ ID NOs 3-6; b) (b 1 ) a variant of any one of SEQ ID NOs: 3-6, wherein the variant has protease activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions; c) (c 1 ) a polypeptide comprising the polypeptide of (a 1 ) or (b 1 ) and a N-terminal and/or C-terminal His- tag and/
  • the carbohydrase of such compositions may be any carbohydrase which can be added into an animal feed for feeding an animal.
  • the carbohydrases include but are not limited to hemicellulases, pectinases, glucanases, amylases (such as a-amylase, b-amylase and y-amylase), xylanases, galactosidases maltases.
  • Preferred xylanases are l,4-R-xylanases, for example endo-l,4-R-xylanases produced by Trichoderma reesei.
  • carbohydrase mixtures are mixtures of different xylanases or mixtures comprising at least two enzymes selected from the group consisting of xylanases, glucanases, hemicellulases, pectinases, amylases, galactosidases, maltases.
  • Mixtures containing a combination of more than 10 active enzymes can be produced by only one non-genetically modified fungus, as for example Talaromyces versatilis.
  • carbohydrases which are covered by the definitions above are Ronozyme ® VP (Glucanase, DSM Nutritional Products AG, Switzerland), Ronozyme ® WX (Xylanase, DSM Nutritional Products Ltd., Switzerland), RONOZYME ® HiStarch (Amylase from Bacillus licheniformis, DSM Nutritional Products AG, Switzerland), Rovabio (Carbohydrase blend, Adisseo, France), ECONASE ® (Xylanase, AB Enzymes, Germany), CIBENZA CSM (mixture of xylanase, b-glucanase and galactosidase, Novus International , USA), AllzymeSSF (Carbohydrase blend, Alltech USA).
  • the feed composition, and/or the components such as the carbohydrase and the protease which the composition contains may be formulated as a liquid formulation or a solid formulation. Accordingly, the feed composition according to the present invention may also comprise one or more formulating agents.
  • the formulating agents may be selected from the group consisting of polyol such as glycerol, sorbitol, ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, dipropylene glycol and polyethylene glycol (PEG); a salt such as organic or inorganic zinc, sodium, potassium, calcium or magnesium salts (for example, magnesium sulfate, 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 and zinc sulfate); and starch or a sugar or sugar derivative such as sucrose
  • the feed composition according to the present invention may also comprise one or more emulsifying agents.
  • the emulsifying agents may be selected advantageously from the group consisting of polyglycerol esters of fatty acids such as esterified ricinoleic acid or propylene glycol esters of fatty acids, saccharo- esters or saccharo-glycerides, polyethylene glycol, lecithins, etc..
  • the protease may be provided in a dosage of between 1,000 units/kg animal feed and 1,000,000 units/kg animal feed, for example in one of the following amounts (dosage ranges): 1,000, 2,000, 4,000, 6,000, 8,000, 10,000, 15,000, 20,000, 30,000, 50,000, 80,000, 100,000, 150,000, 200,000, 250,000, 300,000, 500,000, 600,000, 800,000, 1,000,000 units/kg animal feed.
  • the carbohydrase may be provided in a dosage of between 10 units/kg animal feed and 5,000 units/kg animal feed, for example in one of the following: 10, 20, 40, 50, 60, 80, 100, 200, 500, 800, 1,000, 2,000, 3,000, 4,000 and 5,000 units/kg animal feed.
  • the feed composition according to the present invention may be formulated as a feed additive or an animal feed and thus may further comprise ingredients as defined above suitable for an animal feed.
  • the third aspect of the present invention provides use of one or more proteolytic enzymes, i.e., proteases, and a carbohydrase in an animal feed for improving digestibility of carbohydrates in an animal, wherein the protease, the carbohydrase, the carbohydrate and the animal feed and the relevant dosage are defined as above.
  • Example 1 Compositions comprising protease
  • Compositions 1-3 comprising protease according to the present invention was made by mixing the following ingredients and amounts as shown in Table 1.
  • Example 2 Animal feed comprising protease An animal feed comprising protease according to the present invention was made by adding protease into the feed with the following formulation as shown in Table 2.
  • Example 3 Effect of protease in the animal feed on SBM The effect of a protease for application in the animal feed on soy-bean meal (SBM) was investigated in an in vitro experiment.
  • RONOZYME ® VP breaks down and solubilizes NSPs from SBM, and, when adding protease, 5% more of the NSPs from SBM are hydrolyzed and solubilized.
  • Example 4 Use of protease in the animal feed on destarched com
  • 100 mg of RONOZYME ® WX CT granules were extracted in 25 mL of 0.1 M acetate buffer + 5 mM CaCh pH 6.0. The sample was filtered with a 0.2 pm filter. 100 pL of the obtained enzyme solution was added to 4 mL of 10% destarched corn weight/volume. 11,2 pL of a solution of the purified protease (concentration 21.5 mg/mL) was diluted with 1989 pLof 0.1 M acetate buffer + 5 mM CaCh pH 6.0. 100 pL of the obtained enzyme solution was added to 4 mL of 10% destarched corn weight/volume.
  • RONOZYME ® WX breaks down and solubilizes arabinoxylans from wheat, and, when adding protease, 23% more arabinoxylan is solubilized.
  • Example 5 Use of protease in the animal feed on com

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EP22714769.1A 2021-02-26 2022-02-28 Verfahren zur verbesserung der kohlenhydratverdaulichkeit durch eine carbohydratase in einem tierfutter unter verwendung von serinprotease Pending EP4297583A1 (de)

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