Cellulolytic enzymes for animal feed comprising oilseed material

Abstract

The present invention relates to the use of a cellulolytic enzyme in an animal feed comprising oilseeds, thereby improving the availability of nutrients from the feed and the nutritional value of the animal feed. The invention further relates to an animal feed comprising a cellulolytic enzyme and oilseed material.

Description

[0001] References to sequence listings

[0002] This application contains a sequence listing in computer readable form. This computer readable form is incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of fiber degrading enzymes in animal feeds comprising oilseeds. Background of the Invention

[0005] Oilseed materials make up about 25% of monogastric diets. They are cheap, available in large quantities, and have a high protein content. However, oilseed materials contain substances that are barely digested by monogastric animals, as these animals lack the relevant enzymes in their digestive tract for digesting these substances. If feed ingredients are barely digested, the energy contained in the feed ingredients is not fully utilized. In addition, undigested substances have an effect on the viscosity of the feed in the digestive tract. Increased viscosity will lead to impaired digestibility of other nutrients.

[0006] Maintaining a healthy gut is important in the production of monogastric animals, and diet, along with environmental conditions, are key contributing factors that influence the composition of the microbiota.

[0007] There is a need in the art to improve the nutritional value of animal feeds comprising oilseed material.Furthermore, there is a need in the art to improve the intestinal health of monogastric animals fed a feed comprising oilseed material. Summary of the invention

[0008] The present invention relates to a method of improving the nutritional value of an animal feed comprising oilseed material.

[0009] The present invention further relates to a method for improving the growth performance of an animal, the method comprising administering to the animal a fiber degrading enzyme, an animal feed comprising a fiber degrading enzyme or an animal feed additive; preferably, the growth performance is growth rate, feed conversion ratio and / or body weight gain.

[0010] The present invention relates to a method for improving the nutritional value of an animal feed comprising an oilseed material, the method comprising adding a fiber degrading enzyme to the animal feed. A further aspect relates to a method for improving the growth performance of an animal, the method comprising administering to the animal a fiber degrading enzyme, an animal feed comprising a fiber degrading enzyme or an animal feed additive; preferably, the growth performance is growth rate, feed conversion ratio and / or body weight gain.

[0011] The present invention further relates to a method for the in situ production of prebiotics in an oilseed based animal feed, said method comprising adding a fiber degrading enzyme to said animal feed, including a method for the in situ production of prebiotics in monogastric animals.

[0012] The invention further relates to a method for reducing the insoluble pectin fraction in oilseed based animal feed.

[0013] The present invention further relates to a method of improving the intestinal health of a monogastric animal, the method comprising administering to the animal an oilseed based animal feed, wherein the animal feed comprises a fiber degrading enzyme.

[0014] The present invention further relates to methods of inducing butyrate production in monogastric animals.

[0015] The present invention further relates to an animal feed comprising a fiber degrading enzyme and an oilseed material, such as the following animal feed comprising a fiber degrading enzyme and an oilseed material, wherein the feed comprises an oilseed material in an amount of 10 to 500 g / kg feed and a fiber degrading enzyme in an amount of 0.1 to 500 mg enzyme protein / kg feed. Additional aspects relate to an animal feed additive comprising a fiber degrading enzyme and one or more additional components selected from the group consisting of: one or more vitamins; one or more minerals; one or more amino acids; one or more phytobiotics; one or more prebiotics; one or more organic acids; and one or more other feed ingredients. A related aspect relates to an animal feed comprising the animal feed additive of the present invention and an oilseed material.

[0016] The invention further relates to the use of a fiber degrading enzyme in the preparation of an enzyme-enriched animal feed.

[0017] The present invention further relates to the use of a combination of a polypeptide having rhamnogalacturonan lyase activity and a polypeptide having galactanase activity in animal feed or an animal feed additive; or the use of a combination of rhamnogalacturonan lyase and pectin lyase in animal feed or an animal feed additive; or the use of a combination of endo-β-1,4-galactanase and pectin lyase in animal feed or an animal feed additive; or the use of a combination of xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase and pectin lyase in animal feed or an animal feed additive.

[0018] The present invention further relates to an animal feed additive comprising a polypeptide having rhamnogalacturonan lyase activity and a polypeptide having galactanase activity; preferably an animal feed additive comprising: a combination of rhamnogalacturonan lyase and endo-β-1,4-galactanase; a combination of rhamnogalacturonan lyase and pectin lyase; a combination of endo-β-1,4-galactanase and pectin lyase; or a combination of xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase and pectin lyase; and an animal feed comprising the animal feed additive.

[0019] The invention further relates to a method for improving the average metabolizable energy of a plant-based diet in a monogastric animal.

[0020] The present invention further relates to a polypeptide having xyloglucan-specific endo-1,4-β-glucanase activity, in particular a polypeptide having xyloglucan-specific endo-1,4-β-glucanase activity selected from the group consisting of:

[0021] (a) a polypeptide having at least 99.7%, at least 99.8%, at least 99.9% or 100% sequence identity to SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO: 3;

[0022] (b) a polypeptide derived from SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO: 3 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0023] (c) a polypeptide having at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:4 or the mature polypeptide of SEQ ID NO:4;

[0024] (d) a polypeptide derived from SEQ ID NO: 4 or the mature polypeptide of SEQ ID NO: 4 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0025] (e) a polypeptide derived from the polypeptide of (a), (b), (c) or (d), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0026] (f) a fragment of the polypeptide of (a), (b), (c), (d) or (e);

[0027] The polypeptide has xyloglucan-specific endo-1,4-β-glucanase activity.

[0028] The present invention further relates to a polypeptide having xylose galacturonase activity, in particular a polypeptide having xylose galacturonase activity selected from the group consisting of:

[0029] (a) a polypeptide having at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:6 or the mature polypeptide of SEQ ID NO:6;

[0030] (b) a polypeptide derived from SEQ ID NO:6 or the mature polypeptide of SEQ ID NO:6 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0031] (c) a polypeptide having at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:7 or the mature polypeptide of SEQ ID NO:7;

[0032] (d) a polypeptide derived from SEQ ID NO: 7 or the mature polypeptide of SEQ ID NO: 7 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0033] (e) a polypeptide derived from the polypeptide of (a), (b), (c) or (d), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0034] (f) a fragment of the polypeptide of (a), (b), (c), (d) or (e);

[0035] The polypeptide has xylose galacturonase activity.

[0036] The present invention further relates to a polypeptide having rhamnogalacturonan lyase activity, in particular a polypeptide having rhamnogalacturonan lyase activity selected from the group consisting of:

[0037] (a) a polypeptide having at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:9 or the mature polypeptide of SEQ ID NO:9;

[0038] (b) a polypeptide derived from SEQ ID NO: 9 or the mature polypeptide of SEQ ID NO: 9 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0039] (c) a polypeptide derived from the polypeptide of (a) or (b) wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0040] (d) a fragment of the polypeptide of (a), (b) or (c);

[0041] The polypeptide has rhamnogalacturonan lyase activity.

[0042] The present invention further relates to a polypeptide having xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase activity, in particular a polypeptide having xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase activity selected from the group consisting of:

[0043] (a) a polypeptide having at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10 or the mature polypeptide of SEQ ID NO: 10;

[0044] (b) a polypeptide derived from SEQ ID NO: 10 or the mature polypeptide of SEQ ID NO: 10 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0045] (c) a polypeptide derived from the polypeptide of (a) or (b) wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0046] (d) a fragment of the polypeptide of (a), (b) or (c);

[0047] The polypeptide has xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase activity.

[0048] The present invention further relates to polynucleotides encoding the polypeptides of the present invention.

[0049] The present invention further relates to an animal feed additive comprising the polypeptide of the present invention.

[0050] The present invention further relates to an animal feed comprising the polypeptide of the present invention.

[0051] Overview of Sequence Listing

[0052] SEQ ID NO: 1 is a polypeptide having rhamnogalacturonan lyase activity from Aspergillus aculeatus.

[0053] SEQ ID NO: 2 is a polypeptide having endo-polygalacturonase activity from Aspergillus aculeatus.

[0054] SEQ ID NO: 3 is a polypeptide having xyloglucan-specific endo-1,4-β-glucanase activity from Aspergillus aculeatus.

[0055] SEQ ID NO: 4 is a polypeptide having xyloglucan-specific endo-1,4-β-glucanase activity from Aspergillus luchuensis.

[0056] SEQ ID NO: 5 is a polypeptide having galactanase activity from Cohnella sp-60555.

[0057] SEQ ID NO: 6 is a polypeptide having xylose galacturonase activity from Aspergillus tubingensis.

[0058] SEQ ID NO: 7 is a polypeptide having xylose galacturonase activity from Aspergillus aculeatus.

[0059] SEQ ID NO: 8 is a polypeptide having pectin lyase activity from Aspergillus aculeatus.

[0060] SEQ ID NO: 9 is a polypeptide having rhamnogalacturonan lyase activity from Penicillium oxalicum.

[0061] SEQ ID NO: 10 is a polypeptide having xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase activity from Penicillium rubens.

[0062] SEQ ID NO: 11 is the Bacillus clausii secretion signal. DETAILED DESCRIPTION

[0063] Method for improving the nutritional value of animal feed

[0064] The present invention relates to the use of fiber degrading enzymes in animal feed comprising oilseeds, thereby improving the availability of nutrients from the feed and the nutritional value of the animal feed.

[0065] According to the present invention, an animal feed supplemented with a fiber degrading enzyme, preferably a pectinase, gives additional performance benefits in animals compared to the same animal feed but in the absence of pectinase.In a preferred embodiment, the animal feed comprises oilseed material.

[0066] In a further aspect, the present invention relates to a method of improving the nutritional value of an animal feed comprising oilseed material, the method comprising adding a fiber degrading enzyme to the animal feed.

[0067] In preferred embodiments, the improvement is compared to the same animal feed or animal feed supplement but without the fiber degrading enzyme.

[0068] The term improving the nutritional value of animal feed means increasing the availability of nutrients in the feed. The nutritional value refers in particular to improving the dissolution and degradation of fibers in the oilseed material, thereby increasing the amount of released oligomers containing fucose, rhamnose, arabinose, galactose, glucose, xylose, glucuronic acid and / or galacturonic acid that can be utilized by the animal and the animal microbiota. Thus, an improved release of oligomers containing fucose, rhamnose, arabinose, galactose, glucose, xylose and / or galacturonic acid will result in an improved nutritional value of the feed, thereby resulting in an increase in growth rate and / or weight gain and / or feed conversion (i.e. feed intake relative to weight gain).

[0069] In a further aspect, the present invention relates to a method for improving the growth performance of an animal, the method comprising administering to the animal a fiber degrading enzyme, an animal feed comprising a fiber degrading enzyme, or an animal feed additive. In a preferred embodiment, the growth performance is growth rate, feed conversion ratio (FCR) and / or body weight gain (BWG).

[0070] In preferred embodiments, the improvement is compared to animals fed the same animal feed or animal feed supplement but which does not include the fiber degrading enzyme.

[0071] In one embodiment, the growth rate is improved by at least 1%, such as at least 2%, at least 5%, or at least 10%. In another embodiment, the growth rate is improved by between 1% and 15%, such as between 2% and 10%, between 4% and 8%, or any combination of these intervals.

[0072] In one embodiment, the FCR is improved by at least 1%, such as at least 1.0%, at least 1.5%, or at least 2.0%. In another embodiment, the improvement in FCR is between 1% and 5%, such as between 1.5% and 4%, between 2% and 3%, or any combination of these intervals.

[0073] In one embodiment, the BWG is improved by at least 1%, such as at least 1.0%, at least 1.5%, or at least 2.0%. In another embodiment, the BWG is improved by between 1% and 5%, such as between 1.5% and 4%, between 2% and 3%, or any combination of these intervals.

[0074] Growth rate: The growth rate of an animal can be measured, for example, by the percentage of body weight gain per day.

[0075] Feed Conversion Ratio (FCR): FCR is a measure of the efficiency with which an animal converts a mass of feed into a desired increase in output. In animals raised for meat, such as swine, poultry and fish, the output is the animal's weight gain. Specifically, FCR is calculated as feed intake divided by body weight gain over a specified period of time. An improvement in FCR means a decrease in the FCR value. A 2% improvement in FCR means a 2% decrease in FCR.

[0076] Body Weight Gain (BWG): "Body Weight Gain" means the increase in live weight of an animal during a given period of time, eg, body weight gain from day 1 to day 21.

[0077] In particular embodiments, the oilseed material is selected from the group consisting of soybean, rapeseed, sunflower, pea, lupin, tepary bean, scarlet runner bean, slim jimbean, lima bean, French bean, broad bean (fava bean), chickpea, lentil, peanut, flaxseed, cottonseed or a combination thereof. In particular embodiments, the oilseed material is processed into a processed form, such as oilseed meal, full fat oilseed meal, oilseed protein concentrate, fermented oilseed meal or any combination thereof. In a preferred embodiment, the oilseed material is selected from the group consisting of soybean meal, rapeseed meal, sunflower meal, pea meal, peanut meal, flaxseed meal, cottonseed meal or a combination thereof. In another preferred embodiment, the oilseed material is selected from the group consisting of rapeseed meal and soybean meal. Rapeseed meal and soybean meal are byproducts of bioethanol and food production. In one embodiment, the plant-based material is from the taxonomic subclass Rosa clade. In one aspect, the plant-based material is from the taxonomic order Leguminosae, such as Leguminosae, preferably Caesalpinioideae or Mimosoideae or Fabaceae, or more preferably from the Phaseolus, Garbanzo, Genis, Vicia, Dalbergia or Phaseolus. In one aspect, the plant-based material is from the taxonomic order Brassicales, such as Brassicaceae, preferably Brassiceae, more preferably Brassica family.

[0078] The term "animal" refers to all animals including humans. Examples of animals are non-ruminants and ruminants. Ruminants include, for example, animals such as sheep, goats, cattle (e.g., beef cattle, dairy cows and calves), deer, yaks, camels, llamas and kangaroos. Non-ruminants include monogastric animals, such as pigs or swine (including but not limited to piglets, growing pigs and sows); poultry such as turkeys, ducks and chickens (including but not limited to broilers, laying hens); horses (including but not limited to hot-blooded horses, cold-blooded horses and warm-blooded horses), calves; fish (including but not limited to amberjack, arapaima, barracuda, perch, bluefish, scorpionfish, carp, catfish, katra, milkfish, char, cichlid, cobia, cod, sunfish, gilthead bream, stonefish, eel, goby, goldfish, silk foot fish, grouper, guapot, halibut, java fish, rohu, lai, loach, mackerel, milkfish, silver perch, mudfish, mullet, pago, pearlfish, pegeri, river bass, pike, pompano, roach, salmon, shrimp fish, amberjack, black bass, sea bream, glow fish, sleeper shark, snakehead, snapper, sablefish, flounder, lancefoot, sturgeon, sunfish, sweetfish, tench, troll, tilapia, trout, tuna, turbot, white trout, walleye, and whitefish); and crustaceans (including, but not limited to, shrimp and prawns).

[0079] In a preferred embodiment, the animal is a monogastric animal, preferably the monogastric animal is selected from the group consisting of: pigs or swine (including but not limited to piglets, growing pigs and sows); poultry, such as turkeys, ducks and chickens (including but not limited to broilers, laying hens).

[0080] In a further aspect, the present invention relates to a method for the in situ production of prebiotics in an oilseed based animal feed, the method comprising adding a fiber degrading enzyme to said animal feed.

[0081] Prebiotics are substances that induce the growth or activity of microorganisms (e.g., bacteria and fungi) that contribute to the health of the host. Prebiotics are generally indigestible fiber compounds that pass through the upper part of the gastrointestinal tract in an undigested state and stimulate the growth or activity of beneficial microorganisms that colonize the large intestine by acting as substrates. Typically, prebiotics increase the number or activity of bifidobacteria and lactic acid bacteria in the gastrointestinal tract.

[0082] In a further aspect, the present invention relates to a method for reducing the insoluble pectin fraction in an oilseed-based animal feed, the method comprising adding a fiber degrading enzyme to the animal feed. In the present invention, the fiber degrading enzyme degrades the insoluble pectin fraction of the oilseed-based animal feed, so that prebiotic oligomers and polymers comprising pectin oligosaccharides are generated.

[0083] Methods for improving intestinal health in monogastric animals

[0084] In the present invention, the fiber degrading enzymes of the present invention have a beneficial effect on the accumulation of short chain fatty acids caused by the fermentation of oil seed materials by the cecal microbiota. In one embodiment, the accumulation of butyrate is increased by one, two, three or more times. Increased formation of butyrate can indicate better intestinal health, as butyrate is a well-known intestinal health-promoting molecule with anti-inflammatory properties. In other embodiments, the accumulation of acetate is increased by one, two, three or more times. In other embodiments, the accumulation of propionic acid is increased by one, two, three or more times.

[0085] In a further aspect, the present invention relates to a method for improving the intestinal health of a monogastric animal, the method comprising administering to the animal an oilseed-based animal feed, wherein the animal feed comprises a fiber-degrading enzyme. In one embodiment, in the presence of a fiber-degrading enzyme of the invention, the abundance of Enterococcus and Escherichia / Shigella in the fermentation of rapeseed meal (RSM) by the chicken cecal microbiota is reduced. In a further embodiment, the enzyme treatment favours the growth of Bacteroides (a genus containing bacteria that are well known to degrade pectin), which favours the growth of butyrate producers in a cross-feeding manner. In a further embodiment, in the presence of a fiber-degrading enzyme of the invention, the relative abundance of Bacteroides increases by 0.5%-100%, preferably 1%-50%, more preferably 2%-20% compared to a control treatment. In a particular embodiment, the relative abundance of Bacteroides is increased by 5% in the presence of galactanase compared to the control treatment, and the relative abundance is increased by 10% when combined with rhamnogalacturonan polysaccharide endolyase (RG-I lyase). In a further embodiment, the relative abundance of Propionibacterium (a genus known as a propionic acid producer) is increased by 5%-2000%, preferably 10%-1000%, more preferably 20%-500% in the presence of the fiber degrading enzyme of the present invention. In a particular embodiment, the relative abundance of Propionibacterium is doubled when RG-I lyase is added to the galactanase compared to the galactanase alone. In a further embodiment, the relative abundance of Lactobacillus (as the most common host of probiotics) is increased by 5%-2000%, preferably 10%-1000%, more preferably 20%-500% in the presence of the fiber degrading enzyme of the present invention. In a particular embodiment, when RSM is treated with galactanase, the relative abundance of Lactobacillus increases from 1.9% to 2.7%, and when RG-I lyase is combined with galactanase, the relative abundance of Lactobacillus further increases to 4.5%. In a further embodiment, the relative abundance of Butyricicoccus increases by 1%-5000%, preferably 5%-2000%, more preferably 10%-1000% in the presence of the fiber degrading enzyme of the invention compared to the control treatment. In a particular embodiment, the relative abundance of Butyricicoccus increases by 40% in the presence of galactanase compared to the control treatment, and almost triples when combined with RG-I lyase.

[0086] In a further aspect, the invention relates to a method for in situ production of prebiotics in a monogastric animal, the method comprising administering to the animal an enzyme-enriched oilseed-based animal feed, wherein the animal feed comprises a fiber-degrading enzyme. In a further embodiment, the in situ cecal butyrate level in the animal is increased. In a further embodiment, the microbiota composition in the animal is altered.

[0087] In further embodiments, the present invention is directed to a method of inducing butyrate production in a monogastric animal, the method comprising administering to said animal an oilseed based animal feed, wherein said animal feed comprises a fiber degrading enzyme.

[0088] Fiber degrading enzyme of the present invention

[0089] Enzymes can be classified on the basis of the Enzyme Nomenclature manual from NC-IUBMB, 1992, and can also be found on the ENZYME website at the following website: http: / / www.expasy.ch / enzyme / . ENZYME is an information repository for enzyme nomenclature. It is mainly based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUB-MB) and describes each type of characterized enzyme, providing the enzyme with an EC (Enzyme Commission) number (Bairoch A. The ENZYME database [enzyme database], 2000, Nucleic Acids Res [nucleic acid research] 28: 304-305). This IUB-MB enzyme nomenclature is based on their substrate specificity and sometimes on their molecular mechanism; this classification does not reflect the structural characteristics of these enzymes.

[0090] An alternative classification of certain glycoside hydrolases into families based on amino acid sequence similarity was proposed several years ago. They are currently divided into 90 different families: see the CAZy (ModO) Internet site (Coutinho, PM & Henrissat, B. (1999) Carbohydrate-Active Enzymes server: http: / / afmb.cnrs-mrs.fr / ~cazy / CAZY / index.html (corresponding paper: Coutinho, PM & Henrissat, B. (1999) Carbohydrate-active enzymes: an integrated database approach. "Recent Advances in Carbohydrate Bioengineering", HJ Gilbert, G. Davies, B. Henrissat and B. Svensson, eds., The Royal Society of Chemistry, Cambridge, pp. 3-12; Coutinho, PM & Henrissat, B. (1999) The modular structure of cells and other carbohydrate-active enzymes: an integrated ("Modular structure of cellulases and other carbohydrate-active enzymes: an integrated database approach" in "Genetics, Biochemistry and Ecology of Cellulose Degradation", edited by K. Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, S. Karita and T. Kimura, Uni Publishers Co., Tokyo, pp. 15-23)).

[0091] As used herein, the term "fiber degrading enzyme" may include one or more of the following fiber degrading enzymes selected from the group consisting of pectinase, xyloglucan-specific endo-1,4-β-glucanase, xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase, and combinations thereof. In a preferred embodiment, the fiber degrading enzyme is pectinase. In a preferred embodiment, the fiber degrading enzyme is selected from the group consisting of: pectinase, xyloglucan-specific endo-1,4-β-glucanase, xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase and combinations thereof; preferably, the fiber degrading enzyme is pectinase; more preferably, the fiber degrading enzyme is one or more pectinases selected from the group consisting of: rhamnogalacturonan lyase, endo-β-1,4-galactanase, polygalacturonase, rhamnogalacturonanase, pectin methylesterase, pectin lyase, pectin acetylesterase, galactan endo-β-1,3-galactanase, xylogalacturonase and combinations thereof. In a more preferred embodiment, the fiber degrading enzyme is one or more pectinases selected from the group consisting of: rhamnogalacturonan lyase (α-L-rhamnopyranosyl-1,4-α-D-galactopyranosyluronate endolyase, EC 4.2.2.23), endo-β-1,4-galactanase (EC 3.2.1.89), polygalacturonase (galacturonan glycanohydrolase, EC 3.2.1.15 and EC 3.2.1.67), rhamnogalacturonanase (rhamnogalacturonan α-D-galacturonan-1,2-α-L-rhamnosylhydrolase, EC 3.2.1.171), pectin methylesterase (pectin pectyl hydrolase, EC 3.1.1.11), pectin lyase ((1,4)-6-O-methyl-α-D-galacturonan lyase, EC 4.2.2.10), pectin acetylesterase (acetate acetylhydrolase, EC 3.1.1.6), galactan endo-β-1,3-galactanase (EC 3.2.1.181), xylogalacturonase and combinations thereof.In another preferred embodiment, the fiber degrading enzyme is a pectinase selected from the group consisting of: rhamnogalacturonan lyase, endo-β-1,4-galactanase, pectin lyase and a combination thereof; preferably, the fiber degrading enzyme is selected from the group consisting of: a combination of rhamnogalacturonan lyase and endo-β-1,4-galactanase; a combination of rhamnogalacturonan lyase and pectin lyase; a combination of endo-β-1,4-galactanase and pectin lyase; and a combination of xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase and pectin lyase.

[0092] Pectinase: The term "pectinase" is defined as a large class of enzymes that catalyze the hydrolysis of pectin, a structural plant cell wall acidic heteropolysaccharide whose backbone contains 1,4-linked α-D-galacturonic acid residues. The following are definitions of the different classes of pectinases of the present invention. Activity units are defined for each pectinase class. One skilled in the art can readily determine whether a polypeptide has a particular type of pectinase activity using the following definitions and activity units.

[0093] Rhamnogalacturonan lyase: The term "rhamnogalacturonan lyase" (α-L-rhamnopyranosyl-1,4-α-D-galactopyranosyl endolyase, EC 4.2.2.23) is defined as an enzyme that catalyzes the endo-eliminative cleavage of the L-α-rhamnopyranosyl-1,4-α-D-galactopyranosyl uronic acid bond of rhamnogalacturonan, leaving L-rhamnopyranose at the reducing end and 4-deoxy-4,5-unsaturated D-galactopyranosyl uronic acid at the non-reducing end. A rhamnogalacturonan lyase unit activity is defined as the activity of 4600 M of rhamnogalacturonan at 235 nm under standard reaction conditions of pH 9.0; 37°C; reaction buffer: 25 mM Tris / HCl, 25 mM glycine / NaOH; reaction time: 5 minutes. -1 cm -1 The extinction coefficient of the dimer molecule is equivalent to the absorbance of 1 μmol of unsaturated dimerized galacturonic acid and the amount of enzyme that produces 1 μmol of oligogalacturonic acid per minute.

[0094] Endo-β-1,4-galactanase: The term "endo-β-1,4-galactanase (EC 3.2.1.89)" is defined as an enzyme that specifically hydrolyzes (1->4)-β-D-galactosidic linkages in type I arabinogalactans. Galactanase activity can be determined by reducing end using a colorimetric assay developed by Lever (Analytical Biochemistry 47, 273-279, 1972). Galactanase produces a reducing end sugar that reacts with PAHBAH, producing a color change that is proportional to enzyme activity under the conditions used for the assay.

[0095] Polygalacturonase: The term "polygalacturonase" (1,4-α-D-galacturonan polysaccharide hydrolase, EC 3.2.1.15 and EC 3.2.1.67) is defined as an enzyme that catalyzes the hydrolysis of 1,4-α-D-galactosiduronic acid linkages in pectate and other galacturonans. Polygalacturonases are classified as endo-polygalacturonases or exo-polygalacturonases. Endo-polygalacturonases (EC 3.2.1.15) catalyze the random cleavage of pectic acid, while exo-polygalacturonases (EC 3.2.1.67) catalyze the cleavage of pectic acid in a sequential manner at the non-reducing end of pectic acid to produce mono- or digalacturonates. Classes of polygalacturonases are distinguished by their characteristic amino acid sequences with generally conserved functional domain motifs called SPNTDG (PGI), GDDC (PGII), CGPGHGISIGSLG (PGIII) and RIK (PG IV). The polygalacturonase unit (PGU) is defined as the amount of enzyme that will release 1.0 micromole of galacturonic acid from polygalacturonic acid per hour under standard conditions of pH 4.0 and 25°C (Kertesz, ZI (1955) Methods in Enzymology. 1, 162-164).

[0096] Rhamnogalacturonanase: The term "rhamnogalacturonanase" (rhamnogalacturonan α-D-galacturonan-1,2-α-L-rhamnose hydrolase, EC 3.2.1.171) is defined as an enzyme that catalyzes the endohydrolysis of α-D-galacturonan-1,2-α-L-rhamnose glycosidic bonds in the rhamnogalacturonan backbone, with an initial conversion of the anomeric configuration releasing oligosaccharides with β-D-galacturonic acid at the reducing end. The classes of rhamnogalacturonanase are distinguished by their specificity for either the rhamnogalacturonan I (RG I) pectic heteropolysaccharide or the rhamnogalacturonan II (RGII) pectic heteropolysaccharide. The rhamnogalacturonanase activity unit (RGU) is defined as the amount of dye released (as measured by absorbance change) from a solution of 20 mg / mL AZ-rhamnogalacturonan / mg enzyme per minute under standard reaction conditions: pH 4.5; 40°C; buffer: 25 mM sodium acetate; reaction time: 16 hours (de Vries, RP (2015) Biotechnology for Biofuels. 8:107).

[0097] Pectin methylesterase: The term "pectin methylesterase" (pectin pectylhydrolase, EC 3.1.1.11) is defined as an enzyme which catalyzes the demethoxylation of methyl ester groups in pectin chains to form pectinates and release methanol. The pectinesterase unit (PMU) is defined as the amount of methanol released from a 1.0% pectin solution containing 0.1 M sodium chloride per gram of enzyme in 30 minutes under standard conditions, pH 7.5, 30°C (Kertesz, ZI (1955) Methods in Enzymology. 1, 162-164).

[0098] Pectin lyase: The term "pectin lyase" ((1,4)-6-O-methyl-α-D-galacturonan lyase, EC 4.2.2.10) is defined as an enzyme that catalyzes the eliminative cleavage of 1,4-α-D-galacturonan methyl esters at the non-reducing end into oligosaccharides having a 4-deoxy-6-O-methyl-α-D-galacto-4-enuronyl group. A pectin lyase unit (PLU) is defined as the amount of enzyme that causes a change of 1.0 in absorbance at 235 nm in a 0.5% w / v pectin solution under standard conditions of pH 6.0; 40°C; reaction buffer: 100 mM citric acid, 100 mM sodium phosphate; reaction time: 5 minutes (Albersheim, P. (1966) Methods in Enzymology, Vol. 8, 628-631).

[0099] Pectin acetylesterase: The term "pectin acetylesterase" (acetate acetylhydrolase, EC 3.1.1.6) is defined as an enzyme that catalyzes the deacetylation of acetyl ester groups in pectin chains to form pectinate and release acetic acid. A unit of pectin acetylesterase activity is defined as the activity of pectin acetylesterase as measured by the standard assay conditions (pH 7.4, 37°C, reaction buffer: 25 mM Tris-HCl, 50 mM EDTA and 150 mM MgCl 2 The amount of p-nitrophenol (in mmol) released by 1 mg of enzyme from a 2 mM solution of p-nitrophenol-acetyl in 1 minute, measured by absorbance at 460 nm (Pogorelko, G. (2013) BIOCHEMISTRY AND METABOLISM. 162: 9-23).

[0100] Galactan endo-β-1,3-galactanase: The term "galactan endo-β-1,3-galactanase (EC 3.2.1.181)" is defined as an enzyme that catalyzes the endohydrolysis of β-1,3 bonds (requiring at least three consecutive β-1,3-residues) in arabinogalactan. A unit of β-galactanase activity is defined as the amount of enzyme that releases 1 μmol of galactose per minute from a 1% β-galactan solution under standard reaction conditions pH 4.0; 37°C; reaction buffer: 100 mM sodium acetate / acetic acid with 0.2% bovine serum albumin; reaction time: 4 hours (Carey, AT (1995) Plant Physiol. 108: 1099-1107).

[0101] Xylosegalacturonase: The term "xylosegalacturonase" is defined as an enzyme having the ability to cleave galacturonic acid polymers (eg as found in pectin) which may be at least partially substituted with xylose at internal glycosidic bonds.

[0102] Xyloglucan-specific endo-1,4-β-glucanase: The term "xyloglucan-specific endo-1,4-β-glucanase" is defined as catalyzing the chemical reaction xyloglucan + H2O Enzymes for xyloglucan oligosaccharides.

[0103] Xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase: The term "xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase" is defined as an enzyme having endo-β-1,4-glucanase, endo-β-1,4-xylanase, endo-β-1,3-1,4-glucanase, xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase activity.

[0104] In further embodiments, the fiber degrading enzyme is a rhamnogalacturonan lyase (α-L-rhamnopyranosyl-1,4-α-D-galactopyranosyl endolyase, EC 4.2.2.23).

[0105] In further embodiments, the fiber degrading enzyme is endo-β-1,4-galactanase (EC 3.2.1.89).

[0106] In embodiments, the fiber degrading enzyme is a polygalacturonase (1,4-α-D-galacturonan polysaccharide hydrolase, EC 3.2.1.15 and EC 3.2.1.67).

[0107] In further embodiments, the fiber degrading enzyme is rhamnogalacturonanase (rhamnogalacturonan α-D-galacturonosyl-1,2-α-L-rhamnohydrolase, EC 3.2.1.171).

[0108] In further embodiments, the fiber degrading enzyme is pectin methylesterase (pectin pectylhydrolase, EC 3.1.1.11).

[0109] In further embodiments, the fiber degrading enzyme is pectin lyase ((1,4)-6-O-methyl-α-D-galacturonan lyase, EC 4.2.2.10).

[0110] In further embodiments, the fiber degrading enzyme is pectin acetylesterase (acetate acetylhydrolase, EC 3.1.1.6).

[0111] In further embodiments, the fiber degrading enzyme is endo-β-1,3-galactanase (EC 3.2.1.181).

[0112] In further embodiments, the fiber degrading enzyme is xylogalacturonase.

[0113] In further embodiments, the fiber degrading enzyme is a xyloglucan-specific endo-1,4-beta glucanase.

[0114] In further embodiments, the fiber degrading enzyme is a xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase.

[0115] In further embodiments, the fiber degrading enzyme is a combination of a pectinase and a xyloglucan-specific endo-1,4-beta glucanase.

[0116] In further embodiments, the fiber degrading enzyme is a combination of pectinase and xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase.

[0117] In further embodiments, the fiber degrading enzyme is a combination of a xyloglucan-specific endo-1,4-β glucanase and a xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase.

[0118] In further embodiments, the fiber degrading enzyme is a combination of rhamnogalacturonan lyase and endo-β-1,4-galactanase.

[0119] In further embodiments, the fiber degrading enzyme is a combination of rhamnogalacturonan lyase and polygalacturonase.

[0120] In further embodiments, the fiber degrading enzyme is a combination of rhamnogalacturonan lyase and rhamnogalacturonanase.

[0121] In further embodiments, the fiber degrading enzyme is a combination of rhamnogalacturonan lyase and pectin methylesterase.

[0122] In further embodiments, the fiber degrading enzyme is a combination of rhamnogalacturonan lyase and pectin lyase.

[0123] In further embodiments, the fiber degrading enzyme is a combination of rhamnogalacturonan lyase and pectin acetylesterase.

[0124] In further embodiments, the fiber degrading enzyme is a combination of rhamnogalacturonan lyase and galactan endo-β-1,3-galactanase.

[0125] In further embodiments, the fiber degrading enzyme is a combination of rhamnogalacturonan lyase and xylogalacturonase.

[0126] In further embodiments, the fiber degrading enzyme is a combination of endo-β-1,4-galactanase and polygalacturonase.

[0127] In further embodiments, the fiber degrading enzyme is a combination of endo-β-1,4-galactanase and rhamnogalacturonase.

[0128] In further embodiments, the fiber degrading enzyme is a combination of endo-β-1,4-galactanase and pectin methylesterase.

[0129] In further embodiments, the fiber degrading enzyme is a combination of endo-β-1,4-galactanase and pectin lyase.

[0130] In further embodiments, the fiber degrading enzyme is a combination of endo-β-1,4-galactanase and pectin acetylesterase.

[0131] In further embodiments, the fiber degrading enzyme is a combination of endo-β-1,4-galactanase and endo-β-1,3-galactanase.

[0132] In further embodiments, the fiber degrading enzyme is a combination of endo-β-1,4-galactanase and xylogalacturonase.

[0133] In further embodiments, the fiber degrading enzyme is a combination of polygalacturonase and rhamnogalacturonase.

[0134] In further embodiments, the fiber degrading enzyme is a combination of polygalacturonase and pectin methylesterase.

[0135] In further embodiments, the fiber degrading enzyme is a combination of polygalacturonase and pectin acetylesterase.

[0136] In further embodiments, the fiber degrading enzyme is a combination of polygalacturonase and endo-β-1,3-galactanase.

[0137] In further embodiments, the fiber degrading enzyme is a combination of polygalacturonase and xylogalacturonase.

[0138] In further embodiments, the fiber degrading enzyme is a combination of rhamnogalacturonase and pectin methylesterase.

[0139] In further embodiments, the fiber degrading enzyme is a combination of rhamnogalacturonase and pectin lyase.

[0140] In further embodiments, the fiber degrading enzyme is a combination of rhamnogalacturonase and pectin acetylesterase.

[0141] In further embodiments, the fiber degrading enzyme is a combination of rhamnogalacturonase and endo-β-1,3-galactanase.

[0142] In further embodiments, the fiber degrading enzyme is a combination of rhamnogalacturonase and xylogalacturonase.

[0143] In further embodiments, the fiber degrading enzyme is a combination of pectin methylesterase and pectin lyase.

[0144] In further embodiments, the fiber degrading enzyme is a combination of pectin methylesterase and pectin acetylesterase.

[0145] In further embodiments, the fiber degrading enzyme is a combination of pectin methylesterase and galactan endo-β-1,3-galactanase.

[0146] In further embodiments, the fiber degrading enzyme is a combination of pectin methylesterase and xylogalacturonase.

[0147] In further embodiments, the fiber degrading enzyme is a combination of pectin lyase and pectin acetylesterase.

[0148] In further embodiments, the fiber degrading enzyme is a combination of pectin lyase and endo-β-1,3-galactanase.

[0149] In further embodiments, the fiber degrading enzyme is a combination of pectin lyase and xylogalacturonase.

[0150] In further embodiments, the fiber degrading enzyme is a combination of pectin acetylesterase and galactan endo-β-1,3-galactanase.

[0151] In further embodiments, the fiber degrading enzyme is a combination of pectin acetylesterase and xylogalacturonase.

[0152] In further embodiments, the fiber degrading enzyme is a combination of endo-β-1,3-galactanase and xylogalacturonase.

[0153] Sources of Fibrinolytic Enzymes

[0154] The fiber degrading enzymes of the present invention can be obtained from microorganisms of any genus. For the purposes of the present invention, the term "obtained from" as used herein in conjunction with a given source should mean that the enzyme encoded by the polynucleotide is produced by the source or by a strain into which the polynucleotide of the present invention has been inserted. In one aspect, the enzyme obtained from a given source is secreted outside the cell.

[0155] It should be understood that for the aforementioned species, the present invention encompasses the perfect and imperfect stages and other taxonomic equivalents, such as anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

[0156] The above-mentioned probe can be used from other sources, including from nature (for example, soil, compost, water, etc.) separated microorganisms or directly from natural materials (for example, soil, compost, water, etc.) DNA sample identification and obtaining the enzyme. The technology for directly separating microorganisms and DNA from natural habitats is well known in the art. The polynucleotides encoding the enzyme can then be obtained by similarly screening the genomic DNA or cDNA library of another microorganism or the DNA sample mixed. Once the polynucleotides encoding the enzyme have been detected with probes, the polynucleotides can be separated or cloned by utilizing technology known to those of ordinary skill in the art (see, for example, Davis et al., 2012, Basic Methods in Molecular Biology [basic method of molecular biology], Elsevier [Elsevier]).

[0157] In one embodiment the fiber degrading enzyme is obtained or obtainable from Aspergillus or Penicillium or Cohnella.

[0158] In one embodiment, the fiber degrading enzyme is a rhamnogalacturonan lyase obtained from Aspergillus or Penicillium, such as a rhamnogalacturonan lyase obtained from Aspergillus aculeatus or Penicillium oxalicum.

[0159] In one embodiment, the fiber degrading enzyme is an endo-polygalacturonase obtained from Aspergillus, such as an endo-polygalacturonase obtained from Aspergillus aculeatus.

[0160] In one embodiment, the fiber degrading enzyme is a xyloglucan-specific endo-1,4-β-glucanase obtained or obtainable from Aspergillus, such as a xyloglucan-specific endo-1,4-β-glucanase obtained from Aspergillus aculeatus or Aspergillus ryukyuus.

[0161] In one embodiment, the fiber degrading enzyme is a galactanase obtained or obtainable from Cohenella, such as a galactanase obtained from Cohenella sp.-60555.

[0162] In one embodiment, the fiber degrading enzyme is a xylogalacturonase obtained or obtainable from Aspergillus, such as a xylogalacturonase obtained from Aspergillus tubingensis or Aspergillus aculeatus.

[0163] In one embodiment the fiber degrading enzyme is a pectin lyase obtained or obtainable from Aspergillus, such as a pectin lyase obtained from Aspergillus aculeatus.

[0164] In one embodiment, the fiber degrading enzyme is an endo-β-1,4-glucanase / endo-xyloglucanase obtained or obtainable from Penicillium, such as an endo-β-1,4-glucanase / endo-xyloglucanase obtained from Penicillium rubragenum.

[0165] It should be understood that for the aforementioned species, the present invention encompasses the perfect and imperfect stages and other taxonomic equivalents, such as anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

[0166] The above-mentioned bacterial and fungal strains are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Microbial Culture Collection (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), the Netherlands Culture Collection (Centraalbureau Voor Schimmelcultures, CBS), and the U.S. Agricultural Research Service Patent Culture Collection Northern Regional Research Center (NRRL) .

[0167] Questions related to taxonomy can be solved by consulting taxonomic databases, such as the NCBI taxonomy browser, which can be obtained at the following Internet site: http: / / www.ncbi.nlm.nih.gov / Taxonomy / taxonomyhome.html / . However, it is preferred to refer to the following manuals: Dictionary of the Fungi, 9th edition, edited by Kirk, PM, PF Cannon, JC David & J. A. Stalpers, CAB Publishing, 2001; and Bergey's Manual of Systematic Bacteriology, 2nd edition (2005).

[0168] The term "a / kind" as used herein in any context means "one / kind or more / kind", preferably "at least one / kind". For example, this is the case for the use of "a" fiber degrading enzyme as specified in claim 1, which is considered equivalent to claiming the use of "at least one" or "one or more" of such fiber degrading enzymes.

[0169] The term "variant" refers to a fiber degrading enzyme that contains an artificial mutation (i.e., substitution, insertion (including extension) and / or deletion (e.g., truncation)) at one or more positions. Substitution means replacing the amino acid occupying a certain position with a different amino acid; deletion means removing the amino acid occupying a certain position; and insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, in particular 1 amino acid) adjacent to and immediately following the amino acid occupying a position. Variants can be natural variants (allelic variants) or synthetically prepared. Preferably, the nature of the amino acid changes is minor, for example, conservative amino acid substitutions that do not significantly affect protein folding and / or activity; small deletions; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides; or small extensions that facilitate purification by changing the net charge or another function (such as a polyhistidine segment, an antigenic epitope, or a binding domain).

[0170] A "fragment" of a fiber degrading enzyme is designated as having one or more amino acids deleted from the amino terminus and / or carboxyl terminus of the amino acid sequence of the fiber degrading enzyme.

[0171] For the purposes of the above definitions of variants and fragments, the term "minor" and the term "one or more" refer to up to 30 changes (e.g., 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 or 30) compared to a specified fiber degrading enzyme. In preferred embodiments of any of these definitions, the number of changes is less than 30, 25, 20, 15, 10, or less than 5.

[0172] Examples of conservative substitutions are within the following groups: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). The most commonly occurring exchanges are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly, as well as the reverse of these.

[0173] In addition to the 20 basic amino acids, non-basic amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline and α-methylserine) can replace the amino acid residues of the wild-type polypeptide. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and non-natural amino acids can be used to replace amino acid residues. "Non-natural amino acids" have been modified after protein synthesis and / or have a chemical structure different from that of standard amino acids in one or more of their side chains. Non-natural amino acids can be synthesized chemically and are preferably commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

[0174] Alternatively, these amino acid changes have such a property that the physicochemical properties of the polypeptide are changed. For example, amino acid changes can improve the thermal stability of the polypeptide, change the substrate specificity, change the optimal pH, and the like.

[0175] Essential amino acids can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resulting mutant molecules are tested for biological activity (i.e., fiber degrading enzyme activity) to identify amino acid residues that are critical for the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of an enzyme or other biological interaction can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, together with mutations to putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred from analysis of identities with polypeptides related to the polypeptides according to the invention.

[0176] Single or multiple amino acid substitutions can be made and tested using known mutagenesis, recombination and / or shuffling methods followed by relevant screening procedures, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochem. 30:10832-10837; U.S. Pat. No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0177] Mutagenesis / shuffling methods can be combined with high-throughput, automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells. Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow rapid determination of the importance of each amino acid residue in the target polypeptide and are applicable to polypeptides of unknown structure.

[0178] Mature polypeptide: The term "mature polypeptide" means a polypeptide in its mature form after N-terminal and / or C-terminal processing (eg, removal of a signal peptide). In one embodiment, the mature polypeptide of the rhamnogalacturonan lyase of SEQ ID NO:1 is amino acids 20-527 thereof; the mature polypeptide of the endo-polygalacturonase of SEQ ID NO:2 is amino acids 21-378 thereof; the mature polypeptide of the xyloglucan-specific endo-1,4-β-glucanase of SEQ ID NO:3 is amino acids 15-238 thereof; the mature polypeptide of the xyloglucan-specific endo-1,4-β-glucanase of SEQ ID NO:4 is amino acids 16-241 thereof; the mature polypeptide of the galactanase of SEQ ID NO:5 is amino acids 33-348 thereof; the mature polypeptide of the xylose galacturonase of SEQ ID NO:6 is amino acids 19-406 thereof; the mature polypeptide of the xylose galacturonase of SEQ ID NO:7 is amino acids 19-406 thereof; the mature polypeptide of the pectin lyase of SEQ ID NO:8 is amino acids 20-389 thereof; The mature polypeptide of the rhamnogalacturonan lyase of SEQ ID NO:9 is amino acids 21-530 thereof; or the mature polypeptide of the endo-β-1,4-glucanase / endo-xyloglucanase of SEQ ID NO:10 is amino acids 20-574 thereof.

[0179] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide having fiber degrading enzyme activity.

[0180] In particular embodiments, the fiber degrading enzyme of the present invention is isolated, i.e., substantially free of other polypeptides having enzymatic activity, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by SDS-PAGE. For detection purposes, the SDS-gel can be stained with Coomassie stain or silver stain, as is generally known in the art. It should be ensured that no overloading occurs, e.g., by checking linearity by applying various concentrations in different lanes on the gel. In particular, such polypeptide preparations can be obtained using recombinant production methods; however, when the polypeptides are produced by traditional fermentation methods, it is not easy to obtain these polypeptide preparations; and there will be high variability between batches.

[0181] The polypeptides included in the compositions of the present invention are preferably also purified. The term purified refers to a protein-rich preparation from which a large amount of low-molecular components, typical residual nutrients, and minerals derived from fermentation have been removed. Such purification can be carried out, for example, by conventional chromatography, such as ion exchange chromatography, hydrophobic interaction chromatography, and size exclusion chromatography (see, for example, Protein Purification, Principles, High Resolution Methods, and Applications. Editors: Jan-Christer Janson, Lars Rydén, VCH Publishers, 1989).

[0182] It is advantageous to use the isolated and / or purified polypeptide according to the invention. For example, it is much easier to correctly dose an enzyme that is substantially free of interference or contamination by other enzymes. The term correctly dosed refers in particular to the goal of obtaining consistent and constant animal feeding results, as well as the ability to optimize the dosage based on the desired effect.

[0183] Sequence identity: The relatedness between two amino acid sequences is described by the parameter "sequence identity".

[0184] For purposes of the present invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [Journal of Molecular Biology] 48: 443-453) is used to determine the sequence identity between two amino acid sequences as the output of "longest identity", which is implemented by the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite [European Molecular Biology Open Software Suite], Rice et al., 2000, Trends Genet. [Genetics Trend] 16: 276-277, preferably 6.6.0 version or later). The parameters used are gap opening penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order to make the longest identity reported by the Needle program, the non-simplified (-nobrief) option must be specified in the command line. The output of the "longest identity" marked by Needle is calculated as follows:

[0185] (identical residues × 100) / (alignment length – total number of gaps in the alignment)

[0186] In one embodiment, the fiber degrading enzyme has at least 60% sequence identity with the mature polypeptide of the rhamnogalacturonan lyase of SEQ ID NO: 1. In another embodiment, the fiber degrading enzyme has at least 60% sequence identity with the mature polypeptide of the endo-polygalacturonase of SEQ ID NO: 2. In another embodiment, the fiber degrading enzyme has at least 60% sequence identity with the mature polypeptide of the xyloglucan-specific endo-1,4-β-glucanase of SEQ ID NO: 3. In another embodiment, the fiber degrading enzyme has at least 60% sequence identity with the mature polypeptide of the xyloglucan-specific endo-1,4-β-glucanase of SEQ ID NO: 4. In another embodiment, the fiber degrading enzyme has at least 60% sequence identity with the mature polypeptide of the galactanase of SEQ ID NO: 5. In another embodiment, the fiber degrading enzyme has at least 60% sequence identity with the mature polypeptide of the xyloglucan galacturonase of SEQ ID NO: 6. In another embodiment, the fiber degrading enzyme has at least 60% sequence identity to the mature polypeptide of the xylose galacturonase of SEQ ID NO: 7. In another embodiment, the fiber degrading enzyme has at least 60% sequence identity to the mature polypeptide of the pectin lyase of SEQ ID NO: 8. In another embodiment, the fiber degrading enzyme has at least 60% sequence identity to the mature polypeptide of the rhamnogalacturonan lyase of SEQ ID NO: 9. In another embodiment, the fiber degrading enzyme has at least 60% sequence identity to the mature polypeptide of the endo-β-1,4-glucanase / endo-xyloglucanase of SEQ ID NO: 10.

[0187] In particular embodiments, the degree of sequence identity is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100%. In further embodiments, the degree of sequence identity is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.

[0188] In further particular embodiments, the fiber degrading enzyme is selected from the group consisting of:

[0189] (a) a rhamnogalacturonan lyase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 or the mature polypeptide of SEQ ID NO: 1;

[0190] (b) a rhamnogalacturonan lyase derived from SEQ ID NO: 1 or the mature polypeptide of SEQ ID NO: 1 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0191] (c) a rhamnogalacturonan lyase derived from (a) or (b), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, such as 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids;

[0192] (d) A fragment of the rhamnogalacturonan lyase of (a), (b) or (c), wherein the fragment has rhamnogalacturonan lyase activity;

[0193] (e) an endo-polygalacturonase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO: 2;

[0194] (f) an endo-polygalacturonase derived from SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO: 2 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0195] (g) an endo-polygalacturonase derived from (e) or (f), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids;

[0196] (h) a fragment of the endo-polygalacturonase of (e), (f) or (g), wherein the fragment has endo-polygalacturonase activity;

[0197] (i) a xyloglucan-specific endo-1,4-β-glucanase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO: 3;

[0198] (j) a xyloglucan-specific endo-1,4-beta-glucanase derived from SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO: 3 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0199] (k) a xyloglucan-specific endo-1,4-β-glucanase derived from (i) or (j), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids;

[0200] (l) A fragment of the xyloglucan-specific endo-1,4-β-glucanase of (i), (j) or (k), wherein the fragment has xyloglucan-specific endo-1,4-β-glucanase activity;

[0201] (m) a xyloglucan-specific endo-1,4-β-glucanase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:4 or the mature polypeptide of SEQ ID NO:4;

[0202] (n) a xyloglucan-specific endo-1,4-beta-glucanase derived from SEQ ID NO: 4 or the mature polypeptide of SEQ ID NO: 4 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0203] (o) a xyloglucan-specific endo-1,4-β-glucanase derived from (m) or (n), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0204] A fragment of the xyloglucan-specific endo-1,4-β-glucanase of (p), (m), (n) or (o), wherein the fragment has xyloglucan-specific endo-1,4-β-glucanase activity;

[0205] (q) a galactanase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:5 or the mature polypeptide of SEQ ID NO:5;

[0206] (r) a galactanase derived from SEQ ID NO: 5 or the mature polypeptide of SEQ ID NO: 5 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions, at one or more positions;

[0207] (s) a galactanase derived from (q) or (r), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids;

[0208] (t) a fragment of the galactanase of (q), (r) or (s), wherein the fragment has galactanase activity;

[0209] (u) a xylogalacturonase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:6 or the mature polypeptide of SEQ ID NO:6;

[0210] (v) a xylogalacturonase derived from SEQ ID NO: 6 or the mature polypeptide of SEQ ID NO: 6 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0211] (w) a xylogalacturonase derived from (u) or (v), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids;

[0212] (x) a fragment of the xylose galacturonase of (u), (v) or (w), wherein the fragment has xylose galacturonase activity;

[0213] (y) a xylogalacturonase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:7 or the mature polypeptide of SEQ ID NO:7;

[0214] (z) a xylogalacturonase derived from SEQ ID NO: 7 or the mature polypeptide of SEQ ID NO: 7 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0215] (aa) a xylogalacturonase derived from (y) or (z), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids;

[0216] A fragment of the xylose galacturonase of (bb)(y), (z) or (aa), wherein the fragment has xylose galacturonase activity;

[0217] (cc) a pectin lyase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:8 or the mature polypeptide of SEQ ID NO:8;

[0218] (dd) a pectin lyase derived from SEQ ID NO: 8 or the mature polypeptide of SEQ ID NO: 8 by having 1 to 30 alterations, e.g. substitutions, deletions and / or insertions, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions, at one or more positions;

[0219] (ee) a pectin lyase derived from (cc) or (dd), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids;

[0220] (ff) (cc), (dd) or (ee) a fragment of the pectin lyase, wherein the fragment has pectin lyase activity;

[0221] (gg) a rhamnogalacturonan lyase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:9 or the mature polypeptide of SEQ ID NO:9;

[0222] (hh) a rhamnogalacturonan lyase derived from SEQ ID NO: 9 or the mature polypeptide of SEQ ID NO: 9 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0223] (ii) a rhamnogalacturonan lyase derived from (gg) or (hh), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, such as 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0224] (jj) A fragment of the rhamnogalacturonan lyase of (gg), (hh) or (ii), wherein the fragment has rhamnogalacturonan lyase activity;

[0225] (kk) a xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 10 or the mature polypeptide of SEQ ID NO: 10;

[0226] (ll) a xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase derived from SEQ ID NO: 10 or the mature polypeptide of SEQ ID NO: 10 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0227] (mm) a xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase derived from (kk) or (ll), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0228] A fragment of a xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase of (nn)(kk), (ll) or (mm), wherein the fragment has xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase activity.

[0229] In still other specific embodiments, the fiber degrading enzyme of the present invention comprises (preferably has or consists of) the mature polypeptide of any one of the fiber degrading enzymes of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and / or SEQ ID NO:10; or a variant or fragment thereof having fiber degrading enzyme activity.

[0230] Animal feed and animal feed additives

[0231] The present invention also relates to a method for preparing an enzyme-enriched animal feed using the fiber-degrading enzyme of the present invention, as well as animal feed and feed additives comprising the fiber-degrading enzyme of the present invention.

[0232] The term "animal feed" refers to any compound, preparation or mixture suitable or intended for ingestion by an animal. Animal feeds for monogastric animals typically contain concentrates together with vitamins, minerals, enzymes, direct fed microbials, amino acids and / or other feed ingredients (such as in a premix), while animal feeds for ruminants usually contain forage (including roughage and silage), and may further contain concentrates together with vitamins, minerals, enzymes, direct fed microbials, amino acids and / or other feed ingredients (such as in a premix).

[0233] The term "concentrate" means feeds with a high protein and energy concentration, such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (e.g., from corn, oats, rye, barley, wheat, whole or prepared via cracking, milling, etc.), oilseed cakes (e.g., from cottonseed, safflower, sunflower, soybean (e.g., soybean meal), rapeseed / canola, peanuts or groundnuts), palm kernel cake, yeast-derived materials and distillers grains (e.g., wet distillers grains (WDS) and distillers dried grains with solubles (DDGS)).

[0234] In particular embodiments, the fiber degrading enzyme of the invention is used in feed for: (i) non-ruminant animals; preferably (ii) monogastric animals; more preferably (iii) pigs, poultry, fish and crustaceans; or most preferably (iv) pigs and poultry.

[0235] The fiber degrading enzyme of the present invention may be fed to the animal before, after or simultaneously with the diet. The latter is preferred.

[0236] The term feed, feed composition or diet means any compound, preparation, mixture or composition suitable or intended for ingestion by an animal. See below for more information on animal feed compositions.

[0237] In one embodiment the present invention relates to an animal feed comprising a fiber degrading enzyme and an oilseed material, wherein the feed comprises the oilseed material in an amount of 10 to 500 g / kg feed and the fiber degrading enzyme in an amount of 0.1 to 500 mg enzyme protein / kg feed.

[0238] The dosage of the fiber degrading enzyme of the present invention can be optimized using a simple trial and error method known in the art. Different pectinases may have different optimal dosage ranges. Examples of suitable dosage ranges are: 0.1-500 mg enzyme protein (EP) / kg diet (substrate); preferably 0.2-400, 0.5-300, 1-200 or 2-100 mg EP / kg diet.

[0239] To determine the mg enzyme protein of the fiber degrading enzyme per kg feed, the fiber degrading enzyme is purified from the feed composition and the specific activity of the purified fiber degrading enzyme is determined using the relevant assay. The fiber degrading enzyme activity of a feed composition like this is also determined using the same assay and the dosage in mg enzyme protein fiber degrading enzyme / kg feed is calculated based on these two determinations.

[0240] The same rules apply to determining the mg of enzyme protein fiber degrading enzyme in the feed additive. Of course, if a sample of the fiber degrading enzyme used in the preparation of the feed additive or feed is available, the specific activity can be determined from this sample (without purifying the fiber degrading enzyme from the feed composition or additive).

[0241] In a further aspect, the present invention relates to an animal feed additive comprising a fiber degrading enzyme and one or more additional components selected from the group consisting of: one or more vitamins; one or more minerals; one or more amino acids; one or more phytobiotics; one or more prebiotics; one or more organic acids; and one or more other feed ingredients.

[0242] A non-exclusive list of examples of these components is listed below:

[0243] Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E, and vitamin K, such as vitamin K3.

[0244] Examples of water-soluble vitamins are vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and pantothenates, such as calcium D-pantothenate.

[0245] Examples of trace minerals are manganese, zinc, iron, copper, iodine, selenium and cobalt.

[0246] Examples of macrominerals are calcium, phosphorus and sodium.

[0247] Examples of amino acids used in animal feed are lysine, alanine, beta-alanine, threonine, methionine and tryptophan.

[0248] Phytobiotics are a group of natural growth promoters or non-antibiotic growth promoters derived from herbs, spices or other plants used as feed additives. Phytobiotics can be single substances prepared from essential oils / extracts, essential oils / extracts, single plants and plant mixtures (herbal products) or mixtures of essential oils / extracts / plants (specialty products).

[0249] Examples of phytobiotics are rosemary, sage, oregano, thyme, cloves and lemongrass. Examples of essential oils are thymol, eugenol, m-cresol, vanillin, salicylates, resorcinol, guajacol, gingerol, lavender oil, ionones, irones, eucalyptol, menthol, peppermint oil, α-pinene, limonene, anethole, linalool, methyl dihydrojasmonate, carvacrol, propionic acid / propionic acid esters, acetic acid / acetic acid esters, butyric acid / butyric acid esters, rosemary oil, clove oil, geraniol, terpineol, citronellol, amyl salicylate and / or benzyl salicylate, cinnamaldehyde, plant polyphenols (tannins), turmeric and turmeric extracts.

[0250] Examples of commercial products are (DSM Nutritional Products), Cinergy TM 、Cinergy TM FIT, Biacid TM (Cargill), (R) and rom(R)DC (Biomin) and Envivo EO (DuPont Animal Nutrition).

[0251] Organic acids (C1-C7) are widely distributed in nature as normal components of plant or animal tissues. They are also formed by microbial fermentation of carbohydrates, mainly in the large intestine. They are commonly used as substitutes for antibiotic growth promoters in pig and poultry production because they have a preventive effect on intestinal problems such as necrotic enteritis in chickens and E. coli infections in piglets. Organic acids can be sold as a single component or typically a mixture of 2 or 3 different organic acids. Examples of organic acids are propionic acid, formic acid, citric acid, lactic acid, sorbic acid, malic acid, acetic acid, fumaric acid, benzoic acid, butyric acid and tartaric acid or its salt (typically sodium or potassium salts, such as potassium diformate or sodium butyrate).

[0252] Examples of commercial products are Vitall(R) (DSM Nutritional ), R), R), rain(R), pro-Cid( Lupro-Mix(R), Lupro-Mix(R)NA (BASF), n-Butyric Acid AF (OXEA), Biacid TM 、Prohacid TMClassic and Vance TM (Cargill), Biotronic(R) (Biomin), and Adimix Precision (Nutriad).

[0253] Furthermore, optional feed additive ingredients are colorants, for example carotenoids, such as β-carotene, astaxanthin, and lutein; aromatic compounds; stabilizers; antimicrobial peptides; polyunsaturated fatty acids; reactive oxygen generating substances; and / or at least one other enzyme selected from another pectinase (EC 3.2.1.8); and / or β-glucanase (EC 3.2.1.4 or EC 3.2.1.6).

[0254] Examples of antimicrobial peptides (AMPs) are CAP18, Leucocin A, Tritrpticin, Protegrin-1, Thanatin, Defensins, Lactoferrin, Lactoferrin peptides and Ovispirin such as Novispirin (Robert Lehrer, 2000), Plectasin and Statins (including compounds and polypeptides disclosed in WO 03 / 044049 and WO 03 / 048148) and variants or fragments thereof that retain antimicrobial activity.

[0255] Examples of antifungal polypeptides (AFPs) are Aspergillus giganteus and Aspergillus niger peptides and variants and fragments thereof which retain antifungal activity as disclosed in WO 94 / 01459 and WO 02 / 090384.

[0256] Examples of polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids such as arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid.

[0257] Examples of reactive oxygen generating species are chemicals such as perborates, persulfates or percarbonates; and enzymes such as oxidases, oxygenases or synthases.

[0258] Typically, fat-soluble and water-soluble vitamins and trace minerals form part of a so-called premix intended for addition to feed, whereas macro-minerals are usually added to the feed separately.A premix enriched with the fiber degrading enzyme of the invention is an example of an animal feed additive of the invention.

[0259] In practice, the composition of the feed additive as exemplified above is incorporated into animal feed (e.g., poultry feed) using a concentrate or premix. A premix refers to a preferably uniform mixture of one or more micro-ingredients and a diluent and / or a carrier. A premix is ​​used to promote uniform dispersion of micro-ingredients in a larger mixture. Premixes according to the present invention can be added to feed ingredients or drinking water as a solid (e.g., as a water-soluble powder) or a liquid.

[0260] In a particular embodiment, the animal feed additive of the present invention is intended to be included (or specified to be included) in an animal diet or feed at the following levels: 0.01% to 10.0%; more particularly 0.05% to 5.0%; or 0.2% to 1.0% (% means g additive / 100g feed). In particular, this is also true for premixes.

[0261] The nutritional requirements of these components (exemplified for poultry and piglets / pigs) are listed in Table A of WO 01 / 58275. The nutritional requirements mean that the indicated concentrations of these components should be provided in the diet.

[0262] In an alternative embodiment, the animal feed additive of the present invention comprises at least one of the individual components specified in Table A of WO 01 / 58275. At least one means any one, one or more of one or two or three or four etc. up to all thirteen or up to all fifteen individual components. More particularly, this at least one individual component is contained in the additive of the present invention in an amount to provide an in-feed-concentration within the range indicated in the fourth, fifth, or sixth column of Table A.

[0263] Animal feed compositions or diets have a relatively high protein content. Poultry and pig diets can be characterized as indicated in Table B, columns 2-3 of WO 01 / 58275. Fish diets can be characterized as indicated in column 4 of such Table B. In addition, such fish diets typically have a crude fat content of 200-310 g / kg.

[0264] WO 01 / 58275 corresponds to US Pat. No. 6,960,462, which is hereby incorporated by reference.

[0265] The animal feed composition according to the invention has a crude protein content of 50-800 g / kg (preferably 50-600 g / kg, more preferably 60-500 g / kg, even more preferably 70-500 g / kg, most preferably 80-400 g / kg), and further comprises at least one fiber degrading enzyme as claimed herein. In further preferred embodiments, the crude protein content is 150-800, 160-800, 170-800, 180-800, 190-800 or 200-800, all in g / kg (dry matter). In a particular embodiment, the crude protein content comes from the oilseed material of the invention.

[0266] In addition or in the alternative (to the crude protein content indicated above), the animal feed composition suitably has a metabolizable energy content of 10-30 MJ / kg; and / or a calcium content of 0.1-200 g / kg; and / or an available phosphorus content of 0.1-200 g / kg; and / or a methionine content of 0.1-100 g / kg; and / or a methionine plus cysteine ​​content of 0.1-150 g / kg; and / or a lysine content of 0.5-50 g / kg. In embodiments, the content of metabolizable energy, crude protein, calcium, phosphorus, methionine, methionine plus cysteine, and / or lysine falls within any of ranges 2, 3, 4 or 5 (R.2-5) in Table B of WO 01 / 58275.

[0267] Crude protein is calculated as nitrogen (N) multiplied by a factor of 6.25, i.e., crude protein (g / kg) = N (g / kg) x 6.25. Nitrogen content is determined by the Kjeldahl method (AOAC, 1984, Official Methods of Analysis 14th Edition, Association of Official Analytical Chemists, Washington, D.C.).

[0268] Metabolizable energy can be calculated based on the NRC publication Nutrient requirements in swine, 9th reprint 1988, subcommittee on swine nutrition, committee on animal nutrition, board of agriculture, national research council. National Academy Press, Washington, DC, pp. 2-6; and 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.

[0269] In a further aspect, the present invention relates to a method for improving the average metabolizable energy of a plant-based diet in a monogastric animal, the method comprising administering an animal feed additive according to the invention or an animal feed according to the invention.

[0270] The dietary content of calcium, available phosphorus and amino acids in the animal's complete diet was calculated on the basis of feed tables, e.g. Veevoedertabel 1997, gegevens over chemische samenstelling [Chemical composition data], verteerbaarheid en voederwaarde van voedermiddelen [Digestibility and feed value of feed ingredients], Central Veevoederbureau [Central Animal Feed Bureau], Runderweg 6, 8219 pk, Lelystad. ISBN 90-72839-13-7.

[0271] In particular embodiments, the animal feed compositions of the present invention contain 0-80% oilseed material.

[0272] Animal diets can be prepared, for example, as a powdered feed (non-pelleted) or pelleted feed. Typically, the ground feed is mixed and sufficient amounts of essential vitamins and minerals are added according to the instructions for the species in question. The enzymes are added as solid or liquid enzyme formulations. For example, solid enzyme formulations are typically added before or during the mixing step; liquid enzyme preparations are typically added after the pelleting step. The enzymes can also be incorporated into feed additives or premixes, as described above.

[0273] In a preferred embodiment, the animal feed has been pelletized.The animal feed can be treated with the enzyme of the invention before the pelletizing step or sprayed after the pelletizing step.

[0274] In a further aspect, the present invention relates to the use of a combination of a polypeptide having rhamnogalacturonan lyase activity and a polypeptide having galactanase activity in animal feed or an animal feed additive.

[0275] In a further aspect, the present invention relates to an animal feed additive comprising a polypeptide having rhamnogalacturonan lyase activity and a polypeptide having galactanase activity; preferably, the present invention relates to an animal feed additive comprising: a combination of rhamnogalacturonan lyase and endo-β-1,4-galactanase; a combination of rhamnogalacturonan lyase and pectin lyase; a combination of endo-β-1,4-galactanase and pectin lyase; or a combination of xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase and pectin lyase.

[0276] In one embodiment, the polypeptide having rhamnogalacturonan lyase activity is RGL_1. In a further embodiment, the polypeptide having rhamnogalacturonan lyase activity is selected from the group consisting of:

[0277] (a) a rhamnogalacturonan lyase having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 or the mature polypeptide of SEQ ID NO: 1; preferably a rhamnogalacturonan lyase obtained or obtainable from Aspergillus; more preferably a rhamnogalacturonan lyase obtained or obtainable from Aspergillus aculeatus;

[0278] (b) a rhamnogalacturonan lyase derived from SEQ ID NO: 1 or the mature polypeptide of SEQ ID NO: 1 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0279] (c) a rhamnogalacturonan lyase derived from (a) or (b), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0280] (d) A fragment of the rhamnogalacturonan lyase of (a), (b) or (c), wherein the fragment has rhamnogalacturonan lyase activity.

[0281] In further embodiments, the polypeptide having galactanase activity is glycoside hydrolase family (GH) 53 (GH53). In further embodiments, the polypeptide having galactanase activity is selected from the group consisting of:

[0282] (a) a galactanase having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:5 or the mature polypeptide of SEQ ID NO:5; preferably a galactanase obtained or obtainable from Cohenella species; more preferably a galactanase obtained or obtainable from Cohenella species-60555;

[0283] (b) a galactanase derived from SEQ ID NO: 5 or the mature polypeptide of SEQ ID NO: 5 by having 1-30 alterations, e.g. substitutions, deletions and / or insertions, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0284] (c) a galactanase derived from (a) or (b), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0285] (d) A fragment of the galactanase of (a), (b) or (c), wherein the fragment has galactanase activity.

[0286] In a further aspect, the present invention relates to an animal feed additive comprising a polypeptide having rhamnogalacturonan lyase activity and a polypeptide having galactanase activity. In addition to the polypeptide having rhamnogalacturonan lyase activity and the polypeptide having galactanase activity of the present invention, the animal feed additive of the present invention further comprises one or more additional components selected from the group consisting of: one or more vitamins; one or more minerals; one or more amino acids; one or more phytobiotics; one or more prebiotics; one or more organic acids; and one or more other feed ingredients.

[0287] In a further aspect, the present invention relates to a polypeptide having xyloglucan-specific endo-1,4-β-glucanase activity selected from the group consisting of:

[0288] (a) a polypeptide having at least 99.7%, at least 99.8%, at least 99.9% or 100% sequence identity to SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO: 3;

[0289] (b) a polypeptide derived from SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO: 3 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0290] (c) a polypeptide having at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:4 or the mature polypeptide of SEQ ID NO:4;

[0291] (d) a polypeptide derived from SEQ ID NO: 4 or the mature polypeptide of SEQ ID NO: 4 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0292] (e) a polypeptide derived from the polypeptide of (a), (b), (c) or (d), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0293] (f) a fragment of the polypeptide of (a), (b), (c), (d) or (e);

[0294] The polypeptide has xyloglucan-specific endo-1,4-β-glucanase activity.

[0295] In one embodiment, a polypeptide of the invention comprises, consists essentially of, or consists of SEQ ID NO:3, the mature polypeptide of SEQ ID NO:3, SEQ ID NO:4, or the mature polypeptide of SEQ ID NO:4.

[0296] In a further aspect, the invention relates to a polypeptide having xylose galacturonase activity selected from the group consisting of:

[0297] (a) a polypeptide having at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:6 or the mature polypeptide of SEQ ID NO:6;

[0298] (b) a polypeptide derived from SEQ ID NO:6 or the mature polypeptide of SEQ ID NO:6 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0299] (c) a polypeptide having at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:7 or the mature polypeptide of SEQ ID NO:7;

[0300] (d) a polypeptide derived from SEQ ID NO: 7 or the mature polypeptide of SEQ ID NO: 7 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0301] (e) a polypeptide derived from the polypeptide of (a), (b), (c) or (d), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0302] (f) a fragment of the polypeptide of (a), (b), (c), (d) or (e);

[0303] The polypeptide has xylose galacturonase activity.

[0304] In one embodiment, a polypeptide of the invention comprises, consists essentially of, or consists of SEQ ID NO:6, the mature polypeptide of SEQ ID NO:6, SEQ ID NO:7, or the mature polypeptide of SEQ ID NO:7.

[0305] In a further aspect, the invention relates to a polypeptide having rhamnogalacturonan lyase activity selected from the group consisting of:

[0306] (a) a polypeptide having at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:9 or the mature polypeptide of SEQ ID NO:9;

[0307] (b) a polypeptide derived from SEQ ID NO: 9 or the mature polypeptide of SEQ ID NO: 9 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0308] (c) a polypeptide derived from the polypeptide of (a) or (b) wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0309] (d) a fragment of the polypeptide of (a), (b) or (c);

[0310] The polypeptide has rhamnogalacturonan lyase activity.

[0311] In one embodiment, a polypeptide of the invention comprises, consists essentially of, or consists of SEQ ID NO:9 or the mature polypeptide of SEQ ID NO:9.

[0312] In a further aspect, the present invention relates to a polypeptide having xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase activity selected from the group consisting of:

[0313] (a) a polypeptide having at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10 or the mature polypeptide of SEQ ID NO: 10;

[0314] (b) a polypeptide derived from SEQ ID NO: 10 or the mature polypeptide of SEQ ID NO: 10 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0315] (c) a polypeptide derived from the polypeptide of (a) or (b) wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0316] (d) a fragment of the polypeptide of (a), (b) or (c);

[0317] The polypeptide has xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase activity.

[0318] In one embodiment, a polypeptide of the invention comprises, consists essentially of, or consists of SEQ ID NO:10 or the mature polypeptide of SEQ ID NO:10.

[0319] Polynucleotide

[0320] In a further aspect, the present invention relates to a polynucleotide encoding a polypeptide of the present invention.

[0321] The polynucleotide may be genomic DNA, cDNA, synthetic DNA, synthetic RNA, mRNA or a combination thereof. The polynucleotide may be cloned from a strain of Aspergillus or Penicillium or Cohenella or a related organism, and thus, for example, may be a polynucleotide sequence encoding a variant of a polypeptide of the invention.

[0322] In one embodiment, the polynucleotide encoding the rhamnogalacturonan lyase of the present invention is isolated from an Aspergillus or Penicillium cell.

[0323] In one embodiment, the polynucleotide encoding the xyloglucan-specific endo-1,4-β-glucanase of the invention is isolated from an Aspergillus cell.

[0324] In one embodiment, the polynucleotide encoding a galactanase of the invention is isolated from a Cohenella cell.

[0325] Polynucleotides can also be mutated by introducing nucleotide substitutions that do not result in changes in the amino acid sequence of the polypeptide, but correspond to the codon usage of the host organism intended for producing the enzyme, or by introducing nucleotide substitutions that may produce a different amino acid sequence. For a general description of nucleotide substitutions, see, e.g., Ford et al., 1991, Protein Expression and Purification 2:95-107.

[0326] In one embodiment, the polynucleotide is isolated.

[0327] In another embodiment, the polynucleotide is purified.

[0328] Nucleic acid construct

[0329] The present invention also relates to a nucleic acid construct comprising a polynucleotide of the present invention, wherein the polynucleotide is operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with these control sequences.

[0330] Polynucleotides can be manipulated in a variety of ways to provide expression of a polypeptide. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide prior to its insertion into the vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0331] Promoter

[0332] The control sequence may be a promoter, i.e., a polynucleotide that is recognized by the host cell for expression of a polynucleotide encoding the polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that exhibits transcriptional activity in the host cell, including mutant promoters, truncated promoters, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0333] Examples of suitable promoters for directing transcription of the polynucleotides of the invention in bacterial host cells are described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., New York; Davis et al., 2012, supra; and Song et al., 2016, PLOS One 11(7):e0158447.

[0334] Terminator

[0335] The control sequence may also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator is operably linked to the 3'-end of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.

[0336] Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0337] mRNA stabilizer

[0338] The control sequence may also be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of a gene, which increases the expression of the gene.

[0339] Examples of suitable mRNA stabilizer regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacteriol. 177:3465-3471).

[0340] Examples of mRNA stabilizer regions of fungal cells are described in Geisberg et al., 2014, Cell 156(4):812-824 and Morozov et al., 2006, Eukaryotic Cell 5(11):1838-1846.

[0341] Preamble sequence

[0342] The control sequence may also be a leader sequence, i.e., an untranslated region of an mRNA that is important for translation by the host cell. The leader sequence may be operably linked to the 5'-end of the polynucleotide encoding the polypeptide. Any leader sequence that is functional in the host cell may be used.

[0343] Suitable leader sequences for bacterial host cells are provided by Hambraeus et al., 2000, Microbiology 146(12):3051-3059 and Kaberdin and 2006, FEMS Microbiol. Rev. [FEMS Microbiology Review] 30(6): 967-979 description.

[0344] polyadenylation sequence

[0345] The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3'-terminus of the polynucleotide which, when transcribed, is recognized by the host cell as a signal to add polyadenylic acid residues to transcribed mRNA. Any polyadenylation sequence functional in the host cell may be used.

[0346] Signal peptide

[0347] The control sequence can also be a signal peptide coding region that encodes a signal peptide connected to the N-terminus of the polypeptide and directs the polypeptide to enter the secretory pathway of the cell. The 5'-end of the coding sequence of the polynucleotide itself may contain a signal peptide coding sequence that is naturally connected to the coding sequence segment of the encoded polypeptide in the translation open reading frame. Alternatively, the 5'-end of the coding sequence may contain a signal peptide coding sequence that is heterologous to the coding sequence. In the case where the coding sequence does not naturally contain a signal peptide coding sequence, a heterologous signal peptide coding sequence may be required. Alternatively, a heterologous signal peptide coding sequence can simply replace the natural signal peptide coding sequence to enhance the secretion of the polypeptide. Any signal peptide coding sequence that directs the expressed polypeptide to enter the secretory pathway of the host cell can be used.

[0348] Effective signal peptide coding sequences for bacterial host cells are those obtained from the following genes: Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus α-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM) and Bacillus subtilis prsA. Additional signal peptides are described by Freudl, 2018, Microbial Cell Factories [Microbial Cell Factory] 17:52.

[0349] Propeptide

[0350] The control sequence can also be a propeptide coding sequence encoding a propeptide at the N-terminus of a polypeptide. The resulting polypeptide is referred to as a proenzyme or propolypeptide (or in some cases as a zymogen). Propolypeptides are generally inactive and can be converted into active polypeptides by catalytic cleavage or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence can be obtained from the following genes: Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95 / 33836), Rhizomucor miehei aspartic protease and Saccharomyces cerevisiae α-factor.

[0351] In the case where both the signal peptide sequence and the propeptide sequence are present, the propeptide sequence is located adjacent to the N-terminus of the polypeptide and the signal peptide sequence is located adjacent to the N-terminus of the propeptide sequence. Additionally or alternatively, when both the signal peptide sequence and the propeptide sequence are present, the polypeptide may comprise only a portion of the signal peptide sequence and / or only a portion of the propeptide sequence. Alternatively, the final or separated polypeptide may comprise a mixture of a mature polypeptide and a polypeptide comprising a partial or full-length propeptide sequence and / or a signal peptide sequence.

[0352] Adjustment sequence

[0353] It may also be desirable to add regulatory sequences that regulate the expression of polypeptides associated with host cell growth. Examples of regulatory sequences are regulatory sequences that cause gene expression to turn on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory sequences in prokaryotic systems include lac, tac, and trp operator systems. In yeast, the ADH2 system or the GAL1 system may be used.

[0354] Expression vector

[0355] The present invention further relates to a recombinant expression vector comprising a polynucleotide of the present invention, a promoter and a transcription and translation termination signal. A plurality of nucleotides and control sequences can be linked together to produce a recombinant expression vector, and the recombinant expression vector can include one or more convenient restriction sites to allow the insertion or replacement of the polynucleotide encoding a polypeptide at such a site. Alternatively, the polynucleotide can be expressed by inserting a polynucleotide or a nucleic acid construct comprising the polynucleotide into a suitable vector for expression. When producing an expression vector, the encoding sequence is located in the vector so that the encoding sequence is operably connected to the suitable control sequence for expression.

[0356] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be easily subjected to recombinant DNA procedures and can cause expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0357] The vector can be an autonomously replicating vector, i.e. a vector existing as an extrachromosomal entity, which replicates independently of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The vector can contain any means for ensuring self-replication. Alternatively, the vector can be a vector that is integrated into the genome and replicates with the chromosome into which it has been integrated when it is introduced into the host cell. Moreover, a single vector or plasmid or two or more vectors or plasmids can be used, which contain the total DNA to be introduced into the host cell genome together, or a transposon can be used.

[0358] The vector preferably contains one or more selectable markers that permit easy selection of transformed cells, transfected cells, transduced cells, etc. A selectable marker is a gene whose product provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0359] The vector preferably contains at least one element that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0360] For integration into the host cell genome, the vector may rely on the polynucleotide sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination, such as homology directed repair (HDR), or non-homologous recombination, such as non-homologous end joining (NHEJ).

[0361] For autonomous replication, the vector may further comprise an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicon that mediates autonomous replication that functions in the cell. The term "origin of replication" or "plasmid replicon" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0362] More than one copy of polynucleotides of the present invention can be inserted into host cells to improve the production of polypeptides. For example, 2 or 3 or 4 or 5 or more copies are inserted into host cells. The copy number of the increase of polynucleotides can be obtained by integrating at least one other copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene together with the polynucleotides, wherein cells containing amplified copies of the selectable marker gene and thus other copies of the polynucleotides can be selected by cultivating cells in the presence of an appropriate selective agent.

[0363] Host cells

[0364] The present invention also relates to recombinant host cells comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention.

[0365] The construct or vector comprising the polynucleotide is introduced into the host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector, as described earlier. The selection of the host cell will depend to a large extent on the gene encoding the polypeptide and its source. The polypeptide can be native or heterologous to the recombinant host cell. In addition, at least one of the one or more control sequences can be heterologous to the polynucleotide encoding the polypeptide. The recombinant host cell can contain a single copy or at least two copies of the polynucleotide of the present invention, such as three, four, five or more copies.

[0366] The host cell may be any microbial cell, such as a prokaryotic cell or a fungal cell, that can be used to recombinantly produce a polypeptide of the present invention.

[0367] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0368] The bacterial host cell can be any Bacillus cell, including but not limited to Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells. In an embodiment, the Bacillus cell is a Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus subtilis cell.

[0369] For the purposes of the present invention, the Bacillus genus / species shall be defined as described in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. 70:406-438.

[0370] The bacterial host cell may also be any Streptococcus cell, including but not limited to Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equisubsp. Zooepidemicus cells.

[0371] The bacterial host cell may also be any Streptomyces cell, including but not limited to Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0372] Methods for introducing DNA into prokaryotic host cells are well known in the art, and any suitable method can be used, including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, transduction, wherein DNA is introduced as linearized or circular polynucleotides. Those skilled in the art will be able to easily determine suitable methods for introducing DNA into a given prokaryotic cell according to, for example, the genus. Methods for introducing DNA into prokaryotic host cells are described, for example, in Heinze et al., 2018, BMC Microbiology [BMC Microbiology] 18: 56; Burke et al., 2001, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 98: 6289-6294; Choi et al., 2006, J. Microbiol. Methods [Microbiological Methods Magazine] 64: 391-397 and Donald et al., 2013, J. Bacteriol. [Bacteriology Magazine] 195 (11): 2612-2620.

[0373] In one aspect, the host cell is isolated.

[0374] In another aspect, the host cell is purified.

[0375] Generation method

[0376] The present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) culturing a cell under conditions conducive to production of the polypeptide, the cell producing the polypeptide in its wild-type form; and optionally (b) recovering the polypeptide. In one aspect, the cell is an Aspergillus or Penicillium or Cohen's cell. In another aspect, the cell is an Aspergillus aculeatus, Aspergillus tubingensis, Aspergillus ryukyuus, Penicillium oxalicum or Penicillium erythrogenum cell. In another aspect, the cell is a Cohen's species-60555 cell.

[0377] The present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the polypeptide; and optionally (b) recovering the polypeptide.

[0378] Host cells are cultivated in a nutrient medium suitable for producing polypeptides using methods known in the art. For example, cells can be cultured in suitable medium and under conditions allowing expression and / or separation of polypeptides by shaking flask culture or in a laboratory or industrial fermentor on a small scale or in large scale fermentation (including continuous, batch, fed-batch or solid-state and / or microcarrier-based fermentation). Suitable medium can be obtained from commercial suppliers or can be prepared according to disclosed composition (for example, in the catalog of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be directly recovered from the medium. If the polypeptide is not secreted, it can be recovered from the cell lysate.

[0379] The polypeptide can be detected using methods known in the art that are specific for the polypeptide, including but not limited to assays using specific antibodies, enzyme product formation, disappearance of enzyme substrate, or determining the relative or specific activity of the polypeptide.

[0380] The polypeptide can be recovered from the culture medium using methods known in the art, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation or precipitation. In one aspect, the whole fermentation broth containing the polypeptide is recovered. In another aspect, the cell-free fermentation broth containing the polypeptide is recovered.

[0381] The polypeptides can be purified by a variety of procedures known in the art to obtain substantially pure polypeptides and / or polypeptide fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science; 80(1):6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129:3-10).

[0382] In alternative aspects, the polypeptide is not recovered.

[0383] In a further aspect, the invention relates to an animal feed additive comprising the polypeptide of the invention.

[0384] In a further aspect, the invention relates to an animal feed comprising the polypeptide of the invention or the animal feed additive of the invention.

[0385] The present invention is further defined by the following numbered paragraphs:

[0386] 1. A method of improving the nutritional value of an animal feed comprising oilseed material, the method comprising adding a fiber degrading enzyme to the animal feed.

[0387] 2. A method for improving the growth performance of an animal, the method comprising administering to the animal a fiber degrading enzyme, an animal feed comprising a fiber degrading enzyme, or an animal feed additive; preferably, the growth performance is growth rate, feed conversion rate and / or body weight gain.

[0388] 3. The method according to paragraph 1 or 2, wherein the oil seed material is selected from the group consisting of soybean, rapeseed, sunflower, pea, lupin, broad bean, runner bean, gimme bean, lima bean, French bean, broad bean, chickpea, lentil, peanut, flaxseed, cottonseed or a combination thereof; or wherein the oil seed material is processed; preferably, the oil seed material is selected from the group consisting of soybean meal, rapeseed meal, sunflower meal, pea meal, peanut meal, flaxseed meal, cottonseed meal or a combination thereof.

[0389] 4. The method according to any one of paragraphs 1 to 3, wherein the animal is a monogastric animal, preferably the monogastric animal is selected from the group consisting of: pigs or swine (including but not limited to piglets, growing pigs and sows); poultry, such as turkeys, ducks and chickens (including but not limited to broilers, laying hens).

[0390] 5. The method according to any one of paragraphs 1 to 4, wherein the fiber degrading enzyme is selected from the group consisting of: pectinase, xyloglucan-specific endo-1,4-β-glucanase, xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase and combinations thereof; preferably, the fiber degrading enzyme is pectinase; more preferably, the fiber degrading enzyme is one or more pectinases selected from the group consisting of: rhamnogalacturonan lyase, endo-β-1,4-galactanase, polygalacturonase, rhamnogalacturonanase, pectin methylesterase, pectin lyase, pectin acetyl ester enzyme, galactan endo-β-1,3-galactanase, xylogalacturonase and a combination thereof; or wherein the fiber degrading enzyme is selected from the group consisting of: pectinase, xyloglucan-specific endo-1,4-β-glucanase, xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase and a combination thereof; preferably, the fiber degrading enzyme is pectinase; more preferably, the fiber degrading enzyme is one or more pectinases selected from the group consisting of: rhamnogalacturonan lyase (α-L-rhamnopyranosyl-1,4-α-D-galactopyranosyl ester endo-lyase, EC 4.2.2.23), endo-β-1,4-galactanase (EC 3.2.1.89), polygalacturonase (1,4-α-D-galacturonan polysaccharide hydrolase, EC 3.2.1.15 and EC 3.2.1.67), rhamnogalacturonase (rhamnogalacturonan α-D-galacturonan-1,2-α-L-rhamnosyl hydrolase, EC 3.2.1.171), pectin methylesterase (pectin pectyl hydrolase, EC 3.1.1.11), pectin lyase ((1,4)-6-O-methyl-α-D-galacturonan polysaccharide hydrolase, EC 4.2.2.10), pectin acetylesterase (acetate acetylhydrolase, EC 3.1.1.6), endo-β-1,3-galactan (EC 3.2.1.181), xylogalacturonase and combinations thereof.

[0391] 6. A method according to any one of paragraphs 1-5, wherein the fiber degrading enzyme is a pectinase selected from the group consisting of: rhamnogalacturonan lyase, endo-β-1,4-galactanase, pectin lyase and a combination thereof; preferably, the fiber degrading enzyme is selected from the group consisting of: a combination of rhamnogalacturonan lyase and endo-β-1,4-galactanase; a combination of rhamnogalacturonan lyase and pectin lyase; a combination of endo-β-1,4-galactanase and pectin lyase; and a combination of xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase and pectin lyase.

[0392] 7. The method according to any one of paragraphs 1 to 6, wherein the fiber degrading enzyme is obtained or obtainable from Aspergillus or Penicillium or Cohenella; preferably, the fiber degrading enzyme is a rhamnogalacturonan lyase obtained from Aspergillus or Penicillium, such as a rhamnogalacturonan lyase obtained from Aspergillus or Penicillium oxalicum; the fiber degrading enzyme is an endo-polygalacturonase obtained from Aspergillus, such as an endo-polygalacturonase obtained from Aspergillus aculeatus; the fiber degrading enzyme is a xyloglucan-specific endo-1,4-β-glucanase obtained or obtainable from Aspergillus, such as a xyloglucan-specific endo-1,4-β-glucanase obtained from Aspergillus aculeatus or Aspergillus ryukyuus. β-glucanase; the fiber degrading enzyme is a galactanase obtained or obtainable from Cohenella, such as a galactanase obtained from Cohenella species-60555; the fiber degrading enzyme is a xylogalacturonase obtained or obtainable from Aspergillus, such as a xylogalacturonase obtained from Aspergillus tubingensis or Aspergillus aculeatus; the fiber degrading enzyme is a pectin lyase obtained or obtainable from Aspergillus, such as a pectin lyase obtained from Aspergillus aculeatus; the fiber degrading enzyme is an endo-β-1,4-glucanase / endo-xyloglucanase obtained or obtainable from Penicillium, such as an endo-β-1,4-glucanase / endo-xyloglucanase obtained from Penicillium rubra.

[0393] 8. A method for the in situ production of prebiotics in an oilseed based animal feed, the method comprising adding a fiber degrading enzyme to the animal feed.

[0394] 9. A method of reducing the insoluble pectin fraction in an oilseed based animal feed, the method comprising adding a fiber degrading enzyme to the animal feed.

[0395] 10. A method of improving the intestinal health of a monogastric animal, the method comprising administering to the animal an oilseed based animal feed, wherein the animal feed comprises a fiber degrading enzyme.

[0396] 11. The method according to paragraph 10, wherein the fiber degrading enzyme degrades pectin polysaccharides of the oilseed material so as to generate prebiotic oligomers and polymers comprising pectin oligosaccharides.

[0397] 12. The method of paragraph 11, wherein in situ cecal butyrate levels in the animal are increased.

[0398] 13. The method of paragraph 11, wherein the composition of the microbiota in the animal is altered.

[0399] 14. A method for the in situ production of prebiotics in a monogastric animal, the method comprising administering to the animal an enzyme-enriched oilseed-based animal feed, wherein the animal feed comprises a fiber-degrading enzyme.

[0400] 15. A method of inducing butyrate production in a monogastric animal, the method comprising administering to the animal an oilseed based animal feed, wherein the animal feed comprises a fiber degrading enzyme.

[0401] 16. The method according to any one of paragraphs 8 to 15, wherein the oilseed material is selected from the group consisting of soybean, rapeseed, sunflower, pea, lupin, broad bean, runner bean, gimme bean, lima bean, French bean, broad bean, chickpea, lentil, peanut, flaxseed, cottonseed or a combination thereof; or wherein the oilseed material is processed, preferably the oilseed material is selected from the group consisting of soybean meal, rapeseed meal, sunflower meal, pea meal, peanut meal, flaxseed meal, cottonseed meal or a combination thereof.

[0402] 17. The method according to any one of paragraphs 8 to 16, wherein the animal is a monogastric animal, preferably the monogastric animal is selected from the group consisting of: pigs or swine (including but not limited to piglets, growing pigs and sows); poultry, such as turkeys, ducks and chickens (including but not limited to broilers, laying hens).

[0403] 18. The method according to any one of paragraphs 8 to 17, wherein the fiber degrading enzyme is selected from the group consisting of: pectinase, xyloglucan-specific endo-1,4-β-glucanase, xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase and combinations thereof; preferably, the fiber degrading enzyme is pectinase; more preferably, the fiber degrading enzyme is one or more pectinases selected from the group consisting of: rhamnogalacturonan lyase, endo-β-1,4-galactanase, polygalacturonase, rhamnogalacturonanase, pectin methylesterase, pectin lyase, pectin acetylesterase, galactan endo-β-1,3-galactanase, xylogalacturonase and combinations thereof.

[0404] 19. A method according to any one of paragraphs 8 to 18, wherein the fiber degrading enzyme is a pectinase selected from the group consisting of: rhamnogalacturonan lyase, endo-β-1,4-galactanase, pectin lyase and a combination thereof; preferably, the fiber degrading enzyme is selected from the group consisting of: a combination of rhamnogalacturonan lyase and endo-β-1,4-galactanase; a combination of rhamnogalacturonan lyase and pectin lyase; a combination of endo-β-1,4-galactanase and pectin lyase; and a combination of xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase and pectin lyase.

[0405] 20. The method according to any one of paragraphs 8 to 19, wherein the fiber degrading enzyme is obtained or obtainable from Aspergillus or Penicillium or Cohenella; preferably, the fiber degrading enzyme is a rhamnogalacturonan lyase obtained from Aspergillus or Penicillium, such as a rhamnogalacturonan lyase obtained from Aspergillus or Penicillium oxalicum; the fiber degrading enzyme is an endo-polygalacturonase obtained from Aspergillus, such as an endo-polygalacturonase obtained from Aspergillus aculeatus; the fiber degrading enzyme is a xyloglucan-specific endo-1,4-β-glucanase obtained or obtainable from Aspergillus, such as a xyloglucan-specific endo-1,4-β-glucanase obtained from Aspergillus aculeatus or Aspergillus ryukyuus -β-glucanase; the fiber degrading enzyme is a galactanase obtained or obtainable from Cohenella, such as a galactanase obtained from Cohenella species-60555; the fiber degrading enzyme is a xylogalacturonase obtained or obtainable from Aspergillus, such as a xylogalacturonase obtained from Aspergillus tubingensis or Aspergillus aculeatus; the fiber degrading enzyme is a pectin lyase obtained or obtainable from Aspergillus, such as a pectin lyase obtained from Aspergillus aculeatus; the fiber degrading enzyme is an endo-β-1,4-glucanase / endo-xyloglucanase obtained or obtainable from Penicillium, such as an endo-β-1,4-glucanase / endo-xyloglucanase obtained from Penicillium rubra.

[0406] 21. An animal feed comprising a fiber degrading enzyme and an oilseed material, wherein the feed comprises the oilseed material in an amount of 10 to 500 g / kg feed and the fiber degrading enzyme in an amount of 0.1 to 500 mg enzyme protein / kg feed.

[0407] 22. Use of a fiber degrading enzyme in the preparation of an enzyme-enriched animal feed, wherein the animal feed is an oilseed based animal feed.

[0408] 23. The animal feed according to paragraph 21, the use according to paragraph 22, wherein the oilseed material is selected from the group consisting of soybean, rapeseed, sunflower, pea, peanut, linseed, cottonseed or a combination thereof; preferably, the oilseed material is selected from the group consisting of soybean meal, rapeseed meal, sunflower meal, pea meal, peanut meal, linseed meal, cottonseed meal or a combination thereof.

[0409] 24. An animal feed additive comprising a fiber degrading enzyme and one or more additional components selected from the group consisting of: one or more vitamins; one or more minerals; one or more amino acids; one or more phytobiotics; one or more prebiotics; one or more organic acids; and one or more other feed ingredients.

[0410] 25. The animal feed according to paragraph 21, the use according to paragraph 22, the animal feed additive according to paragraph 24, wherein the animal is a monogastric animal, preferably the monogastric animal is selected from the group consisting of: pigs or swine (including but not limited to piglets, growing pigs and sows); poultry, such as turkeys, ducks and chickens (including but not limited to broilers, laying hens).

[0411] 26. The animal feed according to paragraph 21, the use according to paragraph 22, the animal feed additive according to paragraph 24, wherein the fiber degrading enzyme is selected from the group consisting of pectinase, xyloglucan-specific endo-1,4-β-glucanase, xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase and combinations thereof; preferably, the fiber degrading enzyme is pectinase; more preferably, the fiber degrading enzyme is one or more pectinases selected from the group consisting of rhamnogalacturonan lyase (α-L-rhamnopyranosyl-1,4-α-D-galacturonan ester endo-lyase, EC 4.2.2.23), endo-β-1,4-galactanase (EC 3.2.1.89), polygalacturonase (1,4-α-D-galacturonan polysaccharide hydrolase, EC 3.2.1.15 and EC 3.2.1.67), rhamnogalacturonase (rhamnogalacturonan α-D-galacturonan-1,2-α-L-rhamnohydrolase, EC 3.2.1.171), pectin methylesterase (pectin pectyl hydrolase, EC 3.1.1.11), pectin lyase ((1,4)-6-O-methyl-α-D-galacturonan lyase, EC 4.2.2.10), pectin acetylesterase (acetate acetylhydrolase, EC3.1.1.6), galactan endo-β-1,3-galactan (EC 3.2.1.181), xylogalacturonase and combinations thereof.

[0412] 27. The animal feed according to paragraph 21, the use according to paragraph 22, the animal feed additive according to paragraph 24, wherein the fiber degrading enzyme is a pectinase; preferably, the fiber degrading enzyme is a pectinase selected from the group consisting of: rhamnogalacturonan lyase, endo-β-1,4-galactanase and combinations thereof.

[0413] 28. The animal feed according to paragraph 21, the use according to paragraph 22, the animal feed additive according to paragraph 24, wherein the fiber degrading enzyme is obtained or obtainable from Aspergillus or Penicillium or Cohenella; preferably, the fiber degrading enzyme is a rhamnogalacturonan lyase obtained from Aspergillus or Penicillium, such as a rhamnogalacturonan lyase obtained from Aspergillus or Penicillium oxalicum; the fiber degrading enzyme is an endo-polygalacturonase obtained from Aspergillus, such as an endo-polygalacturonase obtained from Aspergillus aculeatus; the fiber degrading enzyme is a xyloglucan-specific endo-1,4-β-glucanase obtained or obtainable from Aspergillus, such as a xyloglucan lyase obtained from Aspergillus aculeatus or Aspergillus ryukyuus -60555; the fiber-degrading enzyme is a xyloglucan-specific endo-1,4-β-glucanase; the fiber-degrading enzyme is a galactanase obtained or obtainable from Cohenella, such as a galactanase obtained from Cohenella species-60555; the fiber-degrading enzyme is a xyloglucanase obtained or obtainable from Aspergillus, such as a xyloglucanase obtained from Aspergillus tubingensis or Aspergillus aculeatus; the fiber-degrading enzyme is a pectin lyase obtained or obtainable from Aspergillus, such as a pectin lyase obtained from Aspergillus aculeatus; the fiber-degrading enzyme is an endo-β-1,4-glucanase / endo-xyloglucanase obtained or obtainable from Penicillium, such as an endo-β-1,4-glucanase / endo-xyloglucanase obtained from Penicillium rubragenes.

[0414] 29. The animal feed according to paragraph 21, the use according to paragraph 22, the animal feed additive according to paragraph 24, wherein the fiber degrading enzyme is selected from the group consisting of:

[0415] (a) a rhamnogalacturonan lyase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 or the mature polypeptide of SEQ ID NO: 1;

[0416] (b) a rhamnogalacturonan lyase derived from SEQ ID NO: 1 or the mature polypeptide of SEQ ID NO: 1 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0417] (c) a rhamnogalacturonan lyase derived from (a) or (b), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, such as 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids;

[0418] (d) A fragment of the rhamnogalacturonan lyase of (a), (b) or (c), wherein the fragment has rhamnogalacturonan lyase activity;

[0419] (e) an endo-polygalacturonase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO: 2;

[0420] (f) an endo-polygalacturonase derived from SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO: 2 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0421] (g) an endo-polygalacturonase derived from (e) or (f), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids;

[0422] (h) a fragment of the endo-polygalacturonase of (e), (f) or (g), wherein the fragment has endo-polygalacturonase activity;

[0423] (i) a xyloglucan-specific endo-1,4-β-glucanase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO: 3;

[0424] (j) a xyloglucan-specific endo-1,4-beta-glucanase derived from SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO: 3 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0425] (k) a xyloglucan-specific endo-1,4-β-glucanase derived from (i) or (j), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids;

[0426] (l) A fragment of the xyloglucan-specific endo-1,4-β-glucanase of (i), (j) or (k), wherein the fragment has xyloglucan-specific endo-1,4-β-glucanase activity;

[0427] (m) a xyloglucan-specific endo-1,4-β-glucanase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:4 or the mature polypeptide of SEQ ID NO:4;

[0428] (n) a xyloglucan-specific endo-1,4-beta-glucanase derived from SEQ ID NO: 4 or the mature polypeptide of SEQ ID NO: 4 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0429] (o) a xyloglucan-specific endo-1,4-β-glucanase derived from (m) or (n), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0430] A fragment of the xyloglucan-specific endo-1,4-β-glucanase of (p), (m), (n) or (o), wherein the fragment has xyloglucan-specific endo-1,4-β-glucanase activity;

[0431] (q) a galactanase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:5 or the mature polypeptide of SEQ ID NO:5;

[0432] (r) a galactanase derived from SEQ ID NO: 5 or the mature polypeptide of SEQ ID NO: 5 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions, at one or more positions;

[0433] (s) a galactanase derived from (q) or (r), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, such as 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids;

[0434] (t) a fragment of the galactanase of (q), (r) or (s), wherein the fragment has galactanase activity;

[0435] (u) a xylogalacturonase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:6 or the mature polypeptide of SEQ ID NO:6;

[0436] (v) a xylogalacturonase derived from SEQ ID NO: 6 or the mature polypeptide of SEQ ID NO: 6 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0437] (w) a xylogalacturonase derived from (u) or (v), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids;

[0438] (x) a fragment of the xylose galacturonase of (u), (v) or (w), wherein the fragment has xylose galacturonase activity;

[0439] (y) a xylogalacturonase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:7 or the mature polypeptide of SEQ ID NO:7;

[0440] (z) a xylogalacturonase derived from SEQ ID NO: 7 or the mature polypeptide of SEQ ID NO: 7 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0441] (aa) a xylogalacturonase derived from (y) or (z), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids;

[0442] A fragment of the xylose galacturonase of (bb)(y), (z) or (aa), wherein the fragment has xylose galacturonase activity;

[0443] (cc) a pectin lyase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:8 or the mature polypeptide of SEQ ID NO:8;

[0444] (dd) a pectin lyase derived from SEQ ID NO: 8 or the mature polypeptide of SEQ ID NO: 8 by having 1 to 30 alterations, e.g. substitutions, deletions and / or insertions, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions, at one or more positions;

[0445] (ee) a pectin lyase derived from (cc) or (dd), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids;

[0446] (ff) (cc), (dd) or (ee) a fragment of the pectin lyase, wherein the fragment has pectin lyase activity;

[0447] (gg) a rhamnogalacturonan lyase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:9 or the mature polypeptide of SEQ ID NO:9;

[0448] (hh) a rhamnogalacturonan lyase derived from SEQ ID NO: 9 or the mature polypeptide of SEQ ID NO: 9 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0449] (ii) a rhamnogalacturonan lyase derived from (gg) or (hh), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, such as 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0450] (jj) A fragment of the rhamnogalacturonan lyase of (gg), (hh) or (ii), wherein the fragment has rhamnogalacturonan lyase activity;

[0451] (kk) a xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 10 or the mature polypeptide of SEQ ID NO: 10;

[0452] (ll) a xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase derived from SEQ ID NO: 10 or the mature polypeptide of SEQ ID NO: 10 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0453] (mm) a xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase derived from (kk) or (ll), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0454] A fragment of a xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase of (nn)(kk), (ll) or (mm), wherein the fragment has xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase activity.

[0455] 30. Use of a combination of a polypeptide having rhamnogalacturonan lyase activity and a polypeptide having galactanase activity in animal feed or an animal feed additive; use of a combination of rhamnogalacturonan lyase and endo-β-1,4-galactanase in animal feed or an animal feed additive; use of a combination of rhamnogalacturonan lyase and pectin lyase in animal feed or an animal feed additive; use of a combination of endo-β-1,4-galactanase and pectin lyase in animal feed or an animal feed additive; or use of a combination of xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase and pectin lyase in animal feed or an animal feed additive.

[0456] 31. The use according to paragraph 30, wherein the polypeptide having rhamnogalacturonan lyase activity is RGL_1.

[0457] 32. The use according to paragraph 31, wherein the polypeptide having rhamnogalacturonan lyase activity is selected from the group consisting of:

[0458] (a) a rhamnogalacturonan lyase having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 or the mature polypeptide of SEQ ID NO: 1; preferably a rhamnogalacturonan lyase obtained or obtainable from Aspergillus; more preferably a rhamnogalacturonan lyase obtained or obtainable from Aspergillus aculeatus;

[0459] (b) a rhamnogalacturonan lyase derived from SEQ ID NO: 1 or the mature polypeptide of SEQ ID NO: 1 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0460] (c) a rhamnogalacturonan lyase derived from (a) or (b), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0461] (d) A fragment of the rhamnogalacturonan lyase of (a), (b) or (c), wherein the fragment has rhamnogalacturonan lyase activity.

[0462] 33. The use according to paragraph 30, wherein the polypeptide having galactanase activity is GH53.

[0463] 34. The use according to paragraph 30, wherein the polypeptide having galactanase activity is selected from the group consisting of:

[0464] (a) a galactanase having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:5 or the mature polypeptide of SEQ ID NO:5; preferably a galactanase obtained or obtainable from Cohenella species; more preferably a galactanase obtained or obtainable from Cohenella species-60555;

[0465] (b) a galactanase derived from SEQ ID NO: 5 or the mature polypeptide of SEQ ID NO: 5 by having 1-30 alterations, e.g. substitutions, deletions and / or insertions, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0466] (c) a galactanase derived from (a) or (b), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0467] (d) A fragment of the galactanase of (a), (b) or (c), wherein the fragment has galactanase activity.

[0468] 35. An animal feed additive comprising a polypeptide having rhamnogalacturonan lyase activity and a polypeptide having galactanase activity; preferably comprising the following animal feed additives: a combination of rhamnogalacturonan lyase and endo-β-1,4-galactanase; a combination of rhamnogalacturonan lyase and pectin lyase; a combination of endo-β-1,4-galactanase and pectin lyase; or a combination of xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase and pectin lyase.

[0469] 36. The animal feed additive according to paragraph 35, wherein the polypeptide having rhamnogalacturonan lyase activity is RGL_1.

[0470] 37. The animal feed additive according to paragraph 35 or 36, wherein the polypeptide having rhamnogalacturonan lyase activity is selected from the group consisting of:

[0471] (a) a rhamnogalacturonan lyase having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 or the mature polypeptide of SEQ ID NO: 1; preferably a rhamnogalacturonan lyase obtained or obtainable from Aspergillus; more preferably a rhamnogalacturonan lyase obtained or obtainable from Aspergillus aculeatus;

[0472] (b) a rhamnogalacturonan lyase derived from SEQ ID NO: 1 or the mature polypeptide of SEQ ID NO: 1 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0473] (c) a rhamnogalacturonan lyase derived from (a) or (b), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0474] (d) A fragment of the rhamnogalacturonan lyase of (a), (b) or (c), wherein the fragment has rhamnogalacturonan lyase activity.

[0475] 38. The animal feed additive of paragraph 35, wherein the polypeptide having galactanase activity is GH53.

[0476] 39. The animal feed additive according to paragraph 35 or 38, wherein the polypeptide having galactanase activity is selected from the group consisting of:

[0477] (a) a galactanase having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:5 or the mature polypeptide of SEQ ID NO:5; preferably a galactanase obtained or obtainable from Cohenella species; more preferably a galactanase obtained or obtainable from Cohenella species-60555;

[0478] (b) a galactanase derived from SEQ ID NO: 5 or the mature polypeptide of SEQ ID NO: 5 by having 1-30 alterations, e.g. substitutions, deletions and / or insertions, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0479] (c) a galactanase derived from (a) or (b), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0480] (d) A fragment of the galactanase of (a), (b) or (c), wherein the fragment has galactanase activity.

[0481] 40. The animal feed additive of any one of paragraphs 35 to 39, further comprising one or more additional components selected from the group consisting of: one or more vitamins; one or more minerals; one or more amino acids; one or more phytobiotics; one or more prebiotics; one or more organic acids; and one or more other feed ingredients.

[0482] 41. An animal feed comprising the animal feed additive of any of paragraphs 35-40 and an oilseed material.

[0483] 42. A method of improving the average metabolizable energy of a plant-based diet in a monogastric animal, the method comprising administering an animal feed additive according to any one of paragraphs 24, 35 to 40 or an animal feed according to any one of paragraphs 21 to 28 or 41.

[0484] 43. A polypeptide having xyloglucan-specific endo-1,4-β-glucanase activity, the polypeptide being selected from the group consisting of:

[0485] (a) a polypeptide having at least 99.7%, at least 99.8%, at least 99.9% or 100% sequence identity to SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO: 3;

[0486] (b) a polypeptide derived from SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO: 3 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0487] (c) a polypeptide having at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:4 or the mature polypeptide of SEQ ID NO:4;

[0488] (d) a polypeptide derived from SEQ ID NO: 4 or the mature polypeptide of SEQ ID NO: 4 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0489] (e) a polypeptide derived from the polypeptide of (a), (b), (c) or (d), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0490] (f) a fragment of the polypeptide of (a), (b), (c), (d) or (e);

[0491] The polypeptide has xyloglucan-specific endo-1,4-β-glucanase activity.

[0492] 44. The polypeptide of paragraph 43, comprising, consisting essentially of, or consisting of SEQ ID NO: 3, the mature polypeptide of SEQ ID NO: 3, SEQ ID NO: 4, or the mature polypeptide of SEQ ID NO: 4.

[0493] 45. A polypeptide having xylose galacturonase activity, the polypeptide being selected from the group consisting of:

[0494] (a) a polypeptide having at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:6 or the mature polypeptide of SEQ ID NO:6;

[0495] (b) a polypeptide derived from SEQ ID NO:6 or the mature polypeptide of SEQ ID NO:6 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0496] (c) a polypeptide having at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:7 or the mature polypeptide of SEQ ID NO:7;

[0497] (d) a polypeptide derived from SEQ ID NO: 7 or the mature polypeptide of SEQ ID NO: 7 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0498] (e) a polypeptide derived from the polypeptide of (a), (b), (c) or (d), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0499] (f) a fragment of the polypeptide of (a), (b), (c), (d) or (e);

[0500] The polypeptide has xylose galacturonase activity.

[0501] 46. ​​The polypeptide of paragraph 45, comprising, consisting essentially of, or consisting of SEQ ID NO: 6, the mature polypeptide of SEQ ID NO: 6, SEQ ID NO: 7, or the mature polypeptide of SEQ ID NO: 7.

[0502] 47. A polypeptide having rhamnogalacturonan lyase activity, the polypeptide being selected from the group consisting of:

[0503] (a) a polypeptide having at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:9 or the mature polypeptide of SEQ ID NO:9;

[0504] (b) a polypeptide derived from SEQ ID NO: 9 or the mature polypeptide of SEQ ID NO: 9 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0505] (c) a polypeptide derived from the polypeptide of (a) or (b) wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0506] (d) a fragment of the polypeptide of (a), (b) or (c);

[0507] The polypeptide has rhamnogalacturonan lyase activity.

[0508] 48. The polypeptide of paragraph 47, comprising, consisting essentially of, or consisting of SEQ ID NO: 9 or the mature polypeptide of SEQ ID NO: 9.

[0509] 49. A polypeptide having xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase activity, the polypeptide being selected from the group consisting of:

[0510] (a) a polypeptide having at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10 or the mature polypeptide of SEQ ID NO: 10;

[0511] (b) a polypeptide derived from SEQ ID NO: 10 or the mature polypeptide of SEQ ID NO: 10 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions;

[0512] (c) a polypeptide derived from the polypeptide of (a) or (b) wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and

[0513] (d) a fragment of the polypeptide of (a), (b) or (c);

[0514] The polypeptide has xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase activity.

[0515] 50. The polypeptide of paragraph 49, comprising, consisting essentially of, or consisting of SEQ ID NO: 10 or the mature polypeptide of SEQ ID NO: 10.

[0516] 51. A polynucleotide encoding the polypeptide according to any one of paragraphs 43-50.

[0517] 52. A nucleic acid construct or expression vector comprising the polynucleotide according to paragraph 51 operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

[0518] 53. A recombinant host cell comprising the nucleic acid construct or expression vector according to paragraph 52.

[0519] 54. An animal feed additive comprising the polypeptide according to any of paragraphs 43-50.

[0520] 55. An animal feed comprising the polypeptide according to any one of paragraphs 43-50 or the animal feed additive according to paragraph 54.

[0521] The present invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

[0522] Examples

[0523] Chemicals used as buffers and substrates were commercial products of at least reagent grade.

[0524] Enzymes used in the examples

[0525]

[0526]

[0527] Example 1: Effects of rhamnogalacturonan endolyase, endo-β-1,4-galactanase and combination on soybean The influence of powder

[0528] The effects of endo-rhamnogalacturonan lyase (RGL_1, Aspergillus aculeatus) and endo-β-1,4-galactanase (GH53), both used in animal feed, on deproteinized soy flour (SBM) were investigated in vitro.

[0529] By protease SBM samples obtained from DSM Nutritional Products (Villageneve, France) were deproteinized by double incubation with ELISA (Novozymes) at 50°C for 3 h followed by precipitation in 80% ethanol. After the second incubation together, the enzyme was inactivated for 15 min at 80° C. After centrifugation at 3,000 rpm for 15 min at 0° C., the ethanol from the precipitate was evaporated overnight in a fume hood and the sample was freeze-dried.

[0530] Deproteinized SBM (3% dry matter) was incubated with rhamnogalacturonan endolyase (RGL_1), endo-β-1,4-galactanase (GH53), or a combination thereof in acetate buffer at pH 5.0 for 4 h at 40° C. The experiments were run in triplicate.

[0531] After incubation with the enzyme, solids were removed by centrifugation at 4,700 g and 0° C. for 15 min, and 1,000 μL of the supernatant was subjected to acid hydrolysis (0.5 MH 2 SO 4 The hydrolyzate was filtered through a 0.2 μm wwPTFE membrane (AcroPrep Advance 8582, PALL) and analyzed for its monosaccharide content by high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

[0532] Monosaccharide separation was achieved on a CarboPac Analytical PA1 column (4 mm x 250 mm) with a CarboPac PA1 guard column (4 mm x 50 mm) (Thermofisher) at 30°C with the eluent flow rate fixed at 1 mL / min. Elution of neutral monosaccharides was achieved in 14 min using 10 mM NaOH as the eluent. In sequence, the eluent concentration was increased to 500 mM and the acidic monosaccharides were resolved in 7.5 min. The column was re-equilibrated with 10 mM NaOH for 10.5 min before the subsequent samples were injected. The concentrations of monosaccharides were calculated based on standard curves of fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid.

[0533] As shown in Table 1, RG-I lyase alone can dissolve rhamnose, arabinose, galactose and galacturonic acid. Galactanase alone can dissolve arabinose and galactose. Therefore, both enzymes are effective in dissolving pectin polymers by themselves. When the two enzymes are mixed, the effects are synergistic. As demonstrated experimentally, when RG-I lyase is combined with GH53, the breakdown of polysaccharides containing rhamnose, arabinose and galacturonic acid from SBM is increased to approximately 3 times compared to the sum of the effects of the two enzymes separately, illustrating the synergistic effect between the two enzyme products.

[0534] Table 1. Mean values ​​of the amounts of rhamnose, arabinose, galactose and galacturonic acid after treatment without enzyme addition (control), with addition of 20 ppm of RG-I lyase (RGL_1), with addition of 20 ppm of galactanase (GH53), and a combination of both enzymes (20 ppm each). The values ​​are given as % of monosaccharides in soy flour (SBM).

[0535]

[0536]

[0537] Example 2: Effect of rhamnogalacturonan endolyase, endo-β-1,4-galactanase and combination on the growth of chicken blind Effects of the intestinal microbiota on butyrate accumulation in in vitro fermentation of rapeseed meal

[0538] The effects of endo-rhamnogalacturonan lyase (RGL_1, Aspergillus aculeatus) and endo-β-1,4-galactanase (GH53), both used in animal feed, on deproteinized rapeseed meal (RSM) were investigated in vitro.

[0539] By protease RSM samples obtained from DSM Nutritional Products (Villageneve, France) were deproteinized by double incubation with 1,2-dihydro-1,4 ... After the second incubation together, the enzyme was inactivated for 15 min at 80° C. After centrifugation at 3,000 rpm for 15 min at 0° C., the ethanol from the precipitate was evaporated overnight in a fume hood and the sample was freeze-dried.

[0540] Deproteinized RSM (3% dry matter) was diluted in 5% paraformaldehyde as described by Duncan et al. (Duncan, SH, Hold, GL, Barcenilla, A., Stewart, CS & Flint, HJ Roseburia intestinalis sp. nov., a novel saccharolytic, butyrate-producing bacterium from human faeces [Rose ... intestinalis) new species, a new glycolytic, butyrate-producing bacterium from human feces]. Int. J. Syst. Evol. Microbiol. [International Journal of Systematic and Evolutionary Microbiology] 52, 1615-1620 (2002)) in anoxic sterile YCFA medium prepared as described, which was modified so that the carbon source was 1% non-starch polysaccharides from deproteinized RSM and the pH was adjusted to 6.5. Chicken cecal contents from 35-day-old broilers were added to the final 1,000-fold dilution for the first round of fermentation. After fermentation at 37°C for 12 hours, the first fermentation was used as an inoculum for the second fermentation, and so on. The fermentation supernatant was sampled after 12 hours and stored at -20°C until analysis. The fermentation was run in five replicates.

[0541] Quantification of acetate, propionate, and butyrate in the supernatant was achieved by running the samples in a HPLC apparatus equipped with an ion exchange BioRad HPX-87H column (with a BioRad cation H precolumn, 60°C) and a refractive index detector.

[0542] Experiments have shown that treatment with rhamnogalacturonan endolyase and galactanase, respectively, increased butyrate accumulation in RSM caused by fermentation by chicken cecal microbiota from 8.9 mM to approximately 11.5 mM. The combination of these two enzymes further increased butyrate accumulation to 13 mM.

[0543] Table 2. Mean values ​​of the amounts of acetate, propionate and butyrate after four cycles of 12-h fermentation of RSM by chicken cecal microbiota with no enzyme addition (control), 20 ppm of RG-I lyase (RGL_1), 20 ppm of galactanase (GH53), and a combination of both enzymes (20 ppm each). The values ​​are given in mM.

[0544] deal with Acetate Propionic acid Butyrate Comparison 49,234 25,3547 8,88528 Galactanase 52,0745 25,688 11,3802 RG-I lyase 56,3005 32,0729 11,6395 RG-I lyase and galactanase 56,8993 30,9499 13,0665

[0545] Example 3: Effect of xyloglucan-specific β-glucanase on RSM

[0546] The effects of two xyloglucan-specific β-glucanases (GH12_1), one from Aspergillus aculeatus and one from Aspergillus ryukyuensis, applied in animal feed, on deproteinized rapeseed meal (RSM) were investigated in vitro.

[0547] By protease RSM samples obtained from DSM Nutritional Products (Villageneve, France) were deproteinized by double incubation with 1,2-dihydro-1,4 ... After the second incubation together, the enzyme was inactivated for 15 min at 80° C. After centrifugation at 3,000 rpm for 15 min at 0° C., the ethanol from the precipitate was evaporated overnight in a fume hood and the sample was freeze-dried.

[0548] Deproteinized RSM (3% dry matter) was incubated separately with two xyloglucan-specific β-glucanases (GH12_1) in acetate buffer at pH 5.0 at 40° C. for 4 h. The experiments were run in triplicate.

[0549] After incubation with the enzyme, the solids were removed by centrifugation at 4,700 g and 0° C. for 15 min, and 1,000 μL of the supernatant was subjected to acid hydrolysis (0.5 MH 2 SO4 The hydrolyzate was filtered through a 0.2 μm wwPTFE membrane (AcroPrep Advance 8582, PALL) and analyzed for its monosaccharide content by high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

[0550] Monosaccharide separation was achieved on a CarboPac Analytical PA1 column (4 mm x 250 mm) with a CarboPac PA1 guard column (4 mm x 50 mm) (Thermo Fisher Scientific) at 30°C with the eluent flow rate fixed at 1 mL / min. Elution of neutral monosaccharides was achieved in 14 min using 10 mM NaOH as the eluent. In sequence, the eluent concentration was increased to 500 mM and the acidic monosaccharides were resolved in 7.5 min. The column was re-equilibrated with 10 mM NaOH for 10.5 min before the subsequent samples were injected. The concentrations of monosaccharides were calculated based on standard curves of fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid.

[0551] GH12_1 from Aspergillus ryukyuensis increased the amounts of fucose, xylose, and glucose in the supernatant of RSM by 4-fold, 45%, and 21%, respectively.

[0552] GH12_1 from Aspergillus aculeatus increased the amounts of fucose, xylose and glucose in the supernatant of RSM by 5-fold, 73% and 35%, respectively.

[0553] Table 3. Mean values ​​of the amounts of fucose, rhamnose, arabinose, galactose, glucose and xylose after treatment without enzyme addition (control), with addition of 20 ppm of GH12_1 (A. ryukyuensis) and with addition of 20 ppm of GH12_1 (A. aculeatus). The values ​​are given as % of monosaccharides in rapeseed meal (RSM).

[0554] deal with Fucose Rhamnose Arabinose Galactose glucose Xylose Comparison 0,011383 0,040478 1,228275 0,846672 1,297418 0,374021 GH12_1 / Aspergillus Ryukyu 0,041984 0,046149 1,293083 0,962642 1,575253 0,54325 GH12_1 / Aspergillus aculeatus 0,054315 0,053886 1,41049 1,061931 1,75221 0,64836

[0555] Example 4: Effect of endo-polygalacturonase on RSM

[0556] The effects of an endo-polygalacturonase (GH28_9, Aspergillus aculeatus), used in animal feed, on deproteinized rapeseed meal (RSM) were investigated in vitro.

[0557] By protease (Novozymes) were double incubated at 50°C

[0558] RSM samples obtained from DSM Nutritional Products (Villageneve, France) were deproteinized for 3 h and subsequently precipitated in 80% ethanol. After the second incubation together, the enzyme was inactivated for 15 min at 80° C. After centrifugation at 3,000 rpm for 15 min at 0° C., the ethanol from the precipitate was evaporated overnight in a fume hood and the sample was freeze-dried.

[0559] Deproteinized RSM (3% dry matter) was incubated with endo-polygalacturonase (GH28_9) in acetate buffer, pH 5.0, for 4 h at 40° C. The experiment was run in triplicate.

[0560] After incubation with the enzyme, solids were removed by centrifugation at 4,700 g and 0° C. for 15 min, and 1,000 μL of the supernatant was subjected to acid hydrolysis (0.5 MH 2 SO 4 The hydrolyzate was filtered through a 0.2 μm wwPTFE membrane (AcroPrep Advance 8582, PALL) and analyzed for its monosaccharide content by high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

[0561] Monosaccharide separation was achieved on a CarboPac Analytical PA1 column (4 mm x 250 mm) with a CarboPac PA1 guard column (4 mm x 50 mm) (Thermo Fisher Scientific) at 30°C with the eluent flow rate fixed at 1 mL / min. Elution of neutral monosaccharides was achieved in 14 min using 10 mM NaOH as the eluent. In sequence, the eluent concentration was increased to 500 mM and the acidic monosaccharides were resolved in 7.5 min. The column was re-equilibrated with 10 mM NaOH for 10.5 min before the subsequent samples were injected. The concentrations of monosaccharides were calculated based on standard curves of fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid.

[0562] Treatment with endo-polygalacturonase (GH28_9) increased the amount of monosaccharides in the supernatant of RSM by 14%. The amount of fucose and rhamnose in the soluble fraction of the enzyme-treated sample was 70% higher than that of the control, while the solubilization of arabinose, galactose, glucose and xylose was between 11% and 17%.

[0563] Table 4. Mean values ​​of the amount of fucose, rhamnose, arabinose, galactose, glucose, xylose and the total amount of monosaccharides after treatment without enzyme addition (control) and with addition of 20 ppm endo-polygalacturonase (GH28_9). The values ​​are given as % of monosaccharides in rapeseed meal (RSM).

[0564]

[0565]

[0566] Example 5: Effect of pectinase on RSM

[0567] The effects of two pectinases used in animal feed, one from Aspergillus tubingensis and one from Aspergillus aculeatus, on deproteinized rapeseed meal (RSM) were investigated in vitro.

[0568] By protease RSM samples obtained from DSM Nutritional Products (Villageneve, France) were deproteinized by double incubation with 1,2-dihydro-1,4 ... After the second incubation together, the enzyme was inactivated for 15 min at 80° C. After centrifugation at 3,000 rpm for 15 min at 0° C., the ethanol from the precipitate was evaporated overnight in a fume hood and the sample was freeze-dried.

[0569] Deproteinized RSM (3% dry matter) was incubated separately with both pectinases in acetate buffer at pH 5.0 at 40° C. for 4 h. The experiments were run in triplicate.

[0570] After incubation with the enzyme, the solids were removed by centrifugation at 4,700 g and 0°C for 15 min, and 1,000 μL of the supernatant was subjected to acid hydrolysis (0,5 M H 2 SO 4 The hydrolyzate was filtered through a 0,2 μm wwPTFE membrane (AcroPrep Advance 8582, PALL) and analyzed for its monosaccharide content by high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

[0571] Monosaccharide separation was achieved on a CarboPac Analytical PA1 column (4 mm x 250 mm) with a CarboPac PA1 guard column (4 mm x 50 mm) (Thermo Fisher Scientific) at 30°C with the eluent flow rate fixed at 1 mL / min. Elution of neutral monosaccharides was achieved in 14 min using 10 mM NaOH as the eluent. In sequence, the eluent concentration was increased to 500 mM and the acidic monosaccharides were resolved in 7.5 min. The column was re-equilibrated with 10 mM NaOH for 10.5 min before the subsequent samples were injected. The concentrations of monosaccharides were calculated based on standard curves of fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid.

[0572] Pectinase from Aspergillus tubingensis increased the amounts of xylose and rhamnose in the supernatant of RSM by 10% and 35%, respectively.

[0573] Pectinase from Aspergillus aculeatus increased the amounts of xylose and rhamnose in the supernatant of RSM by 13% and 74%, respectively.

[0574] Table 5. Mean values ​​of the amount of fucose, rhamnose, arabinose, xylose and the total amount of monosaccharides after treatment without added enzyme (control), with addition of 20 ppm pectinase (Aspergillus tubingensis) and with addition of 20 ppm pectinase (Aspergillus aculeatus). The values ​​are given as % of monosaccharides in rapeseed meal (RSM).

[0575] deal with Fucose Rhamnose Arabinose Xylose total Comparison 0,01604 0,0418 1,26044 0,46345 3,8607 Pectinase / Aspergillus aculeatus 0,01824 0,07255 1,30973 0,52326 3,93309 Pectinase / Aspergillus tubingensis 0,01823 0,05638 1,31123 0,5046 4,00899

[0576] Example 6: Effect of rhamnogalacturonan endolyase, endo-β-1,4-galactanase and combination on soybean The influence of powder

[0577] The effects of endo-rhamnogalacturonan lyase (RGL_1, Penicillium oxalicum) and endo-β-1,4-galactanase (GH53), both used in animal feed, on deproteinized soybean meal (SBM) were investigated in vitro.

[0578] By protease SBM samples obtained from DSM Nutritional Products (Villageneve, France) were deproteinized by double incubation with 1,2-dihydro-1,4 ... After the second incubation together, the enzyme was inactivated for 15 min at 80° C. After centrifugation at 3,000 rpm for 15 min at 0° C., the ethanol from the precipitate was evaporated overnight in a fume hood and the sample was freeze-dried.

[0579] Deproteinized SBM (3% dry matter) was incubated with rhamnogalacturonan endolyase (RGL_1), endo-β-1,4-galactanase (GH53), or a combination thereof in acetate buffer at pH 5.0 for 4 h at 40° C. The experiments were run in triplicate.

[0580] After incubation with the enzyme, the solids were removed by centrifugation at 4,700 g and 0° C. for 15 min, and 1,000 μL of the supernatant was subjected to acid hydrolysis (0.5 MH 2 SO 4 The hydrolyzate was filtered through a 0.2 μm wwPTFE membrane (AcroPrep Advance 8582, PALL) and analyzed for its monosaccharide content by high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

[0581] Monosaccharide separation was achieved on a CarboPac Analytical PA1 column (4 mm x 250 mm) with a CarboPac PA1 guard column (4 mm x 50 mm) (Thermo Fisher Scientific) at 30°C with the eluent flow rate fixed at 1 mL / min. Elution of neutral monosaccharides was achieved in 14 min using 10 mM NaOH as the eluent. In sequence, the eluent concentration was increased to 500 mM and the acidic monosaccharides were resolved in 7.5 min. The column was re-equilibrated with 10 mM NaOH for 10.5 min before the subsequent samples were injected. The concentrations of monosaccharides were calculated based on standard curves of fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid.

[0582] The experiments demonstrated that when RGL_1 was combined with GH53, polysaccharides from SBM were solubilized by approximately 20% more compared to the sum of the effects of the two enzymes separately, illustrating a synergistic effect between the two enzyme products. The combination of the two enzymes resulted in 11.3% solubilization of insoluble NSPs, while RGL_1 and GH53 resulted in 1.0% and 8.4% solubilization of insoluble NSPs, respectively. The main monosaccharides present in the solubilized NSPs were rhamnose, galacturonic acid, arabinose, galactose, xylose, and fucose.

[0583] Table 6. Mean values ​​of the amount of fucose, rhamnose, arabinose, galactose, xylose, galacturonic acid and the total amount of monosaccharides after treatment without enzyme addition (control), with addition of 20 ppm of RG-I lyase (RGL_1), with addition of 20 ppm of galactanase (GH53), and with a combination of both enzymes (20 ppm each). The values ​​are given as % of monosaccharides in soy flour (SBM).

[0584]

[0585] Example 7: Effect of rhamnogalacturonan endolyase, endo-β-1,4-galactanase and combination on rapeseed Effect of seed powder

[0586] The effects of endo-rhamnogalacturonan lyase (RGL_1, Penicillium oxalicum) and endo-β-1,4-galactanase (GH53), both used in animal feed, on deproteinized rapeseed meal (RSM) were investigated in vitro.

[0587] By protease RSM samples obtained from DSM Nutritional Products (Villageneve, France) were deproteinized by double incubation with 1,2-dihydro-1,4 ... After the second incubation together, the enzyme was inactivated for 15 min at 80° C. After centrifugation at 3,000 rpm for 15 min at 0° C., the ethanol from the precipitate was evaporated overnight in a fume hood and the sample was freeze-dried.

[0588] Deproteinized RSM (3% dry matter) was incubated with rhamnogalacturonan endolyase (RGL_1), endo-β-1,4-galactanase (GH53), or a combination thereof in acetate buffer at pH 5.0 for 4 h at 40° C. The experiments were run in triplicate.

[0589] After incubation with the enzyme, the solids were removed by centrifugation at 4,700 g and 0° C. for 15 min, and 1,000 μL of the supernatant was subjected to acid hydrolysis (0.5 MH 2 SO 4 The hydrolyzate was filtered through a 0.2 μm wwPTFE membrane (AcroPrep Advance 8582, PALL) and analyzed for its monosaccharide content by high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

[0590] Monosaccharide separation was achieved on a CarboPac Analytical PA1 column (4 mm x 250 mm) with a CarboPac PA1 guard column (4 mm x 50 mm) (Thermo Fisher Scientific) at 30°C with the eluent flow rate fixed at 1 mL / min. Elution of neutral monosaccharides was achieved in 14 min using 10 mM NaOH as the eluent. In sequence, the eluent concentration was increased to 500 mM and the acidic monosaccharides were resolved in 7.5 min. The column was re-equilibrated with 10 mM NaOH for 10.5 min before the subsequent samples were injected. The concentrations of monosaccharides were calculated based on standard curves of fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid.

[0591] The experiments demonstrated that when RGL_1 was combined with GH53, polysaccharides from RSM were solubilized by approximately 44% more compared to the sum of the effects of the two enzymes separately, illustrating the synergistic effect between the two enzyme products. The combination of the two enzymes resulted in 3.9% solubilization of insoluble NSPs, while RGL_1 and GH53 resulted in 0.5% and 2.2% solubilization of insoluble NSPs, respectively. The main monosaccharides present in the solubilized NSPs were rhamnose, galacturonic acid, arabinose, galactose, and xylose.

[0592] Table 7. Mean values ​​of the amount of rhamnose, arabinose, galactose, xylose, galacturonic acid and the total amount of monosaccharides after treatment without enzyme addition (control), with addition of 20 ppm of RG-I lyase (RGL_1), with addition of 20 ppm of galactanase (GH53), and with a combination of both enzymes (20 ppm each). The values ​​are given as % of monosaccharides in rapeseed meal (RSM).

[0593]

[0594] Example 8: rhamnogalacturonan endolyase, endo-β-1,4-galactanase, xyloglucanase and Effect of its combination with pectin lyase on rapeseed meal

[0595] The effects of two rhamnogalacturonan endolyases (RGL_1, one from Aspergillus aculeatus and one from Penicillium oxalicum), endo-β-1,4-galactanase (GH53), endo-xyloglucanase (GH5_4) and their combination with pectin lyase (LYA1_4) applied in animal feed on deproteinized rapeseed meal (RSM) were investigated in vitro.

[0596] By protease RSM samples obtained from DSM Nutritional Products (Villageneve, France) were deproteinized by double incubation with 1,2-dihydro-1,4 ... After the second incubation together, the enzyme was inactivated for 15 min at 80° C. After centrifugation at 3,000 rpm for 15 min at 0° C., the ethanol from the precipitate was evaporated overnight in a fume hood and the sample was freeze-dried.

[0597] Deproteinized RSM (3% dry matter) was incubated with rhamnogalacturonan endolyase (RGL_1), endo-β-1,4-galactanase (GH53), xyloglucanase, pectate lyase and their combination in acetate buffer at pH 5.0 for 4 h at 40° C. The experiments were run in triplicate.

[0598] After incubation with the enzyme, the solids were removed by centrifugation at 4,700 g and 0° C. for 15 min, and 1,000 μL of the supernatant was subjected to acid hydrolysis (0.5 MH 2 SO 4 The hydrolyzate was filtered through a 0.2 μm wwPTFE membrane (AcroPrep Advance 8582, PALL) and analyzed for its monosaccharide content by high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

[0599] Monosaccharide separation was achieved on a CarboPac Analytical PA1 column (4 mm x 250 mm) with a CarboPac PA1 guard column (4 mm x 50 mm) (Thermo Fisher Scientific) at 30°C with the eluent flow rate fixed at 1 mL / min. Elution of neutral monosaccharides was achieved in 14 min using 10 mM NaOH as the eluent. In sequence, the eluent concentration was increased to 500 mM and the acidic monosaccharides were resolved in 7.5 min. The column was re-equilibrated with 10 mM NaOH for 10.5 min before the subsequent samples were injected. The concentrations of monosaccharides were calculated based on standard curves of fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid.

[0600] The experiments demonstrated that combining pectin lyase with one of the other enzymes doubled the amount of total monosaccharides in the hydrolysate of the soluble fraction of RSM compared to the control.

[0601] When xyloglucanase was combined with pectin lyase, the total amount of monosaccharides (mg) in the soluble fraction of 100 mg of RSM increased by more than 5 percentage points (pp) compared to the sum of the effects of the two enzymes separately, indicating a synergistic effect between the two enzymes. The combination of xyloglucanase and pectin lyase improved the solubilization of polysaccharides containing fucose, rhamnose, arabinose, galactose, glucose, xylose and galacturonic acid. For polysaccharides containing arabinose, galactose, xylose and galacturonic acid, major improvements in the percentage of soluble monosaccharides in the soluble fraction of RSM treated with the combined enzymes were observed, i.e. increases of 1.5 pp, 0.8 pp, 0.6 pp and 0.3 pp, as compared to the sum of the cases treated with the two enzymes separately.

[0602] When galactanase was combined with pectin lyase, the total amount of monosaccharides (mg) in the soluble fraction of 100 mg of RSM increased by approximately 3 pp compared to the sum of the effects of the two enzymes separately, illustrating a synergistic effect between the two enzymes. The combination of galactanase and pectin lyase improved the solubilization of polysaccharides containing fucose, rhamnose, arabinose, galactose, glucose, xylose and galacturonic acid. For polysaccharides containing arabinose, galactose, xylose and galacturonic acid, major improvements were observed in the percentage of soluble monosaccharides in the soluble fraction of RSM treated with the combined enzymes, i.e. increases of 1.1 pp, 0.2 pp, 0.3 pp and 0.3 pp, respectively, as compared to the sum of treatments with the two enzymes separately.

[0603] When RG-I lyase (A. aculeatus) was combined with pectin lyase, the total amount of monosaccharides (mg) in the soluble fraction of 100 mg of RSM increased by more than 2 pp compared to the sum of the effects of the two enzymes separately, indicating a synergistic effect between the two enzymes. The combination of RG-I lyase (A. aculeatus) and pectin lyase improved the solubilization of polysaccharides containing fucose, rhamnose, arabinose, galactose, glucose, xylose and galacturonic acid. For polysaccharides containing arabinose, galactose, xylose and galacturonic acid, major improvements were observed in the percentage of soluble monosaccharides in the soluble fraction of RSM treated with the combined enzymes, i.e. increases of 0.9 pp, 0.4 pp, 0.2 pp and 0.1 pp, as compared to the sum of treatments with the two enzymes separately.

[0604] When RG-I lyase (Penicillium oxalicum) was combined with pectin lyase, the total amount of monosaccharides (mg) in the soluble fraction of 100 mg of RSM increased by approximately 5 pp compared to the sum of the effects of the two enzymes separately, indicating a synergistic effect between the two enzymes. The combination of RG-I lyase (Penicillium oxalicum) and pectin lyase improved the solubilization of polysaccharides containing fucose, rhamnose, arabinose, galactose, glucose, xylose and galacturonic acid. For polysaccharides containing arabinose, galactose, xylose and galacturonic acid, major improvements were observed in the percentage of soluble monosaccharides in the soluble fraction of RSM treated with the combined enzymes, i.e. increases of 1.6 pp, 0.7 pp, 0.4 pp and 0.4 pp, respectively, compared to the sum of treatments with the two enzymes separately.

[0605] Table 8. Mean values ​​of the amount of fucose, rhamnose, arabinose, galactose, xylose, galacturonic acid and the total amount of monosaccharides after treatment without addition of enzymes (control), addition of 20 ppm pectin lyase (LYA1_4), addition of 20 ppm xyloglucanase (GH5_4), galactanase (GH53), RG-1 lyase from Aspergillus aculeatus and from Penicillium oxalicum (RGL_1) and their combination with pectin lyase (20 ppm each). The values ​​are given as % of monosaccharides in rapeseed meal (RSM).

[0606]

[0607]

[0608] Example 9: Endo-rhamnogalacturonan lyase, endo-β-1,4-galactanase and their interactions with pectin cleavage Effects of a combination of hydrolytic enzymes on short-chain fatty acid accumulation in chicken cecal microbiota during in vitro fermentation of rapeseed meal

[0609] The effects of two rhamnogalacturonan endolyases (RGL_1, one from Aspergillus aculeatus and one from Penicillium oxalicum), an endo-β-1,4-galactanase (GH53) and their combination with a pectin lyase (LYA1_4), applied in animal feed, on deproteinized rapeseed meal (RSM) were investigated in vitro.

[0610] By protease RSM samples obtained from DSM Nutritional Products (Villageneve, France) were deproteinized by double incubation with 1,2-dihydro-1,4 ... After the second incubation together, the enzyme was inactivated for 15 min at 80° C. After centrifugation at 3,000 rpm for 15 min at 0° C., the ethanol from the precipitate was evaporated overnight in a fume hood and the sample was freeze-dried.

[0611] Deproteinized RSM (3% dry matter) was diluted in 5% paraformaldehyde as described by Duncan et al. (Duncan, SH, Hold, GL, Barcenilla, A., Stewart, CS & Flint, HJ Roseburia intestinalis sp. nov., a novel saccharolytic, butyrate-producing bacterium from human faeces [Roseburia intestinalis sp. nov.] with and without endo-rhamnogalacturonan lyase (20 ppm), endo-β-1,4-galactanase (20 ppm) and their combination with pectin lyase (20 ppm). intestinalis) new species, a new saccharolytic, butyrate-producing bacterium from human feces]. Int. J. Syst. Evol. Microbiol. [International Journal of Systematic and Evolutionary Microbiology] 52, 1615-1620 (2002)) in anoxic sterile YCFA medium prepared as described, which was modified so that the carbon source was 1% non-starch polysaccharides from deproteinized RSM and the pH was adjusted to 6.5. The cecal contents pool from four 35-day-old broilers was added to the final 1,000-fold dilution for the first round of fermentation. After fermentation at 37°C for 12 hours, the first fermentation was used as an inoculum for the second fermentation, and so on. The fermentation supernatant was sampled after 12 hours and stored at -20°C until analysis. The fermentation was run in five replicates.

[0612] Quantification of acetate, propionate, and butyrate in the supernatant was achieved by running the samples in a HPLC apparatus equipped with an ion exchange BioRad HPX-87H column (with a BioRad cation H precolumn, 60°C) and a refractive index detector.

[0613] Experiments have shown that treatment with rhamnogalacturonan endolyase (Aspergillus aculeatus), rhamnogalacturonan endolyase (Penicillium oxalicum), galactanase and pectin lyase, respectively, increased the butyrate accumulation of RSM caused by fermentation of chicken cecal microbiota from 2.34 mM to 4.71 mM, 2.99 mM, 7.09 mM and 5.4 mM. The combination of rhamnogalacturonan endolyase (Aspergillus aculeatus) and pectin lyase further increased the accumulation of butyrate to 7.91 mM. The combination of rhamnogalacturonan endolyase (Penicillium oxalicum) and pectin lyase further increased the accumulation of butyrate to 8.64 mM. The combination of galactanase and pectin lyase further increased the accumulation of butyrate to 9.63 mM.

[0614] Experiments have shown that treatment with rhamnogalacturonan endolyase (Aspergillus aculeatus), rhamnogalacturonan endolyase (Penicillium oxalicum), galactanase and pectin lyase, respectively, increased the acetate accumulation of RSM caused by fermentation of chicken cecal microbiota from 71.8 mM to 86.1 mM, 85.2 mM, 88.3 mM and 81.8 mM, respectively. The combination of rhamnogalacturonan endolyase (Aspergillus aculeatus) and pectin lyase further increased the accumulation of acetate to 91.3 mM. The combination of rhamnogalacturonan endolyase (Penicillium oxalicum) and pectin lyase further increased the accumulation of acetate to 94.6 mM. The combination of galactanase and pectin lyase further increased the accumulation of acetate to 96.9 mM.

[0615] Experiments have shown that treatment with rhamnogalacturonan endolyase (Aspergillus aculeatus), rhamnogalacturonan endolyase (Penicillium oxalicum), galactanase and pectin lyase, respectively, increased the accumulation of propionate in RSM caused by fermentation of chicken cecal microbiota from 15.3 mM to 19.8 mM, 17.5 mM, 17.5 mM and 20.5 mM, respectively. The combination of rhamnogalacturonan endolyase (Aspergillus aculeatus) and pectin lyase further increased the accumulation of propionate to 24.3 mM. The combination of rhamnogalacturonan endolyase (Penicillium oxalicum) and pectin lyase further increased the accumulation of propionate to 27.9 mM. The combination of galactanase and pectin lyase further increased the accumulation of propionate to 26.1 mM.

[0616] Table 9. Mean values ​​of the amounts of acetate, butyrate and propionate after four cycles of 12-h fermentation of RSM by chicken cecal microbiota with no enzyme addition (control), 20 ppm RG-I lyase (RGL_1) from Aspergillus aculeatus, 20 ppm RG-I lyase (RGL_1) from Aspergillus aculeatus, 20 ppm galactanase (GH53), 20 ppm pectin lyase and a combination of pectin lyase and three other enzymes (20 ppm each). The values ​​are given in mM.

[0617] Enzyme treatment Acetate Butyrate Propionate Comparison 71,77 2,34 15,27 RG-I lyase (Aspergillus aculeatus) 86,11 4,71 19,85 Galactanase 88,28 7,09 17,55 Pectin lyase 81,77 5,40 20,55 RG-I lyase (Penicillium oxalicum) 85,19 2,99 17,45 RG-I lyase (Aspergillus aculeatus) + pectin lyase 91,34 7,91 24,28 Galactanase + pectin lyase 96,89 9,63 26,09 RG-I lyase (Penicillium oxalicum) + pectin lyase 94,56 8,64 27,90

[0618] Example 10: Endo-β-1,4-galactanase and combination with rhamnogalacturonan endolyase Effects on the taxonomic distribution of chicken cecal microbiota after in vitro fermentation of rapeseed meal

[0619] The effects of endo-rhamnogalacturonan lyase (RGL_1, Aspergillus aculeatus) and endo-β-1,4-galactanase (GH53), both used in animal feed, on deproteinized rapeseed meal (RSM) were investigated in vitro.

[0620] By protease RSM samples obtained from DSM Nutritional Products (Villageneve, France) were deproteinized by double incubation with 1,2-dihydro-1,4 ... After the second incubation together, the enzyme was inactivated for 15 min at 80° C. After centrifugation at 3,000 rpm for 15 min at 0° C., the ethanol from the precipitate was evaporated overnight in a fume hood and the sample was freeze-dried.

[0621] Deproteinized RSM (3% dry matter) was diluted in 5% dm as described by Duncan et al. (Duncan, SH, Hold, GL, Barcenilla, A., Stewart, CS & Flint, HJ Roseburia intestinalis sp. nov., a novel saccharolytic, butyrate-producing bacterium from human faeces [Roseburia intestinalis sp. nov.] without any enzyme, with endo-β-1,4-galactanase (20 ppm) and with a combination of endo-β-1,4-galactanase (20 ppm) and rhamnogalacturonan endolyase. intestinalis) new species, a new glycolytic, butyrate-producing bacterium from human feces]. Int. J. Syst. Evol. Microbiol. [International Journal of Systematic and Evolutionary Microbiology] 52, 1615-1620 (2002)) in anoxic sterile YCFA medium prepared as described, the medium was modified so that the carbon source was 1% non-starch polysaccharides from deproteinized RSM, and the pH was adjusted to 6.5. Chicken cecal contents from 35-day-old broilers were added to the final 1,000-fold dilution for the first round of fermentation. Fermentations were run in five replicates. The fermentation was sampled after 12 hours of fermentation at 37°C and stored at -20°C until analysis. DNA was extracted according to the DNeasy Ultra Clean Microbial Kit (Qiagen, January 2020) protocol.

[0622] For library preparation, 16S rRNA gene amplicons targeting the V3-V4 region of 16S were prepared for sequencing in the Illumina MiSeq system according to the protocol provided by Illumina (Supporting Document, Section 15044223, Revision B).

[0623] This experiment demonstrated that in the presence of enzymes, the abundance of Enterococci and Escherichia / Shigella decreased in the fermentation of RSM by chicken cecal microbiota, both in the presence of galactanase (20 ppm) and in combination with RG-I lyase (20 ppm).

[0624] The enzyme treatment favored the growth of Bacteroides, a genus containing bacteria known to degrade pectin, which in a cross-feeding manner favored the growth of butyrate producers. In the presence of galactanase, the relative abundance of Bacteroides increased by 5% compared to the control treatment, and increased by 10% when combined with RG-I lyase.

[0625] The relative abundance of Propionibacterium (a genus well known for being propionic acid producers) doubled when RG-I lyase was added to galactanase compared to galactanase alone.

[0626] When RSM was treated with galactanase, the relative abundance of Lactobacillus, the host of the most common probiotics, increased from 1.9% to 2.7%, and when RG-I lyase was combined with galactanase, the relative abundance of Lactobacillus increased further to 4.5%.

[0627] In the presence of galactanase, the relative abundance of the genus Butyricoccus increased by 40% compared to the control treatment and almost tripled when combined with the RG-I lyase. This genus contains butyrate producers.

[0628] Table 10. Relative abundance of the top ten bacteria identified using sequencing of the V3-V4 region of 16S rRNA after fermentation of RSM by chicken cecal microbiota without enzyme addition (control), with 20 ppm of galactanase (GH53), and in combination with 20 ppm of RG-I lyase (RGL_1). The values ​​are given in percentage.

[0629]

[0630]

[0631] Example 11: Animal feed and feed additive composition

[0632] A formulation of fiber degrading enzymes containing 0.050 g of enzyme protein was added to the following premix (per kg premix):

[0633]

[0634] Animal Feed

[0635] This is an example of an animal feed (broiler feed) containing 0.5 mg / kg (0.5 ppm) of fiber degrading enzymes (calculated as pectinase protein):

[0636] 65.00% wheat

[0637] 32.35% soybean meal (50% crude protein, CP)

[0638] 1.0% soybean oil

[0639] 0.2% DL-Methionine

[0640] 0.22% DCP (Dicalcium Phosphate)

[0641] 0.76% CaCO3 (Calcium Carbonate)

[0642] 0.32% sand

[0643] 0.15% NaCl (sodium chloride)

[0644] 1% of the above premix

[0645] The ingredients are mixed and the feed is pelletized at the desired temperature (eg, 70°C).

[0646] Example 12: GH53 galactanase from Cohenella sp.-60555 (SEQ ID NO: 1 with His-tag Cloning of the mature polypeptide of NO:5

[0647] The gene encoding galactanase is amplified by PCR and fused to regulatory elements, affinity purification tags and homology regions to be recombined into the Bacillus subtilis genome. The linear integration construct is a SOE-PCR fusion product (Horton, RM, Hunt, HD, Ho, SN, Pullen, JK and Pease, LR (1989) Engineering hybrid genes without the use of restriction enzymes, gene splicing by overlap extension [engineering hybrid genes in the case of gene splicing without using restriction enzymes, by overlapping extension] Gene [Gene] 77: 61-68), which is formed by fusion of genes between two Bacillus subtilis chromosome regions with a strong promoter and a chloramphenicol resistance marker. The SOE PCR method is also described in patent application WO 2003095658.

[0648] The gene was expressed under the control of a triple promoter system (as described in WO 99 / 43835) consisting of the Bacillus licheniformis alpha-amylase gene (amyL) promoter including a stabilizing sequence, the Bacillus amyloliquefaciens alpha-amylase gene (amyQ) promoter, and the Bacillus thuringiensis cryIIIA promoter.

[0649] The gene was expressed using a Bacillus clausii secretion signal (encoding the following amino acid sequence: MKKPLGKIVASTALLISVAFSSSIASA, SEQ ID NO: 11) instead of the native secretion signal. In addition, the expression construct resulted in the addition of an amino-terminal polyhistidine purification tag to the native mature protein, allowing the enzyme to be purified by immobilized metal ion affinity chromatography.

[0650] The SOE-PCR product was transformed into Bacillus subtilis and integrated into the pectate lyase site in the chromosome by homologous recombination. Subsequently, a recombinant Bacillus subtilis clone containing the corresponding galactanase expression construct was selected and cultured on a rotary shaker in 500 ml baffled conical flasks, each containing 100 ml of starch-rich medium. After a 3-5 day culture period at 30° C. to 37° C., the supernatant containing the enzyme was harvested by centrifugation and the enzyme was purified by immobilized metal affinity chromatography.

[0651] Example 13: GH53 galactanase from Cohenella sp.-60555 (SEQ ID NO: 1 with His-tag Purification of the mature polypeptide of NO:5

[0652] The pH of the supernatant from Example 12 was adjusted to pH 8, filtered through a 0.2 μM filter, and then applied to 5 ml of HisTrap TM The protein was loaded onto a HiPrep excel column (GE Healthcare Life Sciences, Pittsburgh, USA). Prior to loading, the column had been equilibrated in 5 column volumes (CV) of 50 mM Tris / HCl pH 8. To remove unbound material, the column was washed with 8 CV of 50 mM Tris / HCl pH 8, and elution of the target was obtained with 50 mM HEPES pH 7 + 10 mM imidazole. The eluted protein was washed with a HiPrep TM Desalting was performed on a 26 / 10 desalting column (GE Healthcare Life Sciences, Pittsburgh, USA) balanced with 3CV of 50 mM HEPES pH 7 + 100 mM NaCl. This buffer was also used for elution of the target, and the flow rate was 10 ml / min. Relevant fractions were selected and merged based on chromatogram and SDS-PAGE analysis.

[0653] Galactanase assay

[0654] Galactanase activity can be determined using a reducing end colorimetric assay. A 10% soybean meal substrate (prepared from soybean meal ground to a particle size of 0.5 mm) was filled into a 96-well format plate using a solid dispenser. The weight was measured before and after the addition of soybean meal, and the mass of substrate per well was estimated assuming equal distribution across the plate.

[0655] The enzyme was stirred in 100 mM activity buffer (100 mM acetate, 100 mM MES, 100 mM glycine in 0.01% Triton X100, 1 mM CaCl 2, pH 6.5) was diluted to 0.6ppm (final enzyme concentration in solution), and the sample was vibrated at 40°C for 2 hours. The sample was centrifuged at 3000xg for 5 minutes, and 75 μl of each sample (supernatant) was transferred to a new PCR plate. In each sample, 75 μl of active buffer was added, the sample was mixed, and then 75 μl of stop solution (15mg / mlPAHBAH (Sigma H-9882) was added, in Ka-Na-tartrate / NaOH solution, pH>10). The solution was mixed at 95°C for 10min, then at 10°C for 1min, and the sample was transferred to a new 96MTP and absorbance was measured at 405nm.

[0656] The invention described and claimed herein is not limited to the scope of the specific aspects disclosed herein, as these aspects are intended to serve as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of the invention. In fact, various modifications of the invention, in addition to those shown and described herein, will become clear to those skilled in the art as a result of the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the event of a conflict, the present disclosure including definitions shall prevail.

Claims

1. A method of improving the nutritional value of an animal feed comprising oilseed material, the method comprising adding a fiber degrading enzyme to the animal feed.

2. A method for improving the growth performance of an animal, the method comprising administering to the animal a fiber degrading enzyme, an animal feed comprising a fiber degrading enzyme, or an animal feed additive; preferably, the growth performance is growth rate, feed conversion rate and / or body weight gain.

3. A method for the in situ production of prebiotics in an oilseed based animal feed, the method comprising adding a fiber degrading enzyme to the animal feed.

4. A method of improving the intestinal health of a monogastric animal, the method comprising administering to the animal an oilseed based animal feed, wherein the animal feed comprises a fiber degrading enzyme.

5. An animal feed comprising a fiber degrading enzyme and an oilseed material, wherein the feed comprises the oilseed material in an amount of 10 to 500 g / kg feed and the fiber degrading enzyme in an amount of 0.1 to 500 mg enzyme protein / kg feed.

6. An animal feed additive comprising a fiber degrading enzyme and one or more additional components selected from the group consisting of: one or more vitamins; one or more minerals; one or more amino acids; one or more phytobiotics; one or more prebiotics; one or more organic acids; and one or more other feed ingredients.

7. The method according to any one of claims 1 to 4, the animal feed according to claim 5, the animal feed additive according to claim 6, wherein the fiber degrading enzyme is selected from the group consisting of: pectinase, xyloglucan-specific endo-1,4-β-glucanase, xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase and combinations thereof; preferably, the fiber degrading enzyme is pectinase; more preferably, the fiber degrading enzyme is one or more pectinases selected from the group consisting of: rhamnogalacturonan lyase (α-L-rhamnopyranosyl-1,4-α-D-galacturonan pyranose ester endo-lyase, EC 4.2.2.23), endo-β-1,4-galactanase (EC 3.2.1.89), polygalacturonase (1,4-α-D-galacturonan polysaccharide hydrolase, EC 3.2.1.15 and EC 3.2.1.67), rhamnogalacturonase (rhamnogalacturonan α-D-galacturonan-1,2-α-L-rhamnosyl hydrolase, EC 3.2.1.171), pectin methylesterase (pectin pectyl hydrolase, EC 3.1.1.11), pectin lyase ((1,4)-6-O-methyl-α-D-galacturonan lyase, EC 4.2.2.10), pectin acetylesterase (acetate acetylhydrolase, EC 3.1.1.6), galactan endo-β-1,3-galactanase (EC 3.2.1.181), xylogalacturonase and combinations thereof; most preferably, the fiber degrading enzyme is selected from the group consisting of: a combination of rhamnogalacturonan lyase and endo-β-1,4-galactanase; a combination of rhamnogalacturonan lyase and pectin lyase; a combination of endo-β-1,4-galactanase and pectin lyase; and a combination of xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase and pectin lyase.

8. The method according to any one of claims 1 to 4, the animal feed according to claim 5, the animal feed additive according to claim 6, wherein the fiber degrading enzyme is selected from the group consisting of: (a) a rhamnogalacturonan lyase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 or the mature polypeptide of SEQ ID NO: 1; (b) a rhamnogalacturonan lyase derived from SEQ ID NO: 1 or the mature polypeptide of SEQ ID NO: 1 by having 1-30 alterations, e.g. substitutions, deletions and / or insertions, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions; (c) a rhamnogalacturonan lyase derived from (a) or (b), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, such as 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; (d) A fragment of the rhamnogalacturonan lyase of (a), (b) or (c), wherein the fragment has rhamnogalacturonan lyase activity; (e) an endo-polygalacturonase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO: 2; (f) an endo-polygalacturonase derived from SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO: 2 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions; (g) an endo-polygalacturonase derived from (e) or (f), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; (h) a fragment of the endo-polygalacturonase of (e), (f) or (g), wherein the fragment has endo-polygalacturonase activity; (i) has at least 60% similarity to SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO: 3, for example, a xyloglucan-specific endo-1,4-beta-glucanase with at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; (j) a xyloglucan-specific endo-1,4-beta-glucanase derived from SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO: 3 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions; (k) a xyloglucan-specific endo-1,4-β-glucanase derived from (i) or (j), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; (l) A fragment of the xyloglucan-specific endo-1,4-β-glucanase of (i), (j) or (k), wherein the fragment has xyloglucan-specific endo-1,4-β-glucanase activity; (m) a xyloglucan-specific endo-1,4-β-glucanase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:4 or the mature polypeptide of SEQ ID NO:4; (n) a xyloglucan-specific endo-1,4-beta-glucanase derived from SEQ ID NO: 4 or the mature polypeptide of SEQ ID NO: 4 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions; (o) a xyloglucan-specific endo-1,4-β-glucanase derived from (m) or (n), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and A fragment of the xyloglucan-specific endo-1,4-β-glucanase of (p), (m), (n) or (o), wherein the fragment has xyloglucan-specific endo-1,4-β-glucanase activity; (q) a galactanase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:5 or the mature polypeptide of SEQ ID NO:5; (r) a galactanase derived from SEQ ID NO: 5 or the mature polypeptide of SEQ ID NO: 5 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, particularly substitutions, at one or more positions; (s) a galactanase derived from (q) or (r), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; (t) a fragment of the galactanase of (q), (r) or (s), wherein the fragment has galactanase activity; (u) a xylogalacturonase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:6 or the mature polypeptide of SEQ ID NO:6; (v) a xylogalacturonase derived from SEQ ID NO: 6 or the mature polypeptide of SEQ ID NO: 6 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions; (w) a xylogalacturonase derived from (u) or (v), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; (x) a fragment of the xylose galacturonase of (u), (v) or (w), wherein the fragment has xylose galacturonase activity; (y) a xylogalacturonase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:7 or the mature polypeptide of SEQ ID NO:7; (z) a xylogalacturonase derived from SEQ ID NO: 7 or the mature polypeptide of SEQ ID NO: 7 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions; (aa) a xylogalacturonase derived from (y) or (z), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; A fragment of the xylose galacturonase of (bb)(y), (z) or (aa), wherein the fragment has xylose galacturonase activity; (cc) a pectin lyase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:8 or the mature polypeptide of SEQ ID NO:8; (dd) a pectin lyase derived from SEQ ID NO: 8 or the mature polypeptide of SEQ ID NO: 8 by having 1 to 30 alterations, e.g. substitutions, deletions and / or insertions, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions; (ee) a pectin lyase derived from (cc) or (dd), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; (ff) (cc), (dd) or (ee) a fragment of the pectin lyase, wherein the fragment has pectin lyase activity; (gg) a rhamnogalacturonan lyase having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:9 or the mature polypeptide of SEQ ID NO:9; (hh) a rhamnogalacturonan lyase derived from SEQ ID NO: 9 or the mature polypeptide of SEQ ID NO: 9 by having 1 to 30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions; (ii) a rhamnogalacturonan lyase derived from (gg) or (hh), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, such as 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and (jj) A fragment of the rhamnogalacturonan lyase of (gg), (hh) or (ii), wherein the fragment has rhamnogalacturonan lyase activity; (kk) has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114 At least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, Xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase with at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity; (ll) a xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase derived from SEQ ID NO: 10 or the mature polypeptide of SEQ ID NO: 10 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions; (mm) a xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase derived from (kk) or (ll), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and A fragment of a xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase of (nn)(kk), (ll) or (mm), wherein the fragment has xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase activity.

9. Use of a combination of a polypeptide having rhamnogalacturonan lyase activity and a polypeptide having galactanase activity in animal feed or an animal feed additive; or use of a combination of rhamnogalacturonan lyase and pectin lyase in animal feed or an animal feed additive; or use of a combination of endo-β-1,4-galactanase and pectin lyase in animal feed or an animal feed additive; or use of a combination of xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase and pectin lyase in animal feed or an animal feed additive.

10. An animal feed additive comprising a polypeptide having rhamnogalacturonan lyase activity and a polypeptide having galactanase activity; or an animal feed additive comprising: a combination of rhamnogalacturonan lyase and endo-β-1,4-galactanase; a combination of rhamnogalacturonan lyase and pectin lyase; a combination of endo-β-1,4-galactanase and pectin lyase; or a combination of xyloglucan-specific endo-β-1,4-glucanase / endo-xyloglucanase and pectin lyase.

11. An animal feed comprising the animal feed additive according to claim 10, and oilseed material.

12. A polypeptide having xyloglucan-specific endo-1,4-β-glucanase activity, wherein the polypeptide is selected from the group consisting of: (a) a polypeptide having at least 99.7%, at least 99.8%, at least 99.9% or 100% sequence identity to SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO: 3; (b) a polypeptide derived from SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO: 3 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions; (c) a polypeptide having at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:4 or the mature polypeptide of SEQ ID NO:4; (d) a polypeptide derived from SEQ ID NO: 4 or the mature polypeptide of SEQ ID NO: 4 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions; (e) a polypeptide derived from the polypeptide of (a), (b), (c) or (d), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and (f) a fragment of the polypeptide of (a), (b), (c), (d) or (e); The polypeptide has xyloglucan-specific endo-1,4-β-glucanase activity.

13. A polypeptide having xylose galacturonase activity, wherein the polypeptide is selected from the group consisting of: (a) a polypeptide having at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:6 or the mature polypeptide of SEQ ID NO:6; (b) a polypeptide derived from SEQ ID NO:6 or the mature polypeptide of SEQ ID NO:6 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions; (c) a polypeptide having at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:7 or the mature polypeptide of SEQ ID NO:7; (d) a polypeptide derived from SEQ ID NO: 7 or the mature polypeptide of SEQ ID NO: 7 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions; (e) a polypeptide derived from the polypeptide of (a), (b), (c) or (d), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and (f) a fragment of the polypeptide of (a), (b), (c), (d) or (e); The polypeptide has xylose galacturonase activity.

14. A polypeptide having rhamnogalacturonan lyase activity, wherein the polypeptide is selected from the group consisting of: (a) a polypeptide having at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:9 or the mature polypeptide of SEQ ID NO:9; (b) a polypeptide derived from SEQ ID NO:9 or the mature polypeptide of SEQ ID NO:9 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions; (c) a polypeptide derived from the polypeptide of (a) or (b) wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and (d) a fragment of the polypeptide of (a), (b) or (c); The polypeptide has rhamnogalacturonan lyase activity.

15. A polypeptide having xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase activity, the polypeptide being selected from the group consisting of: (a) a polypeptide having at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10 or the mature polypeptide of SEQ ID NO: 10; (b) a polypeptide derived from SEQ ID NO: 10 or the mature polypeptide of SEQ ID NO: 10 by having 1-30 alterations, e.g., substitutions, deletions and / or insertions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions, at one or more positions; (c) a polypeptide derived from the polypeptide of (a) or (b) wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids, e.g. 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids; and (d) a fragment of the polypeptide of (a), (b) or (c); The polypeptide has xyloglucan-specific endo-b-1,4-glucanase / endo-xyloglucanase activity.

16. A polynucleotide encoding the polypeptide according to any one of claims 12-15.

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