CN116615110A

CN116615110A - Protease animal feed formulations

Info

Publication number: CN116615110A
Application number: CN202280007741.5A
Authority: CN
Inventors: F·博勒普
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2021-12-16
Filing date: 2022-12-08
Publication date: 2023-08-18

Abstract

The application relates to a method for processing a silicon wafer

Description

Protease animal feed formulations

Reference to sequence Listing

The application comprises a sequence listing in computer readable form. The computer readable form is incorporated herein by reference.

Technical Field

The present application relates to novel pellet formulations for proteases for use in animal feed.

Background

The prospect of climate change is associated with losing fertile farms and thus reducing agricultural yield, forcing producers to find more efficient ways of food production. On the other hand, the price of traditional feed commodities steadily rises, and the purchase price of meat is not in a stagnation state, which means that farmers are continually searching for ways for reducing the production cost of the feed commodities.

Pelletization was introduced into the feed industry in the middle of the 20 th century, 20 s, to improve feed utilization and improve handling characteristics. Early pelleting processes involved mixing the feed ingredients and pelleting them without further treatment. The principle of the method is to prevent the change of vitamins and proteins due to the addition of heat to the feed mixture. Since the 60 s of the 20 th century, the focus of research on pelletization processes has been on improving conditioning operations, focusing on increasing the residence time and temperature to which conditioning pastes are to be subjected.

Animal feeds comprising enzymes are known to have a number of advantages (depending on the enzyme used). Typically, animal feeds are found in one of two forms: a pasty feed consisting of all dietary components mixed together, or a pelleted feed in which the different dietary components are compressed into pellets of approximately the same size. Pelleted feed is generally advantageous for a number of reasons, such as availability of all desired ingredients and ease of storage and handling.

The feed pellets may include one or more enzymes and are typically produced by mixing granules containing the active ingredient (e.g., enzyme) with other ingredients (e.g., cereal and nutrients) and then conditioning and processing the mixture into pellets. It is important that the nutrients and enzymes are evenly distributed in the feed to ensure that all animals get an optimal blend of nutrients and enzymes via the feed.

During the conditioning and pelletization process, the temperature rises, in some cases high temperatures can be reached. Furthermore, the high temperatures during the conditioning and pelleting process may negatively affect the stability of the enzyme and thus its activity. The formulation of enzymes prior to pelleting is an intelligent selection process in which the formulation ensures enzymatic activity after pelleting process, transport and long term storage.

Granulation of enzymes is a difficult task. Despite the fact that there are numerous patent applications concerning different processes for producing particulate non-dusting enzymes, the different granulation processes used today in industrial scale hardly exceed two or three. The most common of these methods are: the enzyme is embedded in spheres of waxy material by a so-called pelletisation process (see german DOS 2,060,095) and the process described in uk patent specification 1,362,365, in which the enzyme is mixed with filler, binder and water, and then extruded and rounded. Enzyme granules with an extremely low dust content can be produced by both methods. However, both of these methods have some drawbacks. In the pelletization process, at least about 50% of the product must be a waxy material, such as an ethoxylated fatty alcohol, which is quite expensive. The other methods described above have the disadvantage of being difficult to produce on an industrial scale.

Commercially available productsProAct is available in two forms: a thermally stable free flowing dust free CT formulation or liquid form (L) for post-pelletization liquid applications. The formulation involves a core (in which the enzyme is absorbed) and an additional coating to ensure stability of the expensive enzyme during the pelleting process, transportation and long-term storage. ProAct is the best performing product in animal feed. Both formulations were intended to be mixed into the premix and/or feed material to obtain a minimum enzyme activity level of 15 000prot/kg in the finishing chicken feed material. The CT formulation is in a particulate coated heat-resistant form, at least to some extent, such that +.>Proact becomes the most stable feed protease. />The procact is stable throughout the intestinal tract, supplementing the performance of other feed enzymes (e.g., carbohydrases and phytases). />The procact has excellent stability in all feed applications, including premix and pellet feeds. The dust-free formulation ensures that no safety problems exist when incorporated into feed.

Proteases are widely used in a variety of applications including cleaning agents, textiles, baking and animal feeds. These applications generally benefit from enzymes that are protected from moisture, temperature, and irritant chemicals. Thus, the enzyme is typically in particulate form and coated with one or more protective coatings.

However, pelleting and coating add significantly to the cost of the enzyme product. It is desirable to provide low cost enzyme granules.

Disclosure of Invention

The invention providesNovel particles or formulations of procact polypeptides and variants thereof. Surprisingly, it was found that- >The polypeptides of procact and variants thereof can be formulated into lower cost and traditionally less robust formulations and maintain about the same level of activity after conditions of exact repetition of pelleting conditions.

The novel granules and formulations of the protease increase the digestibility of the protein and ensure that more amino acids are available to the animal. The amount of nitrogen excreted is reduced. Ultimately, this may increase the chances of using cheaper feed materials, thereby reducing feed costs. Alternatively, the protein content in the diet may be reduced while still maintaining the animal's productivity.

One aspect of the invention relates to an animal feed additive comprising a polypeptide having protease activity, wherein the polypeptide has at least 70% sequence identity to SEQ ID No. 1; characterized in that the enzyme is formulated as a formulation selected from the group consisting of:

i. granules prepared by an extrusion process;

particles prepared by a spray drying process;

particles comprising a salt core (e.g. sodium sulphate or sodium chloride core) and a protease-containing layer; and

granules prepared by a high shear granulation process.

A further aspect of the invention relates to particles comprising a salt core (e.g. sodium sulphate or sodium chloride core) and a protease, typically an acid-stable protease layer, wherein the protease is a polypeptide having protease activity and at least 70% sequence identity to SEQ ID NO. 1. The particles comprising a salt core and a protease-containing layer are typically microparticles.

Still further aspects of the invention relate to particles comprising a polypeptide having protease activity and having at least 70% sequence identity to the polypeptide of SEQ ID NO. 1; the particles are prepared by a spray drying process.

One aspect of the invention relates to a particle comprising a polypeptide having protease activity and having at least 70% sequence identity to the polypeptide of SEQ ID No. 1, said particle being prepared by an extrusion process. A novel formulation of a polypeptide having protease activity and having at least 70% sequence identity to the polypeptide of SEQ ID No. 1 is prepared by extrusion techniques or by extrusion processes. Advantageously, it is relatively inexpensive to prepare and, surprisingly, the polypeptides which can be used as animal feed additives have suitable stability. Unexpectedly, when in useNo significant degree of protein denaturation typically associated with extrusion technology processes was observed with the procact polypeptides.

One aspect of the invention relates to a method comprising extrudingAn animal feed additive that produces an enzyme pellet, wherein the enzyme is a polypeptide having protease activity and at least 70% sequence identity to SEQ ID No. 1. According to one aspect of the invention, the method of the invention comprises (a) combining a polypeptide having protease activity, a solid carrier, optionally water, and a meltable hydrophobic substance to provide a combination product; (b) Optionally applying sufficient heat to the combination product to melt the hydrophobic substance; (c) extruding the product of step (b); and (d) drying and cooling the extruded product of step (c) or actively drying and cooling the extruded product of step (c) to provide a thermostable enzyme product, wherein the polypeptide having protease activity has at least 70% sequence identity with SEQ ID NO. 1, i.e. with The polypeptides of procact have at least 75% sequence identity. A further aspect of the invention relates to a method for preparing an animal feed additive comprising a polypeptide having protease activity and having at least 70% sequence identity with SEQ ID NO. 1, i.e.with>A polypeptide of procact having at least 75% sequence identity, the method comprising an extrusion process comprising extruding a combination comprising said polypeptide, a meltable hydrophobic substance and a solid carrier. In another embodiment, the invention encompasses a method for preparing a thermostable enzyme product for use in animal feed manufacture, the method comprising (a) combining an enzyme, a solid carrier, and a meltable hydrophobic substance to provide a combined product; (b1) Reducing the moisture content by applying heat to the combination product and (b 2) melting the hydrophobic substance; and (c) cooling the combination product to provide a thermostable enzyme product, wherein the thermostable enzyme is a polypeptide having protease activity and at least 70% sequence identity to SEQ ID No. 1.

In yet another embodiment, the invention encompasses a method for preparing a thermostable enzyme product for use in animal feed manufacture, the method comprising (a) combining an enzyme, a solid carrier, and a meltable hydrophobic substance to provide a combined product, and optionally additional water to form a suitable paste; (b) Optionally applying sufficient heat to the combination product to melt the hydrophobic substance; (c) extruding the product of step (b); and (d) drying and cooling the extruded product of step (c) to provide a thermostable enzyme product. In one aspect of this embodiment, the meltable hydrophobic material is added in step (a) as a solid sheet or as a premelted molten liquid. Those skilled in the art will recognize that step (b) may not be required if the fusible hydrophobic substance is added as a premelted molten liquid. The components mentioned in step (a) may be combined in a single step or alternatively in separate steps. For example, the enzyme may be first combined with a solid carrier and optionally water, optionally dried, and then the resulting enzyme/carrier combination combined with a meltable hydrophobic material.

A further aspect of the invention relates to a particle comprising a polypeptide having protease activity and having at least 70% sequence identity to the polypeptide of SEQ ID NO. 1, which particle is prepared by a high shear granulation process. A related aspect of the invention relates to the use of the granulate as defined in the invention in or for the preparation of an animal feed.

A further aspect of the invention relates to a method for preparing a particle comprising a polypeptide having protease activity and having at least 70% sequence identity to the polypeptide of SEQ ID NO. 1, said method comprising a process comprising a formulation process selected from the group consisting of

i. An extrusion process;

ii, spray drying process;

spraying or wetting the salt core with a protease-containing liquid; and

and iv, a high-shear granulation process.

In yet another embodiment, the invention encompasses a method for preparing a thermostable enzyme product for use in animal feed manufacture, the method comprising (a) combining a polypeptide having protease activity and having at least 70% sequence identity to SEQ ID No. 1, a solid carrier and a meltable hydrophobic material to provide a combined product, and optionally additional water to form a suitable paste; (b) Melting the hydrophobic substance, or optionally melting the hydrophobic substance by applying heat to the combination product; (c) extruding the product of step (b); and (d) optionally drying and cooling the extruded product of step (c) to provide a thermostable enzyme product.

In one aspect of this embodiment, the meltable hydrophobic material is added in step (a) as a solid sheet or as a premelted molten liquid. Those skilled in the art will recognize that step (b) may not be required if the fusible hydrophobic substance is added as a premelted molten liquid. The components mentioned in step (a) may be combined in a single step or alternatively in separate steps. For example, the enzyme may be first combined with a solid carrier and optionally water, optionally dried, and then the resulting enzyme/carrier combination combined with a meltable hydrophobic material.

The invention providesNovel formulations of procact polypeptides. The novel formulation is prepared by high shear granulation. Advantageously, it is relatively inexpensive to prepare and, surprisingly, the polypeptides which can be used as animal feed additives have suitable stability.

One aspect of the invention relates to an enzyme particle, the particle being prepared by a method comprising a high shear granulation process, the particle comprising a polypeptide having protease activity, the polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID No. 1. The particles suitably further comprise at least one binder and cellulose or a derivative thereof.

A further aspect of the invention relates to an animal feed additive comprising the enzyme granulate of the invention. A further aspect of the invention relates to an animal feed comprising the enzyme granulate of the invention. A further aspect of the invention relates to an animal feed comprising the animal feed additive of the invention. A further aspect of the invention relates to a method of preparing a granule comprising granules, the method comprising high shear granulation, the high shear granulation process comprising

A. Forming a powder mixture by combining at least the following

i. Cellulose or derivatives thereof

Optionally a binder; and

optionally a filler; and

B. adding liquid phase granulating agent

Wherein the polypeptide having protease activity is added to the powder mixture or to the liquid phase granulator, and wherein the at least one binder is added to the powder mixture or to the liquid phase granulator, or to both; wherein the polypeptide having protease activity is a polypeptide having at least 70% sequence identity to SEQ ID NO. 1,

or wherein the high shear granulation process comprises

A'. Forming a powder mixture by combining at least the following

i. Cellulose or derivatives thereof

ii, an adhesive; and

optionally a filler; and

B'. Adding liquid phase granulating agent

Wherein the polypeptide having protease activity is added to a powder mixture or to a liquid phase granulating agent,

wherein the polypeptide having protease activity is a polypeptide having at least 70% sequence identity to SEQ ID NO. 1.

Detailed Description

One aspect of the invention relates to an animal feed additive comprising a polypeptide having protease activity, wherein the protease comprises a polypeptide having at least 70% sequence identity to SEQ ID No. 1; characterized in that the protease is formulated as a formulation selected from the group consisting of:

i. granules prepared by an extrusion process;

particles prepared by a spray drying process;

granules prepared by a high shear granulation process.

A further aspect of the invention relates to a particle comprising a polypeptide having protease activity, wherein the protease comprises a polypeptide having at least 70% sequence identity to SEQ ID No. 1, characterized in that the particle is selected from the group consisting of: i. a particle comprising a salt core (e.g., sodium sulfate or sodium chloride core) and a protease-containing layer; particles prepared by a spray drying process; granules prepared by an extrusion process; and granules prepared by a high shear granulation process.

Surprisingly, it was found that SEQ ID NO 1 and variants thereof are stable enough to be used as animal feed additives when formulated as particles selected from the group consisting of: microparticles comprising a salt core and a protease-containing layer, particles prepared by a spray drying process, particles prepared by an extrusion process; and granules prepared by a high shear granulation process. Furthermore, it has surprisingly been found that these novel inexpensive SEQ ID NO. 1 particles and variants thereof and commercially available compositions comprising more robust formulationsProAct CT has equivalent stability.

Definition of the definition

Animals: the term "animal" refers to all animals except 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 (pig or swines) (including but not limited to piglets, growing pigs and sows); poultry such as turkeys, ducks, and chickens (including but not limited to broiler chickens and layer chickens); horses (including but not limited to hot, cold and warm-blooded), calves; cobia, cod, small tippler, head porgy, stone head fish eel, goby, goldfish, fillets, fish with feet cobia, cod, small fish, head porgy, head of stone, eel, goby, goldfish, silk foot fish grouper, papaver, halibut, java fish, dace, larix, misgurni anguillicaudati, mackerel, pacific, and Pacific milk fish, silver bass, mud fish, mullet, pergola, pearl spotted fish, peclet, sea bass, dog, pomfret, croaker, salmon, dried shrimp, canadian, sea bass, sea carp, luminescent fish, sleeping shark, snakehead, porgy, saw cover fish, flatfish, spiny foot fish, sturgeon, roll fish, fragrant fish, red sea bream, teleomorpha, tilapia, trout, tuna, ruby, white trout, white spot fish, and white fish); and crustaceans (including but not limited to shrimp and prawns).

Animal feed: the term "animal feed" refers to any compound, formulation or mixture suitable or intended for ingestion by an animal. Animal feed for monogastric animals typically comprises concentrates along with vitamins, minerals, enzymes, direct-fed microorganisms, amino acids and/or other feed ingredients (as in a premix), whereas animal feed for ruminants typically comprises forage grasses (including roughage and silage), and may further comprise concentrates along with vitamins, minerals, enzymes, direct-fed microorganisms, amino acids and/or other feed ingredients (as in a premix).

Weight gain: the term "weight gain" means an increase in the animal's living weight during a given period of time, such as weight gain from day 1 to day 21.

Composition: the term "composition" refers to a composition comprising a carrier and at least one enzyme of the invention. The compositions described herein may be mixed with animal feed and may be referred to as "powdered feed".

Concentrate: the term "concentrate" means a feed with high protein and energy concentration, such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (e.g. whole from corn, oat, rye, barley, wheat or prepared by crushing, milling etc.), oilseed cakes (e.g. from cottonseed, safflower, sunflower, soybean, rapeseed/canola (canola), peanut or groundnut), palm kernel cakes, yeast derived materials and distillers grains (e.g. wet distillers grains (WDS) and distillers dried grains with solubles (DDGS)).

Direct fed microorganisms: the term "direct fed microorganism" means living microorganisms, including spores, which when administered in a suitable amount produce a beneficial effect on a host, such as improving digestion or health.

Effective amount/concentration/dose: the term "effective amount", "effective concentration", or "effective dose" is defined as the amount, concentration, or dose of enzyme sufficient to improve digestion or yield in an animal. The absolute number of actual effective doses depends on factors including: the health of the animal in question, the presence of other components. An "effective amount", "effective concentration", or "effective dose" of an enzyme may be determined by routine assays known to those of skill in the art.

Extrusion (expansion) technology is a modern feed processing technology. Feed processed by extrusion techniques has many desirable and undesirable characteristics such as starch gelatinization and degradation, protein denaturation, reduced anti-nutritional factors, increased palatability, and the like. In extrusion technology, a material having a certain moisture content is fed into a feed extruder under the drive of a screw rod and a screw rod, whereby the material is moved axially forward. The material and screw, material and barrel, and the interior of the material create friction whereby the material is forced to squeeze, agitate and shear, further refining and homogenizing the material as the pressure and temperature in the feed extruder chamber increases and the internal friction between the material and screw, material and barrel increases. With increasing temperature, high pressure and high shear forces, complex physical and chemical changes in the composition of the material occur. Finally, the paste material is ejected from the die orifice, creating a transient pressure differential that expands the material, thereby forming a loose, porous, and brittle extruded product.

Feed conversion rate: the term "feed conversion rate" refers to the amount of feed that is fed to an animal to increase the weight of the animal by a specified amount. Improved feed conversion means lower feed conversion. By "lower feed conversion rate" or "improved feed conversion rate" is meant that the amount of feed required to be fed to an animal in a feed is reduced by the same amount of weight gain of the animal as the amount of feed required to increase the weight of the animal by a specified amount when the feed does not comprise the feed additive composition.

Feed efficiency: the term "feed efficiency" means the amount of weight gain per unit feed when an animal is fed any or a specified amount of food over a period of time. By "increased feed efficiency" it is meant that the use of the feed additive composition according to the invention in a feed results in an increased weight gain per unit feed intake compared to an animal fed a feed in the absence of said feed additive composition.

Forage: the term "forage" as defined herein also includes coarse food grain. Forage is fresh plant material such as hay and silage from forage plants (grasses) and other forage plants (seaweeds, germinated grains, and legumes) or any combination thereof. Examples of forage plants are alfalfa (Alfalfa, lucerne), cranberry, brassica plants (e.g., kale, rapeseed (canola), turnip cabbage (swedish turnip), radish), clover (e.g., clover, red clover, ground clover, white clover), grass (e.g., bermuda grass, brome, false oat grass, festuca, southwest grass (heath grass), prairie grass, duck grass (orcard grass), ryegrass, timothy grass), corn (maize), millet, barley, oat, rye, sorghum, soybean and wheat and vegetables (e.g., sugar beet). Forage further includes crop residues from grain production (e.g., corn stover; straw from wheat, barley, oats, rye, and other grains); residues from vegetables like beet leaf (bean top); residues from oilseed production such as stems and leaves from soybeans, rapeseed and other leguminous crops; and from grain refining for animal or human consumption or from parts of the fuel production or other industries.

Nutrient digestibility: the term "nutrient digestibility" means the fraction of nutrients that disappear from the gastrointestinal tract or from a designated section of the gastrointestinal tract (e.g., the small intestine). Nutrient digestibility can be measured as the difference between the nutrients administered to a subject and those excreted in the subject's stool, or the difference between the nutrients administered to a subject and those in the digesta remaining on a specified section of the gastrointestinal tract (e.g., the ileum).

Nutrient digestibility as used herein can be measured by: the difference between the intake of nutrients over a period of time and the excreted nutrients obtained by total collection of excretions; or using an inert marker that is not absorbed by the animal and allows the researcher to calculate the amount of nutrients that disappear throughout the gastrointestinal tract or section of the gastrointestinal tract. Such inert markers may be titanium dioxide, chromium oxide, or acid insoluble ash. Digestibility can be expressed as a percentage of nutrients in the feed, or as mass units of digestible nutrients per mass units of nutrients in the feed. Nutrient digestibility as used herein encompasses starch digestibility, fat digestibility, protein digestibility, and amino acid digestibility.

Energy digestibility as used herein means the total energy of the feed consumed minus the total energy of the faeces, or the total energy of the feed consumed minus the total energy of the remaining digesta on a specified section of the gastrointestinal tract (e.g. ileum) of an animal. Metabolic energy as used herein refers to apparent metabolic energy and means the total energy of the feed consumed minus the total energy contained in fecal, urinary and digested gas products. The energy digestibility and metabolic energy can be measured as the difference between the total energy intake and total energy in the digesta excreted in the faeces or present in the designated section of the gastrointestinal tract, using the same method as measuring nutrient digesta, with appropriate correction for nitrogen excretion to calculate the metabolic energy of the feed.

And (3) pill preparation: the terms "pellet" and/or "pelletization" refer to solid round, spherical and/or cylindrical tablets or pellets, as well as processes for forming such solid shapes, particularly feed pellets and solid extruded animal feed. As used herein, the term "extrusion" is a term well known in the art and refers to the process of passing a composition under pressure through an orifice as described herein.

Poultry: the term "poultry" means domestic birds raised by humans with respect to their eggs and/or their meat and/or their feathers produced. Poultry includes broiler chickens and layer chickens. Poultry includes members of the class galloanserans (birds), particularly the galliformes (which include chickens, guinea fowl, quail and turkeys) and the family of the anserans, ducks in which they are commonly referred to as "waterfowl" and include ducks and geese. Poultry also includes other birds that are killed for their meat, such as young pigeons. Examples of poultry include chickens (including layer chickens, broilers and chicks), ducks, geese, pigeons, turkeys and quails.

Coarse grain: the term "coarse food grain" means dry plant material with high levels of fibres, such as fibres, bran, bracts from seeds and grains as well as crop residues (e.g. straw, copra, straw, chaff, beet waste).

Ruminant animal: the term "ruminant" means the following mammals: it digests plant-based feed primarily by bacterial action, by first fermenting/degrading the plant-based feed in the first compartment of the animal's stomach, then ruminating the semi-digested pellet (now referred to as "ruminating food (cud)") and chewing again. The process of chewing the ruminant again to further break down plant matter and stimulate digestion is called ruminant. Examples of ruminants are cows, beef cattle, calves, goats, sheep, lambs, deer, yaks, camels and llamas.

Sequence identity: the degree of relatedness between two amino acid sequences is described by the parameter "sequence identity". Sequence identity is determined by one of three methods:

sequence identity determination method 1

Sequence identity between two amino acid sequences was determined as the output of the "longest identity" using the Needman-Wunsch algorithm (Needleman and Wunsch,1970, J.mol. Biol. [ J. Mol. Biol. Mol. 48:443-453) as implemented in the Nidel program of the EMBOSS software package (EMBOSS: european molecular biology open software suite (The European Molecular Biology Open Software Suite), rice et al 2000,Trends Genet. [ genetics trend ] 16:276-277) (version 6.6.0). The parameters used are gap opening penalty of 10, gap extension penalty of 0.5, and EBLOSUM62 (the emoss version of BLOSUM 62) substitution matrix. In order for the nitel program to report the longest identity, a non-reduced (nobrief) option must be specified in the command line. The output of the "longest identity" for the nitel marker is calculated as follows:

(identical residues x 100)/(alignment Length-total number of gaps in the alignment)

Sequence identity determination method 2

Sequence identity between two amino acid sequences was determined using the Needman-Wunsch algorithm (Needleman and Wunsch,1970, supra) as implemented in the Nelder program of the EMBOSS software package (EMBOSS: european molecular biology open software suite (The European Molecular Biology Open Software Suite), rice et al, 2000, supra) (6.6.0 edition). The parameters used are gap opening penalty of 10, gap extension penalty of 0.5, and EBLOSUM62 (the emoss version of BLOSUM 62) substitution matrix. Percent sequence identity is calculated as follows:

(identical residues x 100)/(length of shortest sequence in alignment)

Sequence identity determination method 3

Sequence identity between two amino acid sequences was determined using the Needman-Wunsch algorithm (Needleman and Wunsch,1970, supra) as implemented in the Nidel program of the EMBOSS software package (EMBOSS: european molecular biology open software suite (The European Molecular Biology Open Software Suite), rice et al, 2000, supra) (6.6.0 edition). The parameters used are gap opening penalty of 10, gap extension penalty of 0.5, and EBLOSUM62 (the emoss version of BLOSUM 62) substitution matrix. Percent identity is calculated as follows:

(identical residues x 100)/(length of alignment)

Silaging: the term "silage" means a fermented, high moisture storage feed that can be fed to ruminants (ruminant-chewing (cud-oving) animals, such as cattle and sheep) or used as a biofuel feedstock for anaerobic digesters. It is fermented and stored in a process called silage (ensilage, ensiling or silaging) and is typically made from grass or cereal crops (e.g. forage plants such as maize, sorghum, oats, rye, timothy etc.) or legume crops (e.g. clover/trefoil), alfalfa, field peas) using whole green plants (not just grains). Silage can be made from many field crops and specific terms (oat silage (oatlage) for oat, half-dry silage (hayage) for alfalfa) can be used depending on the type. Silage is produced by placing cut green vegetation in a silage pit, by stacking it in a large pile covered with plastic sheet, or by wrapping a large bag in plastic film.

Particle Size Distribution (PSD): the term "particle size distribution" or "PSD" is used herein for the particles of the present invention and defines the relative amount of particles present according to size, typically by volume. PSD is described as D values D10, D50 and D90, where D10 refers to the 10% percentile of the particle size distribution (meaning that 10% of the particle volume has a particle size equal to or less than a given value), D50 describes the 50% percentile and D90 describes the 90% percentile. The particle size distribution can be measured by laser diffraction or optical digital imaging or sieve analysis. The D values reported herein are measured by laser diffraction, wherein particle size is reported as volume equivalent sphere diameter.

Small enzyme particles: the term "small enzyme particles" refers to particles comprising an enzyme having a median particle size (diameter) of about 100 to 2000 microns, preferably 200 to 1500 microns, more preferably 300 to 1200 microns.

Pig: the term "pig" means a domestic pig raised by humans to obtain food, such as its meat. Pigs include members of the genus pig, such as domestic pigs (Sus scrofa domesticus) or pigs (Sus domastens), as well as piglets, growing pigs and sows.

Thermally stable: the term "thermostable" is a term known in the art and in a preferred aspect, stable is intended to mean the ability of an enzyme to remain active under heat stress (e.g., during an extrusion process). With respect to polypeptides having protease activity, thermostability is intended to mean that the protease in the extruded product retains at least 60% of the 75.000prot/g activity of the polypeptide having protease activity of SEQ ID NO 1, e.g. at least 65% of the 75.000prot/g activity, e.g. at least 70% of the 75.000prot/g activity, e.g. at least 75% of the 75.000prot/g activity, e.g. at least 80% of the 75.000prot/g activity, e.g. at least 85% of the 75.000prot/g activity, e.g. at least 90% of the 75.000prot/g activity, e.g. at least 95% of the 75.000prot/g activity.

Vegetable protein: the term "plant protein" is meant to include any compound, formulation or mixture of at least one protein derived or derived from a plant, including modified proteins and protein derivatives.

According to one aspect of the invention, the method of the invention comprises (a) combining a polypeptide having protease activity, a solid carrier, optionally water, and a meltable hydrophobic substance to provide a combination product; (b) Optionally applying sufficient heat to the combination product to melt the hydrophobic substance; (c) extruding the product of step (b); and (d) drying and cooling the extruded product of step (c) or actively drying and cooling the extruded product of step (c) to provide a thermostable enzyme product, wherein the polypeptide having protease activity has at least 70% sequence identity with SEQ ID NO. 1, i.e. withThe polypeptides of procact have at least 75% sequence identity.

Polypeptides

Polypeptides or proteases with protease activity are sometimes also designated as peptidases, proteases, peptide hydrolases or proteolytic enzymes. The protease may be an exo-type protease starting from a hydrolyzed peptide at either end or an endo-type protease (endopeptidase) that acts within the polypeptide chain. Endopeptidases exhibit activity on N-and C-terminated peptide substrates, which are related to the specificity of the protease in question.

Protease activity may be measured using any assay in which a substrate is employed that includes peptide bonds associated with the specificity of the protease in question. The assay pH and assay temperature are equally applicable to the protease in question. Examples of pH determination are pH 5, 6, 7, 8, 9, 10 or 11. Examples of the measured temperature are 30 ℃, 35 ℃, 37 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃ or 70 ℃, 80 ℃, 90 ℃ or 95 ℃.

Examples of protease substrates are casein and pNA-substrates, such as Suc-AAPF-NA (available, for example, from Sigma S7388). The capital letters in the pNA-substrate refer to the single-letter amino acid code. Another example is Protazyme AK (azurin-dyed crosslinked casein prepared by Megazyme) as a tablet T-PRAK. For pH activity and pH stability studies, pNA-substrates are preferred, whereas for temperature activity studies, protazyme AK substrates are preferred.

For the purposes of the present invention, protease activity is determined using assays described in the art, such as the Suc-AAPF-pNA assay, the Protazyme AK assay, the Suc-AAPX-pNA assay, and the o-phthalaldehyde (OPA). For the Protazyme AK assay, insoluble Protazyme AK (azurin-crosslinked casein) substrate released blue color when incubated with protease and this color was determined as a measure of protease activity. For the Suc-AAPF-pNA assay, the colorless Suc-AAPF-pNA substrate released yellow p-nitroaniline when incubated with protease and the yellow color was determined as a measure of protease activity.

The particles comprise a polypeptide having protease activity, which polypeptide has at least 70% sequence identity to the polypeptide of SEQ ID NO:1 as defined herein:

SEQ ID NO:1

Ala Asp Ile Ile Gly Gly Leu Ala Tyr Thr Met Gly Gly Arg Cys Ser

Val Gly Phe Ala Ala Thr Asn Ala Ala Gly Gln Pro Gly Phe Val ThrAla Gly His Cys Gly Arg Val Gly Thr Gln Val Thr Ile Gly Asn GlyArg Gly Val Phe Glu Gln Ser Val Phe Pro Gly Asn Asp Ala Ala PheVal Arg Gly Thr Ser Asn Phe Thr Leu Thr Asn Leu Val Ser Arg TyrAsn Thr Gly Gly Tyr Ala Thr Val Ala Gly His Asn Gln Ala Pro Ile

Gly Ser Ser Val Cys Arg Ser Gly Ser Thr Thr Gly Trp His Cys Gly

Thr Ile Gln Ala Arg Gly Gln Ser Val Ser Tyr Pro Glu Gly Thr Val

Thr Asn Met Thr Arg Thr Thr Val Cys Ala Glu Pro Gly Asp Ser GlyGly Ser Tyr Ile Ser Gly Thr Gln Ala Gln Gly Val Thr Ser Gly Gly

Ser Gly Asn Cys Arg Thr Gly Gly Thr Thr Phe Tyr Gln Glu Val ThrPro Met Val Asn Ser Trp Gly Val Arg Leu Arg Thr

the polypeptide may be natural or synthetic. It is suitably obtained from, obtainable from, nocardiopsis sp. It may suitably be derived from a polypeptide obtained from the nocardia mimetic species NRRL 18262.

Ronozyme Proact is a preparation of serine protease produced by a genetically modified strain of Bacillus licheniformis (Bacillus licheniformis). It is produced by fermentation of a sporulation-deficient bacillus licheniformis strain Rh 3, which expresses a synthetic gene encoding a serine protease (EC 3.4.21.). Thus, in one aspect of the invention, the polypeptide having protease activity is produced by a genetically modified strain of Bacillus licheniformis, preferably sporulation-deficient Bacillus licheniformis strain Rh 3, and has at least 70% sequence identity with the polypeptide having SEQ ID NO. 1.

Typically, a polypeptide having protease activity has at least 70% sequence identity to a polypeptide of SEQ ID NO.1 and has protease activity, e.g. has at least 75% sequence identity to a polypeptide of SEQ ID NO.1, e.g. has at least 80%, e.g. at least 81%, e.g. at least 82%, e.g. at least 83%, e.g. at least 84%, e.g. at least 85%, e.g. at least 86%, e.g. at least 87%, e.g. at least 88%, e.g. at least 89%, e.g. at least 90%, e.g. at least 91%, e.g. at least 92%, e.g. at least 93%, e.g. at least 94%, e.g. at least 95%, e.g. at least 96%, e.g. at least 97%, e.g. at least 98%, e.g. at least 99%, e.g. 100% sequence identity to a polypeptide of SEQ ID NO. 1.

In another exemplary embodiment, the polypeptide having protease activity comprises a polypeptide sequence having at least 70% sequence identity to the polypeptide of SEQ ID NO. 1 and further comprises an N-terminal sequence of 1 to 30 amino acid residues and/or a C-terminal sequence of 1 to 30 amino acid residues. The polypeptide having protease activity may comprise a polypeptide sequence having at least 75% sequence identity to the polypeptide of SEQ ID No. 1, e.g. having at least 80%, e.g. at least 81%, e.g. at least 82%, e.g. at least 83%, e.g. at least 84%, e.g. at least 85%, e.g. at least 86%, e.g. at least 87%, e.g. at least 88%, e.g. at least 89%, e.g. at least 90%, e.g. at least 91%, e.g. at least 92%, e.g. at least 93%, e.g. at least 94%, e.g. at least 95%, e.g. at least 96%, e.g. at least 97%, e.g. at least 98%, e.g. at least 99%, e.g. 100% sequence identity to the polypeptide of SEQ ID No. 1, and further comprising an N-terminal sequence of 1 to 30 amino acid residues and/or a C-terminal sequence of 1 to 30 amino acid residues.

The polypeptide having protease activity is typically selected from polypeptides having at least 75%, such as at least 80%, such as at least 85%, preferably at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity with SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3. The polypeptide having protease activity is typically selected from the group consisting of

i. An amino acid sequence having at least 80% sequence identity to SEQ ID No. 1;

an amino acid sequence having at least 80% sequence identity to SEQ ID No. 2; and

amino acid sequence of SEQ ID No. 2 having at least 80% sequence identity to SEQ ID No. 3:

ADIIGGLAYT IGGRCSVGFA ATNAAGQPGF VTAGHCGRVG TQVTIGNGRG VFEQSVFPGN DAAFVRGTSNFTLTNLVSRY NTGGYATVAG HNQAPIGSSV CRSGSTTGWH CGTIQARGQS VSYPEGTVTNMTRTTVCAEP GDSGGSYISG TQAQGVTSGG SGNCRTGGTT FYQEVTPMVN SWGVRLRT

SEQ ID NO:3：

MKKPLGKIVASTALLISVAFSSSIASAAPAPVPQTPVADDSAASMTEALKRDLDLTSAEAEELLSAQEAAIETDAEATEAAGEAYGGSLFDTETLELTVLVTDASAVEAVEATGAQATVVSHGTEGLTEVVEDLNGAEVPESVLGWYPDVESDTVVVEVLEGSDADVAALADAGVDSSSVRVEEAEEAPQVYADIIGGLAYT MGGRCSVGFA ATNAAGQPGF VTAGHCGRVG TQVTIGNGRG VFEQSVFPGN DAAFVRGTSN FTLTNLVSRY NTGGYATVAG HNQAPIGSSV CRSGSTTGWH CGTIQARGQS VSYPEGTVTN MTRTTVCAEP GDSGGSYISG TQAQGVTSGG SGNCRTGGTT FYQEVTPMVN SWGVRLRT QSHVQSAP

polypeptides having protease activity typically have a minimum protease activity level of at least 35.000PROT/kg, e.g., at least 50.000PROT/kg, e.g., at least 75.000 PROT/kg. With respect to polypeptides having protease activity, thermostability is intended to mean that the protease in the extruded product retains at least 60% of the 75.000prot/g activity of the polypeptide having protease activity of SEQ ID NO 1, e.g. at least 65% of the 75.000prot/g activity, e.g. at least 70% of the 75.000prot/g activity, e.g. at least 75% of the 75.000prot/g activity, e.g. at least 80% of the 75.000prot/g activity, e.g. at least 85% of the 75.000prot/g activity, e.g. at least 90% of the 75.000prot/g activity, e.g. at least 95% of the 75.000prot/g activity.

Ronozyme Proact is a preparation of serine protease produced by a genetically modified strain of Bacillus licheniformis. It is produced by fermentation of a sporulation-deficient bacillus licheniformis strain Rh 3, which expresses a synthetic gene encoding a serine protease (EC 3.4.21.). Thus, in one aspect of the invention, the polypeptide having protease activity is produced by a genetically modified strain of Bacillus licheniformis, preferably sporulation-deficient Bacillus licheniformis strain Rh 3, and has at least 70% sequence identity with the polypeptide having SEQ ID NO. 1. In typical embodiments, the polypeptides of the invention have at least 60% sequence identity to SEQ ID NO. 1.

Acid-stable proteases

In a preferred embodiment, the protease is an acid stable protease. In the context of the present invention, the term acid-stable means in a dilution corresponding to a280=1.0, and after incubation for 2 hours at 37 ℃ in the following buffer:

100mM succinic acid, 100mM HEPES, 100mM CHES,

·100mM CABS、1mM CaCl2、150mM KCl、0.01％ TritonX-100，pH 3.5，

The protease activity of the purified protease is at least 40% of the reference activity as measured using the assay described herein for pH stability (substrate: suc-AAPF-pNA, pH 9.0, 25 ℃).

In certain embodiments of the above acid stability definition, the protease activity is at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 97% of the reference activity.

The term reference activity refers to the activity after incubation in pure form in a diluent corresponding to a280=1.0 at 5 ℃ for 2 hours in the following buffer: 100mM succinic acid, 100mM HEPES, 100mM CHES, 100mM CABS, 1mM CaCl2, 150mM KCl, 0.01%X-100, pH 9.0, protease activity of the same protease, wherein the activity is determined as described above.

In other words, the method of determining acid stability comprises the steps of:

a) The protease sample to be tested (in pure form, a280=1.0) was split into two aliquots (I and II);

b) Aliquots I were incubated at 37 ℃ and pH 3.5 for 2 hours;

c) The residual activity of aliquot I was measured (pH 9.0 and 25 ℃);

d) Aliquot II was incubated at 5 ℃ and pH 9.0 for 2 hours;

e) The residual activity of aliquot II was measured (pH 9.0 and 25 ℃);

f) The percentage of residual activity of aliquot I relative to the residual activity of aliquot II was calculated.

Alternatively, in the above definition of acid stability, the pH of the buffer of step b) may be 1.0, 1.5, 2.0, 2.5, 3.0, 3.1, 3.2, 3.3 or 3.4.

In other alternative embodiments of the above acid stability definition in relation to the pH value of the buffer of the above alternative step b), the residual protease activity is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 97% compared to the reference value.

In alternative embodiments, a pH of 6.0, 6.5, 7.0, 7.5, 8.0 or 8.5 may be applied to the step d) buffer.

In the above definition of acid stability, the term a280=1.0 means the concentration (dilution) of the pure protease which gives an absorbance of 1.0 at 280nm relative to the buffer blank in a cuvette of 1cm path length.

And in the above definition of acid stability, the term pure protease refers to a sample having an A280/A260 ratio of greater than or equal to 1.70.

Examples of proteases according to the invention are

a) A protease derived from nocardia sp NRRL 18262; or (b)

b) A protease having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the proteases of (a).

(c) Proteases having at least 75%, such as at least 80%, such as at least 85%, preferably at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity with SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.

(d) Protease activity having the amino acid sequence: the amino acid sequence has at least 80% sequence identity with SEQ ID NO. 1, at least 80% sequence identity with SEQ ID NO. 2, or at least 80% sequence identity with SEQ ID NO. 3.

In another specific embodiment, the protease according to the invention is thermostable.

Ronozyme Proact is a preparation of serine protease produced by a genetically modified strain of Bacillus licheniformis. It is produced by fermentation of a sporulation-deficient bacillus licheniformis strain Rh 3, which expresses a synthetic gene encoding a serine protease (EC 3.4.21.). Thus, in one aspect of the invention, the polypeptide having protease activity is produced by a genetically modified strain of Bacillus licheniformis, preferably sporulation-deficient Bacillus licheniformis strain Rh 3, and has at least 70% sequence identity with the polypeptide having SEQ ID NO. 1.

Polypeptides having protease activity typically have a minimum protease activity level of at least 35.000PROT/kg, e.g., at least 50.000PROT/kg, e.g., at least 75.000 PROT/kg. Ronozyme Proact has a protease activity of a minimum of 75,000 prot/g. With respect to polypeptides having protease activity, thermostability is intended to mean that the protease in the extruded product retains at least 60% of the 75.000prot/g activity of the polypeptide having protease activity of SEQ ID NO 1, e.g. at least 65% of the 75.000prot/g activity, e.g. at least 70% of the 75.000prot/g activity, e.g. at least 75% of the 75.000prot/g activity, e.g. at least 80% of the 75.000prot/g activity, e.g. at least 85% of the 75.000prot/g activity, e.g. at least 90% of the 75.000prot/g activity, e.g. at least 95% of the 75.000prot/g activity.

The term thermally stable means one or more of the following: the optimum temperature is at least 50 ℃, 52 ℃, 54 ℃, 56 ℃, 58 ℃, 60 ℃, 62 ℃, 64 ℃, 66 ℃, 68 ℃, or at least 70 ℃.

In a preferred embodiment, the polypeptide having protease activity is selected from the group consisting of:

(a) A polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 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 a polypeptide of SEQ ID NO. 1;

(b) A variant of the polypeptide of SEQ ID NO. 1, which variant comprises a substitution, deletion, and/or insertion at one or more (e.g., several) positions; and

(c) A fragment of the polypeptide of (a) or (b), which fragment has protease activity.

In a more preferred embodiment, the polypeptide comprises or consists of SEQ ID NO. 1.

In another preferred embodiment, the enzyme granulate of the invention comprises or consists of protease, dextrin and water, preferably acid-stable protease, dextrin and water.

Extrusion enzyme granules

One aspect of the invention relates to a particle comprising a polypeptide having protease activity and having at least 70% sequence identity to the polypeptide of SEQ ID No. 1; the particles are prepared by an extrusion process.

One aspect of the invention relates to an animal feed additive comprising pellets prepared by an extrusion process. One aspect of the invention relates to particles prepared by an extrusion process. One embodiment of the invention relates to a formulation comprising a polypeptide of the invention as extruded particles or prepared by a method comprising an extrusion process, typically comprising extruding a combination comprising the polypeptide, a meltable hydrophobic substance and a solid carrier.

One aspect of the invention relates to an animal feed additive comprising an extruded enzyme pellet, wherein the enzyme is a polypeptide having protease activity and having at least 70% sequence identity to SEQ ID No. 1. According to one aspect of the invention, the method of the invention comprises (a) combining a polypeptide having protease activity, a solid carrier, optionally water, and a meltable hydrophobic substance to provide a combination product; (b) Optionally applying sufficient heat to the combination product to melt the hydrophobic substance; (c) extruding the product of step (b); and (d) drying and cooling the extruded product of step (c) or actively drying and cooling the extruded product of step (c) to provide a thermostable enzyme product, wherein the polypeptide having protease activity has at least 70% sequence identity with SEQ ID NO. 1, i.e. with The polypeptides of procact have at least 75% sequence identity. A further aspect of the invention relates to a method for preparing an animal feed additive comprising a polypeptide having protease activity and having at least 70% sequence identity with SEQ ID NO. 1, i.e.with>A polypeptide of procact having at least 75% sequence identity, the method comprising an extrusion process comprising extruding a combination comprising said polypeptide, a meltable hydrophobic substance and a solid carrier. At the position ofIn another embodiment, the invention encompasses a method for preparing a thermostable enzyme product for use in animal feed manufacture, the method comprising (a) combining an enzyme, a solid carrier, and a meltable hydrophobic material to provide a combined product; (b1) Reducing the moisture content by applying heat to the combination product and (b 2) melting the hydrophobic substance; and (c) cooling the combination product to provide a thermostable enzyme product, wherein the thermostable enzyme is a polypeptide having protease activity and at least 70% sequence identity to SEQ ID No. 1.

Fusible hydrophobic substance

The fusible hydrophobic substance is typically selected from oils and waxes, for example from the group consisting of: hydrogenated castor oil, hydrogenated palm kernel oil, hydrogenated rapeseed oil, hydrogenated palm oil, blends of hydrogenated and unhydrogenated vegetable oils, 12-hydroxystearic acid, microcrystalline waxes (e.g., cerit HOT) and high melting point waxes (e.g., mekon White).

Fusible hydrophobic substances according to the present invention include, but are not limited to, oils and waxes, such as hydrogenated vegetable oils (e.g., castor oil (HCO), palm kernel oil (HPKO), palm oil (FHPO or akoflke Palm 58 (AP)) or rapeseed oil (FHRO or akoflke FSR (AFx, where x=f (flakes) or M (melt)))), blends of hydrogenated and unhydrogenated vegetable oils (PB 3), 12-hydroxystearic acid (12-HSA), microcrystalline waxes (e.g., cerit HOT), and high melting point paraffins (e.g., mekon White). Such meltable hydrophobic material may be a single component or derived from a mixture of products designed to produce a desired melting point. This would include a combination with a water-immiscible liquid or a low melting hydrophobic solid, which can produce a mixture with a reduced melting point. These include waxes, C26 (and higher), paraffin waxes, cholesterol, fatty alcohols (e.g., cetyl alcohol), mono-, di-and triglycerides of animal and vegetable origin (e.g., animal fats, hydrogenated castor oil), fatty derivatives (e.g., fatty acids, soaps, esters), hydrophobic starches (e.g., ethylcellulose), lecithins. Waxes may be of natural origin, meaning that they may be of animal, vegetable or mineral origin. Animal waxes include, but are not limited to, beeswax, lanolin, shellac wax and chinese insect wax. Vegetable waxes include, but are not limited to, carnauba (camauba), candelilla (candelilla), bay and sugarcane waxes. Mineral waxes include, but are not limited to, paraffin waxes or ceresin waxes (earth wax), including mineral waxes (ozkerite), pure Bai Dela (ceresin), and montan waxes or petroleum waxes, including paraffin waxes and microcrystalline waxes. Alternatively, the wax may be a synthetic wax or a mixture of natural and synthetic waxes. For example, these may include low molecular weight partially oxidized polyethylenes, which may preferentially eutectic with paraffin wax. The fatty derivatives may be fatty acids, fatty acid amides, fatty alcohols and fatty esters or mixtures thereof. The acid amine may be stearamide. The sterol or long chain sterol ester may also be, for example, cholesterol or ergosterol. One skilled in the art will recognize that combinations of two or more of the waxes and/or oils mentioned above may be used.

"meltable" hydrophobic material means a hydrophobic material that is solid at the typical ambient storage temperature of a feed product but melts above that temperature. In one embodiment, the melting temperature will be in the range of 20 ℃ to 100 ℃. The upper temperature limit is limited by the ability to melt the hydrophobic materials in the process and the stability of the enzyme at these elevated temperatures during processing. In one aspect of this embodiment, the hydrophobic material has a melting point in the range of 20 ℃ to 95 ℃ C. In another aspect of this embodiment, the hydrophobic material has a melting point in the range of 25 ℃ to 90 ℃. In yet another aspect of this embodiment, the hydrophobic substance has a melting point in the range of 20 ℃ to 80 ℃, such as 20 ℃ to 70 ℃, such as 20 ℃ to 65 ℃, such as 20 ℃ to 60 ℃.

HCO is hydrogenated castor oil and typically has a melting point in the range of 82 ℃ to 86 ℃. PB3 is a blend of hydrogenated and unhydrogenated vegetable oils and typically has a melting point in the range of 38℃to 46 ℃. Akoflke Palm58 or FHPO is hydrogenated (fully hardened) Palm oil, typically with a melting point in the range of 58-6O deg.c. HPKO is hardened palm kernel oil with a typical melting point range of 41-44 ℃. Akoflag FSR or FHRO is hydrogenated (fully hardened) rapeseed oil, with a typical melting point range of 66-69 ℃. Those skilled in the art will recognize that the actual melting point may vary depending on the environmental or physical conditions under which the fusible hydrophobic substance is heated or the source of the fusible hydrophobic substance.

In one embodiment, the enzyme-containing product of the present invention may comprise any suitable amount of meltable hydrophobic material that protects the enzyme and maintains bioavailability. In one aspect of this embodiment, the enzyme-containing product comprises from 1% to 30% by weight of the meltable hydrophobic material. In another aspect of this embodiment, the enzyme-containing product comprises 5% -20% by weight of the meltable hydrophobic material. In another aspect of this embodiment, the enzyme-containing product comprises at least 5% or more, such as 7.5%, 10%, 20% or 30% by weight of the meltable hydrophobic material. Without being bound by theory, it is believed that treating the enzyme with a meltable hydrophobic material protects the enzyme product substrate from temperature and moisture during the pelletization process. A fusible hydrophobic substance is added to the matrix in a concentration sufficient to affect the treatment and ensure enhanced retention activity of the enzyme, regardless of the concentration of enzyme present in the matrix.

Solid carrier

In typical embodiments, solid carriers suitable for use in the methods of the invention include, but are not limited to, plant-derived absorbents (e.g., ground seed grains such as ground corn, ground wheat, semolina, soybean meal, rice hulls, corn gluten feed, corn grits, distillers dried grains), mineral-derived absorbents (e.g., silica, diatomaceous earth, or clay). In a more typical embodiment, the solid carrier is ground wheat or corn. In another exemplary embodiment, the solid carrier is wheat or corn flour.

In one embodiment, the solid carrier is an absorbent and/or adsorbent material, such as a plant-based absorbent or a mineral-derived absorbent.

Additional components

Those skilled in the art will recognize that the present invention is applicable to protecting other thermally unstable components of an animal feed concentrate, such as, but not limited to, any of the following groups (alone or in combination): vitamins such as vitamins A, B, C, D, D, E, riboflavin, niacin, choline, folic acid, and the like; nucleic acids and nucleotides, etc., such as guanine, thymidine, cytosine, adenine, etc.; amino acids such as glycine, lysine, threonine, tryptophan, arginine, tyrosine, methionine, and the like; microorganisms such as Aspergillus niger (Aspergillus niger), aspergillus oryzae (A. Oryzae), bacillus subtilis (Bacillus subtilis), bacillus licheniformis (B. Lichenifermis), lactobacillus acidophilus (Lactobacillus acidophilus), lactobacillus bulgaricus (L. Bulgaricum), etc.; medicaments and vaccines such as aureomycin, erythromycin, oxytetracycline, and the like; and flavoring agents such as sugar, perfume, essential oil, and synthetic flavoring.

Extrusion process

According to the invention, the pelletization process for preparing animal feed is an extrusion process. Typical extrusion processes for making feed pellets are known to those skilled in the art. Extruded or pelletized products are products in which the feed mixture (powdered feed) is pressed into pellets or extruded under pressure through a small opening and cut into granules, which are subsequently dried. Such particles are typically of a predetermined size, as the material (typically a plate with a bore) with the extrusion opening is open to limit the allowable pressure drop through the extrusion opening. Furthermore, when small openings are used, very high extrusion pressures increase the heat generation in the powdered feed. (Michael S.Showell (eds.); powdered detergents [ powder detergent ]; surfactant Science Series [ surfactant science series ];1998; volume 71; pages 140-142; marcel Dekker [ Marssel Dekker ]).

In a particular embodiment, the powdered feed is introduced into an extruder to form variable length pellets from the extrudate. The extrusion device may be any screw extruder known in the art. In particular embodiments, the extruder is a twin screw extruder, such as a Werner & Pfleiderer type continuous 37 "extruder. Extrusion parameters (e.g., capacity, screw speed, die diameter, drying temperature, drying time, etc.) depend on the particular extrusion process and/or extrusion apparatus used.

In an embodiment, the extruder screw speed is 1 to 1,000RPM. In a more specific embodiment, the screw speed of the extruder is 100RPM. In an even more particular embodiment, the screw speed of the extruder is 150RPM. In yet even more particular embodiments, the screw speed of the extruder is 200RPM. In still even more particular embodiments, the screw speed of the extruder is 250RPM. In still even more particular embodiments, the screw speed of the extruder is 300RPM.

In an embodiment, the die diameter is 0.5mm-5.0mm. In a more specific embodiment, the die diameter is 0.5mm. In an even more specific embodiment, the die diameter is 1.0mm. In yet an even more particular embodiment, the die diameter is 1.5mm. In the most specific embodiment, the die diameter is 2.0mm.

The pellets are placed and then dried for a specific period of time, for example at least 15 minutes, preferably 20 minutes (at a temperature of 60 ℃ to 100 ℃), preferably 90 ℃ to 100 ℃, more preferably 90 ℃, even more preferably 95 ℃, even more preferably still 100 ℃.

One aspect of the invention relates to a method for preparing an animal feed additive comprising a polypeptide having protease activity and having at least 70% sequence identity with SEQ ID NO. 1, i.e. withPolypeptides of ProAct orA polypeptide having at least 75% sequence identity to a polypeptide as defined herein, the method comprising an extrusion process comprising extruding a combination comprising the polypeptide, a meltable hydrophobic substance and a solid carrier.

In yet another embodiment, a polypeptide having protease activity and at least 70% sequence identity to SEQ ID NO. 1 is substantially stable when subjected to an extrusion process having a pressure of 1 bar to 40 bar and to an extrusion process having an extrusion process temperature of 60℃to 100 ℃.

According to one aspect of the invention, the method of the invention comprises (a) combining a polypeptide having protease activity, a solid carrier, optionally water, and a meltable hydrophobic substance to provide a combination product; (b) Optionally applying sufficient heat to the combination product to melt the hydrophobic substance; (c) extruding the product of step (b); and (d) drying and cooling the extruded product of step (c) or actively drying and cooling the extruded product of step (c) to provide a thermostable enzyme product, wherein the polypeptide having protease activity has at least 70% sequence identity with SEQ ID NO. 1, i.e. with The polypeptides of procact have at least 75% sequence identity.

In another embodiment, the invention encompasses a method for preparing a thermostable enzyme product for use in animal feed manufacture, the method comprising (a) combining an enzyme, a solid carrier, and a meltable hydrophobic substance to provide a combined product; (b1) Reducing the moisture content by applying heat to the combination product and (b 2) melting the hydrophobic substance; and (c) cooling the combination product to provide a thermostable enzyme product, wherein the thermostable enzyme is a polypeptide having protease activity and at least 70% sequence identity to SEQ ID No. 1.

In a further embodiment of the invention, the fusible hydrophobic substance-treated enzyme product of the invention is mixed with a suitable feed agent and compounded via a heating/pelleting process to produce an animal feed containing a specified amount of protease. This process typically involves: 1. mixing all components, i.e. polypeptides having protease activity, meltable hydrophobic material and solid carrier; 2. they are compressed by an extruder, optionally with steam injection as a binder, to produce suitable feed pellets for administration to animals (such as but not limited to poultry or swine).

During this process, the temperature of the feed (also referred to as "meal") may rise to about 90 ℃. At these temperatures, most enzymes may be rapidly deactivated. The product of this process was then subjected to an enzyme recovery assay (expressed as% recovery relative to the equivalent untreated powder used to prepare the pellets). The product of the original granulation process was used as a comparison.

In further embodiments, the solid and liquid components of the feed are pre-mixed, except for the liquid binder component that is last mixed. In a ring die pellet extruder, the resulting powder is extruded with or without steam conditioning, preferably without steam conditioning, and the extruded pellets may be cooled and/or dried as desired. The liquid binder will have tack and adhesive properties and preferably will be a concentrated liquid by-product from the cereal, food or feed processing industry.

As discussed, it has surprisingly been found that the polypeptide of SEQ ID NO:1 can be formulated as an extrudate, wherein the extrusion process may comprise the use of elevated temperatures, including the use of steam, without significant loss of activity. However, the process may alternatively eliminate conditioning steps involving the use of steam and/or elevated temperatures, but involve a "cold" pelletization process. In the cold granulation process of the present invention, a liquid binder is used instead of steam. The binder itself is an animal feed ingredient and has tack and adhesive properties. When the liquid binder is applied to other feed ingredients, free moisture penetrates into the solid particles in the powder, and the tacky adhesive substance in the binder agglomerates the fine particles into large particles, which then remain on the surface of the large solid particles, forming an adhesive surface. When the resulting wet coherent powder is molded through a die, the particles are compacted and bonded together to form pellets with enhanced durability.

In the cold pelletization extrusion process, after compounding, the dry ingredients are mixed in a mixer. Liquid ingredients, such as fat or molasses, are then added and mixed. Typically, a liquid binder is added last, by blending the binder into the mixture, to obtain a uniform cohesive powder. The liquid binder may be used in a ratio of 5% to 25% (by weight) in the formulation, with 10% to 20% being preferred for cold granulation. Liquid feed ingredients are generally a relatively economical source of nutrition, concentrated liquid byproducts from the grain, food or feed processing industries, such as molasses and fat. The liquid binder may be used in conventional extrusion processes involving heat or steam. However, in conventional pelletization processes, the amount of these liquids is typically limited to less than 6%.

In conventional pelletization processes, conditioning the powder with steam is a prerequisite for pressing the powder or powder into pellets. The heat and water from the steam are used to activate the binder in the powder particles (i.e., protein and carbohydrate), soften them, and impart adhesive properties to the particle surfaces. When the powder is compacted through a die, the particles are compacted and bonded together to form a pellet. In the cold granulation process of the present invention, a liquid binder is used instead of steam. The adhesive has tack and adhesive properties. When such a liquid binder is applied, free moisture permeates the solid particles in the powder, and the tacky adhesive substance in the binder aggregates the fine particles into large particles, which then remain on the surface of the large solid particles, forming an adhesive surface. When wet coherent powder is molded through the mouth, the particles are compacted and bonded together to form a stable pellet. The liquid binder used in the cold granulation process may be any concentrated liquid by-product from the cereal, food or feed processing industry. The liquid binder should have a solids content of 20% to 80% (preferably 35% to 65%) by weight and should have tack and adhesive properties. Typical liquid binders include brew (concentrated molasses-like by-product of the brewing industry), corn steep liquor, concentrated pig solubles, concentrated brewery solubles, molasses, desugared molasses, syrup and concentrated liquid whey.

In the cold pelletization process, the pellet temperature exiting the pellet extruder die is from 35 ℃ to 70 ℃, typically from 37 ℃ to 65 ℃, typically less than 55 ℃, depending on the diet formulation, liquid binder type and binder level used. In contrast, in conventional pelletization processes, the pellets may have a temperature of 60 ℃ to 100 ℃. The low temperature of the pellets of the present invention provides the opportunity to incorporate heat sensitive and labile substances and feed ingredients (e.g. other enzymes than SEQ ID NO:1, microorganisms and milk proteins) or other feed ingredients that may be destroyed by heating and/or make nutrients unavailable in conventional pelleting processes.

A further aspect of the invention relates to a method of producing an animal feed pellet by the extrusion process described herein.

The invention further provides a product obtainable by the process of the invention, a process for preparing an animal feed comprising combining the product obtainable by the process of the invention with a suitable animal feed ingredient, and an animal feed so produced.

As can be seen from examples 10 and 12, the pellets produced by the extrusion process according to the present invention have high activity after undergoing pellet model thermal stability studies, which is higher than many commercial products in animal feeds.

Granules prepared by spray drying process

Particles

One aspect of the invention relates to a particle comprising a polypeptide having protease activity and having at least 70% sequence identity to the polypeptide of SEQ ID No. 1; the particles are prepared by a spray drying process.

The corresponding particles typically further comprise a carbohydrate. The carbohydrate is preferably selected from the group consisting of lactose, sucrose, mannitol, alpha-cyclodextrin and dextrin (more preferably dextrin).

A particle according to this aspect, typically comprising a protease selected from the group consisting of:

(a) A polypeptide having at least 75%, at least 80%, 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 the polypeptide of SEQ ID No. 1;

In the particles according to this aspect of the invention, the polypeptide may comprise or consist of SEQ ID NO. 1.

The particles may be produced by a spray drying process comprising (a) preparing a spray solution comprising a polypeptide having protease activity and having at least 70% sequence identity to the polypeptide of SEQ ID No. 1; and carbohydrates. The particles suitably comprise water and typically have a water content of less than 7%. Thus, the particles typically comprise or consist of an acid stable protease, dextrin and water.

The enzyme granules prepared by the spray drying process of the invention have a simple structure comprising a protease and suitably a carbohydrate, such as dextrin. Enzyme granules prepared by the spray drying process have excellent enzyme properties including pH stability and temperature activity while reducing granulation and coating costs (process costs and raw material costs). In preferred embodiments of the particles prepared by the spray drying process, the residual activity of the enzyme particles of the invention retains at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 97% of the reference activity after storage at ambient temperature for at least 5 days, 30 days, 2 months or 1 year. In more preferred embodiments, the residual activity of the enzyme granules of the invention retains at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 97% of the reference activity after storage at ambient temperature for at least 5 days, 30 days, 2 months or 1 year. In preferred embodiments, the acid stability of the enzyme particles of the invention is at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 97% of the reference activity. In more preferred embodiments, the acid stability of the enzyme granules of the invention is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 97% of the reference activity. In preferred embodiments, the temperature activity of the enzyme particles of the invention is at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 97% of the reference activity. In a more preferred embodiment, the temperature activity of the enzyme particles of the invention is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 97% of the reference activity.

Preparation of granules prepared by spray drying process

In one aspect, the invention relates to a method of producing enzyme granules, the method comprising

(a) Preparing a spray solution comprising an acid stable protease and a carbohydrate; and

(b) Spraying the spray liquid in a spray tower.

In one embodiment, a method of producing an enzyme particle comprises

(a) Preparing a spray solution comprising a polypeptide having protease activity and having at least 70% sequence identity to the polypeptide of SEQ ID No. 1; and a carbohydrate; and

(b) Spraying the spray liquid in a spray tower.

In the method of preparing the granules by the spray drying process, the carbohydrate may be selected from the group consisting of lactose, sucrose, mannitol, alpha-cyclodextrin and dextrin, preferably dextrin. Typically, spray towers have an inlet temperature of 100 ℃ to 200 ℃ and/or a product temperature of 50 ℃ to 80 ℃.

Methods for preparing enzyme granules prepared by a spray drying process can be found in c.e. caps, handbook of Powder Technology; particle size enlargement [ powder technical handbook; increased particle size ]; roll 1; 1980; found in Elsevier [ Elsevier inc.). In a preferred embodiment, the enzyme granules are produced by spray drying. The spray is typically a carbohydrate selected from the group consisting of lactose, sucrose, mannitol, alpha-cyclodextrin and dextrin. The dextrin is typically white dextrin.

Spray-drying products, wherein a liquid enzyme-containing solution is atomized in a spray-drying tower to form droplets, which are dried during their descent along the drying tower to form a continuous film layer encapsulating the enzyme-containing particles. Very small particles can be produced in this way (Michael S.Shell (editions); powdered detergents [ powdered detergent ]; surfactant Science Series [ surfactant science series ];1998; volume 71; pages 140-142; marcel Dekker [ Marselde Dekker press ]).

After drying, the enzyme granules preferably contain 0.1% -10% w/w water, preferably 1%, 2%, 3%, 4%, 5%, 6% or 7% w/w water.

One aspect of the invention relates to enzyme granules for use in animal feed, the granules being defined as prepared by a spray drying process. Further aspects relate to animal feeds comprising particles prepared by a spray drying process. Related aspects relate to the use of enzyme granules prepared by a spray drying process in animal feed.

As can be seen from example 11, the granules prepared by the spray drying process according to the present invention have high activity after undergoing the pellet model thermal stability study.

Particles (microparticles) comprising a salt core and a protease-containing layer

Granule (microparticle)

Enzyme particles comprising a salt core and a protease-containing layer typically comprise a sodium sulfate or sodium chloride core and a protease-containing layer. The protease in the protease-containing layer is typically an acid-stable protease. The enzyme granule comprising a salt core and a protease-containing layer has excellent enzyme properties including pH stability and temperature activity, while reducing granulation and coating costs (process costs and raw material costs). In preferred embodiments, the residual activity of the enzyme particle comprising a salt core and an acid-stable protease layer retains at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 97% of the reference activity after storage at ambient temperature for at least 5 days, 30 days, 2 months or 1 year. In more preferred embodiments, the residual activity of the enzyme particle comprising a salt core and an acid-stable protease layer retains at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 97% of the reference activity after storage at ambient temperature for at least 5 days, 30 days, 2 months or 1 year. In preferred embodiments, the acid stability of the enzyme particle comprising a salt core and an acid-stable protease layer is at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 97% of the reference activity. In more preferred embodiments, the acid stability of the enzyme particle comprising a salt core and an acid-stable protease layer is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 97% of the reference activity. In preferred embodiments, the temperature activity of the enzyme particle comprising a salt core and an acid-stable protease layer is at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 97% of the reference activity. In a more preferred embodiment, the temperature activity of the enzyme particles of the invention is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 97% of the reference activity.

Preparation of particles comprising a salt core and a protease-containing layer

In one aspect, the invention relates to an enzyme particle comprising a salt core and a protease-containing layer, preferably comprising a sodium sulphate or sodium chloride core and a protease-containing layer. Methods for preparing enzyme granules can be found in c.e. caps, handbook of Powder Technology; particle size enlargement [ powder technical handbook; increased particle size ]; roll 1; 1980; found in Elsevier [ Elsevier inc.).

In a preferred embodiment, the enzyme granulate comprising a salt core and a protease-containing layer is prepared by fluid bed granulation.

i) Fluidized bed granulation involves suspending particulates in an air stream and spraying a liquid onto the fluidized particles via a nozzle. The particles hit by the spray droplets wet and are tacky.

ii) these cores may be subjected to drying, for example in a fluid bed dryer. Other known methods for drying pellets in the feed or enzyme industry may be used by those skilled in the art. The drying is preferably carried out at a product temperature of from 25 ℃ to 90 ℃. For some enzymes it is important that the enzyme particles contain a small amount of water before being coated with the salt. If the water sensitive enzyme is coated with salt before excess water is removed, the water will have an adverse effect on the activity of the enzyme. After drying, the cores preferably contain 0.1% -10% w/w water, preferably 1%, 2%, 3%, 4% or 5% w/w water.

Sodium sulfate or sodium chloride core

The core may comprise a single salt or a mixture of two or more salts. The salt may be water soluble, in particular having a solubility of at least 0.1 g in 100g of water at 20 ℃, preferably at least 0.5g/100g of water, such as at least 1g/100g of water, such as at least 5g/100g of water.

The salt may be an inorganic salt, such as a sulfate salt. The salt may be in anhydrous form, or it may be a hydrated salt, i.e. a crystalline salt hydrate with one or more bound water of crystallization, as described for example in WO 99/32595. Specific examples include anhydrous sodium sulfate (Na ₂ SO ₄ ) Anhydrous sodium chloride (NaCl). In a preferred embodiment, the salt is selected from the group consisting of sodium sulfate and sodium chloride.

Protease-containing layer

Preferably, the acid-stable protease is applied to the salt core as a granulation liquid or liquid (e.g., protease concentrate, dissolved in buffer or water), e.g., using a fluidized bed, as known in the art.

The protease is typically selected from the group consisting of:

The protease is selected from a polypeptide having at least 75%, such as at least 80%, such as at least 85%, preferably at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity with SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3. The enzyme particles typically comprise SEQ ID NO. 1, SEQ ID NO. 2 or SEQ OD NO. 3.

Optionally additional coating

The particles may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA).

The enzyme particles are typically microparticles having a particle size of 100-2000 microns, preferably 200-1500 microns, more preferably 300-1200 microns. The granules have a moisture content of less than 5%.

The method of producing microparticles suitably comprises

(a) Preparing sodium sulfate or sodium chloride cores; and

(b) The protease solution is distributed on sodium sulfate or sodium chloride cores.

The protease solution is distributed on sodium sulfate or sodium chloride cores by spraying. Typically, the particles are prepared in a fluidized bed apparatus.

High shear particles

One aspect of the invention relates to an animal feed additive comprising a polypeptide having protease activity, i.e. a polypeptide having at least 70% sequence identity to SEQ ID NO. 1 in granules or pellets prepared by a high shear granulation process.

The high shear granulation process can result in a pelleting stable particle of the polypeptide having protease activity (i.e., a polypeptide having at least 70% sequence identity to SEQ ID NO: 1). Polypeptides having at least 70% sequence identity to the polypeptide of SEQ ID NO. 1 are known to be good animal feed additives. One aspect of the invention relates to an animal feed additive comprising a polypeptide having protease activity and at least 70% sequence identity to SEQ ID No. 1 in a granulate prepared by a high shear granulation process.

Particles

One aspect of the invention relates to an enzyme particle, the particle being prepared by a method comprising a high shear granulation process, the particle comprising a polypeptide having protease activity, the polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID No. 1.

The high shear granulation process typically includes

A. Forming a powder mixture by combining at least the following

i. Cellulose or derivatives thereof

ii, an adhesive; and

optionally a filler; and

B. adding liquid phase granulating agent

Wherein the polypeptide having protease activity is added to the powder mixture or to the liquid phase granulating agent.

Enzyme granules prepared by a high shear granulation process typically have a density of from 0.35 to 0.8, such as 0.37 to 0.7, such as 0.40 to 0.6.

Cellulose

The enzyme particles comprise cellulose or a derivative thereof. Many commercial sources of cellulose are suitable and known to those skilled in the art. Typically, the cellulose or derivative thereof is in the form of fibers or is microcrystalline cellulose.

Examples of suitable celluloses include cellulose powder-CEPO S20 (swedish cellulose powder and wood flour mill limited (The Swedish cellulose powder and Wood Flour Mills ltd.)) and cellulose arbor BC200.

There are several brands of cellulose in fiber form, such as CEPO and ARBOCEL, on the market. Svenska Tramjolsfabrikerna AB the publication "Cepo Cellulose Powder [ Cepo cellulose powder ]" indicates that for Cepo S/20 cellulose the approximate maximum fiber length is 500mu, the approximate average fiber length is 160mu, the approximate maximum fiber width is 50mu, and the approximate average fiber width is 30mu. It is also noted that the CEPO SS/200 cellulose has an approximate maximum fiber length of 150mu, an approximate average fiber length of 50mu, an approximate maximum fiber width of 45mu and an approximate average fiber width of 25 mu. Cellulose fibers having these dimensions are well suited for the purposes of the present invention.

The cellulose in fibrous form may be sawdust, pure fibrous cellulose, cotton, or other forms of pure or impure fibrous cellulose.

The cellulose and cellulose derivatives may be selected from the group consisting of hydroxypropyl cellulose, methyl cellulose or carboxymethyl cellulose (CMC).

A preferred embodiment of the process according to the invention comprises the use of between 5% and 30% by weight of cellulose or cellulose derivatives.

Adhesive agent

The enzyme particles comprise a binder. The binder is typically selected from the group consisting of polyvinylpyrrolidone, titanium dioxide, dextrin, polyvinyl alcohol, polyethylene glycol, cellulose and cellulose derivatives (e.g. hydroxypropyl cellulose, methylcellulose or carboxymethylcellulose (CMC)) (e.g. polyvinylpyrrolidone, titanium dioxide, dextrin, polyvinyl alcohol, cellulose and cellulose derivatives).

Dextrin W80 is a suitable dextrin.

Filler (B)

The filler may be any component that does not interfere with the granulation process, such as an inorganic salt. This may include any salt comprising one or more anions selected from the anions CO and cations ₃ ^2- 、SO ₄ ^2- 、HPO ₄ ^2- 、H ₂ PO ₄ ^- 、F ^- 、Cl ^- 、Br ^- 、NO ₃ ^- 、I ^- 、ClO ₄ ^- And SCN ^- Selected from the group consisting of Na ⁺ >K ⁺ >Mg ²⁺ >Ca ²⁺ A group of groups. One exemplary embodiment is selected from NaCl, caCO ₃ 、Na ₂ SO ₄ CaCl and NaHCO ₃ (typically NaCl, caCO) ₃ 、Na ₂ SO ₄ ) A group of groups.

Granulating agent

Using high shear granulation, enzyme granules can be produced without an undesired layer of starting material for granulation on the walls of the rotary drum granulator. When high shear granulation is used, the powder mixture being granulated is less sensitive to the granulating agent.

More particularly, the enzyme granule production process according to the invention suitably comprises introducing 2 to 40% by weight of cellulose in the form of fibres, 0 to 10% by weight of a binder as defined herein, an amount of enzyme and filler that produces the desired enzyme activity in the finished granule, a liquid phase granulator consisting of a waxy substance as defined herein and/or water in an amount of between 5 and 70% by weight, wherein the waxy substance is at a maximum of 40% by weight and the water is at a maximum of 70% by weight, wherein all percentages refer to the total amount of dry substance, the order of introduction of the different materials being arbitrary except that at least a majority of the granulator is introduced after at least a majority of the dry substance is introduced into the granulator, after which the granule is dried in a conventional manner, preferably in a fluidised bed, if necessary.

The binders used in the process according to the invention are binders conventionally used in the pelletization field which have a high melting point or no melting point at all and are of non-waxy nature, such as polyvinylpyrrolidone, dextrin, polyvinyl alcohol and cellulose derivatives (including, for example, hydroxypropylcellulose, methylcellulose or CMC). As described above, if a granulating agent is not used, granules cannot be formed based on cellulose, enzyme, filler and binder.

Fillers are typically used for the purpose of modulating the desired enzymatic activity in the finished granules. Since the enzyme introduced into the granulator already contains a diluent which is considered a filler, an additional filler is not always required to normalize the enzymatic activity of the granules. If a filler is used, it may typically be NaCl, but other components, especially other inorganic salts, which act as fillers, not interfering with the granulation process and subsequent use of the product may also be used.

The liquid-phase granulating agent may be selected from the group consisting of waxy substances and/or water or aqueous solutions. The granulating agent may be water and/or a waxy substance. The granulating agent is always used as a liquid phase in the granulating process; therefore, if the wax-like substance is present, it is dissolved or dispersed in water or melted. Waxy substances are understood as meaning substances having a melting point of between 30℃and 100℃and preferably between 40℃and 60 ℃.

Both water and waxy substances are granulating agents, i.e. they are active during particle formation; the waxy material remains as an ingredient in the finished granule while most of the water is removed during the drying process. Therefore, in order to represent all amounts as finished dry particles, all percentages are calculated based on the total dry matter, which means that water (one of the granulating agents) is not added to the other ingredients when calculating the percentage of water, whereas the waxy substance (the other granulating agent) must be added to the other dry ingredients when calculating the percentage of waxy substance. Examples of waxy substances are polyethylene glycols, fatty alcohols, ethoxylated fatty alcohols, higher fatty acids, mono-, di-and tri-glycerides of higher fatty acids (e.g. glycerol monostearate, alkylaryl ethoxylates and coconut monoethanolamide).

If a large amount of waxy substances is used, relatively little or no water is added, and vice versa. Thus, the granulating agent may be water alone, waxy substance alone or a mixture of water and waxy substance. If a mixture of water and a wax-like substance is used, the water and the wax-like substance may be added in any order, for example, by adding water first and then the wax-like substance, or by adding the wax-like substance first and then the water, or by adding a solution or suspension of the wax-like substance in water. Further, if a mixture of water and a wax-like substance is used, the wax-like substance may be soluble in water or insoluble in water (but dispersible in water).

If no water is used in the granulator, drying is generally not required. In this case, the granulating agent is a molten waxy material that requires only cooling to solidify the particles. In most cases, however, some drying will take place, and the drying is usually done in the form of fluidized bed drying, whereby small amounts of dust and small particles are blown off the particle surface. However, any type of drying may be used. In the case of no water as granulating agent, a flow regulator or anti-caking agent may be added to the granules before or after the cooling step, for example fumed silica, such as the commercial products AEROSIL or CAB-OSIL.

A further aspect of the invention relates to a method of preparing a particle comprising particles, the method comprising

The high shear granulation process comprises the following

A. Forming a powder mixture by combining at least the following

i. Cellulose or derivatives thereof

Optionally a binder; and

optionally a filler; and

B. adding liquid phase granulating agent

Wherein the polypeptide having protease activity is added to the powder mixture or to the liquid phase granulator, and wherein the at least one binder is added to the powder mixture or to the liquid phase granulator, or to both;

Wherein the polypeptide having protease activity is a polypeptide having at least 70% sequence identity to SEQ ID NO. 1,

or wherein the high shear granulation process comprises

A'. Forming a powder mixture by combining at least the following

i. Cellulose or derivatives thereof

ii, an adhesive; and

optionally a filler; and

b'. Adding liquid phase granulating agent

Typically, the polypeptide has at least 70% sequence identity to the polypeptide of SEQ ID NO. 1 and has protease activity, e.g. has at least 75% sequence identity to the polypeptide of SEQ ID NO. 1, e.g. has at least 80%, e.g. at least 81%, e.g. at least 82%, e.g. at least 83%, e.g. at least 84%, e.g. at least 85%, e.g. at least 86%, e.g. at least 87%, e.g. at least 88%, e.g. at least 89%, e.g. at least 90%, e.g. at least 91%, e.g. at least 92%, e.g. at least 93%, e.g. at least 94%, e.g. at least 95%, e.g. at least 96%, e.g. at least 97%, e.g. at least 98%, e.g. at least 99%, e.g. 100% sequence identity to the polypeptide of SEQ ID NO. 1.

Following high shear granulation, proteases typically have a minimum enzyme activity level of 15.000 PROT/kg.

The granulator may be any known type of mixing granulator, rotary drum granulator, pan granulator or variants of these. If a mixing granulator is used, for example a mixing drum granulator from the company Gebr.Lodige Maschinen G.m.b.H. (Padberg 479, elsen street 7-9, DT (479 Paderbonn, elsen trasse7-9, DT)) is used, a small rotating knife is preferably installed in the granulator to compact the granules.

A preferred embodiment of the process according to the invention comprises granulation at 50-70 ℃.

Enzyme granules produced by a high shear granulation process typically provide dried granules of between 0.2 and 2mm (e.g. 0.3 and 1.5 mm) in diameter.

Preferably, all solid material is first added to the granulator, after which a homogeneous mixture is produced, and then the granulating agent is introduced (from one or more nozzles present on the granulator) in the form of a spray.

Typically, the total solid starting material has a fill volume of less than 50% of the total granulator volume, preferably less than 30% of the total granulator volume.

Using granulation according to the practice of the present invention, excessive recirculation of excessively fine and large particles can be avoided; in practice, only about 20% of the particles on average are recycled.

The high shear granulation process typically includes

1. A given composition in dry powder form is composed.

2. Mixing the dry powder composition.

3. The powder mixture is wetted with a granulating agent (e.g., water or a water/binder solution).

4. The wet powder mixture is processed with a granulating device (rotary knife) until the granules have the desired particle distribution and roundness.

5. The wet granules are fluidized bed dried until the dryness meets both the enzyme stability requirements and the free flow characteristics and mechanical strength requirements.

Typically this will correspond to a moisture content of less than 10%, preferably less than 3%.

Animal feed and animal feed additive

The invention also relates to a method of preparing the pellets of the invention in an enzyme-enriched animal feed, and to animal feeds and feed additives comprising the pellets of the invention.

In a particular embodiment, the granulate of the invention is for use in a feed for: (i) a non-ruminant animal; preferably (ii) a monogastric animal; more preferably (iii) pigs, poultry, fish and crustaceans; alternatively, most preferably, (iv) pigs and poultry.

The particles of the invention may be fed to the animal before, after or simultaneously with the diet. The latter is preferred.

The term feed, feed composition or diet means any compound, formulation, mixture or composition suitable for or intended to be ingested by an animal. More information about animal feed compositions is provided below.

In one embodiment, the invention relates to an animal feed comprising the particles of the invention. In addition to endogenous proteases, the particles of the invention provide additional protein digestibility resulting in an increase in amino acid digestibility of 3% -6%. The granules of the invention increase energy (ME) by at least 25kcal/kg diet.

The particles promote sustainable poultry production by supporting the following:

1. natural resources are effectively utilized: lower soybean utilization and more alternative (local product and byproduct) feedstocks

2. Reduce livestock emissions: low nitrogen emissions by reducing crude protein diets

3. Life performance and animal welfare: significantly reduce foot pad lesions

SEQ ID NO 1 was specifically developed for inclusion in animal diets, which significantly increased protein digestion. It supplements naturally occurring proteases and significantly increases peptide supply, thereby improving animal productivity. It can raise the digestibility of extensive protein source and grain so as to save feed cost.

In a preferred embodiment of the invention, the animal feed comprises 100 to 500g protease/mT feed, e.g. 100 to 300g/mT, e.g. 125 to 250g/mT. In a preferred embodiment of broiler chicken, the animal feed comprises pellets to comprise 150 to 250g protease/mT feed, e.g. 175g/mT to 225g/mT, e.g. 200g/mT for broiler chicken.

In a preferred embodiment of broiler chicken, the animal feed comprises pellets to comprise 100 to 200g protease/mT feed, e.g. 125g/mT to 175g/mT, e.g. 150g/mT for layer and breeder chickens.

In a further aspect, the present invention relates to an animal feed additive comprising the particles of the invention and one or more further components selected from the group consisting of: one or more vitamins; one or more minerals; one or more amino acids; one or more phytes; one or more prebiotics; one or more organic acids; and one or more other feed ingredients. A non-exclusive list of examples of these components is set forth below:

examples of fat-soluble vitamins are vitamin a, vitamin D3, vitamin E and vitamin K, such as vitamin K3.

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

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

Examples of macrominerals are calcium, phosphorus and sodium.

Examples of amino acids for animal feed are lysine, alanine, beta-alanine, threonine, methionine and tryptophan.

Phytos is a group of natural or non-antibiotic growth promoters from herbs, spices or other plants used as feed additives. The plant extract may be a single substance prepared from essential oils/extracts, single plants and plant mixtures (herbal products) or mixtures of essential oils/extracts/plants (specialty products). Examples of phytases are rosemary, sage, oregano, thyme, clove and lemon grass. Examples of essential oils are thymol, eugenol, m-cresol, vanillin, salicylates, resorcinol, o-methoxyphenol (guajacol), gingerol, lavender oil, ionone, irone, eucalyptol, menthol, peppermint oil, alpha-pinene, limonene, anethole, linalool, methyl dihydrojasmonate, carvacrol, propionic acid/propionate, acetic acid/acetate, butyric acid/butyrate, rosemary oil, clove oil, geraniol, terpineol, citronellol, amyl salicylate and/or benzyl salicylate, cinnamaldehyde, plant polyphenols (tannins), turmeric and turmeric extracts.

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 primarily in the large intestine. They are often used as substitutes for antibiotic growth promoters in the production of live pigs and poultry, because they have a preventive effect on intestinal problems such as necrotic enteritis in chickens and escherichia coli infection in piglets. The organic acid may be sold as a single component or as a mixture of typically 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 salts thereof (typically sodium or potassium salts, such as potassium diformate or sodium butyrate).

Furthermore, optional feed additive ingredients are colorants, such as carotenoids, e.g. beta-carotene, astaxanthin and lutein; an aromatic compound; a stabilizer; an antimicrobial peptide; polyunsaturated fatty acids; an active oxygen-generating substance; and/or at least one other enzyme (EC 3.2.1.8) selected from another pectase; and/or beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6).

Examples of antimicrobial peptides (AMPs) are CAP18, lincomycin (Leucocin) a, trichromatin (tritptin), proglin-1, mortiferin (Thanatin), defensin, lactoferrin peptides and Ovispirin (Ovispirin) such as Novispirin (Novispirin) (Robert Lehrer, 2000), mycelial mycin (Plectasin) and statins (including the compounds and polypeptides disclosed in WO 03/044049 and WO 03/048148) variants or fragments of the foregoing that retain antimicrobial activity.

Examples of antifungal polypeptides (AFP) are Aspergillus megaterium and Aspergillus niger peptides, as well as variants and fragments thereof which retain antifungal activity, as disclosed in WO 94/01459 and WO 02/090384.

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

Examples of reactive oxygen species are chemicals such as perborate, persulfate, or percarbonate; enzymes such as oxidase, oxygenase or synthase.

Typically, the fat-soluble vitamins and water-soluble vitamins and trace minerals form part of a so-called premix intended for addition to the feed, whereas the bulk minerals are typically added separately to the feed. The premix enriched with the particles of the present invention is an example of an animal feed additive of the present invention.

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

In an alternative, the animal feed additive of the 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, two, three, or four, etc., up to all thirteen or up to all fifteen individual components. More particularly, the at least one individual component is included in the additive of the present invention in an amount that provides a concentration in feed (in-feed-concentration) within the range indicated in the fourth, or fifth, or sixth column of table a.

Animal feed compositions or diets have a relatively high protein content. Poultry and swine diets can be characterized as indicated in Table B, columns 2-3 of WO 01/58275. The fish diet can be characterized as indicated in column 4 of table B. Furthermore, such fish diets typically have a crude fat content of 200-310 g/kg.

WO 01/58275 corresponds to us patent No. 6,960,462, which is hereby incorporated by reference.

The animal feed composition according to the invention has a crude protein content of 50-800g/kg (preferably 50-600g/kg, more preferably 60-500g/kg, even more preferably 70-500g/kg, most preferably 80-400 g/kg), and furthermore 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 certain embodiments, the crude protein content is from the oilseed material of the present invention.

Additionally or in the alternative (of the crude protein content indicated above), the animal feed composition of the invention has a metabolizable energy content of 10-30 MJ/kg; and/or a calcium content of 0.1-200 g/kg; and/or an effective phosphorus content of 0.1-200 g/kg; and/or methionine content of 0.1-100 g/kg; and/or a methionine plus cysteine content of 0.1-150 g/kg; and/or lysine content of 0.5-50 g/kg.

In particular embodiments, the metabolizable energy, crude protein, calcium, phosphorus, methionine plus cysteine, and/or lysine content falls within any of ranges 2, 3, 4, or 5 (R.2-5) in Table B of WO 01/58275.

The crude protein was calculated as nitrogen (N) multiplied by a factor of 6.25, i.e. crude protein (g/kg) =n (g/kg) x 6.25. The nitrogen content was determined by the Kjeldahl method (A.O.A.C., 1984,Official Methods of Analysis [ official analytical methods ] 14 th edition, association of Official Analytical Chemists [ official analytical chemist's collection ], washington, D.C.).

Metabolizable energy may be calculated based on: NRC publication Nutrient requirements in swine [ nutrient requirement for pigs ], ninth representational 1988,subcommittee on swine nutrition,committee on animal nutrition,board of agriculture,national research council [ national institutes of research, agriculture, animal nutrition institute, pig nutrient division, national academy of sciences, usa ] National Academy Press [ national academy of sciences, publishing, washington, d.c., pages 2-6; european Table of Energy Values for Poultry Feed-stuffs [ European poultry feed material energy value Table ], spelderholt centre for poultry research and extension [ Stokes Host poultry research and popularization center ],7361DA Beck Bei Heng, netherlands, grafisch bedrijf Ponsen & looijen bv, wageningen, ISBN 90-71463-12-5.

In a further aspect, the invention relates to a method of improving the average metabolic energy of a plant-based diet in a monogastric animal, the method comprising administering an animal feed additive of the invention or an animal feed of the invention.

The dietary content of calcium, available phosphorus and amino acids in the animal's whole diet was calculated based on a feed table such as Veevoedertabel 1997,gegevens over chemische samenstelling,verteerbaarheid en voederwaarde van voedermiddelen,Central Veevoederbureau,Runderweg 6,8219pk Lelystad.ISBN 90-72839-13-7.

PREFERRED EMBODIMENTS

1. An animal feed additive comprising a polypeptide having protease activity, wherein the protease comprises a polypeptide having at least 70% sequence identity to SEQ ID No. 1; characterized in that the enzyme is formulated as a formulation selected from the group consisting of:

i. granules prepared by an extrusion process;

particles prepared by a spray drying process;

granules prepared by high shear granulation process

2. An animal feed additive according to paragraph 1, wherein the polypeptide having protease activity is obtained or obtainable from nocardia sp NRRL 18262.

3. The animal feed additive according to paragraph 1 or 2, wherein the polypeptide having protease activity has at least 75% sequence identity with the polypeptide of SEQ ID No. 1, e.g. has at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity with the polypeptide of SEQ ID No. 1.

4. An animal feed additive according to paragraphs 1 to 3, wherein the polypeptide having protease activity is selected from polypeptides having at least 75%, such as at least 80%, such as at least 85%, preferably at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3.

5. The animal feed additive of paragraphs 1 to 4, wherein the polypeptide having protease activity is selected from the group consisting of

i. A polypeptide having at least 80% sequence identity to SEQ ID No. 1 and having protease activity;

a polypeptide having at least 80% sequence identity to SEQ ID No. 2 and having protease activity; and

a polypeptide having at least 80% sequence identity with SEQ ID No. 3 having protease activity.

6. The animal feed additive according to any one of paragraphs 1 to 5, wherein the pellets produced by the extrusion process are pellets comprising

i. The polypeptide having protease activity and having at least 70% sequence identity to SEQ ID No. 1;

hydrophobic substances; and

solid carrier.

7. An animal feed additive according to paragraph 6, wherein the hydrophobic substance is selected from the group consisting of oils and waxes, for example from the group consisting of: hydrogenated castor oil, hydrogenated palm kernel oil, hydrogenated rapeseed oil, hydrogenated palm oil, blends of hydrogenated and unhydrogenated vegetable oils, 12-hydroxystearic acid, microcrystalline waxes (e.g., cerit HOT) and high melting point waxes (e.g., mekon White).

8. An animal feed additive according to paragraph 6, wherein the solid carrier is selected from the group consisting of an absorbent and/or an adsorbent material (e.g. a plant-based absorbent or a mineral-derived absorbent).

9. The animal feed additive of any one of paragraphs 1 to 5, wherein the particles prepared by the spray drying process further comprise a carbohydrate.

10. The animal feed additive according to any one of paragraphs 1 to 5, wherein the particles are prepared by a spray drying process comprising the step of (a) preparing a spray liquid comprising a polypeptide having protease activity and having at least 70% sequence identity to the polypeptide of SEQ ID No. 1 and a carbohydrate.

11. The animal feed additive of paragraph 10, wherein the spray drying process comprises the steps of

(a) Preparing a spray solution comprising a polypeptide having protease activity and having at least 70% sequence identity to the polypeptide of SEQ ID No. 1 and a carbohydrate; and

(b) Spraying the spray liquid in a spray tower.

12. The animal feed additive of paragraph 11, wherein the spray tower has an inlet temperature of 100 ℃ to 200 ℃ and/or a product temperature of 50 ℃ to 80 ℃.

13. An animal feed additive according to paragraphs 9 to 12, wherein the carbohydrate is selected from the group consisting of lactose, sucrose, mannitol, alpha-cyclodextrin and dextrin (preferably dextrin).

14. The animal feed additive of any one of paragraphs 1 to 5 and 9 to 13, wherein the particles are prepared by a spray drying process, and wherein the particles have a moisture content of less than 7%.

15. The animal feed additive of any one of paragraphs 1 to 5 and 9 to 14, wherein the particles comprise or consist of protease, dextrin and water.

16. The animal feed additive according to any one of paragraphs 1 to 5, wherein the enzyme granulate comprises a salt core and a protease-containing layer, wherein the protease is a polypeptide having protease activity and at least 70% sequence identity to SEQ ID No. 1.

17. The animal feed additive of paragraph 16, wherein the salt core is a sodium sulfate or sodium chloride core or a combination thereof.

18. An animal feed additive according to any one of paragraphs 16 to 17, wherein the enzyme granules have a particle size of 100-2000 microns, preferably 200-1500 microns, more preferably 300-1200 microns.

19. An animal feed additive according to any one of paragraphs 16 to 18, wherein the pellet has a moisture content of less than 5%.

20. The animal feed additive of any one of paragraphs 16 to 19, wherein the pellet is prepared by a process comprising the steps of: (a) preparing a salt core; and (b) distributing the protease solution onto the salt nuclei.

21. An animal feed additive according to paragraph 20, wherein the protease liquor is distributed by spraying onto sodium sulfate or sodium chloride nuclei.

22. An animal feed additive according to any one of paragraphs 16 to 21, wherein the enzyme granulate is prepared in a fluidised bed apparatus.

23. An animal feed additive according to any one of paragraphs 1 to 5 comprising a polypeptide having protease activity and at least 70% sequence identity to SEQ ID No. 1 in a granulate prepared by a high shear granulation process.

24. An animal feed additive according to paragraph 23, wherein the high shear granulation process comprises the steps of

A. Forming a powder mixture by combining at least the following

i. Cellulose or derivatives thereof

ii, an adhesive; and

optionally a filler; and

B. adding liquid phase granulating agent

25. An enzyme particle comprising a salt core, e.g. a sodium sulphate or sodium chloride core, and a protease-containing layer, wherein the protease is a polypeptide having protease activity and at least 70% sequence identity to SEQ ID No. 1.

26. The enzyme particle of paragraph 25, wherein the core is a sodium sulfate core.

27. The enzyme particle of paragraph 25 or 26, wherein the protease is selected from the group consisting of:

28. An enzyme particle according to any one of paragraphs 25 to 27, wherein the polypeptide comprises or consists of SEQ ID NO. 1.

29. The enzyme particle according to any one of paragraphs 25 to 28, wherein the enzyme particle has a particle size of 100-2000 microns, preferably 200-1500 microns, more preferably 300-1200 microns.

30. The enzyme particle of any one of paragraphs 25 to 29, wherein the particle has a moisture content of less than 5%.

31. A method for producing enzyme granules, the method comprising

(a) Preparing a salt core, such as a sodium sulfate or sodium chloride core; and

32. A method according to paragraph 31, wherein the protease solution is distributed by spraying onto sodium sulfate or sodium chloride nuclei.

33. The method of paragraph 31 or 32, wherein the enzyme granules are prepared in a fluidized bed apparatus.

34. An animal feed comprising the enzyme granulate according to any one of paragraphs 25 to 30.

35. Use of the enzyme granulate of any one of paragraphs 25 to 30 in animal feed.

36. A particle comprising a polypeptide having protease activity and having at least 70% sequence identity to the polypeptide of SEQ ID No. 1; the particles are prepared by a spray drying process.

37. The particle of paragraph 36, further comprising a carbohydrate.

38. The granule according to paragraph 36 or 37, wherein the carbohydrate is selected from the group consisting of lactose, sucrose, mannitol, alpha-cyclodextrin and dextrin (preferably dextrin).

39. The particle of paragraphs 36 to 38, wherein the protease is selected from the group consisting of:

40. The particle of any one of paragraphs 36 to 39, wherein the polypeptide comprises or consists of SEQ ID NO. 1.

41. The particle of any one of paragraphs 36 to 40 produced by a spray drying process comprising (a) preparing a spray solution comprising a polypeptide having protease activity and at least 70% sequence identity to the polypeptide of SEQ ID No. 1; and carbohydrates.

42. The particle of any one of paragraphs 36 to 41, wherein the particle has a moisture content of less than 7%.

43. The particle of any one of paragraphs 36 to 42, wherein the particle comprises or consists of a protease, dextrin and water.

44. A method for producing enzyme granules, the method comprising

(b) Spraying the spray liquid in a spray tower.

45. A method according to paragraph 44, wherein the carbohydrate is selected from the group consisting of lactose, sucrose, mannitol, alpha-cyclodextrin and dextrin (preferably dextrin).

1. A method according to paragraphs 44 to 45, wherein the spray tower has an inlet temperature of 100 ℃ to 200 ℃ and/or a product temperature of 50 ℃ to 80 ℃.

46. An enzyme granulate for use in animal feed, the granulate being as defined in any one of paragraphs 44 to 45.

47. An animal feed comprising the granulate of any one of paragraphs 44 to 45.

48. Use of an enzyme granulate according to any one of paragraphs 44 to 46 in animal feed.

49. A method for producing enzyme granules, the method comprising

(b) Spraying the spray liquid in a spray tower.

50. A method according to paragraph 49, wherein the carbohydrate is selected from the group consisting of lactose, sucrose, mannitol, alpha-cyclodextrin and dextrin (preferably dextrin).

51. A method according to paragraph 49, wherein the spray tower has an inlet temperature of 100℃to 200℃and/or a product temperature of 50℃to 80 ℃.

52. An enzyme particle prepared by a method comprising a high shear granulation process, the particle comprising a polypeptide having protease activity having at least 70% sequence identity to the polypeptide of SEQ ID No. 1.

53. An enzyme particle according to paragraph 52, further comprising at least one binder and cellulose or a derivative thereof.

54. The enzyme granules of paragraph 52, wherein the high shear granulation process comprises forming a powder mixture by combining at least the following

i. Cellulose or derivatives thereof

Optionally a binder; and

optionally a filler; and

adding liquid phase granulating agent

Wherein the polypeptide having protease activity is added to the powder mixture or to the liquid phase granulator, and wherein the at least one binder is added to the powder mixture or to the liquid phase granulator, or to both.

55. The enzyme granules of paragraph 52 or 53, wherein the high shear granulation process comprises

Forming a powder mixture by combining at least the following

i. Cellulose or derivatives thereof

ii, an adhesive; and

optionally a filler; and

adding liquid phase granulating agent

56. The enzyme particle of any one of paragraphs 52 to 55, comprising a binder selected from the group consisting of: polyvinylpyrrolidone, titanium dioxide, dextrin, polyvinyl alcohol, cellulose and cellulose derivatives (e.g. hydroxypropyl cellulose, methylcellulose or carboxymethylcellulose (CMC)).

57. The enzyme granules of any of paragraphs 52 to 56, comprising a filler selected from the group consisting of any component (e.g., inorganic salt) that does not interfere with the granulation process.

58. An enzyme particle according to any one of paragraphs 52 to 57, comprising cellulose or a derivative thereof in fibrous form.

59. The enzyme granules according to any of paragraphs 52 to 58, wherein the liquid-phase granulating agent is selected from the group consisting of a waxy substance and/or water or an aqueous solution.

60. The enzyme particle of paragraph 59, wherein the waxy substance is selected from the group consisting of polyethylene glycol, fatty alcohols, ethoxylated fatty alcohols, higher fatty acids, mono-, di-and tri-glycerides of higher fatty acids (e.g., glyceryl monostearate, alkylaryl ethoxylate, and coconut monoethanolamide).

61. The enzyme granule of paragraph 59, wherein the liquid phase granulating agent is water.

62. The enzyme granule of any one of paragraphs 52 to 58, which is substantially free of a wax coating or a salt coating.

63. The enzyme particle according to any one of paragraphs 52 to 62, having a density of 0.35 to 0.8, such as 0.37 to 0.7, such as 0.40 to 0.6.

64. An animal feed additive comprising the enzyme pellet of any one of paragraphs 52 to 63.

65. An animal feed additive comprising the enzyme pellet of any one of paragraphs 1 to 12.

66. A method of making a granule comprising granules, the method comprising high shear granulation, the high shear granulation process comprising

Forming a powder mixture by combining at least the following

i. Cellulose or derivatives thereof

Optionally a binder; and

Optionally a filler; and

adding liquid phase granulating agent

67. a method of preparing a particle by an extrusion process, the method comprising (a) combining a polypeptide having protease activity, a solid carrier, optionally water, and a meltable hydrophobic substance to provide a combination product; (b) Optionally applying sufficient heat to the combination product to melt the hydrophobic substance; (c) extruding the product of step (b); and (d) drying and cooling the extruded product of step (c) or actively drying and cooling the extruded product of step (c) to provide a thermostable enzyme product, wherein the polypeptide having protease activity has at least 70% sequence identity with SEQ ID NO. 1, i.e. withThe polypeptides of procact have at least 75% sequence identity.

68. A method for preparing a thermostable enzyme product for use in animal feed manufacturing according to paragraph 67, the method comprising (a) combining an enzyme, a solid carrier, and a meltable hydrophobic substance to provide a combined product; (b1) Reducing the moisture content by applying heat to the combination product and (b 2) melting the hydrophobic substance; and (c) cooling the combination product to provide a thermostable enzyme product, wherein the thermostable enzyme is a polypeptide having protease activity and at least 70% sequence identity to SEQ ID No. 1.

69. The method for preparing a thermostable enzyme product for use in animal feed manufacturing according to example 67, the method comprising (a) combining an enzyme, a solid carrier, and a meltable hydrophobic substance to provide a combined product, and optionally additional water to form a suitable paste; (b) Optionally applying sufficient heat to the combination product to melt the hydrophobic substance; (c) extruding the product of step (b); and (d) drying and cooling the extruded product of step (c) to provide a thermostable enzyme product. In one aspect of this embodiment, the meltable hydrophobic material is added in step (a) as a solid sheet or as a premelted molten liquid.

70. The method for preparing a thermostable enzyme product for use in animal feed production according to example 67, the method comprising (a) combining a polypeptide having protease activity and having at least 70% sequence identity to SEQ ID No. 1, a solid carrier and a meltable hydrophobic material to provide a combined product, and optionally additional water to form a suitable paste; (b) Melting the hydrophobic substance, or optionally melting the hydrophobic substance by applying heat to the combination product; (c) extruding the product of step (b); and (d) optionally drying and cooling the extruded product of step (c) to provide a thermostable enzyme product.

71. The method according to any one of embodiments 67 to 71, wherein the extruding step is at a temperature of 60 ℃ to 120 ℃, such as 70 ℃ to 110 ℃, such as 70 ℃ to 100 ℃, such as 80 ℃ to 100 ℃.

72. The method of any one of embodiments 67 to 71, wherein the extruding step further comprises a liquid binder and is performed at a temperature of 25 ℃ to 70 ℃, such as 30 ℃ to 60 ℃, such as 25 ℃ to 55 ℃, such as 30 ℃ to 55 ℃.

73. An animal feed pellet prepared by the method of any one of examples 67 to 73.

Examples

Example 1 extrusion with hydrogenated castor oil.

General process

The preparation of the solid protease-containing extruded product is a pellet extrusion process in which all components are combined with a suitable amount of water to be used as a blend of components in a mixing process. The resulting wet mixture is then extruded through a suitable extrusion device to produce wet granules. These wet granules are then further processed to shape the granules and then dried to a suitable moisture content.

SEQ ID NO. 1 was mixed with wheat flour at about 1:10 wt/wt. When the two components are mixed, molten HCO (approximately the same weight as the protein) is poured into the mixture. Water was then added and the entire premix was blended for an additional about one minute. After this time, the premix is extruded through a 0.5mm to 1mm (e.g., 0.8 mm) screen, the wet strands are broken up and then rounded in a spheronizer. The 0.8mm extrudate produced about 0.8mm spherical or round pellets. After the spheroidization process, the wet particles are transferred to a dryer and dried at about 50-90 ℃ for 20 minutes. The dried product was sieved through about 1.2mm and 0.4-0.5mm sieves. The fraction remaining on the 0.4-0.5mm screen is a suitable extrudate for use as a feed additive.

Other samples were prepared that contained different ratios of hydrogenated castor oil to protein or wheat flour to protein.

Example 2 extrusion with blends of hydrogenated and unhydrogenated vegetable oils.

For these experiments, SEQ ID NO 1 was mixed with wheat flour in a weight ratio of about 1:10. When the two components are mixed, a blend of hydrogenated and unhydrogenated vegetable oils (PB 3) (approximately the same weight as the protein) is added to and blended with the wheat flour as a soft solid. Water (approximately the same weight as protein) was then added and the entire premix was blended for about one minute. After this time, the premix was extruded through a 0.8mm screen and the wet strands broken up and then rounded in a spheronizer. After the spheroidization process, the wet particles are dried at about 50 ℃ to 70 ℃ for 20 minutes. The dried product was sieved through 1.2mm and 0.6mm sieves. The fraction remaining on the 0.6mm screen is a suitable extrudate for use as a feed additive.

Example 3 incorporation of solid hydrogenated castor oil by extrusion process.

A method was devised to incorporate a fusible hydrophobic substance in solid form into the extrusion process, rather than adding molten HCO or softening PB3. HCO flakes were mixed with wheat flour at a 1:10 weight ratio. Then the polypeptide of SEQ ID NO. 1 was added with mixing. Water was added and the whole premix was blended for an additional minute. The premix was extruded through a 0.8mm screen and the wet strands broken up and then spheronized in a spheronizer. The wet granules were dried at 50C to 75C for 20 minutes. The dried product was sieved through 1.2mm and 0.5mm sieves. The retained granules of the 0.5mm sieve were used to prepare animal feed.

Example 4. Incorporation of solid Akoflake FSR by extrusion process.

In another experiment, example 3, a fully hardened rapeseed oil Akoflake FSR was used.

Example 5: protease Activity in extruded pellets

For polypeptides having protease activity and to be effective as animal feed, the protease activity must be maintained at an effective level during the pelleting process. Typically, the feed pellets are extruded through a high temperature nozzle prior to drying and subsequent feeding. The extrusion process described above results in a free flowing product that exhibits a higher degree of enzyme protection. The pellets produced above are used to manufacture animal feed by conventional pelleting processes. The activity of the polypeptide with protease activity in the extruded pellets was comparable to the activity of the same polypeptide of the Ronozyme Proact.

Example 6: particles prepared by a spray drying process: enzyme layer on salt nucleus

The spray liquid has the following composition

80000g SEQ ID NO:1 concentrate

20000g dextrin W80

60000g of water

Spraying in spray tower with inlet temperature of 160 deg.c

C, the product temperature is 65C. The spray dried particles have a water content of less than 7%.

Example 7: enzyme layer on salt nucleus

/>

4000g of Na ₂ SO ₄ The cores were loaded into a Glatt Procell (GF 3, bottom spray) fluidized bed.

Spraying granulation liquid (composed of 1200g protease concentrate derived from Nocardia sp NRRL 18262) at 65deg.C ₂ SO ₄ On the nucleus.

The granules are dried to a moisture content of less than 5% and sieved to obtain a product having a particle size between 300 and 1180 microns.

Example 8: high shear granulation

Uncoated high shear mixer

The powder mixture having the following composition

At the position ofThe mixer FM 50 was granulated with a granulation liquid consisting of:

3544g Ronozyme ProAct (SEQ ID NO: 1) enzyme concentrate.

Granulation is performed as described in U.S. Pat. No. 4,106,991, example 1.

The granules are dried in a fluid bed dryer to a moisture content of less than 1%.

Example 9: high shear granulation II

20% -30% (25%) Ronozyme Proact (SEQ ID NO: 1), 10% cellulose fiber, 1% binder: PVP K30

1. A powder component; 7.5kg of ground proteolytic enzyme Ronozyme Proact, 0.6kg of titanium dioxide, 3.0kg of cellulose powder CEPO S20 (Swedish cellulose powder and Wood flour Co., ltd.), 18.6kg of ground sodium chloride

2. The above components were mixed in a Lodige mixer FM 130 daz for 1 minute at a mixer speed of 160rpm and a single cross-blade granulator at a speed of 3000rpm.

3. Thereafter, during continuous mixing using a mixing-aggregation and granulation apparatus, wetting is performed using an aqueous solution of 3% -6% (e.g., 4.5%) polyvinylpyrrolidone (PVP K30).

4. After spraying the binder solution, the wet mixture is further exposed to the compaction of the granulation device for 7-10 minutes.

The rotational speed of the mixing and agglomerating was maintained at 160rpm and the rotational speed of the granulating device was maintained at 3000rpm.

Example 10: pelleting stability model

The purpose of these tests was to evaluate commercial productsStability of the novel formulation of proteases found in procact CT and stability of commercially available proteases. Pelletization stability model test incubation at 95 ℃ for 90 seconds, parameters used in the industrial pelletization process were applied. Experiments were run in triplicate and the average reported. Enzyme activity and recovery were measured using spectrophotometry (pNA assay) based on the Suc-AAPP-pNA substrate. In this assay, the enzyme product was mixed with the substrate in buffer at pH 7.0 and 37℃for 15 minutes and the kinetic activity was measured and the absorbance of the product reaction at 405nm was monitored.

The residual activity of the protease product after steam treatment was evaluated using the following assay: 250mg of each enzyme product was dispensed into aluminum cups. The stress steam incubation was performed in a closed styropor vessel with an internal size of 27x 18x 20 cm. An liter of boiling water is poured into the steam generator. Steam is transferred from the steam generator to the tank. When the temperature reached 95 ℃, the sample was placed on a plate and inserted into the box through a drawer. The temperature inside the tank was monitored using a thermometer mounted on the container lid. Incubation was performed for 90 seconds from the sample insertion into the box. Immediately after incubation, the samples were cooled on ice, resuspended in 0.1M acetate buffer (5 mM Ca++, pH 5.0) and protease activity was measured using the pNA assay described above. The residual activity was evaluated as the ratio of the activity of the steam box treated sample to the activity of the control sample, comparing each enzyme product to a similar sample not subjected to the steam treatment test. The experimental results are reported in tables 1-4, indicating that the proteases in the novel formulations have similar stability as one of the ProAct CT commercial products and higher stability than the other commercial products.

At the position ofIn large-scale production of ProAct CT, the activity is about 90%, and therefore, in this caseIn the steam stability model, the 90 second test is the best prediction, and the results show that for cheaper protease formulations, i.e. for extruded enzyme pellets, pellets comprising salt nuclei and protease-containing layers and pellets prepared by a method comprising a high shear granulation process, the and & @ was obtained in the 90 second steam stability test at 95 °c>The commercial product of ProAct CT has equivalent stability.

Micro-granulation formulation of SEQ ID NO. 1 (ProAct): particles comprising a salt core and a protease-containing layer (procact)

Table 1. Residual activity after test of pelleting stability model for different protease formulations and commercial products. The enzyme product was stressed for 90s at a temperature of 95 ℃.

High shear granulation formulation of SEQ ID NO. 1 (ProAct):

table 2. Residual activity after test of pelleting stability model for different protease formulations and commercial products. The enzyme product was stressed for 90s at a temperature of 95 ℃.

Extrusion formulation of SEQ ID NO. 1 (ProAct)

Table 3. Residual activity after test of pelleting stability model for different protease formulations and commercial products. The enzyme product was stressed for 90s at a temperature of 95 ℃.

Example 11 pelletization stability model

Pilot granulation, temperature stability test and activity analysis

The purpose of these tests was to evaluate commercial productsStability of novel formulations of proteases found in procact and stability of commercially available proteases procact. Pelleting stability model test was performed at 95 ℃ (simulating the temperature applied in the industrial pelleting process), and incubated for 5 minutes. Experiments were run in triplicate and the average reported. Enzyme activity and recovery were measured using spectrophotometry (pNA assay) based on Suc-Ala-Ala-Pro-Phe-pNA substrates. In this assay, the enzyme product was mixed with the substrate in buffer at pH7.0 and 37℃for 15 minutes and the kinetic activity was measured and the absorbance of the product reaction at 405nm was monitored.

The residual activity of the protease product after temperature treatment was evaluated using the following assay: 25mg of each enzyme product was dispensed into 0.2mL tubes (thin-walled 8-way tube, siemens technologies (Thermo Scientific)). 5 μl of deionized water was added to the lid of each tube to simulate the moisture of the pelletization process. The tube was placed in a PCR apparatus (GeneAmp PCR system 9700, perkin Elmer) and incubated at 95℃for 5 minutes. Immediately after incubation, the samples were cooled on ice, resuspended in 5ml of 0.1M acetate buffer (5 mM Ca++, pH 5.0) and protease activity was measured using the pNA assay described above. The residual activity was evaluated as the ratio of the activity of the steam box treated sample to the activity of the control sample, comparing each enzyme product to a similar sample not subjected to the temperature treatment test. The experimental results are reported in table 1, indicating that the proteases in the novel formulations have similar stability as one of the commercial products of procact.

Table 4. Residual activity after test of the novel protease formulations and commercial product pelleting stability model. The enzyme product was stressed for 5 minutes at a temperature of 95 ℃.

Example 12: determination of purity of protease sample SDS-PAGE

SDS-PAGE purity of protease samples was determined by the following procedure:

mu.l of protease solution (A) ₂₈₀ Concentration = 0.025) was mixed with 10 μl 50% (w/v) TCA (trichloroacetic acid) in Eppendorf tubes on ice. After half an hour on ice, the tube was centrifuged (5 min, 0 ℃,14.000x g) and the supernatant carefully removed. Mu.l of SDS-PAGE sample buffer (200. Mu.l of Tris-glycine SDS sample buffer (2X) (125 mM Tris/HCl (pH 6.8), 4% (w/v) SDS, 50ppm bromophenol blue, 20% (v/v) glycerol from NOVEX) ^TM To the pellet was added 160. Mu.l deionized water + 20. Mu.l beta-mercaptoethanol + 20. Mu.l 3M unbuffered Tris base (Sigma T-1503) and the tube was boiled for 3 minutes. The tube was briefly centrifuged and 10. Mu.l of sample was placed in NOVEX ^TM 4% -20% gradient Tris-glycine pre-gels (polyacrylamide gradient gels based on Laemmli chemistry but without SDS in the gel (Laemmli, UK, (1970) Nature [ Nature ]]Volume 227, pages 680-685), EC 60255). Electrophoresis was performed with Tris-glycine running buffer (2.9 g Tris base, 14.4g glycine, 1.0g SDS, distilled water to 1 liter) in two buffer reservoirs at a constant voltage of 150V until the bromophenol blue tracer stain reached the bottom of the gel. After electrophoresis, the gel was rinsed 3 times with 100ml distilled water for 5 minutes each by gentle shaking. Then use Blue staining reagent (colloidal Comassie G-250 product from PIERCE (PIERCE), cat# 24592) gently shake the gel for one hour, then gently shake wash with distilled water for 8 to 16 hours, and change distilled water several times. Finally, the gel was dried between 2 sheets of cellophane. The dried gel was scanned using an Arcus II scanner of AGFA equipped with foolok 95v2.08 software and imported into image evaluation software CREAM for Windows by a File/acquisition (File/Acquire) command with the following (foolok 95v 2.08) settings ^TM (catalog nos. 990001 and 990005, kem-En-Tec Co., denmark): original = reflection, mode = color RGB, scan resolution = 240ppi, output resolution = 120lpi, scale factor = 100%, range = histogram with global choice and minimum = 0 and maximum = 215, tone curve = none, sharpness = none, dot removal = none and parser = none, to generate SDS-PAGE gel ^TM And (3) evaluating the test pieces. Evaluation using menu command Analysis/1-D (Analysis/1-D). The following two scan lines were placed on the. Img picture file using Lane placement Tool (Lane table Tool): sample scan lines and background scan lines. The sample scan line was placed in the middle of the sample lane (containing the protease in question) from directly below the application well to directly above the bromophenol blue tracer stain location. The background scan line was placed parallel to the sample scan line, but at the location where no sample was applied in the illustrated SDS-PAGE gel, the start and end points of the background scan line were perpendicular to the start and end points of the sample scan line. The background scan line represents the true background of the gel. The width and shape of the scan lines are not adjusted. The intensity along the Scan line is now recorded using the 1-D/Scan (1-D/Scan) menu command and Medium (Medium) sensitivity. The background scan is subtracted from the sample scan using a 1-D/Editor (1-D/Editor) menu command. Then select 1-D/Results (1-D/Results) menu commands and use Area% of protease peak (e.g., by CREAM) ^TM Software calculation) as SDS-PAGE purity of the protease.

All protease samples had SDS-PAGE purity of greater than 95%.

Example 13: determination of pH Activity

Suc-AAPF-pNA(S-7388) is used to obtain a pH activity profile.

Assay buffer: 100mM succinic acid (Merck 1.00682), 100mM HEPES (Sigma H-3375), 100mM CHES (Sigma C-2885), 100mM CABS (Sigma C-5580), 1mM CaCl ₂ ，150mM KCl、0.01％X-100, adjusted to pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 or 11.0 with HCl or NaOH.

Measuring temperature: 25 ℃.

Mu.l of protease sample (diluted in 0.01%)X-100) with 1.5ml of assay buffer of the corresponding pH value, and the pH of the mixture is adjusted to the pH of the assay buffer. By adding 1.5ml of pNA substrate (50 mg dissolved in 1.0ml DMSO and further with 0.01%)>X-100 diluted 45-fold) and after mixing, monitoring A by a spectrophotometer ₄₀₅ As a measure of protease activity at the pH in question. The assay was repeated at other pH values using assay buffer and activity measurements were plotted as relative activity versus pH. The relative activity was normalized to the highest activity (pH optimum), i.e. the activity at pH optimum was set to 1 or 100%. The protease samples were diluted to ensure that all activity measurements were within the linear portion of the measured dose-response curve.

Example 14: determination of pH stability

Suc-AAPF-pNA(S-7388) is used to obtain a pH stability profile.

Assay buffer: 100mM succinic acid, 100mM HEPES, 100mM CHES, 100mM CABS, 1mM CaCl ₂ 、150mM KCl、0.01％X-100, adjusted to pH 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 or 11.0 with HCl or NaOH.

Each protease sample (in 1mM succinic acid, 2mM CaCl ₂ In 100mM NaClpH 6.0, and A ₂₈₀ Absorption of>10 Dilution to a in assay buffer at the pH of each test ₂₈₀ =1.0. The diluted protease samples were incubated at 37℃for 2 hours. After incubation, the protease samples were incubated in 100mM succinic acid, 100mM HEPES, 100mM CHES, 100mM CABS, 1mM CaCl ₂ 、150mM KCl、0.01％X-100, pH 9.0, to bring the pH of all samples to pH 9.0.

In the following activity measurements, the temperature was 25 ℃. Mu.l of the diluted protease sample was mixed with 1.5ml of assay buffer pH 9.0 and purified by adding 1.5ml of pNA substrate (50 mg dissolved in 1.0ml DMSO and further with 0.01%X-100 diluted 45-fold) to initiate the active reaction, and monitoring A by a spectrophotometer after mixing ₄₀₅ As a measure of (residual) protease activity. Incubation at 37 ℃ was performed at different pH values and activity measurements were plotted as residual activity versus pH. Residual activity was normalized to that of parallel incubation (control) in which protease was diluted to a in assay buffer at pH 9.0 ₂₈₀ =1.0 and incubated at 5 ℃ for 2 hours, then activity measurements were performed as with other incubations. Protease samples were diluted prior to activity measurement to ensure that all activity measurements were within the linear portion of the measured dose-response curve.

Example 15 temperature-Activity assay

Protazyme AK tablets were used to obtain temperature profiles. Protazyme AK tablets are azurin-dyed crosslinked casein prepared into tablets by the company Meggese.

Assay buffer: 100mM succinic acid, 100mM HEPES, 100mM CHES, 100mM CABS, 1mM CaCl ₂ 、150mM KCl、0.01％X-100, adjusted to using NaOHpH 9.0。

Protazyme AK tablets were suspended in 2.0ml 0.01% by gentle stirringX-100. Mu.l of this suspension and 500. Mu.l of assay buffer were mixed in an Eppendorf tube and placed on ice. Mu.l protease sample (diluted in 0.01% Triton X-100) was added. The assay was started by transferring the Eppendorf tube to an Eppendorf thermo-mixer set to the assay temperature. The tube was incubated on an Eppendorf homomixer for 15 minutes at the highest shaking rate. The assay incubation was stopped by transferring the tube back to the ice bath. The tube was centrifuged in an ice-cold centrifuge for several minutes and the supernatant a was read by a spectrophotometer ₆₅₀ . Blank buffer (instead of enzyme) was included in the assay. A is that ₆₅₀ (protease) -A ₆₅₀ The (blank) is a measure of protease activity. Assays were performed at different temperatures and activity measurements were plotted as relative activity versus incubation temperature. The relative activity was normalized to the highest activity (optimum temperature). The protease samples were diluted to ensure that all activity measurements were within the near linear portion of the measured dose-response curve.

The optimum activities (pH and temperature activities) are summarized in Table 5, and detailed comparisons of the pH stability data for proteases at acidic pH values are shown in Table 6.

Table 5: optimum pH and temperature of protease

Table 6: pH stability of protease, pH between 2.0 and 5.0

.....................................................................

Example 16: absorption purity of purified protease sample

₂₈₀ ₂₆₀ Determination of A/A ratio

A of purified protease sample ₂₈₀ /A ₂₆₀ The ratio was determined as follows.

A ₂₆₀ Meaning the absorbance of the protease sample at 260nm relative to buffer blank in a 1cm path length cuvette. A is that ₂₈₀ Meaning the absorbance at 280nm of the same protease sample relative to the buffer blank in a 1cm path length cuvette.

Diluting the purified protease sample in buffer until spectrophotometer A ₂₈₀ The reading is within the linear portion of its response curve. Determination of A from the readings ₂₈₀ /A ₂₆₀ Ratio: NRRL 18262 was 1.83 for the Nocardia species.

Example 17: protease-degraded soybean meal (SBM) insolubility derived from nocardia sp NRRL 18262 Capacity of the part

Proteases from Nocardia species NRRL 18262 were tested for their ability to make insoluble/indigestible parts of the SBM accessible to digestive enzymes and/or added exogenous enzymes.

The performance was compared with two aspartic proteases, protease I and protease II, prepared as described in WO 95/02044. The document also discloses its use in feed. Protease I is an Aspergillus pepsin type II protease, and protease II is an Aspergillus pepsin type I protease (both aspartic proteases, i.e., non-subtilisins), derived from Aspergillus aculeatus (Aspergillus aculeatus) (cf. Handbook of Proteolytic Enzymes [ Pron. Handbook ] mentioned above).

The test substrates, so-called soybean residues, are produced in a process that mimics the digestive tract of monogastric animals, including pepsin treatment at pH 2 and pancreatin treatment at pH 7.

In the pancreatin treatment step, a series of commercially available enzymes are added at high doses to degrade SBM components that are accessible to existing commercially available enzymes.

The following enzymes (all commercially available from Novozymes A/S) were added: ALCALAASE ^TM 2.4L、NEUTRASE ^TM 0.5L、FLAVOURZYME ^TM 1000L、ENERGEX ^TM L、BIOFEED ^TM Plus L、PHYTASE NOVO ^TM L. The SBM used was a standard 48% protein SBM for feed, which has been pelleted.

After treatment, only 5% of the total protein was left in the resulting soybean residue.

FITC labelling protocol

The residue was then labeled with FITC (Molecular Probes, F-143), as follows: the soybean residue (wet weight 25g, dry weight about 5 g) was suspended in 100ml of 0.1m carbonate buffer (pH 9) and stirred at 40 ℃ for 1 hour. The suspension was cooled to room temperature and treated overnight with fluorescein 5-isothiocyanate (FITC) in the dark. The uncoupled probe was removed by ultrafiltration (10.000 Mw cut-off).

FITC assay

FITC-labeled soybean residues were used to test the ability of proteases to degrade soybean residues using the following assay: at 37℃0.4ml of protease sample (A ₂₈₀ =0.1) was mixed with 0.4ml FITC-soybean residue (10 mg/ml 0.2M sodium phosphate buffer suspended at pH 6.5), and relative fluorescence units (RFU 485/535nm were measured after 0 hours and after 22 hours incubation; excitation wavelength/monitoring wavelength). Prior to RFU determination, samples were centrifuged at 20.000, 20.000x G for 1min and 250 μl of supernatant was transferred to a black droplet scale. Measurements were performed using a VICTOR 1420 multi-label counter (in vitro, denmark). Iain d johnson generally describes RFU as follows: introduction to Fluorescence Techniques, handbook of Fluorescent Probes and Research Chemicals, [ fluorescence technical theory, fluorescence probes and handbooks of research chemicals ] ]Molecular Probes]Richard P.Haugland, 6 th edition, 1996 (ISBN 0-9652240-0-7).

Blank samples were prepared by adding 0.4ml buffer instead of enzyme sample.

RFU _{Sample of} ＝ΔRFU _{Sample of} -ΔRFU _{Blank space} Wherein DeltaRFu=rfu (22 hours) -RFU (0 hours)

FITC value (RFU) _{Sample of} Values) are shown in table 7 below. The error range of FITC values is typically +/-20.000. In contrast to protease I and protease II, proteases derived from nocardia sp.

TABLE 7 protease degradation ability of soybean residues

Protease enzyme	FITC(+/-20000)
		Nocardia species NRRL 18262	92900
Protease I	-9200
		Protease II	-1200

Example 18: in vitro testing of proteases derived from nocardia sp NRRL 18262

Protease derived from Nocardia species NRRL 18262 was tested together with protease derived from Bacillus species NCIMB 40484 ("PD 498", prepared as described in example 1 of WO 93/24623), together with protease-containing enzyme preparation FLAVOURZYME from Aspergillus oryzae ^TM Energy to solubilize maize-SBM (maize-soybean meal) proteins in an in vitro digestive System (simulated digestion of monogastric animals) commercially available from Novozymes A/S, novozymes of Danish Bagnograd (Bagsvaerd, denmark) Force. For blank treatment, maize-SBM was incubated without exogenous protease.

In vitro model overview

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Substrate(s)

10g of the maize-SBM diet was used, wherein the maize-SBM ratio was 6:4 (w/w). The protein content in SBM was 43% (w/w) and the protein content in maize meal was 8.2% (w/w). The total amount of protein in the 10g maize-SBM diet was 2.21g.

Digestive enzyme

Pepsin (Sigma P-7000;539U/mg, solid), pancreatin (Sigma P-7545;8XU.S.P. (United states Pharmacopeia)).

Enzymatic protein assay

A-based using the principles outlined below ₂₈₀ Values and amino acid sequence (amino acid composition) the amount of protease protein was calculated: S.C.Gill and P.H.von Hippel, analytical Biochemistry [ analytical biochemistry ]]182,319-326,(1989)。

Experimental procedure for in vitro model

1. 10g of substrate was weighed into a 100ml flask.

2. At 0min, 46ml HCl (0.1M) containing pepsin (3000U/g diet) and 1ml protease (0.1 mg enzyme protein/g diet, FLAVOURZYME) ^TM Except for: 3.3mg/g diet) was added to the flask while mixing. The flask was incubated at 40 ℃.

3. At 20-25min, the pH was measured.

4. At 45min, 16ml of H was added ₂ O。

5. At 60min, 7ml NaOH (0.4M) was added.

6. At 80min, 5ml of the mixture containing NaHCO of pancreatin (8.0 mg/g diet) ₃ (1M)。

7. At 90min, the pH was measured.

8. At 300min, the pH was measured.

9. At 320min, 30ml aliquots were removed and centrifuged (10000 Xg, 10min,0 ℃).

10. The total soluble protein in the supernatant was determined.

Protein assay

The supernatants were analyzed for protein content using the Kjeldahl method (determination of nitrogen%; A.O.A.C. (1984), official Methods of Analysis [ official analytical methods ] 14 th edition, association of Official Analytical Chemists [ official analytical chemist's collection ], washington, D.C.).

Calculation of

For all samples, in vitro protein solubility was calculated using the following formula.

Amount of protein in the dietary sample: 10g 22.1% = 2.21g

If all proteins were dissolved in 75ml of liquid, the protein concentration in the supernatant would be: 2.21g/75 ml.apprxeq.2.95%.

Note that the supernatant also includes digestive enzymes and exogenous enzymes. To determine solubility, the protein contributions of digestive and exogenous enzymes should be subtracted as much as possible from the protein concentration of the supernatant.

Protein% = ((X mg pancreatin/g diet X10 g diet X0.69X 100%)/(1000 mg/g X g)) + ((YU pepsin/g diet X10 g diet X0.57x 100%)/(539U/mg X1000 mg/g X g)),

Wherein 0.69 and 0.57 refer to the protein content of the pancreatin and pepsin preparations used (i.e. 69% pancreatin and 57% pepsin as protein measured according to the kjeldahl method mentioned above).

Protein from exogenous enzyme (Z mg EP/g diet) = (Z mg EP/g diet x 10g diet x 100%)/(1000 mg/g x g)

Corrected protein% in supernatant = protein% in supernatant analyzed,% protein from digestive enzymes%o+protein from exogenous enzymes)

Protein solubilization (%) = (corrected protein% x 100% in supernatant)/2.95%

The following results indicate that proteases derived from nocardia sp NRRL 18262 have significantly better effects on protein solubilization than the blank and bacillus sp NCIMB 40484 proteases.

^a、b、c Values within a column that do not share a common superscript letter are significantly different, P<0.05.SD is standard deviation; n is the number of observations.

Example 19: degradation of phytolectin SBA and soybean Bowman-Birk and Kunitz inhibitors

Proteases from Nonomia species NRRL 18262 and Bacillus species NCIMB 40484 were tested for their ability to hydrolyze soybean lectin (SBA) and soybean Bowman-Birk and Kunitz trypsin inhibitors.

Pure SBA (Fluka 61763), bowman-Birk inhibitor (Sigma T-9777) or Kunitz inhibitor (trypsin inhibitor from soybean, boehringer Mannheim 109886) were incubated with protease at pH 6.5 and 37 ℃ for 2 hours (protease: antinutritional factor = 1:10, based on a ₂₈₀ ). Incubation buffer: 50mM dimethyl glutaric acid, 150mM NaCl, 1mM CaCl ₂ 、0.01％ Triton X-100，pH 6.5。

The ability of proteases to degrade SBA and protease inhibitors was estimated based on the disappearance of the native SBA or trypsin inhibitor bands and the appearance of low molecular weight degradation products on SDS-PAGE gels. The gel was stained with coomassie blue and the band intensity was determined by scanning.

The results (expressed as% of degraded antinutritional factors) are shown in table 8 below.

It is expected that the ability to degrade anti-nutritional factors in soybeans can also be estimated by using anti-SBA antibodies, bowman-Birk inhibitors or Kunitz inhibitors by applying Western techniques after incubation of soybean meal with candidate proteases (see WO 98/56260).

TABLE 8

Example 20: influence of acid-stable nocardia protease on growth performance of broiler chickens

The test was performed according to the French official living animal experimental instructions. Day-old broilers ('Ross PM 3') were provided separately by gender from commercial hatcheries.

Chickens were housed in a cable bottom rack chicken cage, and these cages were placed in an environmentally controlled room. Feed and tap water are provided at will.

On day 8, chickens were grouped per 6 chickens by body weight, assigned to a control treatment group (receiving experimental diet without enzyme), or assigned to an enzyme treatment group (receiving experimental diet supplemented with 100mg protease enzyme protein/kg feed).

Each treatment was repeated using 12 groups of 6 groups per sex. Each group was weighed on days 8 and 29. Mid-term feed consumption was determined and weight gain and feed conversion rate were calculated.

The experimental diet was based on cornstarch and soy flour (44% crude protein) as the major ingredients (table 5). The feed was pelleted at about 70℃ (die configuration: 3X 20 mm). An appropriate amount of protease is diluted in a fixed amount of water and sprayed onto the pelleted feed. For the control treatment, the treatment was handled in the same manner using an appropriate amount of water.

For statistical evaluation, a two-factor analysis of variance (factors: treatment and gender) was performed using the GLM procedure of SAS package (SAS Institute inc., 1985). If significant treatment effects were shown (p < 0.05), differences between the treatment averages were analyzed using the Duncan test. An improvement in weight gain, and/or an improvement in feed conversion, and/or an improvement in the nutritional value of the soy flour (considering that cornstarch is a highly digestible ingredient) is expected.

Reference to the literature

EEC(1986):Directive de la Commission du 9 avril 1986 fixant la méthode de calcul de la valeurénérgetique des aliments composés destinés à la volaille.Journal Officiel des Communautés Européennes L130,53-54

SAS institute company (1985):User's Guide[/>user guide]North Carolina Karin, 5 th edition

Table 5 composition of experimental diet

¹ Content analyzed: 90.6% dry matter, 45.3% crude protein, 2.0% crude fat, 4.9% crude fiber

² Feed corresponding to 90mg of rasagilin-Na/kg as anticoccidial

³ Based on the analysis of nutrient content calculation (EC equation; EEC, 1986)

Feed ingredient suppliers

Corn starch: roquettes Frmores, F-62136, lawster France (Lestrem, france)

Soy flour 44: rekasan GmbH, D-07338, kaulsdorf, germany

Animal fat: fondoirs Gachot SA, F-67100, styrasburg, france

Soybean oil: ewoco Sarl, F-68970, french lid Mark (Guemar, france)

DL-methionine: producit Roche SA, F-92521, seine, france, heinae, pair of Paenie-sur-Seine

MCP: brenntag Lorraine, F-54200, chart of France (Toul, france)

Salt: minoterie Moderne, F-68560, irsanger (Hirsinge, france)

And (2) an adhesive: minoterie Moderne, F-68560, irsanger (Hirsinge, france)

Premix (AM vol pair NS 4231): agrobase, F-01007, british area Bragg (Bourg-en-Bresse, france)

Ball An (Avatec): producit Roche SA, F-92521, seine, france, heinae, pair of Paenie-sur-Seine

Example 21: turkeys and salmon premix and diet supplemented with acid stable nocardia protease.

A premix (content per kilogram) was prepared having the following composition:

protease from nocardia sp NRRL 18262 was added to the premix (prepared as described in example 2) in an amount equivalent to 10g protease enzyme protein/kg.

The pelleted turkey initial and growth phase diets were prepared as follows, with the compositions shown in the following table (based on Leeson and Summer (1997), but usingOptimization procedure recalculated without meat meal) and containing 100mg protease enzyme protein/kg:

the milled corn, soy flour, fish meal and vegetable fat are mixed in a cascade mixer. Limestone, calcium phosphate and salt were added together with the above premix in an amount of 10g/kg diet and then mixed. The resulting mixture was subjected to pelletization (first steam conditioning, then pelletization step).

As generally outlined above, two salmon diets were also prepared. The actual composition is shown in the following table (compiled from Refstie et al (1998), aquaculture]Volume 162, pages 301-302). Using The feed optimization program recalculates the estimated nutrient content.

Proteases derived from nocardia albicans prepared as described in example 2 were added to the diet in amounts corresponding to 100mg protease protein/kg.

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Example 22: purity determination of protease-containing enzyme products

The purity of a protease-containing enzyme product (e.g., a protease preparation, such as a commercial multicomponent enzyme product) may be determined by a method based on fractionation of the protease-containing enzyme product on a size exclusion column. Size exclusion chromatography (also known as gel filtration chromatography) is performed on a porous gel matrix (packed in a chromatographic column) with a pore size distribution comparable to the size of the protein molecules to be separated. Relatively small protein molecules may diffuse from the surrounding solution into the gel, whereas larger molecules will not diffuse to the same extent into the gel due to their size. Thus, protein molecules are separated according to their size, with larger molecules eluting from the column before smaller molecules.

And (5) measuring the protein concentration.

Protein concentration in the protease-containing enzyme product was determined using BCA protein assay kit from pierce (same as pierce catalog No. 23225). The sodium salt of bicinchoninic acid (BCA) is a stable water-soluble compound capable of reacting with cuprous ions (Cu ¹⁺ ) A dark purple complex forms. The BCA reagent forms the basis of a BCA protein assay kit, enabling the monitoring of proteins and alkaline Cu ²⁺ The cuprous ions produced in the reaction (biuret reaction). The color produced by this reaction is stable and increases proportionally with increasing protein concentration (Smith, P.K. et al (1985), analytical Biochemistry [ analytical biochemistry ]]Volume 150, pages 76-85). By mixing 50 parts of reagent A with 1 part of reagent B (reagent A is Pierce catalog No. 23223, contains BCA and tartrate in an alkaline carbonate buffer; reagent B is Pierce catalog No. 23224, contains 4% CuSO) ₄ *5H ₂ O) mixing to prepare BCA working solution. Mu.l of the sample was mixed with 3.0ml of BCA working solution. After 30 minutes at 37 ℃, the sample was cooled to room temperature and a was read ₄₉₀ As a measure of the concentration of protein in the sample. Bovine serum albumin dilutions (pierce catalog No. 23209) were included as standard in the assay.

Sample pretreatment.

If the protease-containing enzyme product is solid, the product is first dissolved/suspended in 20 volumes of 100mM H at pH 6 ₃ BO ₃ 10mM 3,3' -dimethyl glutaric acid, 2mM CaCl ₂ (buffer A) at 5℃for at least 15 minutes, and if the enzyme at this stage is a suspension, the suspension is filtered through a 0.45. Mu. Filter to give a clear solution. From this point in time, the solution is treated as a liquid protease-containing enzyme product.

If the protease-containing enzyme product is liquid, it is first dialyzed against 100 volumes of buffer A+150mM NaCl (buffer B) in a 6-8000Da cutoff SpectraPor dialysis tube (catalog No. 132 670, from Spectrum Medical Industries) at 5℃for at least 5 hours to remove formulation chemicals that might give the liquid protease-containing enzyme product a high viscosity, which is detrimental to size exclusion chromatography.

If a precipitate forms during dialysis, the dialyzed protease-containing enzyme product is filtered through a 0.45. Mu. Filter. The protein concentration in the dialyzed enzyme product was determined using the protein concentration measurement described above, and the enzyme product was diluted with buffer B to give a sample having a protein concentration of 5mg/ml which was ready for size exclusion chromatography. If the enzyme product after dialysis has a protein concentration of less than 5mg/ml, it is used as such.

Size exclusion chromatography.

300ml HiLoad26/60 Superdex75pg column (Amersham pharmacia Biotech Co. (Amersham Pharmacia Biotech)) were equilibrated in buffer B (flow rate: 1 ml/min). 1.0ml of the protease-containing enzyme sample was applied to the column, and the column was eluted with buffer B (flow rate: 1 ml/min). 2.0ml fractions were collected from the column outlet until all applied samples eluted from the chromatographic column. The protein content (see protein concentration assays above) and protease activity of the collected fractions were analyzed by appropriate assays. An example of a suitable assay is the Suc-AAPF-pNA assay. Other suitable assays are, for example, CPU assays and Protazyme AK assays. Conditions (e.g., pH) of the protease activity assay are adjusted to measure as much protease as possible in the fractionated sample. The conditions of the above mentioned assays are examples of suitable conditions. Other suitable conditions are mentioned above in the section on measurement of protease activity. Protein peaks that are active in one or more protease assays are defined as protease peaks. The purity of the protease peaks was calculated as follows, dividing the amount of protein in the peak by the amount of total protein in all identified protease peaks. The purity of the protease-containing enzyme product was calculated as follows, using the procedure described above, dividing the amount of protein in the protease peak by the amount of protein in all identified protease peaks.

Claims

1. An animal feed additive comprising a polypeptide having protease activity, wherein the polypeptide has at least 70% sequence identity to SEQ ID No. 1; characterized in that the enzyme is formulated as a formulation selected from the group consisting of:

i. granules prepared by an extrusion process;

particles prepared by a spray drying process;

particles comprising a salt core, e.g. a sodium sulphate or sodium chloride core, and a protease-containing layer; and

granules prepared by a high shear granulation process.

2. An animal feed additive according to claim 1, wherein the polypeptide having protease activity is obtained or obtainable from nocardia sp NRRL 18262.

3. An animal feed additive according to claim 1 or 2, wherein the polypeptide having protease activity has at least 75% sequence identity with the polypeptide of SEQ ID No. 1, e.g. at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity with the polypeptide of SEQ ID No. 1.

4. An animal feed additive according to claims 1 to 3, wherein the polypeptide having protease activity is selected from polypeptides having at least 75%, such as at least 80%, such as at least 85%, preferably at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3.

5. The animal feed additive according to any one of claims 1 to 4, wherein the pellets prepared by the extrusion process are pellets comprising

hydrophobic substances; and

solid carrier.

6. An animal feed additive according to any one of claims 1 to 5, wherein the spray drying process comprises the steps of

(b) Spraying the spray liquid in a spray tower.

7. The animal feed additive of claim 6, wherein the granule comprises or consists of a stable protease, dextrin and water.

8. The animal feed additive according to any one of claims 1 to 5, wherein the enzyme granulate comprises a salt core and a protease-containing layer, wherein the protease is a polypeptide having protease activity and at least 70% sequence identity to SEQ ID No. 1.

9. The animal feed additive according to claim 8, wherein the salt core is a sodium sulfate or sodium chloride core or a combination thereof.

10. An animal feed additive according to any one of claims 8 to 9, wherein the enzyme granules have a particle size of 100-2000 microns, preferably 200-1500 microns, more preferably 300-1200 microns.

11. An animal feed additive according to any one of claims 8 to 10, wherein the enzyme granulate is prepared in a fluidised bed apparatus.

12. The animal feed additive according to any one of claims 1 to 5, comprising a polypeptide having protease activity and at least 70% sequence identity to SEQ ID No. 1 in a granulate prepared by a high shear granulation process.

13. An animal feed additive according to claim 12, wherein the high shear granulation process comprises the steps of

A. Forming a powder mixture by combining at least the following

i. Cellulose or derivatives thereof

ii, an adhesive; and

optionally a filler; and

B. adding liquid phase granulating agent

14. A particle comprising a salt core, e.g. a sodium sulphate or sodium chloride core, and a protease-containing layer, wherein the protease is a polypeptide having protease activity and at least 70% sequence identity to SEQ ID No. 1.

15. A particle comprising a polypeptide having protease activity and having at least 70% sequence identity to the polypeptide of SEQ ID No. 1; the particles are prepared by a spray drying process.

16. A particle comprising a polypeptide having protease activity and having at least 70% sequence identity to the polypeptide of SEQ ID No. 1, said particle being prepared by an extrusion process.

17. A particle comprising a polypeptide having protease activity and having at least 70% sequence identity to the polypeptide of SEQ ID No. 1, said particle being prepared by a high shear granulation process.

18. Use of a granulate as defined in any one of claims 14 to 17 in animal feed.

19. A method of preparing a particle comprising a polypeptide having protease activity and having at least 70% sequence identity to the polypeptide of SEQ ID No. 1, said method comprising a process comprising a formulation process selected from the group consisting of

i. Extruding;

spray drying;

spraying or wetting the salt core with a protease-containing liquid; and

high shear granulation.