MXPA99011240A - High-activity phytase compositions - Google Patents

Abstract

A process is disclosed for preparing aqueous phytase-containing liquids involving culturing microorganisms of the genus Aspergillus or Trichoderma in a medium containing assimilable carbon and nitrogen sources (e.g. glucose and ammonium ions), filtering the medium and subjecting the resulting filtrate to ultra-filtration to give an aqueous composition having at least 14,000 FTU/g. This aqueous liquid can be used to prepare granulates that can be incorporated in animal feedstuffs.

Description

HIGH ACTIVITY FIELD COMPOSITIONS Description of the invention The present invention relates to the preparation and formulation of phytase enzymes and to their use for preparing granulates for food enzymes in animal feeds.

Background of the Invention It is becoming more common to use various enzymes such as phytases in animal feed, for example, in cattle. These enzymes are included in order to improve the uptake of nutrients or minerals from the food by the animal, and can also help the digestion capacity. These are usually produced by the cultivation of microorganisms in large-scale fermenters operated by industrial producers of enzymes. At the end of the fermentation the resulting "broth" is usually subjected to a series of filtration steps to separate the biomass (the microorganisms) from the desired enzyme (in solution) .The enzyme solution is either sold as REF .: 32199 a liquid (often after the addition of various stabilizers) or processed to a dry or anhydrous formulation. The liquid and anhydrous formulations of enzymes are used on a commercial scale by the animal feed industry. Liquid formulations can be added to the feed after pellet formation in order to avoid heat inactivation of the enzyme, which could occur during the pelletizing process. _ Anhydrous formulations usually involve the formation of pellets by steam where the food is subjected to one or more steam injections before the formation of pellets. In the subsequent pellet formation step the feed is forced through a die or die and the resulting strips are cut into suitable pellets of variable length. During this process temperatures can rise to 60-95 ° C. _ Phytases are enzymes that (at least partially) hydrolyse phytate (myo-inositolhexakis-phosphate) to myo-inositol and inorganic phosphate. These enzymes are found in wheat fiber, in vegetable seeds, in the intestines of animals and can be produced by microorganisms. Phytases are provided in animal feeds because, since they are able to degrade phytate, they can increase the bioavailability of phosphorus and other nutritional components to the animal. Phytases may also increase. the capacity of calcium digestion. Phosphorus is an essential element for the development of organisms. For livestock, the feed is "frequently supplemented with inorganic phosphorus in order to obtain good development in monogastric animals." However, there is often no need for this in foods for ruminant animals, because the microorganisms present in the rumen produce enzymes catalyzing the conversion of phytase to inositol and inorganic phosphate Phthalate degradation is often desirable because phytic acid can be antinutritional, since it chelates useful minerals such as calcium, zinc, magnesium and iron, and can also react adversely with proteins, thereby decreasing their bioavailability to the animal.The addition of phytase can also reduce the amount of inorganic food that needs to be added, and thus less phosphorus is excreted in the manure, which is better for the environment. ~ The gene for various phytase enzymes has been cloned and expressed.The European patent EP- A-0,420,358 (Gist-Brocades) describes the expression of microbial phytases. In a recent European patent application EP-A-0, 684, 313 (Hoffman-La Roche) the DNA sequences coding for various polynucleotides having phytase activity are described. European Patent EP-A-0 758, 018 (Gist-Brocades) refers to methods for improving the stability of enzymes, especially for use as animal feeds, and refers to phytases. WO-A / 03612 (Alko) describes the production of phytase degrading enzymes in Tri ch oderma, while WO-A-97/16076 (Novo Nordisk) describes enzyme-containing preparations for use in food manufacturing for animals, which comprise various hydrophobic substances. Animal feed represents one of the largest costs incurred in the maintenance of livestock and other animals. In addition, additives such as enzymes such as phytase can be added significantly to the cost of animal feed. It is an object of the present invention to make it possible to provide phytase compositions which are "cheaper to produce." This can be achieved by being able to manufacture highly concentrated or highly concentrated phytase compositions. the present Applicant is able to make these high activity compositions, and this will be discussed below.An additional advantage to make possible the preparation of high activity phytase compositions, which has been perceived by the Applicant, is that these compositions can show a marked increase in stability, especially during a pelletizing process in the preparation of animal feed (pellets), and thus are more likely to retain the higher ~ phytase activity than the prior art compositions, with the weather.

Description of the invention In a first aspect of the present invention, there is provided a process for the preparation of an aqueous liquid comprising a phytase, the process comprising: (a) the cultivation, in an aqueous medium, of a microorganism of the genus Aspergillus or Tri choderma having a heterologous phytase gene under the control of a glucoamylase promoter (for Aspergi llus) or cellobiohydrolase (for Tri ch oderma) under conditions that allow for "recombinant expression of the phytase, where the medium comprises, as a food for the microorganism, a source of assimilable carbon and a source of assimilable nitrogen, (b) filtration of the aqueous medium to remove the microorganisms, to give an aqueous filtrate, and _ (c) to hold the filtrate of the step (b) to ultrafiltration to give an aqueous liquid having a phytase concentration of at least 14,000 FTU / g.It has been found that this process provides a particularly high concentration of phytase in the composition. n aqueous resulting. This has allowed the preparation of other phytase compositions, also at high levels of activity, which means that not only is the process cheaper (per unit of enzymatic activity), but also that the more concentrated phytase containing compositions have been found. , are much more stable than their "less concentrated counterparts." Microorganisms are preferably of the species Aspergillus niger, Aspergillus oryzae or Tri choderma reesei For the Aspergillus organisms, the phytase gene is suitably under the control of a promoter. of glucoamylase (or amyloglucosidase, AG) For Trichoderma organisms, it is preferred to use a celiobiohydrolase promoter.The assimilable carbon source may comprise glucose and / or maltodextrin, and / or the source of assimilable nitrogen may comprise ammonium ions. and the "ammonium ions, may be the only sources of carbon or nitrogen assimilable in the medium - aqueous. Go, it is contemplated that complex sources of carbon or nitrogen are not used. Ammonium ions can be provided either as ammonia or. like an ammonium salt. Preferred ammonium salts include ammonium nitrate, ammonium sulfate and ammonium phosphate. Preferably, the carbon and / or nitrogen source is supplied to the culture medium during the fermentation process. The rate or rate of supply from any source can be substantially the same as that which is consumed by the microorganisms. In this way, the carbon and / or nitrogen source can be provided in a continuous manner. The carbon and nitrogen sources can be provided separately, or in the same supply. The aqueous-resulting liquid can have a phytase concentration of at least 16,000, and possibly even 18,000 or more FTU / g. By using these particular organisms under these conditions, the filtrate will be relatively concentrated. This allows it to be subject to ultrafiltration. In some methods of the prior art, the resulting filtrate contains too much waste and other substances to allow ultr filtration (filter clogging). However, in the process of the present invention, the filtrate is relatively "clean" which allows the filtrate to be subjected to ultrafiltration, without any further processing, and therefore a particularly high aqueous composition can be obtained by ultrafiltration. concentration. The methods of the prior art have discussed the possibility of subjecting either the filtrate or the aqueous composition to crystallization and / or to color removal steps, for example filtration with mineral carbon. However, these two additional steps (which could be added to the production cost = of the phytase) can be omitted in the present invention. Preferably, the microorganisms do not possess, or at least do not express, a glucoamylase gene. This means that the microorganism can devote more energy to the production of the phytase. The microorganism may possess multiple copies of the phytase gene. It has been found that this increases phytase production levels, because there are more phytase genes to be expressed. The aqueous composition may be substantially free of taka-amylase. In the process of the invention, it is preferred that _ (substantially) all carbon and / or nitrogen sources have been consumed by the microorganisms before the filtration takes place in step (b). This can be achieved by allowing the fermentation to continue for some time after the last supply of the carbon and / or nitrogen sources. Alternatively, fermentation may be allowed to continue beyond the stage when all carbon and / or nitrogen sources have been added. The advantage of this, as will be apparent, is that the aqueous composition can then be (substantially) free of carbon and / or nitrogen sources (e.g., glucose and / or ammonium ions). Again, this may constitute a cleaner aqueous liquid, which may contain fewer by-products. By reducing the number of by-products, the number of processing steps required to be able to use either the aqueous liquid, or to be able to obtain a high phytase activity, can be minimized. The most preferred organism is Aspergillus niger. It also be preferred that the phytase be expressed in a microorganism, with a signal sequence of glucoamylase. - The resulting aqueous liquid containing phytase can then be used for a variety of purposes, although its application in animal feeds is specifically contemplated here. A second aspect of the invention relates to this aqueous liquid, such as is preparable by a process of the first aspect, which comprises the phytase at a concentration of at least 14,000 FTU / g. In the specification a "phytase" means not only the phytase enzymes of natural origin, but any enzyme possessing phytase activity, for example the ability to catalyze the reaction involving the elimination or release of inorganic phosphorus (phosphate) from the io-inositol phosphates Preferably, the phytase will belong to class EC 3.1.3.8 The phytase itself is preferably a fungal phytase, such as that derived from a species of Aspergi llus or Tri choderma. Processes for the preparation of phytase formulations in the form of granules using an edible carbohydrate polymer as a carrier The carrier can be in a particulate or powder form The aqueous liquid containing phytase, such as a solution or a suspension can be mixed with the solid carrier and allowed to absorb on the carrier During or after mixing, the liquid that The phytase and the carrier are processed in a granulate, which can be subsequently dried. The use of the carbohydrate carrier can allow the absorption of large amounts of the composition (and therefore of phytase). The mixture can be used to form a plastic paste or a non-elastic mass which can be easily processed into granules, for example it is extrudable. Suitably, the carrier is not fibrous, which allows for easier granulation: fibrous materials can prevent granulation by extrusion. A number of prior art documents refer to pellets containing various enzymes, but they find use as detergents, often in washing compositions. In contrast, the present application finds use in animal feeds and for that reason the granulates of the invention are edible (by animals) and preferably also digestible. It will therefore not be surprising that the granulates, the granules and the compositions of the invention are free of soap, detergents and bleaching or bleaching compounds, zeolites, binders, fillers (Ti02, kaolin, silicates, talc, etc.) to name but a few. The edible carbohydrate polymer must be chosen such that it is edible by the animal for whom the food is intended, and preferably also digestible. The polymer preferably comprises glucose units (for example, a polymer containing glucose), or (C6H? 0? 5) n.

Preferably, the carbohydrate polymer comprises units of α-D-glucopyranose, amylose (a linear polymer of (1-> 4) -aD-glucan) and / or amylopectin (a branched D-glucan with D- (1-4) -linkages. Starch is the preferred carbohydrate polymer Other suitable polymers containing glucose can be used instead, or in addition to starch, and include α-glucans, β-glucans, pectin (such as protopect ina), and glycogen. Derivatives of these carbohydrate polymers, such as ethers and / or esters thereof, are also contemplated, although gelatinized starch is often avoided. Suitably, the carbohydrate polymer is insoluble in water.

In the examples described herein, corn starch, potato starch and rice starch are used. However, the starch obtained from other sources (for example, plants, such as vegetables or crops), such as tapioca, cassava, wheat, corn, sago, rye, oats, barley, yam, sorghum or maranta is also applicable. Similarly, native or modified types of starch (e.g. dextrin) can be used in the invention. Preferably, the carbohydrate (eg, starch) contains little or no protein, for example, less than 5% (w / w), such as less than 2% (w / w), preferably less than 1% (W / W). • At least 15% (w / w) - of the solid carrier can comprise the carbohydrate polymer (such as starch). Preferably X, however, at least 30% (w / w) of the solid carrier comprises the carbohydrate, optimally at least 40% Ip / p). Advantageously, the major component of the solid carrier is the carbohydrate (for example starch), for example more than 50% (w / w), preferably at least 60% (w / w), suitably at least 70% (w / w) , and optimally at least 80% (w / w). These percentages by weight are based on the total weight of the non-enzymatic components in the final dry granulate.

The amount of phytase containing liquid, which can be absorbed onto the carrier, is usually limited by the amount of water that can be absorbed. For natural, granular starch, this can vary between 25 ~ - 30% (w / w), without using elevated temperatures (which cause the starch to swell). In practice, the percentage of enzyme liquid that is to be added to the carbohydrate will often be much greater than this because the enzyme-containing liquid will usually contain a significant amount of solids. The phytase solution may contain about 25% (w / w) solids, as a result of which the carbohydrate (for example starch) and the phytase solution can be mixed at a rate or ratio of carbohydrate: 0.5: 1 to 2: 1, for example 1.2: 1 to 1.6: 1, such. as a ratio of approximately 60% (w / w): 40% (w / w), respectively, Preferably, the amount of liquid added to the solid carrier is such that (substantially) all the water in the liquid (aqueous) is absorbed by the carbohydrate present in the solid carrier. At elevated temperatures, starch and other carbohydrate polymers can absorb much greater amounts of water under swelling. For this reason, the carbohydrate polymer is desirably capable of absorbing water (or aqueous liquids containing enzymes). For example, corn starch can absorb up to three times its weight of water at 60 ° C and up to ten times at 70 ° C. The use of higher temperatures in order to absorb a liquid containing greater amount of enzyme, is thus contemplated by the present invention, and of course it is preferable especially when dealing with thermostable phytase enzymes. For these enzymes, the mixture of the solid carrier and the liquid can therefore be conducted at elevated temperatures (for example, above ambient temperature), such as above 30 ° C, preferably above __ 40 ° C and optimally above 50 ° C. Alternatively, or in addition, the liquid can be provided at this temperature. However, in general, non-swelling conditions are preferred at lower temperatures (for example at room temperature) to minimize the loss of activity arising from the instability of the phytases (heat sensitive) at higher temperatures. Suitably, the temperature during the mixing of the enzyme and the liquid is 20 to 25 ° C.

The jaechanical processing used in the present invention for the preparation of the mixture of the liquid containing phytase and the solid carrier in granules (in other words, granulation) can employ known techniques frequently used in food, food and enzyme formulation processes. This may comprise expansion, extrusion, spheronization, pelletizing, high-cut granulation, drum granulation, fluidized bed agglomeration or a combination thereof. These processes are usually characterized by a mechanical energy input, such as the drive of a screw, the rotation of a mixing mechanism, the pressure of a rotating mechanism of a pelletizing apparatus, the movement of the particles by a lower plate rotating a fluidized bed agglomerator or the movement of the particles by a gas stream, or a combination thereof. These allow the solid carrier (for example in the form of a powder) to be mixed with the liquid containing phytase (an aqueous solution or suspension), and thus subsequently granulated. In a further embodiment of the invention, the granulate (for example an agglomerate) is formed by spraying or spraying or coating the liquid containing phytase on the carrier, such as in a fluidized bed agglomerator. Here, the resulting granules may include an agglomerate as may be produced in a fluidized bed agglomerator. Preferably, the mixture of the phytase-containing liquid and the solid carrier further comprises kneading the mixture. This can improve the plasticity of the mixture in order to facilitate granulation (for example extrusion). If the granulate is formed by extrusion it is preferably carried out at low pressure. This may offer the advantage that the temperature of the mixture, which is extruded, will not increase, or will only increase slightly. Extrusion at low pressure includes extrusion for example in a basket or dome extruder of the Fuji_ Paudal type. Preferably, the extrusion does not result in the temperature of the material being extruded rising above 40 ° C. Extrusion can naturally produce granules (granules can break after passing through a die) or a cutter can be used.

Suitably, the granules will have a water content of 30 to 40%, such as 33 to 37%. The enzyme content is suitably from 3 to 10%, for example from 5 to 9%. The obtained granules can be subjected to rounding (for example spheronization) such as in a spheronizer, for example a machine MARUMERISERMR and / or compaction. The granules can be spheronized before drying, since this can reduce the formation of dust in the final granulate and / or can facilitate any coating of the granulate. The granules can then be dried, such as in a fluidized bed dryer or, in the case of fluidized bed agglomeration, they can be immediately dried (in the agglomerator) to obtain granulates (dry solid). Other known methods for drying granules in the food, food or enzymes industry can be used by the person skilled in the art. Suitably, the granulate is capable of flowing. Drying preferably takes place at a temperature of 25 to 60 ° C, such as 30 to 50 ° C. Here, the drying can last from 10 minutes to several hours, such as from 15 to 30 minutes. The length of time required will of course depend on the amount of granules that are to be dried, but as a guide this may be 1 to 2 seconds per kg of granules. After drying the granules, the resulting granulate preferably has a water content of 3 to 10%, such as 5 to 9%. A coating may be applied to the granulate to give additional characteristics or properties (eg flavored), as low dust content, color, protection of the enzyme from the neighboring environment, different enzymatic activities in a granulate or a combination thereof. The granules can be coated with a fat, a wax, a polymer, a salt, an ointment and / or an ointment or a coating (for example a liquid) containing a (second) enzyme or a combination thereof. It will be apparent that if desired, several layers of (different) coatings can be applied. To apply the coating or coatings on the granulates, a number of known methods are available, which include the use of a fluidized bed, a high cut granulator, a mixing granulator, or a Nauta type mixer.

In other embodiments, additional ingredients may be incorporated into the granulate, for example, as processing aids, for further improvement of pelletizing stability and / or storage stability of the granulate. A number of such preferred additives are discussed below. Salts may be included in the granulate (for example with the solid carrier or a liquid). Preferably (as suggested in European Patent EP-A-0 758, 018) one or more inorganic salts can be added, which can improve the stability in processing and storage of the anhydrous phytase preparation. Preferred inorganic salts comprise a divalent cation, such as zinc, magnesium, and calcium.Sulfate is the most favored anion.Preferably (as suggested in European Patent EP-A-0 758, 018) a or various inorganic salts, which can improve the stability in the processing and storage of the anhydrous enzyme preparation.The preferred inorganic salts are water soluble.These can comprise a divalent cation, such as zinc (in particular), magnesium, and calcium.Sulfate is the most preferred anion although other anions can be used which result in water solubility.Salts (for example to the mixture) can be added to the solid form. to be dissolved in the water or in the liquid containing enzyme before mixing with the solid carrier Suitably, the salt is provided in an amount that is at least 15% (w / w based on the enzyme) , such as at least 30%. However, this can be as high as at least 60% or even 70% (again, w / w based on the enzyme). These quantities can be applied either to the granules or to the granulate. The granulate can therefore comprise less than 12% (w / w) of the salt, for example from 2.5 to 7.5%, for example from 4 to 6%. "If the salt is provided in the water, then it may be in an amount of 5 to 30% (w / w) such as 15 to 25%." Further improvement of the stability of the pellet formation can It can be obtained by the incorporation of hydrophobic, gel-forming or slow-dissolving compounds (for example in water), these can be provided from 1 to 10%, "such as from 2 to 8%, and preferably from 4 to 6% by weight. weight (based on the weight of the water and the solid carrier ingredients). Suitable substances include cellulose derivatives, such as HPMC (hydroxypropylmethylcellulose), CMC (carboxymethylcellulose), HEC (hydroxyethylcellulose); polyvinyl alcohols (PVA); and / or edible oils. Edible oils such as soybean oils or canola oil can be added (for example to the mixture to be granulated) as a processing aid, although hydrophobic substances (eg palm oil) are preferably absent as a rule. ). Preferably, the granules have a relatively narrow size distribution (for example, these are monodispersed). This can facilitate a homogenous distribution of the phytase in the granules and / or in the enzyme granulate in the animal feed. The process of the invention tends to produce granules with a narrow size distribution. However, if necessary, an additional step in the process can be included to further narrow the size distribution of the granules, such as sieving The size distribution of the granulate is suitably between 100 μm and 2000 μm, preferably between 200 μm and 1800 μm, and optimally between 300 μm and 1600 μm The granules may be irregularly (but preferably _-regular), for example approximately spherical .. Another suitable enzyme may be included in the animal feed which includes pet foods.The function of these enzymes is often to improve - the feed conversion rate, for example by reducing the viscosity or by reducing the antinutritional effect of certain food compounds. Food enzymes may also be used, such as to reduce the amount of compounds that are hazardous to the environment in manure.The preferred enzymes for these purposes are: carbohydratases, such as amylolitic enzymes and cellular phage degrading enzymes. plant, of which are included cellulases such as β-glucanases, hemicellulases, such as xylanases, or galactanases; peptidases, galactosidases, peptinases, esterases; proteases, preferably with an optimum pH neutral and / or acid; and lipases, preferably phospholipases such as mammalian pancreatic phospholipases A2. Preferably, the enzyme does not include starch degrading enzymes (for example amylases). In some modalities, proteases may be excluded, since they may cause damage if they are ingested. If the enzyme is a plant cell wall degrading enzyme, for example a cellulase, and in particular a hemicellulase, such as xylanase, then the final granulate can have an activity of the enzyme in the range of 3,000 to 100,000, preferably 5,000 to 80,000, and optimally from 8,000 to 70,000, EXU / g. If the enzyme is a cellulase, such as β-gluconase, then the final granulate can have an enzymatic activity of 500 to 15,000, preferably 1,000 to 10,000, and optimally of 1,500 to 7,000 BGU / g. The granules may comprise from 5 to 20, for example 7 to 15% of the enzyme (s) The enzyme may be of natural or recombinant origin, A preferred process according to the invention therefore comprises: a. mixture of the aqueous liquid containing the phytase and the solid carrier comprising at least 15% (w / w) or an edible carbohydrate polymer, for example the mixture of the solid carrier with an aqueous liquid containing enzyme; the resulting mixture: c) the granulation, for example by mechanical processing, of the mixture in order to obtain granules containing enzyme, for example by using a granulator or by extrusion, d) optionally spheronization of the granules; e) the drying of the resulting granules to obtain an enzyme-containing granulate During the entire process, the maximum temperature at which the enzymes are exposed will be maintained, below 80 ° C. Two of the invention are suitable for use in the preparation of an animal feed. In its broadest aspect this invention covers a. granulate comprising a phytase and an edible carbohydrate polymer, the granulate having an activity of at least 6,000 FTU / g. In such processes the granulates are mixed with food substances, either as such, or as part of a premix. The characteristics of the granulates according to the invention allow their use as a component of a mixture which is very suitable as an animal feed, especially if the mixture is treated with steam and subsequently formed into pellets. The dry granules may be visible or distinguishable in such pellets. Thus, a third aspect of the invention relates to a process for the preparation of animal feed, or a premix or precursor for an animal feed, the process comprising mixing a composition of the second "aspect, with one or more food substances for animals (eg seeds) or ingredients.This can then be sterilized, for example, subjected to heat treatment.The resulting composition is then suitably processed into pellets.A fourth aspect of the invention relates to a composition that comprises a granulate of the second aspect, which is preferably an edible food composition such as an animal feed.This composition is in the form preferably of pellets (there may be from 1 to 5, for example, from 2 to 4, dry granules per pella).

Suitably, the composition comprises from 0.05 to 2.0, such as from 0.3 to 1.0, optimally from 0.4 to 0.6 FTU / g of the phytase. A xylanase from 0.5 to 50, for example from 1 to 40 EXU / g, may be present. Alternatively, or in addition, a cellulase of 0.1 to 1.0 may be present, for example 0.2 to 0.4 BGU / g. The composition may have a water content of 10 to 20%, for example 12 to 15%. The amount of the enzyme (s) is suitably from 0.0005 to 0.0012%, such as at least 5 ppm. A fifth aspect relates to a process for promoting the development of an animal, the process comprising feeding an animal with a diet comprising a composition of the second aspect, or a composition the fourth aspect. Here, the animal's diet can include either the granulate itself, or the granulate present in a food. A sixth aspect of the present invention relates to the use of the compositions in, or as a component of, an animal feed, or for use in an animal diet. A seventh aspect of the present invention also relates to the use of a composition comprising at least 15% (w / w) of an edible carbohydrate polymer, as a carrier for a phytase, to improve the stability of pellet formation of the phytase. Suitable animals include farm animals (pigs, poultry, cattle), non-ruminants or monogastric animals (pigs, poultry, marine animals such as fish), ruminants (cattle or sheep, for example, cows, sheep, goats). , deer, calves, rams). Poultry includes chickens, chickens and turkeys. The preferred features and characteristics of one aspect of the invention are equally applicable to others, muta tis mu tandi s. The following examples are presented merely to illustrate the invention, and are not intended to be considered as limiting.

EXAMPLES EXAMPLE 1 Fermentation of A. niger CBS 513.8: Preparations of fungal spores of Aspergillus niger were made following standard techniques.

The spores and subsequently the cells were transferred through a series of batch fermentations in Erlenmeyer flasks to a 10 liter fermenter. After the development in the batch culture, the content of this fermentor was used as an inoculum for a final batch fermentation of 500 liters. The medium used contains: 91 g / 1 of corn starch (BDH Chemicals); ammonium 38 g / 1 glucose »H20; 0.6 g / 1 MgSO4 »7H20; 0.6 g / 1 of KC1; 0.2 g / 1 of FeS0 * 7H20 and 12 g / 1 of NK03. The pH was maintained at 4.6 ± 0.3 by automatic titration with either 4 N NaOH or 4 N H2SO4. Cells were grown at 28 ° C at a concentration of dissolved oxygen automatically controlled at a saturation of 25% air. Phytase production reached a maximum level of 5-10 U / ml after 10 days of fermentation. The fermentation was repeated using ammonium sulfate in place of corn starch (to give equivalent assimilable nitrogen content).

EXAMPLE 2 Purification and characterization of phytase: assay of phytase activity 100 μl of broth filtrate (diluted when necessary) or supernatant or 100 μl of demineralized water as reference are added to an incubation mixture having the following composition: 0.25 M sodium acetate buffer, pH 5.5, or buffer. glycine hydrochloride; pH 2.5 - 1 mM phytic acid, sodium salt demineralized water up to 900 μl The resulting mixture was incubated for 30 minutes at 37 ° C. The reaction was stopped by the addition of -1 ml of 10% TCA (trichloroacetic acid). After the reaction was finished, 2 ml of reagent (3.66 g of FeS04 »7H20 in 50 ml of ammonium molybdate solution (2.5 g of (NH4) 4Mo7024 * 4H20 and 8 ml of H2SO4, diluted to 250 ml with demineralised water)).

The intensity of the blue color was measured spectrophotometrically at 750 nm. The measurements are indicative of the amount of phosphate released in relation to a phosphate calibration curve in the range of 0-1 mMol / 1.

EXAMPLE 3 A. Expression of phytase in A. niger CBS 513.86 transfornaaddoo ccoonn vveeccttoorreess of eexxpprresión containing the gene diee l 1a, a ffiittaassaa ddee AA. ffiiccuuuum, fused to the sequencers Prroommoottoorraass yy // oo ddee sseeiñal of the amyloglucosidase gene (AG) of A. niger.

To obtain overexpression of the phytase in A. no ger was derived an expression cassette in which the A. ficuu phytase gene was under the control of the amyloglucosidase (AG) promoter of A. nor ger in combination with a signal sequence. For the longer leader sequence, the AG promoter sequence was fused to the sequence encoding the phytase, including the phytase leader sequence, which was fused to the phytase gene fragment encoding the mature protein (see for reference Example 10 of European patent EP-A-0, 420, 358).

B. Expression of the phytase gene under the control of the AG promoter in A. niger.

The strain of A. niger CBS 513.88 (deposited on October 10, 1988) was transformed with 10 μg of a DNA fragment, by known methods (see for example Example 9 of European patent EP-A-0, 420 , 358). Simple transformants of A. niger from each expression cassette were isolated, and spores were streaked onto selective agar-acetamide plates. The spores of each transformant were harvested from cells developed for 3 days at 37 ° C on 0.4% potato-dextrose agar plates (Oxoid, England). The phytase production was tested in shake flasks under the following conditions: Approximately 1 x 108 spores were inoculated in 100 ml of preculture medium containing (per liter): 1 g of KH2P04; 30 g of maltose; 5 g of yeast extract; 10 g of casein hydrolyzate; 0. 5 g of MgSO4 »7H20 and 3 g of Tween 80. The pH was adjusted to 5.5. After day-to-day development at 34 ° C on a rotary shaker, 1 ml of the growth culture was inoculated into a 100 ml main culture containing (per liter): 2 g KH2P04; 70 g of maltodextrin (maldex MD03, A ylum); 12.5 g of yeast extract; 25 g "" of casein hydrolyzate; 2 g of K2S04; 5 g of MgSO4 «7H20; 0.03 g of ZnCl2; 0.02 g of CaCl2; 0.05 g of MnS0 »4H20 and FeSo4. The pH was adjusted to 5.6. The mycelium developed for at least 140 hours. The phytase production was measured as described in Example 2. The fermentation was repeated using equivalent amounts of glucose and ammonium sulfate as the carbon and nitrogen sources. The broth was filtered to give a filtrate that was separated from the biomass. Using the expression cassette PFYT3 (AG promoter / phytase guide), a maximum phytase activity of 280 U / ml was obtained.

EXAMPLE 4 Purification of phytase from the filtrate The purification to obtain highly purified phytase was as follows: 1. Cation exchange chromatography at pH 4.9 2. Cation exchange chromatography at pH 3.8 3. Anion exchange chromatography at pH 6.3 4. Ultrafiltration 1. The phytase filtrate was diluted to 20 times with -water and the pH was adjusted to 4.9. This material was passed through a Sepharose S Rapid Flow column equilibrated with a 20 mM citric acid buffer / NaOH pH 4.9. The unbound material, with the phytase, was collected and used for the next step. 2. The pH 4.9 material was brought to pH 3.8 and the phytase was bound on a Sepharose S Fast Flow column equilibrated with the 2 mM citric acid buffer / NaOH pH 3.8. The phytase was eluted from the column with a buffer of 20 mM NaP0, 50 mM NaCl, pH 7.6. 3. The combined phytase fractions from the second cation exchange step were adjusted to a pH of 6.3 and the phytase was bound on a Q Sepharose Fast Flow column equilibrated with 10 mM KP0, pH 6.3 as a buffer. The phytase was eluted using a gradient up to 1 M NaCl in the same buffer.

The final product (exchanged in anions) that ^ contained. 10 mg protein / ml, concentrated to one tenth by ultrafiltration using an Amicon stirred cell (2 1 module) with a Kalle E35 membrane at 3 bars. The final z concentration for the purified phytase reached 280-300 g / 1 (28-30%). With a specific activity of 100 FTU / g of protein, this results in a phytase activity of 28,000-30,000 FTU / g.

EXAMPLE 5 Stability Tests of High Activity Phytase To demonstrate - that a higher concentration of enzyme (in granules made using the high activity phytase fluid) gives a higher stability in the formation of pellets, granules with an increasing concentration of enzyme were elaborated and the stability of the Pellet formation of these samples was also tested.

Comparative Sample A: Preparation of a granulate with low enzymatic activity, based on corn starch, by mixing, kneading, extrusion, feronization and drying. A mixture was prepared by mixing and kneading 73% (w / w) of corn starch and phytase ultrafiltrate of low concentration at 4% (w / w), and 23% water (w / w). This mixture was extruded using a Nica E-220 basket extruder to obtain a wet extrudate which was spheronized in a -Marumeriser ™ Fuji Paudal for 2 minutes to obtain round particles with an average diameter of 600 μm. These particles were subsequently dried in a Fluat GPCG 1.1 fluid bed dryer. The final activity of the granulate was 610 FTU / g.

Comparative Sample B: Preparation of a medium activity enzyme granulate, based on corn starch, by mixing, kneading, extruding, spheronizing and drying. A mixture was prepared by mixing and kneading 70% (w / w) of corn starch and 17% - (p / p) of phytase ultrafiltrate and 13% (w / w) of water.

This mixture was extruded using a Nica E-220 basket extruder to obtain a wet extrudate which was spheronized in a Marumeriser ™ Fiji Paudal for 2 minutes to obtain round particles with an average diameter of 600 μm. These particles were subsequently dried in a Glatt GPCG 1.1 fluid bed dryer. The final activity of the granulate was 4170 FTU / g Sample C Preparation of a highly active enzyme granulate, based on corn starch, by mixing, kneading, extruding, spheronizing and drying. A mixture was prepared by mixing and kneading 67% (w / w) of corn starch and 30% (w / w) of the phytase ultrafiltrate prepared in Example 4 (but diluted to 18,400 FTU / g) and 3 % (w / w) of water. This mixture was extruded using a Nica E-220 basket extruder to obtain a wet extrudate which was spheronized in a Marumeriser ™ Fuji Paudal for 2 minutes to obtain round particles with an average diameter of 600 μm. These particles were subsequently dried in a Glatt GPCG 1.1 fluid bed dryer. The final activity of the granulate was 6830 FTU / g.

Comparison of stabilities in pellet training The different enzymatic granules were subsequently placed in a pellet formation test and their stability in pellet formation was compared. The pellet formation test consists of mixing the enzyme granules with a feed premix respectively at 1500, 320 and 200 ppm. These mixtures were pretreated by steam injection to give an elevation of temperature to 75 ° C, after which the mixtures were converted into pellets in a pelletizing machine to obtain the food pellets at a temperature of 82 ° C, which They were subsequently dried. This type of process is typical for the food industry, to obtain food pellets. Table 1 summarizes the results of the pellet formation tests. It is apparent that the two granules with the highest concentration of enzyme had much greater stability in the formation of pellets. z ~ TABLE 1 Results of the pellet formation tests EXAMPLE 6 Preparation of an enzyme granulate based on potato starch, containing soybean oil and additions of MgSO4 by mixing, kneading, pelletizing and drying In a mixer / kneader 30 kg of potato starch and 2.5 kg of soybean oil were added, and mixed. Subsequently, an ultrafiltrate of phytase derived from Aspergill was added (16,840 FTU / g) containing MgS0 * 7H20 (3.5 kg of MgSO4 »7H20 dissolved in 14 kg of ultrafiltrate). The product was mixed thoroughly in the kneader, then extruded and dried in a fluid bed dryer as in Example 1. This resulted in a product of 5870 FTU / g.

EXAMPLE 7 Preparation of an enzyme granulate based on rice starch by mixing, kneading, extrusion, spheronization and drying A mixture was prepared by mixing and kneading 62% (w / w) of rice starch and 38% (w / w) of the same phytase ultrafiltrate used in Example 6. This mixture was extruded using the Fuji basket extruder. Paudal to obtain a wet extrudate which was then spheronized in the MARUMERISERMR for one minute, to obtain round particles with an average diameter of 785 μm. These particles were subsequently dried in a fluidized bed dryer as in Example 1. The final activity of the granulate was 7280 FTU / g.

EXAMPLE 8 Preparation of an enzyme granulate based on corn starch containing an HPMC addition, by mixing, kneading, • extrusion, spheronization and drying An enzyme preparation was obtained by kneading a mixture of 54% (w / w) corn starch , - 5% HPMC (hydroxypropylmethylcellulose) and 41% (w / w) of the phytase ultrafiltrate used in Example 6. This mixture was extruded using a Fuji Paudal basket extruder to obtain a wet extrudate which was spheronized in a MARUMERISERMR for one minute to obtain round particles with an average diameter of 780 μm. These were subsequently dried in a fluid bed dryer for 20 minutes at 40 ° C bed temperature, and inlet temperature of 75 ° C. The dry enzyme granulate obtained in this way had an activity of 8470 FTU / g.

EXAMPLE 9 Preparation of an enzyme granulate based on corn starch containing an HEC addition, by mixing, kneading, extrusion, spheronization and drying An enzyme preparation was obtained by mixing and kneading 54% (w / w) of corn starch, 5% (w / w) of HEC (hydroxyethylcellulose) with "41% (w / w) of the same phytase ultrafiltrate used in Example 6. This mixture was extruded using the Fuji Paudal basket extruder to obtain a wet extrudate which was spheronized in the MARUMERISERMR for one minute to obtain round particles with an average diameter of 780 μm, which were subsequently dried in a dryer fluidized bed for 20 minutes at 40 ° C bed temperature, and inlet temperature of 75 ° C. The dried enzyme granules, obtained in this way had an activity of 8410 FTU / g.

EXAMPLE 10 An ultrafiltrate of 18,000 FTU / g, derived from the ultrafiltrate of Example 4, was used and diluted.

Samples The activity of the 3 samples prepared was "610 (Comparative A), 4170 (Comparative B) and 6830 (C) FTU / g." This gave three active foods of 1,153, 1,685 and 1,745 FTU / g, respectively. sample, 150 g were mixed with 20 kg of feed as described below, after which the premix was mixed with 80 kg of feed and divided into two parts to convert feed for two tests at two different temperatures. was 153.6 g in 20 kg of feed This sample of 20,153.6 g was divided into two equal portions of 10,076 kg each portion was then mixed with 230 kg of feed to obtain the flour for testing. g of granulate with 20 kg of feed and were divided into two portions of 10,048 kg, each portion was then mixed with 230 kg of feed to obtain the flour for the tests.The speed of pellet formation was 600 kg / h.

The food mixture consisted of Corn 20.00% Wheat 30.00% Soybean (hot) 10.00% Soybean (coarse meal 46.7 / 3.7 18.20% Tapioca (65% starch) 6.97% Animal meal (56.5 / 10.9) 4.00% Fish meal (70.6% re) 1.00 % Feather meal, hydraulic _ 1.00% Soybean oil / corn oil 1.30% Animal fat 4.00% Premix of Vit./min. (Corn) 1.00% Calcium carbonate 0.85% Monocalcium phosphate 1.05% Salt 0.26% L hydrochloride -lysine 0.16% DL-methionine 0.21% The three mixtures were then converted into pellets. The food was introduced into a conditioner where steam was added directly to the flour. The temperature rose to 75 ° C. Subsequently the flour came out of the pellet former where it was pushed through a die plate with holes of 5 mm and thickness of 65 mm. The temperature of the food at this point was raised another 4 ° C to 79 ° C. The activity of the three foods was 10.11 (A); 10.04 (B) and 9.81 (C).

The results of this test for residual activity were: 63 (A); 66 (B) and 72% (C) respectively for the original samples of 610; 4170; 6830 and FTU / g. This shows that even with similar activities (B and C) the highest activity formulation (C; 6830 FTU / g, of the invention) gave a much higher pelletizing stability. This was 6% higher than for sample B (comparative), notable since only an increase of 3% was observed (from A to B) with a very large increase in activity (610 to 4170 FTU / g).

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (38)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A process for the preparation of an aqueous liquid comprising a phytase, the process is characterized in that it comprises: (a) the cultivation, in an aqueous medium, of a microorganism of the genus Aspergillus or Tri ch oderma having a phytase gene heterologous under the control of a glucoamylase promoter (for Aspergillus) or cellobiohydrolase (for "Tri ch oderma), under conditions that allow the recombinant expression of phytase, where the medium comprises, as a feed for the microorganism, a source of assimilable carbon and a source of assimilable nitrogen, (b) filtration of the aqueous medium to remove the microorganisms, to give an aqueous filtrate, and (c) attaching the filtrate from step (b) to ultrafiltration to give an aqueous liquid, having a phytase concentration of at least 14,000 FTU / g.

2. A process "according to claim 1, characterized in that the microorganism is Aspergillus niger, Aspergillus oryzae or Tri choderma reesei.

3. A process according to claim 1 or 2, characterized in that the microorganism does not possess, or does not express, a glucoamylase (AG) gene.

4. A process according to any of the preceding claims, characterized in that the microorganism possesses multiple copies of the phytase gene. _

5. A process according to any of the preceding claims, characterized in that the aqueous liquid is substantially free of taka-amylase.

6. "A process according to any of the preceding claims, characterized in that substantially all sources of carbon and nitrogen in the medium have been consumed by the microorganism before filtration in step (b).

7. A process according to any of the preceding claims, characterized in that the aqueous liquid is free of carbon and / or nitrogen sources.

8. A process according to any of the preceding claims, characterized in that the phytase is expressed in the microorganism with a glucoamylase signal sequence.

9. A process according to any of the preceding claims, characterized in that neither the aqueous filtrate nor the aqueous liquid are subject to crystallization and / or to a color removal step.

10. A process according to any of the preceding claims, characterized in that the resulting aqueous liquid has a phytase activity of 18.00 FTU / g or more.

11. An aqueous liquid preparable by a process according to any of the preceding claims, characterized in that it comprises a phytase at a concentration of at least 14,000 FTU / g.

12. An aqueous liquid according to claim 11, characterized in that it is derived from a culture medium in which the phytase was expressed.

13. A process for the preparation of a granulate containing phytase, suitable for use in an animal feed, the process is characterized in that it comprises the processing of a solid carrier comprising at least 15% (w / w) of a carbohydrate polymer edible and an aqueous liquid according to claim 11 or 12, para. get granules that contain phytase.

14. A process according to claim 13, characterized in that the aqueous liquid and the carrier are mixed, and the resulting mixture is kneaded.

15. A process according to claim 14, characterized in that the granules are subsequently dried.

16. A process according to claim 13 or 14, characterized in that the process comprises: a) mixing the aqueous liquid containing the phytase with the solid carrier; b) the mechanical processing of the mixture obtained in a) to obtain granules containing the enzyme; and c) drying the enzyme-containing granules obtained in step b).

17. A process according to claim 15, characterized in that the processing comprises extrusion, pelletization, high-cut granulation, expansion, fluidized-bed agglomeration or a combination thereof.

18. A granulate containing phytase, characterized in that it can be prepared by a process according to any of claims 13 to 17.

19. A granulate comprising dry granules formed from a phytase and a solid carrier, characterized in that it comprises at least 15% (w / w) of an edible carbohydrate polymer.

20. A granulate according to claim 19, characterized in that the granules comprise at least one divalent cation.

21. A granulate according to claim 19 or 20, characterized in that the granules comprise one or more hydrophobic compounds, gel formers or water insoluble.

22. A granulate according to claim 21, characterized in that the hydrophobic, gel-forming or water-insoluble compound comprises a cellulose derivative, polyvinyl alcohol (PVA) or an edible oil.

23. A granulate according to claim 22, characterized in that the cellulose derivative is hydroxypropylmethylcellulose, carboxymethylcellulose or hydroxyethylcellulose and / or the edible oil is soybean oil or canola oil. _

24. A granulate according to any of claims 19 to 23, characterized in that it further comprises an endo-xylanase and / or β-glucanase.

25. A granulate according to any of claims 19 to 24, characterized in that the carrier comprises starch.

26. A granulate according to any of claims 19 to 25, characterized in that the phytase is different from a heat-tolerant phytase (thermostable).

27. A granulate according to any of claims 19 to 26, characterized in that the phytase is a fungal phytase.

28. A composition according to any of claims 19 to 27, characterized in that the fungal phytase is derived from a species of Aspergillus or Tri choderma.

29. A process for the preparation of an animal feed, a premix or a precursor for animal feed, the process is characterized in that it comprises mixing a phytase containing granulate as defined in any of claims 19 to 28. , with one or more substances or food ingredients for animals. _

- 30. A process "according to claim 29, characterized in that the mixture of the food substance and the composition or the granulate is steam treated, pelletized and optionally dried.

31. A composition, characterized in that it comprises a. granulate according to any one of claims 19 to 28 and / or a granulate containing phytase, with an activity of at least 6,000 FTU / g.

32. A composition according to claim 31, characterized in that it is an edible food composition.

T 33. A composition according to claim 32, characterized in that it is an animal feed.

34. A composition according to claim 32 or 33, characterized in that it comprises pellets of one or more food substances or food ingredients mixed with a granulate according to any of claims 19 to 27.

35. A composition according to any of claims 31 to 34, characterized in that it is an animal feed, or a premix or precursor for an animal feed, preparable by a process according to claim 29 or 30.

36. A process for the promotion of the development of an animal, the process is characterized in that it comprises feeding an animal with a diet comprising a granulate according to any of claims 19 to 27, or a composition as defined in accordance with any of claims 31 to 35.

37. The use of a granulate as defined in any of claims 19 to 27 in, or as a component of, an animal feed or for use in an animal diet.

38. The use of a composition comprising at least 15% (w / w) of an edible carbohydrate polymer as a carrier for a phytase to improve the stability in the formation of pellets, of the phytase.