Granulates containing feed-enzymes

ES 2 220 437 T3

DESCRIPTION

Granules containing feed enzymes.

Field of the invention

The present invention relates to the formulation of enzymes, preferably feed enzymes, in granules. These (edible) granules can then be used in animal feed.

Background of the invention

Animal feed represents one of the largest costs used to keep livestock and other animals. The use of various enzymes in animal feed, for example livestock, has become almost common practice. These enzymes are typically produced by growing microorganisms in large-scale fermenters run by industrial enzyme producers. 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). Subsequently, the enzyme solution is concentrated and processed as a liquid (often after the addition of various stabilizers) or to a dry formulation.

Liquid and dry enzyme formulations are used on a commercial scale by the feed industry. Liquid enzyme formulations can be added to the feed after pelleting to avoid thermal inactivation of the enzyme that would occur during the pelletization process. However, the amounts of enzyme in the final feed preparations are usually very small, which makes it difficult to achieve a homogeneous distribution of the enzyme in the feed, and liquids are notably more difficult to mix uniformly in the feed than dry ingredients. . In addition, specialized (expensive) equipment is needed to add liquids to the feed after pelletizing, which is not currently available in most feed mills (due to the additional cost).

Dry enzyme formulations can be added to feed prior to pelleting and are therefore subjected to heat inactivation during pelletization. Preferred manufacturing procedures in the feed industry involve steam pelletizing where the feed is subjected to an injection or injections of steam prior to pelletizing, a procedure called conditioning. In the subsequent pelletization step the feed is forced through a nozzle and the resulting yarns are cut into suitable pellets of varying length. The moisture content immediately prior to pelletizing is generally between 13% and 16%. During this conditioning procedure the temperature can rise up to 60-95 ° C. The combined effect of high moisture content and high temperature is detrimental to most enzymes. These disadvantages are also found in other types of thermomechanical treatments such as extrusion and expansion.

To overcome these problems, EP-A-0.257.996 suggests that the stability of enzymes in feed processing could be increased by preparing an enzyme "premix" in which an enzyme-containing solution is absorbed onto a carrier based on grains consisting of flour, and the premix is ​​subsequently pelletized and dried. However, these flour based premixes are not suitable for milder processing methods (of the dough-like premix) such as granules, such as low pressure extrusion or high shear granulation, due to the stickiness of flour based premixes. .

Various enzyme manufacturers have developed alternative formulation methods to improve the stability of dry enzyme products during pelletization and storage.

EP-A-0,569,468 relates to a formulation consisting of an enzyme-containing granulate which is coated with a high melting point wax or fat which is said to improve resistance to nodulation conditions. The granulate is prepared by mixing a dry inorganic carrier (eg sodium sulfate) with the enzyme solution in a high shear granulator. As a consequence of the fat coating, the dissolution time of the granules is long (approximately one hour). Therefore, the bioavailability of the enzyme to the animal is decreased. Furthermore, the granules have a wide particle size distribution. This makes it difficult to obtain a uniformly distributed enzyme concentration after coating, since small particles absorb a relatively high amount of coating agent compared to large particles. EPA-0.569.468 further teaches that any beneficial effect of the coating with respect to nodulation stability is specific to the type of coated granulate, which in this case is based on a sodium sulfate carrier. However, the absorption capacity of these carriers (sodium sulfate) is much lower than that of carriers such as flour, which is undesirable if more concentrated enzyme-containing granules are to be produced.

WO97 / 39116 describes formulations consisting of a preformed granule that is capable of absorbing at least 5% water. However, with these formulations a satisfactory degree of solubility can only be obtained with very powerful mixing. Therefore, such a formulation may be effective in the area of ​​detergents but not in the intestinal tract of the animal.

WO98 / 54980 describes enzyme-containing granules containing edible carbohydrates, of which starch is preferred. Although these formulations dissolve easily in water and thus ensure a good biodis2

In the wearability, the nodulation stability of these starch-based granules is less than that obtained with the previously described fat-coated granules.

GB 2 167 758 discloses enzyme containing granules of improved strength and disintegration ability which are suitable for use in detergent. The granules are obtained by granulating an enzyme together with fine synthetic fibrous material in the form of pieces or pulp. The resulting granules can be coated with a fusible waxy material and colored.

WO98 / 55599 demonstrates that high concentration phytase compositions that are cheaper to produce show increased stability, especially during a pelletization process in the preparation of animal feed (pellets). However, the nodulation stability of these compositions also still lags behind the stability obtained with the previously described fat-coated granules.

Thus, there remains a need for stable formulations of enzymes for use in animal feed that are inexpensive to produce, that combine satisfactory stability against pelletization with good bioavailability of the enzyme to the animal, and that have optimal storage stability. .

Description of the invention

The present invention provides a process for the preparation of an enzyme-containing granulate suitable for use in an animal feed, the process comprising processing an enzyme, a solid carrier, optionally additives and water in appropriate relative amounts to obtain enzyme-containing granules, drying the granules and coating the dried granules with polyethylene glycol, in which the polyethylene glycol has a molecular weight ranging from 6,000 to 20,000 daltons.

The advantages of coating the granule with this polyethylene glycol are firstly that this coating provides effective protection against the formation of dust. On the other hand, the polyethylene glycol coating can be dissolved in water. Although the dissolution time of coated granules is increased compared to the same uncoated granules, it is still much shorter (20 times) than the dissolution time of granules coated with a fat type coating. A short dissolution time significantly improves the bioavailability of the enzyme to the animal. Finally, a polyethylene glycol coating provides good stability against pelletization of the granule. Surprisingly, the polyethylene glycol coating provides good protection against water vapor and water, although the coating can dissolve in water.

The polyethylene glycol that is used to coat the granules has a molecular weight (average) of 6,000 to 20,000 daltons because the melting temperature of such polyethylene glycols is around 60 ° C.

The percentages used throughout this specification refer to percentages by weight and are based on the weight of the final dry granulate (end product) (unless otherwise indicated).

The polyethylene glycol coating is preferably applied at 1-20%, more preferably at 5-20% and most preferably at about 8-12% of the weight of the granules.

In a preferred embodiment of the invention, polyethylene glycol is dissolved at a concentration of 50% (w / w) in water before it is applied to coat the granules.

Additional coatings can also be applied to the granule to give additional (eg, favored) characteristics or properties, such as low dust content, color, protection of the enzyme from the surrounding environment, different enzyme activities in a granulate, or a combination thereof. The granules can be further coated with a fat, a wax, a polymer, a salt, an ointment and / or an ointment or a coating (eg liquid) containing a (second) enzyme or a combination thereof. It will be apparent that, if desired, several layers of (different) coatings can be applied.

The solid carrier to be used to prepare the granulate according to the present invention is a powder that can be compacted into a granule. The solid carrier to be used preferably has an average particle size ranging between 5 and 20 µm.

In a preferred embodiment of the present invention, the solid carrier consists essentially of an edible carbohydrate polymer. The many advantages of using an edible carbohydrate polymer are presented in patent application WO98 / 54980.

An edible carbohydrate polymer is a carbohydrate polymer that is permitted to be used as a feed additive. The edible carbohydrate polymer should be chosen so that it is edible by the animal for which the feed is intended, and preferably also digestible. The polymer preferably comprises hexose polymer units, more preferably glucose polymer units, most preferably the carbohydrate polymer comprises α-D-glucopyranose units, amylose (a polymer of α-D-glucan (1 ^ 4) linear) and / or amylopectin (a branched D-glucan with aD- (1 ^ 4) and a-D '- (1 ^ 6) bonds). Starch is the preferred carbohydrate polymer. Other suitable hexose-containing polymers that can be used in place of, or in addition to

ES 2 220 437 T3, starch include α-glucans, β-glucans, pectin (such as protopectin) and glycogen. Derivatives of these carbohydrate polymers, such as ethers and / or esters thereof, are also contemplated. Gelatinized starch is best avoided and thus may not be present. Suitably the carbohydrate polymer is insoluble in water.

In the examples described here, corn, potato and rice starch is used. However, it is equally applicable to 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 arrowroot. Similarly, both natural and modified types of starch (eg, dextrin) can be used in the invention. Preferably, the carbohydrate (e.g. starch) contains little or no protein, preferably less than 5% (w / w), more preferably less than 2% (w / w), most preferably less than 1% (w / w ).

In other embodiments of the invention, one or more additional ingredients may be incorporated into the granules, for example as processing aids and / or for further improvement of the pellet stability and / or storage stability of the granulate. A number of such additives are discussed below.

In one embodiment of the invention, the additive comprises a water soluble inorganic salt (as suggested in EP-A-0.758.018). Preferably, the granules comprise at least 0.1% of a water soluble inorganic salt comprising a divalent cation, more preferably zinc. Most preferably the inorganic salt is zinc sulfate. The final product preferably contains 500-1500mg of Zn / kg of final product, more preferably 7001300mg of Zn / kg of final product and most preferably 900-1100mg of Zn / kg of final product. Divalent cations are preferred because they provide the best storage and processing stability. Sulfate is preferred as an anion because it provides the best drying performance. The salts can be added (eg, to the mixture) in solid form. Alternatively, the salt or salts can be dissolved in the enzyme-containing water or liquid prior to mixing with the solid carrier.

A further improvement in stability against anodulization can be obtained by incorporating hydrophobic, gel-forming or slow-dissolving compounds into the formulation. These can be provided by adding at least 0.1%, preferably at least 0.5%, and more preferably at least 1% of the desired compound (w / w, based on the weight of water and solid carrier ingredients) to the mixture to be be processed as granules. Suitable substances include derived celluloses, such as HPMC (hydroxypropyl methyl cellulose), CMC (carboxy methyl cellulose), HEC (hydroxy ethyl cellulose), polyvinyl alcohols (PVA) and / or edible oils. . Edible oils, such as soybean oil or canola oil, can be added (eg, to the mixture to be granulated) as a processing aid.

In yet another embodiment of the invention, the formulation comprises trehalose. Preferably, trehalose is applied at a concentration of 0.1-2.5% (w / w of final product), more preferably at a concentration of 0.25-1%, most preferably at a concentration of 0.4 -0.5%.

In the process of the invention the enzyme and water are preferably provided as an enzyme-containing liquid (preferably aqueous), such as a solution or suspension, which is from, or derived from, a microbial fermentation process. This fermentation procedure will usually be one in which the enzyme is produced. The fermentation procedure can result in a broth containing the microorganisms (which produced the desired enzyme) and an aqueous solution. This aqueous solution, once separated from the microorganisms (for example, by filtration) can be the aqueous enzyme-containing liquid used in the invention. Thus, in preferred embodiments, the aqueous enzyme-containing liquid is a filtrate. Usually the enzyme will be in an active form. Preferably, the liquid is in a concentrated form, such as an ultrafiltrate (UF), which can allow the production of a granulate with a desired level of activity.

The amount of enzyme-containing liquid (and thus enzyme) that can be absorbed onto the carrier is usually limited by the amount of water that can be absorbed. The enzyme solution can contain approximately 25% (w / w) dry matter. The amount of water added to the solid carrier is such that (substantially) all of the water in the aqueous liquid is absorbed by all the components present in the solid carrier. The use of higher temperatures to absorb a greater amount of enzyme-containing liquid is also contemplated by the present invention, and indeed is especially preferable when handling thermostable enzymes. For these enzymes, therefore, the mixing of the solid carrier and the liquid (or enzyme and water) is carried out at a temperature above 30 ° C, preferably above 40 ° C and more preferably above 50 ° C. . Alternatively or in addition, the liquid can be provided at this temperature. In general, non-swelling conditions of the solid carrier (at lower temperatures) are preferred to minimize loss arising from enzyme instability (thermosensitive) at higher temperatures.

The enzyme-containing liquid or water may comprise one or more enzymes. A suitable enzyme or enzymes are feed enzymes to be included in animal feed (including pet food). The function of these feed enzymes is often to improve the conversion rate of the feed, for example by reducing the viscosity or reducing the antinutritional effect of certain compounds in the feed. Feed enzymes (such as phytase) can also be used to reduce the amount of compounds that are harmful to the environment in manure.

In a preferred embodiment, granules containing 4

ES 2 220 437 T3 contain a high concentration of a feed enzyme. The advantages of preparing phytase compositions at a high concentration have already been described in WO98 / 55599.

Feed enzymes include: phosphatases, such as phytases (both 3-phytases and 6-phytases) and / or acid phosphatases; carbohydrates, such as amylolytic enzymes and / or plant cell wall degrading enzymes, including cellulases such as / 1-glucanases and / or hemicellulases such as xylanases or galactanases; proteases or peptidases such as lysozyme; galactosidases, pectinases, esterases, lipases, preferably phospholipases such as the mammary pancreatic phospholipases A2, and glucose oxidase. Preferably, the feed enzymes have a neutral and / or acid pH optimum. More preferably, the feed enzyme comprises at least one enzyme selected from the group consisting of phytases, xylanases, phospholipases, and glucose oxidase. Most preferably, the feed enzyme comprises at least one enzyme selected from the group consisting of phytases and xylanases.

If the enzyme is a phytase, then the final granulate may preferably have a phytase activity ranging from 4,000 to 20,000 FTU / g, more preferably 5,000 to 20,000 FTU / g, most preferably 5,000 to 15,000 FTU / g. One phytase unit (FTU) is therefore defined as the amount of enzyme that liberates 1 mol of inorganic phosphate per minute from sodium phytate (0.0051 mol / liter) at 37 ° C and at a pH of 5.5 under the conditions in which the phytase activity was determined according to the procedure "ISL method 61696" (manual vanadate assay).

If the enzyme is a xylanase, then the final granulate may preferably have a xylanase activity ranging from 5,000 to 100,000 EXU / g, more preferably 10,000 to 100,000 EXU / g, and most preferably 15,000 to 100,000 EXU / g. One endo-xylanase unit (EXU) is therefore defined as the amount of enzyme that releases 4.53 µmoles of reducing sugars, measured as xylose equivalents, per minute under the conditions of the "ISL 61731 method" procedure.

ISL methods are available upon request from DSM, Food Specialties, Agri Ingredients, Wateringseweg 1, PO Box 1, 2600 MA, Delft, The Netherlands.

In addition to these feed enzymes, the invention is equally applicable to non-enzymatic polypeptides with biological activities, such as antigenic determinants to be used as vaccines and / or polypeptides engineered to have an increased content of essential amino acids. The biological activity of these non-enzymatic polypeptides can be sensitive to heat inactivation.

A preferred process according to the invention comprises the steps of:

to. mixing an aqueous liquid containing the enzyme, a solid carrier consisting essentially of an edible carbohydrate polymer, and one or more additive components selected from the group consisting of a slow-dissolving or gel-forming compound, such as polyvinyl alcohol , a water soluble inorganic salt comprising a divalent cation, and trehalose;

b. mechanically processing the mixture simultaneously with or subsequent to mixing, to obtain a granule.

c. dry the granule;

d. coating the granule with polyethylene glycol in a fluid bed coater.

The mechanical processing used in the present invention to prepare and / or granulate the enzyme mixture, water (eg, an enzyme-containing liquid), solid support, and optionally additives comprise known techniques frequently used in formulation procedures. of food, feed and enzymes. This mechanical processing, for example, comprises expansion, extrusion, spheronization, pelletization, high shear granulation, drum granulation, fluid bed agglomeration, or a combination thereof. These processes are usually characterized by a supply of mechanical energy, such as the rotation of a screw or a mixing mechanism, the pressure of a turning mechanism of a pelletizing apparatus, the movement of particles by means of a rotating bottom plate of an agglomerator. fluid bed or the movement of the particles by a gas stream, or a combination thereof. These procedures allow the solid support (eg, in the form of a powder) to be mixed with the enzyme and water, eg, an enzyme-containing liquid (an aqueous solution or suspension) and subsequently granulated. Alternatively, the solid carrier can be mixed with the enzyme (eg, in a powder form) to which is then added water, such as a liquid (or suspension) (which can act as a granulating liquid).

In a still further embodiment of the invention, the granulate (eg, an agglomerate) is formed by spraying or coating the enzyme-containing liquid on a carrier, such as in a fluid bed agglomerator. Here, the resulting granules can include an agglomerate which can be produced in a fluid bed agglomerator. Preferably, mixing the enzyme-containing liquid and the solid carrier further comprises kneading the mixture. This can improve the plasticity of the mixture to facilitate granulation.

If the granulate is formed by extrusion, this is preferably carried out under low pressure. This can offer the

It is advantageous that the temperature of the extruded mixture is not increased, or only slightly. Low pressure extrusion includes extrusion, for example, into a Fuji Paudal type of basket or dome extruder.

The granules obtained can be subjected to rounding (eg spheronization) such as in a Marumeriser ™, and / or compaction. The granules can be spheronized before drying as this can reduce dust formation in the final granulate and / or can facilitate any coating of the granulate.

The granules can then be dried, such as in a fluid bed dryer or, in the case of fluid bed agglomeration, they can be dried immediately (in the agglomerator) to obtain granules (solid dry). Other known methods for drying granules in the food, feed or enzyme industry may be used by the skilled person. Suitably the granulate is fluid. Drying preferably takes place at a product temperature of 25 to 60 ° C, preferably 30 to 50 ° C. Typically, a dried granule contains about 59% moisture.

To apply the polyethylene glycol and optionally other coating or coatings on the granules, a number of known methods are available including the use of a fluidized bed, a high shear granulator, a mixing granulator or a Nauta type of mixer. In a preferred method for applying the polyethylene glycol onto the granulate, the polyethylene glycol is sprayed onto a fluidized bed of the granules to be coated at a temperature that exceeds the melting point of the polyethylene glycol, for example preferably above 60 ° C. Subsequently, the temperature of the fluidized bed is lowered to allow the polyethylene glycol coating to solidify. The coating step may include a simultaneous drying step in which polyethylene glycol dissolved in water is applied.

Coating of the granule can already be started during the drying process. Alternatively, granule coating can take place subsequent to drying. Preferably drying and coating are done in the same apparatus.

Preferably, the granules have a relatively narrow particle size distribution (eg, they are monodisperse). This can facilitate a homogeneous distribution of the enzyme granules in the feed pellets. The process of the invention tends to produce granules with a narrow size distribution. The size distribution of the granulate is suitably between 10 µm and 2,000 µm, preferably between 200 µm and 1,800 µm, more preferably between 400 µm and 1,600 µm and most preferably between 700 and 1,000 µm. The granules can be of irregular (but preferably regular) shape, for example approximately spherical.

If necessary, an additional step may be included in the process to further narrow the size distribution of the granules, such as sieving. For example, this additional screening step will select granules that have a size distribution between 0.7 and 1 mm.

The enzyme-containing granulate obtainable by these procedures (which forms another aspect of the invention) is suitable for use in an animal feed and seeks to solve or at least mitigate the problems encountered in the prior art. The dissolution time of the granules is very short (a few minutes) and therefore the bioavailability of the enzyme to the animal is improved compared to fat-coated granules. The enzyme concentration is higher so that the granule is cheaper to produce and the stabilities against pelletization and storage are improved. Finally, the granule is free of any soap, detergents, bleach or bleaching compounds, zeolites, binders and for that reason it is edible and preferably also digestible.

The invention thus provides an enzyme-containing granulate suitable for use in an animal feed which can be obtained by the previously mentioned processes and which has the following characteristics. The granulate consists of polyethylene glycol coated granules containing a food enzyme, a solid carrier, and optionally one or more additives. Preferably, said additives comprise at least one of a slow dissolving or gelling compound such as polyvinyl alcohol, a water soluble inorganic salt and trehalose.

The granulate of the invention is suitable for use in the preparation of an animal feed. In such processes, the granulate is mixed with feed substances, such as, as part of a premix or as a precursor to an animal feed. The characteristics of the granulate according to the invention allow its use as a component of a mixture which is very suitable as an animal feed, especially if the mixture is treated with steam, subsequently pelletized and optionally dried.

Thus, a further aspect of the present invention relates to a process for the preparation of an animal feed, or a premix or a precursor of an animal feed, the process comprising mixing the granules provided by the present invention with one or more substances or ingredients for animal feed.

The present invention also relates to a method for promoting the growth of an animal, the method comprising feeding an animal a diet comprising the granules provided by the invention. Here, the diet of the animal may include the granulate itself or the granulate present in a feed. Suitable animals include farm animals, such as cattle, pigs, and poultry.

ES 2 220 437 T3

Another aspect of the invention thus relates to a composition comprising the granulate of the invention, which composition is preferably an edible feed composition such as an animal feed.

Yet another aspect of the present invention relates to the use of the granulate of the invention in, or as a component of, an animal feed or for use in an animal diet.

Preferred features and characteristics of one aspect of the invention are equally applicable to another and vice versa.

The following Examples are presented merely to illustrate the invention and are not intended to be, or are not considered to be, limiting.

Examples

General methods

Conditioning stage

50 grams of pellets are mixed in 10 kg of feed of the premix of choice and mixed just before testing with 240 kg of the same recipe. This 250 kg mixture is dosed in a mixer / conditioner by means of a dosing screw, at a speed of 600 kg / h, where it is heated by direct steam up to 55 or 80 ° C.

The residence time is approximately 10-15 seconds and then the hot mix is ​​fed into the pelletizing press. The nodules exiting the nozzle are between 75 and 82 ° C and fall onto a cooling belt. From this strap, samples are taken for stability measurement.

Example 1

Stability against phytase pelletization in pig feed

In a Glatt VG 25 mixer, 3000 grams of cornstarch (C-gel from Cerestar) was mixed with 1380 grams of phytase ultrafiltrate (UF) with a pure enzyme content of 18.0%. After mixing, the mixture was extruded with a NICA E-220 extruder and spheronized on a Fuji Paudal QJ-400G spheronizer. The particles obtained were dried in a Glatt GPCG 1.1 (A) fluid bed drier. A second batch (B) was made by adding 70 grams of glycerol to 1420 grams of UF, corresponding to 27% of glycerol with respect to the pure enzyme, and they were mixed with the starch. A third batch (C) was made by adding 210 grams of sorbitol to 1490 grams of UF, which corresponds to 78% of sorbitol with respect to the pure enzyme, and they were mixed with the starch. A fourth batch (D) was made by adding 210 grams of inositol to 1490 grams of UF, which corresponds to 78% of inositol with respect to the pure enzyme and they were mixed with the starch. The competitive sample is the high speed granulate Phytase Novo^® CT.

TABLE 1

Residual defytase activity in pig feed in% after conditioning / pelletizing at 55/75 ° C

<td>N ° Sample with addition</td><td>Residual activity in%</td>

<td>A standard</td><td> 71</td>

<td>B glycerol</td><td> 58</td>

<td>C sorbitol</td><td> 69</td>

<td>D inositol</td><td> 73</td>

<td>Competitive sample</td><td> 70</td>

Polyols are generally known to increase the activity of proteins. However, in this example it was observed that none of the polyols tested significantly improved the stability against pelletization of phytase. Glycerol even produced a significant decrease in the stability against the nodulation of phytase.

The pig feed (feed premix) used for stability against nodulation consisted of:

Corn (20.7%), barley (40%), cassava (10%), oats (10%), soybeans (13%), fish meal (3%), wheat sharps (0.84%), soybean oil (0.5%), limestone (1.2%), salt (0.2%), trace elements (0.06%), methionine (0.05%), choline chloride 50% ( 0.05%) and calcium propionate (0.4%), up to a total of 100%.

ES 2 220 437 T3

Example 2

Stability against phytase pelletization in broiler chicken feed

In the same way as in Example 1, the following samples were made with a phytase UF with a pure enzyme content of 18.4%. The first batch (E) was made with 1300 grams of phytase UF in the starch. The second batch (F) was made by adding 13 grams of xanthan gum to 1310 grams of UF, which corresponds to 5.4% of xanthan gum with respect to the pure enzyme, and they were mixed with the starch.

TABLE 2

Residual phytase activity in chicken feed for broiler in% after conditioning / pelleting a

80/82 ° C

<td>Sample No. without addition</td><td>Residual activity in%</td>

<td>E standard</td><td> 21</td>

<td>F xanthan gum</td><td> 20</td>

Again, no stability improvement was observed against phytase pelletization.

The chicken feed for roasting consisted of:

Corn (50%), peas (3.5%), soybean meal (28%), tapioca (2.4%), meat meal (3.6%), fish meal (1%), feather meal (1%), soybean oil (1.8%), animal fat (3.5%), vitamin / mineral premix (0.9%), limestone (0.8%), monocalcium phosphate (0.9%), salt (0.3%), Mervit (Premervo, Utrecht, The Netherlands) 394 (0.7%), Mervit 393 (1.5%), up to a total of 100%.

Example 3

Stability against phytase pelletization in broiler chicken feed

In the same way as in Example 1, the following samples were made with a phytase UF with a pure enzyme content of 17.3%. The first batch (G) was made with 1470 grams of phytase UF in the starch. The second batch (H) was made by adding 15 grams of trehalose (Fluka dihydrate) to 1480 grams of UF, which corresponds to 5.9% of trehalose with respect to the pure enzyme, and they were mixed with the starch.

TABLE 3

Residual phytase activity in chicken feed for broiler in% after conditioning / pelleting a

80/82 ° C

<td>Sample No. without addition</td><td>Residual activity in%</td>

<td>G standard</td><td> 36</td>

<td>H trehalose</td><td> 45</td>

<td>Competitive sample, high speed granulation</td><td> 64</td>

Surprisingly, the addition of the polyol trehalose improves the stability against nodulation of the enzyme. Example 4

Stability against phytase pelletization in broiler chicken feed

In the same way as in Example 1, the following samples were made with a phytase UF with a pure enzyme content of 18.6%. The first batch (I) was made with 1330 grams of phytase UF in the starch. The second batch (J) was made by adding 27 grams of trehalose to 1330 grams of UF, which corresponds to 10.9% of trehalose with respect to the pure enzyme, and they were mixed with the starch. The third batch (K) was made by adding 66 grams of trehalose to 1330 grams of UF, which corresponds to 26.7% of trehalose with respect to the pure enzyme, and they were mixed with the starch. The fourth batch (L) was made by adding 13 grams of trehalose, 13 grams of PVA (5105 from Dupont) and 13 grams of ZnSO4.7aq to 1330 grams of UF, which corresponds to 5.3% of the trehalose and the PVA ,

ES 2 220 437 T3 and 2.8% of the dry salt with respect to the pure enzyme, and mixed with the starch. This last sample was coated in a fluid bed coater with 10% PEG 6000 by heating the granulate mixed with the PEG to 62 ° C and cooled again (M). Similarly, a sample from batch (L) was coated with 10% PEG 20,000 (diluted 1: 2 in water to reduce viscosity) by spraying on a granulate in a fluidized bed coater at 62 ° C, dried and subsequently cooled (N).

TABLE 4

Residual phytase activity in chicken feed for broiler in% after conditioning / pelleting a

81/80 ° C

<td>Sample No. without addition</td><td>Residual activity in%</td>

<td>I standard</td><td> 28</td>

<td>J trehalose 2%</td><td> 28</td>

<td>K trehalose at%</td><td> 34</td>

<td>L ZnSO₄ 1% -PVA 1% -trehalose 1%</td><td> 40</td>

<td>M as L with PEG 6000 coating</td><td> 42</td>

<td>M as L with PEG 20,000 coating</td><td> 52</td>

The highest nodulation stability of the enzyme is obtained with PEG-coated granules containing trehalose, ZnSO4 and PVA.

Example 5

Stability against phytase pelletization in broiler chicken feed

In the same way as in Example 1, the following samples were made with a phytase UF with a pure enzyme content of 18.0%. The first batch (O) was made with 13868 grams of phytase UF with 14 grams of ZnSO4.6aq and 14 grams of PVA 5/88 (from ERKOL SA.), Which corresponds to 3.0% of the dry salt and 5.3% of the PVA with respect to the pure enzyme. The second batch (P) was made in a similar way, but with an additional 14 grams of trehalose in the recipe (5.3% trehalose with respect to the pure enzyme). Both recipes were coated in a fluid bed coater (STREA from NIRO-AEROMATIC) with 10% PEG 6000, dissolved in the same amount of water, resulting in samples Q and R. The four samples were tested in an experiment of nodulization.

TABLE 5

Residual phytase activity in chicken feed for broiler in% after conditioning / pelleting a

80/80 ° C

<td>Sample No. with addition</td><td>Residual activity in%</td>

<td>O Product with ZnSO₄ and PVA</td><td> 32</td>

<td>P as O with 5.3% trehalose</td><td> 32</td>

<td>Q as O with 10% PEG 6000 coating</td><td> 44</td>

<td>R as P with 10% PEG 6000 coating</td><td> 44</td>

The polyethylene glycol coating significantly improved the nodulation stability of the samples.

Example 6

Dissolution time of granules containing phytase

Several of the samples prepared in the previous examples were dissolved in buffer and the samples were collected at

ES 2 220 437 T3 regular intervals. The dissolution time of the granules was not increased or only slightly increased as a consequence of the PEG coating.

TABLE 6

Dissolution time expressed as% dissolved after x minutes

<td>Show</td><td>1 min.</td><td>2 min.</td><td>5 min.</td><td>10 minutes.</td><td>15 min.</td><td>30 min.</td><td>60 min.</td>

<td>L</td><td> 100</td><td> 100</td><td> 98</td><td> 99</td><td> 100</td><td> 99</td><td> 100</td>

<td>M</td><td> 98</td><td> 99</td><td> 100</td><td> 98</td><td> 99</td><td> 99</td><td> 100</td>

<td>N</td><td> 82</td><td> 96</td><td> 99</td><td> 100</td><td> 99</td><td> 100</td><td> 99</td>

<td>HS great</td><td> 4</td><td> 6</td><td> 20</td><td> 30</td><td> 38</td><td> 72</td><td> 95</td>

HS great is Phytase Novo^® CT Example 7

Stability of granules containing phytase

For the analysis of the autostability of the granules, several samples were tested at 35 ° C in closed vials. TABLE 7

Self-stability of different samples

<td>Show</td><td>0 weeks</td><td>2 weeks</td><td>4 weeks</td><td>8 weeks</td><td>12 weeks</td><td>16 weeks</td>

<td>TO</td><td> 100</td><td> 87</td><td> 73</td><td> 69</td><td> 67</td><td> 65</td>

<td>B</td><td> 100</td><td> 87</td><td> 77</td><td> 67</td><td> -</td><td> -</td>

<td>C</td><td> 100</td><td> 84</td><td> 64</td><td> 58</td><td> --</td><td> -</td>

<td>D</td><td> 100</td><td> 87</td><td> 72</td><td> 62</td><td></td><td></td>

<td>G</td><td> 100</td><td> 87</td><td> 82</td><td> 78</td><td> --</td><td> --</td>

<td>H</td><td> 100</td><td> 88</td><td> 80</td><td> 76</td><td> -</td><td> -</td>

<td>I</td><td> 100</td><td> 87</td><td> 78</td><td> 76</td><td> 70</td><td> 69</td>

<td>J</td><td> 100</td><td> 86</td><td> 78</td><td> 77</td><td> 69</td><td> 69</td>

<td>K</td><td> 100</td><td> 88</td><td> 81</td><td> 74</td><td> 70</td><td> 69</td>

<td>L</td><td> 100</td><td> 97</td><td> 93</td><td> 96</td><td> 87</td><td> 86</td>

<td>M</td><td> 100</td><td> 94</td><td> 93</td><td> 92</td><td> 90</td><td> 90</td>

Example 8

Phytase bioavailability using different formulations

The following phytase and monocalcium phosphate (MCP) formulations were added to a standard corn / soy broiler chicken feed at three different doses (75, 150 and 225 FTU / kg) and given to the chickens to broil for 28 days. The broiler chicken feed also contained endogenous phytase activity.

ES 2 220 437 T3 * Natuphos^® 5000 G, (standard product)

Composition:

<td> •</td><td>Phytase UF</td><td>94.49 kg</td>

<td> •</td><td>Trehalose</td><td>0.91 kg</td>

<td> •</td><td>Poly (vinyl alcohol)</td><td>0.91 kg</td>

<td> •</td><td>ZnSO4.6H2O</td><td>0.97 kg</td>

<td> •</td><td>Starch</td><td>200.00 kg</td>

<td> •</td><td>Adding water</td><td>7.11 kg</td>

<td colspan="2">Total weight</td><td>304.39 kg</td>

<td colspan="2">Total weight of dried product</td><td>215.02 kg</td>

* Natuphos^® 5000 G, (PEG coated)

Composition:

<td> •</td><td>Phytase SD</td><td>11.54 kg</td>

<td> •</td><td>Phytase UF</td><td>99.88 kg</td>

<td> •</td><td>Trehalose</td><td>1.14 kg</td>

<td> •</td><td>Poly (vinyl alcohol)</td><td>1.14 kg</td>

<td> •</td><td>ZnSO4.6H2O</td><td>1.14 kg</td>

<td> •</td><td>Starch</td><td>200.00 kg</td>

<td> •</td><td>Adding water</td><td>7.10 kg</td>

<td colspan="2">Total weight</td><td>306.85 kg</td>

<td colspan="2">Total weight of dried product</td><td>228.82 kg</td>

Coated with 10% 1/1/2 solution of PEG6000 / PEG20000 / H2O (Phytase SD: Spray Dried Phytase Powder) * Phytase Novo^® CT, Competitor Product, Grease Coated * MCP: Positive Control Groups

After 28 days the growth of the animals was determined (Table 8).

Regression analysis was applied to allow comparison of growth by phytase formulation (Table 9), with which growth was calculated as follows:

Growth = intersection + regression coefficient * phytase activity (FTU / kg).

TABLE 8

Broiler chicken growth after 28 days

<td>Product</td><td>Activity (FTU / kg¹)</td><td>Increase (g)</td>

<td>Control</td><td> 82</td><td> 1292</td>

<td>MCP (0.2 g P / kg)</td><td> 86</td><td> 1459</td>

<td>MCP (0.4 g P / kg)</td><td> 115</td><td> 1581</td>

<td>MCP (0.6 g P / kg)</td><td> 116</td><td> 1674</td>

<td>NPHG 518 RE1</td><td> 185</td><td> 1433</td>

<td>NPHG 518 RE1</td><td> 281</td><td> 1456</td>

<td>NPHG 518 RE1</td><td> 393</td><td> 1528</td>

<td>NPHG 518 RE2A</td><td> 208</td><td> 1452</td>

<td>NPHG 518 RE2A</td><td> 375</td><td> 1475</td>

<td>NPHG 518 RE2A</td><td> 416</td><td> 1629</td>

ES 2 220 437 T3

TABLE 8 (continued)

<td>Product</td><td>Activity (FTU / kg¹)</td><td>Increase (g)</td>

<td>Phytase Novo CT</td><td> 182</td><td> 1331</td>

<td>Phytase Novo CT</td><td> 274</td><td> 1481</td>

<td>Phytase Novo CT</td><td> 401</td><td> 1458</td>

<td>¹ Determined phytase activity</td><td>TABLE 9</td><td></td>

<td>Intersection: 1302</td><td>Estimate</td><td> %</td>

<td>MCP</td><td> -</td><td></td>

<td>NPHG 518 RE1 (ZnSO4)</td><td> 0,583</td><td> 100</td>

<td>NPHG 518 RE2 (ZnSO4 + PEG)</td><td> 0,649</td><td> 111</td>

<td>Phytase Novo CT</td><td> 0,434</td><td> 74</td>

It can be concluded that the bioavailability of phytase in the polyethylene glycol coated formulation is much higher than that of phytase in the formulation containing a fat coating.