Patent Description:
More specifically, the invention provides an additive for fodder for animal nutrition, for example feedlot cattle or poultry, which includes a phytase enzyme and a calcium phosphate source in a single additive.

Feed for feedlot animals and poultry, generally known as compound fodder, is fodder that is mixed from various raw materials and additives. Compound fodder can be prepared as whole feed, which provides all nutrients that are required daily, as concentrates, which provide a part of the ration (protein, energy), or as supplements, which provide additional micronutrients, for example minerals and vitamins.

A main ingredient used in commercial preparations is cereal grains or legumes, for example corn, soybean, sorghum, oats, barley and mixtures thereof.

In the animal feed industry, the sale and manufacture of premixes is also relevant. Premixes can be composed of micro-ingredients that are mixed into commercial rations or produced individually to obtain animal feed. Micro-ingredients may include nutrients, vitamins, minerals, chemical preservatives, antibiotics, fermentation products, enzymes and other essential ingredients.

Thus, fodder mixtures are normally formulated according to the specific requirements of the target animal. Feed manufacturers prepare the mixtures by purchasing the products, in other words, the main ingredients, for example cereals such as corn, soybean, sorghum, oats and barley, and the desired micro-ingredients and mixing or combining them in a fodder mill according to specifications provided by a nutritionist. These mixtures can be obtained in the form of flours, pellets or pastes.

Usually, a phosphorus and calcium source, such as monocalcium phosphate with a phosphorus content of more than <NUM> % or dicalcium phosphate with a phosphorus content of <NUM> %, is added to the fodder.

Although micro-ingredients provide a highly bioavailable source of inorganic phosphorus, organic phosphorus is also present in animal feed in plant tissue which is the main ingredient. Nevertheless, this organic phosphorus is present as phytic acid, inositol hexaphosphate (phytate), which cannot be digested by non-ruminant animals.

Among the mineral macronutrients present in fodder, one of the most important is phosphorus. Although it is usually present in high amounts in diets, it is not properly assimilated in most cases. Of the total amount of this mineral that is consumed, only <NUM> % is retained, while the remaining <NUM> % is discarded in faeces (<NPL>).

The problem in the assimilation of phosphorus and other essential substances is due to the presence of anti-nutritional factors in feed, which act by binding to nutrients, causing a decrease in the availability thereof (<NPL>; <NPL>).

Among the anti-nutritional factors related to phosphorus metabolism is phytic acid, abundant in cereals and legumes, the presence of which can vary from <NUM> % in cereals such as soybean to <NUM>% in corn and wheat (<NPL>).

Phytic acid is a carboxylic acid with a pKa less than <NUM>, which has six strongly coupled protons with a pKa of between <NUM> and <NUM> in its structure, which gives the structure a strong chelation potential on essential minerals such as calcium, as well as the ability to bind to proteins, amino acids and sugars and inhibit some digestive enzymes, as trypsin and chymotrypsin, alpha-amylases, tyrosinases and pepsins. By establishing ionic bonds between phytic acid and nutrients, insoluble chelates called phytates, which are a large group of highly complex compounds that cannot be assimilated by non-ruminant organisms, are formed (<NPL>). Accordingly, everything that is bound to this compound will not be fully used. Moreover, since organic phosphorus is therefore not available to the animal, it is excreted into the surrounding environment, which constitutes a potential danger for the immediate surroundings when it is released into the environment in large quantities.

To avoid this problem, the technique of adding a phytase enzyme to fodder as a separate ingredient is known.

Phytases are part of a subgroup of enzymes of the family of acid phosphatases, which are of the hydrolase type. They act by breaking phosphomonoester bonds, degrading phytates to myo-inositol hexaphosphate and inorganic phosphorus, which have less or no chelating effect (Kim et al. In this manner, organic phosphorus becomes an additional phosphorus source. Phytase activity is measured by FTU units, one FTU unit being the amount of enzyme necessary to release <NUM>µmol of inorganic orthophosphate per minute in a solution containing <NUM> mol/l of sodium phytate at pH <NUM> and at a temperature of <NUM>.

For example, <CIT> describes a thermotolerant phytase and the use thereof in a feed product for cattle.

Likewise, phosphates are important sources of Ca, the content of which is inverse to the content in P; mineral supplementation, in particular calcium, is made in the form of bone meal, which is currently highly questioned, tricalcium, dicalcium and monocalcium phosphates.

Nevertheless, the drawback arises when it comes to mixing some phytases with the different calcium phosphate sources, in particular monocalcium phosphate, dicalcium phosphate or a mixture of both.

Thus, it has been described that Ca<NUM>+ has an inhibitory effect on phytase activity at concentrations above <NUM> (<NPL>). Likewise, <NPL>, indicates that in a study of the possible industrial application of a phytase, phytase activity was inhibited by <NUM> % by the presence of a calcium ion at concentrations of <NUM> and <NUM>.

<NPL>) also indicates that the response to a given level of exogenous phytase can be affected by the amount of calcium and/or the Ca/P ratio, the level of P and the level of phytate in the diet. Thus, a high Ca/phytate molar ratio present in the diet can lead to the formation of highly insoluble Ca-phytate complexes in the intestinal tract. It is also believed that an excess of Ca may reduce enzyme activity by competing with phytases for their preferred site of action (<NPL>). In pig studies, a greater response to phytases, better use of P, is obtained when the total Ca/total P ratio is kept between <NUM>:<NUM> and <NUM>:<NUM>; higher levels of Ca reduce P uptake and phytate utilisation (<NPL>). It has also been noted that an increase in the level of vitamin D in the diet (<NUM> IU/kg) partially reduces the negative effect that a normal level of Ca has on phytase activity, but it does not lead to an improvement when the Ca level is low (Lei et al. Nevertheless, no increase in P utilisation is observed upon adding <NUM>,<NUM> IU of vitamin D/kg to a diet with or without phytases (<NPL>).

The sensitivity of phytases to the presence of soluble phosphorus in the form of phosphate has also been described in the literature (<NPL>. Enzymatic Properties of Phytase from Escherichia coli DFI5α4,<NUM>; <NPL>, Enzymatic evaluation of Bacillus amyloliquefaciens phytase as a feed additive).

Therefore, calcium sources in the form of calcium phosphates and phytases as independent additives are usually incorporated into the fodder, since, as previously mentioned, direct mixing of these two additives is not feasible due to the fact that phosphates inhibit phytase activity.

For example, <CIT> describes an additive for fodder that can include a phytase and macrominerals such as calcium or phosphorous that are added to the fodder separately. <CIT> discloses a liquid enzyme formulation comprising: (a) a phytase; (b) a buffer, wherein the buffer is selected from the group consisting of: sodium citrate, potassium citrate, citric acid, sodium acetate, acetic acid, sodium phosphate, potassium phosphate, and any combination thereof; (c) a stabiliser selected from the group consisting of: sucrose, sorbitol, mannitol, glycerol, trehalose, sodium chloride, sodium sulphate, or any combination thereof; and, (d) an anti-microbial. <CIT> discloses a pig feed that allows the utilisation rate of calcium and phosphorous therein to be improved, the discharge of calcium and phosphorous to be decreased, the environmental pollution to be reduced, the feed/grain ratio of pig feeding to be reduced, and the enterprise operating costs to be decreased. The composite feed comprises, by weight, <NUM>-<NUM> parts of corn, <NUM>-<NUM> parts of wheat bran, <NUM>-<NUM> parts of soybean meal, <NUM>-<NUM> parts of a premix, <NUM>-<NUM> parts of salt, <NUM>-<NUM> parts of stone flour, <NUM>-<NUM> parts of calcium hydrogen phosphate, <NUM>-<NUM> parts of phytase, and <NUM>-<NUM> parts of citric acid.

The present invention solves the drawbacks of the additives currently known in the state of the art, providing an additive that includes both a phosphorus source and a phytase, in particular an additive that includes monocalcium and dicalcium phosphates containing a phytase enzyme, without the phytase losing its activity.

To that end, in general, the invention is based on the inclusion of the phytase enzyme in an organic fibre matrix to which a natural calcium chelating agent is incorporated.

As mentioned, the invention provides a food additive for fodder which includes a phytase enzyme and a calcium phosphate source in a single additive.

Thus, in one embodiment, the additive of the invention comprises:.

Preferably, the polyol included as a liquid phase in the additive of the invention is selected from glycols such as ethylene glycol, propylene glycol, butylene glycol, polyethylene glycol, polypropylene glycol, polybutylene glycol or combinations thereof.

Preferably, the amount of polyol present in the additive is selected from between <NUM> and <NUM> % by weight with respect to the total weight of the liquid mixture, adapting the amount of polyol to the amount of ground vegetable fibre.

Preferably, the amount of ground vegetable fibre present in the additive of the invention ranges between <NUM>-<NUM> % by weight with respect to the total weight of the liquid mixture.

In another embodiment, the additive of the invention comprises:.

In this second embodiment, the amount of ground vegetable fibre present in the additive of the invention ranges between <NUM> and <NUM> % with respect to the total weight of the solid mixture.

In both embodiments, any suitable vegetable fibre containing phytate, for example barley, corn, wheat, wheat bran, rice bran or soybean flour, can be used as vegetable fibre for the described additive. Preferably, cereal bran is used, for example wheat or rice bran.

For the described embodiments, the amount of the liquid or solid mixture to be applied to the phosphate source is selected so that the additive of the invention contains between <NUM> and <NUM>,<NUM> FTU per gram.

Although the phytase used in the invention is not particularly limited, it should maintain its activity in the pH range of the animal's gastrointestinal tract. Therefore, in one embodiment of the invention a thermostable phytase with high specific activity, of at least <NUM> ± <NUM> U/mg, derived from Serratia odorifera, is used, for example that described in patent application <CIT>. Selection of this particular phytase ensures that its activity is maintained within a broad pH range from about <NUM> in the stomach to about <NUM> in the intestine.

Another object of the invention is the use of the additive described above in conventional animal fodder, said additive according to the invention being incorporated in a proportion of between <NUM> and <NUM> for each ton of fodder. The use of the additive of the invention makes it possible to recover <NUM> % of the phytase activity in the final fodder.

In this regard, the additive of the invention is easy to apply in the formulation of the fodder, applied, for example, by direct mixing with the other components of the food ration directly or as part of a premix, or to be mixed directly with fodder already formulated in the form of flour and which must be corrected in terms of its nutritional characteristics.

The additive of the invention is also suited to be applied in feed mixtures (for example corn, wheat, barley, soybean, pea, among others) that will later be ground to make fodder in the form of flour, ground and pelletised to make fodder in pellet form, or ground and moistened to form slurry.

Likewise, the additive of the invention is also suited to be applied at a time after grinding and prior to pelletisation or, when appropriate, to the generation of the slurry.

The invention is illustrated below on the basis of exemplary embodiments thereof. These examples are illustrative of the invention and should not be understood as limiting it.

A solution A is prepared containing <NUM>,<NUM>±<NUM>,<NUM> FTU/ml provided by the phytase of Serratia odorifera described in <CIT>, <NUM> % by weight of ground wheat bran and <NUM> % by weight of citric acid, as well as <NUM> % by weight of polyethylene glycol.

For the purpose of comparison, a control solution (B) is prepared which only contains said phytase in a similar proportion (<NUM>,<NUM>±<NUM>,<NUM> FTU/ml).

Both solutions are added to three sources of granulated phosphate in concentrations of <NUM>, <NUM> and <NUM> %: <NUM>. monocalcium phosphate (with an average particle size of <NUM>±<NUM>), <NUM>. dicalcium phosphate (with an average particle size of <NUM>±<NUM>) and <NUM>. monocalcium phosphate (with an average particle size of <NUM>±<NUM>).

Finally, the real activity of each combination is measured, obtaining the results shown in the following table <NUM>:.

As can be seen from the results obtained, the additive of the invention which includes phytase, ground wheat bran, citric acid and polyethylene glycol, as well as a phosphorus source, retains phytase activity to a much greater extent than phytase alone.

A solution A is prepared containing <NUM>,<NUM>±<NUM>,<NUM> FTU/ml provided by the phytase of Escherichia coli, <NUM> % by weight of ground wheat bran and <NUM> % by weight of citric acid, as well as <NUM> % by weight of polyethylene glycol.

A solution A is prepared containing <NUM>,<NUM>±<NUM> FTU/ml provided by a phytase of Serratia odorifera, <NUM> % by weight of ground wheat bran and <NUM> % by weight of citric acid.

For the purpose of comparison, a control solution B is prepared which only contains said phytase in the same proportion. Solutions A and B are dried by spray-drying methods.

The activity of dry products is quantified:.

Both dry products are added to two sources of powder phosphate in concentrations of <NUM>, <NUM> and <NUM> %: <NUM>. monocalcium phosphate and <NUM>. dicalcium phosphate.

Finally, the real activity of each product is measured, obtaining the results shown in the following table <NUM>:.

Claim 1:
A food additive for fodder for animal nutrition, the additive comprising a liquid mixture of a polyol together with
i) a phytase in an amount suited to provide a final phytase activity of between <NUM>,<NUM> and <NUM>,<NUM> FTU/ml, FTU being the amount of enzyme necessary to release <NUM>µmol of inorganic orthophosphate per minute in a solution containing <NUM> mol/l of sodium phytate at pH <NUM> and at a temperature of <NUM>;
ii) ground vegetable fibre with a maximum particle diameter of <NUM>,
iii) citric acid in a concentration between <NUM>-<NUM> % by weight with respect to the total weight of the liquid mixture as chelating agent, stabilising the pH at pH <NUM> and
a phosphorus source selected among monocalcium phosphate, monodicalcium phosphate or a combination of both in the form of defluorinated or granulated mud.