Feed additives for animal feed are well known. Such feed additives may serve to improve the gut health of animals, especially livestock, or to improve the general health of the animals. Also feed additives are known to preserve animal feed, in particular against bacterial infestation and/or fungal infestation. A commonly used ingredient in such preserving feed additives is propionic acid and salts thereof. Propionic acid may relatively effectively reduce mold formation on animal feed. Under certain conditions, e.g. at higher temperatures and/or more moist conditions, the effectiveness of propionic acid may not be sufficient. Moreover, under such conditions the stability of the feed additive, and the diluted feed additive (with water) before it is applied onto the animal feed may be low, which in turn renders the feed additive to be less effective against molds. The object of the invention to provide novel feed additives.

The present invention pertains to a feed additive comprising an emulsifier, propionic acid and/or salt thereof and at least one fatty acid having 6 to 12 carbon atoms, wherein the weight ratio of propionic acid and fatty acid is at least 2 and at most 25 and the amount of ammonium formate is less than 0.5 wt%. The feed additive of the invention was shown to have a better antifungal efficacy against molds, especially molds that form on animal feed upon storage. The fatty acids generally are more efficacious against mold than propionic acid and/or its salts, i.e. these fatty acids have a lower minimum inhibitory concentration (MIC) value for various molds than propionic acid and/or its salts. It was also found that the feed additive of the invention is effective against xerophilic molds, i.e. molds that also grow under water-poor or water-free environments such as Aspergillus chevalieri or Penicillium lanosocoeruleum. In addition, it was found that the combination of propionic acid and the fatty acid of the invention exhibits a synergistic antifungal efficacy against molds when compared to propionic acid and fatty acid per se, i.e. the actual efficacy of the combination is more than the sum of the individual efficacies of the individual components. Moreover, the presence of the fatty acids, and particularly when the amount of the fatty acid of the invention is at least 2.5 weight percent (wt%), increases the stability of the feed additive per se, and upon dilution with water before being applied to the animal feed. A further advantage is that animal feeds will contain less to no mycotoxins originating from molds such as

Aspergillus flavus and Aspergillus parasiticus. Also the shelf life of animal feed will be improved. Further advantages of the feed additive of the invention are its ability to inhibit bacterial growth, to improve the gut health, improve epithelial integrity and improve the palatability of the animal feed.

The feed additive of the invention comprises propionic acid and/or salt thereof.

Examples of suitable salts include ammonium propionate, sodium propionate and potassium propionate. The feed additive may comprise propionic acid alone, a salt of propionic acid alone or a combination of propionic acid and a salt of propionic acid. Preferably, the feed additive comprises a combination of propionic acid and a salt of propionic acid. The presence of the salt of propionic acid allows the feed additive to have a reduced smell or no smell at all, and reduces the corrosiveness of the feed additive. In one embodiment, the weight ratio between propionic acid and the salt of propionic acid is at most 100, preferably at most 50, more preferably at most 20, even more preferably at most 10, and most preferably at most 5, and generally at least 0.01 , preferably at least 0.02, more preferably at least 0.05, even more preferably at least 0.1 , and most preferably at least 0.2.

In one embodiment of the invention, the amount of propionic acid and/or salts thereof is generally at least 10 wt%, based on the total weight of feed additive. Preferably, the amount of propionic acid and/or salts thereof is at least 20 wt%, more preferably at least 25 wt% and most preferably at least 30 wt%, and generally at most 80 wt%, preferably at most 70 wt%, more preferably at most 60 wt%, and most preferably at most 50 wt%%, based on the total weight of feed additive.

The feed additive of the invention comprises fatty acids having 6 to 12 carbon atoms. Such fatty acids are also referred to as medium chain fatty acids (MCFA). The fatty acid may be any known fatty acid or fatty acid combinations. In a preferred embodiment, the feed additive of the invention comprises at least two fatty acids having 6 to 12 carbon atoms. In a further embodiment, the feed additive of the invention comprises a fatty acid having 6, 8, 10 and/or 12 carbon atoms. Examples of such fatty acids include caproic acid, caprylic acid, capric acid and lauric acid, as well as combinations of two or more of these fatty acids such as caprylic acid and capric acid, and caprylic acid, capric acid and lauric acid. Specific examples include Wilfarin caprylic/capric acid ex Wilmar Europe Trading B.V. (comprising C8 and C10 carboxylic acids) and Lodestar C8-C12 B25/B30 ex Berg + Schmidt Functional Lipids GmbH & Co KG (comprising C8, C10 and C12 carboxylic acids). Such combinations of fatty acids generally have a better efficacy and are generally economically more attractive. When the feed additives comprises a combination of caprylic acid and capric acid, the weight ratio between caprylic acid and capric acid is at least 0.5, preferably at least 0.6 and most preferably at least 0.7, and generally at most 2, preferably at most 1.7, and most preferably at most 1.5. In one embodiment, the fatty acid is not pelargonic acid. Pelargonic acid per se is relatively expensive and consequently less suitable for use in feed additives.

In one embodiment of the invention, the amount of fatty acid is generally at least 2.5 wt%, based on the total weight of feed additive. Preferably, the amount of fatty acid is at least 3 wt%, more preferably at least 4 wt% and most preferably at least 5 wt%, and generally at most 60 wt%, preferably at most 50 wt%, more preferably at most 40 wt%, even more preferably at most 30 wt%, and most preferably at most 20 wt%%, based on the total weight of feed additive. The feed additive of the invention comprises the propionic acid and/or salts thereof and the fatty acid in a weight ratio of at most 25. In the context of this description, the weight of propionic acid and salts thereof should be taken as the total weight of propionic acid and salt(s) of propionic acid; also the weight of fatty acid should be taken as the total weight of the fatty acids of the invention. Preferably, the weight ratio of propionic acid and fatty acid is at most 20, more preferably at most 15, and most preferably at most 10, and preferably the weight ratio of propionic acid and fatty acid is at least 0.1 , more preferably at least 0.5, even more preferably at least 1 , more preferably at least 2, and most preferably at least 3. In one embodiment, the feed additive may further comprise formic acid and/or salt thereof. Examples of suitable salts include ammonium formate, sodium formate and potassium formate, preferably the salt is selected from sodium formate and potassium formate. The feed additive may comprise formic acid alone, a salt of formic acid alone or a combination of formic acid and a salt of formic acid. Preferably, the amount of salt of formic acid is at most 20 wt%, preferably at most 10 wt%, more preferably at most 5 wt% and most preferably at most 1 wt%, and generally at least 0.001 wt%, preferably at most 0.01 wt% and most preferably at most 0.1 wt%. The feed additive of the invention comprises ammonium formate in an amount of at most 0.5 wt%, preferably at most 0.1 wt% and most preferably at most 0.01 wt%. In a preferred embodiment, the feed additive of the invention is substantially free from ammonium formate. More preferably, the feed additive of the invention is completely free from ammonium formate. The term “substantially free from ammonium formate” means that less than 100 parts per million of ammonium formate is present in the feed additive. The term“completely free” means that the feed additive contains less than 20 parts per billion (ppb) of ammonium formate. The feed additive of the invention comprises the propionic acid and/or salts thereof and formic acid in a weight ratio of at most 25. In the context of this description, the weight of formic acid should be taken as the total weight of formic acid and salt(s) of formic acid. Preferably, the weight ratio of propionic acid and formic acid is at most 20, more preferably at most 15, and most preferably at most 10, and preferably the weight ratio of propionic acid and formic acid is at least 0.1 , more preferably at least 0.5, even more preferably at least 1 , more preferably at least 2, and most preferably at least 3.

In one embodiment of the invention, the feed additive further comprises sorbic acid. Preferably, the amount of sorbic acid is generally at least 0.1 wt%, based on the total weight of feed additive. Preferably, the amount of sorbic acid is at least 0.2 wt%, more preferably at least 0.5 wt% and most preferably at least 1 wt%, and generally at most 5 wt%, preferably at most 4 wt%, more preferably at most 3 wt%, and most preferably at most 2 wt%%, based on the total weight of feed additive. In one embodiment of the invention, the feed additive further comprises a neutralizing agent. The neutralizing agent may be any neutralizing agent known in the art. Examples of such neutralizing agent include sodium hydroxide, potassium hydroxide and ammonia (NH ₃ ). Preferably, the neutralizing agent is selected from sodium hydroxide and potassium hydroxide. Most preferably, the neutralizing agent is sodium hydroxide.

The neutralizing agent may generally be present in an amount of at least 1 wt%, based on the total weight of feed additive. Preferably, the amount of neutzralizing agent is at least 2 wt%, more preferably at least 5 wt% and most preferably at least 10 wt%, and generally at most 50 wt%, preferably at most 40 wt%, more preferably at most 30 wt%, and most preferably at most 20 wt%%, based on the total weight of feed additive. In one embodiment of the invention, the feed additive comprises an emulsifier. The emulsifier may be any emulsifier known in the art. Examples of such emulsifiers include glycerol-monolaurate, ethoxylated castor oil, soy lecithin, milk-derived casein, lysolecithin (lecithin), bile salt, glycerol polyethylene glycol ricinoleate, polyethoxylated sorbitan (e.g. Polysorbate® 80), non-ionic surfactants (e.g. Bredol®) and sodium stearoyl-2-lactylate.

The emulsifier may generally be present in an amount of at least 1 wt%, based on the total weight of feed additive. Preferably, the amount of emulsifier is at least 2 wt%, more preferably at least 5 wt% and most preferably at least 10 wt%, and generally at most 50 wt%, preferably at most 40 wt%, more preferably at most 30 wt%, and most preferably at most 20 wt%%, based on the total weight of feed additive.

In one embodiment of the invention, the feed additive further comprises an organic acid with nutritional and/or antibacterial properties. The organic acid may be any organic acid with nutritional and/or antibacterial properties known in the art. Examples of such organic acids include lactic acid, oxalic acid, butyric acid, tartaric acid, acetic acid, citric acid, fumaric acid, valeric acid and benzoic acid.

The organic acid may generally be present in an amount of at least 1 wt%, based on the total weight of feed additive. Preferably, the amount of organic acid is at least 2 wt%, more preferably at least 5 wt% and most preferably at least 10 wt%, and generally at most 50 wt%, preferably at most 40 wt%, more preferably at most 30 wt%, and most preferably at most 20 wt%%, based on the total weight of feed additive.

The remaining part of the feed additive may be comprised of other components commonly or not commonly used in feed additives. With the propionic acid and the fatty acid, the other components add up to 100 wt% of the total weight of the feed additive.

In one embodiment, the feed additive of the invention is a feed preservative.

The invention further pertains to animal feed comprising the feed additive of the invention. More particularly, the invention pertains to animal feed comprising the feed additive comprising propionic acid and/or salt thereof and at least one fatty acid having 6 to 12 carbon atoms, wherein the weight ratio of propionic acid and fatty acid is at most 25 and the amount of ammonium formate is less than 0.5 wt%. The term“animal feed” is defined as a composition comprising animal nutrients such as fats and/or proteins and/or carbohydrates that is fed to an animal to provide in its metabolic requirements. Animal feed can be a nutritional complete feed (i.e. providing all required nutrients to support a normal metabolism of the animal), but it may also be a premix or other composition that contains only part of the required nutrients, to be mixed with other nutrients or fed separately from these other nutrients.

The term“total daily intake of feed” is defined as the complete mass of feed an animal takes per day, excluding drinking water.

In a first embodiment of the feed according to the invention, the feed additive is present in an amount of 0.01 to 10 kg per ton of total daily intake of feed. In other words, the total amount of feed (excluding the drinking water) as is fed to the animal comprises per 1000 kilograms, 0.01 to 10 kg of feed additive. This amount can be present in a nutritional complete feed as such, at a level of 0.01 to 10 kg per ton of that feed material, or may for example be present in a concentrated feed material (exceeding 10 kg/ton feed material) as long as the amount per total daily intake of feed is between 0.01 and 10 kg feed additive per ton. In particular, the feed additive is fed at an amount of 0.05 to 2 kg per ton of total daily intake of feed. These amounts appear to suffice for use according to the current invention.

The invention further pertains to the use of a feed additive comprising propionic acid and/or salt thereof and at least one fatty acid having 6 to 12 carbon atoms, wherein the weight ratio of propionic acid and fatty acid is at most 25 and the amount of ammonium formate is less than 0.5 wt% for inhibiting mold growth in an animal feed. The invention further pertains to the use of a feed additive comprising an emulsifier, propionic acid and/or salt thereof and at least one fatty acid having 6 to 12 carbon atoms, wherein the weight ratio of propionic acid and fatty acid is at most 25 and the amount of ammonium formate is less than 0.5 wt% for inhibiting mold growth in an animal feed.

The invention further pertains to the use of a feed additive comprising propionic acid and/or salt thereof and at least one fatty acid having 6 to 12 carbon atoms, wherein the weight ratio of propionic acid and fatty acid is at most 25 and the amount of ammonium formate is less than 0.5 wt% in drinking water for animals, in particular livestock. The invention further pertains to the use of a feed additive comprising an emulsifier, propionic acid and/or salt thereof and at least one fatty acid having 6 to 12 carbon atoms, wherein the weight ratio of propionic acid and fatty acid is at most 25 and the amount of ammonium formate is less than 0.5 wt% in drinking water for animals, in particular livestock. The invention is exemplified in the following Examples.

Examples

Example 1 : Synergistic effect of propionic acid with MCFA towards fungi

A new test was developed to evaluate the damage of antifungal compounds ingredients on biofilms of germ tubes of the fungus Aspergillus chevalieri. Small 1 cm ² blocks of colonies of a 1 -week-old sporulating culture grown on solid medium were placed in malt extract supplemented with 20% (w/v) sucrose and vortexed at maximal speed to bring the conidia into suspension. The solution was filtered over sterile glass wool and the concentration of conidia was evaluated. A quantity of 1.25 mL medium containing approximately 10 ⁶ spores was placed in a small Erlenmeyer flask (25 mL) and incubated overnight as standing cultures at 25°C.

Indicated percentages of test compound solutions prepared in ME20S medium were gently added to the standing cultures and mixed carefully. This culture was incubated for 30 min at room temperature, and biofilm was loosened by means of a pipetting tip and 10 pL culture was added to 90 pL demi water containing 4 pM TOTO-1

(Quinolinium, 1-T-[1 ,3-propanediylbis[(dimethyliminio)-3,1-propanediyl]]bis[4-[( 3-methyl- 2(3H)-benzothiazolylidene) methyl]]-tetraiodide, Thermo Fisher Scientific, Waltham, MA, USA) as a final concentration. After 15 min staining, microscopical slides were prepared for fluorescence microscopy (Zeis Axioskop, Zeiss, Oberkochen, Germany) equipped with Filterblock II (09), 450-490 nm, FT 510, LP 520. Micrographs were taken with the Axiocam software (Zeiss, Oberkochen, Germany). The total time of exposition to ingredients was 30 min. and germ tubes in diluted solutions were counted afterwards in a time window of 30 min starting 15 min after the beginning of staining.

This test was used to establish possible synergy between the activated propionate (propionic acid and NFU) and the ingredient MCFA (Wilfarin caprylic/capric acid ex Wilmar Europe Trading B.V.) and E484 as an emulsifier. The concentration of propionic acid and ammonia in the premixture was 57.3 % (w/w) and 8.5 % (w/w), respectively. The concentration of MCFA and E484 in premixture was 2 % (w/w) and 1.8 % (w/w), respectively. The premixture concentrations were tested in the range of 0,4 % (w/w) to 0.15 % (w/w), but the ratio between ingredients remained the same across the range. Table 1. Percentage of germ tubes stained with TOTO-1 of A. chevalieri in the presence of propionic acid and ammonia, of MCFA and E484 and in combination of both agents.

*The column“Synergy” shows the difference in % stained cells between the

combination of ingredients minus the sum of the single ingredients.

Aspergillus chevalieri showed a significantly higher percentage of TOTO-1 stained cells when incubated with a combination of propionic acid and NFU and MCFA + pol80, than when incubated with the sole ingredients (see Table 1). Especially at the higher concentrations, 0.4 - 0.2 % (v/v), the synergistic effect of these ingredients is clearly evident.

Example 2

The antifungal activity of compounds was determined using a MIC (minimum inhibitory concentration) assay in microtiter plate format. For this purpose A. chevalieri was grown for 10 days at 25°C on DG18 (Dichloran Glycerol Agar) supplemented with 100 mg/I CAM (Chloramphenicol) plates (pH 5.6). The fungal spores were harvested by adding ± 10-13 ml sterile ice-cold ACES-Buffer (N-(2-Acetamido)-2-aminoethanesulfonic acid)

(pH 6.8) onto the fungus on the plate. The surface of the agar plates were carefully scraped with a spatula to bring the spores into suspension. The spore suspension was filtrated using a syringe with prepared glass wool filter and a plunger. The filtered spore suspension was centrifuged at 4 °C at 3500 rpm and the spores washed three times with 10 ml fresh ice cold ACES-buffer. Finally the spores were resuspended in ice-cold double strength growth medium and spore concentration determined with a BIJRKER- TORK counting chamber (0.01 D/L Marienfeld). In each well of the microtiter plate 10000 spores were added into malt extract broth supplemented with 10 % (w/v) sucrose and 100 mg/L CAM. A concentration range of test compounds was used to determine the MIC value at which no fungal growth was observed. The plates were incubated for 6 days at 25 °C.

Table 2. MIC values of single ingredients against A. chevalieri.

The results in Table 2 show that MCFA and sorbic acid exhibited the highest antifungal activity, followed by propionic acid. In comparison, formic acid and acetic acid showed significant higher MIC values, indicating a lower antifungal activity. Example 3

The antifungal activity was determined of mixtures with varying ratio of propionic acid and MCFA. The effect of a range of concentrations (6 to 0 %) of MCFA in the formulation, but keeping propionic acid fixed at 57,9 % (see Table 3, mix 1 to 7). The results suggested that increasing the MCFA concentration from 2 to 4 % resulted in higher antifungal activity, but that a further increase to 6 % MCFA had no additional effect. As expected, lowering the MCFA concentration to 0 % significantly reduced the antifungal activity.

Table 3. MIC values of various premixtures against A. chevalieri after 6 days incubation at 25 °C.

The stability of the premixtures was tested under conditions that can mimic customer storage conditions of the product. One big issue for customers especially in the tropical areas is the instability of antifungal solution in a diluted form (7 or 10 volume percentage in water) during storage. An unstable diluted premix shows a visible phase separation of the fatty acids (at the top) and water fraction, independent of the presence of an emulsifier. Our results showed that a good stability at room and higher temperatures of the diluted premixtures was achieved when the MCFA concentration was increased to 4 % in the premixture. Lowering of the MCFA concentration to 1 % and lower resulted in a stable diluted premix as well.

Table 4. Stability of various diluted premixtures after storage for 2 days at indicated temperatures .

Percentages s dilutions are on w/w basis