Uses of a feed additive

Abstract

The present invention pertains to use of a composition comprising propionic acid and / or salt thereof and at least one fatty acid having 6 to 12 carbon atoms for reducing dormant spore viability, for inactivating dormant conidia, for eliminating or reducing germination of conidia, for increasing cell damage and / or cell death of hyphae, and / or for reducing or eliminating germ tube formation.

Description

[0001] USES OF A FEED ADDITIVE

[0002] FIELD OF THE INVENTION

[0003] The present disclosure pertains to uses of a composition that may be used as a feed additive for animal feed, in particular as a feed preservative.

[0004] BACKGROUND OF THE INVENTION

[0005] Animal feed products often are maintained at a relatively low water activity (<0.8) that prevents growth of the majority of bacterial and fungal spoilers. However, several fungi species, such as Aspergillus species, are well adapted to growth under these conditions and, as such, shorten the shelf life of feed. Fungal growth on feed decreases its nutritional value, and results in the accumulation of off-flavors and / or secondary metabolites such as mycotoxins that affect the animal health and performance. Mycotoxin contamination causes billions of dollars in losses worldwide due to reduced crop yields, lost trade revenues (local and international), livestock illnesses, and adverse human health effects. To ensure the safety and stability of food- and feed products, agents are added that slow down or prevent the development of these fungi. Organic acids and their salts are globally used in animal nutrition for microbial preservation and supporting animal health. The weak organic acid propionic acid is an important preservative in food and feed and inhibits growth of various spoilage bacteria, yeasts and fungi, including the mycotoxigenic fungi Aspergillus flavus and A. parasiticus.

[0006] Dijksterhuis et al. (2019. International Journal of Food Microbiology 306) assessed the sensitivity of a panel of isolated feed spoilage fungi for the preservative propionic acid and evaluated the viability of treated conidia and germ tubes. It was shown that growth inhibition of fungi by propionic acid was mainly fungicidal in the case of growing germ tubes due to damage of the cell membrane and the internal integrity of the germ tubes, but that also conidia are partly inactivated, albeit that longer incubation times are needed.

[0007] W02021 / 001578 describes a composition comprising propionic acid, medium-chain fatty acids, and emulsifier and its use in inhibiting mold growth in animal feed.

[0008] In feed many organic acids, including propionic acid, are used as salts of ammonium, sodium, potassium, magnesium or calcium, as these are less odorous, are easier to handle during feed manufacture, are less corrosive, and more soluble in water than the free acids. However, the salts negatively impact the efficacy of the organic acid.

[0009] It is an object of the present invention to provide a composition for use in reducing dormant spore viability, for inactivating dormant conidia, for eliminating or reducing germination of conidiae, for increasing cell damage and / or cell death of hyphae, and / or for reducing or eliminating germ tube formation.

[0010] SUMMARY OF THE INVENTION

[0011] In feed, propionic acid is the weak organic acid of choice to prevent growth of certain fungi. For safe and easy industrial handling this antifungal agent is often applied in the presence of neutralizing ammonium, which however has the disadvantage to negatively affect the efficacy of fungus-inhibiting properties of the formulation. The present inventors tested the impact of medium chain fatty acids (MCFA) to the inhibiting effect of a propionic acid / ammonium formulation on dormant and germinating conidia as well as early biofilms as exemplified by use of the fungus Aspergillus chevalieri, which has been isolated from moulded poultry feed, as a model organism. It was found that dormant conidia were not affected by propionic acid supplemented with ammonium (PAA). In the presence of PAA and MCFA, the latter in itself not being active against dormant conidia at all, decreased formation of colonies from these conidia and distortion of the cellular structure was visible with light and electron microscopy. Germination of conidia, characterised by swelling and germ tube formation in the presence of PAA was further decreased when MCFA was present in the formulation, while the latter component itself did not significantly decrease development. The present inventors concluded that a combination of PAA and MCFA had a synergistic effect on dormant and germinating conidia.

[0012] Moreover, it was observed that cell death of hyphae was higher in the presence of PAA and MCFA, which acted synergistically. Treatment of the cells in the biofilm with PAA resulted in aberrant mitochondria as observed by electron microscopy. When MCFA was present, more severe cell damage was observed. Summarized, the present inventors have found evidence that synergism occurs between propionic acid / ammonium and MCFA on survival structures of spores, during germination and after a short sudden treatment of growing cells.

[0013] The present disclosure provides use of a composition comprising propionic acid and / or salt thereof and at least one fatty acid having 6 to 12 carbon atoms for reducing dormant conidia (or spore) viability, for inactivating dormant conidia (or spores), for eliminating or reducing germination of conidia (or spores), for increasing cell damage and / or cell death of hyphae, and / or for reducing or eliminating germ tube formation. In an embodiment, the salt of the propionic acid is selected from the group consisting of an ammonium, sodium, potassium, magnesium or calcium salt of the propionic acid.

[0014] In an embodiment, the amount of the at least one fatty acid is at least 0.05 wt%, based on the total weight of the composition.

[0015] In an embodiment, the amount of the propionic acid is at least 20 wt%, based on the total weight of the composition.

[0016] In an embodiment, the composition comprises both propionic acid and a salt of propionic acid.

[0017] In an embodiment, the composition taught herein further comprises at least one further organic acid selected from the group consisting of sorbic acid and / or a salt thereof, formic acid and / or a salt thereof, acetic acid and / or a salt thereof, and lactic acid and / or a salt thereof.

[0018] In an embodiment, the composition taught herein is a feed additive such as a feed preservative.

[0019] In an embodiment, the composition taught herein is for inhibiting bacterial, yeast, and / or mold growth in an animal feed.

[0020] GENERAL DEFINITIONS

[0021] In the following description and examples, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given to such terms, the following definitions are provided. Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The disclosures of all publications, patent applications, patents and other references cited herein are incorporated herein in their entirety by reference.

[0022] The term “conidia” as used herein equates with the term “spores”.

[0023] The term ‘farming animal’ refers to animals that are kept or raised for agricultural purposes, like for consumption or to generate income by for example wool, meat, eggs or milk products. Farming animals can be grouped based on their digestive system. Such groups include monogastric, ruminant and pseudo-ruminant animals. Alternatively or additionally, they may be grouped based on their ecosystem. Such groups include aquaculture animals (when referring to animals that are reared in an aqueous ecosystem such as fish and shrimp) and livestock animals (when referring to animals that are reared on land such as poultry, swine, and bovines).

[0024] The term “companion animal” refers to domesticated or domestic-bred animals whose physical, emotional, behavioral and social needs can be readily met as companions in the home, or in close daily relationship with humans. Species suitable to be companion animals include dogs, cats, horses, rabbits, ferrets, birds, guinea pigs and select other small mammals, small reptiles and ornamental fish.

[0025] 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.

[0026] The term “premix” as used herein refers to a mixture of ingredients designed to be mixed with other ingredients, usually raw materials to be used for feed, before use. The ingredients within the mixture of ingredients within the premix are usually ingredients which are to be added to animal feed in small quantities, such as vitamins and minerals, and optionally also antioxidants, pigments, and / or organic acids. For example, a premix for fish feed may comprise vitamins, minerals, antioxidants and / or pigments, but does not usually comprise organic acids. In contrast, a premix for poultry or swine feed may comprise vitamins, minerals, and organic acids, and antioxidants, but not pigments.

[0027] The terms “comprising” or “to comprise” and their conjugations, as used herein, refer to a situation wherein said terms are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. It also encompasses the more limiting verb “to consist essentially of’ and “to consist of”. Reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0028] DETAILED DESCRIPTION

[0029] This patent application has been drafted into sections. However, these sections should not be read in isolation. Unless otherwise specified, each section is to be read in combination with the other sections. The various optional and preferred features can also be combined, even when taken from different parts of the specification. Likewise, all “aspects” and “embodiments” can be combined. No separation of embodiments is intended, unless explicitly stated.

[0030] In a first aspect, the present disclosure provides use of a composition comprising propionic acid and / or salt thereof and at least one fatty acid having 6 to 12 carbon atoms for reducing dormant spore (or conidia) viability, for inactivating dormant spores (or conidia), for eliminating or reducing germination of spores (or conidia), for increasing cell damage and / or cell death of hyphae, and / or for reducing or eliminating germ tube formation.

[0031] The composition taught herein comprises propionic acid and / or a salt thereof. Examples of suitable salts include ammonium propionate, sodium propionate, potassium propionate, magnesium propionate and calcium propionate. The composition may comprise propionic acid alone, a salt of propionic acid alone or a combination of propionic acid and a salt of propionic acid.

[0032] The composition may serve as a feed additive.

[0033] In one embodiment of the present disclosure, the amount of propionic acid and / or salt thereof is generally at least 10 wt%, based on the total weight of the composition. Preferably, the amount of propionic acid and / or salts thereof in the composition is at least 20 wt%, more preferably at least 25 wt% and most preferably at least 30 wt%, such as at least 40 wt% or at least 50 wt%, and generally at most 80 wt%, preferably at most 75 wt%, such as at most 70 wt%, based on the total weight of the composition. Preferably, the feed additive comprises a combination of propionic acid and a salt of propionic acid, e.g., ammonium propionate, sodium propionate, potassium propionate, magnesium propionate and calcium propionate, or any combination thereof. In one embodiment, the weight ratio, based on the total weight of the composition, between propionic acid and the salt of propionic acid is in the range of 1:5 to 5:1, preferably 1:4 to 4:1, even more preferably 1 :3 to 3:1.

[0034] The composition taught herein comprises fatty acids having 6 to 12 carbon atoms. Such fatty acids are also referred to herein as medium chain fatty acids (MCFAs). The fatty acid may be any known MCFA or combination of MCFAs. Examples of such MCFAs include, without limitation, caproic acid (C6), caprylic acid (C8), capric acid (C10) and lauric acid (C12), as well as combinations of two or more of these MCFAs, such as combinations of caprylic acid and capric acid, and combinations of caprylic acid, capric acid and lauric acid. When the composition comprises a combination of caprylic acid and capric acid, the weight ratio between caprylic acid and capric acid is at least 0.2, such as at least 0.3, at least 0.4, 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.

[0035] In one embodiment, the amount of MCFAs is generally at least 0.01 wt%, at least 0.05 wt%, such as at least 0.075 wt%, at least 0.1 wt%, at least 0.125 wt%, at least 0.15 wt%, at least 0.175 wt%, at least 0.2 wt%, at least 0.3 wt%, at least 0.4 wt%, at least 0.5 wt%, at least 0.6 wt%, at least 0.7 wt%, at least 0.8 wt%, at least 0.9 wt%, or at least 1 wt%, based on the total weight of the composition, and generally at most 20 wt%, preferably at most 15 wt%, more preferably at most 10 wt%, even more preferably at most 8 wt%, and most preferably at most 6 wt%%, based on the total weight of the composition.

[0036] In an embodiment, the composition is a liquid composition, such as an aqueous composition.

[0037] In one embodiment, the composition may further comprise other organic acids, such as formic acid, acetic acid, butyric acid, lactic acid, sorbic acid, and / or a salt of any such organic acid. Examples of suitable salts include ammonium, sodium, potassium, calcium, or magnesium salts. In an embodiment, the composition comprises a salt selected from sodium formate and potassium formate.

[0038] The composition may comprise such organic acid alone, a salt of such organic acid alone or a combination of such organic acid and a salt of such organic 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%.

[0039] In a preferred embodiment, the composition taught herein is substantially free from ammonium formate. More preferably, the composition taught herein 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 composition taught herein. The term “completely free” means that the composition contains less than 20 parts per billion (ppb) of ammonium formate.

[0040] In an embodiment, the composition taught herein is effective against xerophilic molds, i.e. molds that also grow under water-poor or water-free environments, such as Aspergillus chevalieri, Aspergillus flavus, Aspergillus parasiticus, or Penicillium lanosocoeruleum. Using the composition taught herein, the shelf life of animal feed may be improved. Further advantages of the composition taught herein are its ability to inhibit bacterial, yeast and fungal growth, to improve the gut health, improve epithelial integrity and improve the palatability of animal feed to which it is added.

[0041] In one embodiment the composition comprises sorbic acid and / or a salt thereof. 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 the composition.

[0042] In one embodiment, the composition further comprises an emulsifier. The emulsifier may be any emulsifier known in the art. Examples of such emulsifiers include, without limitation, glycerol-monolaurate, ethoxylated castor oil (E484), 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. Particularly preferred are emulsifiers that may be included in animal feed according to the European Feed Safety Authority (EFSA) or the FDA.

[0043] The emulsifier may generally be present in an amount of at least 0.1 wt%, at least 0.5 wt%, or at least 1 wt%, based on the total weight of the composition. Preferably, the amount of emulsifier is at most 30 wt%, more preferably at most 20 wt%, even more preferably at most 10 wt%, based on the total weight of the composition.

[0044] In an embodiment, the composition further comprises an additional 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, and / or a salt of such organic acids.

[0045] The organic acid may generally be present in an amount of at least 0.1 wt%, based on the total weight of the composition. Preferably, the amount of such organic acid with nutritional and / or antibacterial properties is at least 0.5 wt%, more preferably at least 1 wt% and most preferably at least 2 wt%, and generally at most 30 wt%, preferably at most 25 wt%, more preferably at most 20 wt%, and most preferably at most 15 wt%%, based on the total weight of the composition.

[0046] The remaining part of the composition may be comprised of other components commonly or not commonly used in composition that may be used as feed additives. With the propionic acid and the MCFA, the other components add up to 100 wt% of the total weight of the composition.

[0047] In one embodiment, the composition taught herein is a feed additive, such as a feed preservative. In another embodiment, the composition taught herein is part of a premix. The composition taught herein may be added to an animal feed to provide an animal feed comprising the composition taught herein. In an embodiment, the composition taught herein is included in an animal feed in an amount of 0.01 to 10 kg per ton of feed. In other words, the animal feed may comprise per 1000 kilograms (ton), 0.01 to 10 kg of the composition taught herein. In particular, the composition taught herein may be included in an amount of 0.05 to 8 kg, 0.1 to 6 kg, or 0.5 to 5 kg, per ton of animal feed.

[0048] Methods uses

[0049] The present disclosure provides use of a composition as taught herein for inhibiting microbial growth in an animal feed, for reducing dormant spore viability, for inactivating dormant conidia, for eliminating or reducing germination of conidia, for increasing cell damage and / or cell death of hyphae, and / or for reducing or eliminating germ tube formation, in particular in an animal feed.

[0050] Moreover, the present disclosure provides a method of inhibiting microbial growth in an animal feed, of reducing dormant spore viability, of inactivating dormant conidia, of eliminating or reducing germination of conidia, of increasing cell damage and / or cell death of hyphae, and / or of reducing or eliminating germ tube formation, in particular in an animal feed.

[0051] The spore or conidia may be a spore or conidia from any type of microbe, such as bacteria, mold, yeast, or fungus. The microbe may, for example, be any type of bacteria, mold, yeast or fungus that could be present in animal feed. In an embodiment, the spore or conidia is from a feed spoilage microbe, such as a feed spoilage bacteria, mold, yeast, or fungus, particularly a spore-forming microbe, such as a spore-forming bacteria, spore-forming mold, spore-forming yeast, or spore-forming fungus, or any combination thereof.

[0052] In an embodiment, the spores or conidia are from one or more fungi genera selected from the group consisting of Fusarium, Aspergillus, Penicillium, Neotyphodium, Claviceps, Phomopsis and Pithomyces. In an embodiment, the spores or conidia are from one or more fungi of the species Aspergillus chevalieri, Aspergillus flavus, Aspergillus parasiticus or Aspergillus ochraceus, from Penicillium expansum, Penicillium viridicatum, Penicillium cyclopium, Penicillium citrinum, Penicillium citreoviride or Penicillium verrucosum, or from Fusarium culmorum; Fusarium graminearum, Fusarium sporotrichioides, Fusarium poae, Fusarium moniliforme, Neotyphodium coenophialum, Neotyphodium lolii, Claviceps purpurea, Phomopsis leptostromiformis, or Pithomyces chartarum.

[0053] The invention is exemplified in the following Example.

[0054] 1. Material and methods 1.1. Strain, growth conditions and harvesting of conidia

[0055] A strain of Aspergillus chevalieri (CBS 145381) isolated from moulded poultry feed (Dijksterhuis et al. 2019, supra) was used during this study. A 30% (w / v) glycerol stock kept at -20 °C was used for inoculation of malt extract agar (Oxoid CM059, Basingstoke, UK) containing 20 or 40 percent sucrose (MEA20S and MEA40S). Conidia were obtained from agar surfaces containing sucrose, as fungal colonies grew better and produced more conidia compared to growth on malt extract agar alone. Colonies form asexual conidia on conidiophores and simultaneously start to form self-fertile structures (ascomata) with a yellow colour that produce ascospores. Conidia were harvested from these plates by gentle use of a T-spatulum after addition of 10 ml of demi water or medium to the plates, to remove the conidia, but not ascomata and ascospores. The suspension was filtered through sterile glass wool and washed twice in demi water or growth medium (Dijksterhuis et al. 2019, supra). The presence of conidia was confirmed by light microscopy as their size, shape and ornamentation. Conidia were counted using a Burker-Turk haemocytometer.

[0056] For experiments with dormant and germinating conidia, conidia were washed in demi water and in malt extract broth, respectively. To study the effect on germ tubes and hyphae, conidia were suspended in malt extract broth supplemented with 20% sucrose (MEB20S). For this, approx. 1 cm2blocks of fungal colony on agar were cut out of agar and placed in an approximately 10 ml MEB20S in a 50 ml Costar tube and vortexed and filtered.

[0057] 1.2 Solutions used

[0058] In Table 1 an overview is presented of treatment codes, the composition and concentration of test compounds of the various treatments used during different stages of development of the fungus. Fungal cells were tested with different components added to water, MEB or MEB20S. Control samples did only contain demi-water or growth medium. The medium chain fatty acids (MCFA) consisted of a 50%-50% mixture of octanoic (C8:0, caprylic)- and decanoic (C10:0, capric) acid. The pH was kept at 4.8 in all experiments using hydrochloric acid or sodium hydroxide solutions.

[0059] Table 1. Treatment codes, composition and assay concentration of compounds tested against different stages of development of A. chevalieri Medium hain fatty acid (mM)* Demi-water -

[0060] Malt Extract -

[0061] Broth (MEB)

[0062] *Medium Chain Fatty Acids (MCFA) were a 50%-50% mixture of C8:0 and C10:0 and the concentration was based on the average molecular weight. Grey boxes indicate the treatments used for each developmental stage. Abbreviations: P, propionic acid; PA, ammonium propionate; M, MCFA; PAM, ammonium propionate combined with MCFA

[0063] 1.3 Effect of treatments on the viability of conidia

[0064] Conidia were assayed with the various treatments for 48 hours at 25°C with gentle agitation (60 rpm) at approximately 1000 cells / ml. Subsequently, 100 pl of solution was directly plated out on MEA20S. Two independent experiments were performed, and development of colonies assessed, and colonies counted on MEA20S plates after three days of incubation at 25°C in the dark. For this, the NIKON SMZ100 stereomicroscope (Nikon, Amsterdam, NL) was used at low (approx. 10x) magnification. In addition, conidia in the tested solutions were concentrated by means of an Eppendorf centrifuge and evaluated by means of light microscopy equipped with differential contrast. Micrographs were taken using an Axioskop 2 plus light microscope (Zeiss, Oberkochen, Germany) equipped with a Nikon RSI20D camera (Nikon, Amsterdam, Netherlands) and collected with the NISEIements software (Nikon, Amsterdam, Netherlands). Composition pictures were created in AdobePhotoshop.

[0065] 1.4. Effect of treatments on swelling and germ tube formation

[0066] To analyse the effect of treatments on the germination process of conidia the oCelloScope (BioSense Solutions ApS, Faren, Denmark) was used. This equipment acquires pictures of settled cells in 96-well-flat-bottomed microtiter plates (Sarstedt AG & Co. KG, Numbrecht, Germany) enabling the researcher to monitor the effect of many treatments on swelling and germ tube formation of conidia in one experiment. Conidia were suspended at a density of 105conidia / ml in malt extract medium and all treatments were tested in six-fold. For each measurement 104conidia per well were used in 100 pl and as a control, conidia in control malt extract medium were monitored. The apparatus was placed in a stove at 25 °C and the Uni Explorer software packet 12.0.0.23 (BioSense Solutions ApS, Faren, Denmark) used for acquisition of images, object tracking (including segmentation), counting of objects and growth kinetics analysis. For acquisition, 24 repetitions were selected with 2 hour-time-intervals per well, 10 images were taken from the centre of each well per time point. For segmentation, the fungHmin algorithm was used (contrast = 0.40 and threshold = 25.00) and for growth kinetic analysis the SESAfungi Normalized algorithm was used. The maximal increase in cell expanse was analysed in R (https: / / www.r-project.org / ) for calculation of the maximal slope in growth rate.

[0067] 1.5. Effect of treatments on germ tubes and hyphae

[0068] To evaluate and compare the effect of treatments on germ tubes and hyphae, 1.25 ml of MEB20S was inoculated with approximately 106conidia and incubated in small Erlenmeyers (25 ml) without agitation at 25 °C in the dark for 20 h as described in Dijksterhuis et al. (2019), supra. In this time frame, many conidia had germinated including the formation of germ tubes and hyphae. Another 1.25 ml, containing a double concentration of formulation (or not in case of the control) was added to the hyphae and gently mixed. After 30 min, 48 pl was gently pipetted from this culture and mixed with 2 pl 100 pM TOTO-1 (Thermo Fisher Scientific, Waltham, MA, USA) solution for 15 min and examined with fluorescence microscopy (Dijksterhuis et al. 2019, supra). Stained hyphae and unstained hyphae were counted in six independent experiments per treatment and about 100 hyphae were counted per measurement.

[0069] 1.6 Electron microscopy

[0070] For electron microscopy, conidia diluted in demi water after treatment were fixed in a 2% glutaraldehyde / 2% formaldehyde (GA / FA) solution in 100 mM sodium cacodylate buffer (pH 7.2) for 1 hour. Subsequently, the cells were cooled to approximately 4°C and stored for a week. Cells were embedded in agarose, spun down and small (1 mm) pieces were excised and postfixed. Treated germinated conidia and hyphae were fixed with the GA / FA solution added to MEA20S for 1 hour and hyphae were transferred with a thin glass rod in fresh PA / FA solution before cooling. These cells were also embedded in agarose and postfixed. Conidia and germlings / hyphae were untreated, PA 1 , M 1 , and PAM 1.

[0071] After dehydration in an alcohol series and flat embedding in EPON, spores and germlings / hyphae were cut out and ultrathin coupes were obtained for Nanoscopy using an electron microscope. Pictures of conidia and vegetative cells were collected and processed. 2. Results

[0072] 2.1 Dormant conidia of A. chevalieri are inactivated by propionic acid and ammonium propionate combined with MCFA.

[0073] This study first addressed the effect of various treatments on dormant conidia, the predominantly aerially dispersed cells (Dijksterhuis et al. 2019, supra) of A. chevalieri. The results are summarized in Table 2.

[0074] Table 2. Germination efficiency of conidia of Aspergillus chevalieri after incubation in demi water and different components for 48 h.

[0075] Treatment Germination (%)

[0076] Demi-water 82

[0077] P 0

[0078] PA1 87

[0079] PA2 77

[0080] M1 97

[0081] M2 84

[0082] PAM1 59

[0083] PAM2 3

[0084] An incubation period of 48 hours in 32 mM propionic acid (P) resulted in complete inhibition of germination of the conidia indicating cell death. PAM2 treatment resulted in germination of only 3% of the conidia. Conidia treated with 32 mM ammonium propionate (PA2) or 0.52 mM MCFA (M2) did not result in significantly lower colony formation compared to the demi water control, whereas their combination PAM2 nearly eradicated germination. The treatments PA1 and 2, M1 and 2, as well as PAM1 showed significant higher colony formation compared to PAM2. We conclude that in this experiment the simultaneous use of ammonium propionate and MCFA has a synergistic effect with respect to inactivation of conidia.

[0085] Differential contrast microscopy of selected conidia that were treated with different formulations. M1 and 2, and PA1 demonstrated that treated cells appeared very similar to untreated cells exhibiting dense cytoplasm of a relatively smooth appearance. These treatments also resulted in the highest levels of germination (Table 2). After treatment with P and PAM2, conidia appeared granulated and space between cytoplasm and cell wall seemed to be present. These notable morphological changes were associated with no or very low colony numbers. Treatments with PA2 and PAM1 resulted in some structural effects and medium germination scores. While PA1 and M1 showed the highest germination scores and no structural effects, their combination had a lower germination score (significantly different from the M1 value) and some altered cell morphology.

[0086] 2.2 Development of conidia of A. chevalieri in the presence of various components is significantly decreased in the presence of ammonium propionate and MCFA, while the latter has no effect on its own.

[0087] Conidia of A chevalieri were analysed using an oCelloscope during their initial development in the presence of M1, PA1 and PAM1 compared to the control malt extract broth. The oCelloscope is a microscope that monitors and quantifies the swelling and subsequent germination of conidia. As in the case of other species, conidia of A. chevalieri perform a stage of isotropic growth in which the cell expands “globally” (often designated as swelling) followed by polarized growth in which a local expansion of the cell wall initiates the formation of a germ tube.

[0088] Control conidia and spores treated with M1 developed germ tubes (sometimes two per spore) in the majority of cells, but the latter treatment resulted in slightly shorter germ tubes. Conidia treated with PA1 were delayed to a larger extent, with only a few single germ tubes formed. PAM1 treatment resulted in occasionally swollen conidia and absence of germ tube formation at that stage.

[0089] Table 3 indicates the numbers of conidia that germinated, were swollen and lost their circularity due to the formation of a germ tube, after a period of 36 hours of incubation.

[0090] Table 3. Percentage of germination of conidia after 36 hours in malt extract medium after different treatments.

[0091] Treatment Germination (%)*

[0092] MEB 95

[0093] PA1 88

[0094] M1 94

[0095] PAM1 67

[0096] The germination percentage is 95% in the case of the control medium. Treatment M 1 (94%) and the control did not differ significantly. The other two treatments PA1 and PAM1 were significantly different from each other and from control and M1 treatment. Thus, M1 did not result in a significant reduction of germination, while PA 1 did. However, the combination of the components in treatment PAM 1 significantly delayed germination in comparison to PA 1 treatment. Compared to the control, PA1 has as ratio of germination from 88 / 95 = 0.93 based on the germination values in Table 3. For M1 this is 1.0. The calculated combined effect of M1 and PA1 would be 0.93 times 1 = 0.94. However, the observed germination value is 71% of the control values for the mixture, PAM 1 , clearly lower than the calculated value. This indicates that the effect of the mixture was significantly larger as the effect of the independent components and henceforth suggests a synergistic antifungal effect of the combination MCFA and ammonium propionate.

[0097] Growth curves were obtained from the oCelloscope data using the SESAnormalized algoritm, based on increase in the amount of area encompassed by the growing cells. Calculation of the maximal slope (as a measure of the growth rate) by means of a R- script showed that the control medium and M1 treatment exhibited a similar rate (Table 4). A clear delay in onset of growth is observed with PA1 and an even greater delay was observed with PAM1 In contrast, the growth rate was significantly lower for treatments PA1 and PAM1 in comparison with the control medium.

[0098] Table 4. Growth rate of germinating conidia

[0099] MEB 2.16

[0100] PA1 1.37

[0101] M1 2.14

[0102] PAM1 1.36

[0103] * The growth rate (slope) was calculated from data obtained via the normalized SESA algorithm of the oCelloscope software. A R-script was used using 4 consecutive points

[0104] (of log (1+x) values) of of growth curves displayed in individual wells. Maximal slope are interpreted as an indication of maximal growth rate. The values were statistically tested by ANOVA followed by a post-hoc Tukey test. Numbers were based on six replicates.

[0105] 2.3 The effect of various treatments on cell damage of germ tubes and hyphae of A. chevalieri

[0106] Germ tubes and hyphae were incubated in malt extract broth supplemented with 20% sucrose with or without treatments for 30 min and subsequently investigated using fluorescence live-dead staining TOTO-1. The results are presented in Table 5.

[0107] Table 5. Percentage of TOTO-1 stained germ tubes and hyphae of A. chevalieri after various treatments for 30 min.

[0108] Treatment Intact hyphae (%)

[0109] MEB 20S 95

[0110] P 21

[0111] PA1 81

[0112] PA2 56

[0113] M1 87

[0114] M2 75

[0115] PAM1 52

[0116] PAM2 27 Control cells appeared turgid as observed with light microscopy and an average of 95% of the cells were intact and stained with TOTO-1 is absent. Only some hyphae were damaged due to their transfer to the microscope objective glass.

[0117] In the case of treatment with P, only 21% of the cells were intact and damaged cells generally appeared collapsed (thinner hyphae and collapsed cytoplasm) with light microscopy and fluorescent green staining visible in hyphae (see also Dijksterhuis et al. 2019, supra). This propionic acid treatment was not significantly different from PAM2 (27 % of cells intact). PA 1 and 2 showed a dose dependent increase of the number of intact cells compared to the controls (81 and 56%intact hyphae, respectively). The PA treatments were significantly different from PAM treatments dependent on the used concentration (PA1 compared to PAM1 and PA2 compared to PAM2). M1 and 2 treated germ tubes and hyphae were least affected apart from the controls (namely 89 and 75% intact cells), with M2 significantly different from the control indicating some damaging effect of M treatment on the cells. The PAM 1 treatment has 52% intact cells.

[0118] Can combined treatments PA and M increase damage to germ tubes and hyphae? Table 6 summarizes the data as percentage intact cells and arcsin-transformed values and shows an estimation of the effects of each single treatment as compared to the untreated cells. The calculated PAM1 value would be 0.80; multiplying the PA1 value 0.86 with the M1 value 0.94. However the / observed PAM1 value showed a value of 0.52, which is significantly lower as the observed value (PAM1 is significantly lower as both PA1 and M1 , the latter not significantly different from the control). For PAM2 the calculated value of 0.47 was also markedly higher as the observed treatment value being 0.27 (M2 and PA2 are significantly different from PAM2). This indicates that the effect of the combination in the PAM is greater as the sum of the individual components, indicating a synergistical mode of action.

[0119] Table 6. Synergistic effect of treatments on TOTO-1 staining of germ tubes and hyphae. The data as represented as percentage of intact cells and arcsin-transformed values as compared to the untreated cells. lculati compa compa of ated co trol (ar xpecte (% ) rmed v ratio

[0120] PA1 A 0.86 0.83

[0121] PA2 B 0.59 0.62

[0122] M1 C 0.94 0.90

[0123] M2 D 0.79 0.77

[0124] PAM1 A x C 0.52* (0.80**) 0.46 (0.73)

[0125] PAM2 B x D 0.27 (0.47) 0.31 (0.47) *Values based on the number of intact (not-stained) hyphae in 6 independent experiments. ^Calculations based on the expected combined effects of the single treatments as a ratio of the control.

[0126] 2.4 Evaluation of effect various treatments on conidia and germ tubes by transmission electron microscopic (TEM)

[0127] 2.4.1 Conidia

[0128] TEM demonstrated that the conidia were characterised by a thick cell wall containing large ornamentations. Extended ornamentations occurred at one side of the conidia, the ends of these cells, where spores are connected to each other in a row on the conidiophore. The conidia were characterized by numerous small invaginations of the plasma membrane. Inside the conidia, nuclei, mitochondria with cristae, endoplasmic reticulum and microbody-like organelles were visible. Conidia that were incubated with M1 and PA1 showed no morphological differences with the untreated conidia. The same conclusion was drawn by evaluation of these conidia using light microscopy. A number of morphological abnormalities were visible in PAM 1 -treated conidia, the most notable response being enlargement of the periplasmic space between cell wall and plasma membrane that can occupy a large proportion of the cellular volume. Furthermore, lipid body-like profiles were observed in treated cells.

[0129] 2.4.2 Germ tubes and hyphae

[0130] Fungal germ tubes and hyphae were incubated with and without treatment M1 for 30 min before preparation for TEM. The profiles showed numerous mitochondria in hyphae that mostly had a regular shape and some cristae, and in some cases an electron dense inclusion. Inside the germinating conidia a vacuole, often containing dark inclusions was visible presumably as part of the germination process. The M1 treatment did not result in structural changes in cell morphology in comparison with untreated germ tubes.

[0131] In contrast, following a 30 min treatment with treatments PA1 and PAM1 irregularly shaped and enlarged mitochondria that more often than the controls contained electron dense inclusions were observed.

[0132] In addition, cells treated with PAM1 could be devoid of cytoplasm, or contained cytoplasm without many intracellular structures, or with plasma membrane invaginations. Further, mitochondria were located in close proximity to vacuoles, in as if they will be taken up, which suggests autophagy of these organelles. 3 Conclusions

[0133] When the combination of ammonium propionate and MCFA was tested on hyphae for 30 min, after 20 hours of cultivation, we observed that cell death was significantly increased in comparison to ammonium propionate and MCFA alone. Treatment of the hyphae with 16 mM of ammonium propionate acid caused aberrant mitochondria, as evidenced by irregularly shaped and enlarged mitochondria that contained electron dense inclusions as observed by electron microscopy. When ammonium propionate and MCFA were used, more severe cell damage was observed, with signs of autophagy. Summarised, our results demonstrate synergistic antifungal effects of ammonium propionate and medium chain fatty acids on fungal survival structures, during their germination and after a short (sudden) treatment of growing cells.

Claims

CLAIMS1. Use of a composition comprising propionic acid and / or salt thereof and at least one fatty acid having 6 to 12 carbon atoms for reducing dormant spore viability, for inactivating dormant spores, for eliminating or reducing germination of spores, for increasing cell damage and / or cell death of hyphae, and / or for reducing or eliminating germ tube formation.

2. Use according to claim 1 , wherein the salt of the propionic acid is selected from the group consisting of an ammonium, sodium, potassium, magnesium or calcium salt of the propionic acid.

3. Use according to any one of the preceding claims, wherein the amount of the at least one fatty acid having 6 to 12 carbon atoms is at least 0.05 wt%, based on the total weight of the composition.

4. Use according to any one of the preceding claims wherein the amount of the propionic acid is at least 20 wt%, based on the total weight of the composition.

5. Use according to any one of the preceding claims, wherein the composition comprises propionic acid and a salt of propionic acid.

6. Use according to any one of the preceding claims further, wherein the composition further comprises at least one further organic acid selected from the group consisting of sorbic acid and / or a salt thereof, formic acid and / or a salt thereof, acetic acid and / or a salt thereof, and lactic acid and / or a salt thereof.

7. Use according to any one of the preceding claims wherein the composition is a feed preservative composition.

8. Use according to claim 7, wherein the composition is for inhibiting bacterial, yeast, fungal and / or mold growth in an animal feed.

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