WO2024068620A1

WO2024068620A1 - Method for reducing asperphenamate concentration

Info

Publication number: WO2024068620A1
Application number: PCT/EP2023/076527
Authority: WO
Inventors: Wulf-Dieter Moll; Barbara Katharina NOVAK; Valentina RAINER; Dian SCHATZMAYR
Original assignee: Dsm Ip Assets B.V.
Priority date: 2022-09-27
Filing date: 2023-09-26
Publication date: 2024-04-04

Abstract

The present invention relates to a method for reducing asperphenamate concentration in a nutritional composition comprising asperphenamate, the method comprising the step of providing a microorganism comprising a polynucleotide having at least 95% identity to the nucleotide sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2 to a nutritional composition comprising asperphenamate.

Description

METHOD FOR REDUCING ASPERPHENAMATE CONCENTRATION [001] The present invention relates to a method for reducing asperphenamate concentration by a microorganism and use thereof. [002] Fungi are known to produce a large variety of secondary metabolites. Some of these secondary metabolites are capable of causing disease or even death in humans and animals that consume them. Such metabolites are summarized under the term mycotoxins. [003] Some mycotoxins are well known, such as aflatoxins, fumonisins, deoxynivalenol or zearalenone, and means and measures have been described to counteract their undesirable effects on humans or animals. Unfortunately, the various mycotoxins differ considerably in their molecular structure. As a consequence, there is no overall solution to inactivate all mycotoxins by a single measure. In addition, some other mycotoxins have been studied to a much lesser extent, such as enniatins, beauvericin, apicidin or asperphenamate. Despite considerable toxicity of these molecules, only few or even no measures are known to counteract these fungal metabolites. [004] Asperphenamate (CAS No. 63631-36-7; also referred to as auranamide; [(2S)-2- benzamido-3-phenylpropyl] (2S)-2-benzamido-3-phenylpropanoate; 3-phenyl-2- (benzoylamino)propanoic acid 2-(benzoylamino)-3-phenylpropyl ester; anabellamide; asjanin; N- benzoylphenylalanine-2-benzamido-3-phenyl propyl ester; (2S)-2-benzamido-3-phenylpropyl N- benzoyl-L-phenylalaninate, N-benzoyl-L-phenylalanine; 2S-(benzoylamino)-3-phenylpropyl ester; or (2S)-2-benzamido-3-phenylpropyl (2S)-2-benzamido-3-phenylpropanoate) is a carboxylic ester produced by Aspergillus as well as Penicillium species. In a study involving 6157 analyzed feed samples, asperphenamate was found to occur in some of the tested samples, and at a maximum concentration of 18713 ppb. It has been described to inhibit the proliferation of different cell lines, to possess cytotoxic properties, and is associated with constipation and deaths in dairy cattle (Pozzato et al.2020. Toxicon 187: 122-128; Liu et al.2016. Eur J Med Chem 110: 76-86). [05] Removal of cytotoxic molecules such as asperphenamate is therefore of particular importance in food intended for human consumption, but also along the entire food production chain. Likewise, the presence of mycotoxins in fodder or feed is known to affect the wellbeing of livestock on the one hand, and to cause considerable economic losses due to reduced animal performance on the other hand. It is therefore desirable to eliminate any toxic fungal metabolite from compositions intended for consumption by humans or animals. [06] Yet a further concern to be considered is a potential synergistic toxicity arising from co- occurrence of two or even more mycotoxins, e.g. asperphenamate and deoxynivalenol. Due to such synergistic toxicity, even low concentrations of mycotoxins may have considerable adverse effects. Also in this respect, it is desirable to remove mycotoxin molecules from nutritional compositions to avoid potentiation of the toxic effects of the individual mycotoxins with one another. [007] In view of the prior art as outlined above, it is an objective of the present invention to provide a method for reducing the concentration of asperphenamate in a nutritional composition. [008] This objective was achieved by a method and a use as described in the claims. In particular, reduction of asperphenamate concentration in a nutritional composition is achieved by a method comprising the steps of a) providing the nutritional composition comprising asperphenamate; b) providing at least one microorganism, wherein the at least one microorganism comprises at least one polynucleotide having at least 95%, preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity to the nucleotide sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2; c) forming a mixture comprising water, the nutritional composition of a) and the at least one microorganism of b); and d) incubating the mixture of step c). Hereby, asperphenamate concentration is effectively reduced, resulting in a safer and healthier nutritional composition. Also, the reduction in asperphenamate concentration can improve animal performance, e.g. by lowering the feed conversion ratio (FCR), as a result of negative effects of the asperphenamate on feed intake and/or feed conversion. [009] In one embodiment, the at least one microorganism comprises at least one polynucleotide having 100% identity to the nucleotide sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2. In other words, the at least one microorganism comprises at least one polynucleotide having the nucleotide sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2. In a particular embodiment, the at least one microorganism comprises a first polynucleotide having at least 95%, preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity, or even 100% identity to the nucleotide sequence of SEQ ID NO: 1, and the at least one microorganism comprises a second polynucleotide having at least 95%, preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity, or even 100% identity to the nucleotide sequence of SEQ ID NO: 2. For the sake of clarity, it is considered that the at least one microorganism referred to herein comprises at least one polynucleotide having at least 95%, preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity, or even 100% identity to the nucleotide sequence of SEQ ID NO: 1; and/or at least one polynucleotide having at least 95%, preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity, or even 100% identity to the nucleotide sequence of SEQ ID NO: 2. Said at least one polynucleotide(s) having at least 95%, preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity, or even 100% identity to the nucleotide sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2 may be considered to be comprised by the at least one microorganism by being part(s) of the genome of the at least one microorganism. In this respect, the polynucleotide(s) having at least 95%, preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity, or even 100% identity to the nucleotide sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2, is/are a sub-sequence(s) of the genomic nucleotide sequence of the at least one microorganism. In a particular embodiment the at least one microorganism comprises as part of its genome two polynucleotides, wherein a first polynucleotide of said two polynucleotides as part of the genome of the at least one microorganism has at least 95%, preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity, or even 100% identity to the nucleotide sequence of SEQ ID NO: 1; and wherein a second polynucleotide of said two polynucleotides as part of the genome of the at least one microorganism has at least 95%, preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity, or even 100% identity to the nucleotide sequence of SEQ ID NO: 2. [10] The nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 can be described as internal transcribed spacer (ITS) sequences. The nucleotide sequence of SEQ ID NO: 1 can be described as ITS1 sequence, and the nucleotide sequence of SEQ ID NO: 2 can be described as ITS2 sequence. In the genome, ITS1 and ITS2 sequences separate the 18S from 5.8S ribosomal DNA (rDNA), and 5.8S from 26S rDNA, respectively. Both polynucleotides, the polynucleotide having the nucleotide sequence of SEQ ID NO: 1 as well as the polynucleotide having the nucleotide sequence of SEQ ID NO: 2, are comprised in a polynucleotide sequence deposited in the GenBank database under the identifier AJ601389.1. The nucleotide sequences are shown in Table 1 below. Table 1: Nucleotide sequences of SEQ ID NO: 1 and 2.

[11] A nutritional composition as referred to herein is a composition comprising one or more component(s) having nutritional value. Often such components provide energy to the consumer of the nutritional composition. A nutritional composition may be entirely or at least partly herbal or plant-based, such as commonly used animal feed compositions. The water comprised in the mixture of step c) may be present e.g. in form of moisture, which might come from moisture comprised in the nutritional composition, or which water might be added by the operator of the method, or the water might stem from saliva. [12] The degree of similarity or relatedness of two or more nucleic acid sequences (e.g. DNA or RNA polynucleotides) can be described in terms of sequence identity. The sequence identity can be determined by common methods known to a skilled person. Herein, the preferred method for determination of sequence identity among two polynucleotide sequences is the use of the Clustal Omega nucleotide sequence alignment tool of EMBL-EBI (https://www.ebi.ac.uk/Tools/msa/clustalo/; Sievers et al. 2011. Mol. Syst. Biol. 7: 539) with default settings. Alternatively, the Needleman-Wunsch algorithm for global sequence alignment may be used, e.g. as provided by the National Center for Biotechnology Information (“Needleman- Wunsch Global Align Nucleotide Sequences”) using default settings (Match/Mismatch Scores: 2,- 3; Gap Costs: Existence: 5 Extension 2). [13] Chemical reactions, including reactions catalyzed by biological systems such as enzymes within or obtained from microorganisms, may occur faster or slower depending on environmental conditions. To perform the method of the present invention in a preferred manner, it is therefore considered to perform step d) of the method described above for at least one hour, preferably for at least three hours, more preferably for at least one day; additionally, it is preferred that step d) is performed at a temperature of at least 10 °C, preferably of at least 15 °C, more preferably of at least 20 °C; and at a temperature of at most 50 °C, preferably at most 40 °C, more preferably at most 35 °C. In one embodiment, the method of the present application is performed at common ambient temperature (e.g. 15-35 °C, preferably 18-30 °C, more preferably 20-28 °C). When performing the method described herein under said conditions, a particularly efficient reduction of the asperphenamate concentration or content in a composition can be achieved. The longer the incubation of step d) is performed, the more asperphenamate will be removed from the nutritional composition. Notably, the microorganism can be found capable of removing asperphenamate even throughout such long incubation times. Notwithstanding, a skilled person is aware that a type of reaction as described herein may also be effected outside said preferred conditions. Merely for the sake of clarification, incubation is considered to start immediately upon contact of a microorganism as referred to herein with a composition comprising asperphenamate as referred to herein. Therefore, contacting the microorganism with the composition referred to herein may be considered to start the incubation. The skilled person is well capable of choosing incubation conditions appropriate for reducing an asperphenamate concentration present in a composition to a desired degree. [14] In some embodiments, the at least one microorganism referred to herein belongs to the genus Trichosporon. Preferably, the at least one microorganism is DSM 14153. [015] The nutritional composition referred to herein can consist of, or comprise one or more solid component(s) and/or one or more liquid component(s). In some embodiments, the nutritional composition is selected from the group consisting of foodstuff; fodder; feed; wet distillers grain; dried distillers grain with solubles; intermediate/s thereof; and/or mixture/s thereof. Such a nutritional composition may further comprise foodstuff additive/s; fodder additive/s; feed additive/s; nutritional supplement/s; prebiotic/s; probiotic/s; intermediate/s thereof; and/or mixture/s thereof. Such nutritional compositions are part of the feed/food chain and may thus benefit substantially from a reduction or even removal of asperphenamate. A reduction in asperphenamate concentration in such compositions thus contributes to smaller losses in food production and healthier animals and consumers. Merely for clarification, fodder or feed may e.g. comprise or consist of corn, hay, straw litter, soy, or products obtained therefrom. Also, fodder or feed may comprise or consist of extruded feed products, e.g. pellets. Additives for foodstuff, fodder or feed are used to improve or add properties to the foodstuff, fodder or feed. For instance, such additives may be added to improve organoleptic properties, e.g. to improve taste, smell, appearance, color of the foodstuff, fodder or feed. Also, additives may be added to improve palatability, nutrient availability, or to add probiotic microorganisms to the foodstuff, fodder or feed, or to add or enhance prebiotic activity of the foodstuff, fodder or feed. Also, such additives may be added to counteract potentially undesirable effects of the foodstuff, fodder or feed, such as removal or reduction of one or more undesirable components comprised in the foodstuff, fodder or feed. [016] The at least one microorganism referred to herein may be added to the nutritional composition as part of a composition. Such a composition comprising the at least one microorganism as referred to herein may comprise one or more further components, such as one or more additional microorganism(s) capable of detoxifying one or more myco-/plant-/bacterial- toxin(s); one or more isolated polypeptide(s) capable of detoxifying one or more further myco- /plant-/bacterial-toxins (e.g. fumonisin esterase e.g. as described in UniProtKB: D2D3B6 and/or variant/s thereof; and/or zearalenone lactonase e.g. as described in UniProtKB: Q8NKB0 and/or variant/s thereof); and/or one or more organic absorbent component(s) (e.g. inactivated/dried/lyophilized/live yeast, e.g. Saccharomyces species, such as S. cerevisiae, Pichia pastoris); and/or one or more inorganic absorbent component(s) (e.g. clay product(s), bentonite, zeolite, bentonite-montmorillonite, diatomaceous earth); and/or one or more plant product(s) (e.g. algae product(s), thistle extract); and/or one or more vitamin(s); and/or one or more flavoring compound(s); and/or one or more prebiotic compound(s) (e.g. mannan); and/or one or more probiotic compound(s) as further components. [17] In some embodiments, the method according to the invention is performed in a manner, wherein the at least one microorganism is provided as a dried microorganism formulation, in particular as a spray-dried or freeze-dried formulation, preferably as a spray-dried formulation. Providing the at least one microorganism in the form of such a formulation allows a more convenient handling of the microorganism and facilitates mixing of the microorganism with the nutritional composition. [18] In a preferred embodiment, the microorganism formulation described herein is provided to the nutritional composition in such an amount to achieve a concentration of at least 10 g, e.g. at least 20, 30, 40 or 50 g, of microorganism formulation per ton of nutritional composition. In other words, the final concentration, i.e. the working concentration, of the microorganism formulation in the nutritional composition is at least 10 g (e.g. at least 20, 30, 40 or 50 g, or more) of microorganism formulation per ton of nutritional composition. A person skilled in the art is aware that the present invention may also be performed when using a lower concentration of the microorganism. Notwithstanding, when applying a minimum concentration as described above, asperphenamate reduction or removal may be achieved within a most convenient time frame. [19] Another aspect of the present invention relates to a foodstuff; fodder; feed; wet distillers grain; dried distillers grain with solubles; intermediate/s thereof; and/or mixture/s thereof produced by a method as described herein. Such foodstuff; fodder; feed; wet distillers grain; dried distillers grain with solubles; intermediate/s thereof; and/or mixture/s thereof comprise(s) no or at least lower amounts of asperphenamate compared to foodstuff; fodder; feed; wet distillers grain; dried distillers grain with solubles; intermediate/s thereof; and/or mixture/s thereof that was not produced by a method as described herein. As a consequence, such foodstuff; fodder; feed; wet distillers grain; dried distillers grain with solubles; intermediate/s thereof; and/or mixture/s thereof according to the present invention poses a lower threat to cause asperphenamate-induced toxic effects in a consumer of such foodstuff; fodder; feed; wet distillers grain; dried distillers grain with solubles; intermediate/s thereof; and/or mixture/s thereof. It is considered that a foodstuff; fodder; feed; wet distillers grain; dried distillers grain with solubles; intermediate/s thereof; and/or mixture/s thereof produced by a method as described herein also comprises the at least one microorganism comprising at least one polynucleotide having at least 95%, preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity to the nucleotide sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2. Thus, such foodstuff; fodder; feed; wet distillers grain; dried distillers grain with solubles; intermediate/s thereof; and/or mixture/s thereof is of lower risk of further contamination with asperphenamate by eventual fungal growth compared to foodstuff; fodder; feed; wet distillers grain; dried distillers grain with solubles; intermediate/s thereof; and/or mixture/s thereof which does not comprise the at least one microorganism referred to herein. Preferably, the foodstuff; fodder; feed; wet distillers grain; dried distillers grain with solubles; intermediate/s thereof; and/or mixture/s thereof produced by a method as described herein comprises the microorganism according to the invention at a concentration of at least 10 g, e.g. at least 20, 30, 40 or 50 g, of microorganism formulation per ton of foodstuff; fodder; feed; wet distillers grain; dried distillers grain with solubles; intermediate/s thereof; and/or mixture/s thereof. [20] In some embodiments such a foodstuff; fodder; feed; wet distillers grain; dried distillers grain with solubles; intermediate/s thereof; and/or mixture/s thereof produced by a method as described herein comprises less than 80 ppb of asperphenamate (e.g.50, 40, 30, 20, 10 ppb, or less than 10 ppb, or between 50 and 10 ppb, or between 30 and 10 ppb, or between 30 and 5 ppb of asperphenamate), preferably less than 50 ppb of asperphenamate (e.g. 15 ppb, or between 20 and 5 ppb of asperphenamate), more preferably less than 20 ppb of asperphenamate (e.g. between 10 and 5 ppb of asperphenamate, or 5 ppb, 4 ppb, 3 ppb, 2 ppb etc. of asperphenamate). [21] The microorganism may be administered to a human or to an animal (e.g. pet, poultry, swine, ruminant etc.) by providing a nutritional composition comprising the microorganism. [22] In a further aspect, the invention relates to a use of the at least one microorganism for reducing asperphenamate concentration in a nutritional composition comprising asperphenamate. In such a use, the at least one microorganism comprises at least one polynucleotide having at least 95%, preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity to the nucleotide sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2. In one embodiment, the at least one microorganism comprises one or more polynucleotide(s) having the nucleotide sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2, i.e. the polynucleotide(s) has/have 100% identity to the nucleotide sequence(s) of SEQ ID NO: 1 and/or SEQ ID NO: 2. In other words, in an embodiment wherein the at least one microorganism comprises one polynucleotide, said one polynucleotide has at least 95%, preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity, or even 100% identity to either the nucleotide sequence of SEQ ID NO: 1 or to the nucleotide sequence of SEQ ID NO: 2. In an embodiment wherein the at least one microorganism comprises two polynucleotides, a first polynucleotide has at least 95%, preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity, or even 100% identity to the nucleotide sequence of SEQ ID NO: 1; and a second polynucleotide has at least 95%, preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity, or even 100% identity to the nucleotide sequence of SEQ ID NO: 2. [23] In such a use, said microorganism facilitates a reduction in the concentration of asperphenamate in a nutritional composition comprising asperphenamate and thus reduces the negative effects effected by asperphenamate upon ingestion of the nutritional composition. Preferably, asperphenamate is removed from the nutritional composition entirely or at least to a concentration at which no negative effects are observable any more. [24] In some embodiments of the use described herein, the at least one microorganism is incubated with the nutritional composition and water for at least one hour, preferably for at least three hours, more preferably for at least one day; and said incubation is performed at a temperature of at least 10 °C, preferably of at least 15 °C, more preferably of at least 20 °C; and wherein said incubation is performed at a temperature of at most 40 °C, preferably at most 35 °C, more preferably at most 30 °C. Hereby, most favorable incubation conditions are established which allow a particularly fast reduction of asperphenamate concentration. [025] In a preferred embodiment, the at least one microorganism belongs to the genus Trichosporon, preferably the at least one microorganism is DSM 14153. When choosing the microorganism referred to herein from the genus Trichosporon, in particular when choosing the microorganism to be the microorganism deposited under the identifier number DSM 14153, for reducing asperphenamate concentration in a nutritional composition, said reduction in asperphenamate concentration can be achieved in a particularly robust and reliable manner. [26] In some embodiments of the use according to the invention, the at least one microorganism is provided as a dried microorganism formulation, in particular as a spray-dried or freeze-dried formulation, preferably as a spray-dried formulation. [27] In a preferred embodiment, the microorganism is provided in a microorganism formulation, which microorganism formulation is provided in such an amount to achieve a concentration of at least 10 g of microorganism formulation per ton of nutritional composition, for the use of reducing asperphenamate concentration in a nutritional composition as described herein. [28] In a further aspect, the invention relates to a microorganism for use as a medicament, e.g. in veterinary medicine, and/or in treatment, amelioration, prophylaxis and/or diagnostics of a disease, wherein the at least one microorganism comprises at least one polynucleotide having at least 95%, preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity to the nucleotide sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2. The finding that the at least one microorganism described herein is capable of reducing asperphenamate concentration renders its use as a medicament, in treatment, amelioration, prophylaxis or diagnostics of a disease highly favorable, e.g. in veterinary medicine. In particular, its use as a medicament, in treatment, amelioration, prophylaxis and/or diagnostics of symptoms caused by mycotoxicosis, in particular by asperphenamate toxicosis, provides means for helping individuals subject to or in threat of mycotoxicosis, in particular asperphenamate toxicosis. [029] In the following, the present invention is further described by non-limiting figures and examples, wherein Figure 1 is a graph showing reduction of asperphenamate concentration by microorganism in vitro; Figure 2 is a graph showing reduction of asperphenamate concentration by a spray-dried microorganism formulation; and Figure 3 is a graph showing reduction of asperphenamate concentration by microorganism formulations in a nutritional composition. Examples [030] The present invention as disclosed herein is not limited to specific embodiments, figures, methodology, examples, protocols etc. described herein but solely defined by the claims. Example 1 - Reduction of asperphenamate [031] In order to test the capacities of selected microorganisms for reducing asperphenamate from a solution, strains of the genus Trichosporon deposited by Erber AG (Industriestrasse 21, 3130 Herzogenburg, Austria) at the Leibniz Institute DSMZ-German Collection of microorganisms and Cell Cultures GmbH under the identifier number DSM 14153 were cultivated and contacted with asperphenamate. To this end, a 1 mM stock solution of asperphenamate was prepared using dimethyl sulfoxide (DSMO) as solvent. [032] The microorganisms were grown in YPD media (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose) at 28 °C under agitation to a final OD600 of the microorganism culture of 4.0.38 mL of the thus produced microorganism culture was subjected to centrifugation for 30 min, 500 xg, 4 °C. The supernatant was discarded and the pelleted cells were resuspended in 38 mL Brunner mineral media (2.44 g/L Na₂HPO₄, 1.52 g/L KH₂PO₄, 0.50 g/L (NH₄)₂SO₄, 0.20 g/L MgSO₄ x 7 H₂O, 0.05 g/L CaCl₂ x 2 H₂O, 10.00 mL/L trace element solution SL-4; Trace element solution SL- 4: 0.50 g/L EDTA, 0.20 g/L FeSO₄ x 7 H₂O, 100.00 mL/L trace element solution SL-6; Trace element solution SL-6: 0.10 g/L ZnSO₄ x 7 H₂O, 0.03 g/L MnCl₂ x 4 H₂O, 0.30 g/L H₃BO₃, 0.20 g/L CoCl₂ x 6 H₂O, 0.01 g/L CuCl₂ x 2 H₂O, 0.02 g/L NiCl₂ x 6 H₂O, 0.03 g/L Na₂MoO₄ x 2 H₂O). [033] To start the toxin removal reaction, 980 µL of the microorganism resuspended in Brunner mineral media was mixed with 20 µL of the 1 mM asperphenamate stock solution. As a blank control, 980 µL Brunner mineral media without the microorganisms was mixed with 20 µL of the 1 mM asperphenamate stock solution. The toxin removal reactions were incubated for 60 h at 22 °C. 50 µL samples were taken immediately after mixing (time 0) as well as throughout the incubation. To stop the reaction, the samples were mixed with 200 µL acetonitrile and stored at 4 °C until further analyses. [34] To determine asperphenamate concentration, the samples were mixed thoroughly, incubated at 95 °C for 10 min and then subjected to centrifugation (20 min, 30279 x rcf, 4 °C). The supernatant was analyzed either directly or diluted 1:9 with acetonitrile to achieve a suitable analyte concentration for quantification. The analysis was performed as described in Steiner et al. (2020. J Chromatogr A.1629: 461502 and supplementary material). [35] As shown in Fig. 1, asperphenamate can be found reduced upon contacting with the microorganisms, whereas no such reduction could be observed in the absence of the microorganisms. The y-axis in Fig.1 indicates asperphenamate concentration in %, wherein the initial asperphenamate concentration (i.e. at time 0) is set to 100%. The x-axis indicates the incubation period in hours. Asperphenamate concentrations determined from the reactions comprising the microorganisms are shown as black triangles. Asperphenamate concentrations determined from the blank control are shown as grey-filled circles. Example 2 - Reduction of asperphenamate by spray-dried microorganism [36] In many applications, microorganisms are provided in a spray-dried formulation. To confirm the results obtained in Example 1 for a microorganism formulated in such a manner, the strain DSM 14153 was cultivated as described above. Collected yeast biomass was mixed with maltodextrin to achieve a maltodextrin concentration of 4% shortly before drying. For spray- drying, the inlet temperature was approximately 180 °C, the outlet temperature was approximately 78-80 °C. [37] To start the toxin removal reaction, 1 g of the thus formulated microorganism was mixed in 9 mL buffer solution (2.44 g/L Na2HPO4, 1.52 g/L KH2PO4, 0.5 g/L (NH4)2SO4, 0.2 g/L MgSO4 x 7 H2O, 10 mL/L trace element solution SL-4; 10 mL/L vitamin solution; Vitamin solution: 2.0 mg/L biotin, 2.0 mg/L folic acid, 10.0 mg/L pyridoxine-HCl, 5.0 mg/L thiamin-HCl, 5.0 mg/L riboflavin, 5.0 mg/L nicotinamide, 5.0 mg/L D,L-pantothenate, 0.1 mg/L cyanocobalamin, 100.0 mg/L menadione, 22.0 mg/L phylloquinone, 5.0 mg/L p-aminobenzoic acid, 5.0 mg/L thioctic acid; pH was set to 7, sterilized by autoclaving) additionally containing 40 µM asperphenamate. This solution was diluted further 1:5 with buffer and incubated at 37 °C. Samples were drawn throughout incubation for later analysis as described in Example 1. [38] Already three hours after start of the toxin removal reaction, the asperphenamate concentration was reduced by half of the initial concentration. After 24 h, no asperphenamate could be observed, see Figure 2. The y-axis in Fig.2 indicates asperphenamate concentration in %, wherein the initial asperphenamate concentration (i.e. at time 0) is set to 100%. The x-axis indicates the incubation period in hours. Asperphenamate concentrations determined from the reactions comprising the spray-dried microorganisms are shown as black triangles. Example 3 - Reduction of asperphenamate concentration from nutritional composition [39] To test the applicability of the microorganism for reduction of asperphenamate from a complex matrix, spray-dried formulations of the strain DSM 14153 as prepared in Example 2 were added to a nutritional composition comprising asperphenamate. [40] As an example composition for a nutritional composition, commonly used pig feed was used (e.g. Masching et al. 2016. Toxins 8:(3):84; Schwartz-Zimmermann et al. 2018. World Mycotoxin Journal, DOI 10.3920/WMJ2017.2265). Spray-dried DSM 14153 was applied to this nutritional composition at different concentrations: 0.1 g, 0.01 g, 0.001 g, or 0.0000125 g of spray- dried microorganism formulation was added to 250 mg of pig feed. Thus, concentrations of 400, 40, 4, and 0.05 g of spray-dried microorganism formulation per 1 kg of nutritional composition were tested. As source for water, 10 mL of buffer solution as described in Example 2 was added. Asperphenamate was added to a concentration of 13 µM to start the toxin removal reaction. Reduction of asperphenamate concentration can be seen immediately upon contacting the microorganism with asperphenamate in the nutritional composition. At all tested concentrations of the microorganism formulation, asperphenamate was effectively reduced, see Fig.3. The y- axis in Fig.3 indicates asperphenamate concentration in %, wherein the initial asperphenamate concentration (i.e. at time 0) is set to 100%. The x-axis indicates the incubation period in hours. Asperphenamate concentrations determined from the reactions comprising the microorganisms at 400, 40, 4, or 0.05 kg per ton of nutritional composition are shown as solid grey line, dashed black line, dotted blank line, or solid black line, respectively. Asperphenamate concentrations determined from blank reactions not comprising the microorganism are shown as dashed grey line. After 24 h, asperphenamate was almost entirely removed from the nutritional composition comprising the microorganisms. After 48 h, asperphenamate concentration was reduced to a concentration below the detection limit by all microorganism concentrations.

Claims

1. Method for reducing asperphenamate concentration in a nutritional composition comprising asperphenamate, the method comprising the steps of a) providing the nutritional composition comprising asperphenamate; b) providing at least one microorganism, wherein the at least one microorganism comprises at least one polynucleotide having at least 95%, preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity to the nucleotide sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2; c) forming a mixture comprising water, the nutritional composition of a) and the at least one microorganism of b); d) incubating the mixture of step c).

2. The method according to claim 1 , wherein step d) incubating the mixture of step c) is performed for at least one hour, preferably for at least three hours, more preferably for at least one day; and wherein step d) is performed at a temperature of at least 10 °C, preferably of at least 15 °C, more preferably of at least 20 °C; and wherein step d) is performed at a temperature of at most 50 °C, preferably at most 40 °C, more preferably at most 35 °C.

3. The method according to claim 1 or 2, wherein the at least one microorganism belongs to the genus Trichosporon.

4. The method according to any one of the preceding claims, wherein the at least one microorganism is DSM 14153.

5. The method according to any one of the preceding claims, wherein the nutritional composition is selected from the group consisting of foodstuff; fodder; feed; wet distillers grain; dried distillers grain with solubles; intermediate/s thereof; and/or mixture/s thereof.

6. The method according to any one of the preceding claims, wherein the at least one microorganism is provided as a dried microorganism formulation, in particular as a spray- dried or freeze-dried formulation, preferably as a spray-dried formulation.

7. The method according to claim 6, wherein the microorganism formulation is provided in such an amount to achieve a concentration of at least 10 g, e.g. at least 20, 30, 40 or 50 g, of microorganism formulation per ton of nutritional composition.

8. Foodstuff; fodder; feed; wet distillers grain; dried distillers grain with solubles; intermediate/s thereof; and/or mixture/s thereof produced by a method of any one of the preceding claims.

9. Use of at least one microorganism for reducing asperphenamate concentration in a nutritional composition comprising asperphenamate, wherein the at least one microorganism comprises at least one polynucleotide having at least 95%, preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity to the nucleotide sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2.

10. The use according to claim 9, wherein the at least one microorganism is incubated with the nutritional composition and water for at least one hour, preferably for at least three hours, more preferably for at least one day; and said incubation is performed at a temperature of at least 10 °C, preferably of at least 15 °C, more preferably of at least 20 °C; and wherein said incubation is performed at a temperature of at most 40 °C, preferably at most 35 °C, more preferably at most 30 °C.

11. The use according to claim 9 or 10, wherein the at least one microorganism belongs to the genus Trichosporon, preferably the at least one microorganism is DSM 14153.

12. The use according to any one of claims 9-11 , wherein the at least one microorganism is provided as a dried microorganism formulation, in particular as a spray-dried or freeze-dried formulation, preferably as a spray-dried formulation.

13. The use according to claim 12, wherein the microorganism formulation is provided in such an amount to achieve a concentration of at least 10 g, e.g. at least 20, 30, 40 or 50 g, of microorganism formulation per ton of nutritional composition.

14. Microorganism for use as a medicament, e.g. in veterinary medicine, and/or in treatment, amelioration, prophylaxis and/or diagnostics of a disease, wherein the at least one microorganism comprises at least one polynucleotide having at least 95%, preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity to the nucleotide sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2.

15. Microorganism for use in treatment, amelioration, prophylaxis and/or diagnostics of symptoms caused by mycotoxicosis, in particular by asperphenamate toxicosis, wherein the at least one microorganism comprises at least one polynucleotide having at least 95%, preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity to the nucleotide sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2.