Means and methods for reducing apicidin concentrations in compositions

Abstract

The present invention relates to a method for reducing the concentration of apicidin in a composition and to the use of a microorganism having at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 7.

Description

Detailed Description of the Invention

[0001] The present invention relates to a method for reducing the apicidin concentration in a nutritional composition by providing a microorganism, to a method for treating foodstuffs, feed, etc. and to the use of said microorganism.

[0002] Apicidin (CAS number: 183506-66-3; also referred to as, for example, (3S,6S,9S,15aR)-9-[(2S)-butan-2-yl]-6-[(1-methoxy-1H-indol-3-yl)methyl]-3-(6-oxooctyl)octahydro-2H-pyrido[1,2-a][1,4,7,10]tetraazacyclododecine-1,4,7,10(3H,12H)-tetraone) is a highly toxic metabolite produced by Fusarium fungi, for example F. semitectum. Apicidin is a cyclic tetrapeptide having the molecular structure shown below, and has been described as inhibiting histone deacetylase. [ka]

[0003] Among 28 other tested fungal metabolites, apicidin was found to have the lowest IC50 value, i.e., to exhibit the strongest cytotoxic effect (Novak et al. 2019. Toxins 11:537). The toxic effects of apicidin were shown in rat feeding trials, in brine shrimp, and in human and mouse cell lines (Park et al. 1999. Appl. Environ. Microbiol 65:126). Being a fungal metabolite, apicidin can be found in compositions derived from primary commodities, such as agricultural crops. Notably, apicidin can appear simultaneously with other toxic substances, such as deoxynivalenol. In particular in the case of co-contamination with deoxynivalenol and apicidin, defects in the barrier function of epithelial cells have been observed upon contact with the combined toxins at concentrations of deoxynivalenol that would not affect the cells without the co-presence of apicidin (Springler et al. 2016. Toxins 8:345).

[0004]

[0004] Despite these undesirable effects, and in contrast to other toxic fungal metabolites such as fumonisins, deoxynivalenol or zeralenone, no means have been found to date for counteracting the toxic effects of apicidin.

[0005] In view of the prior art as outlined above, it is therefore an object of the present invention to provide means and measures for reducing the concentration of apicidin in nutritional compositions.

[0006]

[0006] It has been found that this object is achieved by providing a method for reducing the concentration of apicidin in a nutritional composition comprising apicidin, the method comprising the steps of: a) providing a nutritional composition comprising apicidin; b) providing at least one microorganism, wherein the at least one microorganism comprises a 16S rDNA 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 selected from the group consisting of SEQ ID NO: 1-7, preferably a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-4, more preferably a nucleotide sequence of SEQ ID NO: 1; preferably the at least one microorganism belongs to the genus Raoultibacter; 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). According to the present disclosure, the nutritional composition can be modified to contain less or even no apicidin compared to before the treatment, thus reducing or even eliminating apicidin and the toxic effects caused by said apicidin.In one embodiment, the present invention relates to a method for reducing the apicidin concentration in a nutritional composition comprising apicidin, comprising the steps of: a) providing a nutritional composition comprising apicidin; b) providing at least one microorganism, the at least one microorganism belonging to the genus Raoultibacter; 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). [Brief description of the drawings]

[0007] [Figure 1] 1 shows the reduction of apicidin in the composition by microorganisms (1.7*107 cfu / mL starting concentration). [Diagram 2]Figure 1 shows the removal of apicidin from feed using only 1.7*104 cfu (colony forming density) of Raoultibacter species per gram of feed.

[0008] A nutritional composition as referred to herein is a composition comprising one or more ingredients having nutritional value. Often such ingredients provide energy to the consumer of the nutritional composition. The nutritional composition may be entirely or at least partly herbaceous or plant based, such as commonly used animal feed compositions. The water contained in the mixture of step c) may originate from moisture contained in the nutritional composition, e.g. it may be present in the form of moisture, or the water may be added by the operator of the process, or the water could originate from saliva.

[0009]

[0008] The term "16S rDNA" refers to ribosomal deoxyribonucleic acid (rDNA) that encodes the 16S ribonucleic acid component of the 30S subunit of the prokaryotic ribosome. The similarity or relatedness of two or more nucleic acid sequences (e.g., DNA or RNA) can be described in terms of sequence identity. Sequence identity can be determined by common methods known to those skilled in the art. As used herein, a preferred method for determining sequence identity among two or more 16S rDNA sequences is the use of EMBL-EBI's Clustal Omega nucleotide sequence alignment tool (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ; Sievers et al. 2011. Mol. Syst. Biol. 7:539) using default settings. Alternatively, the Needleman-Wunsch algorithm for global sequence alignment ("Needleman-Wunsch Global Align Protein Sequences") may be used, for example, using default settings as provided by the National Center for Biotechnology Information (Match / Mismatch Scores: 2, -3; Gap Costs: Existence: 5 Extension 2).

[0010]

[0009] Chemical reactions, such as reactions catalyzed by biological systems, such as enzymes or microorganisms, can occur faster or slower depending on the environmental conditions. In order to carry out the method of the present application in a preferred manner, it is therefore conceivable to carry out step d) of the method described above for at least 5 minutes, preferably at least 1 hour, more preferably at least 2 hours, even more preferably at least 1 day, most preferably at least 2 days. Furthermore, it is preferred to carry out step d) of the method described above at a temperature of at least 10°C, preferably at least 15°C, more preferably at least 20°C. It is further preferred to carry out said incubation at a temperature of at most 50°C, preferably at most 40°C. In one embodiment, the method of the present application is carried out at normal ambient temperature (i.e. 15-35°C, preferably 18-30°C, more preferably 18-25°C). When carrying out the method described herein under said conditions, a particularly efficient reduction of the apicidin concentration or content in the composition can be achieved. The longer the incubation of step d) is carried out, the more apicidin will be removed from the nutritional composition. In particular, it can be found that even throughout such a long incubation time, the microorganism can remove apicidin.Nevertheless, those skilled in the art know that the type of reaction as described herein can be achieved outside the preferred conditions.Just for clarity, incubation is considered to start as soon as the microorganism as described herein comes into contact with the composition comprising apicidin as described herein.Those skilled in the art can well select the incubation conditions suitable for reducing the apicidin concentration present in the composition to the desired extent.

[0011]

[0010] Surprisingly, it has been found by the present inventors that microorganisms selected from the group consisting of, in particular, the species Raoultibacter species nova, Raoultibacter massiliensis, Raoultibacter timonensis and Raoultibacter phocaeensis are efficient microbial catalysts for achieving a reduction in the apicidin concentration in a nutritional composition. Thus, in one embodiment, the present invention relates to a method as described herein, wherein the at least one microorganism is selected from the group consisting of Raoultibacter species nova, Raoultibacter massiliensis, Raoultibacter timonensis and Raoultibacter phocaeensis. Even more preferably and specifically, the at least one microorganism is selected from the group of strains consisting of DSM 33979, DSM 33980, DSM 33991 and DSM 33992. The labels "DSM 33979, DSM 33980, DSM 33991 and DSM 33992" indicate the identifier numbers of the strains deposited at the Leibniz Institute DSMZ-German Collection of microorganisms and Cell Cultures GmbH. It has been found that outstanding and reliable reduction of apicidin in the composition is achievable when microbial strains selected from DSM 33979, DSM 33980, DSM 33991 and DSM 33992 are used.

[0012]

[0011] The composition may consist of or comprise one or more solid components and / or one or more liquid components. For example, the nutritional composition (as referred to in step a) of the method described above) may be selected from the group consisting of foodstuffs; feed; bait; food additives; feed additives; feed additives; wet distillers grains; dry distillers grains with solubles; dietary supplements; prebiotics; probiotics; intermediates thereof; and / or mixtures thereof. Such compositions are part of the feed / food chain and may therefore substantially benefit from the reduction or even elimination of apicidin. The reduction or even elimination of apicidin in such compositions may therefore contribute to smaller losses in food production and healthier animals and consumers. Just to clarify, the feed or bait may comprise or consist of, for example, corn, hay, straw mulch, soybean or products obtained therefrom. The feed or bait may also comprise or consist of extruded feed products, such as pellets. Foodstuff, feed or feed additives are used to improve or add properties to foodstuffs, feeds or feeds. For example, such additives may be added to improve organoleptic properties, e.g. to improve the taste, smell, appearance, color of the foodstuff, feed or feed. Additives may also be added to improve palatability, nutrient availability, or to add probiotic microorganisms to the foodstuff, feed or feed, or to add or increase the prebiotic activity of the foodstuff, feed or feed. Such additives may also be added to counteract a potentially undesirable effect of the foodstuff, feed or feed, such as removing or reducing one or more undesirable components contained in the foodstuff, feed or feed.

[0013]

[0012] Alternatively, it is believed that a composition selected from a foodstuff; a feed; a bait; an additive (e.g., a foodstuff, a feed or a feed additive); wet distillers grains; dried distillers grains with solubles; a dietary supplement; a prebiotic; a probiotic; intermediates thereof; and / or mixtures thereof may be required such that the composition has a reduced apicidin concentration after suitable treatment compared to the apicidin concentration before said treatment. The object is a method for treating a foodstuff; a feed; a bait; a food additive; a feed additive; a feed additive; a wet distiller's grains; a dried distiller's grains with solubles; a dietary supplement; a prebiotic; a probiotic; an intermediate thereof; and / or a mixture thereof, wherein the foodstuff; a feed; a bait; a food additive; a feed additive; a feed additive; a wet distiller's grains; a dried distiller's grains with solubles; a dietary supplement; a prebiotic; a probiotic; an intermediate thereof; and / or a mixture thereof comprises apicidin, the method comprising the steps of: a) providing at least one microorganism, the at least one microorganism comprising a 16S nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 7, preferably a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 4, more preferably a nucleotide sequence 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. This has been achieved by providing a method comprising the steps of: (a) applying at least one microorganism of a) to a foodstuff; a feed; a bait; a food additive; a feed additive; a feed additive; a wet distillers grains; a dried distillers grains with solubles; a dietary supplement; a prebiotic; a probiotic; an intermediate thereof; and / or a mixture thereof, thereby forming a treatment mixture; and (b) incubating the treatment mixture of step b).Before the above-mentioned treatment method, the foodstuff; feed; feed; food additive; feed additive; feed additive; wet distillers grains; dry distillers grains with solubles; dietary supplement; prebiotic; probiotic; intermediates thereof; and / or mixtures thereof contain apicidin (e.g., at a concentration of more than 30 ppb or even more than 40 ppb, 50 ppb, 60 ppb, 70 ppb, 80 ppb, 90 ppb, 100 ppb or even more). However, the foodstuff; feed; feed; food additive; feed additive; feed additive; wet distillers grains; dry distillers grains with solubles; dietary supplement; prebiotic; probiotic; intermediates thereof; and / or mixtures thereof obtained by such a method no longer contain apicidin or even no apicidin and can therefore be conveniently used for the intended purpose without any further measures required to counteract apicidin-induced adverse effects. If the microorganisms of a) or the foodstuffs; feeds; baits; food additives; feed additives; feed additives; wet distillers grains; dry distillers grains with solubles; dietary supplements; prebiotics; probiotics; their intermediates; and / or mixtures thereof do not contain water (e.g. in the form of moisture), water may be added in step c), in particular before step c).

[0014]

[0013] In one embodiment, the present invention relates to a method for treating foodstuffs; feed; bait; food additives; feed additives; feed additives; wet distillers grains; dried distillers grains with solubles; nutritional supplements; prebiotics; probiotics; intermediates thereof; and / or mixtures thereof, comprising the steps of: a) providing at least one microorganism, wherein the at least one microorganism belongs to the genus Raoultibacter; b) applying the at least one microorganism of a) to foodstuffs; feed; food additives; feed additives; feed additives; wet distillers grains; dried distillers grains with solubles; nutritional supplements; prebiotics; probiotics; intermediates thereof; and / or mixtures thereof, thus forming a treatment mixture; and c) incubating the treatment mixture of step b). Preferably, a method for treating foodstuffs; feedstuffs; baits; additives (e.g. foodstuffs, feedstuffs or feed additives); wet distillers grains; dry distillers grains with solubles; dietary supplements; prebiotics; probiotics; intermediates thereof; and / or mixtures thereof is carried out, wherein step c) of incubating the treated mixture of step b) is carried out for at least 5 minutes, preferably at least 1 hour, more preferably at least 2 hours, even more preferably at least 1 day, most preferably at least 2 days. Preferably, the method is further carried out at a temperature of at least 10°C, preferably at least 15°C, more preferably at least 20°C. It is further preferred that said incubation is carried out at a temperature of at most 50°C, preferably at most 40°C. For example, the method of the present application is carried out at normal ambient temperature (i.e. 15-35°C, preferably 18-30°C, more preferably 18-25°C).

[0015]

[0014] In a preferred embodiment of the method for treating foodstuffs; feedstuffs; food additives; feed additives; feed additives; wet distillers grains; dried distillers grains with solubles; dietary supplements; prebiotics; probiotics; intermediates thereof; and / or mixtures thereof, the method is carried out wherein at least one microorganism is selected from the group consisting of Raoultibacter species nova, Raoultibacter massiliensis, Raoultibacter timonensis and Raoultibacter phocaeensis, preferably wherein at least one microorganism is selected from the group of strains consisting of DSM 33979, DSM 33980, DSM 33991 and DSM 33992.

[0016]

[0015] At least 10 per mL of the liquid composition 4 cfu or at least 10 per gram of solid composition (e.g., foodstuffs; feeds; baits; additives (e.g., foodstuffs, feeds, or feed additives); wet distillers grains; dry distillers grains with solubles; dietary supplements; prebiotics; probiotics; intermediates thereof; and / or mixtures thereof). 4 Concentration of cfu, preferably at least 1.7*10 per mL of liquid composition 4 cfu or at least 1.7*10 per gram of solid composition (e.g., foodstuffs; feeds; baits; additives (e.g., foodstuffs, feeds, or feed additives); wet distillers grains; dry distillers grains with solubles; dietary supplements; prebiotics; probiotics; intermediates thereof; and / or mixtures thereof). 4It is preferred to carry out any of the methods described herein in a manner in which the at least one microorganism is applied in an amount to achieve a concentration of at least 10 cfu. In other words, the final concentration of the at least one microorganism in the composition, i.e., the working concentration, is preferably at least 10 per mL or per g of the composition (e.g., foodstuff; feed; bait; additive (e.g., foodstuff, feed or feed additive); wet distillers grains; dried distillers grains with solubles; dietary supplement; prebiotic; probiotic; intermediates thereof; and / or mixtures thereof). 4 cfu, more preferably at least 1.7*10 per mL or per g of the composition (e.g., foodstuff; feed; bait; additive (e.g., foodstuff, feed or feed additive); wet distillers grains; dry distillers grains with solubles; dietary supplement; prebiotic; probiotic; intermediates thereof; and / or mixtures thereof). 4 The concentration of cfu.

[0017]

[0016] Those skilled in the art will appreciate that the present invention can also be practiced when using lower concentrations of microorganisms, nevertheless, when applying the minimum concentrations as described above, apicidin reduction or elimination can be achieved within the most convenient time frame.

[0018]

[0017] The term "cfu" refers to "colony forming unit" as known in the art. For example, the smallest biological unit capable of reproduction (e.g., a bacterial cell) is a colony forming unit. Typically, a single live bacterial cell is a colony forming unit. In practice, the concentration of colony forming units in a composition can be determined by any commonly suitable method known to those skilled in the art for that purpose, such as counting colonies on a nutrient agar plate or applying flow cytometry suitable for quantifying living microorganisms as known to those skilled in the art.

[0019] Another aspect of the invention relates to a foodstuff; feed; bait; additive (e.g., foodstuff, feed or feed additive); wet distillers grains; dried distillers grains with solubles; dietary supplements; prebiotics; probiotics; intermediates thereof; and / or mixtures thereof that are producible or produced by the method as described herein. The foodstuff; feed; bait; additive (e.g., foodstuff, feed or feed additive); wet distillers grains; dried distillers grains with solubles; dietary supplements; prebiotics; probiotics; intermediates thereof; and / or mixtures thereof that are producible or produced by the method as described herein contain zero or at least a lower amount of apicidin compared to a foodstuff; feed; bait; additive (e.g., foodstuff, feed or feed additive); wet distillers grains; dried distillers grains with solubles; dietary supplements; prebiotics; probiotics; intermediates thereof; and / or mixtures thereof that were not produced by the method as described herein. As a result, such foodstuffs; feeds; baits; additives (e.g., foodstuffs, feeds or feed additives); wet distillers grains; dried distillers grains with solubles; dietary supplements; prebiotics; probiotics; intermediates thereof; and / or mixtures thereof according to the present invention pose a lower threat of causing apicidin-induced toxic effects to consumers of such foodstuffs; feeds; baits; additives (e.g., foodstuffs, feeds or feed additives); wet distillers grains; dried distillers grains with solubles; dietary supplements; prebiotics; probiotics; intermediates thereof; and / or mixtures thereof.Foodstuffs; feeds; feeds; additives (e.g. foodstuffs, feeds or feed additives); wet distillers grains; dry distillers grains with solubles; dietary supplements; prebiotics; probiotics; intermediates thereof; and / or mixtures thereof producible or produced by the methods as described herein comprise at least one microorganism as referred to herein, wherein the at least one microorganism comprises a 16S rDNA having at least 95%, preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identity to the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7, preferably selected from the group consisting of SEQ ID NOs: 1-4, more preferably the nucleotide sequence of SEQ ID NO: 1; preferably, the at least one microorganism is considered to belong to the genus Raoultibacter. Thus, such foodstuffs; feeds; baits; additives (e.g., foodstuffs, feeds or feed additives); wet distillers grains; dried distillers grains with solubles; dietary supplements; prebiotics; probiotics; intermediates thereof; and / or mixtures thereof have a lower risk of further contamination with apicidin due to the resulting growth of bacteria compared to foodstuffs; feeds; baits; additives (e.g., foodstuffs, feeds or feed additives); wet distillers grains; dried distillers grains with solubles; dietary supplements; prebiotics; probiotics; intermediates thereof; and / or mixtures thereof that do not contain at least one microorganism referred to herein.Preferably, the foodstuffs; feeds; baits; additives (e.g., foodstuffs, feeds or feed additives); wet distillers grains; dried distillers grains with solubles; dietary supplements; prebiotics; probiotics; intermediates thereof; and / or mixtures thereof that can be produced or are produced by the method as described herein have a concentration of at least 10 per mL of liquid composition. 4 At least 10 cfu per gram of solid composition 4 cfu per mL of the liquid composition, and more preferably at least 1.7*10 4 At least 1.7*10 cfu / g of solid composition 4The microorganism according to the invention is contained in a concentration of cfu.

[0020]

[0019] In certain embodiments, such foodstuffs; feeds; baits; additives (e.g., foodstuffs, feeds or feed additives); wet distillers grains; dry distillers grains with solubles; dietary supplements; prebiotics; probiotics; intermediates thereof; and / or mixtures thereof that are producible or produced by the methods described herein contain less than 30 ppb apicidin (e.g., 25 ppb or 15 ppb or 30-20 ppb, or 30-10 ppb, or 30-5 ppb apicidin), preferably less than 20 ppb apicidin (e.g., 15 ppb or 20-5 ppb apicidin), more preferably less than 10 ppb apicidin (e.g., 10-5 ppb apicidin or 5 ppb, 4 ppb, 3 ppb, 2 ppb, etc. apicidin).

[0021] In another aspect, the present invention relates to the use (e.g. non-medical use) of at least one microorganism for reducing the concentration of apicidin in a composition (e.g. nutritional composition) comprising apicidin, wherein the at least one microorganism comprises a 16S rDNA having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7, preferably a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-4, more preferably at least 95%, more 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; preferably the at least one microorganism belongs to the genus Raoultibacter. Surprisingly, it has been found that such a microorganism can reduce the concentration of apicidin in a composition, such as foodstuffs; feedstuffs; baits; additives (e.g. foodstuffs, feedstuffs or feed additives); wet distillers grains; dry distillers grains with solubles; dietary supplements; prebiotics; probiotics; intermediates thereof; and / or mixtures thereof. This allows consumers of such compositions with reduced or even zero apicidin to be less or not exposed to the toxic effects of apicidin. In particular, animals (e.g. poultry, pigs, ruminants, etc.) that do not suffer from mycotoxicosis, such as apicidinosis, may be found to show improved performance (e.g. increased weight gain and / or reduced feed conversion ratio). The microorganisms of the present invention may therefore be used to improve the performance of animals when they are fed with a nutritional composition comprising apicidin. In one embodiment, the present invention relates to the use of at least one microorganism to reduce the apicidin concentration in a composition (e.g. nutritional composition), wherein the at least one microorganism belongs to the genus Raoultibacter.

[0022]

[0021] Preferably, the use of at least one microorganism to reduce the apicidin concentration in a composition as described herein is carried out in a manner in which the at least one microorganism is selected from the group consisting of Raoultibacter species nova, Raoultibacter massiliensis, Raoultibacter timonensis and Raoultibacter phocaeensis, preferably the at least one microorganism is selected from the group of strains consisting of DSM 33979, DSM 33980, DSM 33991 and DSM 33992.

[0023] In a further aspect, the present invention relates to a microorganism and for use as a medicament (e.g. in veterinary medicine) and / or in the treatment, amelioration, prevention and / or diagnosis of disease, wherein the microorganism comprises a 16S rDNA having at least 95%, preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identity to the nucleotide sequence of SEQ ID NO: 1 selected from the group consisting of SEQ ID NO: 1-7, preferably a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-4, more preferably the nucleotide sequence of SEQ ID NO: 1, and preferably the microorganism belongs to the genus Raoultibacter. In one embodiment, the present invention relates to a microorganism for use as a medicament (e.g. in veterinary medicine) and / or in the treatment, amelioration, prevention and / or diagnosis of disease, wherein the microorganism belongs to the genus Raoultibacter.

[0024]

[0023] The unexpected discovery that the microorganism of the present invention is able to remove apicidin makes its use as a drug in the treatment, amelioration, prevention or diagnosis of disease highly advantageous. It is therefore a further aspect of the present invention to provide a microorganism for use in the treatment, amelioration, prevention and / or diagnosis of symptoms caused by mycotoxinosis, in particular apicidinosis, comprising a 16S rDNA 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 with the nucleotide sequence of SEQ ID NO: 1-7, preferably a nucleotide sequence of SEQ ID NO: 1-4, more preferably SEQ ID NO: 1; preferably belonging to the genus Raoultibacter. In one embodiment, the present invention relates to a microorganism for use in the treatment, amelioration, prevention and / or diagnosis of symptoms caused by mycotoxinosis, in particular apicidinosis, comprising a 16S rDNA 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 with the nucleotide sequence of SEQ ID NO: 1; preferably belonging to the genus Raoultibacter.

[0025]

[0024] The microorganisms may be administered to humans or to animals (eg, pets, poultry, pigs, ruminants, etc.) by providing a nutritional composition comprising the microorganisms.

[0026]

[0025] A composition comprising at least one microorganism as referred to in this specification may further comprise one or more additional microorganisms capable of detoxifying one or more mushroom / plant / bacterial toxins; one or more isolated polypeptides capable of detoxifying one or more further mushroom / plant / bacterial toxins (e.g., fumonisin esterases and / or variants thereof, e.g. as described in UniProtKB:D2D3B6; and / or zeralenone lactonase and / or variants thereof, e.g. as described in UniProtKB:Q8NKB0); and / or one or more organic absorbent components (e.g., inactivated / dried / lyophilized / live yeast, e.g., Saccharomyces spp., e.g., S. cerevisiae, Pichia pastoris, pastoris); and / or one or more inorganic absorbent ingredients (e.g., clay products, bentonite, zeolites, bentonite-montmorillonite, diatomaceous earth); and / or one or more plant products (e.g., algae products, thistle extract); and / or one or more vitamins; and / or one or more flavor compounds; and / or one or more prebiotic compounds (e.g., mannan); and / or one or more probiotic compounds.

[0027]

[0026] In a preferred embodiment, at least one microorganism for use as a medicine (e.g. in veterinary medicine) and / or for use in the treatment, amelioration, prevention and / or diagnosis of a disease and / or for use in the treatment, amelioration, prevention and / or diagnosis of a symptom caused by a mycotoxigenic disease, particularly as referred to herein (e.g. by apicidinosis), is selected from the group consisting of Raoultibacter species nova, Raoultibacter massiliensis, Raoultibacter timonensis and Raoultibacter phocaeensis, more preferably the microorganism is selected from the group consisting of strains consisting of DSM 33979, DSM 33980, DSM 33991 and DSM 33992. Due to the reliable reduction of apicidin, the microorganisms according to these preferred embodiments have been found to be particularly suitable for use as medicaments, in particular in the treatment, amelioration, prevention and / or diagnosis of symptoms caused by mycotoxime, in particular apicidinotoxicosis.

[0028] Preferably, the microorganism of the invention for use as a medicine (e.g. in veterinary medicine) and / or for the treatment, amelioration, prevention and / or diagnosis of a disease; and / or a composition of the invention for use in the treatment, amelioration, prevention and / or diagnosis of a condition caused by mycotoxinosis (e.g. by apicidinosis) is characterized in that the microorganism contained in such a composition is at least 10 per mL of liquid composition. 4 At least 10 cfu or per gram of solids 4 Concentration of cfu, preferably at least 1.7*10 per mL of liquid composition 4 At least 1.7*10 cfu or per gram of solid composition 4In other words, the final concentration, i.e. the working concentration, of at least one microorganism according to the invention in such a composition is preferably at least 10 per mL or per g of the composition. 4 cfu, more preferably at least 1.7*10 per mL or per g of the composition 4 The concentration of cfu.

[0029]

[0028] In the following the invention is further illustrated by non-limiting figures and examples, in which Figure 1 shows the reduction in apicidin concentration by a microbial strain according to the invention in an experimental set-up; Figure 2 shows the reduction in apicidin concentration in feed by an additive according to the invention.

[0030]

[0029] The figures shown and described herein merely serve as illustrative examples and should not be construed as limiting the embodiments of the present invention.

[0031] [Example]

[0030] The invention as disclosed herein is not limited to the specific embodiments, diagrams, methodologies, examples, protocols, etc. described herein, but is exclusively and explicitly defined by the claims. The examples disclosed below can only be considered to refer to exemplary embodiments of the underlying invention as explicitly defined in the claims.

[0032] Example 1: Apicidin reduction

[0033]

[0032] To experimentally demonstrate the reduction of apicidin concentration in microorganisms according to the present invention, the following steps were performed: Apicidin was dissolved in DMSO (dimethyl sulfoxide) to obtain a 1 ppm stock solution. Precultures were prepared from one of the following microorganisms: Raoultibacter species nova (R. spec. nov.; DSM 33979), Raoultibacter massiliensis (R. massiliensis; DSM 33980), Raoultibacter timonensis (R. timonensis; DSM 33991), Raoultibacter phocaeensis (R. phocaeensis; DSM 33992). For this purpose, viable samples from any of the bacterial species were used to culture the bacteria in airtight, sealable laboratory glass bottles or anaerobic culture tubes (Hungate type) in sterile Wilkins-Chalgren medium (10 g / L tryptone, 10 g / L gelatin peptone, 5 g / L yeast extract, 1 g / L glucose, 5 g / L NaCl, 1 g / L L-arginine, 1 g / L sodium pyruvate, 0.0005 g / L menadione, 0.005 g / l hemin, pH 7 at 25°C; Wilkins and Chalgren. 1976. Antimicrob. Agents. Chemother. 10:926-928), which further contained 0.5 mL / L of 0.1% (w / v) resazurin sodium solution, 0.3 g / L L-cysteine ​​hydrochloride monohydrate, and 10-20 mg / L sodium dithionite, in a hydrogen atmosphere. In detail, the medium broth was prepared by dissolving the components of Wilkins-Chalgren medium in distilled water and then adding resazurin sodium. The solution was boiled and then cooled to room temperature while sparging nitrogen through the solution. L-cysteine ​​hydrochloride monohydrate and sodium dithionite were then added aseptically. These precultures were incubated at 37°C for 3 days. Aliquots of the precultures were used to prepare 1.7*10 4 cfu / mL or 1.7*10 8The main culture was inoculated in the medium as described above to a bacterial concentration of either cfu / mL. The bacterial concentration in cfu / mL can be determined by any suitable method known to those skilled in the art, such as plate counting (i.e., placing an aliquot of the culture on a nutrient agar plate and counting the number of colonies formed after incubation) or flow cytometry. Finally, apicidin was added to the main culture to a target concentration of 83 ppb. The main culture thus prepared was incubated at 37°C. Samples were taken periodically throughout the incubation to measure the residual apicidin. All steps were performed in a hydrogen atmosphere. As a control, the main culture was inoculated with heat-inactivated microorganisms. Heat inactivation was achieved by incubation at 95°C for 15 minutes.

[0034] After incubation, samples were analyzed by LC-MS / MS. For this purpose, 100 μL of sample taken from the main culture was mixed with 100 μL of acetonitrile, followed by centrifugation at 16,600×g for 10 min and further dilution 1:6 with absolute methanol.

[0035] The samples were chromatographically separated on a Zorbax Eclipse Plus C18 RRHD 50×2.1 mm, 1.8 μm column using an Agilent Infinity II 1290 chromatography system. The eluents were A: 5% methanol, 94.9% water, 0.1% formic acid, 1 mM ammonium formate; and B: 99.9% methanol, 0.1% xanthan, 1 mM ammonium formate. The gradient started with 70% B and increased to 100% in 0.7 min. It was then kept constant until 0.9 min. The eluent composition was returned to 70% at 0.91 min. The elution time of apicidin was 0.3 min. The mass spectrometer TripleQuad 5500 from Sciex was equipped with an electrospray ionization source. It was used in negative mode. The ion spray voltage was −4500 V. The ion source temperature was set at 500° C. The curtain gas setting was 40. The mass spectrometer was used in multiple reaction monitoring mode. The quantifier transition was 622 / 462 (collision energy −30 V). The quantifier transition was 622 / 252 (collision energy −46 V). The dwell time was 150 ms.

[0036] In FIG. 1, microorganisms (1.7*10 7 The x-axis shows the reduction of apicidin in the composition by cfu / mL starting concentration. The x-axis shows the time course of incubation. The y-axis shows the concentration of apicidin throughout the incubation, with the initial apicidin concentration set to 100%. The grey line shows the apicidin concentration of the control, the black solid line shows the apicidin concentration of the R. spec. nov. strain, the black dotted line shows the apicidin concentration of the R. timonensis strain, the black dashed line shows the apicidin concentration of the R. massiliensis strain, and the black dashed-dotted line shows the apicidin concentration of the R. phocaeensis strain. Error bars indicate a 15% error to represent unavoidable imprecision during sampling and / or analysis.

[0037] Upon incubation with heat-inactivated Raoultibacter species, a certain drop in apicidin concentration was observed. Without wishing to be bound by theory, the inventors hypothesize that a certain amount of apicidin adsorbs reversibly and / or irreversibly to the experimental tube walls, and thus may explain the drop in apicidin concentration in the control batches. Notably, all tested species effectively and efficiently removed apicidin within approximately 4 days.

[0038] Example 2: Sequence Comparison

[0039]

[0038] In order to compare the microbial strains of the present invention with each other, 16S rDNA sequence comparison was performed. For this purpose, EMBL-EBI's Clustal Omega nucleotide sequence alignment (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ; Sievers et al. 2011 Mol. Syst. Biol. 7:539) was used with default settings. However, any suitable sequence alignment algorithm can be selected by those skilled in the art.

[0040]

[0039] Table 1 below shows the identity of the 16S rDNA sequences of seven exemplary microorganisms according to the invention, where SEQ ID NO:1 is from R. species nova DSM 33979, SEQ ID NO:2 is from R. massiliensis DSM 33980, SEQ ID NO:3 is from R. timonensis DSM 33991, SEQ ID NO:4 is from R. phocaeensis DSM 33992, SEQ ID NO:5 is from R. timonensis strain GAMB-D35-65 (GenBank ID: MT902961.1), SEQ ID NO:6 is from Raoultibacter species strain zg-QD-978 (GenBank ID: SEQ ID NO:7 is the 16S rDNA sequence from Raoultibacter massiliensis strain ban4-RY-D5-23 (GenBank ID: MT902960.1).

[0041] [Table 1]

[0042] Example 3: Feed additive

[0043] To further investigate the ability of the microorganisms according to the invention to reduce the apicidin concentration in complex matrices such as feed, the following experimental steps were carried out: Precultures of one of the following microorganisms were prepared as described in Example 1: Raoultibacter species nova (R. spec. nov.; DSM 33979), Raoultibacter massiliensis (R. massiliensis; DSM 33980), Raoultibacter timonensis (R. timonensis; DSM 33991), Raoultibacter phocaeensis (R. phocaeensis; DSM 33992). 125 mg of sterile typical pig feed (e.g., Masching et al. 2016. Toxins 8:(3):84; Schwartz-Zimmermann et al. 2018. World Mycotoxin Journal, DOI 10.3920 / WMJ2017.2265), which is 1.7*10 per gram of feed. 4 cfu or 1.7*10 per gram of feed 7 The main culture was prepared by inoculating swine feed containing a target concentration of 83 ppb apicidin with an appropriate amount of preculture as an apicidin-reducing additive to achieve any microbial concentration of cfu. The main culture was further incubated at 37°C. Throughout the incubation period, samples were periodically taken and analyzed as described in Example 1. Similar results could be obtained when incubated at ambient temperature, i.e., 15°C to 35°C.

[0044]

[0042] When using the microorganism as an additive to the feed, similar to the setup described in Example 1, it was possible to reduce the apicidin concentration throughout the incubation. In fact, the apicidin reduction was achieved immediately upon addition of the microorganism to the feed and could be further advanced upon longer incubation. Notably, the apicidin concentration was already below 20 ppb in all batches after 24 h of incubation. Table 2 below shows that the apicidin concentration was 1.7*10 ppb per g of feed. 7 1 shows the removal of apicidin by Raoultibacter species applied at a concentration of cfu.

[0045] [Table 2]

[0046] Similar results can be obtained when using even lower concentrations of the microorganism according to the invention. 4 The removal of apicidin from feed using Raoultibacter spp. only cfu (colony forming density) is shown in Figure 2, where the x-axis shows the time course of incubation. The y-axis shows the concentration of apicidin throughout the incubation, with the initial apicidin concentration set to 100%. The solid black line shows the apicidin concentration in the composition treated with the R. spec. nov. strain, the dotted black line in the composition treated with the R. timonensis strain, the dashed black line in the composition treated with the R. massiliensis strain, and the dashed black line in the composition treated with the R. phocaeensis strain. Notably, after only one day of incubation, the apicidin concentration was already reduced by more than 50% in the feed.

[0047]

[0044] 1.7*10 4It is clear to those skilled in the art that a microorganism concentration lower than cfu / g feed may also be suitable for reducing apicidin concentration according to the present invention. When using very low concentrations, those skilled in the art may consider extending the incubation time to remove apicidin. However, in theory, even a single living cell may be able to remove apicidin from the composition if given enough time. When fed with the composition treated as above, animals (e.g., pets, pigs, poultry, ruminants, etc.) show less effect of apicidin poisoning than when fed with the untreated composition.

Claims

**Claim 1** A method for reducing the apicidin concentration in a nutritional composition containing apicidin, comprising: a) providing said nutritional composition containing apicidin; b) providing at least one microorganism, wherein said at least one microorganism comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 7, preferably a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 4, more preferably a nucleotide sequence of SEQ ID NO: 1 and having at least 95%, preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identity with the nucleotide sequence of SEQ ID NO: 1 and containing 16S rDNA; preferably, said at least one microorganism belongs to the genus Raoultibacter; c) forming a mixture containing water, said nutritional composition of a) and said at least one microorganism of b); d) incubating said mixture of step c). A method comprising the above steps. **Claim 2** The method according to claim 1, wherein step d) of incubating said mixture of step c) is carried out for at least 5 minutes, preferably at least 1 hour, more preferably at least 2 hours, even more preferably at least 1 day, most preferably at least 2 days and at a temperature of at least 10°C, preferably at least 15°C, more preferably at least 20°C. **Claim 3** The method according to claim 1 or 2, wherein said at least one microorganism is selected from the group consisting of Raoultibacter species nova, Raoultibacter massiliensis, Raoultibacter timonensis and Raoultibacter phocaensis. **Claim 4** The method according to claim 1 or 2, wherein said at least one microorganism is selected from the group of strains consisting of DSM 33979, DSM 33980, DSM 33991 and DSM 33992. **Claim 5** The nutritional composition of (a) is selected from the group consisting of food products; feeds; baits; food additives; feed additives; bait additives; wet distillers' grains; dry distillers' grains with solubles; dietary supplements; prebiotics; probiotics; their intermediates; and / or mixtures thereof, according to the method of claim 1 or 2.

6. Food products; feeds; baits; food additives; feed additives; bait additives; wet distillers' grains; dry distillers' grains with solubles; dietary supplements ； A method for treating prebiotics; probiotics; their intermediates; and / or mixtures thereof, wherein the food products; feeds; baits; food additives; feed additives; bait additives; wet distillers' grains; dry distillers' grains with solubles; dietary supplements; Prebiotics; probiotics; their intermediates; and / or mixtures thereof contain apicidin, and the method comprises a) providing at least one microorganism, wherein the at least one microorganism comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 7, preferably a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 4, more preferably the nucleotide sequence of SEQ ID NO: 1 and having at least 95%, preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identity with the 16S rDNA; preferably, the at least one microorganism belongs to the genus Raoultibacter, and; b) applying the at least one microorganism of (a) to the food products; feeds; baits; food additives; feed additives; bait additives; wet distillers' grains; dry distillers' grains with solubles; dietary supplements; Prebiotics; probiotics; their intermediates; and / or mixtures thereof to form a treated mixture; c) incubating the treated mixture of step b) A method comprising.

7. The step c) of incubating the treated mixture of step b) is carried out for at least 5 minutes, preferably at least 1 hour, more preferably at least 2 hours, even more preferably at least 1 day, most preferably at least 2 days and at a temperature of at least 10°C, preferably at least 15°C, more preferably at least 20°C, according to the method of claim 6.

8. The at least one microorganism is selected from the group consisting of Raoultibacter species nova, Raoultibacter massiliensis, Raoultibacter timonensis, and Raoultibacter phocaensis; preferably, the at least one microorganism is selected from the group of strains consisting of DSM 33979, DSM 33980, DSM 33991, and DSM 33992, the method according to claim 7.

9. The at least one microorganism is at least 10 per mL of the liquid composition ４ cfu or a solid composition such as food products; feeds; baits; additives such as food additives, feed additives or bait additives; wet distillers' grains; dried distillers' grains with solubles; dietary supplements ； At least 10 per gram of prebiotic; probiotic; their intermediates; and / or their mixtures ４ cfu concentration; preferably at least 1.7 * 10 per mL of liquid composition ４ cfu or solid compositions such as food products; feeds; baits; additives such as food additives, feed additives or bait additives; wet distillers grains; dry distillers grains with soluble components; nutritional supplements The method according to claim 6, 7 or 8, applied in an amount to achieve a concentration of at least 1.7 * 10 ４ cfu per gram of prebiotic; probiotic; intermediate thereof; and / or mixture thereof.

10. Food products; feeds; baits; additives (e.g., food product, feed or bait additives); wet distillers' grains; dry distillers' grains with solubles; dietary supplements; prebiotics; probiotics; their intermediates; and / or mixtures thereof that can be produced or are produced by the method according to claim 6.

11. In the use of at least one microorganism for reducing the apicidin concentration in a composition containing apicidin, the at least one microorganism comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 7, preferably a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 4, more preferably having at least 95%, preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identity with the nucleotide sequence of SEQ ID NO: 1; preferably, the at least one microorganism is characterized by belonging to the genus Raoultibacter.

12. The at least one microorganism is selected from the group consisting of Raoultibacter species nova, Raoultibacter massiliensis, Raoultibacter timonensis, and Raoultibacter phocaensis, and preferably, the at least one microorganism is selected from the group of strains consisting of DSM 33979, DSM 33980, DSM 33991, and DSM 33992, the use according to claim 11.

13. In a microorganism for use in the treatment, amelioration, prevention and / or diagnosis of symptoms caused by mycotoxicosis, particularly apicidin intoxication, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 7, preferably a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 4, more preferably having at least 95%, preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identity with the nucleotide sequence of SEQ ID NO: 1, and containing 16S rDNA; preferably belonging to the genus Raoultibacter.

14. The microorganism according to claim 13, selected from the group consisting of Raoultibacter species nova, Raoultibacter massiliensis, Raoultibacter timonensis, and Raoultibacter phocaensis, and preferably selected from the group of strains consisting of DSM 33979, DSM 33980, DSM 33991, and DSM 33992.

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