WO2023099500A1

WO2023099500A1 - Means and methods to reduce apicidin concentration in a composition

Info

Publication number: WO2023099500A1
Application number: PCT/EP2022/083729
Authority: WO
Inventors: Florian Krainer; Silvia LABUDOVA; Barbara NOVAK; Valentina RAINER; Dian SCHATZMAYR
Original assignee: DSM Austria GmbH
Priority date: 2021-12-02
Filing date: 2022-11-29
Publication date: 2023-06-08

Abstract

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

Description

MEANS AND METHODS TO REDUCE APICIDIN CONCENTRATION IN A COMPOSITION

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

[0002] Apicidin (CAS number: 183506-66-3; also referred to e.g. as (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)-tetrone) is a highly toxic metabolite produced by Fusarium fungi, e.g. F. semitectum. Apicidin is a cyclic tetrapeptide having the molecular structure shown below, and described to inhibit histone deacetylase.

[0003] Among 28 other tested fungal metabolites, apicidin was found to have the lowest IC50 value, i.e. to show the strongest cytotoxic activity (Novak et al. 2019. Toxins 11 : 537). The toxic effects of apicidin were also shown in rat feeding trials, in brine shrimp as well as 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 commodities such as e.g. crops. Notably, apicidin can co-occur with other toxic substances such as deoxynivalenol. Particularly 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 which would not have affected the cells without the co-presence of apicidin (Springier et al. 2016. Toxins 8:345).

[0004] Despite these unfavorable effects and in contrast to other toxic fungal metabolites such as fumonisins, deoxynivalenol or zearalenone, no measures to counteract the toxic effect of apicidin have been found so far. [0005] In view of the prior art as outlined above, it is therefore an objective of the present invention to provide means and measures to reduce the concentration of apicidin in a nutritional composition.

[0006] This objective was found to be achieved by providing a method for reducing apicidin concentration in a nutritional composition comprising apicidin, the method comprising the steps of a) providing the 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 a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7, preferably to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-4, more preferably to the nucleotide sequence of SEQ ID NO: 1 ; and preferably wherein 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). Hereby, the nutritional composition can be altered 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 invention relates to a method for reducing apicidin concentration in a nutritional composition comprising apicidin, the method comprising the steps of a) providing the nutritional composition comprising apicidin; b) providing at least one microorganism, wherein 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).

[0007] 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.

[0008] The term "16S rDNA” refers to the ribosomal desoxyribonucleic acid (rDNA) encoding the 16S ribonucleic acid component of the 30S subunit of the prokaryotic ribosome. The degree of similarity or relatedness of two or more nucleic acid sequences (e.g. DNA or RNA) 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 or more 16S rDNA 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 Protein Sequences”) using default settings (Match/Mismatch Scores: 2,-3; Gap Costs: Existence: 5 Extension 2).

[0009] Chemical reactions, including reactions catalyzed by biological systems such as enzymes or microorganisms, may occur faster or slower depending on environmental conditions. To perform the method of the present application in a preferred manner, it is therefore considered to perform step d) of the method described above for at least five minutes, preferably for at least one hour, more preferably for at least two hours, even more preferably for at least one day, most preferably for at least two days. Additionally it is preferred to perform step d) of the method described above at a temperature of at least 10 °C, preferably of at least 15 °C, more preferably of at least 20 °C. It is further preferred to perform said incubation at a temperature of at most 50 °C, preferably of at most 40 °C. In one embodiment, the method of the present application is performed at common ambient temperature (i.e. 15-35 °C, preferably 18-30 °C, more preferably 18-25 °C). When performing the method described herein under said conditions, a particularly efficient reduction of the apicidin concentration or content in a composition may be achieved. The longer the incubation of step d) is performed, the more apicidin will be removed from the nutritional composition. Notably, the microorganism can be found capable of removing apicidin 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 apicidin as referred to herein. The skilled person is well capable of choosing incubation conditions appropriate to reduce an apicidin concentration present in a composition to a desired degree.

[0010] It was surprisingly found by the inventors that in particular microorganisms selected from the group consisting of the species Raoultibacter species nova, Raoultibacter massiliensis, Raoultibacter timonensis, and Raoultibacter phocaeensis are efficient microbial catalysts to achieve a reduction of apicidin concentration in a nutritional composition. Thus, in one embodiment, the invention relates to the 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. Outstanding and reliable reduction of apicidin in a composition was found to be achievable when a microbial strain selected from DSM 33979, DSM 33980, DSM 33991 and DSM 33992 was used.

[0011] The composition can consist of, or comprise one or more solid component(s) and/or one or more liquid component(s). For instance, the nutritional composition (as referred to in step a) of the method described above) can be selected from the group consisting of foodstuff; fodder; feed; foodstuff additives; fodder additives; feed additives; wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof. Such compositions are part of the feed/food chain and may thus benefit substantially from a reduction or even removal of apicidin. Reduced or even removed apicidin in such compositions may therefore contribute 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.

[0012] Alternatively, it is considered that a composition selected from foodstuff; fodder; feed; additives (e.g. foodstuff-, fodder- or feed additives); wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof may be required, which composition has a reduced apicidin concentration after a suitable treatment compared to the apicidin concentration prior to said treatment. This objective has been achieved by providing a method for processing a foodstuff; fodder; feed; foodstuff additives; fodder additives; feed additives; wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof, wherein the foodstuff; fodder; feed; foodstuff additives; fodder additives; feed additives; wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof comprise(s) apicidin, the method comprising the steps of a) 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 a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7, preferably to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-4, more preferably to the nucleotide sequence of SEQ ID NO: 1; and preferably wherein the at least one microorganism belongs to the genus Raoultibacter, b) applying the at least one microorganism of a) to the foodstuff; fodder; feed; foodstuff additives; fodder additives; feed additives; wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof, thus forming a processing mixture; and c) incubating the processing mixture of step b). Prior to the method for processing described above, the foodstuff; fodder; feed; foodstuff additives; fodder additives; feed additives; wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof comprises apicidin (e.g. at a concentration higher than 30 ppb, or even higher than 40 ppb, 50 ppb, 60 ppb, 70 ppb, 80 ppb, 90 ppb, 100 ppb, or even higher). However, the foodstuff; fodder; feed; foodstuff additives; fodder additives; feed additives; wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof obtained by such a method contains less or even no apicidin any more and may thus conveniently be used for the intended purpose without any further measures necessary to counteract apicidin-induced adverse effects. In the event that the microorganism of a), or the foodstuff; fodder; feed; foodstuff additives; fodder additives; feed additives; wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof comprises no water (e.g. in the form of moisture), water may be added at step c), in particular prior to step c).

[0013] In one embodiment, the invention relates to a method for processing a foodstuff; fodder; feed; foodstuff additives; fodder additives; feed additives; wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof, the method 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 the foodstuff; fodder; feed; foodstuff additives; fodder additives; feed additives; wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof, thus forming a processing mixture; and c) incubating the processing mixture of step b). Preferably, the method for processing a foodstuff; fodder; feed; additives (e.g. foodstuff-, fodder- or feed additives); wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof is performed, wherein step c) incubating the processing mixture of step b) is performed for at least five minutes, preferably for at least one hour, more preferably for at least two hours, even more preferably for at least one day, most preferably for at least two days. Preferably, the method is additionally performed at a temperature of at least 10 °C, preferably of at least 15 °C, more preferably of at least 20 °C. It is further preferred to perform said incubation at a temperature of at most 50 °C, preferably of at most 40 °C. For example, the method of the present application is performed at common ambient temperature (i.e. 15-35 °C, preferably 18-30 °C, more preferably 18-25 °C).

[0014] In a preferred embodiment of the method for processing a foodstuff; fodder; feed; foodstuff additives; fodder additives; feed additives; wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof is performed in a way, wherein 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.

[0015] It is preferred to perform any method described herein in a manner, wherein the at least one microorganism is applied in such an amount to achieve a concentration of at least 10⁴ cfu per mL of liquid composition or at least 10⁴ cfu per g of solid composition (e.g. foodstuff; fodder; feed; additives (e.g. foodstuff-, fodder- or feed additives); wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof), preferably at a concentration of at least 1.7*10⁴ cfu per mL of liquid composition or at least 1.7*10⁴ cfu per g of solid composition (e.g. foodstuff; fodder; feed; additives (e.g. foodstuff-, fodder- or feed additives); wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof). In other words, the final concentration, i.e. the working concentration, of the at least one microorganism in a composition is preferably at least 10⁴ cfu per mL or per g of composition (e.g. foodstuff; fodder; feed; additives (e.g. foodstuff-, fodder- or feed additives); wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof), more preferably at a concentration of at least 1 .7*10⁴ cfu per mL or per g of composition (e.g. foodstuff; fodder; feed; additives (e.g. foodstuff-, fodder- or feed additives); wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof). [0016] 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, apicidin reduction or removal may be achieved within a most convenient time frame.

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

[0018] Another aspect of the present invention relates to a foodstuff; fodder; feed; additives (e.g. foodstuff-, fodder- or feed additives); wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof producible or produced by a method as described herein. Foodstuff; fodder; feed; additives (e.g. foodstuff-, fodder- or feed additives); wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof producible or produced by a method as described herein comprise(s) no or at least lower amounts of apicidin compared to foodstuff; fodder; feed; additives (e.g. foodstuff-, fodder- or feed additives); wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; 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; additives (e.g. foodstuff-, fodder- or feed additives); wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof according to the present invention poses a lower threat to cause apicidin-induced toxic effects in a consumer of such foodstuff; fodder; feed; additives (e.g. foodstuff-, fodder- or feed additives); wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof. It is considered that a foodstuff; fodder; feed; additives (e.g. foodstuff-, fodder- or feed additives); wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof producible or produced by a method as described herein comprises 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% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7, preferably to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-4, more preferably to the nucleotide sequence of SEQ ID NO: 1; and preferably wherein the at least one microorganism belongs to the genus Raoultibacter. Thus, such foodstuff; fodder; feed; additives (e.g. foodstuff-, fodder- or feed additives); wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof is of lower risk of further contamination with apicidin by eventual fungal growth compared to foodstuff; fodder; feed; additives (e.g. foodstuff-, fodder- or feed additives); wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof which does not comprise at least one microorganism referred to herein. Preferably, the foodstuff; fodder; feed; additives (e.g. foodstuff-, fodder- or feed additives); wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof producible or produced by a method as described herein comprises the microorganism according to the invention at a concentration of at least 10⁴ cfu per ml_ of liquid composition or at least 10⁴ cfu per g of solid composition, more preferably at least 1.7*10⁴ cfu per mL of liquid composition or at least 1.7*10⁴ cfu per g of solid composition.

[0019] In an embodiment such a foodstuff; fodder; feed; additives (e.g. foodstuff-, fodder- or feed additives); wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof producible or produced by a method as described herein comprises less than 30 ppb of apicidin (e.g. 25 ppb, or 15 ppb, or between 30 and 20 ppb, or between 30 and 10 ppb, or between 30 and 5 ppb of apicidin), preferably less than 20 ppb of apicidin (e.g. 15 ppb, or between 20 and 5 ppb of apicidin), more preferably less than 10 ppb of apicidin (e.g. between 10 and 5 ppb of apicidin, or 5 ppb, 4 ppb, 3 ppb, 2 ppb etc. of apicidin).

[0020] In another aspect, the present invention relates to a use (e.g. non-medical use) of at least one microorganism for reducing apicidin concentration in a composition (e.g. nutritional composition) comprising apicidin, wherein the at least one microorganism comprises a 16S rDNA having 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 a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7, preferably to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-4, more preferably to the nucleotide sequence of SEQ ID NO: 1; and preferably wherein the at least one microorganism belongs to the genus Raoultibacter. Unexpectedly, such a microorganism was found to be capable of reducing the concentration of apicidin in a composition, such as foodstuff; fodder; feed; additives (e.g. foodstuff-, fodder- or feed additives); wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof. Hereby, consumers of such a composition having reduced or even no apicidin are less or not exposed to the toxic effects of apicidin. In particular, animals (e.g. poultry, swine, ruminants etc.) not suffering from mycotoxicoses, such as apicidin toxicosis, can be found to show improved performance (e.g. increased body mass gain and/or reduced feed conversion ratio). The microorganism of the invention may thus be used to improve animal performance when said animal is fed a nutritional composition comprising apicidin. In one embodiment, the invention relates to a use (e.g. non-medical use) of at least one microorganism for reducing apicidin concentration in a composition (e.g. nutritional composition) comprising apicidin, wherein the at least one microorganism belongs to the genus Raoultibacter.

[0021] Preferably, the use of the at least one microorganism for reducing apicidin concentration in a composition as described herein is performed in a way, wherein 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.

[0022] In a further aspect, the present invention relates to a microorganism and may be used as a medicament (e.g. in veterinary medicine) and/or in treatment, amelioration, prophylaxis and/or diagnostics of a disease, wherein the 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 a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7, preferably to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-4, more preferably to the nucleotide sequence of SEQ ID

NO: 1 ; and preferably wherein the microorganism belongs to the genus Raoultibacter. In one embodiment, 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 microorganism belongs to the genus Raoultibacter.

[0023] Due to the surprising finding that the microorganism of the present invention is capable of removing apicidin, its use as a medicament, in treatment, amelioration, prophylaxis or diagnostics of a disease is highly favorable. It is therefore a further aspect of the present invention, to provide a microorganism for use in treatment, amelioration, prophylaxis and/or diagnostics of symptoms caused by mycotoxicosis, in particular by apicidin toxicosis, wherein the 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 a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7, preferably to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1- 4, more preferably to the nucleotide sequence of SEQ ID NO: 1 ; and preferably wherein the microorganism belongs to the genus Raoultibacter. In one embodiment, the invention relates to a microorganism for use in treatment, amelioration, prophylaxis and/or diagnostics of symptoms caused by mycotoxicosis, in particular by apicidin toxicosis, wherein the microorganism belongs to the genus Raoultibacter.

[0024] 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.

[0025] A composition comprising 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/lyophilyzed/live yeast, e.g. Saccharomyces species, e.g. S. cerevisiae, Pichia pastorisy, and/or one or more inorganic absorbent component(s) (e.g. clay product(s), bentonite, zeolite, bentonitemontmorillonite, diatomaceaous 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).

[0026] In a preferred embodiment, the at least one microorganism for use as a medicament (e.g. in veterinary medicine) and/or in treatment, amelioration, prophylaxis and/or diagnostics of a disease, and/or in particular for use in treatment, amelioration, prophylaxis and/or diagnostics of symptoms caused by mycotoxicosis (e.g. by apicidin toxicosis), as referred to herein 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, microorganisms according to these preferred embodiments were found to be particularly suitable to serve the purpose of a use as medicament, in particular in treatment, amelioration, prophylaxis and/or diagnostics of symptoms caused by mycotoxicosis, in particular by apicidin toxicosis. [0027] Preferably, the microorganism of the present invention for use as a medicament (e.g. in veterinary medicine) and/or in treatment, amelioration, prophylaxis and/or diagnostics of a disease; and/or the composition of the present invention for use in treatment, amelioration, prophylaxis and/or diagnostics of symptoms caused by mycotoxicosis (e.g. by apicidin toxicosis), is applied in a way, wherein the microorganism comprised in such a composition is applied to achieve a concentration of at least 10⁴ cfu per mL of liquid composition or at least 10⁴ cfu per g of solid, preferably at a concentration of at least 1.7*10⁴ cfu per mL of liquid composition or at least 1.7*10⁴ cfu per g of solid composition. In other words, the final concentration, i.e. the working concentration, of the at least one microorganism according to the invention in such a composition, is preferably at least 10⁴ cfu per mL or per g of composition, more preferably at a concentration of at least 1.7*10⁴ cfu per mL or per g of composition.

[0028] In the following, the present invention is further described by non-limiting figures and examples, wherein Figure 1 shows the reduction of apicidin concentration by microbial strains according to the present invention in an experimental setup; and wherein Figure 2 shows the reduction of apicidin concentration in feed by an additive according to the present invention.

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

Examples

[0030] 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. Examples disclosed in the following may only be considered to refer to exemplary embodiments of the underlying invention as defined in the claims.

[0031] Example 1 : Apicidin reduction

[0032] To experimentally demonstrate the reduction of apicidin concentration by 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. Pre-cultures of either of the following microorganisms were prepared: 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). To this end, a viable sample from either of the bacterial species was used to inoculate sterile Wilkins-Chalgren media (10 g/L tryptone, 10 g/L gelatin peptone, 5 g/L yeast extract, 1 g/L glucose, 5 g/L NaCI, 1 g/L L-arginine, 1 g/L sodium pyruvate, 0.0005 g/L menadione, 0.005 g/l haemin, pH 7 at 25 °C; Wilkins and Chalgren. 1976. Antimicrob. Agents Chemother. 10: 926- 928) further comprising 0.5 mL/L of a 0.1% (w/v) sodium resazurin solution, 0.3 g/L of L- cysteine hydrochloride monohydrate, and 10-20 mg/L of sodium dithionite in air-tight sealable laboratory glass bottles or anaerobic culture tubes (Hungate type) in hydrogen atmosphere. In detail, the media broth was prepared by dissolving the components of the Wilkins-Chalgren medium in distilled water, followed by addition of the sodium resazurin. This solution was boiled, then cooled to room temperature while sparging with nitrogen. Then, the L-cysteine hydrochloride monohydrate and sodium dithionite were aseptically added. These pre-cultures were incubated at 37 °C for three days. An aliquot of a pre-culture was used to inoculate a main culture in the media as described above to a microbial concentration of either 1.7 * 10⁴ cfu/mL or to 1.7 * 10⁸ cfu/mL. A microbial concentration in cfu/mL can be determined by any method suitable method known by a person having skill in the art, e.g. by plate counting (i.e. plating of culture aliquots on nutrient agar plates and counting of formed colonies after incubation), or by flow cytometry. Finally, apicidin was added to the main culture to a target concentration of 83 ppb. Thus, prepared main cultures were incubated at 37 °C. Samples were drawn regularly throughout incubation to determine residual apicidin. All steps were performed in hydrogen atmosphere. As a control, a main culture was inoculated with heat-inactivated microorganisms. Heat inactivation was achieved by incubation at 95 °C for 15. min.

[0033] After incubation, the samples were analyzed by LC-MS/MS. To this end, 100 pL of sample drawn from the main culture was mixed with 100 pL of acetonitrile, followed by centrifugation at 16,600 xg, 10 min, and further 1:6 dilution with absolute methanol.

[0034] Samples were chromatographically separated on a Zorbax Eclipse Plus C18 RRHD 50x2.1 mm, 1.8 pm column using Infinity II 1290 chromatography system from Agilent. The eluents were A: 5% methanol, 94.9% water, 0.1% formic acid, 1 mM ammonium formate; and B: 99.9% methanol, 0.1% formic acid, 1 mM ammonium formate. The gradient started at 70% of B and was increased to 100% in 0.7 min. Then, it was kept constant until 0.9 min. At 0.91 min eluent composition returned to 70%. The elution time of apicidin was 0.3 min. Mass spectrometer TripleQuad 5500 from Sciex was equiped with electrospray ionisation ion source. It was used in the negative mode. lonSpray voltage was -4500 V. Temperature of ion source was set to 500 °C. Curtain gas setting was 40. Mass spectrometer was used in multiple reaction monitorin mode. The quantifier transition was 622/462 (collision energy -30 V). The qualifier transition was 622/252 (collision energy -46 V). Dwell time was 150 ms. [0035] In Figure 1 , the reduction of apicidin in the composition by the microorganisms (1.7 * 10⁷ cfu/mL starting concentration) is shown. The x-axis shows the time course of the incubation. The y-axis shows the concentration of apicidin throughout the incubation, wherein the initial apicidin concentration was set to 100%. The grey line shows the apicidin concentrations of the control, the solid black line of the R. spec. nov. strain, the dotted black line of the R. timonensis strain, the dashed black line of the R. massiliensis strain, and the dash-dotted black line of the R. phocaeensis strain. The error bars indicate an error of 15% to represent inaccuracies that could not have been avoided during sampling and/or analyses.

[0036] Upon incubation with heat-inactivated Raoultibacter species, a certain decline of the apicidin concentration was observed. Without wishing to be bound by theory, the inventors hypothesize that a certain amount of apicidin might adsorb reversibly and/or irreversibly to the experimental tube wall, thus explaining the decline of the apicidin concentration in the control batch. Notably, all tested species effectively and efficiently removed apicidin within approximately four days.

[0037] Example 2: Sequence comparison

[0038] To compare microorganism strains of the present invention to one another, 16S rDNA sequence comparison was performed. To this end, the Clustal Omega nucleotide sequence alignment of EMBL-EBI (https://www.ebi.ac.uk/Tools/msa/clustalo/; Sievers et al. 2011. Mol. Syst. Biol. 7: 539) was used with default seetings. However, any suitable sequence alignment algorithm may be chosen by a person having skill in the art.

[0039] Table 1 below shows the identities of the 16S rDNA sequences of seven exemplary microorganisms according to the present invention, wherein SEQ ID NO: 1 is the 16S rDNA sequence from R. species nova DSM 33979, SEQ ID NO: 2 from R. massiliensis DSM 33980, SEQ ID NO: 3 from R. timonensis DSM 33991 , SEQ ID NO: 4 from R. phocaeensis DSM 33992, SEQ ID NO: 5 from R. timonensis strain GAMB-D35-65 (GenBank ID: MT902961.1), SEQ ID NO: 6 from Raoultibacter sp. strain zg-QD-978 (GenBank ID: MN938173.1), and SEQ ID NO: 7 from Raoultibacter massiliensis strain ban4-RY-D5-23 (GenBank ID: MT902960.1).

Table 1 : Identities of 16S rDNA sequences of SEQ ID NOs 1-7 to one another.

[0040] Example 3: Feed additive

[0041] To further study the ability of the microorganisms according to the present invention to reduce apicidin concentration in complex matrices such as feed, the following experimental steps were performed. Pre-cultures of either 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). Main cultures were prepared by inoculating 125 mg of a 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) comprising a target concentration of 83 ppb of apicidin with the appropriate amount of preculture as an apicidin-reducing additive to achieve either a microorganism concentration of 1.7 * 10⁴ cfu per g feed or of 1.7 * 10⁷ cfu per g feed. The main cultures were further incubated at 37 °C. Samples were taken regularly throughout the incubation period and analyzed as described in Example 1. When incubated at ambient temperatures, i.e. between approximately 15 °C and 35 °C, comparable results can be obtained.

[0042] Analogous to the setup described in Example 1 , the apicidin concentration could be reduced throughout the incubation when using the microorganisms as additives to feed. In fact, apicidin reduction could be achieved immediately upon addition of the microorganisms to feed, and progressed further upon longer incubation. Notably, apicidin concentration was already less than 20 ppb in all batches after 24 h of incubation. Table 2 below shows the removal of apicidin by Raoultibacter species when applied at a concentration of 1.7 * 10⁷ cfu per g of feed.

Table 2: Apicidin concentrations in feed treated with a microorganism according to the present invention over time.

[0043] Analogous results can be obtained when using even lower concentrations of microorganisms according to the present invention. In this regard, removal of apicidin from feed using only 1.7 * 10⁴ cfu (colony forming unit) of Raoultibacter species per g of feed is shown in Figure 2. Therein, the x-axis shows the time course of the incubation. The y-axis shows the concentration of apicidin throughout the incubation, wherein the initial apicidin concentration was set to 100%. The solid black line shows the apicidin concentrations 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 dash-dotted black line in the composition treated with the R. phocaeensis strain. Notably, after only one day of incubation, apicidin concentration was already reduced in the feed by more than 50%.

[0044] It is clear to a person having skill in the art, that also microbial concentrations lower than 1.7 * 10⁴ cfu/g feed will be suitable to reduce apicidin concentration according to the present invention. When using very low concentrations, a skilled person may consider prolonging the incubation time to remove apicidin. In theory however, even a single live cell will be capable of removing apicidin from a composition when given sufficient time. When fed a composition treated as described above, animals (e.g. pets, pigs, poultry, ruminants etc.) show fewer effects of apicidin intoxication than when fed a non-treated composition.

Claims

1. Method for reducing apicidin concentration in a nutritional composition comprising apicidin, the method comprising the steps of a) providing the 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 a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7, preferably to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-4, more preferably to the nucleotide sequence of SEQ ID NO: 1; and preferably wherein 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).

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

3. The method according to claim 1 or 2, wherein the at least one microorganism is selected from the group consisting of Raoultibacter species nova, Raoultibacter massiliensis, Raoultibacter timonensis, and Raoultibacter phocaeensis.

4. The method according to any one of the preceding claims, wherein the at least one microorganism is selected from the group of strains consisting of DSM 33979, DSM 33980, DSM 33991 and DSM 33992.

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

6. Method for processing a foodstuff; fodder; feed; foodstuff additives; fodder additives; feed additives; wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof, wherein the foodstuff; fodder; feed; foodstuff additives; fodder additives; feed additives; wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof, and/or mixture/s thereof coprise(s) apicidin, the method comprising the steps of a) 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 a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7, preferably to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-4, more preferably to the nucleotide sequence of SEQ ID NO: 1 ; and preferably wherein the at least one microorganism belongs to the genus Raoultibacter, b) applying the at least one microorganism of a) to the foodstuff; fodder; feed; foodstuff additives; fodder additives; feed additives; wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof, thus forming a processing mixture; and c) incubating the processing mixture of step b).

7. The method according to claim 6, wherein step c) incubating the processing mixture of step b) is performed for at least five minutes, preferably for at least one hour, more preferably for at least two hours, even more preferably for at least one day, most preferably for at least two days; and at a temperature of at least 10 °C, preferably of at least 15 °C, more preferably of at least 20 °C.

8. The method according to claims 6 or 7, wherein 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.

9. The method according to any one of the preceding claims, wherein the at least one microorganism is applied in such an amount to achieve a concentration of at least 10⁴ cfu per mL of liquid composition or at least 10⁴ cfu per g of solid composition, e.g. foodstuff; fodder; feed; additives such as foodstuff additives, fodder additives or feed additives; wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof; preferably at a concentration of at least 1.7*10⁴ cfu per ml_ of liquid composition or at least 1.7*10⁴ cfu per g of solid composition, e.g. foodstuff; fodder; feed; additives such as foodstuff additives, fodder additives or feed additives; wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof.

10. Foodstuff; fodder; feed; additives (e.g. foodstuff-, fodder- or feed additives); wet distillers grain; dried distillers grain with solubles; nutritional supplements; prebiotic; probiotic; intermediate/s thereof; and/or mixture/s thereof producible or produced by a method of any one of the preceding claims.

11. Use of at least one microorganism for reducing apicidin concentration in a composition comprising apicidin, characterized in that 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 a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7, preferably to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-4, more preferably to the nucleotide sequence of SEQ ID NO: 1; and preferably wherein the at least one microorganism belongs to the genus Raoultibacter.

12. The use according to claim 11 , wherein 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.

13. Microorganism for use in treatment, amelioration, prophylaxis and/or diagnostics of symptoms caused by mycotoxicosis, in particular by apicidin toxicosis, characterized in that the 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 a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7, preferably to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-4, more preferably to the nucleotide sequence of SEQ ID NO: 1 ; and preferably wherein the microorganism belongs to the genus Raoultibacter. The microorganism according to claim 13, wherein the 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.