Strain of Eubacterium that detoxyfies trichothenes

Details Strain of Eubacterium that detoxyfies trichothenes Strain of Eubacterium that detoxyfies trichothenes

2000-06-29

2004-09-21

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S252500, C435S034000, C435S244000

Reexamination Certificate

active

06794175

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a microorganism of the genus Eubacterium, which is suitable in pure culture or mixed culture for the detoxification of trichothecenes, and to a process for the isolation thereof, its production and formulation and its use and a feedstuff additive comprising the microorganism.
2. Description of the Prior Art
Trichothecenes which belong to the mycotoxins class are contained in numerous animal feedstuffs, where they are customarily introduced into the feedstuffs via mould fungi found on cereals or grasses. As a result of the undesired administration of mycotoxins, in particular trichothecenes, to animals, both their productivity and, for example, the growth of the animals is inhibited, an increased consumption of feedstuff together with a simultaneously poorer feedstuff utilization rate occurring in addition to damage to the health of the animals. To eliminate the adverse effects of mycotoxins, numerous processes for binding or adsorbing these toxins have already been disclosed.
Thus, in WO 91/13555, for example, a feedstuff additive and a process for the inactivation of mycotoxins is described, where particles of a phyllosilicate mineral are added to the feed in order to inactivate the mycotoxins. To increase the effect of these phyllosilicates, the particles are coated with a sequestering agent in order to accelerate the effect. A feedstuff is furthermore known, for example, from WO 92/05706 in which montmorillonite clay is contained as a feedstuff additive. These natural clay minerals having large internal surface areas should bind the mycotoxins to the surface on account of their porosity and immobilize them in this manner.
Furthermore, a feedstuff additive has been disclosed in the Austrian Utility Model AT-U 504 in which an enzyme preparation is used which is capable of forming epoxidases and lactonases and degrading mycotoxins chemically both in the feedstuff and in the gastro-intestinal tract of animals. According to AT-U 504, the action of this enzyme preparation can be increased by the addition of zeolites and the like.
SUMMARY OF THE INVENTION
The present invention now aims at making available a specific microorganism or a defined mixed culture isolated from a natural habitat, with which it is possible to convert mycotoxins, in particular trichothecenes, in a controlled manner into substances which are physiologically harmless and which are harmless, in particular in animal breeding, by biochemical degradation.
To solve this object, a microorganism of the genus Eubacterium was isolated which is suitable for the detoxification of trichothecenes in pure culture, DSM 11798, or mixed culture with the strain
Enterococcus casseliflavus
, DSM 11799, or other anaerobic microorganisms. According to a novel refinement of the invention, the microorganism is suitable for the detoxification of trichothecenes in mixed culture with other anaerobic microorganisms, in particular from the genus Enterococcus, Streptococcus, Lactococcus, Bacillus or Lactobacillus.
The microorganism of the genus Eubacterium, which is also called Eubacterium sp. on account of its association with the genus Eubacterium, and which was deposited in pure culture in the Collection of German Microorganisms under the number DSM 11798, or in mixed culture with the strain
Enterococcus casseliflavus
, which was deposited in the Collection of German Microorganisms under the number DSM 11799, is in particular suitable according to the invention for the detoxification of, in particular, deoxynivalenol (DON), T-2 toxin, HT-2 toxin, nivalenol, monoacetoxyscirpenol, diacetoxyscirpenol, trichodermol, verrucarin, rorodin, acetyldeoxynivalenol, isotrichodermin, hydroxyisotrichodermin, calonectrin, T-2 tetraol, T-2 triol, deacetylneosolaniol, neosolaniol; acetylneosolaniol, sporotrichiol, trichotriol, sambucinol and culmorin. The microorganism according to the invention detoxifies the trichothecenes by reductive biotransformation of the epoxide group contained in the molecule, which epoxide group is responsible for the toxicity of the mycotoxins, in particular trichothecenes. In the trichothecenes corresponding to the following formula, the degradation of the epoxide group is carried out by reductive cleavage of the toxic 12,13-epoxy ring:
DSM 11798 and DSM 11799 were deposited with DSMZ-DEUTSCHE SAMMLUNG VON MIKROOGANISMEN UND ZELLKULTUREN GmbH, Mascheroder Weg 1b, D-38124 Braumschweig, Germany, on Sep. 17, 1997.
The morphology of the microorganism according to the invention shows preferably that it is an anaerobic gram-positive, rod-like, non-spore-forming bacterium, in particular 0.1 to 3 &mgr;m long, which occurs both individually, in pairs or in long chains, in particular up to approximately 150 &mgr;m. Phylogenetic analysis of the microorganism according to the invention has in particular shown a 16S RNA sequence, namely
1
CCTGGCTCAG GATGAACGCT GGCGGCGTGC TTAACACATG CAAGTCGAAC GGATAACCCG

61
CCTCCGGGCG GTTATAGAGT GGCGAACGGG TGAGTAACAC GTGACCAACC TACCTCCCAC

121
TCCGGGATAA CCCAGGGAAA CCTGCGCTAA TACCGGATAC TCCGGGGCCC CCGCATGGGG

181
GCGCCGGGAA AGCCCCGACG GTGGGAGATG GGGTCGCGGC CTATTAGGTA GTCGGCGGGG

241
TAACGGCCCA CCGAGCCCGC GATAGGTAGC CGGGTTGAGA GACCGATCGG CCACATTGGG

301
ACTGAGATAC GGCCCAGACT CCTACGGGAG GCAGCAGTGG GGAATTTTGC GCAATGGGGG

361
AAACCCTGAC GCAGCAACGC CGCGTGCGGG ACGAAGGCCT TCGGGTTGTA AACCGCTTTC

421
AGCAGGGAAG AAGTTGACGG TACCTGCAGA AGAAGCTCCG GCTAACTACG TGCCAGCAGC

481
CGCGGTAATA CGTAGGGAGC GAGCGTTATC CGGATTTATT GGGCGTAAAG CGCGCGTAGG

541
CGGGCGCTTA AGCGGAATCT CTAATCTGAG GGCTCAACCC CCAGCCGGAT TCCGAACTGG

601
GCGCCTCGAG TTCGGTAGAG GAAGACGGAA TTCCCAGTGT AGCGGTGAAA TGCGCAGATA

661
TTGGGAAGAA CACCGATGGC GAAGGCAGTC TTCTGGGCCG TAACTGACGC TGAGGTGCGA

721
AAGCTAGGGG AGCGAACAGG ATTAGATACC CTGGTAGTCC TAGCCGTAAA CGATGGGCAC

781
TAGGTGTGGG GGGGAATGCC CCTCCGTGCC GCAGCTAACG CATTAAGTGC CCCGCCTGGG

841
GAGTACGGCC GCAAGGCTAA AACTCAAAGG AATTGACGGG GGCCCGCACA AGCAGCGGAG

901
CATGTGGCTT AATTCGAAGC AACGCGAAGA ACCTTACCAG GGCTTGACAT GCAGGTGAAG

961
CGGCGGAAAC GCCGTGGCCG AGAGGAGCCT GCACAGGTGG TGCATGGCTG TCGTCAGCTC

1021
GTGTCGTGAG ATGTTGGGTT AAGTCCCGCA ACGAGCGCAA CCCCTGTCGT ATGTTGCCAT

1081
CATTCAGTTG GGGACTCGTA CGAGACTGCC GGCGTCAAGC CGGAGGAAGG TGGGGACGAC

1141
GTCAAGTCAT CATGCCCTTT ATGCCCTGGG CTGCACACGT GCTACAATGG CCGGTACAAC

1201
GGGCTGCGAG CCAGCGATGG CGAGCGAATC CCTCAAAACC GGTCCCAGTT CGGATCGGAG

1261
GCTGCAACCC GCCTCCGTGA AGTCGGAGTT GCTAGTAATC GCGGATCAGC ATGCCGCGGT

1321
GAATACGTTC CCGGGCCTTG TACACACCGC CCGTCACACC ACCCGAGTTG TCTGCACCCG

1381
AAGTCGACGG CCCAACCCGC GAGGGGGGAG TCGCCGAAGG TGTGGGGAGT AAGGGGGGTG

1441
AAGTCGTAAC AAGGTAGCCG TACCGGAAGG TGCGGCT,
The sequence data of the microorganism being compared with known 16S RNA gene sequences of representative microorganisms which are part of the domain of bacteria. This comparison analysis showed the greatest correspondence to bacteria of the genus Eubacterium. However, it was not possible to find any gene sequence corresponding sufficiently to a known microorganism, from which it results that the microorganism according to the invention is a microorganism within the genus Eubacterium which has still not been isolated and classified to date. Physiological investigations, such as, for example, fermentation spectra, reduction of nitrate to nitrite, also clearly showed the association with the genus Eubacterium.
A further object of the present invention is to make available a process for obtaining both a pure culture of the microorganism DSM 11798 and its mixed culture with the strain
Enterococcus casseliflavus
, DSM 11799, and other anaerobic microorganisms, an optimization of yield both in the economical and in the quantitative respect being aimed at in particular.
To achieve this object, the process according to the invention is carried out in such a way that a mixed culture DSM 11799 is obtained from the microorganism and
Enterococcus casseliflavus
from bovine rumen by culturing or fermenting at least twice in dilution series and anaerobic culturing conditions. To obt

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