Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
  • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
  • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
  • A23L29/065—Microorganisms
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
  • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
  • A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
  • A23L7/10—Cereal-derived products
  • A23L7/104—Fermentation of farinaceous cereal or cereal material; Addition of enzymes or microorganisms
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor

Definitions

• the present invention relates to a microorganism for the biological detoxification of mycotoxins, namely ochratoxins and/or zearalenons, as well as a method for biologically detoxifying mycotoxins, namely ochratoxins and/or zearalenons, in food products and animal feeds by the aid of at least one microorganism, as well as the use of bacteria and/or yeasts to detoxify ochratoxins and/or zearalenons, in food products and animal feeds.
• Mycotoxins are naturally occurring secondary metabolites of mould fungi affecting agricultural products all over the world and causing toxic effects already in small quantities.
• the infestation of agricultural products with mycotoxins involves extremely high damage and also induces mycotoxicoses in men and animals, which partially exhibit dramatic effects.
• Due to the high economic losses and the strain on men and animals caused by mycotoxins and the thus induced mycotoxicoses attempts have been made for long to find measures to combat mycotoxin contaminations, with basically two methods having been known from the literature.
• the first approach aims to prevent the growth of mould fungi on food products and animal feeds, thus simultaneously preventing the production of mycotoxins.
• the second approach is directed at subsequently destroying mycotoxins, or decontaminating food products and/or animal feeds.
• WO 91/13555 describes a feed supplement as well as a method for inactivating mycotoxins, wherein particles of a phyllosilicate mineral are added to the feed in order to inactivate said mycotoxins.
• the particles are coated with a sequestrant intended to accelerate their actions.
• an animal feed became known, for instance, from WO 92/05706, which animal feed contains montmorillonit clay as a feed supplement. These natural clay minerals having large internal surface areas are supposed to bind mycotoxins superficially due to their porosity, thus immobilizing the same.
• a feed supplement is known from Austrian Utility Model AT-U 504, which feed supplement uses an enzyme preparation capable of forming epoxidases and lactonases and chemically degrading mycotoxins both in animal feeds and in the gastrointestinal tract of animals.
• AT-U 504 the activity of this enzyme preparation can be enhanced by the addition of zeolithes and the like.
• mycotoxin binders to animal feeds, which bind to mycotoxins immediately in the digestive tract during digestion, are able to minimize the effects of toxins in livestock.
• applied substances include alfalfa, bentonite, zeolithe, clays, active carbon, hydrogenated sodium calcium aluminum silicates, phyllosilicates and yeast or bacterium cell walls (U.S. Pat. No. 5,165,946; WO 99/57994; U.S. Pat. No. 6,045,834; EP 9721741; U.S. Pat. No. 5,165,946; U.S. Pat. No.
• mycotoxins can be degraded and hence partially detoxified, by microorganisms.
• An example of the detoxification of mycotoxins and, in particular, trichothecenes is contained in AT-B 406 166, in which a special pure culture of a microorganism belonging to the genus Eubacterium and deposited under number DSM 11798 as well as a mixed culture of the genus Eubacterium with an Enterococcus, which was deposited under number 11799, detoxify trichothecenes by cleaving the epoxy ring present on trichothecenes.
• the present invention aims to provide special microorganisms as well as mixed or pure cultures and also combinations thereof, which are able to biochemically degrade mycotoxins, namely ochratoxins and/or zearalenons, in a selective manner and convert the same into physiologically safe substances and, in particular, safe substances for the feeds and food industries.
• the microorganism according to the invention of the initially defined kind is essentially characterized in that a microorganism and, in particular, aerobic or anaerobic detoxifying bacteria or yeasts is/are used, which cleave(s) the phenylalanine group of the ochratoxins and degrade zearalenons, respectively, wherein the mycotoxin-detoxifying bacteria are selected from the species Sphingomonas, Stenotrophomonas, Ochrabactrum, Ralstonia and/or Eubacterium, and/or the detoxifying yeasts are selected from the species Trichosporon, Cryptococcus and/or Rhodotorula.
• This metabolization of ochratoxins is effected by an enzyme similar to carboxypeptidase A, which cleaves the amide bond of the ochratoxin directly or via a multi-enzyme complex by which the ring of the phenylalanine is hydroxylated and subsequently cleaved and degraded. Finally, the remaining aspartame is cleaved, thus yielding another nontoxic ochratoxin metabolite.
• This route can be demonstrated in a simple manner:
• microorganisms according to the invention which are selected from bacteria and/or yeasts, it is feasible to detoxify not only ochratoxin A and ochratoxin B, but also the metabolites 4R-hyroxyochratoxin A, 4S-hydroxyochratoxin A, ochratoxin C, ochratoxin A methyl ester, ochratoxin B methyl ester and ochratoxin B ethyl ester. Furthermore, these microorganisms enable the degradation and hence detoxification of zearalenons.
• the conditions within the gastrointestinal tract from the stomach to the colon are known to be increasingly anaerobic, which means that the redox potential is increasingly reduced such that, upon ingestion of a foodstuff and/or feeds contaminated with the respective mycotoxins or ochratoxins and/or zearalenons, detoxification at first can be started with aerobic bacteria and/or yeasts and continued with the respective anaerobic bacteria and/or yeasts at the end of a digestive process, or if the foodstuff or feed has already reached an intestinal segment where anaerobic conditions prevail.
• a particularly complete detoxification of mycotoxins is feasible if detoxifying bacteria selected from the species Sphingomonas, Stenotrophomonas, Ochrobactrum, Ralstonia and/or Eubacterium, and/or detoxifying yeasts selected from the species Trichosporon, Cryptococcus and/or Rhodotorula are used as said microorganisms.
• DSM 14169, Ralstonia eutropha DSM 14171 and Eubacterium sp. DSM 14197, as well as the detoxifying yeasts selected from Trichosporon spec. nov. DSM 14153, Cryptococcus sp. DSM 14154, Rhodotorula yarrowii DSM 14155, Trichosporon mucoides DSM 14156 and Trichosporon dulcitum DSM 14162 have proved to be particularly efficient, since they not only ensure the complete degradation of mycotoxins, but can additionally be safely used in food products and animal feeds, which is not necessarily the case with a plurality of other mycotoxin-cleaving and/or degrading bacteria and yeasts.
• DSM 30201 20-100% 60-100% Agrobacterium tumefaciens DSM 9674 25-40% 0-60% Pseudomonas putida ATCC 700007 10-50% 0% Comomonas acidovorans ATCC 11299a 20-57% 0-50% Ascomyceten yeast HA 168 95% 40% Cryptococcus flavus HB 402 90% 100% Rhodotorula mucilaginosa HB 403 20% 0% Cryptococcus laurentii HB 404 50% 0% Unknown HB 508 30% 30% Trichosporon spec. nov.
• HB 704 40% 40% Unknown HB 529 100% 95% Asocomycetes yeast HA 1265 90% 0% Asocomycetes yeast HA 1322 0% 95% Trichosporon ovoides HB 519 100% 90% Triosporon dulcitum HB 523 100% 100% Rhodotorula fujisanensis HB 711 30% 0% Cryptococcus curvatus HB 782 20% 95% Trichosporon guehoae HB 892 50% 20% Trichosp.
• Trichosporon mucoides HB 896 40% 20% Trichosporon mucoides HB 900 100% 100% Trichosporon cutaneum ATCC 46446 0% 70% Trichosporon dulcitum ATCC 90777 0% 100% Trichosporon laibachii ATCC 90778 0% 100% Trichosporon moniliiforme ATCC 90779 0% 60% Cryptococcus humicolus ATCC 90770 0% 30% Eubacterium sp. F6 30-70% 100% Eubacterium callanderi Di1_8 90% 100% Streptococcus sp.
• the bacteria and/or yeasts are stabilized particularly by lyophilization, spray-drying or microencapsulation.
• lyophilization, spray-drying or microencapsulation By stabilizing said microorganisms, their viability and life-time are improved or enhanced, and, in addition, they will be applicable on a more universal scale, and hence usable at any time in any desired application, in the stabilized state.
• Stabilization through lyophilization, spray-drying or micro-encapsulation is known per se, these being simple and rapidly realizable methods that yield stable, viable microorganisms.
• the bacteria or yeasts are used as cell-free extracts or crude extracts.
• a further development in using a cell-free extract contemplates that the latter is used in a solution and, in particular, an aqueous solution.
• Aqueous solutions of cell-free extracts offer the advantage that, being sprayed on the food or feed to be treated, they get into contact with the contaminating mycotoxins directly on the surfaces of the same, detoxification thus being achieved already immediately upon spraying of said extract.
• a crude extract of bacteria and yeasts which is obtained by applying ultrasound, enzymatic digestion, a combination of shock-freezing and thawing, a flow homogenizer, a French press, autolysis at a high NaCl concentration, and/or a bead mill, has the advantage that such a crude extract can be obtained in a particularly quick and unproblematic manner such that, in particular, in those cases where the rapid use of detoxifying or mycotoxin-degrading substances is required, the use of a crude extract yields rapid and reliable results.
• crude extracts can be directly used in the production of animal feed such that the feed supplemented with microorganisms will be taken up by the animals, and the microorganisms and, in particular, yeasts or bacteria will enter into action only in the digestive tract of the animal.
• the invention also contemplates the use of a mixture containing the crude extracts of various detoxifying bacteria and/or yeasts.
• microorganisms in an unbuffered or buffered solution containing phosphate or trishydrochloride buffer at a pH value of between 1 and 12 and, in particular, 2 and 8, as in correspondence with a preferred embodiment, offers the advantage that the microorganisms can be administered directly with the foodstuff or feeds, thus entering into action in the gastrointestinal tract in those regions where the redox potential is suitable for the optimum action of the microorganisms employed.
• a buffered solution renders feasible the immediate adaptation to the respective pH prevailing in the gastrointestinal tract such that the food or delicacy good supplemented with the buffered solution of microorganisms will not cause any shift or disturbance of the pH prevailing in the gastrointestinal tract, thus enhancing the easy digestibility of the supplemented food or feeds on the one hand and preventing any digestive disturbance in the gastrointestinal environment.
• Another object of the present invention resides in providing substances or microorganisms which can be used to detoxify food products or delicacy goods without impairing or affecting the living beings ingesting the same and without impairing or affecting said food products or delicacy goods treated therewith, apart from detoxifying the mycotoxins present on said food products or delicacy goods.
• bacteria selected from Sphingomonas sp. DSM 14170 and DSM 14167, Stenotrophomonas nitritreducens DSM 14168, Stenotrophomonas sp. DSM 14169, Ralstonia eutropha DSM 14171 and Eubacterium sp. DSM 14197, and/or yeasts selected from Trichosporon spec. nov. DSM 14153, Cryptococcus sp.
• DSM 14154, Rhodotorula yarrowii DSM 14155, Trichosporon mucoides DSM 14156 and Trichosporon dulcitum DSM 14162 enables the mycotoxins, namely ochratoxins, present on the surfaces of food products or animal feeds to be detoxified by cleaving the phenylalanine group and zearalenons to be cleaved, without affecting or influencing in any manner whatsoever the food products or animal feeds treated therewith.
• Trichosporon spec. nov. DSM 14153, Eubacterium sp. DSM 14197 or Stenotrophomonas nitritreducens DSM 14168 has proved particularly suitable for this purpose, those microorganisms ensuring the, in particular, complete degradation of mycotoxins, namely ochratoxins and/or zearalenons, without entailing any risk.
• Another object of the present invention resides in providing a method for biologically detoxifying by a microorganism, mycotoxins, namely ochratoxins and/or zearalenons, in food products and animal feeds, which enables the contaminating toxins to be completely and rapidly decontaminated directly upon entering into contact with food products or animal feeds, or within the digestive tract of the living beings taking in said food products or animal feeds.
• the method according to the invention is essentially characterized in that a microorganism according to the invention and, in particular, bacteria and/or yeasts according to the invention are mixed with the food product or animal feeds in amounts ranging from 0.01% by weight to 1% by weight and, in particular, 0.05% by weight to 0.5% by weight.
• a food product or animal feed supplemented with the accordingly stabilized microorganism will be obtained in stable form.
• a further development of the invention provides for the mixing of said food products or animal feeds by stirring in an aqueous suspension of said microorganism containing water at 20 to 99% by weight and, in particular, 35 to 85% by weight, at temperatures of from 10 to 60° C. and, in particular, 15 to 45° C., for 2 minutes to 12 hours and, in particular, 5 minutes to 2 hours.
• a pO 2 of 40% and a maximum aeration rate of 3 m 3 /h are adjusted as additional parameters.
• the pH is 5.00 at the beginning, yet changes in the course of the growth process (rising up to 8.5). After about 40 to 44 hours, the contents can be used as an inoculum for the 3.6 m 3 production fermenter.
• the following medium is used for the latter: Malt extract 17 g/l Yeast extract 5 g/l Peptone 2 g/l Antifoaming agent 0.1%
• the aeration rate is 15 m 3 air per hour. After 40 to 48 hours, the cells are concentrated by means of a flow separator. The fermentation broth can be concentrated to about 1:10 by means of a separator with a bacterial count of about 3 ⁇ 10 9 /ml being obtained.
• Entry temperature of drying medium (air) 160° C.
• a pO 2 of 40% and a maximum gassing rate of 3 m 3 /h are adjusted as additional parameters.
• the stirring rate is 200 rpm.
• the pH is about 6.8 to 6.9 at the beginning, yet changes in the course of the growth process, rising up to 8.3.
• the contents can be used as an inoculum for the 3.6 m 3 production fermenter.
• the following medium was used for the latter: Soybean flour 17 g/l Yeast extract 5 g/l Peptone 2 g/l Antifoaming agent 0.1%
• the aeration rate is adjusted to 15 m 3 air per hour.
• the stirring speed is 250 rpm.
• the cells can be concentrated by means of a flow separator.
• the concentration ratio is about 1:100.
• Entry temperature of drying medium (air) 160° C.
• Cultivation takes place in a 100 ml Pyrex flask with a silicone septum. 80 ml of the autoclaved medium are decanted and mixed with KH 2 PO 4 /Na 2 HPO 4 buffer (pH 7). After the addition of the contents of a cryovial from the working cell bank, the headspace of the flask is gassed with N 2 (1 min). Upon closure of the vial, the latter is incubated at 37° C. for 72 hours.
• 4.5 liters of the above-mentioned culture solution are autoclaved in a 5-liter Schott flask.
• the latter comprises a bleeder connection and two tubes with sterile filters (for gassing the inoculum).
• the buffer solution 1% of the phosphate buffer
• 80 ml inoculum are added.
• the openings are closed by means of tube clamps and the inoculum is incubated at 37° C. for about 64 hours. After a purity test, it can be used as an inoculum for a 1 m 3 fermenter (700 liter capacity).
• the following medium is used for production: Glucose 10 g/l Yeast extract 5 g/l Peptone 2 g/l Cystein HCl 0.5 g/l pH 7.00
• the inoculum is added after the sterilization of the medium in a fermentation tank (40 min, 121° C., 1.21 bar) and recooling to 37° C.
• the headspace of the fermenter is flushed with N 2 .
• the stirring rate is 100 rpm, soda lye (8 mol/l) is used for pH adjustment.
• the redox potential is about ⁇ 240 mV at the beginning, decreasing to more than ⁇ 500 mV during growth.
• the fermentation time is about 48 hours. Concentration is effected by means of a flow separator.
• Subsequent stabilization is effected by freeze-drying, micro-encapsulation or spray-drying.
• Whey powder serves as a cryoprotector in freeze-drying.
• the microorganism is protected from unfavorable living conditions during storage by fluidized-bed granulation using a vegetable fat (Holtmelt process, top spray).
• Spraying rate Ca. 80-150 g/min Temperature of incoming air: 50° C.
• Spraying pressure 3 bar Air amount: 750-1500 m 3 /h Product temperature: ⁇ 47° C.
• a logarithmic dilution series to stage 10 ⁇ 4 is prepared in physiological saline solution from the products obtained in Examples 1 to 3.
• 2 ml are each pipetted into 18 ml of the respective medium (minimal medium (Na 2 HPO 4 2.44 g/l; KH 2 PO 4 1.52 g/l; (NH 3 ) 2 SO 4 0.50 g/l; MgSO 4 ⁇ 7H 2 O 0.20 g/l, CaCl 2 ⁇ 2H 2 O 0.05 g/l), yeast medium or nutrient broth (Oxoid CM001B)), supplemented with 200 ppb OTA.
• minimal medium Na 2 HPO 4 2.44 g/l
• yeast medium or nutrient broth Oxoid
• the used flasks are incubated on a horizontal shaker under suitable conditions. After 2.5, 5 and 24 hours, samples are taken and examined for OTA cleavage by means of high-pressure liquid chromatography. At a dilution stage of 10 ⁇ 3 (corresponding to product bacterial counts of 10 5 ), the yeasts in minimal medium have cleaved 90% of ochratoxin A after 5 hours and 100% after 24 hours.
• the complex yeast medium is used as a test matrix, the products exhibit a cleavage rate of 90% after 6 hours at a dilution stage of 10 ⁇ 2 . After 24 hours, all of the OTA is detoxified.
• This model serves to investigate lyophilisates of the yeast strains DSM 14153, DSM 14154, DSM 15155, DSM 14156 and DSM 14162 as well as the aerobic (DSM 14170, DSM 14167, DSM 14168 und DSM 14169) and anaerobic (DSM 14197) bacterial strains.
• the small bowel of a freshly slaughtered pig is cut into pieces of about 15 cm length, which are each added to 100 ml minimal medium containing OTA [200 ppb].
• the batches were finally inoculated directly with 1 g lyophilisate and incubated at 35° C. After 0, 6, 24 and 48 hours, samples were drawn for a subsequent OTA analysis by means of HPLC and stored in a deep-frozen state ( ⁇ 20° C.) until said analysis.
• germ DSM 14168 was the best; after 6 hours, 50 to 100% of the present toxin had already been reacted, after 24 hours 80 to 100%. DSM 14169 too turned out to be absolutely “acceptable”: after 6 hours, 0 to 90% OTA had been detoxified, after 24 hours 70 to 95%. The two remaining germs clearly performed less well (0-40% after 6 h; 50 to 60% after 24 h; 60-80% after 48 h).
• the anaerobic small-bowel isolate DSM 14197 degraded the present mycotoxin after 6 hours of incubation at a ratio of 0 to 60%; after 24 hours, between 50 and 100% OTA had been reacted.
• This model served to examine lyophilisates of the yeast strains DSM 14153, DSM 14156 and DSM 14162 as well as the bacterial strains DSM 14168 (aerobic), DSM 14169 (aerobic) and DSM 14197 (anaerobic).
• DSM 14156 100 (+) 95 (+) 96 (+) 100 (+) 30 (+) DSM 14162 83 (+) 12 (+) 100 (+) 100 (+) 0 ( ⁇ ) DSM 14170 75 (+) 0 ( ⁇ ) 0 ( ⁇ ) 89 (+) 0 ( ⁇ ) DSM 14167 100 (+) 4 (+) 39 (+) ⁇ 90 (+) 0 ( ⁇ ) DSM 14168 100 (+) 0 ( ⁇ ) 50 (+) 94 (+) 0 ( ⁇ ) DSM 14169 100 (+) 0 ( ⁇ ) 0 ( ⁇ ) 91 (+) 0 ( ⁇ ) DSM 14171 100 (+) 0 ( ⁇ ) 79 (+) 81 (+) 0 ( ⁇ )
• the microorganisms were cultivated for about 66 hours. After this, they were centrifuged (3210 ⁇ g, 15 min, room temperature) and the pellets obtained were resuspended in minimal medium. To the minimal medium were added 1 ppm desoxynivalenol, 1 ppm fumonisin B 1 , 1 ppm zearalenon, 200 ppb aflatoxin B 1 and 2 ppm citrinin. Before incubating the batches at 30° C., a sample was taken (“zero sample”). The incubation time was 96 hours. The batches were determined in duplicate by examining for the HPLC analysis once the supernatant (after centrifugation) and once the whole batch.
• microorganisms according to the invention readily enable, in particular, the degradation of ochratoxins in food products and animal feeds and also in intestinal environment, with the degradation of zearalenon, citrinin and the like yet partially yielding good results.

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