DE19912439C2 - Killer toxins from killer yeast for pharmaceutical use - Google Patents

Description

The invention relates to a so-called fermentation product Killer yeast used to treat certain diseases, especially diarrheal diseases in the veterinary field, especially for the treatment of diarrhea in piglets and calves.

It has long been known that certain strains of yeast during the exponential growth phase one for other yeast strains excrete toxic substance into the fermentation medium. For such yeast strains has the name "killer yeast" enforced, and this term is also used, for example, in "Römpp Lexikon Biotechnologie, 1992" used. killer yeasts excrete toxins during the fermentation process which are usually small, hydrophobic proteins acts against which the killer yeasts themselves are immune, but who kill yeasts during fermentation. The one from the Killer yeast excreted toxins with regard to Description "killer yeast" often also as "killer toxins" designated.

The appearance of killer yeasts in the brewery and Ways to solve the problems that arise  are widely described in the literature, and it can for example on the Brauwelt publication, 40, 1993, 2020 to get expelled.

Certain killer toxins have also been used with different Diseases, e.g. B. associated with enteritis. Here can for example on Gastroenterology, 109, 1995, 1900-1906 to get expelled.

DE 36 42 050 A1 describes the structural formula of a certain killer toxin which was obtained from a yeast strain belonging to the genus Hansenula, namely from Hansenula mrakii IFO 0895 (FERM BP-1197). The killer toxin described there is of the K ₉ type and is said to be useful as an antifungal agent. An application as a drug is not described in this document.

There is already research into whether killer toxins are therapeutic Possess applicability. So in Mycopathol. 96, 1986, 103- 107 topical use of concentrated killer toxin (Hansenula anomala UCSC 25F) in animals. hereby should consider the possible use of killer toxins Skin fungal diseases are tested. Furthermore, in addition antifungal also certain antibiotic properties of Killer toxins described. With killer toxins and their possible uses are concerned, for example Clinical Microbiology Reviews, July 1997, 386/387, Critical Reviews in Microbiology, 18 (1), 1991, 47-87, The Journal of Immunology 1996, 1880-1885, Molecular Medicine, Volume 3, No. 8, August 1997, 544-552, JID Concise Communications, 177, 1988, 807-811, to name a few. In Critical Reviews in Microbiology, 18 (1), 1991, 47-87, the authors come to the Conclusion that the therapeutic use of killer toxins is only possible to a limited extent. Indeed it has none of the previous scientific studies Killer toxins for effective therapeutic use of a killer toxin.  

Especially when raising pigs, but also in other areas of veterinary medicine, the problem often occurs of diarrheal diseases. Such diarrheal diseases which in particular of Escherichia coli bacteria (e.g. "EHEC" - Bacteria) are caused, z. B. at Weaned piglets, i.e. in piglets that are fed by a diet Breast milk can be switched to a different diet, too high Weight loss and often death. Also in human medicine provide such diarrheal diseases, in particular debilitated patients and young children, a significant one Problem.

To treat diarrhea in animals a number of medicines are available and it can for example on Kraftfutter / Feed Magazine 9/1998, 356; Concentrate / Feed Magazine 4/98, 140; Concentrate / Feed Magazine 5/96, 200; The Practical Veterinarian 79, 2, 1998, 171; Collegium Veterinarium XXIV, 1993, 87 and Lohmann Information, September-December 1994, page 5. In the They are also used to treat such diarrheal diseases Antibiotics used for cost reasons, their use in the Veterinary medicine, however, is increasingly criticized as the There is a risk that harmful bacterial strains against Antibiotics become resistant. The publication Kraftfutter / Feed Magazine 4/98, 140 describes the use of Probiotics, also from yeast, but these are not about killer yeast, and killer toxins are not mentioned.

For the prophylaxis and treatment of diarrheal diseases, especially in veterinary medicine, become effective Medicines needed that are easy and inexpensive to manufacture and do not pose the risk of bacterial strains become immune to it.

The invention has for its object a new To provide medicines, especially in the Veterinary medicine for the treatment of diarrhea  can be used. The drug is especially intended for Young animals and particularly preferred for newborn piglets and can be used in weaners, but also in calves.

This task is solved due to the surprising finding that killer toxins by fermentation of killer yeast arise in veterinary medicine and especially in Weaners that prevent severe diarrhea. This was especially true with regard to the conflicting revelation Gastroenterology, 109, 1995, 1900-1906 are not expected.

The invention therefore provides the use of a killer toxin, which can be obtained by fermentation of killer yeast, for Treatment of diarrheal diseases in veterinary medicine and especially in newborn piglets, weaners and Calves available. The administration of the killer toxin can as a common medicine (tablets), but also over a Feed or a feed additive for animals that the Killer toxin in a veterinary effective dose contains. The following is about a drug spoken, this is also a corresponding one Animal feed additives understood.

In addition, the invention provides a feed additive in Meaning of a growth promoter, especially for pigs and Piglet, which is a fermentation product from Killer yeast, especially from Hanseniaspora valbyensis, contains.

The killer yeasts which can be used according to the invention are not particularly restricted. However, it turns out that some Strains of killer yeast are either particularly effective Generate killer toxins or a higher level of killer toxins Generate concentration than other strains of killer yeast. Such killer yeasts are preferred. These are special known to the wine industry. According to the invention  preferred to produce the killer toxins yeast of the type To ferment Ascomycetes or Asporogenous.

The following genera can be named as yeasts of the type Ascomycetes:

• - Saccharomyces (particularly preferred, in particular Saccharomyces globosus, e.g. B. y 438 BKM (BKM Russia, russian trunk collection))
• - Saccharomycodes
• - Zygosaccharomyces
• - Schizosaccharomyces
• - Kluyveromyces
• - Hansenula
• - Pichia (particularly preferred, in particular Pichia anomala, z. B. UCSC 25F and Pichia subpelliculosa e.g. B. NCYC 16)
• - Williopsis (particularly preferred, especially Williopsis mrakii z. B. NCYC 500)
• - Hanseniaspora (particularly preferred, in particular Hanseniaspora valbyensis z. B. CBS 311)
• - Endomycopsis
• - Debaromyces

The following genera can be mentioned as yeasts of the type Asporogenous:

• - Candida
• - Cryptococcus
• - Torulopsis
• - Rhodotorula
• - Trichosporon

The following strains are particularly preferred according to the invention:

• - Hanseniaspora valbyensis 13cs / 6ρ ^- , available from Hanseniaspora valbyensis CBS 311 (Centraalbureau voor Schimmelcultures, Baarn, Netherlands), after mutation of mitochondria (rho ^- or ρ ^- ) with ethidium bromide (30 µg / ml) [Rickwood D., B. Dujon, VM Darley - Usmar, Yeast Mitochondria, In: Yeast - a Practical Approach, Ed. I. Campbell JH Duffus, published in Practical Approach Series 1988, IRL Press Ltd.].
• - Williopsis mrakii AS / 15ρ ^- , available from Williopsis mrakii NCYC 500 (National Collection of Yeast Cultures, Norwich, UK), after mutation of mitochondria (rho ^- or ρ ^- ) with ethidium bromide solution (30 µg / ml) [Rickwood et al. , 1988].
• - Saccharomyces globosus 6 / ρ ⁺ , available from Saccharomyces globosus y 438 BKM (HO / HO, pet / pet, trp / trp, [KIL-K ₄ ], Russian strain collection), after rice isolation of the active killer strain colony No. 6.
• - Pichia anomala 25F-5, available from Pichia anomala UCSC 25F (Universita Cattolica del Sacro Cuore, Rome, Italy), after Rice isolation of the active killer tribe, colony No. 5.
• - Pichia subpelliculosa 16/22, available from Pichia subpelliculosa NCYC 16 (National Collection of Yeast Cultures, Norwich, UK), after rice isolation of the active Killerstamms, colony No. 22.

The most preferred yeast strain according to the invention Hanseniaspora valbyensis 13cs / 6ρ ^- was deposited with the German Collection of Microorganisms and Cell Cultures GmbH (DSM) according to the Budapest Treaty and received the deposit number 12718.

Killer yeasts as such are known and in principle by everyone Depositors, e.g. B. the above, available. The commercially available killer yeast strains are separated into individual colonies, and the colonies are preferred in YPG agar medium for killer activity  tested. Suitably the colony will be the largest Killer activity for the fermentation and production of the Killertoxins used.

It may be advantageous to use the commercial to mutate available strains. The one described below The method is suitable, for example, for producing the particularly preferred killer yeast strains of the Hanseniaspora type valbyensis and Williopsis mrakii. Tribes from Hanseniaspora valbyensis and Williopsis mrakii are commercially available, for example from the National Collection of Yeast Cultures, Norwich, UK, Williopsis mrakii NCYC 500 and the Centraalbureau voor Schimmelcultures, Baarn, Netherlands, Hanseniaspora valbyensis CBS 311.

The strains are suitably mutated with ethidium bromide as described in the literature (Rickwood et al., 1988). The commercially available strains are grown in a rich medium, e.g. B. YPG (1% yeast extract, 2% peptone, 2% glucose) e.g. B. cultivated for three to four hours at 30 ° C with shaking (growth to the logarithmic phase). Then an ethidium bromide solution (up to 30 µg / ml medium) is added to the medium. After 15 hours of cultivation in rich medium, the yeast cells are z. B. YPG agar applied in a dilution so that individual colonies can be isolated. Individual colonies are then transferred in parallel to YPG agar and to RHO agar (0.67% YNB, that is to say Yeast Nitrogen Base, with 1% glycerol). The cells that cannot grow on RHO agar are called rho ^- (or ρ ^- ), that is, "respiratory deficient". Colonies that grow on YPG agar but not on RHO agar are isolated and further examined for killer activity.

The killer activity can be done in a manner known per se z. B. can be determined as follows.  

10 ⁵ cells / ml of the yeast strain Saccharomyces cerevisiae S-6/1 (a killer yeast-sensitive strain, diploid, from the University of Prague, Department of Genetics and Microbiology Charles University, Faculty of Science CSFR - 12844 Prague 2, Vinicna 5, Czech Republic ) are applied to J medium agar (0.25% yeast extract, 0.25% peptone, 0.5% glucose and mineral substances, 50 mM buffer citrate / phosphate, 2% agar, pH 4.7) with 0.003% methylene blue , A well with a diameter of 6 mm is then made in the agar, into which 100 μl of a sample are added. It is incubated for one day at 28 to 30 ° C, and then the area of the zone of inhibition is determined. If necessary, the method can be used with other pathogenic strains, e.g. BE coli, Streptococcus agalactiae, Staphylococcus aureus, Salmonella goldcoast or Pseudomonas aeruginosa can be repeated. If the zone of inhibition reaches an area of 10 mm ² , this corresponds to 1 unit (= 1 unit) of killer activity.

According to the invention, the strain Hansenula mrakii, in particular FERM BP-1197 can be used, which is described in DE 36 42 050 A1 is described in detail. That from this tribe Killer toxin produced (the production is also in the DE 36 42 050 A1) has that in DE 36 42 050 A1 described structure.

As a rule, those described in DE 36 42 050 A1 Process for the production and isolation of a killer toxin from a yeast strain, to clarify the structure of such Killer toxins and for determining killer activity also for the present invention usable. To that extent, therefore, in order Avoid repetitions on the appropriate revelation referred to DE 36 42 050 A1.

The killer yeasts can be fermented in the usual way, as has long been known and used in particular in the brewery industry. For the five particularly preferred killer yeast strains of the present invention, preferred fermentation conditions are as follows:

• - Hanseniaspora valbyensis 13cs / 6ρ ^- :
  The strain is cultured in a medium containing 5% glucose, 2% malic acid, 0.05% trisodium citrate 2-hydrate, 0.004% myo-inositol, 0.1% yeast extract and 0.1% peptone. The pH of the medium is adjusted to approximately 5.0. It is first cultivated for 18 hours with vigorous shaking at 28 ° C. The fermentation broth is then cultivated without shaking for a further 20 to 24 hours.
• - Williopsis mrakii AS / 15ρ ^- :
  The strain is cultured in a medium containing 0.67% YNB (Yeast Nitrogen Base) plus 2% glucose. The pH of the medium is adjusted to approximately 4.7. Another suitable medium contains YPG, ie 1% yeast extract, 2% peptone and 2% glucose. The pH of this medium is also adjusted to about 4.7. It is first cultivated for 18 hours with vigorous shaking at 28 ° C. The fermentation broth is then cultivated with gentle shaking for a further 18 hours and then without shaking for a further 24 hours.
• - Saccharomyces globosus 6 / ρ ⁺ :
  The strain is cultured in a medium containing 0.67% YNB (Yeast Nitrogen Base), 0.5% yeast extract, 0.5% peptone and 2% glucose. The pH of the medium is adjusted to approximately 5.0. It is first cultivated for 18 hours with vigorous shaking at 28 ° C. The fermentation broth is then cultivated without shaking for a further 48 hours.
• - Pichia anomala 25F-5:
  The strain is cultured in a medium containing 0.67% YNB (Yeast Nitrogen Base) and 2% glucose. The pH of the medium is adjusted to approximately 4.7. Another suitable medium contains YPG, ie 1% yeast extract, 2% peptone and 2% glucose. The pH of this medium is also adjusted to about 4.7. It is first cultivated for 18 hours with vigorous shaking at 28 ° C. The fermentation broth is then cultivated with shaking gently for a further 18 hours and then without shaking for a further 20 to 24 hours.
• - Pichia subpelliculosa 16/22:
  The strain is cultured in a medium containing 0.67% YNB (Yeast Nitrogen Base), 0.5% yeast extract, 0.5% peptone and 2% glucose. The pH of the medium is adjusted to approximately 5.0. It is first cultivated for 18 hours with vigorous shaking at 28 ° C. The fermentation broth is then cultivated without shaking for a further 36 hours.

The killer toxin can then be obtained from the fermentation broth be, for example by chromatographic methods. On Process for fermentation and isolation of killer toxins in Gastroenterology, 109, 1995, 1900-1906. Other known methods can also be used.

In particular in the use preferred according to the invention of killer toxin in veterinary medicine is the isolation of the Toxins from the fermentation broth, however, from economic Not preferred for reasons. Rather, the Fermentation broth with the killer toxin, if necessary after suitable processing, used. Is particularly preferred it, the fermentation broth, for example by centrifugation or filtration to get rid of the yeast cells and the obtained solution then freeze-drying undergo. The powder obtained can then in the usual way and way z. B. pressed into tablets, in capsules be filled or used as such.  

With the preferred use of veterinary medicine, it is on most economical, the animals before the fermentation broth Meal mixed with water to give to drink or a To allow permanent admission. One can also in Lyophilisat Dissolve water and add to drink. One can Also add the fermentation broth to the liquid feed. For others Animal species (small animals) can also kill toxin from lyophilisate microencapsulated and / or formulated into tablets and then be added to the potions or the feed.

The most suitable dosage depends on the animal species, the age of the Animals, the severity of diarrhea, the Stage of disease and the specially used killer toxins from. The dosage may be suitable from a veterinarian be determined.

In the case of weaned piglets, a usual daily dose is generally given Killer toxin about 100 to 2000 units, preferably 200 to 1200, more preferably 200 to 1000 units. This matches with usually about 10 to 50 ml of fermentation broth per animal and day. The usual dosage for prevention is about 50 to 1000 units. To prevent diarrhea is a period of use from 10 to 14 days makes sense.

With newborn piglets the cheapest dosage is a little bit lower, for example from 50 to 1000 units per day, preferably from 100 to 600 units per day. The activity (unit, unit) was on pathogenic coli strains as above described, determined.

The following values serve as a reference point and relate to the use of the particularly preferred killer yeast strain Hanseniaspora valbyensis 13cs / 6ρ ^- , as described above.

To prevent diarrhea in piglets in the Weaning stage, that is, in piglets, which lasts about 4 to 5 weeks  are old and weigh approximately 12 to 15 kg, per animal and Approx. 0.5 l water plus 20 ml fermentation broth per day administered. The level of killer activity in the Fermentation broth is about 20 units per ml the total dosage per animal and day is approximately 400 units Killer toxin. The duration of use has been 2 to 3 weeks proven to be cheap.

For acute therapy for diarrhea, newborn piglets, that is 1 to 5 day old piglets, the approximately 1.2 to 1.5 kg weigh 1 to 2 ml suspension of a 10-fold concentrate of Fermentation broth can be administered orally. this will preferably repeated after 8 to 24 hours. The suspension contains 100 to 300 units per ml, so that there is a daily dose from 100 to 600 units per piglet.

For 4 to 6 week olds, weighing approximately 12 to 15 kg Settling piglets with diarrhea have been given oral administration from 2 to 4 ml suspension of a 10-fold concentrate of Fermentation broth proved to be cheap. The administration is repeated 1 to 2 times at intervals of 8 to 24 hours. This results in a daily dose per animal of 200 to 1200 Units of killer toxin.

The medicament according to the invention is generally against Diarrheal diseases, especially of young animals, such as calves or piglets, can be used, regardless of the specific pathogen, however, there is some pathogen selectivity, of which the Diarrhea. Especially the use of the drug is preferred for Diarrhea caused by E. coli (EHEC bacteria) are caused because of such diarrheal diseases often run particularly serious and in the piglet and Calf rearing has the most casualty or death losses Take care (developmental disorders). Without that Invention on a specific mechanism of action is to be restricted, according to the previous  Knowledge assumes that the killer toxins are not the bacteria that cause the diarrhea, fight directly (damage or kill), but rather prevent the pathogens from adhering to intestinal cells. Thereby disease-causing coli can no longer penetrate the intestinal mucosa colonize what, based on current knowledge, be the first Prerequisite for the emergence of lossy E. coli Ruhr and coli enterotoxemia applies. This mechanism seems particularly effective in young animals (calves and piglets) in the first weeks after birth and especially when Weaned piglets. The mechanism of action mentioned also makes clear why the microorganisms practically none Can develop resistance to killer toxins and problems as they occur when antibiotics are administered are to be expected.

This shows, in particular, in young animals Medicines in addition to the treatment or prevention of Bowel disorders also have a growth-promoting effectiveness especially with a longer period of use. This is a Useful duration of 4 to 6 weeks. The dosage here corresponds to the dosage used to prevent Diarrhea is administered.

The following examples illustrate the invention.

example 1

To carry out the fermentation process, 200 l of B _m medium with 10 kg of D (+) glucose, 4 kg of DL-malic acid, 1 kg of ammonium sulfate, 0.1 kg of trisodium citrate-2-hydrate, 8 g of myo-inositol, 200 g of yeast extract and 200 g peptone provided. The pH of the medium was adjusted to approximately 5.0 with approximately 3.35 kg of KOH. The medium was pasteurized at 80 ° C for about 1 hour and then immediately cooled.

In parallel, an inoculum of Hanseniaspora valbyensis 13cs / 6ρ ^- with rich medium YPG (2% glucose, 1% yeast extract, 2% peptone, pH about 5.0) was prepared. 3 × 500 ml medium were inoculated from the slant agar (with 3 × 50 ml pre-inoculum) and incubated for 20 hours. The cultures were transferred to 3 x 4500 ml cultures and incubated for 36 hours at 25 to 28 ° C with vigorous stirring. The pH of the inoculum was about 5, with 10 ⁸ cells per ml.

After the pasteurized medium had cooled, 15 l of the Hanseniaspora valbyensis 13cs / 6ρ ^- inoculum were added. This strain was cultured at 28 ° C for 18 hours with vigorous stirring. Subsequently, cultivation was carried out with slow stirring for 6 hours, and finally the fermentation broth was cultivated for 20 to 24 hours without stirring. The fermentation broth was cooled after the cultivation process (the pH was approximately 5 and the fermentation broth contained approximately ¹⁰⁷ cells / ml) and filtered through a Sartorius membrane of 0.45 μm. The fermentation broth had a killer activity of approximately 20 units / ml and could be stored at 8 ° C.

The fermentation broth with the killer toxin but without the filtered yeast cells was lyophilized in the usual way. About 200 to 250 g of powder could be obtained from 10 l of fermentation broth from Hanseniaspora valbyensis 13cs / 6ρ ^- . The powder was storable in plastic cans at 8 ° C.

The killer activity of the lyophilizate or Fermentation broth was determined as follows.

Pathogenic strains of E. coli, Streptococcus agalactiae, Staphylococcus aureus, Salmonella goldcoast, Enterobacter faecalis and Pseudomonas aeruginosa were used as sensitive strains for Hanseniaspora valbyensis 13cs / 6ρ ^- . The killer activity was determined after application of 10 ⁵ cells / ml of one of the strains mentioned to a standard agar medium for bacteria. A well with a diameter of 6 mm was introduced into the agar plate, into which 100 μl of sample were then added (either the fermentation broth or the 10-fold fermentation broth after lyophilization). It was incubated at 37 ° C for 24 hours and the area of the zone of inhibition was determined. An inhibition zone of 10 mm ² corresponds to 1 unit (= 1 unit) of killer activity.

In the following table the killer activity is in units / ml the 10-fold concentrated solution for various pathogenic Tribes listed.

Example 2

For the production of a feed or feed additive are from the fermentation broth prepared in Example 1 the yeast cells by filtration or centrifugation separated. The procedure is then as described in Example 1 determines the activity. The fermentation broth as such or a concentrate or lyophilisate made from it is in  suitable containers filled and can be used directly as Additive used to water the animals.

Example 3

The effectiveness of the killer toxins according to the present Invention was made on a stock of 1500 piglets per year, in an endemic coli infection with ongoing diseases and deaths prevailed, tested. A test group from 20 piglets at the age of 5 weeks were treated with 20 ml of the 12 days Fermentation broth from Example 1 mixed in water treated. No diseases occurred in the test group or losses on.

In a control group of 20 piglets at 5 weeks of age, the same in parallel to the test group for 12 days Received food like the test group, but without Fermentation broth or other medication, 12 piglets and 2 animals fell ill within 6 days dead. 4 other animals showed coli edema symptoms.

Example 4

If the disease has already been identified by E. coli (Diarrhea, coli dysentery, enterotoxemia) is the effect of Killertoxins according to Example 1 most strongly in direct oral Administration in the form of a lyophilisate as a suspension or after additional cleaning, as a solution. A 10- fold concentration in an aqueous carrier system. The suspension can be used for both calves and piglets, especially in early adolescence.

The effectiveness of the killer toxins has been over a period of time tested by 4 years. During this period, a total of about 3000 to 4000 newborn piglets with the Fermentation broth or treated with lyophilisate. Both newborn piglets aged 1 to 5 days was included  diarrhea in 60% of the animals after a single treatment healed in 35% of the animals after two treatments within 1 to 3 days. The dosage was 1 to 2 ml of the suspension, which is 200 to 400 units per piglet and per Administration corresponds. The administration was after 12 to Repeated 24 hours.

In older piglets from 3 to 5 weeks (here were total tested about 1500 animals), the success rate was similar to that of newborn piglets, however, the dosage was twice as high like the dosage for newborn piglets, that is for one treatment was performed 1 to 3 times at an interval of 8 to 24 Hours 2 to 4 ml of the suspension with the killer toxin administered according to Example 1.

Example 5

From a herd of piglets in which 30 to 40% deaths from diarrhea are observed a test group was selected from 10 piglets. The Piglets were 5 weeks old, in the weaning phase and showed severe diarrhea, edema and severe disorder of general well-being. On the first day, 4 ml Suspension (as described in Example 4) with a dosing device administered directly into the mouth. On the second day the Treatment repeated with the same dose after 12 hours. At the The third day was the disease symptoms in all animals disappeared and all animals showed good appetite. On the fourth Day treatment was repeated with 2 ml suspension to prevent relapse. All animals were then healthy and showed good growth.

Example 6

A 5 day old calf with severe diarrhea and general weakness was with the invention  Treated killer toxin. For this, a suspension was used used as described in Example 4.

On the first day, 12 ml of the suspension were mixed with a Electrolyte dietary as adjuvants directly into the mouth injected. On the second day, another 10 ml suspension administered orally. After 36 hours everyone was gone Signs of illness disappeared.

As described above, 2 calves (twins) were in the Age of 4 days, who were suffering from coli dysentery, with the Killer toxin from Example 1 treated. On 2nd consecutive days were each 10 ml of the suspension administered orally. Both animals were healed after 36 hours.

Example 7

Example 1 was repeated, with the modification that instead of Hanseniaspora valbyensis 13cs / 6ρ ^- the strain Williopsis mrakii AS / 15ρ ^{- was} used. After the fermentation broth had been separated from the yeast cells, lyophilization was carried out. A 20-fold concentrate is prepared from the powder obtained with water and dialyzed using a 3.5 kD MWCO membrane at pH 4.7 in 3 mM citrate / phosphate buffer. After dialysis, the product is lyophilized again and the product can optionally be subjected to further purification steps such as isoelectric focusing, ion exchange chromatography or gel filtration. In the present example, the killer proteins were separated by means of ion exchange chromatography (FPLC - Fast Performance Liquid Chromatography) with a strongly cationic ion exchanger of the Resource Mono S® type (from Pharmacia). The program for the separation of the killer proteins can be found in the following table.

The collector was operated at 1 ml / 2 min, i.e. 25 Test tubes with fractions of 2 ml each were decreased.

Equilibration of the column and separation of the killer proteins was carried out with 10 mM buffer citric acid / sodium citrate at pH 3.2. It was used with a gradient of NaCl between 0 to 0.5 M eluted with the sodium chloride solution using the buffer system had been brought to pH 3.2.

After each fraction was separated, the biological Killer activity tested. The highest biological activity was between Fractions Nos. 9 and 10 and Fraction Nos. 14 as shown in the table below.  

Fractions Nos. 9, 10 and 14 were collected and again undergo dialysis. After dialysis, 50 ml are obtained Solution that have been freeze-dried. 15.6 mg was obtained Powder with 5 mg of killer proteins (measuring the Protein concentration was carried out according to the Lowry method). The The performance of this cleaning system was 90%. To the cleaning contains 1 ml preparation 0.1 mg killer protein 320 units of killer activity.

The degree of purity of the killer toxin was determined with a Polyacrylamide gel electrophoresis under native and denaturing conditions checked and it became a isoelectric focusing performed.

The physicochemical and biochemical properties of the killer protein from Williopsis mrakii AS / 15ρ ^- can be found in the following table.

Diarrhea is common with E. coli at the same time a mixed infection with rota or corona viruses has been detected. Treatment with the killer toxin according to the present Invention, in particular with the killer toxin of Example 1 showed particularly good results and is Consider antibiotic use. Bowel disorders and General health is regenerated faster.

Claims (16)

1. Using a killer toxin obtained by Fermentation of killer yeast, for prophylaxis and / or Treatment of diarrhea. 2. Use according to claim 1, wherein it is in the Killer yeasts for yeasts from Genera Saccharomyces, Pichia, Williopsis and Hanseniaspora acts. 3. Use according to claim 2, wherein it is in the yeast for yeasts from the strains Hanseniaspora valbyensis, Williopsis mrakii, Saccharomyces globosus, Pichia anomala and Pichia subpelliculosa. 4. Use according to one of claims 1 to 3 for veterinary application. 5. Use according to claim 4 for prophylaxis and / or Treatment of pigs and cattle. 6. Use according to claim 5 for prophylaxis and / or Treatment of piglets and calves. 7. Use according to claim 6 for prophylaxis and / or Treatment of diarrhea in weaners. 8. Use according to one of claims 1 to 7, wherein the Killer toxin in the form of a fermentation broth or as Lyophilisate is used. 9. Use according to one of claims 1 to 8, wherein the Killer toxin via a feed additive or a drinker is administered.   10. Use of a killer toxin produced by fermentation of Killer yeast is available as a feed additive. 11. Use according to claim 10, wherein it is in the Killer yeast for the yeasts of Genera Saccharomyces, Pichia, Williopsis and Hanseniaspora acts. 12. Use according to claim 11, wherein it is in the yeast for yeasts from the strains Hanseniaspora valbyensis, Williopsis mrakii, Saccharomyces globosus, Pichia anomala and Pichia subpelliculosa. 13. Use according to one of claims 10 to 12, wherein the Feed additives as fermentation broth or triggered Lyophilisate is present. 14. Yeast strain Hanseniaspora valbyensis 13cs / 6ρ ^- , deposited with the deposit number 12718 at the German Collection of Microorganisms and Cell Cultures GmbH. 15. Killer toxin obtained by fermentation of the Yeast strain according to claim 14. 16. Medicament containing a killer toxin according to claim 15 and a pharmaceutically acceptable carrier.