Compositions containing bacillaene-producing bacteria or preparations thereof

Abstract

The present invention relates to the use of bacillaene-producing bacteria as a probiotic feed ingredient and to compositions containing bacillaene-producing bacteria, as well as the use of bacillaene or derivatives thereof for the treatment of bacterial diseases.

Description

[Technical field]

[0001] The present invention relates to the use of bacillaene-producing bacteria as a probiotic feed ingredient and to compositions containing bacillaene-producing bacteria, as well as the use of bacillaene or a derivative thereof for the treatment of bacterial diseases.

[0002] The use of certain bacterial strains as probiotic ingredients in the feed industry has been previously disclosed in the state of the art. The function of probiotics (also called "direct-fed live microorganisms" or "DFM") is to positively affect the intestinal microflora by supporting the growth of beneficial bacteria and / or inhibiting the growth of pathogenic bacteria. Ideally, the use of probiotics would make the use of antimicrobial growth promoters (AGPs) unnecessary. However, in addition, it is desirable for probiotics to perform further functions, such as aiding in the digestion of certain feed ingredients.

[0003] Therefore, in view of the current state of the art, there is a need for probiotics which not only have a positive effect on the intestinal microflora, but also preferably perform at least one additional function.

[0004] Bacillaene is a secondary metabolite belonging to the polyene class that was discovered and isolated from the fermentation broth of a Bacillus subtilis strain (Patel et al., The Journal of Antibiotics (1995), Vol. 48(9), 997-1003).

[0005] According to the present invention, it has surprisingly been found, by carrying out experiments with knockout mutants, that bacillaene is capable of effectively inhibiting the growth of commercially important pathogens, in particular toxigenic Clostridium perfringens, responsible for necrotic enteritis, Vibrio parahaemolyticus, responsible for shellfish diseases such as early mortality syndrome, and Salmonella enterica, one of the main causes of food poisoning, making it possible to prove that bacteria producing bacillaene are suitable for use as probiotic feed and food ingredients and that bacillaene or its derivatives are suitable for the treatment of bacterial diseases.

[0006] A first subject of the present invention is therefore compositions comprising bacillaene or a derivative thereof, in particular bacillaene-producing microorganisms and / or bacillaene-containing preparations thereof, in particular food, feed and therapeutic compositions, as well as compositions for the treatment of plants.

[0007] According to the present invention, "derivatives of bacillaene" refers in particular to hydrogenated variants of bacillaene, in particular dihydrobacillaene, which have been reported in the literature.

[0008] A further subject of the present invention is therefore also the use of bacillaene-producing microorganisms or preparations thereof as probiotic feed or food ingredients.

[0009] Further subject of the present invention are in particular bacillaene-producing microorganisms suitable as probiotics. To be suitable as probiotics, the microorganisms according to the invention must preferably fulfil certain criteria, such as bile tolerance, heat tolerance and / or the ability to grow under anaerobic conditions.

[0010] The bacillaene producing bacteria according to the invention are in particular capable of inhibiting strains selected from Clostridium, preferably C. perfringens, in particular C. perfringens ATCC 13124, Salmonella, preferably Salmonella enterica, in particular Salmonella enterica subsp. enterica enteritidis DSM 14221, E. coli, in particular E. coli ATCC 11775, and Vibrio, preferably Vibrio parahaemolyticus, in particular Vibrio parahaemolyticus DSM 10027 and / or Vibrio parahaemolyticus TW01.

[0011] The bacillaene producing microorganism according to the invention is preferably a bacterium, more preferably selected from Bacillus, in particular B. subtilis, B. amyloliquefaciens, B. methylotrophicus and B. atropheus, and Paenibacillus, in particular P. polymixa and P. durus. In a highly preferred embodiment of the invention, the bacillaene producing microorganism is B. amyloliquefaciens or B. subtilis.

[0012] The bacillaene producing microorganism according to the invention is preferably further characterized in that it is capable of growing in the presence of 2 mM bile, preferably in the presence of 4 mM bile, in particular in the presence of 2 mM bile with an AUC5 performance value of at least 0.5, preferably at least 0.65, in particular at least 0.8, and an AUC10 performance value of at least 1.2, preferably at least 1.4, in particular at least 1.6; and / or is further characterized in that it is capable of growing in the presence of 0.3 wt.% bile, in particular in the presence of 0.3 wt.% chicken bile and / or in the presence of 0.3 wt.% porcine bile, preferably being able to withstand exposure to 0.3 wt.% bile, in particular 0.3 wt.% chicken bile and / or 0.3 wt.% porcine bile for at least 3 hours, preferably at least 5 or 8 hours.

[0013] The bacillaene producing microorganism according to the invention is preferably further characterized in that it is capable of growing anaerobically and in particular capable of degrading water-insoluble cellulose and proteins under anaerobic conditions.

[0014] The bacillaene producing microorganism according to the invention is preferably further characterized in that at least 50%, preferably at least 70 or 90% of the spores survive exposure to 99° C. for 20 minutes.

[0015] The Bacillus amyloliquefaciens strain that proved to be a suitable bacillaene-producing probiotic was identified by screening of naturally occurring isolates and has been deposited in the name of Evonik Degussa GmbH under the accession number DSM 33014 at the DSMZ (Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstrasse 7B, 38124 Braunschweig, Germany) on 7 February 2019 under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0016] Thus, in a preferred embodiment of the invention, the bacillaene producing microorganism is selected from the following group: a) Bacillus amyloliquefaciens strain DSM 33014; b) A mutant of the Bacillus strain deposited in DSM 33014, which preferably has at least 98%, in particular 99 or 99.5%, sequence identity to the genomic sequence of the Bacillus strain deposited in DSM 33014, and which preferably exhibits bile tolerance and / or thermotolerance and / or is capable of growing under anaerobic conditions.

[0017] The B. amyloliquefaciens strain DSM 33014 shows the 16S rDNA according to SEQ ID NO:1, the yqfD sequence according to SEQ ID NO:2, the gyrB sequence according to SEQ ID NO:3, the rpoB sequence according to SEQ ID NO:4 and the groEL sequence according to SEQ ID NO:5.

[0018] Thus, in a preferred embodiment of the present invention, the preferred bacillaene producing Bacillus strain, in particular Bacillus amyloliquefaciens strain DSM 33014, has at least one, preferably at least three, more preferably all of the following characteristics: a) a 16 S rDNA sequence having at least 98, preferably at least 99, 99.5 or 99.8%, more preferably 100% sequence identity to the 16 S rDNA sequence of strain DSM 33014 and / or SEQ ID NO:1; a) a yqfD sequence having at least 98, preferably at least 99, 99.5 or 99.8%, more preferably 100% sequence identity to the yqfD sequence of strain DSM 33014 and / or SEQ ID NO:2; b) a gyrB sequence having at least 98, preferably at least 99, 99.5 or 99.8%, more preferably 100% sequence identity to the gyrB sequence of strain DSM 33014 and / or SEQ ID NO:3; c) an rpoB sequence having at least 98, preferably at least 99, 99.5 or 99.8%, more preferably 100% sequence identity to the rpoB sequence of strain DSM 33014 and / or SEQ ID NO: 4; d) a groEL sequence having at least 98, preferably at least 99, 99.5 or 99.8%, more preferably 100% sequence identity to the groEL sequence of strain DSM 33014 and / or SEQ ID NO:5.

[0019] The bacillaene producing bacteria according to the invention comprises a gene cluster that allows the production of bacillaene. The gene cluster preferably comprises the genes baeJ, baeL, baeM, baeN and baeR, which code for subunits of the bacillaene synthesis protein complex, also referred to as polyketide synthase or PKS proteins.

[0020] The B. amyloliquefaciens strain DSM 33014 exhibits the baeJ sequence according to SEQ ID NO:6, the baeL sequence according to SEQ ID NO:7, the baeM sequence according to SEQ ID NO:8, the baeN sequence according to SEQ ID NO:9 and the baeR sequence according to SEQ ID NO:10.

[0021] Thus, according to the invention, the gene cluster of PKS proteins preferably comprises the following genes: a) a baeJ gene having a sequence identity of at least 80%, preferably at least 85, 90 or 95%, more preferably at least 98 or 99%, especially 100% to the sequence of the baeJ sequence of strain DSM 33014 and / or to SEQ ID NO: 6; b) a baeL gene having a sequence identity of at least 80%, preferably at least 85, 90 or 95%, more preferably at least 98 or 99%, especially 100% to the sequence of the baeL sequence of strain DSM 33014 and / or to SEQ ID NO: 7; a) a baeM gene having a sequence identity of at least 80%, preferably at least 85, 90 or 95%, more preferably at least 98 or 99%, especially 100% to the sequence of the baeM sequence of strain DSM 33014 and / or to SEQ ID NO: 8; c) a baeN gene having a sequence identity of at least 80%, preferably at least 85, 90 or 95%, more preferably at least 98 or 99%, especially 100% to the sequence of the baeN sequence of strain DSM 33014 and / or to SEQ ID NO: 9; d) a baeR gene having at least 80%, preferably at least 85, 90 or 95%, more preferably at least 98 or 99%, especially 100% sequence identity to the sequence of the baeR sequence of strain DSM 33014 and / or to SEQ ID NO: 10.

[0022] The microorganisms according to the invention are preferably natural isolates, but may also be mutants of natural isolates, in particular natural mutants.Furthermore, the microorganisms according to the invention can also be obtained by genetic engineering, in particular by incorporating the genes encoding the bacillaene producing enzyme subunits, in particular the above mentioned genes baeJ, baeL, baeM, baeN and baeR, into a microorganism, preferably a above mentioned microorganism, more preferably a B. subtilis or B. amyloliquefaciens strain, in which case, preferably, before the incorporation of the genes, the microorganism is not capable of producing bacillaene.

[0023] The term "spontaneous mutant" refers to a mutant that arises from a natural isolate without the deliberate use of a mutagen. Such a natural mutant can be obtained by growing the natural isolate in classical methods, for example in the presence of UV light and / or by applying high temperature or protoplast formation and / or in the presence of a certain antibiotic to which the parent strain is sensitive, and testing any resistant mutant for improved biological activity or improved ability to improve one or more indicators of animal health, especially intestinal health. Other methods for identifying natural mutants are known to those skilled in the art. However, in addition to these preferred natural mutants, any other type of mutant of a natural isolate, such as a mutant obtained by genetic engineering, is also included in the present invention.

[0024] Thus, one particular embodiment of the present invention is a non-naturally occurring mutant of a naturally occurring bacillaene producing strain, in particular a naturally occurring mutant as defined above, preferably characterized by the features described herein above.

[0025] Thus, a further particular embodiment of the present invention is a bacillaene-producing microorganism obtained by integration of the genes encoding the subunits of the bacillaene-producing enzyme, in particular the genes baeJ, baeL, baeM, baeN and baeR mentioned hereinbefore.

[0026] In a preferred embodiment of the invention, the microorganisms and preparations of the invention are administered orally to animals or humans.

[0027] Further subject matter of the present invention are therefore bacillaene-producing microorganisms and / or preparations thereof, and / or compositions such as feed, food, drinking water and rearing water containing bacillaene or its derivatives, as well as therapeutic compositions.

[0028] A further subject of the present invention is also the use of bacillaene-producing microorganisms and / or preparations thereof as probiotic ingredients (DFM) in feed or food products.

[0029] Preferred food products according to the invention are dairy products, in particular yoghurt, cheese, milk, butter and quark.

[0030] The cells of the microorganisms of the present invention may be present, in particular in the compositions of the present invention, as spores (dormant), vegetative cells (growing), transitional cells (in a transition stage from the vegetative phase to the sporulation phase), or a combination of at least two, in particular all, of these cell types. In a preferred embodiment, the compositions of the present invention mainly or exclusively comprise spores.

[0031] Additionally or alternatively, microbial cells may be used in a non-living, inactivated form, since even non-viable cells are expected to have a probiotic effect. Methods for inactivating cells are known to those skilled in the art.

[0032] The bacillaene producing microorganisms of the invention and compositions containing them, when administered to an animal, preferably promote the health of such animal, and / or improve the general body condition of such animal, and / or improve the feed conversion ratio of such animal, and / or reduce the mortality rate of such animal, and / or increase the survival rate of such animal, and / or improve the weight gain of such animal, and / or increase the productivity of such animal, and / or increase the disease resistance of such animal, and / or increase the immune response of such animal, and / or establish or maintain a healthy gut microbiota in such animal, and / or reduce the shedding of pathogens in the feces of such animal. In particular, the microorganisms and compositions of the invention may be used to help re-establish a healthy balance of the gut microbiota following the administration of antibiotics for therapeutic purposes.

[0033] A further subject of the present invention is therefore a method for promoting the health of an animal and / or improving the general physical condition of an animal and / or improving the feed conversion rate of an animal and / or reducing the mortality rate of an animal and / or increasing the survival rate of an animal and / or improving the weight gain of an animal and / or increasing the productivity of an animal and / or increasing the disease resistance of an animal and / or increasing the immune response of an animal and / or establishing or maintaining a healthy intestinal microflora in an animal and / or reducing the shedding of pathogens in the faeces of an animal, which comprises administering a microorganism of the invention and / or a preparation thereof and / or a composition of the invention, and / or bacillaene or a derivative thereof to an animal, in particular an aquatic animal.

[0034] A further subject of the present invention is therefore also the use of the microorganism and / or preparation and / or composition of the invention for promoting the health of an animal and / or improving the general physical condition of an animal and / or improving the feed conversion rate of an animal and / or reducing the mortality rate of an animal and / or increasing the survival rate of an animal and / or improving the weight gain of an animal and / or increasing the productivity of an animal and / or increasing the disease resistance of an animal and / or increasing the immune response of an animal and / or establishing or maintaining a healthy intestinal microflora in an animal and / or reducing the shedding of pathogens in the faeces of an animal, wherein the microorganism and / or preparation and / or composition of the invention, and / or bacillaene or a derivative thereof, is administered to the animal, which animal is in particular an aquatic animal.

[0035] A further subject of the present invention is therefore also the above mentioned microorganisms and / or preparations and / or compositions of the invention, and / or bacillaene or derivatives thereof, which promote the health of an animal and / or improve the general physical condition of an animal and / or improve the feed conversion ratio of an animal and / or reduce the mortality rate of an animal and / or increase the survival rate of an animal and / or improve the weight gain of an animal and / or increase the productivity of an animal and / or increase the disease resistance of an animal and / or increase the immune response of an animal and / or establish or maintain a healthy intestinal microflora in an animal and / or reduce the shedding of pathogens in the faeces of an animal.

[0036] "Increasing animal productivity" refers specifically to any of the following: production of more or better quality eggs, milk or meat, or increased production of weanlings.

[0037] The methods and uses of the microorganisms, preparations and compositions of the invention may be therapeutic or non-therapeutic. In particularly preferred embodiments of the invention, the methods and uses are non-therapeutic, in particular feeding applications.

[0038] Since untreated animal manure, due to pathogenic bacteria and other components, can have harmful environmental effects, in particular on the animals themselves and / or on humans who come into contact with the manure, which can be avoided by feeding the animals with the microorganisms, compositions or preparations of the invention or by directly treating the manure or animal litter with the microorganisms, compositions or preparations of the invention, a further subject of the invention is a method for controlling and / or avoiding the harmful environmental effects of manure or contaminated liquids, comprising the step of applying to the manure, contaminated liquid, litter, pit or manure pit at least one bacillaene producing microorganism and / or its preparation and / or composition according to the invention. Preferably, the microorganism, preparation or composition is applied in liquid form, for example by spraying, or as a powder, for example by dusting.

[0039] Since harmful bacteria can have a negative effect on the consistency of the litter and can result in a slightly or very runny litter which can cause footpad lesions in particular in poultry, which can be avoided by feeding the animal a microorganism, composition or preparation of the invention, a further subject of the invention is a method for controlling and / or improving the consistency of the litter, in particular a method for ensuring a solid consistency of the litter and / or a method for avoiding footpad lesions, which comprises feeding an animal, in particular poultry, with at least one microorganism, a preparation and / or a composition according to the invention.

[0040] The microorganisms and preparations according to the invention can also be used to improve the quality of feed and food compositions, as well as the quality of water and aqueous solutions.A further subject of the invention is therefore also a method for controlling and / or improving the quality of a feed or food composition, as well as the quality of water or aqueous solutions, in particular drinking water and / or rearing water, comprising the step of applying at least one microorganism and / or at least one preparation and / or at least one composition according to the invention to the feed, food, water or aqueous solution.

[0041] Furthermore, the microorganisms and preparations according to the invention can also be used for the treatment of microbial diseases in plants.A further subject of the invention is therefore also a method for treating and / or preventing microbial diseases in plants, in particular in cultivated plants, which comprises a step of applying to the plant at least one microorganism and / or at least one preparation and / or at least one composition according to the invention.Application can be carried out in liquid form, for example by spraying, or in solid form, in particular as a powder, preferably as a formulated powder.

[0042] By using the microorganisms, preparations and compositions of the invention, preferably an improvement in at least one of the above mentioned characteristics is achieved, where the achievement of a characteristic preferably means an improvement of at least 1%, more preferably at least 3 or at least 5%, compared to a suitable negative control. As negative control, any average known in the animal husbandry field can be used, but preferably, as negative control, an animal is used that is subjected to the same treatment as the test animals, but is not administered the microorganism and / or preparation of the invention.

[0043] In addition to the ability to inhibit the growth of the previously mentioned pathogenic bacteria through the production of bacillaene, the microorganisms and preparations used according to the invention are preferably capable of inhibiting the growth of further pathogenic bacteria.

[0044] In particular, the microorganisms, preparations and compositions of the invention can be administered or fed to an animal in an amount effective to inhibit and / or reduce the growth of pathogenic bacteria in the intestine of the animal. Such pathogenic bacteria can include, in addition to Clostridium, Salmonella, Vibrio and Escherichia coli, Listeria, Enterococcus, Staphylococcus, Aeromonas, Streptococcus, Campylobacter, Shigella, Haemophilus and Brachyspira, among others. In this regard, the method of the invention can be used to reduce the amount of pathogenic bacteria, viruses and protozoa excreted in the feces of an animal. The method of the invention can also be used to maintain or increase the growth of beneficial bacteria, such as lactic acid bacteria, in the intestine of an animal. By reducing pathogenic bacteria and / or increasing or maintaining beneficial bacteria, the composition of the invention can maintain an overall healthy intestinal microflora.

[0045] Thus, a further subject of the present invention is a method for inhibiting and / or reducing the growth of pathogenic bacteria, and / or maintaining and / or increasing the growth of beneficial bacteria, in particular in the intestine of an animal or human, comprising administering to the animal or human a microorganism, preparation and / or composition according to the invention, wherein the pathogenic bacteria are preferably Clostridium, in particular C. perfringens, C. difficile, C. novyi, C. septicum and C. colinum, Listeria, in particular L. monocytogenes, L. seeligeri and L. welshimeri, Salmonella, in particular the subspecies enterica, arizonae, bongori, in particular the serovar S. gallinarum ... gallinarum, S. pullorum, S. typhimurium, S. enteritidis, S. cholerasuis, S. heidelberg, S. dublin, S. hadar, S. typhi, S. paratyphi and S. infantis, S. enterica, Enterococcus, in particular E. faecalis, E. faecium and E. cecorum, Staphylococcus, in particular S. aureus, Aeromonas, Streptococcus, in particular S. suis. suis and S. gallinaceus, Campylobacter, especially C. jejuni and C. coli.coli, Escherichia coli, Haemophilus, particularly Haemophilus parasuis, Brachyspira, particularly Brachyspira hyodysenteriae, and Vibrio, particularly V. parahaemolyticus and V. harveyi, and the beneficial bacteria are preferably selected from Lactobacillus, particularly Lactobacillus and Bifidobacterium. The pathogenic microorganism is in a preferred embodiment selected from Clostridium, particularly C. perfringens, Salmonella, particularly S. enterica, and Vibrio, particularly V. parahaemolyticus.

[0046] In a preferred embodiment of the invention, the amount of at least one pathogenic bacterium, in particular the amount of C. perfringens, S. enterica and / or V. parahaemolyticus, is reduced by at least 0.5 log, more preferably by at least 1 log, 2 log or 3 log.

[0047] Thus, further subject of the present invention are also microorganisms, preparations and compositions according to the invention for inhibiting and / or reducing the growth of pathogenic bacteria and / or maintaining and / or increasing the growth of beneficial bacteria, in particular in the intestine of animals or humans, whereby the pathogenic bacteria are preferably Clostridium, in particular C. perfringens, C. difficile, C. novyi, C. septicum and C. colinum, Listeria, in particular L. monocytogenes, L. seeligeri and L. welshimeri, Salmonella, in particular the subspecies enterica, arizonae, bongori, in particular the serovar S. gallinarum, in particular the serovar S. gallinarum, S. pullorum, S. typhimurium, S. enteritidis, S. cholerasuis, S. heidelberg, S. dublin, S. hadar, S. typhi, S. paratyphi and S. infantis, S. enterica, Enterococcus, in particular E. faecalis, E. faecium and E. cecorum, Staphylococcus, in particular S. aureus, Aeromonas, Streptococcus, in particular S. suis. suis and S. gallinaceus, Campylobacter, especially C. jejuni and C. coli.coli, Escherichia coli, Haemophilus, in particular Haemophilus parasuis, Brachyspira, in particular Brachyspira hyodysenteriae, and Vibrio, in particular V. parahaemolyticus and V. harveyi, and the beneficial bacteria are preferably selected from Lactobacillus, in particular Lactobacillus and Bifidobacterium. The pathogenic microorganisms are in a preferred embodiment selected from Clostridium, in particular C. perfringens, Salmonella, in particular S. enterica, and Vibrio, in particular V. parahaemolyticus.

[0048] The occurrence and / or increased proliferation of pathogenic bacteria can cause or may cause the occurrence of certain diseases. For example, the occurrence and / or increased proliferation of Clostridium perfringens can cause the occurrence of enteric diseases in pigs and poultry, especially the occurrence of necrotic enteritis. The occurrence and / or increased proliferation of C. perfringens can also cause the occurrence of further diseases such as bacterial enteritis, gangrenous dermatitis and colangiohepatitis. Infection with C. perfringens, even in its mildest form, can already be accompanied by diarrhea, which can cause the litter to become wet and cause secondary diseases such as foot and plantar dermatitis. C. perfringens type C is generally considered to be the primary cause of necrotizing enteritis and necrotizing hemorrhagic enteritis in piglets, while type A has been associated with enteric disease in suckling and fed pigs with milder necrotizing enterocolitis and villous atrophy.

[0049] Clostridium difficile is an important emerging pathogen causing diarrhea primarily in neonatal piglets. Affected piglets may have respiratory distress, abdominal distension and scrotal edema.

[0050] Staphylococcus aureus subsp. aureus can cause digital bumps in chickens and streptococcal mastitis in sows, and can produce toxins that cause food poisoning in humans.

[0051] E. cecorum is known to cause lameness, arthritis and osteomyelitis in broilers, usually due to inflammation of the joints and / or bone tissue. Furthermore, E. cecorum can cause inflammation of the pericardium.

[0052] Salmonella bacteria are an important cause of human food poisoning, often associated with the consumption of meat, such as poultry, pork or products derived from them. Therefore, the control of Salmonella is an important challenge for the meat production industry. In Europe, a baseline study carried out in 2005 on the prevalence of Salmonella in laying hen flocks showed that at EU-wide level, 20.3% of large laying hen holdings were bacteriologically positive for S. enteritidis. In some countries, the prevalence was even higher than 80% [European Food Safety Authority (2006), “Preliminary report: analysis of the baseline study on the prevalence of salmonella in laying hen flocks of Gallus gallus”].

[0053] S. gallinarum is the cause of fowl typhoid fever and S. pullorum is the cause of pullorum fever, both diseases which cause great damage to the poultry industry.

[0054] C. coli is a food-borne bacterium that most people become infected with by eating pork, which usually contains the bacteria. It causes gastroenteritis and acute enteritis in humans, as well as acute diarrhea. Pigs are the primary host, but it can also infect humans, birds, and a wide range of other animals.

[0055] S. gallinaceus can cause septicemia in poultry. Gross lesions included splenomegaly, hepatomegaly, renal enlargement and congestion. Multiple areas of necrosis and / or infarction in the liver and spleen, accompanied by valvular endocarditis, were also observed.

[0056] S. suis is an important pathogen in pigs and one of the most important causes of bacterial mortality in postweaning piglets, causing septicaemia, meningitis and many other infectious diseases.

[0057] Vibrio parahaemolyticus is particularly responsible for shellfish diseases such as early mortality syndrome (EMS), also known as acute hepatopancreatic necrosis (AHPND), which affects both black prawn (Penaeus monodon) and white shrimp (Penaeus vannamei).

[0058] Pathogens may cause further diseases such as polyarthritis, fibrous polyserositis, postweaning intestinal disorders such as postweaning diarrhea and edema disease, and swine dysentery.

[0059] A further subject of the present invention is therefore also a therapeutic composition comprising a microorganism and / or a preparation and / or a composition according to the invention and / or bacillaene or a derivative thereof, in particular a therapeutic composition for the treatment of aquatic animals.

[0060] A preferred subject of the present invention is therefore a therapeutic composition for the treatment of diseases, in particular enteric diseases, associated with bacterial infections, in particular with Clostridium, in particular C. perfringens, and / or Salmonella, in particular S. enterica, preferably S. enterica subsp. enterica enteritidis, and / or Vibrio, in particular Vibrio parahaemolyticus.

[0061] A further preferred subject in this connection is therefore a therapeutic composition comprising the strain and / or preparation and / or composition of the invention and / or bacillaene or a derivative thereof for the treatment and / or prevention of necrotic enteritis and / or necrotic hemorrhagic enteritis, in particular asymptomatic necrotic enteritis and / or necrotic hemorrhagic enteritis, in animals, preferably pigs or poultry.

[0062] A further preferred subject in this connection is therefore a therapeutic composition comprising the microorganism and / or preparation and / or composition of the invention and / or bacillaene or a derivative thereof for treating and / or preventing premature mortality syndrome in animals, preferably animals kept in aquaculture, more preferably crustaceans, in particular shrimps and prawns.

[0063] A further preferred subject in this connection is therefore a therapeutic composition for treating and / or preventing diseases resulting from food poisoning, comprising a microorganism and / or a preparation and / or a composition according to the invention and / or bacillaene or a derivative thereof.

[0064] Another preferred subject in this connection is therefore a therapeutic composition comprising the microorganism and / or preparation and / or composition of the invention and / or bacillaene or a derivative thereof for the treatment and / or prevention of bacterial enteritis, necrotic dermatitis, cholangiohepatitis, clostridiosis, diarrhoea, dyspnea, abdominal distension, scrotal oedema, digital bumps, digital plantar dermatitis, streptococcal mastitis, lameness, arthritis, polyarthritis, fibrous polyserositis, post-weaning intestinal disorders such as post-weaning diarrhoea and oedema disease, dysentery, osteomyelitis, inflammation of the joints and / or bone tissue, inflammation of the pericardium, splenomegaly, hepatomegaly, nephromegaly, congestion, necrosis of the liver or spleen, infarction, valvular endocarditis, sepsis and / or meningitis in animals, preferably pigs or poultry.

[0065] A further subject of the present invention is therefore also the treatment and / or prevention of diseases, in particular enteric diseases, associated with bacterial infections by Clostridium, in particular C. perfringens, and / or Salmonella, in particular S. enterica, preferably S. enterica subsp. enterica enteritidis, and / or Vibrio, in particular Vibrio parahaemolyticus, by administering a microorganism and / or a preparation and / or a composition according to the invention and / or bacillaene or a derivative thereof to an animal in need thereof, the animal being preferably a pig or a poultry.

[0066] A further subject of the present invention is therefore also the treatment and / or prevention of diseases, in particular intestinal diseases, preferably necrotizing enteritis or necrotic hemorrhagic enteritis, in particular asymptomatic necrotizing enteritis or asymptomatic necrotic hemorrhagic enteritis, by administering a microorganism and / or a preparation and / or a composition according to the invention and / or bacillaene or a derivative thereof to an animal in need thereof, which animal is preferably a pig or a poultry.

[0067] A further subject of the present invention is therefore also the treatment and / or prevention of diseases in aquatic animals, in particular early mortality syndrome, by administering the microorganisms and / or preparations and / or compositions of the invention, and / or bacillaene or a derivative thereof, to an animal in need thereof, which is preferably an animal reared in aquaculture, more preferably a crustacean, in particular shrimps and prawns.

[0068] A further subject of the present invention is therefore also the treatment and / or prevention of diseases resulting from food poisoning, by administering the microorganisms and / or preparations and / or compositions of the invention and / or bacillaene or a derivative thereof to an animal or human in need thereof.

[0069] A further subject of the present invention is therefore also the treatment and / or prevention of diseases resulting from food poisoning, in which the microorganisms and / or preparations and / or compositions according to the invention and / or bacillaene or its derivatives are applied to animal food products, in particular meat or eggs, in order to avoid human poisoning.

[0070] A further subject of the present invention is therefore also the treatment and / or prevention of diseases, preferably of pigs or poultry, selected from bacterial enteritis, necrotic dermatitis, cholangiohepatitis, clostridiosis, diarrhoea, bloating, abdominal distension, scrotal oedema, digital swelling, digital plantar dermatitis, streptococcal mastitis, lameness, arthritis, polyarthritis, fibrous polyserositis, postweaning intestinal disorders such as postweaning diarrhoea and oedema disease, dysentery, osteomyelitis, inflammation of the joints and / or bone tissue, inflammation of the pericardium, splenomegaly, hepatomegaly, nephromegaly, congestion, necrosis of the liver or spleen, infarction, valvular endocarditis, septicemia and / or meningitis, by administering a microorganism and / or a preparation and / or a composition according to the invention and / or bacillaene or a derivative thereof to an animal in need thereof.

[0071] The microorganisms and / or preparations and / or compositions of the invention can be administered to the animal in the feed and / or drinking water over several days throughout the animal's life or at a particular stage or part of the animal's life. For example, the microorganisms and / or preparations and / or compositions can be administered only in the weaner feed or only in the finisher feed of livestock.

[0072] A particular subject of the present invention is also a method for promoting human health and / or improving the general physical condition of a human and / or increasing the disease resistance of a human and / or increasing the immune response of a human and / or establishing or maintaining a healthy intestinal microflora in a human, which comprises administering to a human a microorganism and / or a preparation and / or a composition of the invention, and / or bacillaene or a derivative thereof.

[0073] A further subject of the present invention is therefore also the use of the microorganisms and / or preparations and / or compositions of the invention for promoting human health and / or improving the general physical condition of a human and / or increasing the disease resistance of a human and / or increasing the immune response of a human and / or establishing or maintaining a healthy intestinal microflora in a human, by administering to a human the microorganisms and / or preparations and / or compositions of the invention, and / or bacillaene or a derivative thereof.

[0074] The compositions of the invention, in particular feed, food and pharmaceutical compositions, as well as drinking or rearing water, preferably contain the microorganisms of the invention, and preferably contain up to about 1×10 3 ~about 2×10 12 The rate of CFU / g feed or ml water is approximately 1 × 10 3 or about 1 x 10 4 or about 1 x 10 5 or about 1 x 10 6 or about 1 x 10 7 or about 1 x 10 8 or about 1 x 10 9 or about 1 x 10 10 or about 1 x 10 11 or about 1 x 1012 CFU / g feed or ml water, preferably about 1×10 4 ~Approx. 1×10 10 CFU / g feed or ml water, more preferably 1×10 4 ~1×10 7 The animals are dosed with CFU / g feed or ml water.

[0075] Accordingly, preferred amounts of the strains and / or preparations of the invention in the feed, food and water compositions of the invention are preferably in the range of 0.1 wt% to 10 wt%, more preferably 0.2 wt% to 5 wt%, especially 0.3 wt% to 3 wt%.

[0076] The method of the present invention can be used with any type of animal, particularly any type of non-human and non-insect animal, more preferably any type of vertebrate animal, such as mammals, aquatic animals and birds.

[0077] Animals that may benefit from the present invention include, but are not limited to, farm animals, pets, exotic animals, zoo animals, aquatic animals, and animals used for sport, recreation or work.

[0078] Pets are preferably selected from dogs, cats, domestic birds and domestic exotic animals.

[0079] The aquatic animals are preferably selected from fin fish and crustaceans, preferably intended for human nutrition. These include, in particular, carp, tilapia, catfish, tuna, salmon, trout, barramundi, bream, perch, cod, shrimp, lobster, crab, prawn and crayfish. Preferred salmon species in this context are Atlantic salmon, sockeye salmon, cherry salmon, Chinook salmon, salmon, coho salmon, Donau salmon, Pacific salmon and pink salmon.

[0080] Further preferred aquatic animals are farmed fish which are subsequently processed to obtain fishmeal or fish oil. In this context, the fish are preferably herring, pollack, menhaden, anchovies, capelin or cod.

[0081] In a further preferred embodiment, the animals are livestock kept for food consumption or to produce food, such as poultry, pigs and ruminants.

[0082] Poultry may be selected from productive or domesticated poultry, as well as ornamental poultry or wild birds.

[0083] Preferred productive poultry in this context are chickens, turkeys, ducks and geese. The productive livestock in this context are preferably poultry optimized for young stock production or poultry optimized for meat production.

[0084] Preferred ornamental poultry or wild birds are peacocks, pheasants, partridges, chukkars, guineafowl, quail, capercaillie, ptarmigan, pigeons and swans, with quail being particularly preferred.

[0085] Further preferred poultry are ratites, especially ostriches and emus, and parrots.

[0086] The ruminant according to the present invention is preferably selected from cows, goats and sheep.In one embodiment, the composition of the present invention can be fed to pre-weaned animals to improve their health, in particular to reduce the incidence of diarrhea in these animals.Pre-weaned animals are ruminant animals including calves ranging from just after birth to about 12 weeks of age.

[0087] A particularly preferred embodiment of the present invention is therefore a method of feeding animals, preferably terrestrial animals, in particular pigs or birds, or aquatic animals, in particular crustaceans such as shrimps and prawns, comprising administering to the animal a bacillaene-producing microorganism or a preparation thereof, or a feed composition containing a bacillaene-producing microorganism or a preparation thereof.

[0088] The compositions of the present invention may include at least one carrier or typical feed ingredient, or a combination thereof.

[0089] Suitable carriers are inert formulation ingredients added to improve recovery, efficacy or physical properties and / or aid in packaging and administration. Such carriers can be added individually or in combination. These carriers can be selected from anti-caking agents, antioxidants, bulking agents and / or protectants. Examples of useful carriers include polysaccharides (especially starch, maltodextrin, methylcellulose, gums, chitosan and / or inulin), protein sources (especially skimmed milk powder and / or sweet whey powder), peptides, sugars (especially lactose, trehalose, sucrose and / or dextrose), lipids (especially lecithin, vegetable oils and / or mineral oils), salts (especially sodium chloride, sodium carbonate, calcium carbonate, chalk, limestone, magnesium carbonate, sodium phosphate, calcium phosphate, magnesium phosphate and / or sodium citrate), and silicates (especially clays, especially zeolitic clays, amorphous silica, fumed / precipitated silica, zeolites, fuller's earth, baylith, clintpolite, montmorillonite, diatomaceous earth, talc, bentonite, and / or silicates such as aluminium, magnesium and / or calcium silicate). Suitable carriers for animal feed additives are described in the official publication of American Feed Control Officials, Inc., published annually. See, for example, Official Publication of American Feed Control Officials, Sharon Krebs, editor, 2006 edition, ISBN 1-878341-18-9. The carrier can be added after concentrating the fermentation broth and / or during and / or after drying. Preferred carriers according to the present invention are selected from calcium carbonate, diatomaceous earth and vegetable oil.

[0090] A preferred embodiment of the present invention is a concentrate composition, in particular a feed additive composition, i.e. a composition suitable for the preparation of a feed composition comprising at least one microorganism according to the invention and at least one carrier as mentioned above, wherein the at least one microorganism is preferably present in an amount of 0.1-10 wt %, more preferably in an amount of 0.2-5 wt %, in particular in an amount of 0.3-3 wt %, especially in an amount of 0.4-2.2 wt %, and the at least one carrier is preferably present in an amount of at least 90 wt %, preferably in an amount of 90-99.9 wt %, more preferably in an amount of 95-99.8 wt %, in particular in an amount of 97-99.7 wt %, especially in an amount of 97.8-99.6 wt %, and the carrier preferably consists essentially of limestone, in particular limestone containing small amounts of diatomaceous earth and / or vegetable oil.

[0091] These preferred compositions of the invention containing stabilized microorganisms can be used for the preparation of feed and pharmaceutical compositions, as well as drinking water and rearing water, preferably comprising the strain according to the invention in the amounts mentioned herein above. In a preferred embodiment, 50 to 1000 grams of such concentrated composition, in particular 50, 100, 250, 500 or 1000 grams of such concentrated composition per ton of feed, drinking water or rearing water, are used to obtain a composition that can be used for feeding animals. These concentrated compositions preferably contain at least one strain of the invention in an amount of 1 x 10 per gram of concentrated composition. 9 ~2×10 11 CFU, specifically 2 × 10 9 ~1×10 11 Contains CFU amounts.

[0092] Starting from these concentrate compositions, feed and food compositions can be prepared by mixing the concentrate compositions with typical feed or food ingredients, respectively.

[0093] Suitable typical animal feed ingredients which may be contained in the composition according to the invention and / or which may be used for the preparation of a feed composition starting from the concentrated composition according to the invention include one or more of the following: proteins, carbohydrates, fats, further probiotics, prebiotics, enzymes, vitamins, immunomodulators, milk replacers, minerals, amino acids, anticoccidial agents, acid-based products and / or drugs such as antibiotics.

[0094] Carbohydrate-containing ingredients which can be used according to the invention are, for example, forage, roughage, whole wheat flour, sunflower meal or soybean meal, and mixtures thereof.

[0095] Protein-containing ingredients which can be used according to the invention are, for example, soy protein, pea protein, wheat gluten or corn gluten, and mixtures thereof.

[0096] Fat-containing ingredients which can be used according to the invention are in particular oils of both animal and vegetable origin, such as vegetable oils, for example soybean oil, rapeseed oil, sunflower seed oil, linseed oil or palm oil, fish oils, and mixtures thereof.

[0097] Protein-containing ingredients which additionally contain fat and which can be used according to the invention are, for example, fish meal, krill meal, bivalve meal, squid meal or shrimp shells, and combinations thereof.

[0098] Further probiotics (DFM) which can be used in accordance with the invention in combination with the microorganisms and preparations of the invention are preferably Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus pumilus, Bacillus laterosporus, Bacillus coagulans, Bacillus alevi, Bacillus cereus, Bacillus badius, Bacillus thurigiensis, Enterococcus faecium and Pediococcus acidilactici. Preferred examples are bacteria selected from the species Bacillus subtilis DSM 32539 (deposited at the DSMZ on June 14, 2017 under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure) and its derivatives, Bacillus licheniformis DSM 32314 and Bacillus subtilis DSM 32315 (both deposited at the DSMZ on May 12, 2016 under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure) and their derivatives, Bacillus subtilis sold under the trademark CLOSTAT® by Kemin. subtilis PB6 (described in U.S. Pat. No. 7,247,299 and deposited under ATCC Accession No. PTA-6737), Bacillus subtilis C-3102 (U.S. Pat. No. 4,919,No. 936 and deposited at the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan, as FERM BP-1096, Bacillus subtilis DSM 17299 sold under the trademark GalliPro® by Chr. Hansen, Bacillus licheniformis DSM 17236 sold under the trademark GalliProTect® by Chr. Hansen, Bacillus licheniformis DSM Z 5749 and Bacillus subtilis DSM Z 5751 sold under the trademark BioPlus® YC by Chr. Hansen. 5750 spore mixture, B. subtilis DSM 29784 sold under the trademark Alterion® by Adisseo / Novozymes, Bacillus subtilis sold under the trademark PORCBOOST® by Chr. Hansen, or U.S. Pat. No. 6,849,Bacillus coagulans strains described in US Pat. No. 256, ...

[0099] The prebiotics which can be used according to the invention are preferably selected from oligosaccharides, in particular galactooligosaccharides, sialyloligosaccharides, lactulose, lactofructose oligosaccharides, fructooligosaccharides, palatinose or isomaltose oligosaccharides, glycosylsucrose, maltooligosaccharides, isomaltooligosaccharides, cyclodextrins, gentiooligosaccharides, soybean oligosaccharides, xylooligosaccharides, dextran, pectin, polygalacturonan, rhamnogalacturonan, mannan, hemicellulose, arabinogalactan, arabinan, arabinoxylan, resistant starch, mebiose, chitosan, agarose, inulin, tagatose, polydextrose and alginates.

[0100] Enzymes which may be used in the feed composition according to the invention and which may aid in the digestion of the feed are preferably phytases (EC 3.1.3.8 or 3.1.3.26), xylanases (EC 3.2.1.8), galactanases (EC 3.2.1.89), galactosidases, in particular α-galactosidase (EC 3.2.1.22), proteases (EC 3.4), phospholipases, in particular phospholipases A1 (EC 3.1.1.32), A2 (EC 3.1.1.4), C (EC 3.1.4.3) and D (EC 3.1.4.4), lysophospholipases (EC 3.1.1.5), amylases, in particular α-amylase (EC 3.2.1.1); lysozyme (EC 3.2.1.17), glucanases, in particular β-glucanases (EC EC 3.2.1.4 or EC 3.2.1.6), glucoamylase, cellulase, pectinase, or any mixture thereof.

[0101] Examples of commercially available phytases include Bio-Feed™ Phytase (Novozymes), Ronozyme® P and HiPhos™ (DSM Nutritional Products), Natuphos™ (BASF), Finase® and Quantum® Blue (AB Enzymes), Phyzyme® XP (Verenium / DuPont), and Axtra® PHY (DuPont). Other preferred phytases include, for example, those described in WO 98 / 28408, WO 00 / 43503 and WO 03 / 066847.

[0102] Examples of commercially available xylanases include Ronozyme® WX and G2 (DSM Nutritional Products), Econase® XT and Barley (AB Vista), Xylathin® (Verenium) and Axtra® XB (xylanase / β-glucanase, DuPont). Examples of commercially available proteases include Ronozyme® ProAct (DSM Nutritional Products).

[0103] Vitamins that can be used according to the invention are, for example, vitamin A, vitamin D3, vitamin E, vitamin K, e.g. vitamin K3, vitamin B12, biotin, choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and pantothenate, e.g. calcium D-pantothenate, or combinations thereof.

[0104] Immunomodulators that can be used are, for example, antibodies, cytokines, spray-dried plasma, interleukins or interferons, or combinations thereof.

[0105] Minerals that can be used according to the invention are, for example, boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, zinc, calcium, magnesium, potassium or sodium, or combinations thereof.

[0106] Amino acids that can be used according to the invention are, for example, lysine, alanine, threonine, methionine or tryptophan, or combinations thereof.

[0107] Thus, a further embodiment of the present invention is a method for preparing an animal feed composition, comprising mixing at least one microorganism and / or at least one preparation and / or at least one concentrated composition of the present invention, in particular in an amount effective for promoting animal health, in particular intestinal health, with feed ingredients such as proteins, lipids and / or carbohydrates, and optionally further beneficial substances, preferably as mentioned above, to obtain a feeding product, which may also include, for example, a pelleting step.

[0108] Standard pelleting processes known to those skilled in the art can be used, including extrusion of dry or semi-moist feed. A preferred pelleting temperature is from about 65°C to about 120°C.

[0109] The microorganisms and compositions of the present invention can be obtained by culturing the microorganisms of the present invention according to methods known in the art, including using media and other methods described in, for example, US Pat. No. 6,060,051, EP 0287699 or US 2014 / 0010792. Conventional large-scale microbial culture processes include submerged fermentation, solid-state fermentation or liquid surface culture. As nutrients are exhausted toward the end of fermentation, the cells begin to transition from the growth phase to the sporulation phase, so that the end products of fermentation are primarily spores, metabolic products and residual fermentation medium. Sporulation is part of the natural life cycle of these microorganisms and is generally initiated by cells in response to nutrient limitation. Fermentation is configured to obtain high levels of colony-forming units of cells and promote sporulation. The bacterial cells, spores and metabolic products in the culture medium resulting from fermentation can be used directly or concentrated by conventional industrial methods such as centrifugation, tangential flow filtration, depth filtration and evaporation. The concentrated fermentation broth can be washed, for example, by a diafiltration process, to remove residual fermentation broth and metabolic products.

[0110] The fermentation broth or broth concentrate can be dried using conventional drying processes or methods such as spray drying, freeze drying, tray drying, fluid bed drying, drum drying or evaporation, with or without the addition of a carrier. The resulting dried product can be further processed, such as by grinding or granulation, to achieve a particular particle size or physical shape. The above-mentioned carriers may be added after drying.

[0111] The preparations of the microorganisms of the invention, which are a particular subject of the invention, may be cell-free preparations or preparations containing cell debris or preparations containing a mixture of intact cells and cell debris. Particular examples of preparations of microorganisms are the supernatants of the fermentation broths obtained after the end of the fermentation, and the cytosolic preparations which can be obtained by disrupting the microbial cells.

[0112] The cell-free preparations of the microorganisms of the invention can be obtained, for example, by centrifugation and / or filtration of the fermentation broth and / or centrifugation and / or filtration of the suspension obtained after disruption of the microbial cells. Depending on the technique used, these cell-free preparations may not be completely devoid of cells, but may still contain small amounts of cells or cell debris. Since the cells secrete compounds such as metabolites, enzymes and / or peptides into the surrounding medium, the cell supernatant contains a mixture of such compounds, in particular metabolites, enzymes and / or peptides, secreted by the cells. Thus, in a preferred embodiment of the invention, the preparation of the microorganism is the supernatant of the fermentation broth.

[0113] Compositions containing microbial cell debris can be obtained by disrupting the cells using techniques known to those skilled in the art, for example by mechanical means or by applying high pressure. Depending on the degree of force applied, compositions containing only disrupted cells or a mixture of cell debris and intact cells are obtained. Homogenization of the cells can be achieved, for example, using a French cell press, an ultrasonic disrupter, a homogenizer, a microfluidizer, a ball mill, a rod mill, a pebble mill, a bead mill, a high pressure grinding roll, a vertical shaft impactor, an industrial blender, a high shear mixer, a paddle mixer and / or a polytron homogenizer. A suitable alternative is the enzymatic and / or chemical treatment of the cells.

[0114] The cell-free preparations of the invention also include preparations obtained by disrupting cells using the techniques mentioned above at the beginning, followed by removal of cell debris and remaining intact cells, which can be carried out in particular by centrifugation and / or filtration.

[0115] The microbial preparation of the present invention may contain, in addition to bacillaene as active compound, at least one further metabolic product, preferably a mixture of further metabolic products as further described below, and / or at least one enzyme selected from proteases, in particular subtilisins, xylanases and / or cellulases, and / or at least one peptide, and / or combinations thereof.

[0116] A preparation containing an effective mixture of metabolites contained in the microorganism of the present invention and / or contained in the cell preparation mentioned above can be obtained, for example, according to the method described in U.S. Pat. No. 6,060,051. In particular, the preparation can be obtained by precipitating the metabolites contained in the preparation mentioned above with an organic solvent such as ethyl acetate, and then redissolving the precipitated metabolites in a suitable solvent. The metabolites can then be purified by size-exclusion filtration, which separates the metabolites into different fractions based on molecular weight cutoffs.

[0117] A preparation containing an effective mixture of bacillaene and further metabolites of the invention preferably comprises at least 3, more preferably at least 4, 5, 6, 8, 10 or 12, in particular all, metabolites of the microorganisms of the invention. The metabolites preferably have a molecular weight of 400-4000 daltons, more preferably 500-3500 daltons.

[0118] Preferably, according to the invention, an effective amount of the microorganism and / or preparation and / or composition of the invention and / or bacillaene or its derivatives is always used in the embodiments of the invention. The term "effective amount" refers to an amount that brings about at least one beneficial effect on the animal and / or the environment, in particular with regard to the characteristics already mentioned above, compared to an animal not administered the strain and / or preparation and / or composition of the invention but administered the same diet (including feed and other compounds).

[0119] For therapeutic use, preferably use therapeutic amount of the microorganism and / or preparation and / or composition of the present invention.The term "therapeutic amount" refers to an amount sufficient to improve, ameliorate or prevent disease in animals.Those skilled in the art can easily determine the optimal dosage level for different animals, particularly by evaluating the ability of the composition to (i) inhibit or reduce pathogenic bacteria in the intestine at different doses, (ii) increase or maintain the level of beneficial bacteria, and / or (iii) promote animal health, particularly intestinal health, at different doses.

[0120] Working Example Example 1. Evaluation of the pathogen-inhibiting potential of probiotic B. amyloliquefaciens strains encoding the bacillaene biosynthetic cluster The potential effect of bacillaene on pathogen inhibition by probiotic Bacillus strains was evaluated using a well diffusion antagonism test (Parente et al. 1995). Strain DSM 33014 and an additional probiotic bacillaene-producing B. amyloliquefaciens strain ("B. amyloliquefaciens strain A") were used in the test. A knockout mutant was constructed for B. amyloliquefaciens strain A, disrupting the baeJ gene and rendering the strain unable to produce bacillaene (Chen et al., 2006). In addition, a B. subtilis strain lacking the bacillaene gene cluster ("B. subtilis strain B") was tested.

[0121] Well diffusion competition tests were performed with the various pathogens S. enterica subsp. enterica enteritidis DSM 14221, E. coli ATCC 11775, Vibrio parahaemolyticus DSM 10027 and C. perfringens ATCC 13124.

[0122] Bacillus strains were cultured in 100 mL shake flasks containing 40 g soy peptone, 40 g dextrin 10, 1.8 g KH per liter. 2 PO 4 , 4.5g of K 2 HPO 4 , 0.3g MgSO 4 7H 2Pathogens were grown in 10 ml of LB Kelly medium containing OD and 0.2 ml of KellyT trace metal solution (described in Scholz et al., 2011) for 16 h at 37 °C and 200 rpm. Pathogenic strains were grown under suitable conditions as liquid cultures until an optical density at 600 nm of at least 1 was reached, after which 130 μl was spread on the surface of the agar plate with a sterile spatula. For all pathogens, TSBYE (30 g / l TSB + 6 g / l yeast extract) agar plates were used. Wells with a diameter of 9 mm were cut into the dried plates. The first well was used as a non-inoculated medium control without culture, the other wells were inoculated with OD 600 Plates were inoculated with 100 μL of Bacillus culture adjusted to OD 5. For assays with E. coli ATCC 11775 and S. enterica subsp. enterica enteritidis DSM 14221, after 24 h incubation under anaerobic conditions, the plates were incubated for an additional 16 h under aerobic conditions. Each plate was analyzed for the appearance of inhibition halos around the incised well. Exclusion zones were determined in mm by measuring from the edge of the incised well to the border of the excluded lawn. Each inhibition circle was measured twice (horizontally and vertically) and then averaged. The results can be seen in Tables 1 and 2 below. For assays with V. parahaemolyticus DSM 10027, Bacillus cultures were grown in LB Kelly medium for 10 h only and reached an OD 500 of 100 μL. 600 OD 600 The OD was adjusted to 10 and the agar plates were analyzed directly after 24 h of incubation under anaerobic conditions (see Table 3). For inhibition assays with C. perfringens ATCC 13124 on agar plates, the Bacillus cultures were grown for 16 h and the OD was adjusted to 10 and the agar plates were analyzed directly after 24 h of incubation under anaerobic conditions (see Table 3). 600 OD 600 5 and the agar plates were analyzed directly after 24 h of incubation under anaerobic conditions (Table 4).

[0123] [Table 1]

[0124] Data indicate that bacillaene-producing B. amyloliquefaciens strains inhibit the growth of S. enterica subsp. enterica enteritidis DSM 14221, whereas a baeJ knockout mutant and a non-bacillaene-producing B. subtilis strain do not inhibit the pathogen.

[0125] [Table 2]

[0126] The data indicate that bacillaene-producing B. amyloliquefaciens strains inhibit the growth of E. coli ATCC 11775, whereas a baeJ knockout mutant and a non-bacillaene-producing B. subtilis strain do not inhibit the pathogen.

[0127] [Table 3]

[0128] The data indicate that bacillaene-producing B. amyloliquefaciens strains inhibit the growth of V. parahaemolyticus DSM 10027, whereas a baeJ knockout mutant that does not produce bacillaene does not inhibit the pathogen.

[0129] [Table 4]

[0130] The data indicate that bacillaene-producing B. amyloliquefaciens strains inhibit the growth of C. perfringens ATCC 13124, while reduced inhibition against C. perfringens ATCC 13124 is observed for the baeJ knockout mutant, which does not produce bacillaene.

[0131] The inhibition of C. perfringens ATCC 13124 by B. amyloliquefaciens DSM 33014 was analyzed in a liquid environment. B. amyloliquefaciens DSM 33014 was grown overnight in 10 mL of LB Kelly medium. The culture was centrifuged and filtered through a 0.2 micron cellulose acetate membrane to remove cells and spores. The cell- and spore-free supernatant was mixed 1:10 with double-strength Brain Heart Infusion (GranuCult™ BHI Broth, Merck). As a negative control, LB Kelly was used instead of the cell- and spore-free supernatant. C. perfringens ATCC 13124 was grown overnight at 37° C. in 2×BHI under anaerobic conditions and inoculated at 1:100 dilution of the supernatant / 2×BHI mixture and the negative control at 1:10. After overnight growth at 37° C., the culture of C. perfringens ATCC 13124 showed no growth in the presence of the cell- and spore-free supernatant of B. amyloliquefaciens DSM 33014 (clear medium) but showed extensive growth in the negative control (turbid medium). Thus, B. amyloliquefaciens DSM 33014 encoding the bacillarene cluster is capable of inhibiting C. perfringens ATCC 13124 in liquid.

[0132] literature Parente, E., Brienza, C., Moles, M., & Ricciardi, A. 1995: A comparison of methods for the measurement of bacteriocin activity. Journal of microbiological methods, 22(1), 95-108. Chen, X.-H., Vater, J., Piel, J., Franke, P., Scholz, R., Schneider, K., Koumoutsi, A., Hitzeroth, G., Grammel, N., Strittmatter, AW, Gottschalk, G., Suessmuth, R. and Borriss, R. 2006: Structural and Functional Characterization of Three Polyketide Synthase Gene Clusters in Bacillus amyloliquefaciens FZB 42. Journal of Bacteriology, 188(11): 4024-4036.

[0133] Example 2: Strain characteristics for survival in the gastrointestinal tract B. amyloliquefaciens DSM 33014 was screened for its ability to withstand a variety of environmental and gut-related conditions and exert its full potential in the intestine of target animals, characteristics that make it a good strain for a direct-fed live agent / probiotic for animals.

[0134] B. amyloliquefaciens DSM 33014 was cultured in Difco sporulation medium (DSM, composition per liter: 8 g bacto nutrient broth, 10 ml 10% (w / v) KCl, 10 ml 1.2% (w / v) MgSO. 4 7H 2O and 1 ml of 1M Ca(NO 3 ) 2 1 ml of 0.01 M MnCl 2 , 1 ml of 1 mM FeSO 4 ) overnight at 37°C to produce spores (Monteiro et al., 2005). This solution was used to assess spore heat stability and to determine pelleting stability by exposing spores to 80°C for 10 min (Leuschner and Bew, 2003) and bile resistance. Samples of the spore suspension were spotted onto VIB plates (Difco™ Veal Infusion Broth, BD) at pH 7. Plates were incubated at 37°C and analyzed for spore growth.

[0135] In addition, spore survival and qualitative assessment of vegetative cell bile resistance were analyzed. 3 μl of heat-treated spore solution was spotted onto VIB plates containing 0.3% chicken bile or 0.3% porcine bile (Sigma). Plates were incubated overnight at 37° C. and analyzed for strain growth.

[0136] Spores of strain DSM 33014 were viable after heat treatment for 10 minutes at 80° C. In addition, strain DSM 33014 was able to grow in the presence of 0.3% chicken bile or 0.3% porcine bile.

[0137] literature Monteiro, SM, Clemente, JJ, Henriques, AO, Gomes, RJ, Carrondo, MJ and Cunha, AE 2005: A procedure for high-yield spore production by Bacillus subtilis. Biotechnology Progress, 21(4):1026-31. Scholz, R., Molohon, K.J., Nachtigall, J., Vater, J., Markley, A.L., Suessmuth, R.D., Mitchell, D.A. and Borriss, R. 2011: Plantazolicin, a novel microcin B17 / streptolysin S-like natural product from Bacillus amyloliquefaciens FZB42. Journal of Bacteriology, 193(1):215-24. Leuschner, R.G.K. and Bew, J. 2003: Enumeration of Probiotic Bacilli Spores in Animal Feed: Interlaboratory Study. Journal of AOAC International, 86(3):568-75.

Claims

1. A composition comprising bacillaene for the treatment of a disease, in particular an intestinal disease, associated with a bacterial infection by Clostridium, in particular C. perfringens, and / or Salmonella, in particular S. enterica, preferably S. enterica subsp. enterica enteritidis, and / or Vibrio, in particular Vibrio parahaemolyticus, wherein the disease associated with the bacterial infection is preferably selected from necrotizing enterocolitis and early mortality syndrome.

2. The composition of claim 1 , wherein the composition is a feed composition, a food composition, a therapeutic composition, or a composition for treating plants.

3. 3. The composition of claim 1 or 2, wherein the composition comprises at least one feed or food ingredient selected from proteins, carbohydrates, fats, probiotics, prebiotics, enzymes, vitamins, immunomodulators, milk replacers, minerals, amino acids, anticoccidial agents, acid-based products, and / or drugs such as antibiotics.

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