Composition containing bactillane-producing bacteria or preparation thereof

Abstract

The present invention relates to the use of a bacillene-producing bacterium as a probiotic feed ingredient and to compositions containing a bacillene-producing bacterium, and to the use of bacillene or a derivative thereof for the treatment of bacterial diseases.

Description

[0001] This application is a divisional application of patent application filed on February 10, 2020, with application number 202080013452.7 and invention title "Composition or preparation thereof containing bacillaene-producing bacteria".

[0002] This invention relates to the use of bacillaene-producing bacteria as a probiotic feed ingredient, compositions containing bacillaene-producing bacteria, and the use of bacillaene or its derivatives for the treatment of bacterial diseases.

[0003] The use of certain bacterial strains as probiotic ingredients in the feed industry has been previously disclosed in the art. Probiotics (also known as "direct feed microbes" or "DFM") function by positively influencing the gut microbiota by supporting the growth of beneficial bacteria and / or inhibiting the growth of pathogenic bacteria. Ideally, the use of antibiotic growth promoters (AGPs) becomes redundant with the use of probiotics. However, probiotics also need to have other functions, such as aiding in the digestion of specific feed ingredients.

[0004] Therefore, given the existing technology, there is a need for probiotics to positively influence the gut microbiota and, on this basis, fulfill at least one additional function.

[0005] Bacillaene is a polyene-based secondary metabolite that was discovered and isolated from the fermentation broth of Bacillus subtilis strains (Patel et al., The Journal of Antibiotics (1995), Vol. 48(9), 997-1003).

[0006] According to the present invention, experiments using knockout mutant strains unexpectedly revealed that bacillaene effectively inhibits the growth of commercially relevant pathogens, particularly toxin-producing *Clostridium perfringens* (a cause of necrotizing enterocolitis), *Vibrio parahaemolyticus* (a cause of crustacean diseases such as Early Mortality Syndrome), and *Salmonella enterica* (a major cause of food poisoning). Therefore, the results demonstrate that bacillaene-producing bacteria are suitable for use as probiotic feed and food ingredients, and that bacillaene or its derivatives are suitable for treating bacterial diseases.

[0007] Therefore, the first subject of the present invention is compositions, particularly food, feed and therapeutic compositions and compositions for treating plants, which contain bacillaene or derivatives thereof, particularly bacillaene-producing microorganisms and / or preparations containing bacillaene.

[0008] According to the present invention, "derivatives of bacillaene" specifically refers to hydrogenated variants of bacillaene, particularly dihydrobacillaene as described in the literature.

[0009] Therefore, another subject of the present invention is the use of bacillaene-producing microorganisms or preparations thereof as probiotic feed or food ingredients.

[0010] Another subject of the invention is particularly bacillaene-producing microorganisms suitable as probiotics. To be suitable as probiotics, the microorganisms according to the invention should preferably meet certain criteria, such as bile resistance, heat resistance, and / or the ability to grow under anaerobic conditions.

[0011] The bacillaene-producing bacteria according to the invention are particularly effective at inhibiting strains selected from: Clostridium, preferably Clostridium perfringens (Clostridium perfringens). C. perfringens ), especially Clostridium perfringens ATCC 13124, Salmonella, preferably enteric Salmonella ( Salmonella enterica ), especially Salmonella enteritis subspecies DSM 14221 ( Salmonella enterica subsp. enteric enteritis DSM 14221), Escherichia coli (E. coli), especially Escherichia coli ATCC11775, and Vibrio, preferably Vibrio parahaemolyticus. Vibrio parahaemolyticus (especially Vibrio parahaemolyticus DSM10027 and / or Vibrio parahaemolyticus TW01).

[0012] The bacillaene-producing microorganism according to the present invention is preferably a bacterium, and more preferably selected from: Bacillus, especially Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus methylotrophicus, and Bacillus atropheus, as well as Bacillus-like organisms. PaeniBacillus(Specifically, *P. polymixa* and *P. durus*). In a very preferred embodiment of the invention, the bacillaene-producing microorganism is *Bacillus amyloliquefaciens* or *Bacillus subtilis*.

[0013] 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, and is particularly characterized in that, in the presence of 2 mM bile, the AUC5 performance value is at least 0.5, preferably at least 0.65, especially at least 0.8, and the AUC10 performance value is at least 1.2, preferably at least 1.4, especially at least 1.6, and / or it is capable of growing in the presence of 0.3 wt.-% bile, particularly in the presence of 0.3 wt.-% chicken bile and / or in the presence of 0.3 wt.-% porcine bile, preferably, it is characterized in that it is capable of surviving for at least 3 hours, preferably at least 5 or 8 hours, when exposed to 0.3 wt.-% bile, particularly 0.3 wt.-% chicken bile and / or 0.3 wt.-% porcine bile.

[0014] The bacillaene-producing microorganisms according to the invention are preferably further characterized by their ability to grow anaerobically, particularly to degrade water-insoluble cellulose and proteins under anaerobic conditions.

[0015] The bacillaene-producing microorganisms according to the invention are preferably further characterized in that at least 50%, preferably at least 70% or 90% of the spores survive for 20 minutes when exposed to 99°C.

[0016] A strain of Bacillus amyloliquefaciens has been identified as a suitable bacillaene-producing probiotic through screening of naturally occurring isolates and was deposited on February 7, 2019, in the name of Evonik Degussa GmbH at DSMZ (Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstraße 7B, 38124 Braunschweig, Germany), with accession number DSM 33014, in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure.

[0017] Therefore, in a preferred embodiment of the present invention, the bacillaene-producing microorganism is selected from: a) Bacillus amyloliquefaciens strain DSM 33014; b) A mutant strain of Bacillus strain deposited in DSM 33014, preferably having at least 98%, particularly 99% or 99.5% sequence identity with the genome sequence of the Bacillus strain deposited in DSM 33014, wherein the mutant strain preferably exhibits bile resistance and / or heat resistance and / or is able to grow under anaerobic conditions.

[0018] Bacillus amyloliquefaciens strain DSM 33014 exhibits 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.

[0019] Therefore, in a preferred embodiment of the invention, the preferred bacillaene-producing Bacillus strain, particularly Bacillus amyloliquefaciens strain DSM 33014, has at least one, preferably at least three, and more preferably all of the following characteristics: a) A 16S rDNA sequence having at least 98%, preferably at least 99%, 99.5% or 99.8%, more preferably 100% sequence identity with the 16S rDNA sequence of strain DSM 33014 and / or with SEQ ID NO: 1; a) The yqfD sequence having at least 98%, preferably at least 99%, 99.5% or 99.8%, more preferably 100% sequence identity with the yqfD sequence of strain DSM 33014 and / or with SEQ ID NO: 2; b) The gyrB sequence having at least 98%, preferably at least 99%, 99.5% or 99.8%, more preferably 100% sequence identity with the gyrB sequence of strain DSM 33014 and / or with SEQ ID NO: 3; c) The rpoB sequence having at least 98%, preferably at least 99%, 99.5% or 99.8%, more preferably 100% sequence identity with the rpoB sequence of strain DSM 33014 and / or with SEQ ID NO: 4; d) The groEL sequence having at least 98%, preferably at least 99%, 99.5% or 99.8%, more preferably 100% sequence identity with the groEL sequence of strain DSM 33014 and / or with SEQ ID NO: 5.

[0020] The baciallene-producing bacteria according to the present invention contain a gene cluster that allows the production of baciallene. This gene cluster preferably includes genes baeJ, baeL, baeM, baeN, and baeR, which encode subunits of the baciallene synthesis protein complex, also known as polyketide synthase or PKS protein.

[0021] Bacillus 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.

[0022] Therefore, according to the present invention, the gene cluster of the PKS protein preferably includes the following genes: a) The baeJ gene, which has at least 80%, preferably at least 85%, 90% or 95%, more preferably at least 98% or 99%, and most preferably at least 100% sequence identity with the baeJ sequence of strain DSM 33014 and / or the sequence of SEQ ID NO: 6; b) The baeL gene, which has at least 80%, preferably at least 85%, 90% or 95%, more preferably at least 98% or 99%, and most preferably at least 100% sequence identity with the baeL sequence of strain DSM 33014 and / or the sequence of SEQ ID NO: 7. a) The baeM gene, which has at least 80%, preferably at least 85%, 90% or 95%, more preferably at least 98% or 99%, and most preferably 100% sequence identity with the baeM sequence of strain DSM 33014 and / or the sequence of SEQ ID NO: 8. c) The baeN gene, which has at least 80%, preferably at least 85%, 90% or 95%, more preferably at least 98% or 99%, and most preferably at least 100% sequence identity with the baeN sequence of strain DSM 33014 and / or the sequence of SEQ ID NO: 9. d) The baeR gene, which has at least 80%, preferably at least 85%, 90% or 95%, more preferably at least 98% or 99%, and most preferably at least 100% sequence identity with the baeR sequence of strain DSM 33014 and / or the sequence of SEQ ID NO: 10.

[0023] The microorganisms according to the invention are preferably natural isolates, but they can also be mutants of natural isolates, especially spontaneous mutants. Furthermore, the microorganisms according to the invention can also be obtained through genetic engineering, particularly by integrating genes encoding the bacillaene-producing enzyme subunit (especially the genes baeJ, baeL, baeM, baeN, and baeR as described above) into the microorganism, preferably into the microorganism described above, more preferably into strains of Bacillus subtilis or Bacillus amyloliquefaciens, wherein the microorganism preferably cannot produce bacillaene before the integration of this gene.

[0024] The term "spontaneous mutant" refers to a mutant strain generated from a natural isolate without the intentional use of a mutagen. Such spontaneous mutants can be obtained by classical methods, such as growing natural isolates in the presence of UV light and / or by applying high temperatures or protoplast formation and / or in the presence of an antibiotic to which the parent strain is susceptible, and testing the ability of any resistant mutant to target one or more indicators of improved biological activity or improved animal health (particularly gut health). Other methods for identifying spontaneous mutants are known to those skilled in the art. However, in addition to these preferred spontaneous mutants, the present invention also includes all other types of mutants from natural isolates, such as mutants obtained through genetic engineering.

[0025] Therefore, one specific embodiment of the present invention is a non-naturally occurring mutant strain of a naturally occurring bacillaene-producing strain, particularly a spontaneously occurring mutant strain as previously defined, preferably characterized by the features mentioned above in the specification.

[0026] Therefore, another specific embodiment of the present invention is a bacillaene-producing microorganism obtained by integrating genes encoding subunits of bacillaene-producing enzymes (especially genes baeJ, baeL, baeM, baeN, and baeR, as previously mentioned in the specification).

[0027] In a preferred embodiment of the invention, the microorganisms and formulations of the invention are administered orally to animals or humans.

[0028] Therefore, another subject of the present invention is compositions, such as feed, foodstuffs, drinking water and water for raising animals, and therapeutic compositions, which contain bacillaene-producing microorganisms and / or their preparations and / or bacillaene or its derivatives.

[0029] Another subject of this invention is the use of bacillaene-producing microorganisms and / or their preparations as probiotic components (DFM) in feed or food.

[0030] The preferred food ingredients according to the present invention are dairy products, particularly yogurt, cheese, milk, butter and quark cheese.

[0031] The cells of the microorganisms of the present invention can exist as spores (which are dormant), as vegetative cells (which are growing), as transitional cells (which are transitioning from the growth phase to the sporulation phase), or as at least two, and especially as a combination of all these types of cells, particularly in the compositions of the present invention. In a preferred embodiment, the compositions of the present invention mainly comprise or only comprise spores.

[0032] Alternatively, microbial cells can also be used in a non-living, inactivated form, as non-living cells are expected to still have probiotic effects. Methods for inactivating cells are known to those skilled in the art.

[0033] When administered to animals, the bacillaene-producing microorganisms and compositions comprising them of the present invention preferably enhance the health of such animals and / or improve their overall physical condition and / or improve their feed conversion rate and / or reduce their mortality and / or increase their survival rate and / or improve their weight gain and / or increase their productivity and / or enhance their disease resistance and / or increase their immune response and / or establish or maintain a healthy gut microbiota in such animals and / or reduce pathogens excreted through their feces. In particular, the microorganisms and compositions of the present invention can be used to help restore a healthy balance of the gut microbiota after administration of antibiotics for therapeutic purposes.

[0034] Therefore, another subject of the present invention is to enhance animal health and / or improve the overall physical condition of animals and / or improve feed conversion ratios and / or reduce animal mortality and / or increase animal survival and / or improve animal weight gain and / or increase animal productivity and / or improve animal disease resistance and / or increase animal immune response and / or establish or maintain a healthy gut microbiota in animals and / or reduce pathogens excreted through animal feces, wherein the microorganisms of the present invention and / or their preparations and / or the compositions of the present invention and / or bacillaene or their derivatives are applied to animals, particularly aquatic animals.

[0035] Therefore, another subject of the invention is the use of the microorganisms and / or preparations and / or compositions of the invention for enhancing animal health and / or improving the overall physical condition of animals and / or improving feed conversion ratios and / or reducing animal mortality and / or increasing animal survival and / or improving animal weight gain and / or increasing animal productivity and / or increasing animal disease resistance and / or increasing animal immune responses and / or establishing or maintaining a healthy gut microbiota in animals and / or reducing pathogens excreted through animal feces, wherein the microorganisms and / or preparations and / or compositions of the invention and / or bacillaene or derivatives thereof are applied to animals, and wherein the animals are particularly aquatic animals.

[0036] Therefore, another subject of the present invention is the microorganisms and / or preparations and / or compositions and / or bacillaene or derivatives thereof as described above, for enhancing animal health and / or improving the overall physical condition of animals and / or improving feed conversion ratios and / or reducing animal mortality and / or increasing animal survival rates and / or improving animal weight gain and / or increasing animal productivity and / or increasing animal disease resistance and / or increasing animal immune responses and / or establishing or maintaining a healthy gut microbiota in animals and / or reducing pathogens excreted through animal feces.

[0037] "Increasing animal productivity" specifically refers to any of the following: producing more or higher quality eggs, milk, or meat, or increasing the yield of weaned offspring.

[0038] The methods and uses of the microorganisms, formulations, and compositions of the present invention can be therapeutic or non-therapeutic. In a particularly preferred embodiment of the invention, the methods and uses are non-therapeutic, particularly for animal husbandry applications.

[0039] Untreated animal manure can have adverse environmental effects due to pathogenic bacteria and other components, particularly for the animals themselves and / or humans in contact with it. This can be avoided by feeding animals or directly treating animal manure or bedding with the microorganisms, compositions, or preparations of the present invention. Therefore, another subject of the present invention is a method for controlling and / or avoiding the harmful environmental effects of manure or contaminated liquids, comprising the step of applying at least one bacillaene-producing microorganism and / or its preparations and / or compositions according to the present invention to manure, contaminated liquids, litter, pits, or septic tanks. Preferably, the microorganism, preparation, or composition is applied in liquid form (e.g., by spraying) or as a powder (e.g., by spreading).

[0040] Since harmful bacteria can negatively affect the consistency of waste, and especially can affect fairly fluid or highly fluid waste, which can lead to footpad lesions in poultry, this is another subject of the invention: a method for controlling and / or improving the consistency of waste, particularly a method for ensuring the solid consistency of waste and / or a method for avoiding footpad lesions, which can be avoided by feeding animals with at least one microorganism, a preparation, and / or a composition according to the invention.

[0041] The microorganisms and preparations according to the invention can also be used to improve the quality of feed and food compositions, as well as to improve the quality of water and aqueous solutions. Therefore, another subject of the invention is a method for controlling and / or improving the quality of feed or food compositions and the quality of water or aqueous solutions (particularly drinking water and / or animal husbandry water), comprising the step of applying at least one microorganism and / or at least one preparation and / or at least one composition of the invention to feed, food, water, or aqueous solutions.

[0042] Furthermore, the microorganisms and preparations according to the invention can also be used to treat microbial diseases of plants. Therefore, another subject of the invention is a method for treating and / or preventing microbial diseases of plants (particularly cultivated plants), comprising the step of applying at least one microorganism and / or at least one preparation and / or at least one composition of the invention to the plant. This application can be in liquid form (such as by spraying) or in solid form (particularly as a powder, preferably as a formulated powder).

[0043] By using the microorganisms, formulations, and compositions of the present invention, it is preferable to achieve an improvement in at least one of the foregoing characteristics, wherein achieving such 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. The average value of negative controls known in the livestock industry can be used, but it is preferable to use negative control animals that have undergone the same treatment as the tested animals but have not been administered the microorganisms and / or formulations of the present invention.

[0044] In addition to the ability to inhibit the growth of pathogenic bacteria due to the production of bacillaene as described above, the microorganisms and preparations used according to the present invention are preferably able to inhibit the growth of other pathogenic bacteria.

[0045] In particular, the microorganisms, formulations, and compositions of the present invention can effectively inhibit and / or reduce the amount of pathogenic bacteria growing in the animal gut when applied to or fed to animals. In addition to Clostridium, Salmonella, Vibrio, and Escherichia coli, such pathogenic bacteria include Listeria, Enterococci, Staphylococci, Aeromonas, Streptococci, Campylobacter, Shigella, Haemophilus, and Brachyspira. Relatedly, the methods of the present invention can be used to reduce the amount of pathogenic bacteria, viruses, and protozoa excreted in animal feces. The methods of the present invention can also be used to maintain or increase the growth of beneficial bacteria such as lactic acid bacteria in the animal gut. By reducing pathogenic bacteria and / or increasing or maintaining beneficial bacteria, the compositions of the present invention are able to maintain an overall healthy gut microbiota.

[0046] Therefore, another subject of the invention is a method, particularly in the intestines of animals or humans, for inhibiting and / or reducing the growth of pathogenic bacteria and / or maintaining and / or increasing the growth of beneficial bacteria, wherein the microorganisms, preparations, and / or compositions of the invention are applied to the animals or humans, and wherein the pathogenic bacteria are preferably selected from Clostridium, particularly Clostridium perfringens, Clostridium difficile, Clostridium novyi, Clostridium septicum, and Clostridium colinum; Listeria, particularly Listeria monocytogenes, Listeria seeligeri, and Listeria welshimeri; Salmonella, particularly Salmonella enterica, including the enterica subspecies, Arizona subspecies, Bongori subspecies, and particularly the serovars subspecies; and Salmonella gallinarum (S.). Salmonella gallinarum, Salmonella pullorum, Salmonella typhimurium, Salmonella enteritidis, Salmonella choleraesuis, Salmonella heidelbergensis, Salmonella dublin, Salmonella hadar, Salmonella typhi, Salmonella paratyphi, and Salmonella infantis; Enterococci, especially Enterococcus faecalis, Enterococcus faecium, and Enterococcus cecorum; Staphylococci, especially Staphylococcus aureus; Aeromonas; Streptococci, especially Streptococcus suis and Streptococcus chickenis. *Gallinaceus*, Campylobacter, especially *Campylobacter jejuni* and *Campylobacter coli*, *Escherichia coli*, Haemophilus, especially *Haemophilus parasuis*, short spirochetes ( BrachyspiraThe pathogenic microorganisms are selected from the following: *Brachyspira hyodysenteriae*, particularly *Vibrio parahaemolyticus* and *Vibrio harveyi*, and beneficial bacteria preferably selected from lactic acid bacteria, particularly lactobacilli and bifidobacteria. In a preferred embodiment, the pathogenic microorganisms are selected from *Clostridium*, particularly *Clostridium perfringens*, *Salmonella*, particularly *Salmonella enterica*, and *Vibrio*, particularly *Vibrio parahaemolyticus*.

[0047] In a preferred embodiment of the invention, the amount of at least one pathogenic bacterium, particularly Clostridium perfringens, Salmonella enterica, and / or Vibrio parahaemolyticus, is reduced by at least 0.5 log, more preferably by at least 1 log, 2 log, or 3 log.

[0048] Therefore, another subject of the present invention is the microorganisms, preparations, and compositions thereof for inhibiting and / or reducing the growth of pathogenic bacteria and / or for maintaining and / or increasing the growth of beneficial bacteria, particularly in the intestines of animals or humans, wherein the pathogenic bacteria are preferably selected from Clostridium, particularly Clostridium perfringens, Clostridium difficile, Clostridium novyi, Clostridium septicum, and Clostridium colinum; Listeria, particularly Listeria monocytogenes, Listeria seeligeri, and Listeria welshimeri; Salmonella, particularly Salmonella enterica, including the enterica subspecies, Arizona subspecies, and Bongori subspecies, and particularly the serovars subspecies; Salmonella gallinarum; and Salmonella pullorum. Salmonella pullorum, Salmonella typhimurium, Salmonella enteritidis, Salmonella choleraesuis, Salmonella heidelbergensis, Salmonella dublin, Salmonella hadar, Salmonella typhi, Salmonella paratyphi, and Salmonella infantis; Enterococci, especially Enterococcus faecalis, Enterococcus faecium, and Enterococcus cecorum; Staphylococci, especially Staphylococcus aureus; Aeromonas; Streptococci, especially Streptococcus suis and Streptococcus gallinaceus; Campylobacter, especially Campylobacter jejuni and Campylobacter coli. *C. coli*, *Escherichia coli*, *Haemophilus*, especially *Haemophilus parasuis*, and short spirochetes ( BrachyspiraThe pathogenic microorganisms are selected from the following: *Brachyspira hyodysenteriae*, particularly *Vibrio parahaemolyticus* and *Vibrio harveyi*, and beneficial bacteria preferably selected from lactic acid bacteria, particularly lactobacilli and bifidobacteria. In a preferred embodiment, the pathogenic microorganisms are selected from *Clostridium perfringens*, particularly *Clostridium perfringens*, *Salmonella*, particularly *Salmonella enterica*, and *Vibrio*, particularly *Vibrio parahaemolyticus*.

[0049] The presence and / or increased growth of pathogenic bacteria can indeed lead to outbreaks of certain diseases. For example, the presence and / or increased growth of *Clostridium perfringens* can lead to outbreaks of intestinal diseases, particularly necrotic enteritis in pigs and poultry. The presence and / or increased growth of *Clostridium perfringens* can also lead to outbreaks of other diseases such as bacterial enteritis, gangrenous dermatitis, and colangiohepatitis. Even the mildest forms of *Clostridium perfringens* infection can be accompanied by diarrhea, leading to wet garbage and subsequently secondary diseases such as footpad dermatitis. *Clostridium perfringens* type C is generally considered a major cause of necrotic enteritis and necrotic hemorrhagic enteritis in piglets, while type A is associated with mild necrotic enterocolitis and intestinal diseases in suckling and fed pigs with villous atrophy.

[0050] Clostridium difficile is a significant pathogen causing diarrhea in newborn piglets. Affected piglets may experience difficulty breathing, abdominal distension, and scrotal edema.

[0051] Staphylococcus aureus subsp. aureus can cause fowl eczema in chickens and streptococcal mastitis in sows, and can produce toxins that can cause food poisoning in humans.

[0052] Enterococcus cecum is known to cause lameness, arthritis, and osteomyelitis in broilers, usually due to inflammation of the joints and / or bone tissue. In addition, Enterococcus cecum can cause pericarditis.

[0053] Salmonella bacteria are a major cause of food poisoning in humans, typically linked to the consumption of meat, such as poultry, pork, or its derivatives. Therefore, controlling Salmonella is a significant challenge for the meat production industry. In Europe, a baseline study in 2005 on the prevalence of Salmonella in laying hens showed that across the EU, 20.3% of large-scale laying hen farms were bacteriologically positive for Salmonella 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”].

[0054] Salmonella fowleri is the cause of fowl typhoid, while Salmonella pullorum is the cause of pullorum disease. Both diseases have caused enormous damage to the poultry industry.

[0055] Campylobacter coli is a foodborne bacterium, and most people are infected by consuming pork contaminated with this bacterium. It can cause gastroenteritis and acute enterocolitis, as well as acute diarrhea in humans. Pigs are the primary host, but it can also infect humans, birds, and a variety of other animals.

[0056] Streptococcus fowleri can cause septicemia in poultry. Gross lesions include splenomegaly, hepatomegaly, renal enlargement, and congestion. Multiple necrotic and / or infarcted areas in the liver and spleen, associated with valvular endocarditis, have also been observed.

[0057] Streptococcus suis is an important pathogen in pigs and one of the most important causes of bacterial death in weaned piglets, causing septicemia, meningitis and a variety of other infections.

[0058] Vibrio parahaemolyticus is particularly responsible for diseases in crustaceans, such as early fatal syndrome (EMS), also known as acute hepatopancreatic necrosis disease (AHPND), which affects Penaeus monodon and Penaeus vannamei.

[0059] The pathogen can cause other diseases, such as polyarthritis, fibrinous polyserositis, post-weaning intestinal disorders such as post-weaning diarrhea and edema, and swine dysentery.

[0060] Therefore, another subject of the present invention is a therapeutic composition comprising the microorganisms and / or preparations and / or compositions of the present invention and / or bacillaene or derivatives thereof, particularly a therapeutic composition for treating aquatic animals.

[0061] Therefore, the preferred subject of the present invention is a therapeutic composition for treating diseases, particularly intestinal diseases, especially those related to infection with the following bacteria: Clostridium, particularly Clostridium perfringens, and / or Salmonella, particularly Salmonella enterica, preferably Salmonella enterica subsp. enterica, and / or Vibrio, particularly Vibrio parahaemolyticus.

[0062] Therefore, a further preferred subject matter herein is a therapeutic composition for treating and / or preventing necrotizing enteritis and / or necrotizing hemorrhagic enteritis, particularly subclinical necrotizing enteritis and / or necrotizing hemorrhagic enteritis, in animals (preferably pigs or poultry), the therapeutic composition comprising the strains and / or preparations and / or compositions of the present invention and / or bacillaene or derivatives thereof.

[0063] Therefore, a further preferred subject herein is a therapeutic composition for treating and / or preventing early mortality syndrome in animals (preferably aquaculture animals, more preferably crustaceans, especially shrimp and prawns), the therapeutic composition comprising the microorganisms and / or preparations and / or compositions of the present invention and / or bacillaene or derivatives thereof.

[0064] Therefore, a further preferred subject matter herein is a therapeutic composition for treating and / or preventing illnesses caused by food poisoning, the therapeutic composition comprising the microorganisms and / or preparations and / or compositions of the present invention and / or bacillaene or derivatives thereof.

[0065] Therefore, another preferred subject matter herein is a therapeutic composition for treating and / or preventing bacterial enteritis, gangrenous dermatitis, biliary hepatitis, clostridial disease, diarrhea, dyspnea, bloating, scrotal edema, fowl tinea, footpad dermatitis, streptococcal mastitis, lameness, arthritis, polyarthritis, fibrinous polyserositis, post-weaning intestinal disorders such as post-weaning diarrhea and edema, dysentery, osteomyelitis, inflammation of joints and / or bone tissue, pericarditis, splenomegaly, hepatomegaly, renal enlargement, congestion, necrosis, hepatic or splenic infarction, valvular endocarditis, septicemia, and / or meningitis of the present invention, wherein the therapeutic composition comprises the microorganisms and / or preparations and / or compositions of the present invention and / or bacillaene or derivatives thereof.

[0066] Therefore, another subject of the invention is the treatment and / or prevention of diseases, particularly intestinal diseases, which are associated with infection by the following bacteria: Clostridium, particularly Clostridium perfringens, and / or Salmonella, particularly Salmonella enterica, preferably Salmonella enterica subsp. enterica, and / or Vibrio, particularly Vibrio parahaemolyticus, wherein the microorganisms and / or preparations and / or compositions and / or bacillaene or derivatives thereof of the invention are administered to animals in need of them, wherein the animals are preferably pigs or poultry.

[0067] Therefore, another subject of the invention is the treatment and / or prevention of diseases, particularly intestinal diseases, preferably necrotizing enteritis or necrotizing hemorrhagic enteritis, especially subclinical necrotizing enteritis or subclinical necrotizing hemorrhagic enteritis, wherein the microorganisms and / or preparations and / or compositions and / or bacillaene or derivatives thereof of the invention are administered to animals in need of them, wherein the animals are preferably pigs or poultry.

[0068] Therefore, another subject of the present invention is the treatment and / or prevention of diseases in aquatic animals, particularly early fatal syndromes, wherein the microorganisms and / or preparations and / or compositions and / or bacillaene or derivatives thereof of the present invention are applied to animals in need of them, wherein the animals are preferably aquaculture animals, more preferably crustaceans, particularly shrimp and prawns.

[0069] Therefore, another subject of the invention is the treatment and / or prevention of diseases caused by food poisoning, wherein the microorganisms and / or preparations and / or compositions and / or bacillaene or their derivatives of the invention are applied to animals or humans in need of them.

[0070] Therefore, another subject of the invention is the treatment and / or prevention of diseases caused by food poisoning, wherein the microorganisms and / or preparations and / or compositions and / or bacillaene or their derivatives of the invention are applied to animal products, particularly meat or eggs, to avoid human poisoning.

[0071] Therefore, another subject of the invention is the treatment and / or prevention of diseases, preferably diseases of pigs or poultry, selected from: bacterial enteritis, gangrenous dermatitis, biliary hepatitis, clostridial disease, diarrhea, dyspnea, bloating, scrotal edema, fowl tinea, footpad dermatitis, streptococcal mastitis, lameness, arthritis, polyarthritis, fibrinous polyserositis, post-weaning intestinal disorders such as post-weaning diarrhea and edema, dysentery, osteomyelitis, inflammation of joints and / or bone tissue, pericarditis, splenomegaly, hepatomegaly, renal enlargement, congestion, necrosis, hepatic or splenic infarction, valvular endocarditis, septicemia and / or meningitis, wherein the microorganisms and / or preparations and / or compositions and / or bacillaene or derivatives thereof of the invention are administered to the animals in need of them.

[0072] The microorganisms and / or preparations and / or compositions of the present invention can be administered to animals in feed and / or drinking water over multiple days throughout the animal's life or during a specific stage or part of the animal's life. For example, the microorganisms and / or preparations and / or compositions can be administered only in the starter diet of farm animals or only in the finisher diet.

[0073] A particular subject of the invention is also a method for enhancing human health and / or improving overall human physical condition and / or increasing human disease resistance and / or enhancing human immune response and / or establishing or maintaining a healthy gut microbiota in humans, wherein the microorganisms and / or preparations and / or compositions and / or bacillaene or derivatives thereof of the invention are administered to humans.

[0074] Therefore, another subject of the present invention is the use of the microorganisms and / or preparations and / or compositions of the present invention for enhancing human health and / or improving overall human physical condition and / or increasing human disease resistance and / or enhancing human immune response and / or establishing or maintaining a healthy gut microbiota in humans, wherein the microorganisms and / or preparations and / or compositions of the present invention and / or bacillaene or derivatives thereof are administered to humans.

[0075] The compositions of the present invention, particularly feed, food, and pharmaceutical compositions, as well as drinking water or rearing water, preferably contain the microorganisms of the present invention, in an amount of about 1 × 10⁻⁶. 3 CFU to approximately 2×10 12 Administer to animals at a ratio of CFU / g feed or ml water, especially at approximately 1×10⁻⁶. 3 CFU or approximately 1×10 4 CFU or approximately 1×10 5 CFU or approximately 1×10 6 CFU or approximately 1×10 7 CFU or approximately 1×10 8 CFU or approximately 1×10 9 CFU or approximately 1×10 10 CFU or approximately 1×10 11 CFU or approximately 1×10 12 Administer to animals at a ratio of CFU / g feed or ml water, preferably at about 1×10⁻⁶. 4 CFU to approximately 1×10 10 The amount of CFU / g feed or ml water is more preferably 1×10 4 CFU to 1×10 7Administer to animals at a rate of CFU / g feed or ml water.

[0076] Accordingly, the preferred amount of the strains and / or preparations of the present invention in the feed, food and water compositions of the present invention is preferably from 0.1 wt.% to 10 wt.%, more preferably from 0.2 wt.% to 5 wt.%, and particularly from 0.3 wt.% to 3 wt.%.

[0077] The method of the present invention can be used for all kinds of animals, especially all kinds of non-human and non-insect animals, and more preferably all kinds of vertebrates such as mammals, aquatic animals and birds.

[0078] Animals that may benefit from this invention include, but are not limited to, farm animals, pets, exotic animals, zoo animals, aquatic animals, and animals used for exercise, recreation, or work.

[0079] Pets are preferred from dogs, cats, domestic birds, and domesticated exotic animals.

[0080] Aquatic animals are preferably selected from finfish and crustaceans, which are preferred for human nutrition. These include, in particular, carp, tilapia, catfish, tuna, salmon, trout, barramundi, bream, bass, cod, shrimp, lobster, crab, prawns, and crayfish. In this paper, preferred types of salmon are Atlantic salmon, sockeye salmon, masu salmon, king salmon, keta salmon, coho salmon, Danube salmon, Pacific salmon, and pink salmon.

[0081] Further preferred aquatic animals are farmed fish, which are subsequently processed to produce fishmeal or fish oil. In this regard, the fish are preferably herring, pollack, menhaden, anchovies, capelin, or cod.

[0082] In a further preferred embodiment, the animals are farm animals that are raised for consumption or as food producers, such as poultry, pigs, and ruminants.

[0083] Poultry can be selected from productive poultry or domesticated poultry, but can also be selected from fancy poultry or wild birds.

[0084] In this paper, the preferred productive poultry are chickens, turkeys, ducks, and geese. In this paper, productive livestock are preferably poultry optimized for producing young animals or poultry optimized for meat production.

[0085] Preferred ornamental poultry or wildfowl include peacocks, pheasants, partridges, chukkar, guinea fowl, quails, ptarmigans, grouse, pigeons, and swans, with quails being particularly preferred.

[0086] Further preferred poultry are ratites, especially ostriches and emus, as well as parrots.

[0087] The ruminants according to the invention are preferably selected from cattle, goats, and sheep. In one embodiment, the compositions of the invention can be fed to preruminants to enhance their health, particularly to reduce the incidence of diarrhea in these animals. Preruminants are ruminants ranging in age from birth to about 12 weeks, including calves.

[0088] Therefore, a particularly preferred embodiment of the present invention is a method of feeding animals (preferably terrestrial animals, especially pigs or poultry, or aquatic animals, especially crustaceans, such as shrimp and prawns) comprising administering to the animals a bacillaene-producing microorganism or a preparation thereof, or a feed composition containing a bacillaene-producing microorganism or a preparation thereof.

[0089] The compositions of the present invention may contain at least one carrier or typical feed ingredient or a combination thereof.

[0090] Suitable carriers are inert formulation components added to improve recovery, efficacy, or physical properties and / or facilitate packaging and application. These carriers can be added alone or in combination. They can be selected from anti-caking agents, antioxidants, fillers, and / or protectants. Examples of useful carriers include polysaccharides (especially starch, maltodextrin, methylcellulose, gum, chitosan, and / or inulin), protein sources (especially skim 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, particularly beolite clay, amorphous silica, pyrolytic / precipitated silica, zeolite, fuller's earth, baylith, clintpolite, montmorillonite, diatomaceous earth, talc, bentonite, and / or silicates such as aluminum silicate, magnesium silicate, and / or calcium silicate). Suitable carriers for animal feed additives are listed in American Feed Control Officials, Inc.'s Official Publication, published annually. See, for example, Official Publication of American Feed Control Officials, edited by Sharon Krebs, 2006 edition, ISBN 1-878341-18-9. The carrier may be added after the concentrated fermentation broth and / or during and / or after drying. Preferred carriers according to the invention are selected from calcium carbonate, diatomaceous earth, and vegetable oils.

[0091] A preferred embodiment of the present invention is a concentrated composition, particularly a feed additive composition, i.e., a composition suitable for preparing a feed composition, comprising at least one microorganism of the present invention and at least one carrier as described above, wherein the content of the at least one microorganism is preferably 0.1 wt.-% to 10 wt.-%, more preferably 0.2 wt.-% to 5 wt.-%, particularly 0.3 wt.-% to 3 wt.-%, and most preferably 0.4 wt.-% to 2.2 wt.-%, and the content of the at least one carrier is preferably at least 90 wt.-%, preferably 90 wt.-% to 99.9 wt.-%, more preferably 95 wt.-% to 99.8 wt.-%, particularly 97 wt.-% to 99.7 wt.-%, and most preferably 97.8 wt.-% to 99.6 wt.-%, and wherein the carrier is preferably substantially composed of limestone, particularly limestone having a small portion of diatomaceous earth and / or vegetable oil.

[0092] These preferred compositions of the present invention, containing stable microorganisms, can be used to prepare feed and pharmaceutical compositions, as well as drinking water and animal husbandry water, preferably containing the strains according to the invention in the amounts mentioned in the foregoing description. In a preferred embodiment, 50 to 1000 grams of such concentrated composition, particularly 50, 100, 250, 500, or 1000 grams, are used per ton of feed, drinking water, or animal husbandry water to provide compositions suitable for animal husbandry. These concentrated compositions preferably contain at least one strain of the present invention in an amount of 1 × 10⁻⁶. 9 CFU to 2×10 11 CFU / g concentrated composition, especially 2×10 9 Up to 1×10 11 CFU / g concentrated composition.

[0093] Starting with these concentrated compositions, feed and food compositions can be prepared by mixing the concentrated compositions separately with typical feed or food ingredients.

[0094] Suitable typical animal feed ingredients that can be included in the compositions according to the invention and / or used to prepare feed compositions starting from the concentrated compositions according to the invention include one or more of the following: proteins, carbohydrates, fats, other probiotics, prebiotics, enzymes, vitamins, immunomodulators, milk replacers, minerals, amino acids, coccidiostats, acid-based products, and / or drugs, such as antibiotics.

[0095] The carbohydrate-containing components that can be used according to the present invention are, for example, hay, roughage, wheat meal, sunflower meal, or soy meal, and mixtures thereof.

[0096] The protein-containing components that can be used according to the present invention are, for example, soy protein, pea protein, wheat gluten or corn gluten, and mixtures thereof.

[0097] Fatty components that can be used according to the invention are, in particular, oils of animal and plant origin, such as vegetable oils, for example, soybean oil, rapeseed oil, sunflower oil, flaxseed oil or palm oil, fish oil, and mixtures thereof.

[0098] The proteins containing additional fatty components that can be used according to the present invention are, for example, fish meal, krill meal, bivalve meal, squid meal, or shrimp shells, and combinations thereof.

[0099] Other probiotics (DFM) that can be used in combination with the microorganisms and formulations of the present invention are preferably selected from the following bacteria: 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 include Bacillus subtilis DSM 32539 (deposited in DSMZ on June 14, 2017, pursuant to the Budapest Treaty on the International Recognition of Microbial Deposits for Patent Proceedings) and its derivatives, Bacillus licheniformis DSM 32314 and Bacillus subtilis DSM 32315 (both deposited in DSMZ on May 12, 2016, pursuant to the Budapest Treaty on the International Recognition of Microbial Deposits for Patent Proceedings) and their derivatives, Bacillus subtilis PB6 (as described in U.S. Patent No. 7,247,299 and deposited with ATCC accession number PTA-6737), sold by Kemin under the trademark CLOSTAT®, and Bacillus subtilis C-3102 (as described in U.S. Patent No. 4,919,936 and deposited with FERM BP-1096 at the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan), which was developed by Calpis Bacillus subtilis DSM 17299, sold by Chr. Hansen under the trademark GalliPro®; Bacillus licheniformis DSM 17236, sold by Chr. Hansen under the trademark GalliProTect®; a mixture of Bacillus licheniformis DSMZ 5749 and Bacillus subtilis DSMZ 5750 spores, sold by Chr. Hansen under the trademark BioPlus®YC; Bacillus subtilis DSM 29784, sold by Adisseo / Novozymes under the trademark Alterion®; Bacillus subtilis, sold by Chr. Hansen under the trademark PORCBOOST®; or the Bacillus coagulans strain described in U.S. Patent No. 6,849,256. Other non-spore-forming probiotics, such as Saccharomyces cerevisiae and Pichia pastoris. Shepherd's pie Aspergillus niger, Aspergillus oryzae, or Hansenula polymorpha may also be used in the compositions of the present invention. In particular, other probiotics known to be beneficial to human health, such as lactic acid-producing bacteria, especially Lactobacillus or Bifidobacterium, may be used in food compositions. If these other probiotics are not formulated as part of the compositions of the present invention, they may be administered together with (simultaneously or at different times) the compositions of the present invention.

[0100] The prebiotics that can be used according to the present invention are preferably oligosaccharides, particularly selected from galactooligosaccharides, silayloligosaccharide, lactulose, lactosucrose, fructooligosaccharides, paraginose or isomaltooligosaccharides, glycosyl sucrose, maltose, isomaltooligosaccharides, cyclodextrin, gentianose, soybean oligosaccharides, xylooligosaccharides, dextran, pectin, polygalacturonic acid, rhamnogalacturonic acid, mannan, hemicellulose, arabinogalactan, arabinogalactan, arabinoxylan, resistant starch, mehbiose, chitosan, agarose, inulin, tagatose, polydextrose, and alginate.

[0101] Enzymes that can be used in the feed compositions according to the invention and that can aid in feed digestion are preferably selected from phytase (EC 3.1.3.8 or 3.1.3.26), xylanase (EC 3.2.1.8), galactanase (EC 3.2.1.89), galactosidase, especially α-galactosidase (EC 3.2.1.22), protease (EC 3.4), phospholipase, especially phospholipase 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), lysophospholipase (EC 3.1.1.5), amylase, especially α-amylase (EC 3.2.1.1); lysozyme (EC 3.2.1.17), and glucanase, especially β-glucanase (EC 3.2.1.4 or EC 3.1.1.17). 3.2.1.6), glucosylamylase, cellulase, pectinase or any mixture thereof.

[0102] 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 those described, for example, in WO 98 / 28408, WO 00 / 43503, and WO 03 / 066847.

[0103] Examples of commercially available xylanases include Ronozyme® WX and GZ (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 (DSMN Nutritional Products).

[0104] The vitamins that can be used according to the present invention are, for example, vitamin A, vitamin D3, vitamin E, vitamin K, such as vitamin K3, vitamin B12, biotin, choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and pantothenate, such as Ca-D-pantothenate, or combinations thereof.

[0105] Immunomodulators that can be used include, for example, antibodies, cytokines, spray-dried plasma, interleukins or interferons, or combinations thereof.

[0106] The minerals that can be used according to the present invention are, for example, boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, zinc, calcium, magnesium, potassium or sodium, or combinations thereof.

[0107] The amino acids that can be used according to the present invention are, for example, lysine, alanine, threonine, methionine, or tryptophan, or combinations thereof.

[0108] Therefore, another embodiment of the invention is a method for preparing an animal feed composition comprising mixing at least one microorganism and / or at least one formulation and / or at least one concentrated composition of the invention, particularly in an amount that effectively enhances animal health (especially gut health), with feed ingredients (such as proteins, lipids, and / or carbohydrates), and optionally other beneficial substances (preferably, as described above), to provide a feed product. The method may also include, for example, a granulation step.

[0109] Standard granulation methods known to those skilled in the art can be used, including extrusion processing of dry or semi-wet feed. The preferred granulation temperature is from about 65°C to about 120°C.

[0110] The microorganisms and compositions of the present invention can be obtained by culturing the microorganisms of the present invention according to methods well known in the art, including using culture media described, for example, in US 6,060,051, EP0287699, or US 2014 / 0010792, and other methods. Conventional large-scale microbial culture methods include submerged fermentation, solid-state fermentation, or liquid surface culture. Near the end of fermentation, as nutrients are depleted, cells begin to transition from the growth phase to the sporulation phase, such that the final products of fermentation are mostly spores, metabolites, and residual fermentation medium. Sporulation is part of the natural life cycle of these microorganisms and is usually initiated by cells in response to nutrient limitations. Fermentation is configured to obtain high levels of cell colony-forming units and promote sporulation. The bacterial cells, spores, and metabolites in the culture medium produced by fermentation can be used directly or concentrated by conventional industrial methods such as centrifugation, tangential flow filtration, submerged filtration, and evaporation. The concentrated fermentation broth can be washed, for example, via a percolation process to remove residual fermentation broth and metabolites.

[0111] Fermentation broth or concentrate can be dried with or without a carrier using conventional drying processes or methods (such as spray drying, freeze drying, disc drying, fluidized bed drying, drum drying, or evaporation). The resulting dried product can be further processed, for example by grinding or granulation, to obtain a specific particle size or physical form. As mentioned above, a carrier can also be added after drying.

[0112] The microbial formulations of the present invention, which are a specific subject of the invention, can be cell-free formulations, formulations containing cell debris, or formulations containing a mixture of intact cells and cell debris. Specific examples of microbial formulations include the supernatant of the fermentation broth obtained after fermentation, and cytosol formulations obtained by disrupting microbial cells.

[0113] The cell-free preparations of the microorganisms of the present invention can be obtained, for example, by centrifuging and / or filtering the fermentation broth and / or by centrifuging and / or filtering the suspension obtained after rupturing microbial cells. Depending on the technology used, these cell-free preparations may not be completely cell-free, but may still contain a small amount of cells or cell debris. Since cells secrete compounds such as metabolites, enzymes, and / or peptides into the surrounding culture medium, the cell supernatant contains a mixture of such compounds, particularly metabolites, enzymes, and / or peptides secreted by the cells. Therefore, in a preferred embodiment of the invention, the microbial preparation is the supernatant of the fermentation broth.

[0114] Compositions containing microbial cell fragments can be obtained by applying techniques known to those skilled in the art, such as mechanical methods or by applying high pressure to rupture the cells. Depending on the degree of force applied, compositions containing only ruptured cells or compositions containing a mixture of cell fragments and intact cells are obtained. Cell homogenization can be achieved, for example, by using a French cell press, an ultrasonic mixer, a homogenizer, a microfluidic apparatus, a ball mill, a rod mill, a pebble mill, a bead mill, a high-pressure grinding roller, a vertical shaft impactor, an industrial mixer, a high-shear mixer, a paddle mixer, and / or a polytron homogenizer. Suitable alternatives are enzymatic and / or chemical treatment of the cells.

[0115] The cell-free formulation of the present invention also comprises a formulation obtained by first rupturing cells using the technique described above, and subsequently removing cell debris and the remaining intact cells. Removal of cell debris and the remaining intact cells can be specifically performed by centrifugation and / or filtration.

[0116] In addition to containing bacillaene as the active compound, the microbial preparations of the present invention may also contain at least one other metabolite, preferably a mixture of other metabolites as further described below, and / or at least one enzyme selected from proteases, particularly subtilisin, xylanase and / or cellulase, and / or at least one peptide, and / or combinations thereof.

[0117] A formulation comprising an effective mixture of metabolites contained in the microorganisms of the present invention and / or in the aforementioned cell preparations can be obtained, for example, according to the method described in U.S. Patent No. 6,060,051. In particular, the formulation can be obtained by precipitating the metabolites contained in the aforementioned formulation using an organic solvent such as ethyl acetate and subsequently redissolving the precipitated metabolites in a suitable solvent. These metabolites can then be purified by size exclusion filtration, which groups the metabolites into different fractions based on the molecular weight cutoff.

[0118] Formulations containing an effective mixture of bacillaene and other metabolites of the present invention preferably contain at least three metabolites of the microorganisms of the present invention, more preferably at least four, five, six, eight, ten, or twelve metabolites of the microorganisms of the present invention, and particularly all metabolites of the microorganisms of the present invention. The metabolites preferably have a molecular weight of 400 to 4000 Daltons, more preferably 500 to 3500 Daltons.

[0119] Preferably, according to the invention, effective amounts of the microorganisms and / or preparations and / or compositions and / or bacillaene or their derivatives are always used in embodiments of the invention. The term "effective amount" refers to the amount that produces at least one beneficial effect (particularly the features already mentioned above) on animals and / or the environment compared to animals that have not been given the strains and / or preparations and / or compositions of the invention, but have been given the same diet (including feed and other compounds).

[0120] In therapeutic applications, therapeutic amounts of the microorganisms and / or formulations and / or compositions of the present invention are preferred. The term "therapeutic amount" refers to an amount sufficient to improve, reverse, or prevent a disease state in an animal. The optimal dosage level for various animals can be readily determined by those skilled in the art by evaluating the composition's ability to: (i) inhibit or reduce pathogenic bacteria in the gut at different dosages, (ii) increase or maintain the level of beneficial bacteria, and / or (iii) enhance animal health, particularly gut health, at different dosages.

[0121] Working Example Example 1. Evaluation of the pathogen-inhibiting ability of a probiotic strain of Bacillaria amyloliquefaciens encoding a cluster of bacillaene biosynthesis. The potential inhibitory effect of Bacillaene on pathogens by probiotic Bacillus strains was assessed using a well diffusion antagonism test (Parente et al. 1995). The test used strain DSM 33014 and another probiotic Bacillaene-producing Bacillus amyloliquefaciens strain (“Bacillus amyloliquefaciens strain A”). For Bacillus amyloliquefaciens strain A, a knockout mutant was constructed in which the baeJ gene was destroyed, preventing the strain from producing bacillaene (Chen et al., 2006). Additionally, a Bacillus subtilis strain (“Bacillus subtilis strain B”) that does not carry the bacillaene gene cluster was also tested.

[0122] Pore ​​diffusion antagonism assays were performed on different pathogens: Salmonella enteritidis subsp. enteritidis DSM 14221, Escherichia coli ATCC 11775, Vibrio parahaemolyticus DSM 10027, and Clostridium perfringens ATCC 13124.

[0123] Bacillus strains were grown in 10 mL shake flasks in 10 mL LB Kelly medium at 37 °C and 200 rpm for 16 h. This medium contained 40 g soybean peptone, 40 g dextrin 10, 1.8 g KH₂PO₄, 4.5 g K₂HPO₄, 0.3 g MgSO₄·7H₂O, and 0.2 mL Kelly T trace metal solution (as described in Scholz et al., 2011). The pathogenic strains were grown under suitable conditions, such as liquid culture, until the optical density at 600 nm was at least 1, and then 130 μL was spread onto agar plates using a sterile spatula. For all pathogens, TSBYE (30 g / L TSB + 6 g / L yeast extract) agar plates were used. 9 mm diameter wells were cut into the dried plates. The first well served as an uninoculated control without culture, and the other wells were inoculated with 100 μL of medium adjusted to OD₂O₅. 600 5% Bacillus cultures. For the tests of Escherichia coli ATCC 11775 and Salmonella enteritidis subsp. enteritidis DSM 14221, plates were incubated anaerobic for 24 hours, followed by aerobic incubation for 16 hours. The inhibition halo around each well was analyzed. The clearance zone (mm) from the edge of the well to the boundary of the cleared lawn was measured. Each halo was measured twice (horizontal and vertical), and the average value was taken. The results are shown in Tables 1 and 2 below. For the test of Vibrio parahaemolyticus DM 10027, Bacillus cultures were grown only in LB Kelly medium for 10 hours, and OD... 600 Adjust to OD 600 The value was 10, and the agar plates were directly analyzed after anaerobic incubation for 24 hours (see Table 3). For the inhibition test of Clostridium perfringens ATCC 13124, Bacillus cultures were cultured on agar plates for 16 hours, and the OD600 was... 00 Adjust to OD 600 The value was 5, and the agar plates were directly analyzed after being cultured under anaerobic conditions for 24 hours (Table 4).

[0124] Table 1: Comparison of the inhibitory effects of Bacillus amyloliquefaciens strains encoding the bacillaene cluster, the baeJ knockout mutant strain, and Bacillus subtilis strains whose genomes do not carry the bacillaene cluster on pathogenic Salmonella enteritis subsp. DSM 14221 strain in a pore diffusion antagonism assay on TSBYE medium. Pathogen clearance is expressed in mm.

[0125] Data showed that bacillaene-producing Bacillus amyloliquefaciens strains inhibited the growth of Salmonella enteritidis subsp. DSM14221, while the baeJ knockout mutant and non-bacillaene-producing Bacillus subtilis strains did not inhibit the pathogen.

[0126] Table 2: Comparison of the inhibitory effects of Bacillus amyloliquefaciens strains encoding the bacillaene cluster, the baeJ knockout mutant strain, and Bacillus subtilis strains whose genomes do not carry the bacillaene cluster on pathogenic Escherichia coli ATCC 11775 strain in a pore diffusion antagonism assay on TSBYE medium. Pathogen clearance is expressed in mm.

[0127] Data showed that bacillaene-producing Bacillus amyloliquefaciens strains inhibited the growth of Escherichia coli ATCC 11775, while the baeJ knockout mutant and non-bacillaene-producing Bacillus subtilis strains did not inhibit the pathogen.

[0128] Table 3: Comparison of the inhibitory effects of Bacillaria amyloliquefaciens strains encoding the bacillaene cluster and the baeJ knockout mutant strain on pathogenic Vibrio parahaemolyticus strain DSM 10027 in a pore diffusion antagonism assay on TSBYE medium. Pathogen clearance is expressed in mm.

[0129] Data showed that bacillaene-producing Bacillus amyloliquefaciens strains inhibited the growth of Vibrio parahaemolyticus DSM 10027, while the non-bacillaene-producing baeJ knockout mutant strain did not inhibit the pathogen.

[0130] Table 4: Comparison of the inhibitory effects of Bacillus amyloliquefaciens strains encoding the bacillaene cluster and the corresponding baeJ knockout mutants on Clostridium perfringens ATCC 13124 in pore diffusion antagonism assays on TSBYE medium. Pathogen clearance is expressed in mm.

[0131] Data showed that bacillaene-producing Bacillus amyloliquefaciens strains inhibited the growth of Clostridium perfringens ATCC 13124, while the non-bacillaene-producing baeJ knockout mutant strain showed reduced inhibition of Clostridium perfringens ATCC 13124.

[0132] The inhibitory effect of *Bacillus amyloliquefaciens* DSM 33014 on *Clostridium perfringens* ATCC 13124 was analyzed in a liquid environment. *Bacillus amyloliquefaciens* DSM 33014 was grown overnight in 10 mL LB Kelly medium. The culture was centrifuged and filtered through a 0.2 μm cellulose acetate membrane to remove cells and spores. The cell- and spore-free supernatant was mixed with double-strength brain heart infusion (GranuCult). TM BHI broth (Merck) was mixed at a 1:10 ratio. LB Kelly was used as a negative control instead of the cell-free and spore-free supernatant. *Clostridium perfringens* ATCC 13124 was grown overnight in 2xBHI under anaerobic conditions at 37°C and inoculated at a 1:10 ratio into the supernatant / 2xBHI mixture, and then into the negative control at a 1:100 dilution. After overnight growth at 37°C, the *Clostridium perfringens* ATCC 13124 culture showed no growth in the presence of the cell-free and spore-free supernatant of *Bacillus amyloliquefaciens* DSM 33014 (clear medium), but showed abundant growth in the negative control (turbid medium). Therefore, *Bacillus amyloliquefaciens* DSM 33014, encoding the bacillaene cluster, was able to inhibit *Clostridium perfringens* ATCC 13124 in liquid.

[0133] References Parente, E., Brienza, C., Moles, M., & Ricciardi, A. 1995: Acomparison of methods for the measurement of bacteriocin activity. Journal of microbiological methods, 22(1), 95-108. Chen, Synthase Gene Clusters in Bacillusamyloliquefaciens FZB 42. Journal of Bacteriology, 188(11): 4024–4036. Example 2: Strains with characteristics associated with survival in the gastrointestinal tract.

[0134] The goal was to screen Bacillus amyloliquefaciens DSM 33014 for tolerance to a variety of environmental and gut-related conditions, thereby enabling it to fully realize its potential in the gut of target animals as a superior strain of microorganisms / probiotics for direct animal feeding.

[0135] Bacillus amyloliquefaciens DSM 33014 was grown overnight at 37°C in Difco sporulation medium to produce spores (DSM, composition per liter: 8 g bacto nutrient broth, 10 ml 10% (w / v) KCl, 10 ml 1.2% (w / v) MgSO4·7H2O, 1 ml 1 M Ca(NO3)2, 1 ml 0.01 M MnCl2, 1 ml 1 mM FeSO4) (Monteiro et al., 2005). This solution was used to assess the thermal stability of the spores to determine their pelleting stability (Leuschner and Bew, 2003) and bile resistance by exposing them to 80°C for 10 min. Samples of the spore suspension were spotted onto pH 7 VIB plates (Difco™ Veal Infusion Broth, BD). The plates were incubated at 37°C and spore growth was analyzed.

[0136] In addition, qualitative assessments of spore survival rate and bile tolerance of vegetative cells 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). The plates were incubated overnight at 37°C, and the growth of the strains was analyzed.

[0137] Spores of strain DSM 33014 remained viable after heat treatment at 80°C for 10 minutes. Furthermore, strain DSM 33014 could grow in the presence of 0.3% chicken bile or 0.3% porcine bile.

[0138] References Monteiro, SM, Clemente, JJ, Henriques, AO, Gomes, RJ,Carrondo, MJ and Cunha, AE 2005: A procedure for high-yield sporeproduction by Bacillus subtilis. Biotechnology Progress, 21(4):1026-31. Scholz, R., Molohon, KJ, Nachtigall, J., Vater, J., Markley, AL, Süssmuth, RD, Mitchell, DA and Borriss, R. 2011: Plantazolicin, a novelmicrocin B17 / streptolysin S-like natural product from Bacillusamyloliquefaciens FZB42. Journal of Bacteriology, 193(1):215-24. Leuschner, RGK and Bew, J. 2003: Enumeration of Probiotic BacilliSpores in Animal Feed: Interlaboratory Study. Journal of AOAC International,86(3):568-75.

Claims

1. Use of bacillaene in the preparation of compositions for treating diseases associated with bacterial infections of Clostridia, and / or Salmonella, and / or Vibrio, wherein the Clostridia is Clostridium perfringens (Clostridium perfringens). C. perfringens The Salmonella mentioned is *Salmonella enterica* (…). S. enterica ), and / or the Vibrio is Vibrio parahaemolyticus ( Vibrio parahaemolyticus ).

2. The use according to claim 1, wherein the enteric Salmonella is enteric Salmonella enteritidis (S. enterica subsp. enterica enteritidis).

3. The use according to claim 1, wherein the disease is an intestinal disease.

4. The use according to claim 1, wherein the disease is selected from necrotizing enterocolitis and early fatal syndrome.

5. The use according to claim 1, wherein the composition further comprises a bacillaene-producing microorganism selected from bacteria, preferably, in particular, from Bacillus subtilis (Bacillus subtilis). B. subtilis ), Bacillus amyloliquefaciens ( B. amyloliquefaciens ), Methyltrophic Bacillus ( B. methylotrophicus ) and Bacillus atrophus ( B. atropheus ), or Bacillus subtilis ( Paeni Bacillus ), especially those selected from Polymyxin Bacillus ( P. polymixa ) and tough Bacillus ( P. durus ).

6. The use according to claim 5, wherein the microorganism is selected from Bacillus (… Bacillus ) or Bacillus subtilis ( Paeni Bacillus ), and its mixtures.

7. The use according to claim 6, wherein the Bacillus is selected from Bacillus subtilis (… B. subtilis ), Bacillus amyloliquefaciens ( B. amyloliquefaciens ), Methyltrophic Bacillus ( B. methylotrophicus ) and Bacillus atrophus ( B. atropheus ).

8. The use according to claim 6, wherein the Bacillus subtilis is selected from Bacillus polymyxa (… P. polymixa ) and tough Bacillus ( P. durus ).

9. The use according to any one of claims 1 to 8, wherein the composition is a feed composition or a therapeutic composition.

10. The use according to any one of claims 1 to 8, 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, coccidiostats, acid-based products, and / or pharmaceuticals.

11. The use according to claim 10, wherein the drug is an antibiotic.

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