Fermented broth and its use

Abstract

According to the present invention, it has been found that fermentation broths of probiotic microorganisms exhibit beneficial properties that make them suitable not only as feed additives but also as a means of improving the properties of other feed additives.

Description

[Technical Field]

[0001] According to the present invention, it has been found that fermented broths of probiotic microorganisms exhibit beneficial properties that make them suitable not only as feed additives but also as a means of improving the properties of other feed additives.

[0002] Probiotic microorganisms are obtained by culturing a sample of probiotic microorganisms in a fermentation medium, and then isolating the probiotic microorganisms from the resulting fermentation broth.

[0003] The remaining fermentation broth is usually discarded. However, alternative uses for fermentation broth have also been disclosed in the literature. For example, U.S. Patent No. 6,060,051 discloses the use of remaining fermentation broth to isolate metabolites that are useful against fungal and bacterial plant diseases, and in particular active against corn rootworms.

[0004] Surprisingly, according to the present invention, it has been found that drying of residual fermentation broth does not have a harmful effect on the activity of the active substances contained in the residual fermentation broth, particularly enzymatic activity and antimicrobial activity.

[0005] Furthermore, drying the fermented broth makes it easy to handle, and therefore a product that can be applied as a feed additive and mixed with further feed additives is obtained.

[0006] In particular, it has been found that by mixing various dried fermented broths and / or by mixing dried fermented broths with probiotic microorganisms, feed products with excellent properties, especially in terms of protein degradation activity, antimicrobial activity against pathogens, and prebiotic activity against beneficial bacteria, can be obtained.

[0007] Therefore, the first subject matter of the present invention is a method for producing dry fermentation broth, comprising the following steps: a) A step of culturing microorganisms in a fermentation medium to obtain a fermentation broth containing microorganisms, b) A step of separating at least 20% of the microorganisms from the fermentation broth. c) A step of drying the fermentation broth obtained in this way to obtain dried fermentation broth, This method includes [something].

[0008] In the separation step, at least 20%, preferably at least 50%, 60%, 70%, 80%, 90%, or 95%, and more preferably at least 98%, 99%, or 99.5%, of the microorganisms are removed from the fermentation broth. In a very preferred embodiment of the present invention, the fermentation broth contains no microorganisms or very few microorganisms.

[0009] The isolation of microorganisms from fermentation broth can be carried out, in particular by centrifugation, flotation, filtration, especially ultrafiltration or microfiltration and / or decantation.

[0010] Drying of the fermentation broth is preferably carried out by freeze-drying, spray-drying, vacuum-drying, tray-drying, drum-drying, fluidized bed-drying, or spray-granulation of the fermentation broth.

[0011] Freeze-drying of fermentation broth can be carried out by first freezing the broth using liquid nitrogen or dry ice, or by freezing it at -20°C and then drying it under high vacuum (Ananta et al., 2004, "Microbial Ecology in Health and Disease", 16(2-3):113-124). Freeze-drying may also include evaporative cooling of fermentation broth (Bond, 2007, "Methods in Molecular Biology" No. 368, Humana Press, New York, USA, pp. 99-107).

[0012] For spray-drying a fermentation broth, fine droplets of the broth atomized by spraying through a heated nozzle are sprayed into a drying chamber against hot air. The contents of the fermentation broth are collected at the bottom of the chamber (Masters, 1972, "Spray drying". Leonard Hill Books, London, UK).

[0013] Spray granulation is a preferred process in which the fermentation broth is directly converted into free-flowing granule particles of suitable particle size.

[0014] In certain embodiments of the present invention, a concentration step can be carried out either after culturing the microorganism, and before isolating the microorganism, and / or after isolating the microorganism, to increase the total dry matter content of the fermentation broth. Concentration of the fermentation broth can be carried out in particular by solvent evaporation. Solvent evaporation is preferably carried out in a single-stage or multi-stage process using a rotary evaporator, a thin-film evaporator or a falling-film evaporator, as applicable.

[0015] After removing microorganisms from the fermentation broth, the fermentation broth preferably has a solids content (total dry matter) of 1% by weight to 10% by weight, particularly 1% by weight to 6% by weight, and / or preferably 1×10 per 1 ml 10 cfu or less, more preferably 1×10 per 1 ml 9 cfu or less, particularly 1×10 per 1 ml 4 cfu to 1×10 9 cfu per ml, or 1×10 per 1 ml 4 cfu to 1×10 8 cfu of microorganisms per ml.

[0016] After removing microorganisms and before starting drying of the fermentation broth, a specific substance may be added, particularly to preserve enzymes. These substances may in particular be selected from anti-caking agents, antioxidants, bulking agents, and / or protecting agents. Examples of useful substances include polysaccharides (in particular starch, cellulose, methyl cellulose, maltodextrin, gum, dextran, chitosan and / or inulin), polyethylene glycol, amino acids (in particular proline, glycine and / or glutamic acid), protein sources (in particular peptone, skim milk powder and / or sweet whey powder), peptides, saccharides (in particular lactose, xylose, fructose, trehalose, sucrose and / or dextrose), polyols (in particular mannitol, glycerol and / or sorbitol), yeast extract, malt extract, soybean meal, fats (in particular lecithin, vegetable oil and / or mineral oil), salts (in particular sodium chloride, sodium carbonate, calcium carbonate, chalk, limestone, magnesium carbonate, sodium phosphate, calcium phosphate, magnesium phosphate and / or sodium citrate), and silicates (in particular clays, especially beolite clay, amorphous silica, fumed silica / precipitated silica, zeolites, fuller's earth, baylith, clintpolite, montmorillonite, diatomaceous earth, talc, bentonite, and / or silicates such as aluminum silicate, magnesium silicate and / or calcium silicate).

[0017] Preferably, if at least one of these substances and / or a combination of these substances is used, they are added to the supplemented fermentation broth suspension in an amount that is between 1 / 10 and 2 times, preferably between 1 / 5 and an equal amount, relative to the total dry matter content contained in the fermentation broth before addition of said substances.

[0018] The resulting dried product can be further processed, for example by milling or granulation, to achieve a specific particle size or physical form.

[0019] Furthermore, the microorganisms used in the production of the fermentation broth are preferably probiotic microorganisms, especially probiotic bacteria, preferably Bacillus, especially B. subtilis, B. licheniformis, B. amyloliquefaciens, B. atrophaeus, B. clausii, B. coagulans, B. flexus, B. fusiformis, B. lentus, B. megaterium, B. mesentricus, B. mojavensis, B. polymixa, B. pmyrus pumilus), B. smithii, B. toyonensis and B. vallismortis, Enterococcus, especially E. faecium and E. faecalis, Geobacillus, especially G. stearothermophilus, Clostridium, especially C. butyricum, and Streptococcus, especially S. faecalis, S. faecium, S. gallolyticus, S. salivarius (S. Lactobacillus, especially L. acidophilus, L. amylolyticus, L. amylovorus, L. alimentarius, L. aviaries, L. brevis, L.L. buchneri, L. casei, L. cellobiosus, L. coryniformis, L. crispatus, L. curvatus, L. delbrueckii, L. farciminis, L. fermentum, L. gallinarum, L. gasseri, L. helveticus, L. hilgardii, L. johnsonii, L. kefiranofaciens, L. kefiri L. kefiri), L. mucosae, L. panis, L. collinoides, L. paracasei, L. paraplantarum, L. pentosus, L. plantarum, L. pontis, L. reuteri, L. rhamnosus, L. sakei, L. salivarius and L. sanfranciscensis, Pediococcus, especially P. acidilactici, P. dextrinicus (P. The species selected are *P. dextrinicus* and *P. pentosaceus*, *Streptococcus*, especially *S. lactis* and *S. thermophiles*, *Bifidibacterium*, especially *S. adolescentis*, *B. animalis*, *B. bifidum*, *B. breve*, and *B. longum*.

[0020] In a very preferred embodiment of the present invention, the probiotic microorganism is a probiotic microorganism of the genus Bacillus, specifically selected from the following strains: B. subtilis DSM 32315, B. subtilis DSM 32540, B. subtilis DSM 32592, B. licheniformis DSM 32314, B. pumilus DSM 32539, B. amyloliquefaciens CECT 5940, and combinations thereof.

[0021] Accordingly, a further subject matter of the present invention is also a dried fermentation broth obtainable by the method according to the present invention.

[0022] The dried fermentation broth of the present invention preferably contains 1×10 per gram of dried fermentation broth 11 (cfu) or less, more preferably 1×10 per gram of dried fermentation broth 10 (cfu) or less, in particular 1×10 per gram of dried fermentation broth 4 to 1×10 11 (cfu), or 1×10 per gram of dried fermentation broth 4 to 1×10 10 (cfu), or 1×10 per gram of dried fermentation broth 4 to 1×10 9 (cfu) of the microorganism.

[0023] Accordingly, a further subject matter of the present invention is also 1×10 per gram of dried fermentation broth 11 (cfu) or less, more preferably 1×10 per gram of dried fermentation broth 10 (cfu) or less, in particular 1×10 per gram of dried fermentation broth 4 to 1×10 11 (cfu), or 1×10 per gram of dried fermentation broth 4 to 1×10 10 (cfu), or 1×10 per gram of dried fermentation broth 4 to 1×10 9A dry fermentation broth containing microorganisms in an amount of (cfu), wherein the microorganisms are preferably selected from B. subtilis and B. amyloricephaciens.

[0024] Therefore, the amount of cells (cfu) in the dry fermentation broth of the present invention is preferably less than 1% by weight, particularly less than 0.5% or 0.2% by weight, more preferably less than 0.1% by weight, particularly less than 0.05% or 0.02% by weight. In certain embodiments, the amount of cells (cfu) in the dry fermentation broth is even less than 0.01% by weight, particularly less than 0.005% by weight, 0.002% by weight, or even less than 0.001% by weight.

[0025] Therefore, a further subject of the present invention is also a dry fermentation broth containing cells (cfu) in an amount of less than 1% by weight, particularly less than 0.5% by weight or 0.2% by weight, more preferably less than 0.1% by weight, particularly less than 0.05% by weight or 0.02% by weight, in particular embodiments the amount of cells (cfu) in the dry fermentation broth is even less than 0.01% by weight, particularly less than 0.005% by weight, 0.002% by weight or even less than 0.001% by weight, where the microorganism is preferably selected from B. subtilis and B. amyloricephasiensis. The amount of cells (cfu) by weight percentage is preferably calculated based on the cell count (cfu) disclosed by Jeong et al. (1990) in "Biotechnology and Bioengineering", Vol. 35, pp. 160-184.

[0026] Therefore, further inventive subjects of the present invention are selected from indetergents, antioxidants, fillers, and / or protective agents, particularly polysaccharides (especially starch, cellulose, methylcellulose, maltodextrin, gum, dextran, chitosan and / or inulin), polyethylene glycol, amino acids (especially proline, glycine and / or glutamic acid), protein sources (especially peptone, skim milk powder and / or sweet whey powder), peptides, sugars (especially lactose, xylose, fructose, trehalose, sucrose and / or dextrose), polyols (especially mannitol, glycerol and / or sorbitol), yeast extracts, malt extracts, soy flour, fats (especially lecithin, This is a dry fermentation broth of microorganisms from which at least 20%, preferably at least 50%, 70%, or 90% of the microorganisms have been removed, comprising at least one substance selected from 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 beolite clay, amorphous silica, fumed silica / sedimented silica, zeolite, fuller's earth, pylit, clintopolite, montmorillonite, diatomaceous earth, talc, bentonite, and / or silicates such as aluminum silicate, magnesium silicate, and / or calcium silicate). At least one substance and / or a mixture of such substances contained in the dry fermentation broth are present in the dry fermentation broth preferably in an amount of at least 0.1% by weight, more preferably at least 0.5% by weight or at least 1% by weight, particularly in an amount of 0.1% to 67% by weight or 10% to 67% by weight, preferably in an amount of 0.5% to 50% by weight or 10% to 50% by weight, more preferably in an amount of 1% to 30% by weight or 10% to 30% by weight.

[0027] According to the present invention, "dried fermentation broth" means fermentation broth having a total dry matter content of at least 70% by weight, more preferably at least 80% by weight, particularly more than 90% by weight, and especially at least 95% by weight.

[0028] A further subject of the present invention is also a composition comprising at least two, preferably at least three, and especially two, three, four, or five different types of dried fermentation broth of the aforementioned bacteria.

[0029] According to the present invention, it has been surprisingly discovered that the fermented broth of Bacillus amyloricephasiens exhibits unexpected beneficial properties, such as very high proteolytic activity.

[0030] Therefore, a further aspect of the present invention is a fermented broth of Bacillus amyloricefaciens. Thus, a further subject of the present invention is a composition comprising a fermented broth of B. amyloricefaciens, particularly a feed composition, wherein the fermented broth is preferably a fermented broth of B. amyloricefaciens CECT 5940.

[0031] Fermented broth of B. amyloricefaciens is preferably obtained by culturing probiotic microorganisms of the B. amyloricefaciens species in a fermentation medium to obtain a fermented broth containing the above probiotic microorganisms, and then separating at least 20%, preferably at least 50%, 60%, 70%, or 80%, more preferably at least 90%, 95%, or 98% of the microorganisms from the fermented broth. Therefore, B. amyloricefaciens, and especially the fermented product of B. amyloricefaciens, is one in which at least 20%, preferably at least 50%, 60%, 70%, 80%, 90%, or 95% of the microorganisms have been removed.

[0032] After removing / separating microorganisms, the fermentation broth preferably has a solid content (total dry matter) of 1% to 10% by weight, particularly 1% to 6% by weight, and / or preferably 1 × 10⁶ per ml. 10 A concentration of 1 × 10 or less per 1 ml, more preferably 1 × 10 9 Concentrations of 1 x 10 or less, especially 1 x 10 per 1 ml. 4 pieces~1×10 9 A concentration of 1 × 10⁶ per 1 ml.4 pieces~1×10 8 It contains microorganisms at this concentration.

[0033] In preferred embodiments of the present invention, the fermentation broth of B. amyloricephasiensis is used in concentrated or dried form, where the concentration and / or drying is preferably carried out as disclosed in the detailed description above, and / or the concentrated or dried fermentation broth has the characteristics described above in the detailed description.

[0034] Therefore, a specific inventive subject of this invention is also concentrated fermentation broth and / or dry fermentation broth of B. amyloricefaciens, particularly B. amyloricefaciens CECT 5940.

[0035] Therefore, a further subject of the present invention is also 1 × 10 per gram of dried fermentation broth. 11 (cfu) or less, more preferably 1 × 10 per gram of dry fermentation broth. 10 (cfu) or less, especially 1 × 10 per gram of dried fermented broth. 4 ~1 × 10 11 (CFU) amount, or 1 × 10 per gram of dry fermented broth 4 ~1 × 10 10 (CFU) amount, or 1 × 10 per gram of dry fermented broth 4 ~1 × 10 9 This is a dry fermentation broth containing B. amyloricephasiensis, particularly B. amyloricephasiensis CECT 5940, in (cfu) amounts.

[0036] Therefore, a further subject of the present invention is also a dry fermentation broth containing cells (cfu) of B. amyloricephasiensis, particularly B. amyloricephasiensis CECT 5940, in an amount of less than 1% by weight, particularly less than 0.5% by weight or 0.2% by weight, more preferably less than 0.1% by weight, particularly less than 0.05% by weight or 0.02% by weight, and in certain embodiments, a dry fermentation broth in which the amount of cells (cfu) is even less than 0.01% by weight, particularly less than 0.005% by weight, 0.002% by weight or 0.001% by weight.

[0037] The fermented broth of B. amyloricephaciens according to the present invention preferably has a proteolytic activity of at least 500 mU / ml, more preferably at least 1000 mU / ml, as measured by the method disclosed in the examples.

[0038] Surprisingly, according to the present invention, it has been further found that treating animal feed or its additives with microbial fermentation broth before preparing the final feed can improve the properties of the feed.

[0039] Therefore, another subject of the present invention is a method for improving the properties of a feed or feed additive, wherein the animal feed or feed additive is treated with / incubated with at least one fermentation broth of at least one microorganism, preferably with at least 20%, preferably at least 50%, 70%, or 90% of the microorganisms removed, and the properties of the final feed product are improved by carrying out the treatment / incubation.

[0040] The microorganisms used in the production of the fermented broth are, here again, preferably probiotic microorganisms, especially probiotic bacteria, preferably Bacillus, particularly B. subtilis, B. licheniformis, B. amyloricephasiensis, B. atropaeus, B. clauzii, B. coagulans, B. flexus, B. fusiformis, B. lentus, B. megatherium, B. mecentricus, B. mojavensis, B. polymixia, B. pumilus, B. smithii, B. toyonensis and B. vallismolti Enterococcus, especially E. faecium and E. phaecalis; Geobacillus, especially G. stearothermophilus; Clostridium, especially C. butyricum; and Streptococcus, especially S. phaecalis, S. faecium, S. galloriticus, S. salivarius subspecies thermophilus and S. bovis; Lactobacillus, especially L. acidophilus, L. amyloriticus, L. amyroborus, L. arimentarius, L. avialies, L. brevis, L. buchneri, L. casei, L. cerovii L. osas, L. coliniformis, L. crispatus, L. curbatus, L. delbrueckii, L. falsiminis, L. fermentum, L. gallinarum, L. gasseri, L. helveticus, L. hilgardii, L. johnsonii, L. kephyranofasiens, L. kephiri, L. mucosae, L. panis, L. corinoides, L. paracasei, L. paraplantarum, L. pentosus, L. plantarum, L. pontis, L. reuteri, L. rhamnosus, L. sakei, L. salivarius and L. . Selected from .sanfrancisensis, Pediococcus, especially P. acidilactici, P. dextrinicus and P. pentosaceus, Streptococcus, especially S. lactis and S. thermophilus, Bifidibacterium, especially S. adolescentis, B. animalis, B. bifidum, B. breve and B. longum, where in a very preferred embodiment of the present invention, the probiotic microorganism is a probiotic microorganism of the genus Bacillus, in particular the following strains: B. subtilis DSM 32315, B. subtilis DSM 32540, B. subtilis DSM 32592, B. licheniformis DSM 32314, B. pumilus DSM 32539, B.Selected from Amyloricephasiensis CECT 5940 and combinations thereof.

[0041] Fermentation broth can also be obtained for this purpose, preferably by first culturing microorganisms in a suitable fermentation medium, and then separating at least 20%, preferably at least 50%, 70%, or 90% of the microorganisms from the fermentation broth.

[0042] After removing / separating microorganisms, the fermentation broth preferably has a solid content (total dry matter) of 1% to 10% by weight, particularly 1% to 6% by weight, and / or preferably 1 × 10⁶ per ml. 10 A concentration of 1 × 10 or less per 1 ml, more preferably 1 × 10 9 Concentrations of 1 x 10 or less, especially 1 x 10 per 1 ml. 4 pieces~1×10 9 A concentration of 1 × 10⁶ per 1 ml. 4 pieces~1×10 8 It contains microorganisms at this concentration.

[0043] In preferred embodiments of the present invention, the fermentation broth is used in a concentrated or dried form, where the concentration and / or drying is preferably carried out as disclosed in the detailed description above, and / or the concentrated or dried fermentation broth has the characteristics described above in the detailed description.

[0044] In particular, it was found that treating the feed or its additives with fermented broth before preparing the final feed could significantly reduce the amount of antitrophic factors (ANFs).

[0045] ANFs (antigenic proteins) are present in animal feed ingredients, particularly corn, especially soybean meal (SBM). SBM is a primary source of dietary protein in poultry and pigs, but is also becoming increasingly important as a feed additive for aquaculture. ANFs present in feed ingredients (e.g., protein inhibitors, non-starch polysaccharides, lectins, antigenic proteins) can interfere with the utilization of feed nutrients and cause health problems in animals. Therefore, it is important to reduce or remove ANFs before feed consumption.

[0046] Fermentation of soybean meal by Lactobacillus and bacilli is known to reduce ANF and increase the nutritional value of feed. For example, there is suggestion that fermentation of SBM by certain bacterial strains may lead to the degradation of the antigenic proteins β-conglycinin and glycinin. These ANFs are thought to be the cause of abnormal morphological changes in the intestines and liver, as observed in groupers fed SBM. In addition to reducing ANF in raw materials, antimicrobial peptides (AMPs) may be produced during the fermentation of raw materials, which can inhibit pathogenic bacteria in the host.

[0047] Surprisingly, it has been found that the properties of feed additives can be improved by using microbial fermentation broth, and in particular, ANF can be effectively reduced and / or eliminated. Therefore, a further subject of the present invention is a method for improving the properties of animal feed or animal feed additives, which involves treating the animal feed or animal feed additive with fermentation broth of at least one type of microorganism, preferably from which at least 20%, more preferably at least 50%, 70%, or 90% of the microorganisms have been removed.

[0048] A preferred subject matter of this aspect of the present invention is a method for reducing the amount of antitrophic factors (ANFs), particularly β-conglycinin and / or glycinin, in animal feed or animal feed additives, the method comprising treating the animal feed or animal feed additive with a fermentation broth of at least one microorganism.

[0049] A further preferred subject matter of this aspect of the present invention is a method for degrading mycotoxins in animal feed or animal feed additives, the method comprising treating the animal feed or animal feed additive with a fermentation broth of at least one microorganism.

[0050] Further improvements in the properties of feed or feed additives, which can be achieved by incubation of feed or feed additives with fermented broth, include better usability of the proteins contained in the feed or feed additives to animals, and preservation of the feed or feed additives, particularly by lowering the pH and / or reducing the amount of contaminating microorganisms in the feed or feed additives. Therefore, methods for improving such properties are further preferred embodiments of the present invention.

[0051] Feed additives whose properties and / or the properties of the final feed are improved by treatment with fermented broth are preferably selected from corn, soybeans, barley, rice, oats, sorghum, soybean meal, rapeseed meal, and cottonseed meal.

[0052] The fermented broth of microorganisms more preferably used in the present invention for processing animal feed or animal feed additives is a fermented broth of probiotic microorganisms, particularly probiotic bacteria, preferably a fermented broth of probiotic microorganisms already disclosed in the detailed description above, namely Bacillus, particularly B. subtilis, B. licheniformis, B. amyloricephasiensis, B. atropaeus, B. clausii, B. coagulans, B. flexus, B. fusiformis, B. lentus, B. megatherium, B. mecentricus, B. Mojavensis, B. polymyxia, B. pumilus, B. smisi, B. toyonensis and B. vallismortis, Enterococcus, especially E. faecium and E. phaecalis, Geobacillus, especially G. stearothermophilus, Clostridium, especially C. butyricum, and Streptococcus, especially S. phaecalis, S. faecium, S. galloriticus, S. salivarius subspecies thermophilus and S. bovis, Lactobacillus, especially L. acidophilus, L. amyloriticus, L. amyroborus, L. arimentarius L. avialies, L. brevis, L. buchneri, L. casei, L. cerobiosus, L. coliniformis, L. crispatus, L. curbatus, L. delbrueckii, L. falsiminis, L. fermentum, L. gallinarum, L. gasseri, L. helveticus, L. hilgardii, L. johnsonii, L. kephyranofaciens, L. kephiri, L. mucosae, L. panis, L. corinoides, L. paracasei, L. paraplantarum, L. pentosus, L. plantarum, L. pontis, L. reuteri, L. rhamnosus, Fermentation broths are selected from L. sakey, L. salivarius and L. sanfrancisensis, Pediococcus, especially P. acidilactici, P. dextrinicus and P. pentosaceus, Streptococcus, especially S. lactis and S. thermophilus, Bifidibacterium, especially S. adolescentis, B. animalis, B. bifidum, B. breve and B. longum, where, in a very preferred embodiment of the present invention, the fermentation broth is selected from probiotic microorganisms of the genus Bacillus, especially the following strains: B. subtilis DSM 32315, B. subtilis DSM 32540, B.This is a fermentation broth from Bacillus probiotic microorganisms selected from Bacillus subtilis DSM 32592, B. licheniformis DSM 32314, B. pumilus DSM 32539, B. amyloricephaciens CECT 5940, and combinations thereof.

[0053] To pre-treat feed or feed additives, the feed or feed additives and fermented broth are mixed in a ratio of preferably 1:2 to 20:1, more preferably 1:1 to 10:1. The preferred mixing ratio depends on whether the fermented broth is used in liquid, concentrated, or dry form, as the active substances are present at higher concentrations in concentrated and dry forms. For unconcentrated fermented broth, the preferred mixing ratio of feed additives to fermented broth is 1:2 to 2:1 (on a weight / weight basis), while for dry fermented broth, the preferred mixing ratio is 5:1 to 20:1 (on a weight / weight basis).

[0054] To enable efficient improvement of the properties of the feed or feed additive, incubation of the feed or feed additive with the fermented broth is preferably carried out for at least 1 hour, particularly 1 to 100 hours, more preferably at least 2 hours, particularly 2 to 80 hours, and especially at least 4 hours, preferably 4 to 50 hours.

[0055] The fermented broth of the present invention, particularly the dried fermented broth, has the following characteristics: a) Protease activity, b) Cellulase activity, c) Xylanase activity, d) Amylase activity, e) Phytase activity, f) Catalase activity, g) Superoxide dismutase activity, h) lactonase activity, i) Antitrophic factors (ANFs), particularly their activity against β-conglycinin and / or glycinin, j) Activity against mycotoxins, k) Activity against pathogenic microorganisms, especially C. perfringens and / or S. suis, l) Quorum quenching activity, m) Prebiotic activity related to beneficial microorganisms, Preferably, at least one of these, more preferably at least two, three, four, five, six, seven, eight, nine, or ten, and especially all of them.

[0056] In a preferred embodiment of the present invention, the fermentation broth of the present invention has at least the following characteristics: a) Protease activity, b) Cellulase activity, c) Xylanase activity, d) Amylase activity, e) Antitrophic factors (ANFs), particularly their activity against β-conglycinin and / or glycinin, f) Activity against pathogenic microorganisms, especially C. perflingens and / or S. swiss, It holds.

[0057] The fermented broth of the present invention, particularly the dried fermented broth, preferably contains at least 5, more preferably at least 6, 7, 8, 9, 10, or 12 metabolites. The metabolites preferably have a molecular weight between 200 daltons and 5000 daltons, more preferably between 300 daltons and 4000 daltons.

[0058] Further inventive subjects of the present invention are also compositions comprising at least one fermented broth, in particular at least one dried fermented broth, in particular a feed composition, wherein the feed composition preferably comprises at least one further feed additive, in particular a feed additive further disclosed below.

[0059] Further inventive subjects of the present invention are compositions comprising various types of fermented broths as described in the detailed description above, in particular various types of dried fermented broths, and especially feed compositions, wherein the feed composition preferably comprises at least one further feed additive, in particular the feed additives further disclosed below.

[0060] The fermented broths and compositions containing them of the present invention, when administered to animals, preferably promote the health of such animals, and / or improve their overall health, and / or improve their feed conversion ratio, 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 the amount of pathogens released through the feces of such animals. In particular, the use of the fermented broths and compositions of the present invention can help re-establish a healthy balance of the gut microbiota after the administration of antibiotics for therapeutic purposes.

[0061] Accordingly, a further subject of the present invention is a method for promoting animal health and / or improving the overall health status of animals and / or improving the feed conversion ratio of animals and / or reducing animal mortality and / or increasing animal survival rates and / or improving animal weight gain and / or increasing animal productivity and / or enhancing animal disease resistance and / or increasing animal immune response and / or establishing or maintaining a healthy intestinal microbiota in animals and / or reducing pathogens released through animal feces, the method comprising administering to animals a fermented broth of the present invention or a composition of the present invention comprising such fermented broth.

[0062] "Increasing animal productivity" specifically refers to increasing the production of more or higher-quality eggs, milk, or meat, or increasing the production of weaned offspring.

[0063] The fermented broth according to the present invention may also be used to improve water quality. Therefore, a further subject of the present invention is also a method for controlling and / or improving the quality of water or aqueous solutions, in particular drinking water and / or livestock water, comprising the step of applying the fermented broth according to the present invention to water.

[0064] Furthermore, the fermented broth according to the present invention may also be used to treat plants, particularly to treat microbial diseases of plants. Therefore, a further subject of the present invention is also a method for treating plants, particularly a method for treating and / or preventing microbial diseases of plants, particularly cultivated plants, comprising the step of applying at least one of the fermented broths of the present invention to the plants. The application may be carried out in liquid form by spraying, etc., or in solid form, particularly as a powder.

[0065] In particular, the fermented broth of the present invention can be administered or supplied to animals in amounts effective in inhibiting and / or reducing the growth of pathogenic bacteria in the animal's intestines. Such pathogenic bacteria include Clostridium, Listeria, Salmonella, Enterococci, Staphylococci, Aeromonas, Streptococci, Campylobacter, Escherichia coli, and Vibrio. In connection therewith, the methods of the present invention can be used to reduce the amount of pathogenic bacteria released into the feces of animals. 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's intestines. By reducing pathogenic bacteria and / or increasing or maintaining beneficial bacteria, the compositions of the present invention can maintain an overall healthy gut microbiota.

[0066] Therefore, a further subject of the present invention is a method for inhibiting and / or reducing the growth of harmful or pathogenic bacteria, and / or maintaining and / or increasing the growth of beneficial bacteria in the intestines of animals, wherein the fermented broth of the present invention is administered to animals, and the pathogenic bacteria are preferably Clostridium, in particular C. perflingens and C. difficile, Listeria, in particular L. monocytogenes, L. seeligeri and L. welshimeri, Salmonella, in particular S. enterica, S. gallinarum, S. pullorum, S. arizonae, S. typhimurium, S. enteritidis and S. bongoli The method involves selecting beneficial bacteria from the following: Enterococcus bongori, particularly E. faecalis, E. faecium, and E. cecorum; Staphylococcus, particularly S. aureus; Aeromonas; Streptococcus, particularly S. suis and S. gallinaceus; Campylobacter, particularly C. jejuni and C. coli; Escherichia coli; and Vibrio, particularly V. parahemolyticus and V. harveyi, wherein the beneficial bacteria are preferably selected from lactic acid bacteria, particularly Lactobacillus and Bifidobacteria.

[0067] In a preferred embodiment of the present invention, the amount of at least one pathogenic bacterium, particularly C. perfringens, is reduced by at least 0.5 log, more preferably at least 1 log, 2 log, or 3 log.

[0068] Therefore, a further subject of the present invention is also a fermented broth of the present invention for inhibiting and / or reducing the growth of pathogenic bacteria and / or maintaining and / or increasing the growth of beneficial bacteria in the intestines of animals, wherein the pathogenic bacteria are preferably Clostridium, in particular C. perfringens and C. difficile; Listeria, in particular L. monocytogenes, L. sieligeri and L. vercimeri; Salmonella, in particular S. enterica, S. gallinarum, S. prorum, S. arizae, and S. tiphimurium The fermentation broth is selected from S. enteritidis and S. bongoli, Enterococcus bacteria, especially E. faecalis, E. faecium and E. secolam, Staphylococcus bacteria, especially S. aureus, Aeromonas bacteria, Streptococcus bacteria, especially S. suisse and S. gallinaceus, Campylobacter bacteria, especially C. jejuni and C. coli, Escherichia coli, and Vibrio bacteria, especially V. parahaemoricus and V. harvey, and the above beneficial bacteria are preferably selected from lactic acid bacteria, especially Lactobacillus and Bifidobacterium.

[0069] An increase in the occurrence and / or proliferation of pathogenic bacteria can cause or lead to an outbreak of a particular disease. For example, an increase in the occurrence and / or proliferation of Clostridium perfringens can lead to an outbreak of intestinal disease, particularly necrotizing enterocolitis in poultry. An increase in the occurrence and / or proliferation of Clostridium perfringens can also lead to outbreaks of further diseases such as bacterial enteritis, gangrenous dermatitis, and hepatobiliary cholangitis. Even the mildest forms of infection with C. perfringens may already be accompanied by diarrhea, resulting in wet bedding, which can lead to secondary conditions such as plantar dermatitis.

[0070] Therefore, a further subject of the present invention is also a therapeutic composition comprising at least one of the fermented broths of the present invention described above.

[0071] Therefore, a preferred subject in this context is a therapeutic composition for the treatment and / or prevention of necrotizing enterocolitis, particularly asymptomatic necrotizing enterocolitis, in animals, preferably poultry, comprising at least one of the fermented broths of the present invention as described above.

[0072] Therefore, another preferred subject in this context is a therapeutic composition comprising at least one of the fermented broths described above for the treatment and / or prevention of bacterial enteritis, gangrenous dermatitis, hepatic cholangitis, clostridium disease, diarrhea and / or plantar dermatitis in animals, preferably poultry.

[0073] Therefore, a further subject of the present invention is also the treatment and / or prevention of diseases in poultry, in particular intestinal diseases, preferably necrotizing enterocolitis, in particular asymptomatic necrotizing enterocolitis, by administering at least one fermented broth of the present invention to animals in need thereof.

[0074] Therefore, a further subject of the present invention is also the treatment and / or prevention of diseases selected from bacterial enteritis, gangrenous dermatitis, hepatic cholangitis, clostridium disease, diarrhea and / or digitorum dermatitis, preferably poultry diseases, by administering at least one fermented broth of the present invention to the animal in need.

[0075] The fermented broth 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 specific stages or parts of the animal's life. For example, the strain and / or composition can be administered only in weaning feed or only in finishing feed for livestock.

[0076] The compositions of the present invention, particularly feed, food and pharmaceutical compositions, and drinking water or livestock water, preferably contain the fermented broth of the present invention in an amount of 0.1% to 10% by weight, more preferably 0.2% to 5% by weight, and particularly 0.3% to 3% by weight.

[0077] The method of the present invention can be used on all kinds of animals, in particular 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 the present invention include, but are not limited to, livestock, pets, exotic animals, zoo animals, aquatic animals, and animals used for sports, entertainment, or work.

[0079] Pets are preferably selected from dogs, cats, domesticated birds, and domesticated exotic animals.

[0080] Preferably, the aquatic animals are selected from fish and shellfish intended for human nutrition. Aquatic animals include, in particular, carp, tilapia, catfish, tuna, salmon, trout, barramundi, bream, perch, cod, shrimp, lobster, crab, prawn, and crayfish. Preferred types of salmon in this context are Atlantic salmon, sockeye salmon, cherry salmon, king salmon, keta salmon, coho salmon, Danube salmon, Pacific salmon, and pink salmon.

[0081] Further preferred aquatic animals are farmed fish, which are later processed to obtain fishmeal or fish oil. In this context, the fish are preferably herring, pollock, menhaden, anchovies, capelin, or cod.

[0082] In a further preferred embodiment, the animal is livestock such as poultry, pigs, and ruminants, which are raised for consumption or as food-producing animals.

[0083] Poultry can be selected from production poultry or farm poultry, but can also be selected from pet poultry or wild birds. Preferred production poultry in this context are chickens, turkeys, ducks, and geese. Production livestock in this context are preferably poultry optimized for producing young livestock or poultry optimized for producing meat. Preferred pet poultry or wild birds are peacocks, pheasants, partridges, rock partridges, guinea fowl, quail, yellow-billed grouse, grouse, pigeons, and swans, with quail being particularly preferred. Further preferred poultry are flightless birds, especially ostriches and emus, as well as parrots.

[0084] The ruminants according to the present invention are preferably selected from cattle, goats, and sheep. In one embodiment, the composition of the present invention can be fed to preruminants to improve their health and, in particular, reduce the incidence of diarrhea in these animals. Preruminants include calves in the age range from birth to about 12 weeks.

[0085] The composition of the present invention may include at least one carrier or typical feed additive or a combination thereof.

[0086] Suitable carriers are inert formulation additives added to improve recovery, potency, or physical properties, and / or to aid in packaging and administration. Such carriers may be added individually or in combination. These carriers may be selected from anti-caking agents, antioxidants, bulking agents, and / or protective agents. 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), fats (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 clay, especially beolite clay, amorphous silica, fumed silica / sedimented silica, zeolite, Fuller's earth, pylit, clintopolite, 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 indicated in the official publications of American Feed Control Officials, Inc., published annually. See, for example, American Feed Control Officials Official Publications, editor Sharon Krebs, 2006 edition, ISBN 1-878341-18-9. Carriers may be added after the concentration of 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.

[0087] The compositions of the present invention, in particular the feed compositions, may also include probiotics as additional feed additives, where the probiotics are preferably from the above-mentioned list of probiotics, namely Bacillus, in particular B. subtilis, B. licheniformis, B. amyloricephasiensis, B. atropaeus, B. clausii, B. coagulans, B. flexus, B. fusiformis, B. lentus, B. megatherium, B. mecentricus, B. mojavensis, B. polymixia, B. p Myrs, B. smithii, B. toyonensis and B. vallismortis, Enterococcus, especially E. faecium and E. phaecalis, Geobacillus, especially G. stearothermophilus, Clostridium, especially C. butyricum, and Streptococcus, especially S. phaecalis, S. faecium, S. galloriticus, S. salivarius subspecies thermophilus and S. bovis, Lactobacillus, especially L. acidophilus, L. amyloriticus, L. amyroborus, L. arimentarius, L. amyroborus L. villies, L. brevis, L. buchneri, L. casei, L. cerobiosus, L. coliniformis, L. crispatus, L. curbatus, L. delbrueckii, L. farciminis, L. fermentum, L. gallinarum, L. gasseri, L. helveticus, L. hilgardii, L. johnsonii, L. kephyranofaciens, L. kephiri, L. mucosae, L. panis, L. corinoides, L. paracasei, L. paraplantarum, L. pentosus, L. plantarum, L. pontis, L. ro Herein, in a very preferred embodiment of the present invention, the fermentation broth is selected from the following strains: B. subtilis DSM 32315, B. subtilis DSM 32540, B. subtilis DSM 32592, B. licheniformis DSM 32314, B. pumila DSM 32539, B.This is a fermentation broth from Bacillus probiotic microorganisms selected from Amyloricephaciens CECT 5940 and combinations thereof. Further suitable probiotics include Bacillus subtilis PB6 (listed in U.S. Patent No. 7,247,299 and deposited as ATCC accession number PTA-6737), sold by Kemin under the trademark CLOSTAT®; Bacillus subtilis C-3102 (listed in U.S. Patent No. 4,919,936 and deposited as FERM BP-1096 by the National Institute of Microbial Science, National Institute of Advanced Industrial Science and Technology, Japan), sold by Calpis Inc. under the trademark GalliPro®; Bacillus subtilis DSM 17299, sold by Chr. Hansen under the trademark GalliProTect®; and Bacillus licheniformis DSMZ, sold by Chr. Hansen under the trademark BioPlus® YC. A mixture of spores of 5749 and Bacillus subtilis DSMZ 5750, B. subtilis DSM 29784 sold by Addisseo / Novozymes under the trademark Alterion®, Bacillus subtilis sold by Chr. Hansen under the trademark PORCBOOST®, or a Bacillus coagulans strain described in U.S. Patent No. 6,849,256 may be used in the composition of the present invention. Other non-bacillus probiotics such as Saccharomyces cerevisiae, Pichia pastoris, Aspergillus niger, Aspergillus oryzae, or Hansenula may also be used.

[0088] Furthermore, suitable typical animal feed additives that may be included in the compositions according to the present invention and / or used in the preparation of feed compositions starting from concentrated fermented broth or dried fermented broth according to the present invention include one or more of the following: proteins, carbohydrates, fats, prebiotics, enzymes, vitamins, immunomodulators, milk replacers, minerals, amino acids, coccidiosis inhibitors, acid-based products and / or pharmaceuticals, such as antibiotics.

[0089] Carbohydrate-containing materials that can be used in the present invention include, for example, fodder, roughage, whole wheat flour, sunflower meal or soybean meal, and mixtures thereof.

[0090] Protein-containing materials that can be used in the present invention include, for example, soy protein, pea protein, wheat gluten or corn gluten, and mixtures thereof.

[0091] Fat-containing components that can be used in the present invention are, in particular, both animal-derived and plant-derived oils such as vegetable oils, for example, soybean oil, rapeseed oil, sunflower seed oil, linseed oil, or palm oil, fish oil, and mixtures thereof.

[0092] Further protein-containing materials containing fat that may be used in the present invention include, for example, fish meal, krill powder, bivalve powder, squid powder, or shrimp shells, as well as combinations thereof.

[0093] The prebiotics that can be used in the present invention are preferably oligosaccharides, and are particularly selected from galactooligosaccharides, sialyl oligosaccharides, lactulose, lactosucrose, fructooligosaccharides, palatinose or isomaltose oligosaccharides, glycosylsucrose, maltooligosaccharides, isomaltoligosaccharides, cyclodextrin, gentiooligosaccharides, soybean oligosaccharides, xylooligosaccharides, dextran, pectin, polygalacturonan, rhamnogalacturonan, mannan, hemicellulose, arabinogalactan, arabinan, arabinoxylan, resistant starch, mehbiose, chitosan, agarose, inulin, tagatose, polydextrose, and alginates.

[0094] Enzymes that can be used in the feed composition according to the present invention and that can aid in the digestion of feed are preferably phytase (EC 3.1.3.8 or EC 3.1.3.26), xylanase (EC 3.2.1.8), galactosidase (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), phospholipase A2 (EC 3.1.1.4), phospholipase C (EC 3.1.4.3), and phospholipase D (EC 3.1.4.4), lysophospholipase (EC 3.1.1.5), amylase, especially α-amylase (EC 3.2.1.1), and lysozyme (EC 3.2.1.17) Selected from glucanases, particularly β-glucanase (EC 3.2.1.4 or EC 3.2.1.6), glucoamylase, cellulase, pectinase, or any mixture thereof.

[0095] 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 International Publication No. 98 / 28408, International Publication No. 00 / 43503, and International Publication No. 03 / 066847.

[0096] 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). An example of a commercially available protease is Ronozyme® ProAct (DSM Nutritional Products).

[0097] Vitamins that may be used in the present invention include, for example, vitamin A, vitamin D3, vitamin E, vitamin K, for example vitamin K3, vitamin B12, biotin, choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid, and pantothenates, for example calcium D-pantothenate, or combinations thereof.

[0098] Possible immunomodulatory agents include, for example, antibodies, cytokines, sprayed dried plasma, interleukins, or interferons, or combinations thereof.

[0099] Minerals that can be used in this invention include, for example, boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, zinc, calcium, magnesium, potassium, or sodium, or combinations thereof.

[0100] The amino acids that can be used in this invention are, for example, lysine, alanine, threonine, methionine, valine, or tryptophan, or combinations thereof.

[0101] Accordingly, further embodiments of the present invention are methods for preparing animal feed compositions, comprising mixing at least one fermented broth, particularly dry fermented broth, or a mixture of fermented broths according to the present invention, with feed additives such as proteins, fats and / or carbohydrates, and optionally, preferably the beneficial substances described above, in an amount particularly effective for promoting animal health, to obtain a feed product. This method may also include, for example, a pelletizing step.

[0102] A standard pelletizing process known to those skilled in the art, including the extrusion of dry or semi-moist feed, can be used. The preferred pelletizing temperature is between approximately 65°C and approximately 120°C.

[0103] In a particularly preferred embodiment of the present invention, when preparing the feed composition of the present invention, the fermented broth according to the present invention is added in a subsequent step to an already prepared feed product, particularly feed pellets, which is preferably done by spray coating or vacuum coating. This method has the particular advantage of completely avoiding the thermal decomposition of enzymes contained in the fermented broth.

[0104] Furthermore, other susceptible materials such as oils and / or enzymes can be added to feed products, particularly feed pellets, by spray coating or vacuum coating.

[0105] The fermentation broth of the present invention can be obtained by culturing the strain of the present invention according to methods well known in the art, including, for example, using media, conditions, and methods described in U.S. Patent No. 6,060,051, European Patent No. 0287699, U.S. Patent Application Publication No. 2014 / 0010792, or FAO Report 179 (2016): “Probiotics in Animal Nutrition”. Conventional large-scale microbial culture processes include deep fermentation, solid fermentation, or liquid surface culture. As nutrients are depleted towards the end of fermentation, the cells of the Bacillus strain begin to transition from the growth phase to the sporogenesis phase, so the final products of fermentation are mainly spores, metabolites, and residual fermentation medium. Sporogenesis is part of the natural life cycle of these strains and is generally initiated by cells in response to nutrient restriction. Fermentation is configured to yield high levels of colony-forming units (colony-forming units) of probiotic cells, which promotes sporogenesis.

[0106] Preferably, according to the present invention, in embodiments of the present invention, an effective amount of the fermented broth of the present invention is always used. The term "effective amount" refers to an amount that produces at least one beneficial effect on the animal and / or the environment, particularly with respect to the characteristics already described above, compared to an animal that is not administered the fermented broth of the present invention but is otherwise administered the same diet (including feed and other compounds).

[0107] In therapeutic applications, preferably, a therapeutic amount of the fermented broth of the present invention is used. The term "therapeutic amount" refers to an amount sufficient to improve, reverse, or prevent a disease condition in an animal. The optimal dosage level for different animals can be readily determined by those skilled in the art, among other things, by evaluating the composition's ability to (i) inhibit or reduce pathogenic bacteria in the intestines at various doses, (ii) increase or maintain levels of beneficial bacteria, and / or (iii) promote the health of animals at various doses. [Brief explanation of the drawing]

[0108] [Figure 1] This figure shows the cellulase activity of a) vegetative cells of B. subtilis strain DSM 32540, b) vegetative cells of B. amyloricephasiens strain CECT 5940, c) sterile filtered non-dried supernatant of B. amyloricephasiens CECT 5940 ferment, and d) sterile filtered non-dried supernatant of B. amyloricephasiens CECT 5940 ferment. Cellulase activity causes clarification around the area of ​​cellulose hydrolysis on the agar plate. [Figure 2] The diagram shows the cellulase activity of a) vegetative cells of B. subtilis strain DSM 32540, b) sterile filtered supernatant of B. subtilis DSM 32540 ferment, c) sterile filtered non-dried supernatant of B. subtilis DSM 32540 ferment, d) non-dried supernatant of B. subtilis DSM 32540 ferment, and e) non-sterile filtered supernatant of B. subtilis DSM 32540 ferment. Cellulase activity causes clarification around the cellulose hydrolysis area on the agar plate. [Figure 3] a)-d) Sterilized filtered (freeze-dried and dissolved) supernatant of the B. amyloricephasiensis CECT 5940 fermentation, and e) Cellulase activity of vegetative cells of B. amyloricephasiensis CECT 5940. Cellulase activity causes clarification around the area of ​​cellulose hydrolysis on the agar plate. [Figure 4] This figure shows the xylanase activity of a) vegetative cells of B. amyloricephasiensis CECT 5940, b) vegetative cells of B. subtilis DSM 32540, c) sterile filtered non-dried supernatant of B. amyloricephasiensis CECT 5940 ferment, and d) sterile filtered non-dried supernatant of B. amyloricephasiensis CECT 5940 ferment. Xylanase activity causes clarification around the xylan hydrolysis area on agar plates. [Figure 5]a)~d) Sterilized filtered (freeze-dried and dissolved) supernatant of the B. amyloricephaciens CECT 5940 fermentation, and e) Amylase activity of vegetative cells of the B. amyloricephaciens CECT 5940 strain. Amylase activity causes clarification around the starch hydrolysis area on the agar plate. [Figure 6] This figure shows the protease activity of a) vegetative cells of B. subtilis DSM 32540, b) vegetative cells of B. amyloricephasiensis CECT 5940, c) sterile filtered non-dried supernatant of B. amyloricephasiensis CECT 5940 ferment, and d) sterile filtered non-dried supernatant of B. amyloricephasiensis CECT 5940 ferment. Protease activity causes clarification around the area of ​​substrate hydrolysis on agar plates. [Figure 7] a)-d) Sterilized filtered (lyophilized and dissolved) supernatant of the B. amyloricephaciens CECT 5940 fermentation, and e) Protease activity of vegetative cells of B. amyloricephaciens CECT 5940. Protease activity causes clarification around the area of ​​substrate hydrolysis on the agar plate. [Figure 8] This figure shows the SDS-PAGE pattern of soy protein extract treated for 24 hours with the sterile filtered, non-dried supernatant of the fermentation of B. subtilis DSM 32540. [Figure 9] This figure shows the SDS-PAGE pattern of soy protein extract treated for 24 hours with the sterile filtered, non-dried supernatant of a fermentation of B. amyloricephaciens CECT 5940. The protein bands shown were identified by nLC / MS analysis as follows: 1-α' chain of β-conglycinin from soybean (Glycine max), 2-α subunit of β-conglycinin from soybean, 3-α subunit of β-conglycinin from soybean, 4-glycinin from soybean, 5-α' subunit of β-conglycinin from soybean, 6-α' subunit of β-conglycinin from soybean, and 7-α subunit of β-conglycinin from soybean.

[0109] Examples Example 1. Qualitative and quantitative evaluation of digestive enzyme activity. The supernatants of fermentation products of the probiotics B. subtilis DSM 32540 and B. amyloricephasiens strain CECT 5940 were evaluated for digestive enzyme activity, particularly aerobic cellulose degradation activity (Figures 1-3), xylan degradation activity (Figure 4), amylase activity (Figure 5), and proteolytic activity (Figures 6-7) in standard media.

[0110] To evaluate cellulase activity, 3 μl of sterile filtered, non-dried supernatant and / or lyophilized and dissolved supernatant of probiotic bacillus fermentation was spotted onto LB agar containing 5 g / l Sigmacell cellulose. To screen for protease activity, 3 μl of sterile filtered, non-dried supernatant and / or lyophilized and dissolved supernatant of probiotic bacillus strain fermentation was spotted onto LB agar containing 10% skim milk. Xylanase activity was similarly analyzed on LB agar containing 0.5% xylan. Amylase activity was analyzed on LB agar containing 10 g / l soluble starch.

[0111] As a positive control, the enzymatic activity of vegetative cells of each probiotic strain was analyzed. Therefore, 3 μl of liquid culture was spotted directly onto each agar plate and incubated under aerobic conditions at 37°C. The parameter read was the appearance of hydrolysis regions attributable to enzymatic activity. Plates used in the cellulase and amylase assays were stained with Lugol's iodine solution (Figures 1-3, 5).

[0112] Qualitative analysis of digestive enzyme activity revealed that the activity of each enzyme could be found not only in the sterile filtered supernatant but also in the freeze-dried and dissolved sterile filtered supernatant.

[0113] Furthermore, the proteolytic activity of the sterile filtered, non-dried supernatant of the Bacillus strain fermentation was quantitatively evaluated. 10 μL of sterile filtered supernatant was added to 20 μL of 0.5% fluorescein isothiocyanate casein (FITC; C3777, Sigma-Aldrich) solution (all components from Sigma-Aldrich) containing 20 μL of buffer consisting of 20 mM sodium phosphate (dibasic, anhydrous) and 150 mM sodium chloride, and incubated at 37°C for 1 hour. 150 μL of 10% (vol / vol) trichloroacetic acid (Sigma-Aldrich) was added and incubated at 37°C for a further 30 minutes. The sample was then centrifuged at 19000 rpm for 15 minutes, and 2 μL of supernatant was transferred to 200 μL of 500 mM TRIS HCl solution (Trizma BaseTRIS, Sigma-Aldrich). The fluorescence of soluble peptides released via proteolytic degradation was measured at excitation 494 nm and emission 518 nm (TECAN GENios microplate reader, Tecan Group Ltd., Mönnedorf, Switzerland). Analysis was performed in two independent runs, and the results were averaged as milliliters per microliter of solution. The results are shown in Table 1.

[0114] [Table 1]

[0115] In a direct comparison, the supernatant of the fermentation product of B. amyloricephaciens CECT 5940 exhibits more than 10 times higher protease activity than the supernatant of the fermentation product of B. subtilis DSM 32540.

[0116] Example 2. Inhibition of pathogens by supernatant of fermented probiotic Bacillus strains. Pathogen inhibition by the supernatant via secondary metabolites produced by probiotic Bacillus strains during fermentation was evaluated using the well diffusion antagonism test (Parente et al., 1995).

[0117] Well diffusion antagonism tests were conducted using various pathogens, namely Clostridium perfringens reference strains ATCC 13124 and Streptococcus swiss ATCC 43765 from Teo and Tan (2005) (assays were performed using lyophilized and lyseptic filtered supernatants). Strain ATCC 13124 is known to be an α-toxin-producing type A strain that serves as a reference strain for Clostridium.

[0118] S. swiss is a significant pathogen in pigs and one of the most important causes of bacterial death in post-weaned piglets, leading to sepsis, meningitis, and many other infections (Goyette-Desjardins et al., 2014). ATCC 43765 belongs to serogroup R; serotype 2 and was isolated from a pig.

[0119] Pathogenic strains were grown in liquid culture under appropriate conditions to an optical density of at least 1 / 600 nm, and then 130 μl was spread onto the surface of an agar plate using a sterile spatula. TSBYE agar plates were used for all pathogens. A 9 mm diameter well was cut into the dry plate. The first well was used as an uninoculated medium control without culture, and the other well was inoculated with 100 μL of sterile filtered supernatant (with or without heat treatment) of a fermentation of a probiotic Bacillus strain. After incubation at 37°C for 24 hours under appropriate conditions, the clarified area was measured in mm from the edge of the cut well to the boundary of the clarified colony. Each colony was measured twice (horizontally and vertically) and the average was taken. The results are shown in Tables 2 and 3 below.

[0120] [Table 2]

[0121] The data shows that the non-dried supernatant (which has also been heat-treated) of the B. amyloricephaciens CECT 5940 fermentation highly effectively inhibits the growth of C. perfringens.

[0122] [Table 3]

[0123] The data show that the supernatant (which has also been heat-treated) of the B. subtilis DSM 32540 fermentation highly effectively inhibits the growth of S. swiss ATCC 43765. Furthermore, the inhibitory effect was still observed after freeze-drying.

[0124] Teo, AY-L. and Tan, H.-M. (2005). "Inhibition of Clostridium perfringens by a novel strain of Bacillus subtilis from the gastrointestinal tracts of healthy chickens." Appl. Environm. Microbiol., 71:4185~90. 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. Goyette-Desjardins, G., Auger, J., Xu, J., Segura, M., and Gottschalk, M. (2014). "Streptococcus suis, an important pig pathogen and emerging zoonotic agent - an update on the worldwide distribution based on serotyping and sequence typing." Emerg Microbes Infect. June 2014;3(6):e45.

[0125] Example 3. Evaluation of the hydrolytic activity of the sterile filtered supernatant of a probiotic Bacillus strain fermentation product against anti-nutrient factors of soybean meal. Proteins were extracted from defatted soybean meal using a method modified from Iwabuchi and Yamauchi (1987). Defatted soybean meal was extracted at room temperature for 1 hour with stirring using 100 ml of 0.03 M Tris-HCl (pH 8) containing 10 mM β-mercaptoethanol. The sample was centrifuged, the supernatant was filtered to sterile, and the sample was stored at -20°C.

[0126] Sterile, filtered, non-dried supernatants of probiotic B. subtilis DSM 32540 fermentates and B. amyloricephaciens CECT 5940 fermentates were incubated with soy protein extract in a 2:1 ratio at 37°C. Samples were collected at 0, 6, and 24 hours, centrifuged, and the supernatants were stored at -20°C for further analysis. Control samples with unused culture medium added were analyzed in parallel.

[0127] Protein concentrations were measured using the Bio-Rad Protein Assay Kit (Bio-Rad, USA). Protein hydrolysis was confirmed using SDS-Page. Protein concentrations were adjusted, and the proteins were denatured by heat treatment at 95°C for 5 minutes before being loaded onto the gel. 20 μg of extracted protein was loaded into each well of a 10% Mini Protean TGX precast SDS gel. Precision Plus Protein® Dual Color Standards protein ladders were used as markers (10 kDa to 250 kDa). Proteins were separated at 40 mA for 1 hour. The gels were stained with Coomassie Brilliant Blue G250 solution and Coomassie Brilliant Blue R250 solution, and destained with acetic acid solution. The degradation of soy protein could be observed by the disappearance of protein bands over time (Figures 8 and 9).

[0128] Proteolysis of soy protein extract was not detectable in control samples containing only the culture medium or soy protein extract alone. Significant protein hydrolysis was detectable after incubation of soy protein extract for 6 and 24 hours with sterile filtered supernatants of Bacillus subtilis DSM 32540 fermentation and Bacillus amyloricephaciens CECT 5940 fermentation, respectively. Multiple larger protein bands (25kDa–75kDa) decreased with increasing smaller peptides (<25kDa). Analysis of the protein bands that degraded over time and specific bands from the control sample of soy extract incubated with only the culture medium by nanoLC / MS revealed that these were identified as soybean β-conglycinin and glycinin by high-resolution mass spectrometry. Therefore, β-conglycinin and glycinin, which are soybean antigenic proteins, were degraded during incubation with sterile filtered non-dried supernatants of Bacillus fermentations.

[0129] Iwabuchi, S. and Yamauchi, F. (1987): "Determination of glycinin and β-conglycinin in soybean proteins by immunological methods." J.Agric. Food Chem. 35, 200-205.

Claims

1. A method for producing a dry fermentation broth of B. subtilis DSM 32540 or B. amyloricephaciens CECT 5940, comprising the following steps: a) A step of culturing microorganisms from B. subtilis DSM 32540 or B. amyloricephaciens CECT 5940 in a fermentation medium to obtain a fermentation broth containing the said microorganisms. b) A step of removing at least 90% of the microorganisms from the fermentation broth, c) A step of drying the fermentation broth obtained in this way to obtain dried fermentation broth, A method including, The aforementioned fermented broth has the following characteristics: d) Protease activity, e) Cellulase activity, f) Xylanase activity, g) Amylase activity, Having at least one of the following, method.

2. The method according to claim 1, wherein the drying of the fermentation broth is carried out by freeze-drying, spray-drying, vacuum-drying, fluidized bed drying, or spray granulation.

3. A dry fermentation broth of B. subtilis DSM 32540 or B. amyloricephaciens CECT 5940, wherein the dry fermentation broth contains microorganisms in an amount of less than 1% by weight and has the following characteristics: d) Protease activity, e) Cellulase activity, f) Xylanase activity, g) Amylase activity, Having at least one of the following, Dried fermented broth.

4. The dried fermented broth contains, selected from anti-caking agents, antioxidants, fillers, and / or protective agents, particularly polysaccharides (especially starch, cellulose, methylcellulose, maltodextrin, gum, dextran, chitosan and / or inulin), polyethylene glycol, amino acids (especially proline, glycine and / or glutamic acid), protein sources (especially peptone, skim milk powder and / or sweet whey powder), peptides, sugars (especially lactose, xylose, fructose, trehalose, sucrose and / or dextrose), polyols (especially mannitol, glycerol and / or sorbitol), yeast extracts, and malt extracts. The dried fermentation broth according to claim 3, comprising at least one substance selected from soy flour, lipids (especially lecithin, vegetable oil and / or mineral oil), 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 clay, especially beolite clay, amorphous silica, fumed silica / sedimented silica, zeolite, fuller's earth, pylit, clintopolite, montmorillonite, diatomaceous earth, talc, bentonite and / or silicates such as aluminum silicate, magnesium silicate and / or calcium silicate).

5. A composition comprising the dried fermented broth according to claim 3 or 4, and at least one further feed additive selected from probiotics and mixtures of probiotics, carbohydrates, fats, prebiotics, enzymes, vitamins, immunomodulators, milk replacers, minerals, amino acids, coccidia inhibitors, acid-based products and / or pharmaceuticals, such as antibiotics, and mixtures thereof.

6. A method for feeding animals, comprising feeding the animals the dried fermented broth according to claim 3 or 4 or the composition according to claim 5.

7. A method for promoting animal health and / or improving the overall health status of an animal and / or improving the feed conversion ratio 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 disease resistance of an animal and / or increasing the immune response of an animal and / or establishing or maintaining a healthy intestinal microbiota in an animal and / or reducing pathogens released through the feces of an animal, the method comprising administering to an animal at least one dried fermented broth according to claim 3 or 4 and / or at least one composition according to claim 5.

8. A method for controlling and / or improving the quality of water or aqueous solutions, particularly drinking water and / or livestock water, comprising the step of applying at least one dry fermentation broth according to claim 3 or 4 and / or at least one composition according to claim 5 to water or aqueous solution.

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