FERMENTATION BROTHS AND THEIR USE

Abstract

Summary

According to the invention it was found out that fermentation broths of probiotic microorganisms show beneficial characteristics which make them suitable as feed additives as well as means for improving the properties of other feed additives.

Claims

1-19. (canceled)

20. A method of producing a dried fermentation broth, comprising the following steps: a) cultivating microorganisms in a fermentation medium to obtain a fermentation broth containing the microorganisms; b) separating at least 20% of the microorganisms from the fermentation broth; and c) drying the fermentation broth of step b) to obtain the dried fermentation broth.

21. The method of claim 20, wherein drying of the fermentation broth is carried out by freeze-drying, spray drying, vacuum drying, fluidized bed drying or spray granulation.

22. The method of claim 20, wherein the microorganisms are selected from the group consisting of: 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. pumilus, B. smithii, B. toyonensis and B. vallismortis, E. faecium, E. faecalis, G. stearothermophilus, C. butyricum, S. faecalis, S. faecium, S. gallolyticus, S. salivarius subsp. thermophiles, S. bovis, L. acidophilus, L. amylolyticus, L. amylovorus, L. alimentarius, L. aviaries, L. brevis, 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. mucosae, L. panis, L. collinoides, L. paracasei, L. paraplantarum, L. pentosus, L. plantarum, L. pontis, L. reuteri, L. rhamnosus, L. sakei, L. salivarius, L. sanfranciscensis, P. acidilactici, P. dextrinicus, P. pentosaceus, S. lactis, S. thermophiles, S. adolescentis, B. animalis, B. bifidum, B. breve and B. longum.

23. Dried fermentation broth of microorganisms, obtainable by cultivating at least one microorganism in a fermentation medium, removing at least 20%, of the microorganisms from the fermentation broth, and subsequently drying the fermentation broth thus obtained, wherein the dried fermentation broth contains microorganisms in an amount of less than 1 wt.

24. The dried fermentation broth of claim 23, further comprising at least one substance selected from the group consisting of: anti-caking agents, anti-oxidation agents, bulking agents, protectants, polyethylene glycol, amino acids, protein sources, peptides, sugars, polyols, yeast extract, malt extract, soybean flour, lipids, salts, and silicates.

25. The dried fermentation broth of claim 23, wherein the broth is from a fermentation of Bacillus, Enterococcus, Geobacillus, Clostridium, and Streptococcus, Lactobacillus, B. animalis, B. bifidum, B. breve and B. longum, or from mixtures of at least two of such fermentation broths.

26. The dried fermentation broth of claim 25, wherein the broth is from fermentation of Bacillus amyloliquefaciens.

27. The died fermentation broth of claim 23, comprising at least one of 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) Activity against antinutritional factors (ANFs); j) Activity against mycotoxins; k) Activity against pathogenic microorganisms, in particular against C. perfringens and/or S. suis; l) Quorum quenching activity; m) Prebiotic activity with respect to beneficial microorganisms.

28. The dried fermentation broth of claim 23, further comprising a feed additive selected from the group consisting of: probiotics and mixtures of probiotics, carbohydrates, fats, prebiotics, enzymes, vitamins, immune modulators, milk replacers, minerals, amino acids, coccidiostats, acid-based products and/or medicines, antibiotics, and mixtures thereof.

29. A method of improving the health or nutrition of animals, plants or the quality of aqueous solutions, comprising administering the dried fermentation broth of claim 4 or a composition comprising the dried fermentation.

30. The method of claim 29, wherein said method comprises feeding animals the dried fermentation broth or the composition comprising the dried fermentation broth.

31. The method of claim 29, wherein said method comprises enhancing the health of animals, and/or improving the general physical condition of animals, and/or improving the feed conversion rate of animals, and/or decreasing the mortality rate of animals, and/or increasing the survival rates of animals, and/or improving the weight gain of animals, and/or increasing the disease resistance of animals, and/or increasing the immune response of animals, and/or establishing or maintaining a healthy gut microflora in animals, and/or reducing pathogen shedding through the feces of animals, and wherein the method comprises administering the dried fermentation broth or a composition comprising the dried fermentation to the animals.

32. The method of claim 29, wherein said method comprises controlling and/or improving the quality of water or an aqueous solution by applying the dried fermentation broth or a composition comprising the dried fermentation broth to the water or aqueous solution.

33. The method of claim 29, wherein said method comprises treating and/or preventing a microbial disease of a cultivated plant by applying the dried fermentation broth or a composition comprising the dried fermentation to the plant.

34. The method of claim 29, wherein said method comprises improving the properties of an animal feed or feed additive, by adding the dried fermentation broth or a composition comprising the dried fermentation to the animal feed or feed additive.

35. The method of claim 34, wherein the properties which are improved are selected from: reducing the amount of antinutritional factors (ANFs), and/or degrading mycotoxins; and/or increasing the usability of proteins by the animals; and/or preserving the feed or feed additive by lowering the pH and/or lowering the amount of contaminating microorganisms in the feed or feed additive.

36. The method of claim 34, wherein the feed additive is selected from corn, soy, barley, rice, oats, sorghum, soybean meal, rapeseed meal and cotton meal.

37. The method of claim 34, wherein the animal feed or feed additive and the fermentation broth are mixed in a ratio of 2:1 to 1:20.

38. The method of claim 34, wherein the animal feed or feed additive and the fermentation broth are incubated for at least 1 hour.

39. The method of claim 34, wherein the fermentation broth is selected from fermentation broths of: 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. pumilus, B. smithii, B. toyonensis, B. vallismortis, E. faecium, E. faecalis, G. stearothermophilus, C. butyricum, S. faecalis, S. faecium, S. gallolyticus, S. salivarius subsp. thermophilus and S. bovis; L. acidophilus, L. amylolyticus, L. amylovorus, L. alimentarius, L. aviaries, L. brevis, 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. mucosae, L. panis, L. collinoides, L. paracasei, L. paraplantarum, L. pentosus, L. plantarum, L. pontis, L. reuteri, L. rhamnosus, L. sakei, L. salivarius, L. sanfranciscensis, P. acidilactici, P. dextrinicus, P. pentosaceus, S. adolescentis, B. animalis, B. bifidum, B. breve, B. longum, and mixtures of such fermentation broths.

Description

EXPLANATION OF THE FIGURES

[0130] FIG. 1 shows the cellulase activity of a) vegetative cells of the B. subtilis strain DSM 32540; b) vegetative cells of B. amyloliquefaciens strain CECT 5940; c) sterile filtrated non-dried supernatant of B. amyloliquefaciens CECT 5940 fermentation; d) sterile filtrated non-dried supernatant of B. amyloliquefaciens CECT 5940 fermentation. Cellulase activity leads to a clearance on the agar plate around the zone of cellulose hydrolysis.

[0131] FIG. 2 shows the cellulase activity of a) vegetative cells of B. subtilis strain DSM 32540; b) sterile filtrated supernatant of B. subtilis DSM 32540 fermentation; c) sterile filtrated non-dried supernatant of a B. subtilis DSM 32540 fermentation; d) non-sterile filtrated non-dried supernatant of B. subtilis DSM 32540 fermentation; e) non-sterile filtrated supernatant of B. subtilis DSM 32540 fermentation. Cellulase activity leads to a clearance on the agar plate around the zone of cellulose hydrolysis.

[0132] FIG. 3 shows cellulase activity of a)-d) sterile filtrated (freeze-dried and dissolved) supernatant of B. amyloliquefaciens CECT 5940 fermentation; e) vegetative cells of B. amyloliquefaciens CECT 5940. Cellulase activity leads to a clearance on the agar plate around the zone of cellulose hydrolysis.

[0133] FIG. 4 shows the xylanase activity of a) vegetative cells of B. amyloliquefaciens CECT 5940; b) vegetative cells of B. subtilis DSM 32540; c) sterile filtrated non-dried supernatant of B. amyloliquefaciens CECT 5940 fermentation; d) sterile filtrated non-dried supernatant of a B. amyloliquefaciens CECT 5940 fermentation. Xylanase activity leads to a clearance on the agar plate around the zone of xylan hydrolysis.

[0134] FIG. 5 shows the amylase activity of a)-d) sterile filtrated (freeze-dried and dissolved) supernatant of B. amyloliquefaciens CECT 5940 fermentation; e) vegetative cells of B. amyloliquefaciens CECT 5940 strain. Amylase activity leads to a clearance on the agar plate around the zone of starch hydrolysis.

[0135] FIG. 6 shows the protease activity of a) vegetative cells of a B. subtilis DSM 32540; b) vegetative cells of B. amyloliquefaciens CECT 5940; c) sterile filtrated non-dried supernatant of B. amyloliquefaciens CECT 5940 fermentation; d) sterile filtrated non-dried supernatant of B. amyloliquefaciens CECT 5940 fermentation. Protease activity leads to a clearance on the agar plate around the zone of substrate hydrolysis.

[0136] FIG. 7 shows the protease activity of a)-d) sterile filtrated (freeze-dried and dissolved) supernatant of B. amyloliquefaciens CECT 5940 fermentation; e) vegetative cells of a B. amyloliquefaciens CECT 5940. Protease activity leads to a clearance on the agar plate around the zone of substrate hydrolysis.

[0137] FIG. 8 shows the SDS-PAGE pattern of soy protein extracts treated for 24 h with sterile filtrated non-dried supernatants of fermentation of B. subtilis DSM 32540.

[0138] FIG. 9 shows the SDS-PAGE pattern of soy protein extracts treated for 24 h with sterile filtrated non-dried supernatants of fermentation of B. amyloliquefaciens CECT 5940. Indicated protein bands were identified via nLC/MS analyses as: 1-Beta-conglycinin, alpha′ chain of Glycine max; 2-Beta-conglycinin alpha subunit of Glycine max; 3-Beta-conglycinin alpha subunit of Glycine max; 4-Glycinin of Glycine max; 5-Beta-conglycinin alpha prime subunit of Glycine max; 6-Beta-conglycinin alpha prime subunit of Glycine max; 7-Beta-conglycinin alpha subunit of Glycine max.

WORKING EXAMPLES

Example 1. Qualitative and Quantitative Assessment of Digestive Enzymatic Activities

[0139] Supernatants of a fermentation in a standard medium of a probiotic B. subtilis DSM 32540 and B. amyloliquefaciens strain CECT 5940 were evaluated for digestive enzyme activities, in particular aerobic cellulytic (FIG. 1-3), xylanolytic (FIG. 4), amylase (FIG. 5) and proteolytic activity (FIG. 6-7).

[0140] For the evaluation of cellulase activity, 3 μl of sterile filtrated non-dried and/or freeze-dried and dissolved supernatant of probiotic Bacillus fermentations were spotted onto LB agar containing 5 g/I Sigmacell Cellulose. To screen for protease activity, 3 μl sterile filtrated non-dried and/or freeze-dried and dissolved supernatant of probiotic Bacillus strain fermentations were spotted onto LB agar containing 10% skim milk. Xylanase activity was analyzed in a similar way on LB agar containing 0.5% xylan; amylase activity was analyzed on LB agar containing 10 g/I soluble starch.

[0141] As positive control, enzymatic activities of vegetative cells of the respective probiotic strains were analyzed. Therefore, 3 μl of a liquid culture were spotted directly onto the respective agar plates, which were incubated at 37° C. under aerobic conditions. The read out parameter was the appearance of hydrolysis zones resulting from the enzymatic activities. The plates used in the cellulase and amylase assay were stained with Lugol's iodine solution (FIG. 1-3; 5).

[0142] Analyses of digestive enzyme activities in a qualitative way have shown that respective activities can be found in sterile filtrated supernatants as well as in freeze-dried and dissolved sterile filtrated supernatants.

[0143] In addition, proteolytic activity of sterile filtrated non-dried supernatants of Bacillus strain fermentations was assessed in a quantitative way. 10 μL sterile filtrated supernatant were added to 20 μL 0.5% Fluorescein Isothiocyanate Casein (FITC; C3777, Sigma-Aldrich) solution with 20 μL buffer consisting of 20 mM sodium phospate (dibasic, anhydrous) with 150 mM sodium chloride (all components from Sigma-Aldrich), then incubated for 1 h at 37° C. After addition of 150 μL of 10% (v/v) trichlor acetic acid (Sigma-Aldrich) and another 30 min incubation at 37° C., samples were centrifuged at 19,000 rpm for 15 min, then 2 μL of supernatant transferred to 200 μL 500 mM TRIS HCl Solution (Trizma BaseTRIS, Sigma-Aldrich). Fluorescence of soluble peptides due to proteolytic release were determined (TECAN GENios Microplate Reader, Tecan Group Ltd., Mannedorf, Switzerland) at excitation 494 nm, emission 518 nm. Analysis was performed in two independent runs, then averaged as milliunits per microliter solution. Results can be found in Table 1.

TABLE-US-00001 TABLE 1 Protease activity of sterile filtrated supernatants of B. subtilis DSM 32540 and a B. amyloliquefaciens CECT 5940 fermentation. Protease activity Sterile filtrated supernatant of (mU/mL) a fermentation of a B. subtilis strain 107.56 ± 1.98  a fermentation of a B. amyloliquefaciens strain 1551.97 ± 118.06

[0144] In direct comparison, the supernatant of the fermentation of the B. amyloliquefaciens CECT 5940 has a more than 10 fold increased protease activity than the supernatant of the B. subtilis DSM 32540 fermentation.

Example 2. Pathogen Inhibition by Supernatants of Fermentations of Probiotic Bacillus Strains

[0145] Pathogen inhibition by the supernatant via secondary metabolites produced by probiotic Bacillus strains during fermentation was assessed using well diffusion antagonism tests (Parente et al. 1995).

[0146] A well diffusion antagonism test with different pathogens, Clostridium perfringens type strain ATCC 13124 from Teo and Tan (2005) and Streptococcus suis ATCC 43765 was performed (assay performed with freeze-dried dissolved sterile filtrated supernatant). Strain ATCC 13124 is known to be a alpha-toxigenic Type A strain serving as a type strain for Clostridia.

[0147] S. suis is an important pathogen in pigs and one of the most important causes of bacterial mortality in piglets after weaning causing septicemia, meningitis and many other infections (Goyette-Desjardins et al. 2014). ATCC 43765 belongs to Serological group: R; serovar 2 and was isolated from pigs.

[0148] The pathogenic strains were grown under suitable conditions as liquid culture to an optical density of 600 nm of at least 1, then 130 μl were spread with a sterile spatula on the surface of agar plates. For all pathogens TSBYE agar plates are used. 9 mm diameter wells were cut into the dried plates.

[0149] The 1st well was used as non-inoculated media control without culture, the other wells were inoculated with 100 μL sterile filtrated supernatant (with or without heat treatment) of fermentations of probiotic Bacillus strains. After 24 h incubation under suitable conditions at 37° C., the zone of clearance in mm was determined measuring from the edge of the cut well to the border of the cleared lawn. Each colony was measured twice (horizontally, vertically), then averaged. The results can be found in the following tables 2 and 3.

TABLE-US-00002 TABLE 2 Comparison of heat treated and non-heat treated sterile filtrated non-dried supernatant of B. amyloliquefaciens CECT 5940 fermentation in inhibitory capacity on a pathogenic Clostridium perfringens strain in a well diffusion antagonism assays on TSBYE medium, values in mm clearance of pathogen. Pathogen C. perfringens ATCC Supernatant 13124 (mm) 2 min 85° C. heat treated sterile 29.15 filtrated supernatant of a fermentation of the B. amyloliquefaciens strain CECT 5940 sterile filtrated supernatant of a 28.38 fermentation of a B. amyloliquefaciens strain CECT 5940

[0150] The data show that the non-dried supernatant of a B. amyloliquefaciens CECT 5940 fermentation—also heat treated-inhibits the growth of C. perfringens very effectively.

TABLE-US-00003 TABLE 3 Comparison of heat treated and non-heat treated sterile filtrated supernatant of B. subtilis DSM 32540 fermentation in inhibitory capacity on a pathogenic S. suis strain in a well diffusion antagonism assays on TSBYE medium, values in mm clearance of pathogen. Pathogen S. suis ATCC Supernatant 43765 (mm) 2 min 85° C. heat treated sterile filtrated 22.64 supernatant (freeze dried and dissolved in water again) of a fermentation of B. subtilis strain DSM 32540 sterile filtrated supernatant (freeze dried and 22.60 dissolved in water again) of a fermentation of B. subtilis strain DSM 32540

[0151] The data show that the supernatant of a B. subtilis DSM 32540 fermentation—also heat treated-inhibits the growth of S. suis ATCC 43765 very effectively. Additionally, also after freeze-drying an inhibitory effect could still be observed. [0152] Teo, A. Y.-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. [0153] 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. [0154] Goyette-Desjardins, G., Auger, J. P., 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. 2014 June; 3(6):e45.

Example 3. Assessment of Hydrolytic Activity of Sterile Filtrated Supernatants of Probiotic Bacillus Strain Fermentations on Antinutritional Factors of Soybean Meal

[0155] Proteins were extracted from defatted soybean meal using a method adapted from Iwabuchi and Yamauchi (1987). Defatted soybean meal was extracted using 100 ml 0.03 M Tris-HCl (pH 8) containing 10 mM R-mercaptoethanol with agitation for 1 h at room temperature. Samples were centrifuged, the supernatant sterile filtrated and samples stored at −20° C.

[0156] Sterile filtrated non-dried supernatants of probiotic B. subtilis DSM 32540 and B. amyloliquefaciens CECT 5940 fermentations were incubated in a 2:1 ratio with soy protein extracts at 37° C. Samples were taken at 0 hrs, 6 hrs and 24 hrs, centrifuged and the supernatant stored at −20° C. for further analyses. Control samples with the addition of non-spend medium were analyzed in parallel.

[0157] Protein concentrations were determined using the Bio-Rad Protein Assay Kit (Bio-Rad, USA). Protein hydrolysis was monitored using SDS-Page. Protein concentrations were adjusted and proteins denatured by 5 min 95° C. heat treatment before loading onto the gel. 20 μg of extracted proteins were loaded into each well of a 10% Mini Protean TGX Precast SDS Gel. The Precision Plus Protein™ Dual Color Standards protein ladder was used as marker (10-250 kDa). Proteins were separated at 40 mA for an hour. Gels were stained with a Coomassie Brilliant Blue G250 and Coomassie Brilliant Blue R250 solution and destained with an acetic acid solution. Degradation of soy proteins could be observed by the disappearance of proteins bands over time (FIGS. 8 and 9).

[0158] Proteolytic degradation of soy protein extracts was undetectable in control samples containing medium and soy protein extract only. Hydrolysis of prominent proteins could be detected after 6 and 24 h incubation of soy protein extract with a sterile filtrated supernatant of a B. subtilis DSM 32540 fermentation and B. amyloliquefaciens CECT 5940 fermentation, respectively. An increase in of smaller peptides (<25 kDa) accompanied the decrease of multiple bigger protein bands 25-75 kDa). Protein bands that were degraded over time and specific bands from the control sample of the soy extract incubated with medium only were analyzed by nano-LC/MS and identified via high-resolution mass spectrometry to be ß-conglycinine and glycinine of Glycine max. Thus, the antigenic proteins R-conglycinin and glycinin of soy were degraded during the incubation with non-dried sterile filtrated supernatant of a Bacillus fermentations. [0159] Iwabuchi, S. and Yamauchi, F. (1987): Determination of glycinin and R-conglycinin in soybean proteins by immunological methods. J. Agric. Food Chem. 35, 200-205.