BACILLUS SUBTILIS STRAIN WITH PROBIOTIC ACTIVITY

Abstract

The current invention concerns a new B. subtilis strain with strong inhibition of swine and poultry related pathogens and its use as probiotic.

Claims

1-19. (canceled)

20. A Bacillus subtilis strain or a preparation thereof selected from the following group consisting of: a) a B. subtilis strain deposited as DSM 32540; b) a mutant of the B. subtilis strain deposited as DSM 32540 and having all identifying characteristics of the deposited strain; c) a preparation of the B. subtilis strain of paragraph (a) or the mutant B. subtilis strain of paragraph (b); and d) a preparation containing a mixture of compounds from the B. subtilis strain of paragraph (a), and/or the mutant B. subtilis strain of paragraph (b) and/or the preparation of paragraph (c).

21. The Bacillus subtilis strain or a preparation thereof of claim 20, wherein the Bacillus subtilis strain or a preparation thereof is a mutant comprising a DNA sequence identity to the strain DSM 32540 of at least 95%.

22. The Bacillus subtilis strain or a preparation thereof of claim 20, wherein the Bacillus subtilis strain or a preparation thereof is a strain exhibiting at least one of the following characteristics: a) a yqfD sequence with a sequence identity of at least 99.5% to the polynucleotide sequence of SEQ ID NO:2; b) a gyrB sequence with a sequence identity of at least 99.5% to the polynucleotide sequence of SEQ ID NO:3; c) an rpoB sequence with a sequence identity of at least 99.5% to the polynucleotide sequence of SEQ ID NO:4; d) a groEL sequence with a sequence identity of at least 99.5% to the polynucleotide sequence of SEQ ID NO:5.

23. The Bacillus subtilis strain or a preparation thereof of claim 20, wherein the Bacillus subtilis strain or a preparation thereof comprises a strain with a 16S rDNA sequence comprising a sequence identity of at least 99.5% to the polynucleotide sequence of SEQ ID NO:1.

24. The B. subtilis strain or a preparation thereof of claim 20, wherein the B. subtilis strain or a preparation thereof comprises a strain able to grow anaerobically.

25. The B. subtilis strain or a preparation thereof of claim 20, wherein the B. subtilis strain or a preparation thereof is able to inhibit the growth of at least one strain selected from the group consisting of: C. perfringens ATCC 13124; C. perfringens BB-081 Cpe; C. perfringens BB 031 Cpe; C. difficile DSM 1296; S. aureus subsp. aureus DSM 20231; S. gallinaceus DSM 15349; S. suis DSM 9682; C. coli DSM 4689; and E. cecorum DSM 20683.

26. The B. subtilis strain or a preparation thereof of claim 20, wherein the B. subtilis strain or a preparation thereof is a strain able to grow in the presence of 0.05 wt % acetic acid, 0.05 wt % propionic acid, and/or 0.2 wt % lactic acid.

27. The B. subtilis strain or a preparation thereof of claim 20, wherein the B. subtilis strain or a preparation thereof comprises one or more activities selected from the group consisting of: cellulase activity; xylanase activity; catalase activity; and superoxide dismutase activity.

28. The B. subtilis strain or a preparation thereof of claim 20, wherein the B. subtilis strain or a preparation thereof is a strain able to grow in presence of 2 mM bile.

29. A composition comprising the B. subtilis strain or a preparation thereof claim 20 and at least one further component.

30. The composition of claim 29, wherein the composition is a feed or food and the additional component comprises a protein, carbohydrate, fat, probiotic, prebiotic, enzyme, vitamin, immune modulator, milk replacer, mineral, amino acid, coccidiostat, acid-based product, medicine, or combination thereof.

31. The composition of claim 29, wherein the composition is a pharmaceutical composition and the additional component is a pharmaceutically acceptable carrier.

32. The composition of claim 29, wherein the composition is for improving the health status of an animal or a human being.

33. A method of providing nutrients or improving the health of a human or animal, inhibiting the growth of pathogenic bacteria or providing a healthier environment, comprising administering or applying a composition comprising the Bacillus subtilis strain or a preparation thereof of claim 20.

34. The method of claim 33, wherein the composition is administered as a feed or pharmaceutical to an animal or human.

35. The method of claim 33, wherein the composition is administered to an animal or a human being to improve the gut health status of the animal or a human being.

36. The method of claim 33, wherein the composition is administered to animals: to enhance the health of the animals; and/or to improve the general physical condition of the animals; and/or to improve the feed conversion rate of the animals; and/or to decrease the mortality rate of the animals; and/or to increase the survival rate of the animals; and/or to improve the weight gain of the animals; and/or to increase the disease resistance of the animals; and/or to increase the immune response of the animals; and/or to establish or maintain a healthy gut microflora in the animals; and/or to reduce pathogen shedding through the feces of the animals.

37. The method of claim 33, wherein the composition is applied to manure, contaminated liquids, litter, a pit, or a manure pond to control and/or avoid detrimental environmental effects of the manure, contaminated liquids, litter, pit, or manure pond.

38. The method of claim 33, wherein the composition is applied to water or an aqueous solution to improve the quality of the water or aqueous solution.

39. The method of claim 33, wherein the composition is applied to a cultivated plant to treat and/or prevent a microbial disease.

Description

WORKING EXAMPLES

Example 1. Strain Characteristics Relevant to Survival in the Gastrointestinal Tract

[0134] Bacillus subtilis strains were screened from various environmental samples in order to obtain a superior strain as animal direct-fed microbial/probiotic. As the strain is intended to reach its full potential in the intestine of the target animal, the strain was pre-screened to withstand various environmental and gut related conditions. Strain spores were generated (Nicholson and Setlow 1990), washed and incubated at 80 C. for 20 minutes (pasteurization), then titrated in logarithmic/1 in 10 dilutions using veal infusion broth agar (VI, Difco, no. 234420, Becton Dickinson GmbH, Heidelberg, Germany). The second highest dilution prior to no growth was stored at 80 C. and used as standardized starting point for all further assessments from spore state. To simulate gastric passage (Argenzio 2004a; Trampel and Duke 2004), survival of acid exposure was assessed based on Larsen et al. (2014). Growth of vegetative cells was furthermore assessed at low pH indicating growth under stomach/proventriculus and gizzard conditions, as well as in presence of up to 4 mM bile (B8631, CAS 8008-63-8, Sigma-Aldrich) at pH 7 in order to confirm strain growth at the proximal part of the small intestine right after clearance of the stomach or gizzard (Argenzio 2004b; Trampel and Duke 2004). Strain fitness in the anaerobe intestine (Argenzio 2004b; Trampel and Duke 2004) was assessed by inoculating standardized spore solutions under anaerobic conditions (AnaeroPak, Thermo Fisher Scientific) in VI medium supplemented with 2.5 mM KNO.sub.3 (Glaser et al. 1995). Furthermore was the anaerobe proteolytic and cellulytic activity of strains assessed on VI agar plates supplemented with 1% skim milk powder (70166, Sigma-Aldrich) or 0.1% water insoluble AZCL-HE cellulose (I-AZCEL, Megazyme International, Bray, Ireland). Furthermore the aerobe cellulytic activity of strains was assessed using VI agar supplemented with 0.1% water insoluble AZCL-HE cellulose (I-AZCEL, Megazyme International, Bray, Ireland). After 24 h under aerobe conditions, a blue coloration of agar indicated cellulase activity.

[0135] Osmotic stress, as also found in the gut (Argenzio 2004b; Trampel and Duke 2004), was assessed by determining growth on VI agar with addition of 10 wt.-% NaCl (den Besten et al. 2009). Finally, spore heat stability was assessed to determine pelleting stability by exposing spores to 99 C. for 20 min (Palop et al. 1996) and subsequent inoculation on VI agar.

[0136] Bacillus subtilis strain DSM 32540 survived simulated gastric passage, growth of the strain was observed starting at pH 6. Strain DSM 32540 grew anaerobically and was able to degrade water-insoluble cellulose and protein under anaerobic conditions. Further, it was able to degrade water-insoluble cellulose under aerobic conditions. Strain DSM 32540 was able to grow in presence of 2 and 4 mM bile, in presence of 0.3 wt.-% porcine bile and in presence of 0.3 wt.-% chicken bile as well as in presence of 10 wt.-% NaCl. Strain DSM 32540 reached an average spore count of 9.110.sup.8 CFU/mL, and spores of strain DSM 32540 were viable after exposure to 99 C. for 20 min.

REFERENCES

[0137] Argenzio, R. A. (2004a). Secretion of the Stomach and Accessory Glands, p. 405-418. In: Reece, W. O. (ed.), Duke's Physiology of Domestic Animals; Twelfth Edition, Chapter 25; Cornell University Press, Ithaca, N.Y., USA. [0138] Argenzio, R. A. (2004b). Digestive and Absorptive Functions of the Intestines, p. 419-437. In: Reece, W. O. (ed.), Duke's Physiology of Domestic Animals; Twelfth Edition, Chapter 26; Cornell University Press, Ithaca, N.Y., USA. [0139] Dawson, R. M. C.; Elliot, D. C.; Elliot, W. H.; Jones, K. M. (1986). Data for Biochemical Research; 3.sup.rd edition, Oxford Science Publishing, United Kingdom. [0140] Den Besten H M W, Mols M, Moezelaar R, Zwietering M H, Abee T. (2009). Phenotypic and transcriptomic analyses of mildly and severely salt-stressed Bacillus cereus ATCC 14579 cells. Appl Environ Microbiol. 75:4111-9. [0141] Glaser, P., A. Danchin, F. Kunst, P. Zuber, and M. M. Nakano. (1995). Identification and isolation of a gene required for nitrate assimilation and anaerobic growth of Bacillus subtilis. J. Bacteriol. 177:1112-1115 [0142] Nicholson W. L., Setlow P. Sporulation, germination and outgrowth. In: Harwood C R, Cutting S M, editors. Molecular biological methods for Bacillus. Chichester, England: John Wiley & Sons Ltd.; 1990. pp. 27-74. [0143] Palop, A., Raso, J., Pagan, R., Condon, S. and Sala, F. J. (1996). Influence of pH on heat resistance of Bacillus licheniformis in buffer and homogenized foods. International Journal of Food Microbiology 29, 1-10. [0144] Trampel, D. W. and Duke, G. E. (2004). Avian Digestion, p. 488-500. In: Reece, W. O. (ed.), Duke's Physiology of Domestic Animals; Twelfth Edition, Chapter 29; Cornell University Press, Ithaca, N.Y., USA.

Example 2. Comparative Strain Performance Relative to State of the Art Direct-Fed Microbial (DFM)/Probiotic for Animal NutritionQuantitative Assessment of Bile Tolerance

[0145] In order to assess the competitiveness of the Bacillus subtilis strain DSM 32540 selected from example 1, benchmarked analysis was performed using commercially available Bacillus subtilis strain DSM 17299 and/or Bacillus licheniformis strain DSM 17236. Readiness of strains to perform in the proximal small intestine in presence of bile at neutral pH right after gastric passage (Argenzio 2004b; Trampel and Duke 2004) was determined by strain growth in VIB media with addition 0.3 wt.-% porcine bile (Sigma Aldrich). Overnight culture with 50 uL candidate strain cell suspension and 10 mL VIB in 100 mL conical flask was incubated at 37 C. and 200 rpm, then approximately 50 uL of overnight culture was transferred to 100 well honeycomb plates (Oy Growth Curves Ab Ltd, former Thermo Labsystems, Helsinki, Finland) with 1 mL VIB at pH 7 with 0.3 wt.-% porcine bile in order to obtain OD 0.2 per mL.

[0146] Strain specific growth at 37 C. and 200 rpm was observed for 48 h with OD determined every 15 min using Bioscreeen C MBR with BioLink software package (Oy Growth Curves Ab Ltd). Quantitative assessment for each strain was compared as area under the curve between 0-5 h (AUC5, in ODtime in h), area under the curve between 0-10 h (AUC10 in ODtime in h), and time until strains reached its maximum optical density (Tmax in h). Results can be found in Table 2.1.

TABLE-US-00001 TABLE 2.1 Growth of Bacillus subtilis strain DSM 32540 and benchmark strains DSM 17299 and DSM 17236 in presence of 0.3 wt.-% porcine bile. Strain Tmax AUC5 AUC10 AUC30 DSM 32540 9.25 3.33 10.35 31.68 DSM 17299 26 1.57 3.68 22.67 DSM 17236 36.75 1.19 3.27 15.02

[0147] AUC5, area under the curve between time point 0 and 5 h in optical densityh; AUC10, area under the curve between time point 0 and 10 h in optical densityh; Tmax, time in h until maximum optical density was reached.

[0148] In direct comparison, strain DSM 32540 reached its maximum OD in presence of 0.3 wt.-% porcine bile 16.75 h faster than the benchmark strain DSM 17299 and 27.5 h faster than DSM 17236. In addition, strain DSM 32540 grew 2.7 times faster during the first 5 hours, 3.2 times faster during the first 10 h and 2.1 times faster during the first 30 h compared to the growth of DSM 17236, respectively. Furthermore DSM 32540 is able to grow in the presence of 0.3% chicken bile.

REFERENCES

[0149] Argenzio, R. A. (2004b). Digestive and Absorptive Functions of the Intestines, p. 419-437. In: Reece, W. O. (ed.), Duke's Physiology of Domestic Animals; Twelfth Edition, Chapter 26; Cornell University Press, Ithaca, N.Y., USA. [0150] Trampel, D. W. and Duke, G. E. (2004). Avian Digestion, p. 488-500. In: Reece, W. O. (ed.), Duke's Physiology of Domestic Animals; Twelfth Edition, Chapter 29; Cornell University Press, Ithaca, N.Y., USA.

Example 3. Comparative Strain Performance Relative to State of the Art Direct-Fed Microbial (DFM)/Probiotic for Animal NutritionGrowth in Presence of Short Chain Fatty Acids (SCFA)

[0151] Comparative growth of strains DSM 32540 and DSM 17299 was assessed in presence of short chain fatty acids as those are observed in the gut with increasing importance towards the large intestine (Argenzio 2004b; Trampel and Duke 2004). Tests were initiated using standardized spore solution as described in example 1 testing aerobe growth in VI medium at 37 C. and pH 6, read-out parameter was growth versus no growth. For this test, VI medium was adjusted to pH 6 using Mcllvaine buffer (Palop et al. 1996) and subsequently supplemented with 0.05% acetic acid (HA, 537020, CAS 64-19-7, Sigma-Aldrich), 0.05% propionic acid (HP, P1386, CAS 79-09-4, Sigma-Aldrich) or 0.2% lactic acid (HL, W261106, CAS 50-21-5, Sigma-Aldrich). Results can be found in Table 3.1.

TABLE-US-00002 TABLE 3.1 Assessment of growth of Bacillus subtilis strains DSM 32540 and benchmark strain DSM 17299 in presence of short chain fatty acids at pH 6. Strain ID Acetic acid Propionic acid Lactic acid DSM 32540 Yes Yes Yes DSM 17299 No growth No growth No growth

[0152] Bacillus subtilis strain DSM 32540 was able to grow at pH 6 in the presence of acetic, propionic and lactic acid, whereas strain DSM 17299 was unable to grow from spore stage under these conditions.

REFERENCES

[0153] Argenzio, R. A. (2004b). Digestive and Absorptive Functions of the Intestines, p. 419-437. In: Reece, W. O. (ed.), Duke's Physiology of Domestic Animals; Twelfth Edition, Chapter 26; Cornell University Press, Ithaca, N.Y., USA. [0154] Palop, A., Raso, J., Pagan, R., Condon, S. and Sala, F. J. (1996). Influence of pH on heat resistance of Bacillus licheniformis in buffer and homogenized foods. International Journal of Food Microbiology 29, 1-10. [0155] Trampel, D. W. and Duke, G. E. (2004). Avian Digestion, p. 488-500. In: Reece, W. O. (ed.), Duke's Physiology of Domestic Animals; Twelfth Edition, Chapter 29; Cornell University Press, Ithaca, N.Y., USA.

Example 4. Comparative Strain Performance Relative to State of the Art Direct-Fed Microbial (DFM)/Probiotic for Animal NutritionQuantitative Assessment of Enzymatic Activity

[0156] Similar to test conducted in Example 2, strains DSM 32540, DSM 17299 and DSM 17236 were compared evaluating the respective aerobe xylanolytic activity. Xylanase activity was determined as described in Larsen et al. (2014). Analysis was performed in three independent runs, then averaged as milliunits per microliter solution.

TABLE-US-00003 TABLE 4.1 Xylanase activity of strains DSM 32540, DSM 17299 and DSM 17236. Xylanase activity Strain ID (mU/mL) DSM 32540 11.8 0.3 DSM 17299 11.8 1.5 DSM 17236 7.9 1.3

[0157] In direct comparison, strain DSM 32540 has a 1.5 fold increased xylanase activity compared to benchmark strain DSM 17236 and a similar xylanase activity compared to benchmark strain DSM 17299.

REFERENCE

[0158] Larsen, N., Thorsen, L., Kpikpi, E. N., Stuer-Lauridsen, B., Cantor, M. D., Nielsen, B., Brockmann, E., Derkx, E. M. F. and Jespersen, L. (2014). Characterization of Bacillus spp. strains for use as probiotic additives in pig feed. Applied microbiology and biotechnology, 98(3), 1105-1118.

Example 5. Comparative Strain Performance Relative to State of the Art Direct-Fed Microbial (DFM)/Probiotic for Animal NutritionExpression of Metabolites and Pathogen Inhibition

[0159] Similar to tests conducted in Example 2, strains DSM 32540 and wildtype strain DSM 10 were compared evaluating the respective number of metabolites expressed and pathogens inhibited in the respective media. For metabolite expression analysis, starter cultures were grown and tests performed as described in Scholz et al. (2011). From 10 mL Luria Bertami broth (LB, Thermo Fisher Scientific) culture grown for 24 h at 37 C. and 160 rpm in 100 mL flask, 100 uL were transferred to main culture. Main culture was grown either in 10 mL LB containing 0.2 mL/L KellyT trace metal solution (LBKelly, Scholz et al. 2011), or 10 mL Trypticase Soy Broth (Oxoid, Thermo Fisher Scientific) with 0.6% yeast extract (Y1625, CAS 8013-1-2, Sigma-Aldrich; resulting broth abbreviated TSBYE), both for 24 h at 37 C. at 160 rpm in 100 mL flask. Of the main culture, 4 mL were combined with 2 mL n-Butanol in 15 mL test tube, vortexed for 3 min, then sonicated for 15 min. After centrifugation for 1 min at 5000 rpm, organic phase was transferred, vacuum dried and analyzed using High-performance liquid chromatographyelectrospray ionization mass spectrometry (HPLC-ESI-MS; Chen et al. 2006). Every sample was measured in two different modes, negative and positive mode, and mass spectra were acquired. Resulting peaks as similarly reported in Teo and Tan (2005) were converted to molecular mass in Da. Results for comparison can be found in Table 5.1.

TABLE-US-00004 TABLE 5.1 (a) Comparison of metabolites expressed by strains DSM 32540 and wildtype strain DSM 10 in LB-Kelly and TSBYE, respectively (n/d means not detected) 330 350 352 423 424 477 480 572 993 994 1008 1143 Strain Da Da Da Da Da Da Da Da Da Da Da Da DSM 10 n/d n/d n/d n/d n/d n/d n/d n/d yes n/d yes n/d DSM 32540 yes yes yes yes yes yes yes yes yes yes yes yes

TABLE-US-00005 TABLE 5.1 (b) Comparison of metabolites expressed by strains DSM 32540 and wildtype strain DSM 10 in LB-Kelly and TSBYE, respectively (n/d means not detected) 1022 1026 1036 1040 1050 1460 1462 1476 1489 1504 1505 3401 Strain Da Da Da Da Da Da Da Da Da Da Da Da DSM 10 yes n/d yes n/d yes yes yes yes n/d yes n/d yes DSM 32540 yes yes yes yes yes yes n/d yes yes yes yes yes

[0160] In addition, pathogen inhibition via Bacillus subtilis secondary metabolite production, as part of metabolites from table 5.1. but not closer investigated, was assessed using well diffusion antagonism tests (Parente et al. 1995).

[0161] A well diffusion antagonisms test with 7 different pathogens, Clostridium perfringens, Clostridium difficile DSM 1296, Enterococcus cecorum DSM 20683, Staphylococcus aureus subsp. aureus DSM 20231, Streptococcus gallinaceus DSM 15349, Streptococcus suis ATCC 43765 and Campylobacter coli ATCC 33559 was performed.

[0162] Three pathogenic C. perfringens candidates were tested being C. perfringens type strain ATCC 13124 from Teo and Tan (2005), as well as two pathogenic C. perfringens field isolates from swine, obtained from RIPAC-LABOR GmbH, Potsdam-Golm, Germany. The C. perfringens type C-strains from Ripac describe as follows: Strains BB-081_Cpe and BB-031_Cpe were isolated from necrotic enteritis positive swine digestive tract. Strain BB-081_Cpe is cpb2 positive (Songer et al. 2005) and netB negative and Strain BB-031_Cpe tested positive for 2-toxin (Allaart et al. 2012). Strain ATCC 13124 is known to be alpha-toxigenic Type A strain serving as a type strain for Clostridia.

[0163] Clostridium difficile is an important emerging pathogen that causes diarrhea primarily in neonatal swine (Songer et al. 200). Affected piglets may have dyspnea, abdominal distention, and scrotal edema. Diarrhea may not be present in all pigs affected. DSM 1296 is a known type strain for C. difficile and produces cytotoxin.

[0164] Staphylococcus aureus subsp. aureus can cause bumblefoot in chickens (McMullin 2004), streptococcal mastitis in sows (Contreras et al. 2011) and it is capable of generating toxins that produce food poisoning in the human body (2016 Centers for Disease Control and Prevention). DSM 20231 is a serotype 3 type strain.

[0165] E. cecorum is known to cause lameness, arthritis and osteomyelitis in broilers usually caused by an inflammation of a joint and/or bone tissue. Additional E. cecorum can cause an inflammation of the pericardium [Kense et al. 2011]. DSM 20683 was isolated from caecum of a chicken.

[0166] S. gallinaceus can cause septicaemia in poultry. The gross lesions included splenomegaly, hepatomegaly, renomegaly and congestion. Multiple areas of necrosis and/or infarction in the liver and spleen associated with valvular endocarditis were also observed [Collins et al. 2002].

[0167] 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.

[0168] C. coli is a foodborne bacterium, most people usually get infected by eating pig meat that contained the bacteria. It causes gastroenteritis and acute enterocolitis in humans, and also of acute diarrheal illnesses [Fitzgerald et al. 2007]. Pigs are the main host, but it can also infect humans, avian species and a wide range of other animals. ATCC 33559 was isolated from pig feces.

[0169] Bacillus strains were grown in 10 mL TSBYE (30 g/l TSB+6 g/l Yeast extract) or LB-Kelly (LB-Media supplemented with trace elements solution of DSMZ media 1032) for 16 hat 37 C. and 200 rpm in 100 mL shaking flask. The pathogenic strains were grown under suitable conditions as liquid culture to an optical density of 595 nm of at least 1, then 100 l were spread with sterile spatula on the surface of agar plates. For S. gallinaceus BHI agar plates, all other pathogens TSBYE agar plates are used. Three 9 mm diameter wells were cut into the dried plates. 1.sup.st well was used as non-inoculated media control without culture, 2.sup.nd well was inoculated with 100 uL not-inhibiting Bacillus strain (B. cereus var. toyoi, NCIMB 40112), the 3.sup.rd well was inoculated with 100 uL of Bacillus subtilis DSM 32540 or DSM 17299 culture. 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.

TABLE-US-00006 TABLE 5.2 Comparison of Bacillus subtilis DSM 32540, DSM 17236 and DSM 17299 inhibitory capacity on pathogenic Clostridium strains in well diffusion antagonism assays on LB Kelly medium, values in mm clearance of pathogen. Pathogen C. perfringens C. perfringens C. perfringens C. difficile Probiotic ATCC 13124 BB-081_Cpe BB_031_Cpe DSM 1296 DSM 32540 20.3 26.3 18.7 12.3 DSM 17299 8 8 8 n/d DSM 17236 17 15.5 8 8

[0170] The data show that DSM 32540 is able to inhibit the growth of C. perfringens and S. difficile very effectively, in particular in comparison to DSM 17299.

TABLE-US-00007 TABLE 5.3 Comparison of Bacillus subtilis DSM 32540, DSM 17236 and DSM 17299 inhibitory capacity on pathogenic Staphylococcus, Streptococcus and Campylobacter strains in well diffusion antagonism assays on LB Kelly medium, values in mm clearance of pathogen. Pathogen S. aureus S. subsp. aureus gallinaceus S. suis C. coli Probiotic DSM 20231 DSM 15349 DSM 9682 DSM 4689 DSM 32540 21 21.8 30.4 28.4 DSM 17299 8 8 13.5 8 DSM 17236 n/d n/d 20.7 8

[0171] The data show that DSM 32540 is able to inhibit the growth of S. aureus subsp. aureus, S. gallinaceus, S. suis and C. coli very effectively, in particular in comparison to DSM 17299.

TABLE-US-00008 TABLE 5.4 Comparison of Bacillus subtilis DSM 32540, DSM 17236 and DSM 17299 inhibitory capacity on pathogenic Enterococcus cecorum in well diffusion antagonism assays on TSBYE medium, values in mm clearance of pathogen. Pathogen E. cecorum Probiotic DSM 20683 DSM 32540 16.9 DSM 17299 8 DSM 17236 13.7

[0172] The data show that DSM 32540 is able to inhibit the growth of E. cecorum very effectively, in particular in comparison to DSM 17299.

REFERENCES

[0173] 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. Paul McMullin (2004) Chapter: Staphylococcus aureus subsp. aureus infections in chickens. Book: A pocket guide to poultry health and disease. Publisher: 5m Publishing. [0174] 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. [0175] G A Contreras, J M Rodriguez (2011) Mastitis: Comparative Etiology and Epidemiology Journal of Mammary Gland Biology and Neoplasia, Volume 16, Issue 4, pp 339-356 (2016) U.S. Department of Health & Human Services (https://www.cdc.gov/foodsafety/diseases/staphylococcal.html) [0176] Allaart, J. G., de Bruijn, N. D., van Asten, A. J., Fabri, T. H., and Grone, A. (2012). NetB-producing and beta2-producing Clostridium perfringens associated with subclinical necrotic enteritis in laying hens in the Netherlands. Avian Pathol., 41:541-546 JG Songer, F A Uzal (2005) Clostridial Enteric Infections in Pigs. Volume: 17 issue: 6, page(s): 528-536 Journal of Veterinary Diagnostic Investigation [0177] Songer J G, Post K W, Larson D J, et al. (2000) Infection of neonatal swine with Clostridium difficile. Swine Health Prod. 2000; 8(4):185-189 [0178] MJ Kense, W J M Landman (2011) Enterococcus cecorum infections in broiler breeders and their offspring: molecular epidemiology. Avian Pathology, Volume 40, Issue 6 [0179] MD Collins, R A Hutson, E Falsen, E Ingana, M Bisgaard (2002) Streptococcus gallinaceus sp. nov., from chickens. International Journal of Systematic and Evolutionary Microbiology, 52, 1161-1164 [0180] G Goyette-Desjardins, J-P Auger, J Xu, M Segura, M Gottschalk (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. [0181] C Fitzgerald, I Nachamkin (2007). Campylobacter and Arcobacter. In P. R. Murray (Ed.), Manual of Clinical Microbiology (9th ed., pp. 933-946). Washington D.C.: ASM Press.

Example 6. Qualitative Assessment of Antioxidant Enzymatic Activity

[0182] The presence of reactive oxygen species during oxidative stress, which can be caused by stressful conditions as for example heat stress (Lin et al., 2006), can lead to a damage of DNA, proteins or lipids. Probiotics can support the host's oxidative defense system by increasing the antioxidant enzyme activities (Aluwong et al., 2013, Mishra et al., 2015). Therefore, the strain DSM 32540 was screened for antioxidant enzyme activities, in particular for superoxide dismutase and catalase activity. For the evaluation of catalase activity of grown biofilms of the strain, the strain was grown in LB medium supplemented with glucose for 15 hrs at 37 C. and 200 rpm in shaking flasks. The cultures were adjusted to an optical density OD.sub.600 of 1.0 and 10 l of the cultures were spotted onto TSBYE (30 g/l TSB+6 g/l Yeast extract) or LB-Kelly (LB-Media supplemented with trace elements solution of DSMZ media 1032) agar plates, which were incubated at 37 C. under aerobic conditions and at 37 C. under 0.2% oxygen for 15 hrs. 3% H.sub.2O.sub.2 (hydrogen peroxide solution 3%, Sigma-Aldrich) was dropped onto the colonies and catalase activity was analyzed. The read out parameter was the production of 02 and H.sub.2O from H.sub.2O.sub.2 resulting in the formation of foam on the colonies.

TABLE-US-00009 TABLE 6.1 Assessment of catalase activity of colonies of Bacillus subtilis strain DSM 32540 grown as biofilm on LB-Kelly or TSBYE medium under aerobic conditions. Strain ID TSBYE LB-Kelly DSM 32540 Yes Yes

[0183] Bacillus subtilis strain DSM 32540 displayed catalase activity when grown under aerobic conditions.

TABLE-US-00010 TABLE 6.2 Assessment of catalase activity of colonies of Bacillus subtilis strain DSM 32540 grown as biofilm on LB-Kelly or TSBYE medium under 0.2% oxygen. Strain ID TSBYE LB-Kelly DSM 32540 Yes Yes

[0184] Bacillus subtilis strain DSM 32540 displayed catalase activity also when grown under 0.2% oxygen.

[0185] Catalase activity was analyzed in protein extracts obtained from planktonic cells grown under aerobic conditions as well. The strain DSM 32540 was grown for 15 hrs in LB medium containing glucose at 37 C. and 200 rpm in shaking flasks. 8 ml of the cell cultures were harvested by centrifugation for 10 min at 4 C. and 3000 rpm and the pellet was resuspended in PBS pH 7.3. Soluble cell extracts were obtained by using a ribolyser. The disrupted cells were centrifuged for 10 min at 4 C. and 13000 rpm and the supernatant was used for further steps. The protein concentration of the protein extracts was determined by the method of Bradford with bovine serum albumin as a standard (Bradford, 1976). The concentration of the protein extracts was adjusted with PBS pH 7.3, the protein extracts were mixed with native sample loading buffer (2, VWR) and native gel electrophoresis (10% non-denaturing polyacrylamide gels, Biorad) was applied to separate the proteins at 4 C. Catalase activity in cell extracts from planktonic cells was then detected by staining the gel in a staining solution of 1% FeCl.sub.3 and 1% K3Fe(CN).sub.6 (Woodbury et al., 1971). Catalase activity can be seen as bright bands.

TABLE-US-00011 Strain ID LB with glucose DSM 32540 Yes

[0186] Bacillus subtilis strain DSM 32540 displayed catalase activity also under these specific conditions as tested.

[0187] Superoxide dismutase activity was analyzed in protein extracts from cells grown under aerobic conditions as well. Cell extracts were obtained by the method described above and proteins were separated by performing native gel electrophoresis at 4 C. Superoxide dismutase activity was detected by staining the gel with a nitroblue tetrazolium staining method adapted from Beauchamp and Fridovich (19711

TABLE-US-00012 Strain ID LB with glucose DSM 32540 Yes

[0188] Bacillus subtilis strain DSM 32540 displayed superoxide dismutase activity under the conditions tested.

REFERENCES

[0189] Aluwong, T.; Kawu, M.; Raji, M.; Dzenda, T.; Govwang, F.; Sinkalu, V. and Ayo, J. (2013). Effect of Yeast Probiotic on Growth, Antioxidant Enzyme Activities and Malondialdehyde Concentration of Broiler Chickens. Antioxidants 2: 326-339 [0190] Beauchamp, C. and Fridovich, I. (1971). Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44: 276-287. [0191] Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. [0192] Lin, H.; Decuvpere, E. and Buyse, J. (2006). Acute heat stress incuces oxidative stress in broiler chickens. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 144: 11-17. [0193] Mishra, V.; Shah, C.; Mokashe, N.; Chavan, R.; Yadav, H. and Prajapati, J. (2015). Probiotics as potential antioxidants: a systematic review. J. Agric. Food Chem 63: 3615-3626 [0194] Woodbury, W.; Spencer, A. K. and Stahmann, M. A. (1971). An improved procedure using ferricyanide for detecting catalase isozymes. Anal. Biochem. 44: 301-305.

Example 7. Detection of Lactate Production

[0195] It has been shown in in vitro studies that inhibition of pathogen replication can be mediated by low-molecular-weight substances (Oelschlager 2010). Top of this list are short chain fatty acids, e.g. lactic acid (Oelschlager 2010). Possible explanation for the inhibition of pathogens can be decreasing the pH by production of lactic acid (Fuller 1992). Lactic acid bacteria, which are also used as probiotic in animal feed are known to produce lactic acid as major end-product during fermentation of cabohydrates (Halasz 2009). It is shown that B. subtilis strain DSM 32540 can produce lactate in vitro.

[0196] B. subtilis strain DSM 32540 and Bacillus toyonensisa probiotic bacillus known to produce lactic acidwere compared evaluating the anaerobe lactate production. Lactate production was determined as follows: Precultures of the strains were grown over night at 37 C. in 10 mL TSBYE (30 g/l TSB+6 g/l Yeast extract) under aerobic conditions. Main culture was inoculated with an Moo of 0.2 in 10 ml TSBYE complemented with 25 g/l sucrose and 5 mM KNO.sub.3 in 15 ml Falcon Tubes at 37 C. without shaking for 48 h. Lactate was measured in the supernatant with HPLC-UV.

TABLE-US-00013 % Lactate production Bacillus toyonensis 100.00 DSM 32540 147.47

[0197] Strain B. subtilis DSM 32540 produces almost 50% more lactate compared to probiotic strain of Bacillus toyonensis.

REFERENCES

[0198] A Halasz (2009) Book: Food quality and standards Volume III; EOLSS Publishers Co Ltd. Chapter: Lactic acid bacteria. [0199] TA Oelschlager (2010) Mechanisms of probiotic actionsA review. International Journal of Medical Microbiology Volume 300, Issue 1, January 2010, Pages 57-62. [0200] R Fuller 1992 Publisher: Springer science and business media Dordrecht Book: Probiotics: The scientific basis Chapter 9.8 Antimicrobial activity.

Example 8. Comparison of Performance of Swine Reared in China Fed the Novel Bacillus subtilis Strain DSM 32540

[0201] The experiment comprises 168 25-day weaned piglets (LandraceYorkshire), randomly allocated to three treatments (Table 8.1), with 8 replicates, 7 piglets with average 6.50.5 kg for each replicate. Three treatments were mainly based on corn-soybean meal (Table 8.2) and included; 1. Basal control (Control), 2. Control+30 g of Virginiamycin/MT of feed (AGP), 3. Control+Bacillus subtilis strain DSM 32540 at 250 g/MT containing 2.0*10.sup.9 cfu/g (DSM 32540). Experimental treatments were fed ad libitum in mash form from 1-42 days of age.

TABLE-US-00014 TABLE 8.1 Experimental Treatments Additive Treat Diet type Inclusion Level 1 Control diet (without AGP) 2 Control diet + antibiotic (AGP, virginamycin (0.04 kg/t)) 3 Control diet + DSM 32540 500 g/t

TABLE-US-00015 TABLE 8.2 Ingredient and nutrient composition of basal diet. wt.-% Ingredients Corn 48 SBM, 46% 11.68 Barley 10 Fermented SBM, 50% 6 Whey, 3.5% 5.3 Full fat Soya 4 Soya oil 3 Fish meal 3 Glucose 2.5 Cane Sugar 2.5 lS, 38% 0.98 CPM, 23% 0.45 Lys, 98% 0.45 Acidifier 0.3 Thr 0.25 Salt 0.24 Metamino 0.2 Valine 0.08 Try 0.07 Prexim 1 Nutrient composition Crude protein 22.00 Crude fiber 3.89 ME, MJ/kg 14.23 ME, kcal/kg 3402 NE, MJ/kg 10.40 NE, kcal/kg 2485 Ca (%) 0.80 Av P (%) 0.40 SID Lys 1.35 SID Met 0.57 SID Cys 0.24 SID M + C 0.81 SID Thr 0.85 SID Trp 0.30 SID Arg 0.97 SID Ile 0.74 SID Leu 1.45 SID Val 0.92

[0202] The results of the treatments on body weight gain, feed conversion ratio, feed intake and diarrhea of the piglets are reported in Tables 3 and 4.

TABLE-US-00016 TABLE 8.3 Performance data from 1 to 42 d post-weaning. Treatments Age Negative Positive DSM 32540 ADG 1-42 0.460 0.482 0.476 Av. Daily Feed 0.770 0.772 0.773 intake, kg FCR 1-42 2.42 2.34 2.11

TABLE-US-00017 TABLE 8.4 Diarrhea score in percent of piglets suffering diarrhea Negative Treatment control Positive control DSM 32540 1~7 d 8.99 7.72 7.72 8~21 d 6.16 4.69 4.43 22~42 d 2.05 1.73 1.95

[0203] Average daily weight gain, feed conversion ratio, feed intake as well as the diarrhea score were significantly improved by addition of the B. subtilis strain DSM 32540 in comparison to the negative control but similar to positive control containing AGP.

Example 9. Comparison of Performance of Swine Reared in Spain Fed the Novel Bacillus subtilis DSM 32540

[0204] The experiment comprises 64 21-day post-weaned piglets with an average body weight of 7.30.35 kg randomly allocated to 2 treatments (Table 9.1), with 6 replicates, 4 piglets for each replicate. Three treatments were mainly based on corn-soybean meal (Table 9.2) and included; 1. Basal control (Control), 2. Control+Bacillus subtilis strain DSM 32540 at 250 g/MT containing 2.0*10.sup.9 cfu/g (DSM 32540). Experimental treatments were fed ad libitum in mash form from 1-42 days of age post-weaning.

TABLE-US-00018 TABLE 9.1 Ingredient and nutrient composition of basal diet. wt.-% Ingredient Corn 34.63 Soybean meal, 44% CP 26.10 Barley 10.00 Whey powder 9.00 Soybean meal, full fat 8.40 Rapeseed meal 5.00 Soybean oil 3.00 Monocalciumphosphate 1.09 Limestone (CaCO.sub.3) 0.95 Premix Swine 0.50 L-Lys HCl (78%) 0.42 Salt (NaCl) 0.38 MetAMINO 0.24 ThreAMINO 0.15 ValAMINO 0.06 TrpAMINO 0.07 Total 100.0000 Nutrient composition Crude protein 22.00 Crude fiber 3.89 ME, MJ/kg 14.23 ME, kcal/kg 3402 NE, MJ/kg 10.40 NE, kcal/kg 2485 Ca (%) 0.80 Av P (%) 0.40 SID Lys 1.35 SID Met 0.57 SID Cys 0.24 SID M + C 0.81 SID Thr 0.85 SID Trp 0.30 SID Arg 0.97 SID Ile 0.74 SID Leu 1.45

TABLE-US-00019 TABLE 9.2 Animal performance between days 0 and 42, with and without diet supplementation with B. subtilis DSM 32540 based feed additive or antibiotic growth promoter. Treatments Age Negative DSM 32540 ADG, kg 0.263 0.297 Av. Daily Feed intake, kg 0.389 0.429 FCR 1.507 1.473

[0205] B. subtilis strain DSM 32540 significantly improved all tested parameters in comparison to the negative control.