Bacillus subtilis for animal feed

Abstract

Described are the Bacillus subtilis strain deposited as DSM 32685 and mutant strains thereof which are susceptible to relevant antibiotics, have inhibitory activity against, for example, Staphylococcus, Streptococcus, E. coli and Salmonella, and have the ability to facilitate degradation of non-starch polysaccharides and thereby increase the amount of available oligosaccharides (sugar) in animal feed. Also described are compositions comprising at least one Bacillus subtilis strain of the disclosure and optionally further bacteria and/or one or more types of yeast and methods of using such compositions.

Claims

1. A method for inhibiting a bacterial colonization or bacterial infection by one or more of Staphylococcus pseudintermedius, Staphylococcus carnosus, Salmonella enterica, Salmonella Newport, Streptococcus canis, Escherichia coli, and Clostridium perfringens, the method comprising administering to an animal in need thereof an effective amount of a Bacillus subtilis strain deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany (DSMZ) under accession number DSM 32685, wherein strain DSM 32685: (i) exhibits susceptibility to ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol when tested under CLSI guidelines M07-A8 and M45-A2; (ii) exhibits an inhibition zone of at least 1 mm for Staphylococcus pseudintermedius, Escherichia coli, and Salmonella enterica; (iii) reduces the growth rate of Streptococcus canis by at least one log.sub.10 growth in 24 hours; and (iv) inhibits growth of Staphylococcus carnosus, Salmonella Newport, and Clostridium perfringens.

2. The method according to claim 1, wherein the method is effective for improving one or more animal performance parameters selected from the group consisting of i) increased weight gain (WG), ii) lower feed conversion ratio (FCR), iii) increased digestibility of the feed, iv) lower enteritis frequency, v) increased European Production Efficacy Factor (EPEF), and vi) lower mortality.

3. The method according to claim 1, wherein the administered Bacillus subtilis strain is in the form of spores.

4. The method according to claim 1, wherein the Bacillus subtilis strain is administered in a composition that further comprises one or more bacteria of one or more genera selected from Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium, and Megasphaera.

5. The method according to claim 1, wherein the Bacillus subtilis strain is administered in a composition that further comprises one or more types of yeast.

6. The method according to claim 1, wherein the Bacillus subtilis strain is administered in a format selected from a direct fed microbial, an animal feed premix, an animal feed additive, and a complete animal feed.

7. A method for feeding an animal, comprising administering to an animal a Bacillus subtilis strain deposited at DSMZ under accession number DSM 32685, wherein strain DSM 32685: (i) exhibits susceptibility to ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol when tested under CLSI guidelines M07-A8 and M45-A2; (ii) exhibits an inhibition zone at least 1 mm for Staphylococcus pseudintermedius, Escherichia coli, and Salmonella enterica; (iii) reduces the growth rate of Streptococcus canis by at least one log.sub.10 growth in 24 hours; and (iv) inhibits growth of Staphylococcus carnosus, Salmonella Newport, and Clostridium perfringens.

8. The method according to claim 7, wherein the animal is a monogastric animal.

9. The method according to claim 7, wherein the administered Bacillus subtilis strain is in the form of spores.

10. The method according to claim 7, wherein the Bacillus subtilis strain is administered in a composition that further comprises one or more bacteria of one or more genera selected from Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium, and Megasphaera.

11. The method according to claim 7, wherein the Bacillus subtilis strain is administered in a composition that further comprises one or more types of yeast.

12. The method according to claim 7, wherein the Bacillus subtilis strain is administered in an animal feed composition.

Description

LEGEND TO FIGURES

(1) FIG. 1

(2) Inhibition zones of four Bacillus subtilis strains against Staphylococcus pseudintermedius KU ID30618).

(3) Well B2, B4, B6: DSM 32685

(4) Well B8, B10, B12: DSM 19489

(5) Well F2, F4, F6: DSM 32686

(6) Well F8, F10, F12: Calsporin

(7) Inhibition is only seen in well B2, B4 and B6 containing Bacillus subtilis deposited as DSM 32685.

(8) FIG. 2

(9) Inhibition zones of four Bacillus subtilis strains against E. coli O147:K89 F4 H19.

(10) Well B2, B4, B6: DSM 32685

(11) Well B8, B10, B12: DSM 19489

(12) Well E2, E4, E6: DSM 32686

(13) Well E8, E10, E12: Calsporin

(14) Inhibition is seen in well B2, B4 and B6 containing Bacillus subtilis deposited as DSM 32685. Inhibition is also seen in well H12 containing a positive control (the antimicrobial ciprofloxacin).

DEPOSIT AND EXPERT SOLUTION

(15) The Bacillus subtilis strain DSM 19489 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) on Jun. 27, 2007 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

(16) The Bacillus subtilis strain DSM 32685 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) on Nov. 15, 2017 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

(17) The Bacillus subtilis strain DSM 32686 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) on Nov. 15, 2017 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

(18) For all of the above-identified deposited microorganisms, the following additional indications apply:

(19) As regards the respective Patent Offices of the respective designated states, the applicants request that a sample of the deposited microorganisms stated above only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn.

EXAMPLES

Example 1

(20) Pathogen Inhibition

(21) Materials

(22) Veal Infusion Broth (VIB) (BD, Franklin lakes, NJ USA; Difco catalogue number 234420) Brain heart infusion broth (BHI) (Thermo Fischer, Waltham, MA, USA; Oxoid catalogue number CM1135)

(23) Luria-bertani agar (LB) (Per liter: 10 g peptone, 5 g yeast extract, 5 g sodium chloride) Maximum recovery diluent (MRD) (Thermo Fischer, Waltham, MA, USA; Oxoid catalogue number TV5016D)

(24) Tryptone soya agar+5% Sheep blood (TSA+SB) (Thermo Fischer, Waltham, MA, USA; Oxoid catalogue number PB5012A)

(25) Omni tray/single well plates N 242811) (Thermo Fischer, Waltham, MA, USA)/NUNC Denmark

(26) Immuno TSP sterile plates (VWR, Radnor, PA, USA) catalogue number 735-0022 Syringe filters 0.2 m

(27) Pathogen Strains:

(28) Streptococcus canis DTU837 (Technical University of Denmark)

(29) Staphylococcus pseudintermedius DTU4438 (Technical University of Denmark)

(30) Staphylococcus pseudintermedius KU ID30243 (University of Copenhagen, Denmark)

(31) Staphylococcus pseudintermedius KU ID30618 (University of Copenhagen, Denmark)

(32) Staphylococcus pseudintermedius KU ID30377 (University of Copenhagen, Denmark)

(33) The pathogen strains were maintained in BHI with 20% glycerol at 80 C.

(34) Bacillus subtilis Strains:

(35) The Bacillus subtilis strain DSM 32685 was isolated from feces from a dog born and raised in Denmark and the Bacillus subtilis strain DSM 32686 was isolated from feces from a cat born and raised in Denmark. DSM 19489 and a Bacillus subtilis strain isolated from the product Calsporin brought on the market by Asahi Calpis Co., Ltd. Japan, in the following termed Calsporin were used as reference strains.

(36) The Bacillus subtilis strains were maintained in VIB with 20% glycerol at 80 C.

(37) DSM 32685 and DSM 32686 were screened for antibiotic susceptibility according to Guidance on the assessment of bacterial susceptibility to inhibitorys of human and veterinary importance. EFSA Journal 2012; 10(6):2740 and found to be susceptible to all antibiotics and that growth was below the EFSA cut-off values in all cases. Screening for biogenic amine production and cytotoxicity was also done with negative results.

(38) Screening for Inhibition of Staphylococcus Pseudintermedius

(39) The four Staphylococcus pseudintermedius strains were suspended in MRD until McFarland 0.5 was obtained and 60 L of the pathogen suspension was diluted in approximately 100 ml melted LB agar (max. 42 C.). The inoculated agar was poured into Omni Tray Single-well plates, Immuno TSP plates were attached. After drying the immune TSP plates were removed and the wells were inoculated with 5 L overnight culture (triplicates) of Bacillus test strains. The plates were incubated aerobically at 30 C., n=2 for 48 hours.

(40) For each of the pathogens two replicate plates were used with the following positioning of the Bacillus subtilis strains:

(41) Well B2, B4, B6: DSM 32685

(42) Well B8, B10, B12: DSM 19489

(43) Well F2, F4, F6: DSM 32686

(44) Well F8, F10, F12: Calsporin

(45) The concentration of Staphylococcus pseudintermedius was determined to be 110.sup.8 CFU/ml, based on the McFarland 0.5 suspension by serial dilution and plating on TSA+SB agar with 24 hour incubation at 37 C. The concentration of Staphylococcus pseudintermedius in the inoculated agar was therefore estimated to be 110.sup.8 CFU/ml/(100 ml/0.06 ml)=610.sup.4 CFU/ml.

(46) Screening for Inhibition of Streptococcus canis

(47) BHI broth (20 ml) was inoculated with Streptococcus canis (aiming for 510.sup.1 CFU/ml broth) and test samples were added (2 ml sterile filtered culture). The samples were incubated at 37 C. under aerobic conditions for up to 48 hours. Samples for CFU counts were collected at T.sub.0 (immediately after the pathogen and test sample was added) and after 24 h and 48 h of incubation, (n=3). TSA+SB agar plates were used for enumeration of Streptococcus canis. Plates were incubated aerobically at 37 C. for 1-2 days.

(48) Results

(49) TABLE-US-00001 TABLE 1 Inhibition of Staphylococcus pseudintermedius by Bacillus subtilis strains (n = 6) Staphylococcsus pseudintermedius (radii of clearing zone) (mm) Strain DSM # DTU4438 KU ID30243 KU ID30618 KU ID30377 B. subtilis 32685 3 3 3 3 B. subtilis 19489 0 0 0 0 B. subtilis 32686 0 0 0 0 B. subtilis Calsporin 0 0 0 0

(50) The results in Table 1 show that Bacillus subtilis DSM 32685 inhibited the growth of Staphylococcus pseudintermedius. No inhibition was observed with the three other Bacillus subtilis strains tested. A representative example of the inhibition zones is shown in FIG. 1 and shows a clear difference between inhibition and no inhibition.

(51) TABLE-US-00002 TABLE 2 Inhibition of Streptococcus canis DTU837 Log10 CFU Log10 CFU Log10 growth (T.sub.0) (T.sub.24) (T.sub.24 T.sub.0) DSM 32685 1.8 5.1 3.3 DSM 32686 1.8 8.5 6.7 DSM 19489 1.7 9.2 7.5 Calsporin 1.8 8.6 6.8 Control tube 1.7 8.4 6.7

(52) The results in table 2 show that Bacillus subtilis strains DSM 19489 and Calsporin had comparable or higher number of Streptococcus canis DTU837 than the control tube after 24 hours of incubation. In the presence of Bacillus subtilis strain DSM 32685 supernatant Streptococcus canis DTU837 grew 1000 fold less than the control tube, clearly demonstrating an inhibitory effect.

Example 2

(53) Measurement of Amount of Reducing Sugars in Feed Incubated with a Bacillus Composition

(54) The objective of this experiment was to examine the ability of different Bacillus subtilis strains to degrade NSP in commercial dog feed and increase the available sugar amount.

(55) Materials

(56) Sodium phosphate buffer 100 mM pH=6.7 (Merck, Darmstadt, Germany; catalogue number: 1.06586)

(57) Pierce BCA protein Assay Kit (Thermo Fischer, Waltham, MA, USA, catalogue number: PIE-23225)

(58) Dog feed (Carrier Chicken and Rice) composition: Chicken 26%, rice 20%, maize, barley, fat of animal and vegetable origin, linseed, beet fibre, dryed egg components, yeast, fructo-oligosaccarides (FOS), glucoseamine, rosemary and minerals (Svenska Hundfoder, Stenstorp, Sweden)

(59) Method

(60) The dog feed was autoclaved at 121 C. for 15 min for sterilization. Then triplicate feed samples were diluted 20 fold with sodium phosphate buffer to ensure a pH at about 6-6.5 throughout the whole experiment. Bacillus subtilis inoculation was done by adding 2% overnight culture of the Bacillus subtilis strains, grown in VIB. A sample was taken for analysis for reducing sugar (DNS) (T=0). After incubation at 37 C. for 24 hours the samples were centrifuged and the supernatant used for determining DNS.

(61) Reducing sugar was analyzed by 3.5-dinitrosalicylic acid (DNS) assay as follows: Na-acetate buffer (100 mM, pH 6) was mixed with sterile filtered Bacillus subtilis supernatant and incubated at 40 C. for 10 min. DNS reagent was added to the test tube, mixed and incubated in a boiling water bath for 5 min. After cooling, absorbance was measured at 540 nm in a spectrophotometer.

(62) A standard curve was established with a glucose stock solution for presenting results in reducing sugar or enzyme units (amount of enzyme needed to release 1 mol reducing glucose equivalent in 1 ml per time unit).

(63) The experiment was repeated twice and results are reported as an average.

(64) Results

(65) TABLE-US-00003 TABLE 3 Reducing sugar increase over control in presence of three Bacillus subtilis strains (n = 2) Kj/Kg Standard feed deviation Dog feed + DSM 32685 685 27 Dog feed + DSM 19489 438 37 Dog feed + Calsporin 117 9

(66) The results in table 3 show that Bacillus subtilis DSM 32685 was superior to the 2 other strains tested in releasing sugar from dog feed based on the DNS method.

Example 3

(67) Pathogen Inhibition

(68) Pathogen Strains:

(69) Escherichia coli O147:K89 F4 H19 (Statens Serum Institute, Copenhagen, Denmark)

(70) Escherichia coli O149:k91, k88a, c:h10 NCTC10650 (National Collection of Type Cultures,

(71) England)

(72) Salmonella enterica serovar Heidelberg, found in DK 2011, from imported chicken meat from Brazil (Technical University of Denmark, Kgs. Lyngby)

(73) Salmonella enterica serovar Infantis (S. inf. 1; SGSC2483 received from St. Hycinth, Canada; Salmonella Genetic Stock Centre, Department of Biological Sciences 2500 University Dr. N.W., Calgary, Alberta, Canada)

(74) Salmonella enterica serovar Enteritidis (IMASDE, Madrid, Spain)

(75) Salmonella enterica serovar Schwarzengrund (S. sch. 1; received from St. Hycinth, Canada; Salmonella Genetic Stock Centre, Department of Biological Sciences 2500 University Dr. N.W., Calgary, Alberta, Canada)

(76) Salmonella enterica serovar Typhimurium (I 4,5,12:i:1,2), ATCC14028

(77) The pathogen strains were maintained in BHI with 20% glycerol at 80 C.

(78) Screening for Inhibition of E. coli and Salmonella Spp.:

(79) The seven pathogenic E. coli and Salmonella spp. strains were each suspended in MRD until McFarland 0.5 was obtained and 10 L of each pathogen suspension was diluted in approximately 35 ml melted LB agar (max. 42 C.). The inoculated agar was poured into Omni Tray Single-well plates, Immuno TSP plates were attached. After drying the immune TSP plates were removed and the wells were inoculated with 5 L overnight culture (triplicates) of Bacillus test strains. The plates were incubated aerobically at 30 C. for 48 hours. For the E. coli plates, two replicate plates were used (n=2); for the 10 Salmonella spp. plates, one replicate plate was used (n=1).

(80) The following positioning of the Bacillus subtilis strains on the plates containing the pathogens was used:

(81) Well B2, B4, B6: DSM 32685

(82) Well B8, B10, B12: DSM 19489

(83) Well E2, E4, E6: DSM 32686

(84) Well E8, E10, E12: Calsporin

(85) The concentration of E. coli and Salmonella spp. in the inoculated agar was estimated to be 2.910.sup.4 CFU/ml.

(86) Results

(87) TABLE-US-00004 TABLE 4 Inhibition of E. coli O147, E. coli 0149, Salmonella Heidelberg, S. Infantis, S. Enteritidis, S. Schwarzengrund, & S. Typhimurium by Bacillus subtilis strains. Radii of clearing zone (mm) E. coli E. coli S. S. S. S. S. Strain DSM # O147 O149 Heidelberg Infantis Enteritidis Schwarzengrund Typhimurium B. subtilis 32685 2 2 2 2 2 2 2 B. subtilis 19489 0 0 0 0 0 0 0 B. subtilis 32686 0 0 0 0 0 0 0 B. subtilis Calsporin 0 0 0 0 0 0 0

(88) The results in Table 4 show that Bacillus subtilis DSM 32685 inhibited the growth of E. coli O147, E. coli O149, Salmonella Heidelberg, S. infantis, S. Enteritidis, S. Schwarzengrund, & S. Typhimurium. No inhibition was observed with the three other Bacillus subtilis strains tested. A representative example of the inhibition zones is shown in FIG. 2 and shows a clear difference between inhibition and no inhibition.