US20230372413A1 - Bacillus strains improving health and performance of production animals - Google Patents
Bacillus strains improving health and performance of production animals Download PDFInfo
- Publication number
- US20230372413A1 US20230372413A1 US17/746,731 US202217746731A US2023372413A1 US 20230372413 A1 US20230372413 A1 US 20230372413A1 US 202217746731 A US202217746731 A US 202217746731A US 2023372413 A1 US2023372413 A1 US 2023372413A1
- Authority
- US
- United States
- Prior art keywords
- animal feed
- animal
- dsm
- bacillus
- strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241001465754 Metazoa Species 0.000 title claims abstract description 501
- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 292
- 238000004519 manufacturing process Methods 0.000 title abstract description 26
- 230000036541 health Effects 0.000 title abstract description 19
- 235000019730 animal feed additive Nutrition 0.000 claims abstract description 202
- 241000193468 Clostridium perfringens Species 0.000 claims abstract description 102
- 238000000034 method Methods 0.000 claims abstract description 93
- 244000144977 poultry Species 0.000 claims abstract description 61
- 230000000845 anti-microbial effect Effects 0.000 claims abstract description 47
- 241000282898 Sus scrofa Species 0.000 claims abstract description 31
- 241000588724 Escherichia coli Species 0.000 claims abstract description 19
- 102000004190 Enzymes Human genes 0.000 claims description 51
- 108090000790 Enzymes Proteins 0.000 claims description 51
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 42
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 claims description 38
- 230000002949 hemolytic effect Effects 0.000 claims description 27
- 239000011782 vitamin Substances 0.000 claims description 27
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 26
- 239000004615 ingredient Substances 0.000 claims description 26
- 240000008042 Zea mays Species 0.000 claims description 25
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 25
- 229940088594 vitamin Drugs 0.000 claims description 24
- 229930003231 vitamin Natural products 0.000 claims description 24
- 235000013343 vitamin Nutrition 0.000 claims description 24
- 150000001413 amino acids Chemical class 0.000 claims description 22
- 235000002639 sodium chloride Nutrition 0.000 claims description 22
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 21
- 235000001014 amino acid Nutrition 0.000 claims description 21
- 229910052500 inorganic mineral Inorganic materials 0.000 claims description 21
- 235000010755 mineral Nutrition 0.000 claims description 21
- 239000011707 mineral Substances 0.000 claims description 21
- 229960005322 streptomycin Drugs 0.000 claims description 21
- 239000004098 Tetracycline Substances 0.000 claims description 20
- 229930101283 tetracycline Natural products 0.000 claims description 20
- 229960002180 tetracycline Drugs 0.000 claims description 20
- 235000019364 tetracycline Nutrition 0.000 claims description 20
- 150000003522 tetracyclines Chemical class 0.000 claims description 20
- 229930182566 Gentamicin Natural products 0.000 claims description 19
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 claims description 19
- 108010059993 Vancomycin Proteins 0.000 claims description 19
- 229960005091 chloramphenicol Drugs 0.000 claims description 19
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 claims description 19
- 229960002227 clindamycin Drugs 0.000 claims description 19
- KDLRVYVGXIQJDK-AWPVFWJPSA-N clindamycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@H](C)Cl)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 KDLRVYVGXIQJDK-AWPVFWJPSA-N 0.000 claims description 19
- 229960003276 erythromycin Drugs 0.000 claims description 19
- 229930027917 kanamycin Natural products 0.000 claims description 19
- 229960000318 kanamycin Drugs 0.000 claims description 19
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 19
- 229930182823 kanamycin A Natural products 0.000 claims description 19
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 claims description 19
- 229960003165 vancomycin Drugs 0.000 claims description 19
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 claims description 19
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 18
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 18
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 18
- 239000001913 cellulose Substances 0.000 claims description 18
- 229920002678 cellulose Polymers 0.000 claims description 18
- 235000005822 corn Nutrition 0.000 claims description 18
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 16
- 235000021307 Triticum Nutrition 0.000 claims description 15
- 241000209140 Triticum Species 0.000 claims description 15
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 13
- 229960002518 gentamicin Drugs 0.000 claims description 13
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 12
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 11
- YPFDHNVEDLHUCE-UHFFFAOYSA-N propane-1,3-diol Chemical compound OCCCO YPFDHNVEDLHUCE-UHFFFAOYSA-N 0.000 claims description 10
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 8
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 7
- 229920002472 Starch Polymers 0.000 claims description 7
- 235000010216 calcium carbonate Nutrition 0.000 claims description 7
- 235000012054 meals Nutrition 0.000 claims description 7
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 7
- 239000008107 starch Substances 0.000 claims description 7
- 235000019698 starch Nutrition 0.000 claims description 7
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 6
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 6
- 229930006000 Sucrose Natural products 0.000 claims description 6
- 235000013312 flour Nutrition 0.000 claims description 6
- 239000008101 lactose Substances 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims description 6
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 6
- 229960004063 propylene glycol Drugs 0.000 claims description 6
- 239000001509 sodium citrate Substances 0.000 claims description 6
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 6
- 239000005720 sucrose Substances 0.000 claims description 6
- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 claims description 5
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 5
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims description 5
- 239000004375 Dextrin Substances 0.000 claims description 5
- 229920001353 Dextrin Polymers 0.000 claims description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
- 108010068370 Glutens Proteins 0.000 claims description 5
- 239000005913 Maltodextrin Substances 0.000 claims description 5
- 229920002774 Maltodextrin Polymers 0.000 claims description 5
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 5
- 235000019425 dextrin Nutrition 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- 235000021312 gluten Nutrition 0.000 claims description 5
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 5
- 229940035034 maltodextrin Drugs 0.000 claims description 5
- 239000004302 potassium sorbate Substances 0.000 claims description 5
- 235000010241 potassium sorbate Nutrition 0.000 claims description 5
- 229940069338 potassium sorbate Drugs 0.000 claims description 5
- 235000011151 potassium sulphates Nutrition 0.000 claims description 5
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 claims description 5
- 239000004299 sodium benzoate Substances 0.000 claims description 5
- 235000010234 sodium benzoate Nutrition 0.000 claims description 5
- 235000011083 sodium citrates Nutrition 0.000 claims description 5
- 235000011152 sodium sulphate Nutrition 0.000 claims description 5
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 claims description 5
- 235000019345 sodium thiosulphate Nutrition 0.000 claims description 5
- 239000000600 sorbitol Substances 0.000 claims description 5
- 235000015099 wheat brans Nutrition 0.000 claims description 5
- 230000001332 colony forming effect Effects 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 3
- 240000003183 Manihot esculenta Species 0.000 claims 1
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 claims 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims 1
- 229920002125 Sokalan® Polymers 0.000 claims 1
- DNEHKUCSURWDGO-UHFFFAOYSA-N aluminum sodium Chemical compound [Na].[Al] DNEHKUCSURWDGO-UHFFFAOYSA-N 0.000 claims 1
- IJKVHSBPTUYDLN-UHFFFAOYSA-N dihydroxy(oxo)silane Chemical compound O[Si](O)=O IJKVHSBPTUYDLN-UHFFFAOYSA-N 0.000 claims 1
- 229940113116 polyethylene glycol 1000 Drugs 0.000 claims 1
- 229940093430 polyethylene glycol 1500 Drugs 0.000 claims 1
- 229940113115 polyethylene glycol 200 Drugs 0.000 claims 1
- 229940068918 polyethylene glycol 400 Drugs 0.000 claims 1
- 229940057838 polyethylene glycol 4000 Drugs 0.000 claims 1
- 229940057847 polyethylene glycol 600 Drugs 0.000 claims 1
- 244000052769 pathogen Species 0.000 abstract 1
- 230000001580 bacterial effect Effects 0.000 description 102
- 241000287828 Gallus gallus Species 0.000 description 66
- 230000037396 body weight Effects 0.000 description 64
- 235000013594 poultry meat Nutrition 0.000 description 58
- 235000019786 weight gain Nutrition 0.000 description 56
- 229940088598 enzyme Drugs 0.000 description 50
- 201000006747 infectious mononucleosis Diseases 0.000 description 48
- 238000006243 chemical reaction Methods 0.000 description 47
- 239000000203 mixture Substances 0.000 description 47
- 235000014590 basal diet Nutrition 0.000 description 44
- 238000011282 treatment Methods 0.000 description 43
- 208000004232 Enteritis Diseases 0.000 description 40
- 230000001338 necrotic effect Effects 0.000 description 40
- 230000000694 effects Effects 0.000 description 39
- 210000004215 spore Anatomy 0.000 description 39
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 38
- 235000014469 Bacillus subtilis Nutrition 0.000 description 37
- 239000000758 substrate Substances 0.000 description 36
- GAOZTHIDHYLHMS-KEOBGNEYSA-N monensin A Chemical compound C([C@@](O1)(C)[C@H]2CC[C@@](O2)(CC)[C@H]2[C@H](C[C@@H](O2)[C@@H]2[C@H](C[C@@H](C)[C@](O)(CO)O2)C)C)C[C@@]21C[C@H](O)[C@@H](C)[C@@H]([C@@H](C)[C@@H](OC)[C@H](C)C(O)=O)O2 GAOZTHIDHYLHMS-KEOBGNEYSA-N 0.000 description 35
- 229930191564 Monensin Natural products 0.000 description 34
- GAOZTHIDHYLHMS-UHFFFAOYSA-N Monensin A Natural products O1C(CC)(C2C(CC(O2)C2C(CC(C)C(O)(CO)O2)C)C)CCC1C(O1)(C)CCC21CC(O)C(C)C(C(C)C(OC)C(C)C(O)=O)O2 GAOZTHIDHYLHMS-UHFFFAOYSA-N 0.000 description 34
- 239000004459 forage Substances 0.000 description 34
- 229960005358 monensin Drugs 0.000 description 34
- 241000282887 Suidae Species 0.000 description 33
- 235000013330 chicken meat Nutrition 0.000 description 33
- 244000063299 Bacillus subtilis Species 0.000 description 32
- 235000005911 diet Nutrition 0.000 description 29
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 26
- 241000271566 Aves Species 0.000 description 26
- 230000037213 diet Effects 0.000 description 26
- 239000003242 anti bacterial agent Substances 0.000 description 25
- 229940088710 antibiotic agent Drugs 0.000 description 25
- 235000013339 cereals Nutrition 0.000 description 23
- 230000006872 improvement Effects 0.000 description 21
- 241000894006 Bacteria Species 0.000 description 20
- 229940024606 amino acid Drugs 0.000 description 20
- 208000015181 infectious disease Diseases 0.000 description 20
- 239000000463 material Substances 0.000 description 20
- 229920000617 arabinoxylan Polymers 0.000 description 19
- -1 Arabinan Polymers 0.000 description 18
- 241000272517 Anseriformes Species 0.000 description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 17
- 229920001817 Agar Polymers 0.000 description 16
- 241000286209 Phasianidae Species 0.000 description 16
- 239000008272 agar Substances 0.000 description 16
- 229920001221 xylan Polymers 0.000 description 16
- UGXQOOQUZRUVSS-ZZXKWVIFSA-N [5-[3,5-dihydroxy-2-(1,3,4-trihydroxy-5-oxopentan-2-yl)oxyoxan-4-yl]oxy-3,4-dihydroxyoxolan-2-yl]methyl (e)-3-(4-hydroxyphenyl)prop-2-enoate Chemical compound OC1C(OC(CO)C(O)C(O)C=O)OCC(O)C1OC1C(O)C(O)C(COC(=O)\C=C\C=2C=CC(O)=CC=2)O1 UGXQOOQUZRUVSS-ZZXKWVIFSA-N 0.000 description 15
- 239000005018 casein Substances 0.000 description 15
- 235000019621 digestibility Nutrition 0.000 description 15
- 229920000856 Amylose Polymers 0.000 description 14
- 241000196324 Embryophyta Species 0.000 description 14
- 230000012010 growth Effects 0.000 description 14
- 235000015097 nutrients Nutrition 0.000 description 14
- 150000004823 xylans Chemical class 0.000 description 14
- 108010076119 Caseins Proteins 0.000 description 13
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 13
- 235000021240 caseins Nutrition 0.000 description 13
- 150000001875 compounds Chemical class 0.000 description 13
- 235000021050 feed intake Nutrition 0.000 description 13
- 229910052757 nitrogen Inorganic materials 0.000 description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 239000003674 animal food additive Substances 0.000 description 12
- 238000003556 assay Methods 0.000 description 12
- 239000012141 concentrate Substances 0.000 description 12
- 238000010790 dilution Methods 0.000 description 12
- 239000012895 dilution Substances 0.000 description 12
- 238000002474 experimental method Methods 0.000 description 12
- 238000002360 preparation method Methods 0.000 description 12
- 235000007319 Avena orientalis Nutrition 0.000 description 11
- 241000194108 Bacillus licheniformis Species 0.000 description 11
- 240000002791 Brassica napus Species 0.000 description 11
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 11
- 108010082495 Dietary Plant Proteins Proteins 0.000 description 11
- 235000019764 Soybean Meal Nutrition 0.000 description 11
- 235000010980 cellulose Nutrition 0.000 description 11
- 230000002255 enzymatic effect Effects 0.000 description 11
- 210000001035 gastrointestinal tract Anatomy 0.000 description 11
- 230000003902 lesion Effects 0.000 description 11
- 229930182817 methionine Natural products 0.000 description 11
- 235000006109 methionine Nutrition 0.000 description 11
- 239000004455 soybean meal Substances 0.000 description 11
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 10
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 10
- 230000003115 biocidal effect Effects 0.000 description 10
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 10
- 150000003839 salts Chemical class 0.000 description 10
- 206010018910 Haemolysis Diseases 0.000 description 9
- 235000007340 Hordeum vulgare Nutrition 0.000 description 9
- 240000005979 Hordeum vulgare Species 0.000 description 9
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 9
- 241000209056 Secale Species 0.000 description 9
- 235000007238 Secale cereale Nutrition 0.000 description 9
- 244000062793 Sorghum vulgare Species 0.000 description 9
- 230000008588 hemolysis Effects 0.000 description 9
- 239000007858 starting material Substances 0.000 description 9
- 108010011619 6-Phytase Proteins 0.000 description 8
- 241000209761 Avena Species 0.000 description 8
- 206010061043 Clostridial infection Diseases 0.000 description 8
- 208000037384 Clostridium Infections Diseases 0.000 description 8
- 241000238557 Decapoda Species 0.000 description 8
- 235000010469 Glycine max Nutrition 0.000 description 8
- 244000068988 Glycine max Species 0.000 description 8
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 8
- 239000004472 Lysine Substances 0.000 description 8
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 8
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 8
- 235000021374 legumes Nutrition 0.000 description 8
- 239000013642 negative control Substances 0.000 description 8
- 235000018102 proteins Nutrition 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 239000011669 selenium Substances 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 239000011573 trace mineral Substances 0.000 description 8
- 241000251468 Actinopterygii Species 0.000 description 7
- 244000025254 Cannabis sativa Species 0.000 description 7
- 241000272201 Columbiformes Species 0.000 description 7
- 241000238424 Crustacea Species 0.000 description 7
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 7
- 240000004658 Medicago sativa Species 0.000 description 7
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 7
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 7
- 239000004473 Threonine Substances 0.000 description 7
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 7
- 239000011575 calcium Substances 0.000 description 7
- 229910052791 calcium Inorganic materials 0.000 description 7
- FDJOLVPMNUYSCM-WZHZPDAFSA-L cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+3].N#[C-].N([C@@H]([C@]1(C)[N-]\C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C(\C)/C1=N/C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C\C1=N\C([C@H](C1(C)C)CCC(N)=O)=C/1C)[C@@H]2CC(N)=O)=C\1[C@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]1[C@@H](O)[C@@H](N2C3=CC(C)=C(C)C=C3N=C2)O[C@@H]1CO FDJOLVPMNUYSCM-WZHZPDAFSA-L 0.000 description 7
- 239000010949 copper Substances 0.000 description 7
- 235000019688 fish Nutrition 0.000 description 7
- 235000009973 maize Nutrition 0.000 description 7
- 235000016709 nutrition Nutrition 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 229910052711 selenium Inorganic materials 0.000 description 7
- 238000012163 sequencing technique Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 241000894007 species Species 0.000 description 7
- 229960002898 threonine Drugs 0.000 description 7
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 6
- 241000272525 Anas platyrhynchos Species 0.000 description 6
- 108010001478 Bacitracin Proteins 0.000 description 6
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 6
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 description 6
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 6
- MJVAVZPDRWSRRC-UHFFFAOYSA-N Menadione Chemical compound C1=CC=C2C(=O)C(C)=CC(=O)C2=C1 MJVAVZPDRWSRRC-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 235000019770 animal feed premixes Nutrition 0.000 description 6
- 229960003071 bacitracin Drugs 0.000 description 6
- 229930184125 bacitracin Natural products 0.000 description 6
- CLKOFPXJLQSYAH-ABRJDSQDSA-N bacitracin A Chemical compound C1SC([C@@H](N)[C@@H](C)CC)=N[C@@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]1C(=O)N[C@H](CCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2N=CNC=2)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCCCC1 CLKOFPXJLQSYAH-ABRJDSQDSA-N 0.000 description 6
- 239000012620 biological material Substances 0.000 description 6
- 210000003608 fece Anatomy 0.000 description 6
- 235000018977 lysine Nutrition 0.000 description 6
- 239000011572 manganese Substances 0.000 description 6
- 239000008363 phosphate buffer Substances 0.000 description 6
- 229940085127 phytase Drugs 0.000 description 6
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 6
- 108090000765 processed proteins & peptides Proteins 0.000 description 6
- 102000004196 processed proteins & peptides Human genes 0.000 description 6
- 239000004460 silage Substances 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- 235000013619 trace mineral Nutrition 0.000 description 6
- 239000006150 trypticase soy agar Substances 0.000 description 6
- 235000013311 vegetables Nutrition 0.000 description 6
- 235000016068 Berberis vulgaris Nutrition 0.000 description 5
- 241000335053 Beta vulgaris Species 0.000 description 5
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 5
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 description 5
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 5
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 5
- 108091005804 Peptidases Proteins 0.000 description 5
- 239000004365 Protease Substances 0.000 description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 5
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 5
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 description 5
- 238000000540 analysis of variance Methods 0.000 description 5
- 238000009635 antibiotic susceptibility testing Methods 0.000 description 5
- GHPGOEFPKIHBNM-UHFFFAOYSA-N antimony(3+);oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[Sb+3].[Sb+3] GHPGOEFPKIHBNM-UHFFFAOYSA-N 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 239000003224 coccidiostatic agent Substances 0.000 description 5
- 229910052802 copper Inorganic materials 0.000 description 5
- 235000013325 dietary fiber Nutrition 0.000 description 5
- 235000015872 dietary supplement Nutrition 0.000 description 5
- 229960000304 folic acid Drugs 0.000 description 5
- 235000019152 folic acid Nutrition 0.000 description 5
- 239000011724 folic acid Substances 0.000 description 5
- 235000013372 meat Nutrition 0.000 description 5
- 238000010197 meta-analysis Methods 0.000 description 5
- 230000000813 microbial effect Effects 0.000 description 5
- 229960003512 nicotinic acid Drugs 0.000 description 5
- 235000001968 nicotinic acid Nutrition 0.000 description 5
- 239000011664 nicotinic acid Substances 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 239000008188 pellet Substances 0.000 description 5
- 229910052698 phosphorus Inorganic materials 0.000 description 5
- 229920001184 polypeptide Polymers 0.000 description 5
- 239000006041 probiotic Substances 0.000 description 5
- 230000000529 probiotic effect Effects 0.000 description 5
- 235000018291 probiotics Nutrition 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 229960002477 riboflavin Drugs 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- JZRWCGZRTZMZEH-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 5
- 239000011715 vitamin B12 Substances 0.000 description 5
- 235000005282 vitamin D3 Nutrition 0.000 description 5
- 239000011647 vitamin D3 Substances 0.000 description 5
- QYSXJUFSXHHAJI-YRZJJWOYSA-N vitamin D3 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-YRZJJWOYSA-N 0.000 description 5
- 229940011671 vitamin b6 Drugs 0.000 description 5
- 229940021056 vitamin d3 Drugs 0.000 description 5
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 4
- 239000004382 Amylase Substances 0.000 description 4
- 102000013142 Amylases Human genes 0.000 description 4
- 108010065511 Amylases Proteins 0.000 description 4
- 244000105624 Arachis hypogaea Species 0.000 description 4
- 235000010777 Arachis hypogaea Nutrition 0.000 description 4
- 241000972773 Aulopiformes Species 0.000 description 4
- 241001249119 Bacillus vallismortis Species 0.000 description 4
- 235000014698 Brassica juncea var multisecta Nutrition 0.000 description 4
- 235000011297 Brassica napobrassica Nutrition 0.000 description 4
- 235000006008 Brassica napus var napus Nutrition 0.000 description 4
- 240000000385 Brassica napus var. napus Species 0.000 description 4
- 235000006618 Brassica rapa subsp oleifera Nutrition 0.000 description 4
- 241000252210 Cyprinidae Species 0.000 description 4
- 235000019739 Dicalciumphosphate Nutrition 0.000 description 4
- 208000015580 Increased body weight Diseases 0.000 description 4
- 102100037611 Lysophospholipase Human genes 0.000 description 4
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 4
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 4
- 241000277331 Salmonidae Species 0.000 description 4
- 241000276707 Tilapia Species 0.000 description 4
- 241000219793 Trifolium Species 0.000 description 4
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 4
- 235000019418 amylase Nutrition 0.000 description 4
- 230000000843 anti-fungal effect Effects 0.000 description 4
- 239000006030 antibiotic growth promoter Substances 0.000 description 4
- UCMIRNVEIXFBKS-UHFFFAOYSA-N beta-alanine Chemical compound NCCC(O)=O UCMIRNVEIXFBKS-UHFFFAOYSA-N 0.000 description 4
- 239000001506 calcium phosphate Substances 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 241001233037 catfish Species 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 238000003053 completely randomized design Methods 0.000 description 4
- NEFBYIFKOOEVPA-UHFFFAOYSA-K dicalcium phosphate Chemical compound [Ca+2].[Ca+2].[O-]P([O-])([O-])=O NEFBYIFKOOEVPA-UHFFFAOYSA-K 0.000 description 4
- 229940038472 dicalcium phosphate Drugs 0.000 description 4
- 229910000390 dicalcium phosphate Inorganic materials 0.000 description 4
- 230000007613 environmental effect Effects 0.000 description 4
- 235000019197 fats Nutrition 0.000 description 4
- 239000000835 fiber Substances 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 239000011630 iodine Substances 0.000 description 4
- 229910052740 iodine Inorganic materials 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- 229910052748 manganese Inorganic materials 0.000 description 4
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 4
- 230000017066 negative regulation of growth Effects 0.000 description 4
- 239000011574 phosphorus Substances 0.000 description 4
- 239000010908 plant waste Substances 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 235000019515 salmon Nutrition 0.000 description 4
- 238000003307 slaughter Methods 0.000 description 4
- 239000010907 stover Substances 0.000 description 4
- 239000010902 straw Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000001974 tryptic soy broth Substances 0.000 description 4
- 108010050327 trypticase-soy broth Proteins 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- 239000011701 zinc Substances 0.000 description 4
- 229910052725 zinc Inorganic materials 0.000 description 4
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 description 3
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 3
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 3
- 244000075850 Avena orientalis Species 0.000 description 3
- 235000007558 Avena sp Nutrition 0.000 description 3
- 241000219310 Beta vulgaris subsp. vulgaris Species 0.000 description 3
- 240000007124 Brassica oleracea Species 0.000 description 3
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 3
- 108010059892 Cellulase Proteins 0.000 description 3
- 235000019750 Crude protein Nutrition 0.000 description 3
- 239000004470 DL Methionine Substances 0.000 description 3
- 239000004150 EU approved colour Substances 0.000 description 3
- 229920003290 Eviva® Polymers 0.000 description 3
- 241000220485 Fabaceae Species 0.000 description 3
- 235000019733 Fish meal Nutrition 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 241001494479 Pecora Species 0.000 description 3
- 235000021536 Sugar beet Nutrition 0.000 description 3
- 235000015724 Trifolium pratense Nutrition 0.000 description 3
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 description 3
- 229930003427 Vitamin E Natural products 0.000 description 3
- 239000000654 additive Substances 0.000 description 3
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 3
- 108010030291 alpha-Galactosidase Proteins 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 235000019751 broiler diet Nutrition 0.000 description 3
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000011436 cob Substances 0.000 description 3
- 239000010941 cobalt Substances 0.000 description 3
- 229910017052 cobalt Inorganic materials 0.000 description 3
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 3
- 239000006027 corn-soybean meal Substances 0.000 description 3
- 230000000378 dietary effect Effects 0.000 description 3
- 239000006047 digesta Substances 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 235000013601 eggs Nutrition 0.000 description 3
- 238000001125 extrusion Methods 0.000 description 3
- 239000004467 fishmeal Substances 0.000 description 3
- 244000144992 flock Species 0.000 description 3
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 3
- 230000002496 gastric effect Effects 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 229910052742 iron Inorganic materials 0.000 description 3
- 239000011777 magnesium Substances 0.000 description 3
- 239000011738 major mineral Substances 0.000 description 3
- 235000011963 major mineral Nutrition 0.000 description 3
- FFEARJCKVFRZRR-UHFFFAOYSA-N methionine Chemical compound CSCCC(N)C(O)=O FFEARJCKVFRZRR-UHFFFAOYSA-N 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 229940006093 opthalmologic coloring agent diagnostic Drugs 0.000 description 3
- 239000001967 plate count agar Substances 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 235000008160 pyridoxine Nutrition 0.000 description 3
- 239000011677 pyridoxine Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 239000002151 riboflavin Substances 0.000 description 3
- 235000019192 riboflavin Nutrition 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000013589 supplement Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 235000019157 thiamine Nutrition 0.000 description 3
- 239000011721 thiamine Substances 0.000 description 3
- 235000019155 vitamin A Nutrition 0.000 description 3
- 239000011719 vitamin A Substances 0.000 description 3
- 235000019165 vitamin E Nutrition 0.000 description 3
- 239000011709 vitamin E Substances 0.000 description 3
- 229940046009 vitamin E Drugs 0.000 description 3
- 235000012711 vitamin K3 Nutrition 0.000 description 3
- 239000011652 vitamin K3 Substances 0.000 description 3
- 229940045997 vitamin a Drugs 0.000 description 3
- PETRWTHZSKVLRE-UHFFFAOYSA-N 2-Methoxy-4-methylphenol Chemical compound COC1=CC(C)=CC=C1O PETRWTHZSKVLRE-UHFFFAOYSA-N 0.000 description 2
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 description 2
- 235000003276 Apios tuberosa Nutrition 0.000 description 2
- 101710152845 Arabinogalactan endo-beta-1,4-galactanase Proteins 0.000 description 2
- 235000017060 Arachis glabrata Nutrition 0.000 description 2
- 235000018262 Arachis monticola Nutrition 0.000 description 2
- 235000010744 Arachis villosulicarpa Nutrition 0.000 description 2
- 241000871666 Arrhenatherum elatius subsp. baeticum Species 0.000 description 2
- 241000194103 Bacillus pumilus Species 0.000 description 2
- 241000276408 Bacillus subtilis subsp. subtilis str. 168 Species 0.000 description 2
- 241001474374 Blennius Species 0.000 description 2
- 235000011331 Brassica Nutrition 0.000 description 2
- 241000219198 Brassica Species 0.000 description 2
- 244000178924 Brassica napobrassica Species 0.000 description 2
- 235000011293 Brassica napus Nutrition 0.000 description 2
- 235000012905 Brassica oleracea var viridis Nutrition 0.000 description 2
- 235000000540 Brassica rapa subsp rapa Nutrition 0.000 description 2
- 235000003255 Carthamus tinctorius Nutrition 0.000 description 2
- 244000020518 Carthamus tinctorius Species 0.000 description 2
- 241000871189 Chenopodiaceae Species 0.000 description 2
- 235000019743 Choline chloride Nutrition 0.000 description 2
- 241000193403 Clostridium Species 0.000 description 2
- 244000060011 Cocos nucifera Species 0.000 description 2
- 235000013162 Cocos nucifera Nutrition 0.000 description 2
- 244000052363 Cynodon dactylon Species 0.000 description 2
- 235000000638 D-biotin Nutrition 0.000 description 2
- 239000011665 D-biotin Substances 0.000 description 2
- 235000001809 DL-alpha-tocopherylacetate Nutrition 0.000 description 2
- 239000011626 DL-alpha-tocopherylacetate Substances 0.000 description 2
- ZAKOWWREFLAJOT-UHFFFAOYSA-N DL-alpha-tocopherylacetate Chemical compound CC(=O)OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C ZAKOWWREFLAJOT-UHFFFAOYSA-N 0.000 description 2
- 240000004585 Dactylis glomerata Species 0.000 description 2
- 241000288297 Danthonia decumbens Species 0.000 description 2
- ZQZFYGIXNQKOAV-OCEACIFDSA-N Droloxifene Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=C(O)C=CC=1)\C1=CC=C(OCCN(C)C)C=C1 ZQZFYGIXNQKOAV-OCEACIFDSA-N 0.000 description 2
- 101710147028 Endo-beta-1,4-galactanase Proteins 0.000 description 2
- 239000004258 Ethoxyquin Substances 0.000 description 2
- 241000234642 Festuca Species 0.000 description 2
- 201000000628 Gas Gangrene Diseases 0.000 description 2
- 235000003222 Helianthus annuus Nutrition 0.000 description 2
- 244000020551 Helianthus annuus Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 235000019766 L-Lysine Nutrition 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- 235000019738 Limestone Nutrition 0.000 description 2
- 241000209082 Lolium Species 0.000 description 2
- 241000215452 Lotus corniculatus Species 0.000 description 2
- 241000219745 Lupinus Species 0.000 description 2
- 239000006137 Luria-Bertani broth Substances 0.000 description 2
- 108020002496 Lysophospholipase Proteins 0.000 description 2
- 102100033468 Lysozyme C Human genes 0.000 description 2
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 2
- 235000010624 Medicago sativa Nutrition 0.000 description 2
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 2
- 239000007832 Na2SO4 Substances 0.000 description 2
- 241000746983 Phleum pratense Species 0.000 description 2
- 108090000553 Phospholipase D Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- 241000209049 Poa pratensis Species 0.000 description 2
- 241000209504 Poaceae Species 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 241000282894 Sus scrofa domesticus Species 0.000 description 2
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 2
- 240000006345 Trifolium hybridum Species 0.000 description 2
- 235000000598 Trifolium hybridum Nutrition 0.000 description 2
- 244000042324 Trifolium repens Species 0.000 description 2
- 235000013540 Trifolium repens var repens Nutrition 0.000 description 2
- 241000219870 Trifolium subterraneum Species 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- 241000219873 Vicia Species 0.000 description 2
- 229930003779 Vitamin B12 Natural products 0.000 description 2
- 229930003471 Vitamin B2 Natural products 0.000 description 2
- 235000019742 Vitamins premix Nutrition 0.000 description 2
- 235000019740 Vitamins/micromineral premix Nutrition 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000000853 adhesive Substances 0.000 description 2
- 230000001070 adhesive effect Effects 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 108090000637 alpha-Amylases Proteins 0.000 description 2
- 102000005840 alpha-Galactosidase Human genes 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 229940000635 beta-alanine Drugs 0.000 description 2
- 239000006161 blood agar Substances 0.000 description 2
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Chemical compound BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 2
- 238000002815 broth microdilution Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- YKPUWZUDDOIDPM-SOFGYWHQSA-N capsaicin Chemical compound COC1=CC(CNC(=O)CCCC\C=C\C(C)C)=CC=C1O YKPUWZUDDOIDPM-SOFGYWHQSA-N 0.000 description 2
- 229940106157 cellulase Drugs 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 description 2
- 229960003178 choline chloride Drugs 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 239000013256 coordination polymer Substances 0.000 description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 2
- 239000007771 core particle Substances 0.000 description 2
- 235000012343 cottonseed oil Nutrition 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 235000019285 ethoxyquin Nutrition 0.000 description 2
- DECIPOUIJURFOJ-UHFFFAOYSA-N ethoxyquin Chemical compound N1C(C)(C)C=C(C)C2=CC(OCC)=CC=C21 DECIPOUIJURFOJ-UHFFFAOYSA-N 0.000 description 2
- 229940093500 ethoxyquin Drugs 0.000 description 2
- 230000029142 excretion Effects 0.000 description 2
- 238000013100 final test Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000000446 fuel Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 239000010903 husk Substances 0.000 description 2
- 210000003405 ileum Anatomy 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000036512 infertility Effects 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 2
- 239000006028 limestone Substances 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 239000012669 liquid formulation Substances 0.000 description 2
- 229910052749 magnesium Inorganic materials 0.000 description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 229960004051 menadione sodium bisulfite Drugs 0.000 description 2
- XDPFHGWVCTXHDX-UHFFFAOYSA-M menadione sodium sulfonate Chemical compound [Na+].C1=CC=C2C(=O)C(C)(S([O-])(=O)=O)CC(=O)C2=C1 XDPFHGWVCTXHDX-UHFFFAOYSA-M 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 235000019713 millet Nutrition 0.000 description 2
- 238000003801 milling Methods 0.000 description 2
- 235000013379 molasses Nutrition 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- GHOKWGTUZJEAQD-UHFFFAOYSA-N pantothenic acid Chemical compound OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 235000020232 peanut Nutrition 0.000 description 2
- 239000004466 pelleted feed Substances 0.000 description 2
- 239000002985 plastic film Substances 0.000 description 2
- 230000008092 positive effect Effects 0.000 description 2
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 2
- 238000003044 randomized block design Methods 0.000 description 2
- 235000013526 red clover Nutrition 0.000 description 2
- 238000007670 refining Methods 0.000 description 2
- QGNJRVVDBSJHIZ-QHLGVNSISA-N retinyl acetate Chemical compound CC(=O)OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C QGNJRVVDBSJHIZ-QHLGVNSISA-N 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- JBQYATWDVHIOAR-UHFFFAOYSA-N tellanylidenegermanium Chemical compound [Te]=[Ge] JBQYATWDVHIOAR-UHFFFAOYSA-N 0.000 description 2
- 229960003495 thiamine Drugs 0.000 description 2
- 239000007201 ts agar Substances 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 235000019163 vitamin B12 Nutrition 0.000 description 2
- 235000019164 vitamin B2 Nutrition 0.000 description 2
- 239000011716 vitamin B2 Substances 0.000 description 2
- 235000019158 vitamin B6 Nutrition 0.000 description 2
- 239000011726 vitamin B6 Substances 0.000 description 2
- 150000003722 vitamin derivatives Chemical class 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 230000004584 weight gain Effects 0.000 description 2
- 239000001074 1-methoxy-4-[(E)-prop-1-enyl]benzene Substances 0.000 description 1
- 241000272521 Anatidae Species 0.000 description 1
- 108010024976 Asparaginase Proteins 0.000 description 1
- 241000228243 Aspergillus giganteus Species 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- JEBFVOLFMLUKLF-IFPLVEIFSA-N Astaxanthin Natural products CC(=C/C=C/C(=C/C=C/C1=C(C)C(=O)C(O)CC1(C)C)/C)C=CC=C(/C)C=CC=C(/C)C=CC2=C(C)C(=O)C(O)CC2(C)C JEBFVOLFMLUKLF-IFPLVEIFSA-N 0.000 description 1
- 241000193752 Bacillus circulans Species 0.000 description 1
- 241000193749 Bacillus coagulans Species 0.000 description 1
- 241000194107 Bacillus megaterium Species 0.000 description 1
- 208000031729 Bacteremia Diseases 0.000 description 1
- 241000221198 Basidiomycota Species 0.000 description 1
- 101710130006 Beta-glucanase Proteins 0.000 description 1
- 102100032487 Beta-mannosidase Human genes 0.000 description 1
- 241000186000 Bifidobacterium Species 0.000 description 1
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 1
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 1
- 238000009631 Broth culture Methods 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 241000206594 Carnobacterium Species 0.000 description 1
- 108010084185 Cellulases Proteins 0.000 description 1
- 102000005575 Cellulases Human genes 0.000 description 1
- 240000006162 Chenopodium quinoa Species 0.000 description 1
- 241000177202 Chimonobambusa utilis Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- 241001112695 Clostridiales Species 0.000 description 1
- 235000004035 Cryptotaenia japonica Nutrition 0.000 description 1
- 102100028717 Cytosolic 5'-nucleotidase 3A Human genes 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 108010002069 Defensins Proteins 0.000 description 1
- 102000000541 Defensins Human genes 0.000 description 1
- 108010001682 Dextranase Proteins 0.000 description 1
- 241000223934 Eimeria maxima Species 0.000 description 1
- 241000194033 Enterococcus Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 description 1
- 241000272496 Galliformes Species 0.000 description 1
- 241001598647 Galloanserae Species 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 229940121710 HMGCoA reductase inhibitor Drugs 0.000 description 1
- 102100031415 Hepatic triacylglycerol lipase Human genes 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 241000235649 Kluyveromyces Species 0.000 description 1
- 241001138401 Kluyveromyces lactis Species 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 241000194036 Lactococcus Species 0.000 description 1
- 101800004361 Lactoferricin-B Proteins 0.000 description 1
- 108010063045 Lactoferrin Proteins 0.000 description 1
- 102000010445 Lactoferrin Human genes 0.000 description 1
- 241000192132 Leuconostoc Species 0.000 description 1
- 108010013563 Lipoprotein Lipase Proteins 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- 241000604449 Megasphaera Species 0.000 description 1
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 241000272458 Numididae Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004020 Oxygenases Human genes 0.000 description 1
- 108090000417 Oxygenases Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000194105 Paenibacillus polymyxa Species 0.000 description 1
- 102100026367 Pancreatic alpha-amylase Human genes 0.000 description 1
- 241000192001 Pediococcus Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 102100035200 Phospholipase A and acyltransferase 4 Human genes 0.000 description 1
- 102000011420 Phospholipase D Human genes 0.000 description 1
- 102100032967 Phospholipase D1 Human genes 0.000 description 1
- 108010058864 Phospholipases A2 Proteins 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 240000004713 Pisum sativum Species 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 241000186429 Propionibacterium Species 0.000 description 1
- 235000019779 Rapeseed Meal Nutrition 0.000 description 1
- 241000282849 Ruminantia Species 0.000 description 1
- 101000948929 Ruminococcus flavefaciens Endo-beta-1,3-1,4 glucanase Proteins 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 244000253897 Saccharomyces delbrueckii Species 0.000 description 1
- 244000253911 Saccharomyces fragilis Species 0.000 description 1
- 241000235344 Saccharomycetaceae Species 0.000 description 1
- 235000009337 Spinacia oleracea Nutrition 0.000 description 1
- 244000300264 Spinacia oleracea Species 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 241000282890 Sus Species 0.000 description 1
- 241000006364 Torula Species 0.000 description 1
- 241000235006 Torulaspora Species 0.000 description 1
- 240000002913 Trifolium pratense Species 0.000 description 1
- WGLPBDUCMAPZCE-UHFFFAOYSA-N Trioxochromium Chemical compound O=[Cr](=O)=O WGLPBDUCMAPZCE-UHFFFAOYSA-N 0.000 description 1
- 241001314440 Triphora trianthophoros Species 0.000 description 1
- 101800003783 Tritrpticin Proteins 0.000 description 1
- 102000014384 Type C Phospholipases Human genes 0.000 description 1
- 108010079194 Type C Phospholipases Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 241001464837 Viridiplantae Species 0.000 description 1
- 229930003270 Vitamin B Natural products 0.000 description 1
- 229930003451 Vitamin B1 Natural products 0.000 description 1
- 229930003448 Vitamin K Natural products 0.000 description 1
- 235000019752 Wheat Middilings Nutrition 0.000 description 1
- 239000005862 Whey Substances 0.000 description 1
- 108010046377 Whey Proteins Proteins 0.000 description 1
- 102000007544 Whey Proteins Human genes 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- JAZBEHYOTPTENJ-JLNKQSITSA-N all-cis-5,8,11,14,17-icosapentaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O JAZBEHYOTPTENJ-JLNKQSITSA-N 0.000 description 1
- OENHQHLEOONYIE-UKMVMLAPSA-N all-trans beta-carotene Natural products CC=1CCCC(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C OENHQHLEOONYIE-UKMVMLAPSA-N 0.000 description 1
- 102000004139 alpha-Amylases Human genes 0.000 description 1
- 229940024171 alpha-amylase Drugs 0.000 description 1
- JZQOJFLIJNRDHK-CMDGGOBGSA-N alpha-irone Chemical compound CC1CC=C(C)C(\C=C\C(C)=O)C1(C)C JZQOJFLIJNRDHK-CMDGGOBGSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000009604 anaerobic growth Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 239000006053 animal diet Substances 0.000 description 1
- 235000019728 animal nutrition Nutrition 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 229940114079 arachidonic acid Drugs 0.000 description 1
- 235000021342 arachidonic acid Nutrition 0.000 description 1
- 235000013793 astaxanthin Nutrition 0.000 description 1
- 239000001168 astaxanthin Substances 0.000 description 1
- MQZIGYBFDRPAKN-ZWAPEEGVSA-N astaxanthin Chemical compound C([C@H](O)C(=O)C=1C)C(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)C(=O)[C@@H](O)CC1(C)C MQZIGYBFDRPAKN-ZWAPEEGVSA-N 0.000 description 1
- 229940022405 astaxanthin Drugs 0.000 description 1
- 229940054340 bacillus coagulans Drugs 0.000 description 1
- 108010055059 beta-Mannosidase Proteins 0.000 description 1
- 239000011648 beta-carotene Substances 0.000 description 1
- 235000013734 beta-carotene Nutrition 0.000 description 1
- TUPZEYHYWIEDIH-WAIFQNFQSA-N beta-carotene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CCCC1(C)C)C=CC=C(/C)C=CC2=CCCCC2(C)C TUPZEYHYWIEDIH-WAIFQNFQSA-N 0.000 description 1
- 229960002747 betacarotene Drugs 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910052796 boron Inorganic materials 0.000 description 1
- 239000005388 borosilicate glass Substances 0.000 description 1
- ZPFKRQXYKULZKP-UHFFFAOYSA-N butylidene Chemical group [CH2+]CC[CH-] ZPFKRQXYKULZKP-UHFFFAOYSA-N 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229960002079 calcium pantothenate Drugs 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 235000017663 capsaicin Nutrition 0.000 description 1
- 229960002504 capsaicin Drugs 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 235000021466 carotenoid Nutrition 0.000 description 1
- 150000001747 carotenoids Chemical class 0.000 description 1
- POIUWJQBRNEFGX-XAMSXPGMSA-N cathelicidin Chemical compound C([C@@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(C)C)C1=CC=CC=C1 POIUWJQBRNEFGX-XAMSXPGMSA-N 0.000 description 1
- KAFGYXORACVKTE-UEDJBKKJSA-N chembl503567 Chemical compound C([C@H]1C(=O)N[C@H]2CSSC[C@H](NC(=O)[C@H](CC=3C=CC=CC=3)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@H](C(=O)N[C@@H](CSSC[C@@H](C(N1)=O)NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)CNC(=O)CNC(=O)[C@@H](N)CCCNC(N)=N)CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)C1=CC=C(O)C=C1 KAFGYXORACVKTE-UEDJBKKJSA-N 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 229910052804 chromium Inorganic materials 0.000 description 1
- 239000011651 chromium Substances 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000013024 dilution buffer Substances 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 229960005135 eicosapentaenoic acid Drugs 0.000 description 1
- JAZBEHYOTPTENJ-UHFFFAOYSA-N eicosapentaenoic acid Natural products CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O JAZBEHYOTPTENJ-UHFFFAOYSA-N 0.000 description 1
- 235000020673 eicosapentaenoic acid Nutrition 0.000 description 1
- 201000007869 emphysematous cholecystitis Diseases 0.000 description 1
- 108010091371 endoglucanase 1 Proteins 0.000 description 1
- 108010091384 endoglucanase 2 Proteins 0.000 description 1
- 108010092450 endoglucanase Z Proteins 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- ULGZDMOVFRHVEP-UHFFFAOYSA-N erythromycin Chemical compound CC1C(OC2C(C(CC(C)O2)N(C)C)O)C(C)(O)CC(C)C(=O)C(C)C(O)C(O)(C)C(CC)OC(=O)C(C)C1OC1CC(C)(OC)C(O)C(C)O1 ULGZDMOVFRHVEP-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 235000019620 fat digestibility Nutrition 0.000 description 1
- 210000003746 feather Anatomy 0.000 description 1
- 244000037666 field crops Species 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- AWJWCTOOIBYHON-UHFFFAOYSA-N furo[3,4-b]pyrazine-5,7-dione Chemical class C1=CN=C2C(=O)OC(=O)C2=N1 AWJWCTOOIBYHON-UHFFFAOYSA-N 0.000 description 1
- 229940098330 gamma linoleic acid Drugs 0.000 description 1
- VZCCETWTMQHEPK-UHFFFAOYSA-N gamma-Linolensaeure Natural products CCCCCC=CCC=CCC=CCCCCC(O)=O VZCCETWTMQHEPK-UHFFFAOYSA-N 0.000 description 1
- VZCCETWTMQHEPK-QNEBEIHSSA-N gamma-linolenic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/CCCCC(O)=O VZCCETWTMQHEPK-QNEBEIHSSA-N 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- NLDDIKRKFXEWBK-AWEZNQCLSA-N gingerol Chemical compound CCCCC[C@H](O)CC(=O)CCC1=CC=C(O)C(OC)=C1 NLDDIKRKFXEWBK-AWEZNQCLSA-N 0.000 description 1
- JZLXEKNVCWMYHI-UHFFFAOYSA-N gingerol Natural products CCCCC(O)CC(=O)CCC1=CC=C(O)C(OC)=C1 JZLXEKNVCWMYHI-UHFFFAOYSA-N 0.000 description 1
- 235000002780 gingerol Nutrition 0.000 description 1
- 230000008821 health effect Effects 0.000 description 1
- 239000002471 hydroxymethylglutaryl coenzyme A reductase inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- 229930002839 ionone Natural products 0.000 description 1
- 150000002499 ionone derivatives Chemical class 0.000 description 1
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- CFFMZOZGXDAXHP-HOKBLYKWSA-N lactoferricin Chemical compound C([C@H](NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CSSC[C@@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N2CCC[C@H]2C(=O)N[C@@H](CO)C(=O)N[C@H](C(N[C@H](C(=O)N1)[C@@H](C)O)=O)[C@@H](C)CC)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CFFMZOZGXDAXHP-HOKBLYKWSA-N 0.000 description 1
- 235000021242 lactoferrin Nutrition 0.000 description 1
- 229940078795 lactoferrin Drugs 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 235000012680 lutein Nutrition 0.000 description 1
- 239000001656 lutein Substances 0.000 description 1
- 229960005375 lutein Drugs 0.000 description 1
- KBPHJBAIARWVSC-RGZFRNHPSA-N lutein Chemical compound C([C@H](O)CC=1C)C(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\[C@H]1C(C)=C[C@H](O)CC1(C)C KBPHJBAIARWVSC-RGZFRNHPSA-N 0.000 description 1
- ORAKUVXRZWMARG-WZLJTJAWSA-N lutein Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CCCC1(C)C)C=CC=C(/C)C=CC2C(=CC(O)CC2(C)C)C ORAKUVXRZWMARG-WZLJTJAWSA-N 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 102000035118 modified proteins Human genes 0.000 description 1
- 108091005573 modified proteins Proteins 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 229910052750 molybdenum Inorganic materials 0.000 description 1
- 239000011733 molybdenum Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000037257 muscle growth Effects 0.000 description 1
- 206010028320 muscle necrosis Diseases 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 210000003250 oocyst Anatomy 0.000 description 1
- 238000003305 oral gavage Methods 0.000 description 1
- 108010073895 ovispirin Proteins 0.000 description 1
- RUVINXPYWBROJD-UHFFFAOYSA-N para-methoxyphenyl Natural products COC1=CC=C(C=CC)C=C1 RUVINXPYWBROJD-UHFFFAOYSA-N 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- JRKICGRDRMAZLK-UHFFFAOYSA-L persulfate group Chemical group S(=O)(=O)([O-])OOS(=O)(=O)[O-] JRKICGRDRMAZLK-UHFFFAOYSA-L 0.000 description 1
- 235000013970 phaffia yeast Nutrition 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- SHUZOJHMOBOZST-UHFFFAOYSA-N phylloquinone Natural products CC(C)CCCCC(C)CCC(C)CCCC(=CCC1=C(C)C(=O)c2ccccc2C1=O)C SHUZOJHMOBOZST-UHFFFAOYSA-N 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920006255 plastic film Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 235000013406 prebiotics Nutrition 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- OSFBJERFMQCEQY-UHFFFAOYSA-N propylidene Chemical group [CH]CC OSFBJERFMQCEQY-UHFFFAOYSA-N 0.000 description 1
- 108010032966 protegrin-1 Proteins 0.000 description 1
- 239000004456 rapeseed meal Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- MWNQXXOSWHCCOZ-UHFFFAOYSA-L sodium;oxido carbonate Chemical compound [Na+].[O-]OC([O-])=O MWNQXXOSWHCCOZ-UHFFFAOYSA-L 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000008174 sterile solution Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 235000018553 tannin Nutrition 0.000 description 1
- 239000001648 tannin Substances 0.000 description 1
- 229920001864 tannin Polymers 0.000 description 1
- 108010032153 thanatin Proteins 0.000 description 1
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000004408 titanium dioxide Substances 0.000 description 1
- 235000019756 total sulphur amino acid Nutrition 0.000 description 1
- 231100000033 toxigenic Toxicity 0.000 description 1
- 230000001551 toxigenic effect Effects 0.000 description 1
- RUVINXPYWBROJD-ONEGZZNKSA-N trans-anethole Chemical compound COC1=CC=C(\C=C\C)C=C1 RUVINXPYWBROJD-ONEGZZNKSA-N 0.000 description 1
- KBPHJBAIARWVSC-XQIHNALSSA-N trans-lutein Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2C(=CC(O)CC2(C)C)C KBPHJBAIARWVSC-XQIHNALSSA-N 0.000 description 1
- FTKYRNHHOBRIOY-HQUBJAAMSA-N tritrptcin Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](N)C(C)C)C1=CC=CC=C1 FTKYRNHHOBRIOY-HQUBJAAMSA-N 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 235000010374 vitamin B1 Nutrition 0.000 description 1
- 239000011691 vitamin B1 Substances 0.000 description 1
- 235000019168 vitamin K Nutrition 0.000 description 1
- 239000011712 vitamin K Substances 0.000 description 1
- 150000003721 vitamin K derivatives Chemical class 0.000 description 1
- 229940046010 vitamin k Drugs 0.000 description 1
- 229940041603 vitamin k 3 Drugs 0.000 description 1
- FJHBOVDFOQMZRV-XQIHNALSSA-N xanthophyll Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2C=C(C)C(O)CC2(C)C FJHBOVDFOQMZRV-XQIHNALSSA-N 0.000 description 1
- OENHQHLEOONYIE-JLTXGRSLSA-N β-Carotene Chemical compound CC=1CCCC(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C OENHQHLEOONYIE-JLTXGRSLSA-N 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
- A23K20/24—Compounds of alkaline earth metals, e.g. magnesium
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
- A23K20/28—Silicates, e.g. perlites, zeolites or bentonites
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
- A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
Definitions
- the present invention relates to an animal feed or an animal feed additive comprising Bacillus strains which improve the health and performance of production animals.
- the invention further relates to use of the Bacillus strains in animal feed and animal feed additives.
- Clostridium perfringens ( C. perfringens ) is a Gram-positive, rod-shaped, anaerobic, spore-forming bacterium of the genus Clostridium.
- C. perfringens is widely present in nature and can be found as a component of decaying vegetation, marine sediment, the intestinal tract of humans and other vertebrates, insects, and soil.
- C. perfringens Infections due to C. perfringens show evidence of tissue necrosis, bacteremia, emphysematous cholecystitis, and gas gangrene, which is also known as clostridial myonecrosis. C. perfringens can also result in polymicrobial anaerobic infections.
- necrotic enteritis is an enterotoxemic disease that results in significant economic losses in the poultry industry.
- a further object of the invention is to provide a solution that does not present a risk for the health of the animal.
- the solution to this problem is use of non-hemolytic Bacillus strains with activity against Clostridium perfringens.
- a challenge of delivering Bacillus spp. in feed is the common use of antibiotics as growth promoters in feed. Therefore it is necessary to determine the compatibility of strains with commonly-used feed antibiotics such as monensin in order to identify any potential conflicts with use as a direct fed microbial.
- the present invention relates in one embodiment to an animal feed or feed additive comprising a Bacillus strain with high compatibility with monensin.
- Clostat (alias Bacillus PB6) is described in WO2007/064741. Bacillus PB6 has antagonistic effect against C. perfringens. Bacillus PB6 is hemolytic as described in Example 1.
- DFM direct fed microbes
- Bacillus species can be used to prevent and/or control C. perfringens infections and/or necrotic enteritis in mono-gastric animal such as pigs and/or poultry.
- the Bacillus species can also improve the body weight gain and/or feed conversion rate (in both Clostridium perfringens challenged and unchallenged chickens).
- the present invention relates to animal feed or animal feed additive comprising one or more of the Bacillus strains according to the invention which improves health and performance of production animals.
- Methods of feeding an animal with the animal feed or animal feed additive according to the invention and methods of treating a Clostridium perfringens infection and/or for treating necrotic enteritis in an animal is also part of the invention.
- Use of the animal feed or animal feed additive according to the invention to improve the performance of an animal, such as improving the feed conversion rate, improving the body weight gain and/or improving the feed efficiency and/or improving the health is also part of this invention.
- the invention further relates to a method for preventing and/or controlling C. perfringens infections and/or necrotic enteritis in a mono-gastric animal such as swine or poultry by use of an animal feed, wherein the animal feed comprises one or more Bacillus strains selected from the group consisting of the strain having the deposit accession number DSM 29869, DSM 29870, DSM 29871, and DSM 29872, or any combination thereof, wherein the Bacillus strain has antimicrobial activity against Clostridium perfringens and/or E. coli.
- the invention relates to an animal feed that is fed to a mono-gastric animal such as poultry or swine; comprising one or more Bacillus strains selected from the group consisting of the strain having the deposit accession number DSM 29869, DSM 29870, DSM 29871, and DSM 29872, or any combination thereof, wherein the Bacillus strain has antimicrobial activity against Clostridium perfringens and/or E. coli.
- the invention relates to a method for preventing and/or controlling C. perfringens infections and/or necrotic enteritis in an animal such as pigs or poultry, comprising the steps of:
- the method relates to an animal feed that is fed to a mono-gastric animal such as pigs and/or poultry.
- the poultry can, e.g., be chickens (such as broilers and/or layers).
- the animal feed or animal feed additive comprises one of more additional bacteria; is fed to a mono-gastric animal such as pigs and/or poultry; and further comprises concentrate and/or one or more enzymes and/or one or more additional microbes and/or one or more vitamins and/or one or more minerals and/or one or more amino acids and/or one or more other feed ingredients.
- FIG. 1 Alignment of 16 S rDNA sequences.
- SEQ ID NO: 1 (DSM 29869), SEQ ID NO: 2 (DSM 29870), SEQ ID NO: 3 (DSM 29871), SEQ ID NO: 4 (DSM 29872), SEQ ID NO: 5 ( Bacillus vallismortis AB021198), SEQ ID NO: 6 ( Bacillus subtilis from AJ276351), and SEQ ID NO: 7 ( Bacillus amyloliquefaciens AB255669) have been aligned.
- the region covering position 481-1200 is not shown in FIG. 1 (in this region all sequences are identical).
- Animal feed refers to any compound, preparation, or mixture suitable for, or intended for intake by an animal.
- Animal feed for a mono-gastric animal comprises concentrates as well as for example vitamins, minerals, enzymes, amino acids and/or other feed ingredients (such as in a premix).
- the animal feed may further comprise forage.
- An example of poultry feed is given in Example 7.
- Antimicrobial activity against Clostridium perfringens means that the growth of Clostridium perfringens is inhibited and/or that some or all of the Clostridium perfringens are killed. This can be determined by the assay described in Example 2.
- Blend means more than one of the bacterial strains described herein.
- Body Weight Gain The Body Weight Gain of an animal is the increase of body weight of the animal over a specified time period. An example of Body Weight Gain determination is given in Example 8.
- composition refers to a composition comprising a carrier and at least one bacterial strain as described herein.
- compositions described herein may be mixed with an animal feed(s) and referred to as a “mash feed.”
- Concentrates means feed with high protein and energy concentrations, such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (either whole or prepared by crushing, milling, etc. from, e.g., corn, oats, rye, barley, wheat), oilseed press cake (e.g., from cottonseed, safflower, sunflower, soybean (such as soybean meal), rapeseed/canola, peanut or groundnut), palm kernel cake, yeast derived material and distillers grains (such as wet distillers grains (WDS) and dried distillers grains with solubles (DDGS)).
- high protein and energy concentrations such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (either whole or prepared by crushing, milling, etc. from, e.g., corn, oats, rye, barley, wheat), oilseed press cake (e.g., from cottonseed, safflower, sunflower,
- control C. perfringens infections and/or necrotic enteritis means a method and/or composition that partly or completely inhibits C. perfringens infections and/or necrotic enteritis in an animal. Accordingly, the term “control C. perfringens infections and/or necrotic enteritis” means the C. perfringens infections and/or the necrotic enteritis is reduced or completely eliminated.
- Direct Fed Microbial means live micro-organisms including spores which, when administered in adequate amounts, confer a benefit, such as improved digestion or health, on the host.
- Enzymatic activity under aerobic conditions means that the polypeptides produced by a Bacillus strain during growth under aerobic conditions have one of more enzyme activities.
- the substrates tested are amylose, arabinan, cellulose, casein, arabinoxylan and xylan and degradation of the substrate indicates amylase, arabinase, cellulase, protease or xylanase activity respectively. Such an assay is performed as described in Example 6.
- Enzymatic activity under anaerobic conditions means activity of enzymes produced by a Bacillus strain during growth under anaerobic conditions as described in Example 6.
- European Production Efficacy Factor The European Production Efficacy Factor is a way of comparing the live-bird performance of flocks. This single-figure facilitates comparison of performance within and among farms and can be used to assess environmental, climatic and managemental variables.
- the EPEF is calculated as [(liveability (%) ⁇ Liveweight (kg))/(Age at depletion (days) ⁇ FCR)] ⁇ 100, wherein livability is the percentage of birds alive at slaughter, Liveweight is the average weight of the birds at slaughter, age of depletion is the age of the birds at slaughter and FCR is the feed conversion ratio at slaughter.
- Effective amount/concentration/dosage The terms “effective amount”, “effective concentration”, or “effective dosage” are defined as the amount, concentration, or dosage of the bacterial strain(s) sufficient to improve the digestion or yield of an animal. The actual effective dosage in absolute numbers depends on factors including: the state of health of the animal in question, other ingredients present. The “effective amount”, “effective concentration”, or “effective dosage” of the bacterial strains may be determined by routine assays known to those skilled in the art. An example of an effective amount for poultry is given in Example 7.
- FCR Fee Conversion Rate
- FCR is a measure of an animal's efficiency in converting feed mass into increases of the desired output. Animals raised for meat—such as swine, poultry and fish—the output is the mass gained by the animal. Specifically FCR is the mass of the food eaten divided by the output, all over a specified period. FCR can be determined as described in Example 8. Improvement in FCR means reduction of the FCR value. A FCR improvement of 2% means that the FCR was reduced by 2%.
- Feeding an animal means that the composition of the present invention is administered orally to the animal one or more times in an effective amount.
- the oral administration is typically repeated, e.g., one or more times daily over a specified time period such as several days, one week, several weeks, one months or several months.
- Feeding of poultry can, e.g., be performed as described in Example 7. Accordingly, the terms “feeding” or “fed” mean any type of oral administration such as administration via an animal feed or via drinking water.
- Forage is fresh plant material such as hay and silage from forage plants, grass and other forage plants, seaweed, sprouted grains and legumes, or any combination thereof.
- forage plants are Alfalfa (lucerne), birdsfoot trefoil, brassica (e.g., kale, rapeseed (canola), rutabaga (swede), turnip), clover (e.g., alsike clover, red clover, subterranean clover, white clover), grass (e.g., Bermuda grass, brome, false oat grass, fescue, heath grass, meadow grasses, orchard grass, ryegrass, Timothy-grass), corn (maize), millet, barley, oats, rye, sorghum, soybeans and wheat and vegetables such as beets.
- Alfalfa lucerne
- brassica e.g., kale, rapeseed (canola), rutabag
- Forage further includes crop residues from grain production (such as corn stover; straw from wheat, barley, oat, rye and other grains); residues from vegetables like beet tops; residues from oilseed production like stems and leaves form soy beans, rapeseed and other legumes; and fractions from the refining of grains for animal or human consumption or from fuel production or other industries.
- grain production such as corn stover; straw from wheat, barley, oat, rye and other grains
- residues from vegetables like beet tops residues from oilseed production like stems and leaves form soy beans, rapeseed and other legumes
- fractions from the refining of grains for animal or human consumption or from fuel production or other industries such as corn stover; straw from wheat, barley, oat, rye and other grains.
- Isolated means that the one or more bacterial strains described herein are in a form or environment which does not occur in nature, that is, the one or more bacterial strains are at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature.
- Non-hemolytic Hemolysis is the breakdown of red blood cells. The ability of bacterial colonies to induce hemolysis when grown on blood agar is used to classify bacterial strains into hemolytic and non-hemolytic strains. In this context hemolysis is defined as described in EFSA Journal 2011; 9(11):2445, using Bacillus subtilis 168 (BGSC-1A1, Bacillus Genetic Stock Center) as a negative control. A Bacillus strain can be classified as non-hemolytic using the assay described in Example 1.
- Pellet refers to solid rounded, spherical and/or cylindrical tablets or pellets and the processes for forming such solid shapes, particularly feed pellets and solid extruded animal feed.
- extrusion or “extruding” are terms well known in the art and refer to a process of forcing a composition, as described herein, through an orifice under pressure.
- Poultry means domesticated birds kept by humans for the eggs they produce and/or their meat and/or their feathers.
- Poultry includes broilers and layers.
- Poultry include members of the superorder Galloanserae (fowl), especially the order Galliformes (which includes chickens, Guineafowls, quails and turkeys) and the family Anatidae, in order Anseriformes, commonly known as “waterfowl” and including domestic ducks and domestic geese.
- Poultry also includes other birds that are killed for their meat, such as the young of pigeons. Examples of poultry include chickens (including layers, broilers and chicks), ducks, geese, pigeons, turkeys and quail.
- Prevent C. perfringens infections and/or necrotic enteritis means a method and/or composition that prevents development of a C. perfringens infection and/or necrotic enteritis in an animal.
- Roughage means dry plant material with high levels of fiber, such as fiber, bran, husks from seeds and grains and crop residues (such as stover, copra, straw, chaff, sugar beet waste).
- Sensitive to antibiotics means the phenotypic property of a bacterial strain, that growth of said bacterial strain is inhibited under conditions where the bacterial strain would otherwise grow. In this context sensitivity to antibiotics is tested after the CLSI guidelines (M07-A9 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; 2012). The S. aureus ATCC 29213 is used as reference strain, which means that it should be included in the test, and that the results are only valid if S.
- aureus ATCC 29213 show results in compliance with the breakpoints of the CLSI guideline (see example Table 5.5) (M100-S24 Performance Standards for Antimicrobial Susceptibility Testing; informational Supplement, 2014).
- a strain of Bacillus is considered sensitive if the growth is detected at or below the breakpoint concentration of the appropriate antibiotic specified in EFSA journal 2012; 10(6):2740.
- Silage means fermented, high-moisture stored fodder which can be fed to ruminants (cud-chewing animals such as cattle and sheep) or used as a biofuel feedstock for anaerobic digesters. It is fermented and stored in a process called ensilage, ensiling or silaging, and is usually made from grass or cereal crops (e.g., maize, sorghum, oats, rye, timothy etc. forage grass plants),) or legume crops like clovers/trefoils, alfalfa, vetches, using the entire green plant (not just the grain).
- grass or cereal crops e.g., maize, sorghum, oats, rye, timothy etc. forage grass plants
- legume crops like clovers/trefoils, alfalfa, vetches, using the entire green plant (not just the grain).
- Silage can be made from many field crops, and special terms may be used depending on type (oatlage for oats, haylage for alfalfa). Silage is made either by placing cut green vegetation in a silo, by piling it in a large heap covered with plastic sheet, or by wrapping large bales in plastic film.
- spore and “endospore” are interchangeable and have their normal meaning which is well known and understood by those of skill in the art.
- spore refers to a microorganism in its dormant, protected state.
- Stable is a term that is known in the art, and in a preferred aspect, stable is intended to mean the ability of the microorganism to remain in a spore form until it is administered to an animal to improve the health of the animal.
- Swine The term “swine” or “pigs” means domesticated pigs kept by humans for food, such as their meat. Swine includes members of the genus Sus , such as Sus scrofa domesticus or Sus domesticus and include piglets, growing pigs, and sows.
- Vegetable protein refers to any compound, preparation or mixture that includes at least one protein derived from or originating from a vegetable, including modified proteins and protein-derivatives.
- DFM direct fed microbes
- the invention relates to an animal feed or an animal feed additive comprising spores of one or more Bacillus strains according to invention.
- the spores are preferable stable spores. More specifically the invention relates to the following aspects with respect to an animal feed or an animal feed additive comprising Bacillus strains:
- Aspect 1 An animal feed or an animal feed additive comprising spores of a Bacillus strain characterized in that:
- Aspect 2 An animal feed or an animal feed additive comprising spores of a Bacillus strain characterized in that:
- Aspect 3 An animal feed or an animal feed additive comprising spores of a Bacillus strain characterized in that:
- Aspect 4 An animal feed or an animal feed additive comprising spores of a Bacillus strain characterized in that:
- Sensitivity to Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline can, e.g., be determined as described in Example 5.
- Enzymatic activity under aerobic and/or anaerobic conditions that hydrolyzes one or more of the substrates selected from the group consisting of Amylose, Arabinan, Arabinoxylan, Casein and Xylan can, e.g., be determined as described in Example 6.
- Bacillus strain of the animal feed or the animal feed additive according to Aspect 2, 3 or 4 has enzymatic activity under aerobic and/or anaerobic conditions that hydrolyzes one or more of the substrates selected from the group consisting of Amylose, Arabinan, Arabinoxylan, Casein and Xylan.
- Bacillus strain of the animal feed or the animal feed additive according to Aspect 1 or 3 improves one or more performance parameters in poultry selected from the list consisting of body weight gain, European Production Efficacy Factor and feed conversion rate in chickens fed with the Bacillus strain.
- Bacillus strain of the animal feed or the animal feed additive according to Aspect 4 has antimicrobial activity against Clostridium perfringens.
- Bacillus strain of the animal feed or the animal feed additive according to Aspect 1 or 2 is sensitive to at least seven such as eight of the antibiotics selected from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline.
- the improvement of feed conversion rate (FCR) for the animal feed or the animal feed additive of Aspect 1, 2, 3 or 4 results in a FCR of ⁇ 2.5% or less than ⁇ 2.5%, such as less than ⁇ 2.6%, such as less than ⁇ 2.7%, such as less than ⁇ 2.8%, such less than ⁇ 2.9%, such as less than ⁇ 3.0%.
- the improvement of FCR results in a FCR of from ⁇ 5% to ⁇ 2% such as a FCR from ⁇ 4% to ⁇ 2%, such as a FCR of from ⁇ 3.5% to ⁇ 2.5%.
- the improvement of FCR for Aspect 1, 2, 3 or 4 results in a FCR within an interval selected from the group consisting of from ⁇ 5% to ⁇ 4.5%, from ⁇ 4.5% to ⁇ 4%, from ⁇ 4% to ⁇ 3.8%, from ⁇ 3.8% to ⁇ 3.6%, from ⁇ 3.6% to ⁇ 3.4%, from ⁇ 3.4% to ⁇ 3.2%, from ⁇ 3.2 to ⁇ 3.0%, from ⁇ 3.0% to ⁇ 2.8% and from ⁇ 2.8 to ⁇ 2.5%, or any combination of these intervals.
- the FCR can be determined as described in Example 8.
- the improvement in body weight gain for the animal feed or the animal feed additive of Aspect 1, 2, 3 or 4 results in a body weight gain of at least 0.5%, such as at least 0.8%, such as at least 1.5%, such as at least 1.8%, such as at least 2.0%, such as at least 2.3%, such as at least 3.5%, such as at least 4.2%, such as at least 5.2%, such as at least 6.5%, such as at least 7.3%.
- the improvement in body weight gain for Aspect 1, 2, 3 or 4 results in a body weight gain selected from the group consisting of from 1.8% to 2.0%, from 2.0% to 2.2%, from 2.2% to 2.4%, from 2.4% to 2.6%, from 2.6% to 2.8%, from 2.8% to 3.0%, from 3.0% to 3.2%, from 3.2% to 3.4%, from 3.4% to 3.6%, from 3.6% to 3.8%, from 3.8% to 4.0%, from 4% to 5%, from 5% to 7%, from 7% to 10%, or any combination thereof.
- the body weight gain can be determined as described in Example 8.
- the Bacillus strain comprises 16S rDNA that is more than 98% (such as more than 98.5%, such as more than 99%, such as more than 99.5%) sequence identity to SEQ ID NO: 1 and/or
- the Bacillus strain of the animal feed or the animal feed additive according to Aspect 1, 2, 3 or 4 embodiments has a high compatibility with monensin such as being compatible with at least 2.0 ⁇ g/ml monensin as determined in Example 12. It is even more preferred that the Bacillus strain is compatible with at least 2.4 ⁇ g/ml monensin as determined in Example 12 (such as at least 2.5 ⁇ g/ml monensin as determined in Example 12, such as at least 2.6 ⁇ g/ml monensin as determined in Example 12 or such as at least 2.7 ⁇ g/ml monensin as determined in Example 12).
- Bacillus strain of the animal feed or the animal feed additive according to Aspect 1, 2, 3 or 4 is a Bacillus subtilis strain, or a Bacillus amyloliquefaciens strain.
- the Bacillus strain of the animal feed or the animal feed additive according to Aspect 1, 2, 3 or 4 has antimicrobial effect against E. coli .
- the effect against E. coli can, e.g., be determined as described in Example 4.
- Bacillus strain of the animal feed or the animal feed additive according to Aspect 1, 2, 3 or 4 is the Bacillus strain having deposit accession number DSM 29869, or a strain having all of the identifying characteristics of Bacillus DSM 29869 or a mutant thereof that has antimicrobial activity against Clostridium perfringens .
- Bacillus strain of the animal feed or the animal feed additive according to Aspect 1, 2, 3 or 4 is the Bacillus strain having deposit accession number DSM 29870, or a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof that has antimicrobial activity against Clostridium perfringens .
- Bacillus strain of the animal feed or the animal feed additive according to Aspect 1, 2, 3 or 4 is the Bacillus strain having deposit accession number DSM 29871, or a strain having all of the identifying characteristics of Bacillus DSM 29871 or a mutant thereof that has antimicrobial activity against Clostridium perfringens .
- Bacillus strain of the animal feed or the animal feed additive according to Aspect 1, 2, 3 or 4 is the Bacillus strain having deposit accession number DSM 29872, or a strain having all of the identifying characteristics of Bacillus DSM 29872 or a mutant thereof that has antimicrobial activity against Clostridium perfringens.
- the invention relates in one embodiment to an animal feed or an animal feed additive comprising a Bacillus having deposit accession number DSM 29869 or a strain having all of the identifying characteristics of Bacillus DSM 29869 or a mutant thereof.
- the invention relates in another embodiment to an animal feed or an animal feed additive comprising a Bacillus having deposit accession number DSM 29870 or a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof.
- the invention relates in another embodiment to an animal feed or an animal feed additive comprising a Bacillus having deposit accession number DSM 29871 or a strain having all of the identifying characteristics of Bacillus DSM 29871 or a mutant thereof.
- the invention relates in a further embodiment to an animal feed or an animal feed additive comprising a Bacillus having deposit accession number DSM 29872 or a strain having all of the identifying characteristics of Bacillus DSM 29872 or a mutant thereof.
- the bacillus spores of the animal feed or the animal feed additive are present as dried spores.
- the animal feed or the animal feed additive according to Aspect 1, 2, 3 or 4 can also be a liquid composition and/or comprise culture supernatant comprising one or more Bacillus strain(s) of the invention.
- the animal feed or the animal feed additive further comprises a carrier.
- the carrier can comprise one or more of the following compounds: water, glycerol, ethylene glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose, sucrose, sorbitol, lactose, wheat flour, wheat bran, corn gluten meal, starch and cellulose.
- the animal feed or the animal feed additive comprises one or more coccidiostats wherein the composition is, e.g., a premix.
- the animal feed or the animal feed additive according to Aspect 1, 2, 3 or 4 comprises from 10 5 to 10 12 CFU/g of isolated Bacillus spores.
- the animal feed or animal feed additive is characterized in that at least 70% (such as at least 80% or at least 90%) of the Bacillus spores survive gastric stability in a mono-gastric animal such as chickens.
- the animal feed or the animal feed additive according to Aspect 1, 2, 3 or 4 which further comprises one or more components selected from the list consisting of: one or more enzymes; one or more additional microbes; one or more vitamins; one or more minerals; one or more amino acids; and one or more other feed ingredients.
- the animal feed according to Aspect 1, 2, 3 or 4 can be fed to an animal wherein the bacterial count of each Bacillus spore is between 1 ⁇ 10 4 and 1 ⁇ 10 14 CFU/kg of dry matter, preferably between 1 ⁇ 10 6 and 1 ⁇ 10 12 CFU/kg of dry matter, and more preferably between 1 ⁇ 10 7 and 1 ⁇ 10 11 CFU/kg of dry matter.
- the bacterial count of each of the bacterial strains in the animal feed composition is between 1 ⁇ 10 8 and 1 ⁇ 10 10 CFU/kg of dry matter.
- the bacterial count of each of the bacterial strains in the animal feed additive is between 1 ⁇ 10 7 and 1 ⁇ 10 18 CFU/kg of composition, preferably between 1 ⁇ 10 9 and 1 ⁇ 10 16 CFU/kg of composition, more preferably between 1 ⁇ 10 19 and 1 ⁇ 10 15 CFU/kg of composition and most preferably between 1 ⁇ 10 11 and 1 ⁇ 10 14 CFU/kg of dry matter.
- the animal feed or the animal feed additive according to Aspect 1, 2, 3 or 4 can be an animal feed or an animal feed additive wherein the bacterial count of each Bacillus spore is between 1 ⁇ 10 3 and 1 ⁇ 10 13 CFU/animal/day, preferably between 1 ⁇ 10 5 and 1 ⁇ 10 11 CFU/animal/day, and more preferably between 1 ⁇ 10 6 and 1 ⁇ 10 19 CFU/animal/day and even more preferably between 1 ⁇ 10 7 and 1 ⁇ 10 9 CFU/animal/day.
- the animal feed or the animal feed additive according to Aspect 1, 2, 3 or 4 can be a mono-gastric animal feed.
- the mono-gastric animal can be selected from the group consisting of pigs, swine, piglets, sows, poultry, turkeys, ducks, chicken, broilers, layers, chicks, fish and crustaceans. In one embodiment the animal is not a human being.
- Mono-gastric animals include in one embodiment, but are not limited to, pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chicken (including but not limited to broilers, chicks, layers); horses (including but not limited to hotbloods, coldbloods and warm bloods) and fish (including but not limited to salmon, trout, tilapia, catfish and carps; and crustaceans (including but not limited to shrimps and prawns). Pigs and/or poultry are preferred mono-gastric animals.
- the animal feed or the animal feed additive according to Aspect 1, 2, 3 or 4 is an animal feed or animal feed additive wherein the Bacillus strain improves gut health of chickens infected with Clostridium perfringens , e.g., by having antimicrobial activity against Clostridium perfringens.
- the animal feed or the animal feed additive according to Aspect 1, 2, 3 or 4 is an animal feed or animal feed additive for treatment of necrotic enteritis or treatment of a Clostridium perfringens infection (e.g., for treatment of mono-gastric animals including swine and poultry such as chickens).
- the animal feed or animal feed additive comprises one or more bacterial strains such as at least two of the above strains up to and including all of the strains in the group consisting of DSM 29869, DSM 29870, DSM 29871 and DSM 29872.
- the Bacillus spore kills/inhibits at least 40% (such as at least 45%, at least 50%, at least 60%, at least 70% or at least 80%) of Clostridium perfringens after, e.g., 24 hours, e.g., determined as described in Example 2.
- the animal feed or animal feed additive further comprises concentrate. In another embodiment of the invention the animal feed or animal feed additive further comprises forage. In another embodiment of the invention the animal feed or animal feed additive such as the animal feed further comprises one or more additional microbes. In another embodiment of the invention the animal feed or animal feed additive further comprises one or more enzymes. In another embodiment of the invention the animal feed or animal feed additive further comprises one or more vitamins. In another embodiment of the invention the animal feed or animal feed additive further comprises one or more minerals. In another embodiment of the invention the animal feed or animal feed additive such as the animal feed further comprises one or more amino acids. In another embodiment of the invention the animal feed or animal feed additive further comprises one or more other feed ingredients.
- the animal feed or the animal feed additive also improves the health of the mono-gastric animal when fed to said animal. In another embodiment to any of the aforementioned embodiments, the animal feed or the animal feed additive also increases the egg yield of poultry when fed said poultry. In an embodiment to any of the aforementioned embodiments, the animal feed or the animal feed additive increases the meat yield of the mono-gastric animal when fed to said animal.
- the animal feed comprises one or more bacterial strains described herein, wherein the bacterial count of each of the bacterial strains is between 1 ⁇ 10 4 and 1 ⁇ 10 14 CFU/kg of animal feed, preferably between 1 ⁇ 10 6 and 1 ⁇ 10 12 CFU/kg of animal feed, and more preferably between 1 ⁇ 10 7 and 1 ⁇ 10 11 CFU/kg of animal feed. In a more preferred embodiment the bacterial count of each of the bacterial strains described herein is between 1 ⁇ 10 8 and 1 ⁇ 10 10 CFU/kg of animal feed.
- the animal feed additive comprises one or more bacterial strains described herein, wherein the bacterial count of each of the bacterial strains is between 1 ⁇ 10 7 and 1 ⁇ 10 18 CFU/kg of animal feed additive, preferably between 1 ⁇ 10 9 and 1 ⁇ 10 16 CFU/kg of animal feed additive, and more preferably between 1 ⁇ 10 10 and 1 ⁇ 10 15 CFU/kg of animal feed additive. In a more preferred embodiment the bacterial count of each of the bacterial strains described herein is between 1 ⁇ 10 11 and 1 ⁇ 10 14 CFU/kg of animal feed additive.
- the bacterial count of each of the bacterial strains in the animal feed is between 1 ⁇ 10 4 and 1 ⁇ 10 14 CFU/kg of dry matter, preferably between 1 ⁇ 10 6 and 1 ⁇ 10 12 CFU/kg of dry matter, and more preferably between 1 ⁇ 10 7 and 1 ⁇ 10 11 CFU/kg of dry matter. In a more preferred embodiment the bacterial count of each of the bacterial strains in the animal feed is between 1 ⁇ 10 8 and 1 ⁇ 10 10 CFU/kg of dry matter.
- the animal feed or the animal feed additive according to Aspect 1, 2, 3 or 4 can be an animal feed or an animal feed additive wherein the bacterial count of each Bacillus spore is between 1 ⁇ 10 3 and 1 ⁇ 10 13 CFU/animal/day, preferably between 1 ⁇ 10 5 and 1 ⁇ 10 11 CFU/animal/day, and more preferably between 1 ⁇ 10 6 and 1 ⁇ 10 10 CFU/animal/day and even more preferably between 1 ⁇ 10 7 and 1 ⁇ 10 9 CFU/animal/day.
- the one or more bacterial strains are present in the animal feed or the animal feed additive in form of a spore such as a stable spore.
- the stable spore will germinate in the intestine and/or stomach of the mono-gastric animal.
- the one or more bacterial strains are stable when subjected to pressures applied/achieved during an extrusion process for pelleting.
- the one or more bacterial strains are stable at pressures ranging from 1 bar to bar, particularly 10 bar to 40 bar, more particularly 15 bar to 40 bar, even more particularly bar to 40 bar, still even more particularly 35 bar to 37 bar, even still more particularly 36 bar.
- the one or more bacterial strains are stable at high temperatures.
- the bacterial strains are stable when they are subjected to temperatures achieved during an extrusion process for pelleting.
- the one or more bacterial strains are stable at temperatures ranging from 80° C. to 120° C., particularly temperatures ranging from, 90° C. to 120° C., even more particularly temperatures ranging from 95° C. to 120° C.
- the invention relates to an animal feed or animal feed additive comprising a carrier, such as forage, and one or more of the bacteria cultures having characteristics substantially identical to that of a strain selected from the group consisting of:
- the animal feed or animal feed additive comprises a carrier and the strain having the deposit accession number DSM 29869, or a strain having all of the identifying characteristics of Bacillus DSM 29869 or a mutant thereof.
- the animal feed or animal feed additive comprises a carrier and the strain having the deposit accession number DSM 29870, or a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof.
- the animal feed or animal feed additive comprises a carrier and the strain having the deposit accession number DSM 29871, or a strain having all of the identifying characteristics of Bacillus DSM 29871 or a mutant thereof.
- the animal feed or animal feed additive comprises a carrier and the strain having the deposit accession number DSM 29872, or a strain having all of the identifying characteristics of Bacillus DSM 29872 or a mutant thereof.
- the animal feed or animal feed additive is for feeding to a mono-gastric animal.
- Mono-gastric animals include, but are not limited to, pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chicken (including but not limited to broiler chicks, layers); horses (including but not limited to hotbloods, coldbloods and warm bloods) and fish (including but not limited to salmon, trout, tilapia, catfish and carps; and crustaceans (including but not limited to shrimps and prawns). Pigs and/or poultry are preferred mono-gastric animals.
- the animal feed or animal feed additive further comprises one or more additional microbes.
- the animal feed further comprises a bacterium from one or more of the following genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium and Megasphaera or any combination thereof.
- animal feed or animal feed additive further comprises a bacterium from one or more of the following strains of Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus pumilus, Bacillus polymyxa, Bacillus licheniformis, Bacillus megaterium, Bacillus coagulans, Bacillus circulans , or any combination thereof.
- the animal feed or animal feed additive further comprises one or more types of yeast.
- the one or more types of yeast can be selected from the group consisting of Saccharomycetaceae, Saccharomyces (such as S. cerevisiae and/or S. boulardii ), Kluyveromyces (such as K. marxianus and K. lactis ), Candida (such as C. utilis , also called Torula yeast), Pichia (such as P. pastoris ), Torulaspora (such as T. delbrueckii ), Phaffia yeasts and Basidiomycota.
- Saccharomycetaceae Saccharomyces (such as S. cerevisiae and/or S. boulardii ), Kluyveromyces (such as K. marxianus and K. lactis ), Candida (such as C. utilis , also called Torula yeast), Pichia (such as P. pastoris ), Torulaspora (such as T. delbrueck
- the animal feed or the animal feed additive further comprises a carrier.
- the carrier can comprise one or more of the following compounds: water, glycerol, ethylene glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose, sucrose, sorbitol, lactose, wheat flour, wheat bran, corn gluten meal, starch and cellulose.
- the animal feed comprises the Bacillus strain according to Aspect 1, 2, 3 or 4 or the Bacillus strain having deposit accession number DSM 29869 and/or DSM 29870 and/or DSM 29871 and/or DSM 29872, or a strain having all of the identifying characteristics of Bacillus DSM 29869 and/or DSM 29870 and/or DSM 29871 and/or DSM 29872 or a mutant of DSM 29869 and/or DSM 29870 and/or DSM 29871 and/or DSM 29872 and further comprises one or more of concentrate(s), vitamin(s), mineral(s), enzyme(s), amino acid(s) and/or other feed ingredient(s).
- the animal feed further comprises forage.
- Forage as defined herein also includes roughage.
- Forage is fresh plant material such as hay and silage from forage plants, grass and other forage plants, grass and other forage plants, seaweed, sprouted grains and legumes, or any combination thereof.
- forage plants are Alfalfa (lucerne), birdsfoot trefoil, brassica (e.g., kale, rapeseed (canola), rutabaga (swede), turnip), clover (e.g., alsike clover, red clover, subterranean clover, white clover), grass (e.g., Bermuda grass, brome, false oat grass, fescue, heath grass, meadow grasses, orchard grass, ryegrass, Timothy-grass), corn (maize), millet, barley, oats, rye, sorghum, soybeans and wheat and vegetables such as beets.
- Alfalfa lucerne
- brassica e.g
- Crops suitable for ensilage are the ordinary grasses, clovers, alfalfa, vetches, oats, rye and maize.
- Forage further includes crop residues from grain production (such as corn stover; straw from wheat, barley, oat, rye and other grains); residues from vegetables like beet tops; residues from oilseed production like stems and leaves form soy beans, rapeseed and other legumes; and fractions from the refining of grains for animal or human consumption or from fuel production or other industries.
- Roughage is generally dry plant material with high levels of fiber, such as fiber, bran, husks from seeds and grains and crop residues (such as stover, copra, straw, chaff, sugar beet waste).
- concentrates are feed with high protein and energy concentrations, such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (either whole or prepared by crushing, milling, etc. from, e.g., corn, oats, rye, barley, wheat), oilseed press cake (e.g., from cottonseed, safflower, sunflower, soybean (such as soybean meal), rapeseed/canola, peanut or groundnut), palm kernel cake, yeast derived material and distillers grains (such as wet distillers grains (WDS) and dried distillers grains with solubles (DDGS)).
- high protein and energy concentrations such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (either whole or prepared by crushing, milling, etc. from, e.g., corn, oats, rye, barley, wheat), oilseed press cake (e.g., from cottonseed, safflower, sunflower,
- the forage and one or more microbes are mixed with a concentrate. In another embodiment, the forage and one or more microbes are mixed with a premix. In a further embodiment, the forage and one or more microbes are mixed with vitamins and/or minerals. In a further embodiment, the forage and one or more microbes are mixed with one or more enzymes. In a further embodiment, the forage and one or more microbes are mixed with other feed ingredients, such as colouring agents, stabilisers, growth improving additives and aroma compounds/flavorings, polyunsaturated fatty acids (PUFAs); reactive oxygen generating species, anti-microbial peptides, anti-fungal polypeptides and amino acids.
- PUFAs polyunsaturated fatty acids
- the animal feed may comprise Bacillus stain DSM 29869 and/or DSM 29870 and/or DSM 29871 and/or DSM 29872 or a strain having all of the identifying characteristics of Bacillus DSM 29869 and/or DSM 29870 and/or DSM 29871 and/or DSM 29872 or a mutant of DSM 29869 and/or DSM 29870 and/or DSM 29871 and/or DSM 29872 and 0-80% maize; and/or 0-80% sorghum; and/or 0-70% wheat; and/or 0-70% barley; and/or 0-30% oats; and/or 0-40% soybean meal; and/or 0-10% fish meal; and/or 0-20% whey.
- the animal feed may comprise Bacillus stain DSM 29869 and/or DSM 29870 and/or DSM 29871 and/or DSM 29872 or a strain having all of the identifying characteristics of Bacillus DSM 29869 and/or DSM 29870 and/or DSM 29871 and/or DSM 29872 or a mutant of DSM 29869 and/or DSM 29870 and/or DSM 29871 and/or DSM 29872 and vegetable proteins.
- the protein content of the vegetable proteins is at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% (w/w).
- Vegetable proteins may be derived from vegetable protein sources, such as legumes and cereals, for example, materials from plants of the families Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, and Poaceae, such as soy bean meal, lupin meal, rapeseed meal, and combinations thereof.
- Fabaceae Leguminosae
- Cruciferaceae Chenopodiaceae
- Poaceae such as soy bean meal, lupin meal, rapeseed meal, and combinations thereof.
- the vegetable protein source is material from one or more plants of the family Fabaceae, e.g., soybean, lupine, pea, or bean.
- the vegetable protein source is material from one or more plants of the family Chenopodiaceae, e.g., beet, sugar beet, spinach or quinoa.
- Other examples of vegetable protein sources are rapeseed, and cabbage.
- soybean is a preferred vegetable protein source.
- Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oat, maize (corn), rice, and sorghum.
- the animal feed consists of or comprises milk (e.g., from sow), e.g., for feeding of piglets.
- the animal feed consists of or comprises milk replacement, e.g., for feeding of piglets.
- the animal feed may include a premix, comprising, e.g., vitamins, minerals, enzymes, preservatives, antibiotics, other feed ingredients or any combination thereof which are mixed into the animal feed.
- a premix comprising, e.g., vitamins, minerals, enzymes, preservatives, antibiotics, other feed ingredients or any combination thereof which are mixed into the animal feed.
- the invention related to a premix for feeding an animal infected by Clostridium perfringens , the premix comprising at least the Bacillus strain DSM 29869 described above.
- the premix may comprise one or more bacterial strains, in addition to DSM 29869.
- the invention related to a premix for feeding an animal infected by Clostridium perfringens , the premix comprising at least the Bacillus strain DSM 29870 described above.
- the premix may comprise one or more bacterial strains, in addition to DSM 29870.
- the invention related to a premix for feeding an animal infected by Clostridium perfringens , the premix comprising at least the Bacillus strain DSM 29871 described above.
- the premix may comprise one or more bacterial strains, in addition to DSM 29871.
- the invention related to a premix for feeding an animal infected by Clostridium perfringens , the premix comprising at least the Bacillus strain DSM 29872 described above.
- the premix may comprise one or more bacterial strains, in addition to DSM 29872.
- the animal feed may include one or more vitamins, such as one or more fat-soluble vitamins and/or one or more water-soluble vitamins.
- the animal feed may optionally include one or more minerals, such as one or more trace minerals and/or one or more macro minerals.
- fat- and water-soluble vitamins, as well as trace minerals form part of a so-called premix intended for addition to the feed, whereas macro minerals are usually separately added to the feed.
- Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, e.g., vitamin K3.
- Non-limiting examples of water-soluble vitamins include vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g., Ca-D-panthothenate.
- Non-limiting examples of trace minerals include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium and zinc.
- Non-limiting examples of macro minerals include calcium, magnesium, potassium and sodium.
- the animal feed or animal feed additive described herein optionally include one or more enzymes.
- Enzymes can be classified on the basis of the handbook Enzyme Nomenclature from NC-IUBMB, 1992), see also the ENZYME site at the internet: expasy.ch/enzyme/.
- ENZYME is a repository of information relative to the nomenclature of enzymes. It is primarily based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUB-MB), Academic Press, Inc., 1992, and it describes each type of characterized enzyme for which an EC (Enzyme Commission) number has been provided (Bairoch, 2000, The ENZYME database, Nucleic Acids Res. 28:304-305). This IUB-MB Enzyme nomenclature is based on their substrate specificity and occasionally on their molecular mechanism; such a classification does not reflect the structural features of these enzymes.
- glycoside hydrolase enzymes such as endoglucanase, xylanase, galactanase, mannanase, dextranase and alpha-galactosidase
- endoglucanase xylanase
- galactanase galactanase
- mannanase mannanase
- dextranase alpha-galactosidase
- alpha-galactosidase alpha-galactosidase
- the animal feed or animal feed additive of the invention may also comprise at least one other enzyme selected from the group comprising of phytase (EC 3.1.3.8 or 3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC 3.2.1.22); protease (EC 3.4); phospholipase A1 (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1.1.5); phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4); amylase such as, for example, alpha-amylase (EC 3.2.1.1); lysozyme (EC 3.2.1.17); and beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6), or any mixture thereof.
- phytase EC 3.1.3.8 or 3.1.3.26
- the animal feed or animal feed additive of the invention comprises a phytase (EC 3.1.3.8 or 3.1.3.26).
- phytases include Bio-FeedTM Phytase (Novozymes), Ronozyme® P and HiPhosTM (DSM Nutritional Products), NatuphosTM (BASF), Finase® and Quantum® Blue (AB Enzymes), the Phyzyme® XP (Verenium/DuPont) and Axtra® PHY (DuPont).
- Other preferred phytases include those described in, e.g., WO 98/28408, WO 00/43503, and WO 03/066847.
- the animal feed or animal feed additive of the invention comprises a xylanase (EC 3.2.1.8).
- xylanase EC 3.2.1.8
- 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/beta-glucanase, DuPont).
- the animal feed or animal feed additive of the invention comprises a protease (EC 3.4).
- protease examples include Ronozyme® ProAct (DSM Nutritional Products).
- the animal feed or animal feed additive of the invention may further comprise one or more amino acids.
- amino acids which are used in animal feed are lysine, alanine, beta-alanine, threonine, methionine and tryptophan.
- the animal feed or animal feed additive of the invention may further comprise colouring agents, stabilisers, growth improving additives and aroma compounds/flavorings, polyunsaturated fatty acids (PUFAs); reactive oxygen generating species, anti-microbial peptides and anti-fungal polypeptides.
- colouring agents are carotenoids such as beta-carotene, astaxanthin, and lutein.
- aroma compounds/flavorings are creosol, anethol, deca-, undeca- and/or dodeca-lactones, ionones, irone, gingerol, piperidine, propylidene phatalide, butylidene phatalide, capsaicin and tannin.
- antimicrobial peptides examples include CAP18, Leucocin A, Tritrpticin, Protegrin-1, Thanatin, Defensin, Lactoferrin, Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000), Plectasins, and Statins, including the compounds and polypeptides disclosed in WO 03/044049 and WO 03/048148, as well as variants or fragments of the above that retain antimicrobial activity.
- AFP's antifungal polypeptides
- Aspergillus giganteus and Aspergillus niger peptides, as well as variants and fragments thereof which retain antifungal activity, as disclosed in WO 94/01459 and WO 02/090384.
- polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid.
- reactive oxygen generating species are chemicals such as perborate, persulphate, or percarbonate; and enzymes such as an oxidase, an oxygenase or a syntethase.
- the animal feed or animal feed additive of the invention may further comprise at least one amino acid.
- amino acids which are used in animal feed are lysine, alanine, beta-alanine, threonine, methionine and tryptophan.
- Animal diets can, e.g., be manufactured as mash feed (non-pelleted) or pelleted feed.
- the milled feed-stuffs are mixed and sufficient amounts of essential vitamins and minerals are added according to the specifications for the species in question.
- the bacteria cultures and optionally enzymes can be added as solid or liquid formulations.
- mash feed a solid or liquid culture formulation may be added before or during the ingredient mixing step.
- pelleted feed the (liquid or solid) culture preparation may also be added before or during the feed ingredient step.
- a liquid culture preparation comprises the culture of the invention optionally with a polyol, such as glycerol, ethylene glycol or propylene glycol, and is added after the pelleting step, such as by spraying the liquid formulation onto the pellets.
- a polyol such as glycerol, ethylene glycol or propylene glycol
- the enzyme may also be incorporated in a feed additive or premix.
- the enzyme may be added to the feed mix as a granule, which is optionally pelleted or extruded.
- the granule typically comprises a core particle and one or more coatings, which typically are salt and/or wax coatings.
- the core particle can either be a homogeneous blend of an active compound optionally together with salts (e.g., organic or inorganic zinc or calcium salt) or an inert particle with an active compound applied onto it.
- the active compound is the culture of the invention optionally combined with one or more enzymes.
- the inert particle may be water soluble or water insoluble, e.g., starch, a sugar (such as sucrose or lactose), or a salt (such as NaCl, Na 2 SO 4 ).
- the salt coating is typically at least 1 ⁇ m thick and can either be one particular salt or a mixture of salts, such as Na 2 SO 4 , K 2 SO 4 , MgSO 4 and/or sodium citrate.
- salts such as Na 2 SO 4 , K 2 SO 4 , MgSO 4 and/or sodium citrate.
- Other examples are those described in, e.g., WO 2008/017659, WO 2006/034710, WO 97/05245, WO 98/54980, WO 98/55599, WO 2000/70034 or polymer coating such as described in WO 2001/00042.
- the invention relates to a method for treatment of C. perfringens infections and/or necrotic enteritis in an animal such as a mono-gastric animal including poultry using the animal feed or animal feed additive according to Aspect 1, 2, 3 or 4 (in an effective amount).
- the invention relates to a method for treatment of C.
- perfringens infections and/or necrotic enteritis in an animal such as an mono-gastric animal including poultry using an animal feed or animal feed additive comprising the Bacillus strain having deposit accession number DSM 29869 and/or DSM 29870 and/or DSM 29871 and/or DSM 29872, or a strain having all of the identifying characteristics of Bacillus DSM 29869 and/or DSM 29870 and/or DSM 29871 and/or DSM 29872 or a mutant of DSM 29869 and/or DSM 29870 and/or DSM 29871 and/or DSM 29872 (in an effective amount).
- the animal is not a human being.
- the animal feed is fed to a mono-gastric animal.
- Mono-gastric animals include, but are not limited to, pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chicken (including but not limited to broiler chicks, layers); horses (including but not limited to hotbloods, coldbloods and warm bloods) and fish (including but not limited to salmon, trout, tilapia, catfish and carps; and crustaceans (including but not limited to shrimps and prawns). Pigs and/or poultry are preferred mono-gastric animals.
- the animal feed can further comprise one or more components selected from the list consisting of concentrate; forage; one or more enzymes; one or more additional microbes; one or more vitamins; one or more minerals; one or more amino acids; and one or more other feed ingredients.
- the method comprises administering to the animal feed one or more bacterial strains such as at least two of the above strains, at least three of the above strains, or up to and including all of the above strains.
- the method comprises administering to the animal feed the Bacillus strains having the deposit accession number DSM 29869 or a strain having all the identifying characteristics of Bacillus DSM 29869 or a mutant thereof.
- the method comprises administering to the animal feed the Bacillus strains having the deposit accession number DSM 29870 or a strain having all the identifying characteristics of Bacillus DSM 29870 or a mutant thereof.
- the method comprises administering to the animal feed the Bacillus strains having the deposit accession number DSM 29871 or a strain having all the identifying characteristics of Bacillus DSM 29871 or a mutant thereof.
- the method comprises administering to the animal feed the Bacillus strains having the deposit accession number DSM 29872 or a strain having all the identifying characteristics of Bacillus DSM 29872 or a mutant thereof.
- the animal feed further comprises concentrate. In another embodiment of the method, the animal feed further comprises forage. In another embodiment of the method, the animal feed further comprises one or more additional microbes. In another embodiment of the method, the animal feed further comprises one or more enzymes. In another embodiment of the method, the animal feed further comprises one or more vitamins. In another embodiment of the method, the animal feed further comprises one or more minerals. In another embodiment of the method, the animal feed further comprises one or more amino acids. In another embodiment of the method, the animal feed further comprises one or more other feed ingredients.
- the method also improves the health of the mono-gastric animal feed. In another embodiment to any of the aforementioned embodiments, the method also increases the egg yield of poultry. In an embodiment to any of the aforementioned embodiments, the method also increases the meat yield of the mono-gastric animal.
- the method comprises administering to a mono-gastric animal one or more bacterial strains described herein, wherein the bacterial count of each of the bacterial strains is between 1 ⁇ 10 4 and 1 ⁇ 10 14 CFU/kg of forage, preferably between 1 ⁇ 10 6 and 1 ⁇ 10 12 CFU/kg of forage, and more preferably between 1 ⁇ 10 7 and 1 ⁇ 10 11 CFU/kg of animal feed. In a more preferred embodiment the bacterial count of each of the bacterial strains described herein is between 1 ⁇ 10 8 and 1 ⁇ 10 16 CFU/kg of animal feed.
- the method comprises administering to a mono-gastric animal one or more bacterial strains described herein, wherein the bacterial count of each of the bacterial strains is between 1 ⁇ 10 5 and 1 ⁇ 10 15 CFU/animal/day, preferably between 1 ⁇ 10 7 and 1 ⁇ 10 13 CFU/animal/day, and more preferably between 1 ⁇ 10 8 and 1 ⁇ 10 12 CFU/animal/day. In a more preferred embodiment the bacterial count of each of the bacterial strains described herein is between 1 ⁇ 10 9 and 1 ⁇ 10 11 CFU/animal/day.
- the invention covers the method for treatment of C. perfringens infections comprising:
- the method comprises administering to the animal feed one or more bacterial strains such as at least two of the above strains, at least three of the above strains, or up to and including all of the above strains.
- the animal feed is fed to a mono-gastric animal.
- the mono-gastric animal is, e.g., pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chicken (including but not limited to broiler chicks, layers); horses (including but not limited to hotbloods, coldbloods and warm bloods), or fish (including but not limited to salmon, trout, tilapia, catfish and carps; and crustaceans (including but not limited to shrimps and prawns).
- pigs or swine including, but not limited to, piglets, growing pigs, and sows
- poultry such as turkeys, ducks and chicken (including but not limited to broiler chicks, layers)
- horses including but not limited to hotbloods, coldbloods and warm bloods
- fish including but not limited to salmon, trout, tilapia, catfish and carps
- the animal feed further comprises one or more components selected from the list consisting of concentrate; forage; one or more enzymes; one or more additional microbes; one or more vitamins; one or more minerals; one or more amino acids; and one or more other feed ingredients.
- the animal feed further comprises concentrate. In another embodiment of the method, the animal feed further comprises forage. In another embodiment of the method, the animal feed further comprises one or more additional microbes. In another embodiment of the method, the animal feed further comprises one or more enzymes. In another embodiment of the method, the animal feed further comprises one or more vitamins. In another embodiment of the method, the animal feed further comprises one or more minerals. In another embodiment of the method, the animal feed further comprises one or more amino acids. In another embodiment of the method, the animal feed further comprises one or more other feed ingredients.
- the one or more bacterial strains are present in the form of a stable spore.
- the stable spore will germinate in the intestine and/or stomach of the mono-gastric animal.
- the method comprises administering to a mono-gastric animal one or more bacterial strains described herein, wherein the bacterial count of each of the bacterial strains is between 1 ⁇ 10 4 and 1 ⁇ 10 14 CFU/kg of forage, preferably between 1 ⁇ 10 6 and 1 ⁇ 1012 CFU/kg of forage, and more preferably between 1 ⁇ 10 7 and 1 ⁇ 10 11 CFU/kg of forage. In a more preferred embodiment the bacterial count of each of the bacterial strains described herein is between 1 ⁇ 10 8 and 1 ⁇ 10 10 CFU/kg of forage.
- the method comprises administering to a mono-gastric animal one or more bacterial strains described herein, wherein the bacterial count of each of the bacterial strains is between 1 ⁇ 10 5 and 1 ⁇ 10 15 CFU/animal/day, preferably between 1 ⁇ 10 7 and 1 ⁇ 10 13 CFU/animal/day, and more preferably between 1 ⁇ 10 8 and 1 ⁇ 10 12 CFU/animal/day. In a more preferred embodiment the bacterial count of each of the bacterial strains described herein is between 1 ⁇ 10 9 and 1 ⁇ 10 11 CFU/animal/day.
- the method comprises administering to a mono-gastric animal one or more bacterial strains described herein, wherein the bacterial count of each Bacillus spore is between 1 ⁇ 10 5 and 1 ⁇ 10 15 CFU/animal/day, preferably between 1 ⁇ 10 7 and 1 ⁇ 10 13 CFU/animal/day, and more preferably between 1 ⁇ 10 8 and 1 ⁇ 10 12 CFU/animal/day.
- the invention relates in a further embodiment to use of the animal feed or animal feed additive to improve the performance of an animal, such as improving the feed conversion rate and/or improving the European Production Efficacy Factor and/or improving the body weight gain and/or improving the feed efficiency and/or improving the health.
- the improvement of feed conversion rate (FCR) for Aspect 1, 2, 3 or 4 results in a FCR of ⁇ 2.5% or less than ⁇ 2.5%, such as less than ⁇ 2.6%, such as less than ⁇ 2.7%, such as less than ⁇ 2.8%, such less than ⁇ 2.9%, such as less than ⁇ 3.0%.
- the improvement of FCR results in a FCR of from ⁇ 5% to ⁇ 2% such as a FCR from ⁇ 4% to ⁇ 2%, such as a FCR of from ⁇ 3.5% to ⁇ 2.5%.
- the improvement of FCR for Aspect 1, 2, 3 or 4 results in a FCR within an interval selected from the group consisting of from ⁇ 5% to ⁇ 4.5%, from ⁇ 4.5% to ⁇ 4%, from ⁇ 4% to ⁇ 3.8%, from ⁇ 3.8% to ⁇ 3.6%, from ⁇ 3.6% to ⁇ 3.4%, from ⁇ 3.4% to ⁇ 3.2%, from ⁇ 3.2 to ⁇ 3.0%, from ⁇ 3.0% to ⁇ 2.8% and from ⁇ 2.8 to ⁇ 2.5%, or any combination of these intervals.
- the FCR can be determined as described in Example 8.
- the improvement in body weight gain for Aspect 1, 2, 3 or 4 results in a body weight gain of at least 0.5%, such as at least 0.8%, such as at least 1.5%, such as at least 1.8%, such as at least 2.0%, such as at least 2.3%, such as at least 3.5%, such as at least 4.2%, such as at least 5.2%, such as at least 6.5%, such as at least 7.3%.
- the improvement in body weight gain for Aspect 1, 2, 3 or 4 results in a body weight gain selected from the group consisting of from 1.8% to 2.0%, from 2.0% to 2.2%, from 2.2% to 2.4%, from 2.4% to 2.6%, from 2.6% to 2.8%, from 2.8% to 3.0%, from 3.0% to 3.2%, from 3.2% to 3.4%, from 3.4% to 3.6%, from 3.6% to 3.8%, from 3.8% to 4.0%, from 4% to 5%, from 5% to 7%, from 7% to 10%, or any combination thereof.
- the body weight gain can be determined as described in Example 8.
- the invention relates to the use of a feed or feed premix comprising at least the spores of a Bacillus subspecies to improve the performance of an animal infected by C. perfringens .
- a feed or feed premix described within the present comprise a Bacillus DSM 29870 (hereafter DSM 29870).
- DSM 29870 Bacillus DSM 29870
- This strain is characterized in that is non-hemolytic, has antimicrobial activity, e.g., against Clostridium perfringens and/or E.
- coli is sensitive to Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline, improves body weight gain and/or feed conversion ratio in animal, hydrolyzes one or more of the substrates selected from the group consisting of amylase, arabinan, arabinoxylan, casein and xylan.
- the present invention relates to the use of a feed or feed premix comprising at least the DSM 29870 as described above to improve the performance of an animal infected by C. perfringens .
- animal performance may be determined by the body weight gain of the animal and/or by the feed conversion ratio and/or by the feed efficiency and/or by the digestibility of a nutrient in a feed (e.g., amino acid digestibility) and/or digestible energy or metabolizable energy in a feed and/or by nitrogen retention.
- animal performance is determined by the body weight gain of the animal and/or by the feed conversion ratio.
- improved animal performance it is meant that there is increased body weight gain and/or reduced feed conversion ratio and/or improved digestibility of nutrients or energy in a feed and/or by improved nitrogen retention and/or increased feed efficiency resulting from the use of feed or feed premix of the present invention in feed in comparison to feed which does not comprise said feed or feed premix.
- improved animal performance it is meant that there is increased body weight gain and/or reduced feed conversion ratio.
- the animal feed or animal feed premix comprises at least the DSM 29870 bacterial strain described herein is used, wherein the bacterial count of the bacterial strains is between 1 ⁇ 10 4 and 1 ⁇ 10 14 CFU/kg of animal feed or feed premix, preferably between 1 ⁇ 10 6 and 1 ⁇ 10 12 CFU/kg of feed or feed premix, and more preferably between 1 ⁇ 10 7 and 1 ⁇ 10 11 CFU/kg of feed or feed premix. In a more preferred embodiment the bacterial count of the bacterial strains described herein is between 1 ⁇ 10 8 and 1 ⁇ 10 10 CFU/kg of feed or feed premix.
- feed conversion ratio refers to the amount of feed fed to an animal to increase the weight of the animal by a specified amount.
- FCR is the mass of the food eaten divided by the output, all over a specified period. FCR can be determined as described in the Examples.
- An improved feed conversion ratio means a lower feed conversion ratio.
- lower feed conversion ratio or “improved feed conversion ratio” it is meant that the use of a feed additive composition in feed results in a lower amount of feed being required to be fed to an animal to increase the weight of the animal by a specified amount compared to the amount of feed required to increase the weight of the animal by the same amount when the feed does not comprise said feed additive composition.
- the improvement of feed conversion rate (FCR) for Aspect 5, 6, 7 or 8 results in a FCR of ⁇ 2.5% or less than ⁇ 2.5%, such as less than ⁇ 2.6%, such as less than ⁇ 2.7%, such as less than ⁇ 2.8%, such less than ⁇ 2.9%, such as less than ⁇ 3.0%.
- the improvement of FCR results in a FCR of from ⁇ 5% to ⁇ 2% such as a FCR from ⁇ 4% to ⁇ 2%, such as a FCR of from ⁇ 3.5% to ⁇ 2.5%.
- the improvement of FCR for Aspect 1, 2, 3 or 4 results in a FCR within an interval selected from the group consisting of from ⁇ 5% to ⁇ 4.5%, from ⁇ 4.5% to ⁇ 4%, from ⁇ 4% to ⁇ 3.8%, from ⁇ 3.8% to ⁇ 3.6%, from ⁇ 3.6% to ⁇ 3.4%, from ⁇ 3.4% to ⁇ 3.2%, from ⁇ 3.2 to ⁇ 3.0%, from ⁇ 3.0% to ⁇ 2.8% and from ⁇ 2.8 to ⁇ 2.5%, or any combination of these intervals.
- an “increased body weight gain” refers to an animal having increased body weight on being fed feed comprising a feed composition compared with an animal being fed a feed without said feed composition of the invention.
- the Body Weight Gain of an animal is the increase of body weight of the animal over a specified time period.
- the improvement in body weight gain if of at least 0.5% such at least 1%, such at least 2%, such at least 2,3%, such at least 3%, such at least 4%, such at least 5%, such at least 6%, such at least 6.5%, such at least 7%, such at least 8%, such at least 8%, such at least 10%.
- the improvement in body weight gain for Aspect 5, 6, 7 or 8 results in a body weight gain of at least 0.5%, such as at least 0.8%, such as at least 1.5%, such as at least 1.8%, such as at least 2.0%, such as at least 2.3%, such as at least %, such as at least 4.2%, such as at least 5.2%, such as at least 6.5%, such as at least 7.3%.
- the improvement in body weight gain for Aspect 1, 2, 3 or 4 results in a body weight gain selected from the group consisting of from 1.8% to 2.0%, from 2.0% to 2.2%, from 2.2% to 2.4%, from 2.4% to 2.6%, from 2.6% to 2.8%, from 2.8% to 3.0%, from 3.0% to 3.2%, from 3.2% to 3.4%, from 3.4% to 3.6%, from 3.6% to 3.8%, from 3.8% to 4.0%, from 4% to 5%, from 5% to 7%, from 7% to 10%, or any combination thereof.
- feed efficiency refers to the amount of weight gain in an animal that occurs when the animal is fed ad-libitum or a specified amount of food during a period of time.
- increased feed efficiency it is meant that the use of the feed or the feed premix according the present invention in feed results in an increased weight gain per unit of feed intake compared with an animal fed without said feed or feed premix being present.
- Nutrient digestibility as used herein means the fraction of a nutrient that disappears from the gastro-intestinal tract or a specified segment of the gastro-intestinal tract, e.g., the small intestine. Nutrient digestibility may be measured as the difference between what is administered to the subject and what comes out in the faeces of the subject, or between what is administered to the subject and what remains in the digesta on a specified segment of the gastro intestinal tract, e.g., the ileum.
- Nutrient digestibility as used herein may be measured by the difference between the intake of a nutrient and the excreted nutrient by means of the total collection of excreta during a period of time; or with the use of an inert marker that is not absorbed by the animal, and allows the researcher calculating the amount of nutrient that disappeared in the entire gastro-intestinal tract or a segment of the gastro-intestinal tract.
- Such an inert marker may be titanium dioxide, chromic oxide or acid insoluble ash.
- Digestibility may be expressed as a percentage of the nutrient in the feed, or as mass units of digestible nutrient per mass units of nutrient in the feed.
- Nutrient digestibility as used herein encompasses starch digestibility, fat digestibility, protein digestibility, mineral digestibility and amino acid digestibility.
- Energy digestibility means the gross energy of the feed consumed minus the gross energy of the faeces or the gross energy of the feed consumed minus the gross energy of the remaining digesta on a specified segment of the gastro-intestinal tract of the animal, e.g., the ileum.
- Metabolizable energy refers to apparent metabolizable energy and means the gross energy of the feed consumed minus the gross energy contained in the faeces, urine, and gaseous products of digestion.
- Energy digestibility and metabolizable energy may be measured as the difference between the intake of gross energy and the gross energy excreted in the faeces or the digesta present in specified segment of the gastro-intestinal tract using the same methods to measure the digestibility of nutrients, with appropriate corrections for nitrogen excretion to calculate metabolizable energy of feed.
- Nitrogen retention as used herein means a subject's ability to retain nitrogen from the diet as body mass.
- a negative nitrogen balance occurs when the excretion of nitrogen exceeds the daily intake and is often seen when the muscle is being lost.
- a positive nitrogen balance is often associated with muscle growth, particularly in growing animals.
- Nitrogen retention may be measured as the difference between the intake of nitrogen and the excreted nitrogen by means of the total collection of excreta and urine during a period of time. It is understood that excreted nitrogen includes undigested protein from the feed, endogenous proteinaceous secretions, microbial protein, and urinary nitrogen.
- the present invention relates to a method for improving the performance of an animal infected by C. perfringens comprising the step of administering to said animal a feed or feed premix comprising at least the Bacillus strain DSM 29870.
- the feed or feed premix comprises at least the DSMZ 29870 bacterial strain, wherein the bacterial count of each of the bacterial strains is between 1 ⁇ 10 4 and 1 ⁇ 10 14 CFU/kg of feed, preferably between 1 ⁇ 10 6 and 1 ⁇ 10 12 CFU/kg of feed, and more preferably between 1 ⁇ 10 7 and 1 ⁇ 10 11 CFU/kg of feed.
- the bacterial count of the bacterial strains described herein is between 1 ⁇ 10 8 and 1 ⁇ 10 10 CFU/kg of feed.
- the invention deals with a method for improving the body weight gain and/or the feed conversion ratio of an animal infected by C. perfringens comprising the step of administering to said animal a feed composition comprising at least the Bacillus strain DSM 29870.
- the animal is selected from the group consisting of swine and poultry. More specifically, the animal is selected from the group comprising chickens, broilers, layers, quails and turkey, domestic ducks or domestic geese or the young of pigeons, pigs, swine, piglets, growing pigs, or sows
- the present invention relates to a method for feeding an animal infected by C. perfringens comprising administering a feed or feed premix comprising at least the Bacillus strain DSM29870 to said animal optionally in conjunction with other animal feed ingredients.
- the terms “improving animal performance” are defined as above.
- the animal is selected from the group consisting of swine and poultry. More specifically, the animal is selected from the group comprising chickens, broilers, layers, quails and turkey, domestic ducks or domestic geese or the young of pigeons, pigs, swine, piglets, growing pigs, or sows.
- the composition such as a feed comprises at least the DSMZ 29870 bacterial strain, wherein the bacterial count of each of the bacterial strains is between 1 ⁇ 10 4 and 1 ⁇ 10 14 CFU/kg of feed, preferably between 1 ⁇ 10 6 and 1 ⁇ 10 12 CFU/kg of feed, and more preferably between 1 ⁇ 10 7 and 1 ⁇ 10 11 CFU/kg of feed.
- the bacterial count of the bacterial strains described herein is between 1 ⁇ 10 8 and 1 ⁇ 10 10 CFU/kg of feed.
- Hemolysis was tested according to the Technical Guidance on the assessment of the toxigenic potential of Bacillus species used in animal nutrition, EFSA Journal 2011; 9(11):2445.
- Sheep blood agar plates were purchased as ready to use (Becton Dickenson art 254053 or 254087). Alternatively, the agar plates can be prepared by adding 5% defibrinated sheep blood (obtained from Statens Serum Institute, Denmark) to TS-agar (Oxoid CM 131). Agar should be autoclaved at 121° C. for 20 minutes and cooled down to about 40° C. before adding the blood immediately before pouring the plates.
- Bacillus strains were taken from the preservation at ⁇ 80° C. and streaked on TS-agar plate, which was incubated at 30° C. overnight or until growth appeared. From a single colony as little as possible of the material was used to streak a line on 1 ⁇ 4 of an agar plate. The plate was incubated at 30° C. for 72 hours. Hemolysis/clearing zones of the Bacillus strains to be tested were compared with the positive and negative control. As positive control Bacillus subtilis ATCC 21332 was used. As negative control Bacillus subtilis 168 (BGSC-1A1, Bacillus Genetic Stock Center) was used.
- the non-Hemolytic Bacillus strains therefore seem to be common and fairly abundant in nature, but only comprising a minority of the natural strains.
- the non-hemolytic strains seem to be more abundant in Bacillus licheniformis , while most Bacillus amyloliquefaciens strains appear hemolytic. All the non-hemolytic strains were tested for inhibition of growth of Clostridium perfringens . The following strains from screening were all hemolysis negative and among those selected for further studies: DSM 29869, DSM 29870, DSM 29871 and DSM 29872. Clostat ( Bacillus PB6) was determined to be hemolytic.
- Clostridium perfringens strains 23 and 48 All the non-hemolytic strains were tested for inhibition of growth of Clostridium perfringens strains 23 and 48 (both are netB positive) [Gholamiandekhordi et al., 2006, Molecular and phenotypical characterization of Clostridium perfringens isolates from poultry flocks with different diseasestatus, Vet. Microbiol. 113:143-152], were grown overnight in tryptic soy broth (BD part 211822) supplemented with 0.6% yeast extract (BD part 212750) at 35° C. under static anaerobic conditions. 250 ⁇ L of the overnight culture of Clostridium perfringens was added to 250 mL of tryptic soy agar supplemented with 0.6% yeast extract at 40° C. and poured into rectangular petri plates (Nunc part 267060). The inoculated agar was allowed to cool at room temperature after which an 8 mm diameter well was made in the agar.
- the Bacillus strain DSM 29869, DSM 29870, DSM 29871, or DSM 29872 was grown overnight in tryptic soy broth at 35° C. under aerobic conditions. 1000 ⁇ L of Bacillus culture was collected and fractionated into cell-free supernatant and cells by centrifugation. 20 ⁇ L of cell-free supernatant or 100 ⁇ diluted cells in phosphate buffered saline were added directly to the wells in the Clostridium perfringens inoculated agar plates. A control well contained 20 ⁇ L of phosphate buffer saline. The plates were incubated for 18 hours at 35° C. under anaerobic conditions.
- Cell-free supernatant and 100 ⁇ diluted cells of Bacillus strains DSM 29869, DSM 29870, DSM 29871, and DSM 29872 were able to consistently inhibit growth of C. perfringens strains 23 and 48 in vitro. Inhibition was also seen by competitor strain “CloSTAT”, for both supernatant and cells, but was not seen with competitor strain GalliproTect.
- CloSTAT is a strain of Bacillus amyloliquefaciens that was isolated from the commercial DFM product CloSTAT, Kemin.
- Gamin is a strain of Bacillus licheniformis that was isolated from the commercial product Gallipro Tect, Chr Hansen.
- the strains have been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by foreign patent laws to be entitled thereto.
- the deposits represent a substantially pure culture of the deposited strain.
- the deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
- DNA was extracted from a culture of DSM 29869, DSM 29870, DSM 29871 and DSM 29872 using QiaAmp DNA Blood Mini Kit (Qiagen art 51106). The kit was used as recommended for extraction of DNA from gram positive bacteria.
- 16S rDNA was amplified in a total volume of 50 ⁇ l by mixing: 10 pmol of each of Primer 16S F and 16S R, 0.2 mM of each nucleotide, 2.5 units Ampli taq, 1 ⁇ Ampli taq buffer, 5 ⁇ l DNA template and by using the following PCR program: 94° C. 2 min (94° C. 30 s, 52° C. 30 S, 72° C. 1 min) ⁇ 35, 72° C. 10 min on a Perkin Elmer PCR machine. The PCR product was sequenced by Novozymes DNA sequencing facility using primer 530R, 357F, 1390R and 1100F.
- R is A or G.
- the 16 S rDNA sequences from DSM 29869, DSM 29870, DSM 29871 and DSM 29872 are shown as SEQ ID NO: 1-4 in the sequence listing respectively.
- the 16 S rDNA sequences from DSM 29869, DSM 29870, DSM 29871 and DSM 29872 were analyzed by BLAST against EMBL database and showed identity to 16 S rDNA sequences of Bacillus subtilis (SEQ ID NO: 2 and SEQ ID NO: 3) and to Bacillus amyloliquefaciens (SEQ ID NO: 1 and SEQ ID NO: 4).
- SEQ ID NO: 1 In order to study the phylogenetic affiliation of SEQ ID NO: 1 to SEQ ID NO: 4 the sequences were analyzed by a ClustalW alignment in MegAlign (DNASTAR) using SEQ ID NO: 5 to SEQ ID NO: 7 as benchmark. These sequences are reference 16S rDNA sequences of the type strains of Bacillus vallismortis taken from AB021198 (SEQ ID NO: 5), Bacillus subtilis taken from AJ276351 (SEQ ID NO: 6) and Bacillus amyloliquefaciens taken from AB255669 (SEQ ID NO: 7).
- FIG. 1 The ClustalW alignment of SEQ ID NO: 1 to SEQ ID NO: 7 ( FIG. 1 ) shows 7 nucleotide positions where 2 or more sequences have a nucleotide that deviates from the other. For numbering the positions in SEQ ID NO: 6 are used.
- SEQ ID NO: 2 is identical to Bacillus amyloliquefaciens and Bacillus vallismortis , but different from the rest that are identical to each other.
- SEQ ID NO: 1 and SEQ ID NO: 4 are identical to Bacillus amyloliquefaciens , but different from the rest that are identical to each other.
- SEQ ID NO: 4 is identical to Bacillus amyloliquefaciens , but different from the rest that are identical to each other.
- SEQ ID NO: 2 and SEQ ID NO: 3 are identical to Bacillus subtilis , but different from the rest that are identical to each other.
- SEQ ID NO: 1 is identical to Bacillus amyloliquefaciens , but different from the rest that are identical to each other.
- the variation in the 16S rDNA genes support the species affiliation seen in the BLAST report.
- Bacillus amyloliquefaciens DSM 29869 was isolated by Novozymes (Novo Nordisk) from an environmental sample collected at Jamaica in 1990. The strain was identified as Bacillus amyloliquefaciens based on 16S rDNA sequencing
- Bacillus subtilis DSM 29870 was isolated by Novozymes (Novo Nordisk) from an environmental sample collected at Jamaica in 1990. The strain was identified as Bacillus subtilis based on 16S rDNA sequencing.
- Bacillus amyloliquefaciens DSM 29872 was isolated from an environmental sample from the USA in 2008. The strain was identified as Bacillus amyloliquefaciens based on 16S rDNA sequencing.
- Escherichia coli strains ATCC10536 or ATCC25922, were grown overnight in tryptic soy broth (BD part 211822) supplemented with 0.6% yeast extract (BD part 212750) at 35° C. under static anaerobic conditions. 100 ⁇ L of the overnight culture of Escherichia coli was added to 250 mL of tryptic soy agar supplemented with 0.6% yeast extract at 40° C. and poured into rectangular petri plates (Nunc part 267060). The inoculated agar was then allowed to cool at room temperature after which an 8 mm diameter well was made in the agar.
- the Bacillus strain DSM 29869, DSM 29870, DSM 29871, or DSM 29872 was grown overnight in tryptic soy broth at 35° C. under aerobic conditions. 1000 ⁇ L of Bacillus subtilis culture was collected and fractionated into cell-free supernatant and cells by centrifugation. 20 ⁇ L of cell-free supernatant or 100 ⁇ diluted cells in phosphate buffer saline were added directly to the wells in the Escherichia coli inoculated agar plates. A control well contained 20 ⁇ L of phosphate buffer saline. The plates were incubated for 18 hours at 30° C. under aerobic conditions.
- the minimal inhibitory concentrations (MIC) of eight antibiotics against Bacillus strains DSM 29869, DSM 29870, DSM 29871, and DSM 29872 were determined using broth micro dilution essentially as described in the CLSI guidelines (M07-A9 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; 2012). Only modification was that volumes were changed; 90 ⁇ l Mueller Hinton Broth 2 with bacteria was added to 10 ⁇ l antibiotics dilutions, cfu's of bacteria and concentration of antibiotics were changed so final concentrations and final cfu's matched the guideline. The plates were incubated for 20-24 h instead of 16-20 h. The control strain recommended in the CLSI standard Staphylococcus aureus ATCC 29213 was used as control strain.
- Antibiotics were diluted to the concentration of 640 ⁇ g/mL in MHB. A two fold dilution series was prepared in MHB down to the concentration 0.625 ⁇ g/ml. 10 ⁇ l of each dilution and of each antibiotic was pipetted into a micro-titre plate. Later, when the antibiotics were mixed with the suspension of bacteria, the samples were diluted 10 ⁇ (10 ⁇ L sample in a total volume of 100 ⁇ l). This resulted in the final test range of 0.06-64 ⁇ g/ml.
- the assay plates were incubated in a plastic bag with at wet cloth at 37° C. for 20-24 h.
- the MIC was determined as the lowest concentration of antibiotic that completely inhibited growth of bacteria as detected by the unaided eye.
- a 10-fold dilution series in 0.9% NaCl was made to the 10 ⁇ 3 of the cultures inoculated into the micro-titre plate. 2 ⁇ 100 ul from the 10 ⁇ 3 dilution were plated onto two TSA plates. The plates were incubated overnight at 37° C. Number of CFU/ml was counted.
- CFU/ml The amount of bacteria inoculated into the assay plates was measured (CFU/ml). In general the CFU/ml was very close to the target value of 5*10 5 CFU/ml. However, the CFU/ml for Bacillus strains may be associated with some uncertainty, since the bacteria tend to aggregate and once aggregated will only result in one colony forming unit (Tables 5.1 and 5.2).
- SMA plates were streaked from the overnight cultures using a 10 ⁇ L inoculating loop. Up to four strains were streaked per plate, into separate quadrants.
- AZCL substrates which can be autoclaved AZCL-cellulose (AZCL-HE-Cellulose, I-AZCEL, Megazyme), arabinoxylan (AZCL-Arabinoxylan, wheat, I-AZWAX, Megazyme), arabinan (AZCL-Arabinan, debranched, I-AZDAR, Megazyme):
- the media were autoclaved to sterilize. Note: Only these three AZCL substrates can be autoclaved without obvious dye release indicating substrate instability. After autoclaving, media is kept at 50° C. until ready to pour plates. The pH was checked, and if necessary adjusted to pH 7 using 10% HCl or 2N NaOH, maintaining sterility.
- AZCL-amylose I-AZAMY, Megazyme
- AZCL-casein I-AZCAS, Megazyme
- AZCL-cellulose, AZCL-arabinoxylan, AZCL-arabinan For substrates which could be autoclaved (AZCL-cellulose, AZCL-arabinoxylan, AZCL-arabinan): The warm ( ⁇ 50° C.), autoclaved liquid agar media was magnetically stirred in order to suspend the insoluble AZCL substrate. An aliquot was transferred to a sterile solution basin (Periodic mixing of the material in the solution basin was necessary to keep the substrate suspended.) A repeating, multi-channel pipette (e.g., Matrix 1250 ⁇ L pipet along with the corresponding 1250 ⁇ L sterile pipet tips) was used to dispense 180 ⁇ L into each well of a sterile 96-well plate. A set of tips would typically last for up to six columns per plate.
- pipette e.g., Matrix 1250 ⁇ L pipet along with the corresponding 1250 ⁇ L sterile pipet tips
- AZCL-amylose, AZCL-casein, AZCL-xylan For the substrates that could not be autoclaved (AZCL-amylose, AZCL-casein, AZCL-xylan): Into a sterile 250 mL beaker with magnetic stir-bar was added 100 mL of warm, sterile 0.1 ⁇ or 1 ⁇ LB media (no AZCL substrate added). With stirring, add 0.05 mg of AZCL substrate, and continue stirring until re-suspended. It may help to add the substrate slowly at first. Once substrate was re-suspended, it was poured into solution basin and added to plates as described above. One plate was made for each substrate (AZCL-cellulose, AZCL-arabinoxylan, AZCL-arabinan) and condition (0.1 ⁇ and 1 ⁇ LB).
- AZCL substrate agar plates were inoculated by applying 2 ⁇ L of overnight culture that was dispensed on top of the agar. The added liquid should be visible on top of the agar. When moving to the next plate, be sure that there are no drops left at the end of the pipet tips to ensure that each well has been inoculated. Repeat for all AZCL substrate plates. Control enzyme dilutions were prepared, each at 1/100, diluting in sterile phosphate dilution buffer: 10 ⁇ L enzyme preparation+990 ⁇ L buffer. Control enzyme wells were then inoculated with 10 ⁇ L of the appropriate enzyme dilution for each substrate.
- the 4 experimental groups consisted in (Table 7.2): T1, non-infected and non-treated animals; T2, C. perfringens infected and non-treated animals; T3, C. perfringens infected animals fed with the basal diet containing bacitracin (included at 50 ppm, from d1 to d28); T4 , C. perfringens infected animals fed with the basal diet containing the DSM 29870 strain (included at 1.10 9 CFU/kg of feed in trial 1 and 5.10 8 CFU/kg of feed in trials 2 and 3, from d1 to d28).
- Body Weight Gain (BWG) (Live Body Weight) d28 ⁇ (Live Body Weight) d1
- Feed Conversion Ratio ratio between feed consumed and weight gained
- Necrotic enteritis lesion scores total mortality and % of dead birds with necrotic enteritis lesions were also calculated
- strain DSM 29870 can counteract the negative impact of an induced necrotic enteritis on the production parameters with a significant effect in all challenge trials on the FCR and the mortality due to necrotic enteritis, relative to the positive control group (non-infected, non-treated animals).
- the 5 experimental treatments consisted in (Table 8.1): T1, negative control basal diets; T2, basal diets containing the strain DSM 29870 (included at 5.10 8 CFU/kg of feed, from d1 to d35); T3, basal diets containing the strain DSM 29871 (included at 5.10 8 CFU/kg of feed, from d1 to d35); T4 basal diets containing the strain DSM 29872 (included at 5.10 8 CFU/kg of feed, from d1 to d35); T5, basal diet containing a marketed probiotic, GalliPro Max (included at the commercial dose, 8.10 8 CFU/kg of feed, from d1 to d35).
- Basal diets consisted in 3 phases feeding program: starter phase (from 1 to day 12), grower phase (from 13 to day 28) and finisher phase (from 29 to day 35). Every phase was formulated to meet or exceed animal requirement and agree with standard commercial US corn-soybean meal-based broiler diets. The dietary and raw material composition of these diets is given in Table 8.2. All experimental diets include phytase (Ronozyme P, 250 FYT/kg) and did not contain any coccidiostat, NSPase or growth promoting substance. Feed and water were available ad libitum throughout the trial.
- Vitamin mix will provide the following (per kg of diet): thiamin•mononitrate, 2.4 mg; nicotinic acid, 44 mg; riboflavin, 4.4 mg; D-Ca pantothenate, 12 mg; vitamin B 12 (cobalamin), 12.0 ⁇ g; pyridoxine•HCL, 4.7 mg; D-biotin, 0.11 mg; folic acid, 5.5 mg; menadione sodium bisulfite complex, 3.34 mg; choline chloride, 220 mg; cholecalciferol, 27.5 ug; trans-retinyl acetate, 1,892 ug; all-rac ⁇ tocopheryl acetate, 11 mg; ethoxyquin, 125 mg.
- Trace mineral mix provided the following (per kg of diet): manganese (MnSO 4 •H 2 O), 60 mg; iron (FeSO 4 •7H 2 O), 30 mg; zinc (ZnO), 50 mg; copper (CuSO 4 •5H 2 O), 5 mg; iodine (ethylene diamine dihydroiodide), 0.15 mg; selenium (NaSe0 3 ), 0.3 mg.
- Feed Intake (FI) (Remaining Feed) d35 ⁇ (Issued Feed) d1
- Body Weight Gain (BWG) (Live Body Weight) d35 ⁇ (Live Body Weight) d1
- Feed Conversion Ratio ratio between feed consumed and weight gained
- Feed additives did not significantly affect animal feed intake relative to the control. Body weight was slightly increased using the NZB strains whereas GalliPro Max tended to decrease this parameter.
- the DSM 29870, DSM 29871 and DSM 29872 strains also improved significantly the feed conversion ratio by 1.6%, 1.5% and 1.9%, respectively, with no significant differences between the groups fed with NZB strains.
- GalliPro Max had only a slight and non-significant effect on the feed conversion ratio.
- a total of 900 one day-old male broiler chickens Ross 708 were individually weighed (body weight 47.1 g ⁇ 1.1 g) and allocated in floor pens (15 birds/pens). Pens were used in factorial and completely randomized design with 12 pens per treatment (i.e., 180 animals/treatment).
- the 5 experimental treatments consisted in (Table 9.1): T1, negative control basal diets; T2, basal diets containing the strain DSM 29870 (included at 5.10 8 CFU/kg of feed, from d1 to d35); T3, basal diets containing the strain DSM 29871 (included at 5.10 8 CFU/kg of feed, from d1 to d35); T4 basal diets containing the strain DSM 29872 (included at 5.10 8 CFU/kg of feed, from d1 to d35); T5, basal diet containing a marketed probiotic, GalliPro Max (included at the commercial dose, 8.10 8 CFU/kg of feed, from d1 to d35).
- Basal diets consisted in 3 phases feeding program: starter phase (from 1 to day 14), grower phase (from 15 to day 21) and finisher phase (from 22 to day 35). Every phases were formulated to meet or exceed animal requirement and agree with standard commercial US corn-soybean meal-based broiler diets. The dietary and raw material composition of these diets is given in Table 9.2. All experimental diets included vitamin and mineral premixes, but did not contain any coccidiostat, NSPase, phytase or growth promoting substance. Feed and water were available ad libitum throughout the trial.
- Vitamin mix will provide the following (per kg of diet): vitamin A, 13 227 513 IU; vitamin D3, 3 968 254 IU; vitamin E, 66 138 IU; vitamin B 12 , 40 mg; biotin, 254 mg; menadione, 3 968 mg; thiamine, 3 968 mg; riboflavin, 13 228 mg; d-Pantothenic Acid, 22 046 mg; pyridoxine, 7 937 mg; niacin, 110 229 mg; folic acid, 2 205 mg; Selenium Premix: selenium, 600 ppm + calcium, 36% 2 Trace mineral mix provided the following (per kg of diet): calcium, Min: 15.7% and Max: 18.7%; manganese (Mn), 6.0%; zinc (Zn), 6.0%; iron (Fe), 4.0%; copper (Cu), 5000 ppm; iodine (I), 1250 ppm; cobalt (Co), 500 ppm
- the strains DSM 29870 and DSM 29872 improved significantly the feed conversion ratio by 3.6% and 4.2%, respectively.
- DSM 29871 and GalliPro Max also improved (slightly for DSM 29871) the feed conversion ratio in this experiment, but not significantly.
- the strain DSM 29870 allowed a decrease of the mortality level in a numerically higher manner than GalliPro Max.
- the 3 experimental treatments consisted in (Table 10.1): T1, negative control basal diets; T2, basal diets containing the strain DSM 29870 (included at 5.10 8 CFU/kg of feed, from d1 to d35); T3, basal diets containing the strain DSM 29871 (included at 5.10 8 CFU/kg of feed, from d1 to d35); T4 basal diets containing the strain DSM 29872 (included at 5.10 8 CFU/kg of feed, from d1 to d35); T5, basal diet containing a marketed probiotic, GalliPro Max (included at the commercial dose, 8.10 8 CFU/kg of feed, from d1 to d35).
- Basal diets consisted in 3 phases feeding program: starter phase (from 1 to day 21) and grower phase (from 22 to day 35). Every phase was formulated to meet or exceed animal requirement and agree with standard commercial EU corn-soybean meal-based broiler diets. The dietary and raw material composition of these diets is given in Table 10.2. All experimental diets included vitamin and mineral premixes, but did not contain any coccidiostat, NSPase, phytase or growth promoting substance. Feed and water were available ad libitum throughout the trial.
- strain DSM 29870 has been demonstrated on broiler performances (i.e., body weight gain and feed conversion ratio) fed a corn/soybean meal-based diet.
- GalliPro Max showed no significant effect on the body weight gain in any of the working examples above and showed significant and positive effect on feed conversion ratio in one third of the experiments.
- Monensin compatibility of Bacillus strains DSM 29869, DSM 29870, DSM 29871, and DSM 29872 was determined using a modified broth micro dilution similar to the method described in the Example 5. Briefly, a single colony of Bacillus spp. (from overnight tryptic soy agar plates) was inoculated into Mueller Hinton Broth (MHB) and cultured overnight. Sterile media was the inoculated with the overnight culture and allowed to grow for 4 hours to test bacteria in log growth phase. Cultures were then diluted once more 1:200 into fresh MHB and 90 ⁇ L of this inoculated broth was added to the diluted monensin at the indicated concentrations.
- MHB Mueller Hinton Broth
- Bacillus spp. were grown overnight on tryptic soy agar plates (40 g/L) at 37° C.
- Mueller Hinton broth 21 g/L was dissolved in water and autoclaved in glass tubes containing 5 mL of broth each.
- a single colony of Bacillus spp. was inoculated into Mueller Hinton Broth (MHB) and incubated overnight at 37° C. shaking at 200 rpm.
- a 5 mL glass tube of fresh, sterile media was then inoculated with 25 mL of overnight culture and allowed to grow for 4 hours at 37° C. Cultures were then diluted once more 1:200 into fresh MHB. 90 ⁇ L of this inoculated broth was then added to the diluted antibiotic at the indicated concentrations.
- Monensin was diluted into 96% ethanol to a concentration of 800 ⁇ g/mL. This solution was then diluted 10-fold into sterile phosphate buffer to a concentration of 80 ⁇ g/mL. A two fold dilution series was prepared in MHB down to the concentration 2.5 ⁇ g/mL. 10 ⁇ l of each dilution and of each antibiotic was pipetted into a micro titer plate. Later, when the antibiotics were mixed with the suspension of bacteria, the samples were diluted 10 ⁇ (10 ⁇ L sample in a total volume of 100 ⁇ l). This resulted in the final test range of 0.25-8 ⁇ g/ml.
- a potential challenge of delivering Bacillus spp. in feed is the common use of antibiotics as growth promoters in feed. Therefore it is necessary to determine the compatibility of strains with commonly-used feed antibiotics in order to identify any potential conflicts with use as a direct fed microbial. Therefore, the monensin compatibility of Bacillus strains DSM 29869, DSM 29870, DSM 29871, and DSM 29872 were determined along with prior art strains.
- DSM 29869, DSM 29870, DSM 29871, and DSM 29872 indicated a higher level of compatibility with monensin than most of the prior art strains included herein: NN019785, NN062266 (NRRL B-50013), NN062267 (NRRL B-50104), NN062278 (PTA-6507), NN062319 (FERM BP-1096), NN062440, NN062441 (DSM 17236), NN062439.
Abstract
The present disclosure provides novel Bacillus strains that exhibit antimicrobial activity against pathogens, such as Clostridium perfringens and Escherichia coli, as well as animal feeds and animal feed additives comprising such Bacillus strains, and methods of using said strains, said animal feeds and said animal feed additives to improve the health and performance of production animals, such as poultry and swine.
Description
- This application is a divisional of U.S. application Ser. No. 15/543,663, filed on Jul. 14, 2017, and issued as U.S. patent Ser. No. 11/331,351 on May 17, 2022, which is a 35 U.S.C. 371 national application of international application no. PCT/US2016/014492, filed Jan. 22, 2016, which claims priority or the benefit under 35 U.S.C. 119 of U.S. provisional application Nos. 62/106,869, 62/109,210 and 62/260,892 filed on Jan. 23, 2015, Jan. 29, 2015, and Nov. 30, 2015, respectively, the contents of each of which is fully incorporated herein by reference.
- This application contains a reference to a deposit of biological material, which deposit is incorporated herein by reference.
- This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.
- Index to sequence listing:
-
- SEQ ID NO: 1 is 16S rDNA of DSM 29869.
- SEQ ID NO: 2 is 16S rDNA of DSM 29870.
- SEQ ID NO: 3 is 16S rDNA of DSM 29871.
- SEQ ID NO: 4 is 16S rDNA of DSM 29872.
- SEQ ID NO: 5 is 16S rDNA of Bacillus vallismortis from AB021198.
- SEQ ID NO: 6 is 16S rDNA of Bacillus subtilis from AJ276351.
- SEQ ID NO: 7 is 16S rDNA of Bacillus amyloliquefaciens from AB255669.
- SEQ ID NO: 8 to SEQ ID NO: 13: PCR and sequencing primers.
- The present invention relates to an animal feed or an animal feed additive comprising Bacillus strains which improve the health and performance of production animals. The invention further relates to use of the Bacillus strains in animal feed and animal feed additives.
- Clostridium perfringens (C. perfringens) is a Gram-positive, rod-shaped, anaerobic, spore-forming bacterium of the genus Clostridium. C. perfringens is widely present in nature and can be found as a component of decaying vegetation, marine sediment, the intestinal tract of humans and other vertebrates, insects, and soil.
- Infections due to C. perfringens show evidence of tissue necrosis, bacteremia, emphysematous cholecystitis, and gas gangrene, which is also known as clostridial myonecrosis. C. perfringens can also result in polymicrobial anaerobic infections.
- The incidence of Clostridium perfringens-associated necrotic enteritis in poultry has increased in countries that stopped using antibiotic growth promoters. Necrotic enteritis is an enterotoxemic disease that results in significant economic losses in the poultry industry.
- There is a need to develop tools and strategies for the prevention and control of C. perfringens in mono-gastric animals such as poultry. Whilst the vaccination of animals has been suggested, there are challenges associated with vaccinating thousands of animals. Thus discovering a solution which could be administered as an additive in an animal feed would be advantageous.
- It is thus an object of the invention to provide solutions which prevents and/or controls C. perfringens in mono-gastric animals such as poultry by use of an animal feed comprising one or more bacteria with activity against Clostridium perfringens.
- A further object of the invention is to provide a solution that does not present a risk for the health of the animal. The solution to this problem is use of non-hemolytic Bacillus strains with activity against Clostridium perfringens.
- A challenge of delivering Bacillus spp. in feed is the common use of antibiotics as growth promoters in feed. Therefore it is necessary to determine the compatibility of strains with commonly-used feed antibiotics such as monensin in order to identify any potential conflicts with use as a direct fed microbial. The present invention relates in one embodiment to an animal feed or feed additive comprising a Bacillus strain with high compatibility with monensin.
- Knap et al. (2010) describes that Bacillus licheniformis has an effect on necrotic enteritis in broiler chickens (Knap, Lund, Kehlet, Hofacre, and Mathis; Bacillus licheniformis Prevents Necrotic Enteritis in Broiler Chickens; Avian Diseases 54(2):931-935. 2010). This Bacillus licheniformis strain has no effect on Clostridium perfringens in vitro as demonstrated in Example 2.
- Clostat (alias Bacillus PB6) is described in WO2007/064741. Bacillus PB6 has antagonistic effect against C. perfringens. Bacillus PB6 is hemolytic as described in Example 1.
- It has been surprisingly found that the addition of direct fed microbes (DFM) from Bacillus species to animal feed can be used to prevent and/or control C. perfringens infections and/or necrotic enteritis in mono-gastric animal such as pigs and/or poultry. The Bacillus species can also improve the body weight gain and/or feed conversion rate (in both Clostridium perfringens challenged and unchallenged chickens).
- In a first aspect the invention relates to an animal feed or an animal feed additive comprising a Bacillus strain characterized in that:
-
- i) the Bacillus strain is non-hemolytic,
- ii) the Bacillus strain has antimicrobial activity against Clostridium perfringens and
- iii) the Bacillus strain improves body weight gain and/or feed conversion rate in chickens fed with the Bacillus strain.
- In a second aspect the invention relates to an animal feed or an animal feed additive comprising a Bacillus strain characterized in that:
-
- i) the Bacillus strain is non-hemolytic,
- ii) the Bacillus strain has antimicrobial activity against Clostridium perfringens and
- iii) the Bacillus strain is sensitive to at least seven such as eight of the antibiotics selected from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline.
- In a third aspect the invention relates to an animal feed or an animal feed additive comprising a Bacillus strain characterized in that:
-
- i) the Bacillus strain is non-hemolytic,
- ii) the Bacillus strain is sensitive to at least seven such as eight of the antibiotics selected from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline and
- iii) the Bacillus strain improves body weight gain and/or feed conversion rate in chickens fed with the Bacillus strain.
- In a fourth aspect the invention relates to an animal feed or an animal feed additive comprising a Bacillus strain characterized in that:
-
- i) the Bacillus strain is non-hemolytic,
- ii) the Bacillus strain has antimicrobial activity against Clostridium perfringens and
- iii) the Bacillus strain has enzymatic activity under aerobic and/or anaerobic conditions that hydrolyzes one or more of the substrates selected from the group consisting of Amylose, Arabinan, Arabinoxylan, Casein and Xylan.
- The present invention relates to animal feed or animal feed additive comprising one or more of the Bacillus strains according to the invention which improves health and performance of production animals.
- Methods of feeding an animal with the animal feed or animal feed additive according to the invention and methods of treating a Clostridium perfringens infection and/or for treating necrotic enteritis in an animal is also part of the invention. Use of the animal feed or animal feed additive according to the invention to improve the performance of an animal, such as improving the feed conversion rate, improving the body weight gain and/or improving the feed efficiency and/or improving the health is also part of this invention.
- The invention further relates to a method for preventing and/or controlling C. perfringens infections and/or necrotic enteritis in a mono-gastric animal such as swine or poultry by use of an animal feed, wherein the animal feed comprises one or more Bacillus strains selected from the group consisting of the strain having the deposit accession number DSM 29869, DSM 29870, DSM 29871, and DSM 29872, or any combination thereof, wherein the Bacillus strain has antimicrobial activity against Clostridium perfringens and/or E. coli.
- In an embodiment, the invention relates to an animal feed that is fed to a mono-gastric animal such as poultry or swine; comprising one or more Bacillus strains selected from the group consisting of the strain having the deposit accession number DSM 29869, DSM 29870, DSM 29871, and DSM 29872, or any combination thereof, wherein the Bacillus strain has antimicrobial activity against Clostridium perfringens and/or E. coli.
- In a preferred embodiment the invention relates to an animal feed or an animal feed additive comprising a Bacillus strain wherein:
-
- i. the Bacillus strain is selected from the group consisting of:
- the strain having the deposit accession number DSM 29869; a strain having all of the identifying characteristics of Bacillus DSM 29869 or a mutant thereof,
- the strain having the deposit accession number DSM 29870; a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof,
- the strain having the deposit accession number DSM 29871; a strain having all of the identifying characteristics of Bacillus DSM 29871 or a mutant thereof,
- the strain having the deposit accession number DSM 29872; and a strain having all of the identifying characteristics of Bacillus DSM 29872 or a mutant thereof, and
- ii. the Bacillus strain has antimicrobial activity against Clostridium perfringens (such as Clostridium perfringens strains 23 or 48 [Gholamiandekhordi et al., 2006. Molecular and phenotypical characterization of Clostridium perfringens isolates from poultry flocks with different disease status, Vet. Microbiol. 113:143-152] and/or E. coli (such as ATCC10536 or ATCC25922).
- In a further aspect, the invention relates to a method for preventing and/or controlling C. perfringens infections and/or necrotic enteritis in an animal such as pigs or poultry, comprising the steps of:
-
- (a) feeding an animal such as a pig or poultry; and
- (b) administering to an animal such as pigs or poultry one or more Bacillus strains or spores of said strains selected from the group consisting of
- the strain having the deposit accession number DSM 29869; a strain having all of the identifying characteristics of Bacillus DSM 29869 or a mutant thereof,
- the strain having the deposit accession number DSM 29870; a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof,
- the strain having the deposit accession number DSM 29871; a strain having all of the identifying characteristics of Bacillus DSM 29871 or a mutant thereof, and
- the strain having the deposit accession number DSM 29872; a strain having all of the identifying characteristics of Bacillus DSM 29872 or a mutant thereof, or any combination thereof, wherein the Bacillus strain has antimicrobial activity against Clostridium perfringens and/or E. coli, and wherein step (a) occurs before, after, or simultaneously with step (b).
- In an embodiment, the method relates to an animal feed that is fed to a mono-gastric animal such as pigs and/or poultry. The poultry can, e.g., be chickens (such as broilers and/or layers).
- In an embodiment, the animal feed or animal feed additive comprises one of more additional bacteria; is fed to a mono-gastric animal such as pigs and/or poultry; and further comprises concentrate and/or one or more enzymes and/or one or more additional microbes and/or one or more vitamins and/or one or more minerals and/or one or more amino acids and/or one or more other feed ingredients.
-
FIG. 1 Alignment of 16 S rDNA sequences. SEQ ID NO: 1 (DSM 29869), SEQ ID NO: 2 (DSM 29870), SEQ ID NO: 3 (DSM 29871), SEQ ID NO: 4 (DSM 29872), SEQ ID NO: 5 (Bacillus vallismortis AB021198), SEQ ID NO: 6 (Bacillus subtilis from AJ276351), and SEQ ID NO: 7 (Bacillus amyloliquefaciens AB255669) have been aligned. The region covering position 481-1200 is not shown inFIG. 1 (in this region all sequences are identical). - In general, the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references, and context known to those skilled in the art. The following definitions are provided to clarify their specific use in context of the disclosure.
- As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
- Animal feed: The term “animal feed” refers to any compound, preparation, or mixture suitable for, or intended for intake by an animal. Animal feed for a mono-gastric animal comprises concentrates as well as for example vitamins, minerals, enzymes, amino acids and/or other feed ingredients (such as in a premix). The animal feed may further comprise forage. An example of poultry feed is given in Example 7.
- Antimicrobial activity against Clostridium perfringens: The term “Antimicrobial activity against Clostridium perfringens” means that the growth of Clostridium perfringens is inhibited and/or that some or all of the Clostridium perfringens are killed. This can be determined by the assay described in Example 2.
- Blend: the term “blend” means more than one of the bacterial strains described herein.
- Body Weight Gain: The Body Weight Gain of an animal is the increase of body weight of the animal over a specified time period. An example of Body Weight Gain determination is given in Example 8.
- Composition: The term “composition” refers to a composition comprising a carrier and at least one bacterial strain as described herein. The compositions described herein may be mixed with an animal feed(s) and referred to as a “mash feed.”
- Concentrates: The term “concentrates” means feed with high protein and energy concentrations, such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (either whole or prepared by crushing, milling, etc. from, e.g., corn, oats, rye, barley, wheat), oilseed press cake (e.g., from cottonseed, safflower, sunflower, soybean (such as soybean meal), rapeseed/canola, peanut or groundnut), palm kernel cake, yeast derived material and distillers grains (such as wet distillers grains (WDS) and dried distillers grains with solubles (DDGS)).
- Control C. perfringens infections and/or necrotic enteritis: The term “control C. perfringens infections and/or necrotic enteritis” means a method and/or composition that partly or completely inhibits C. perfringens infections and/or necrotic enteritis in an animal. Accordingly, the term “control C. perfringens infections and/or necrotic enteritis” means the C. perfringens infections and/or the necrotic enteritis is reduced or completely eliminated.
- Direct Fed Microbial: The term “direct fed microbial” means live micro-organisms including spores which, when administered in adequate amounts, confer a benefit, such as improved digestion or health, on the host.
- Enzymatic activity under aerobic conditions: The term “enzymatic activity under aerobic conditions” means that the polypeptides produced by a Bacillus strain during growth under aerobic conditions have one of more enzyme activities. The substrates tested are amylose, arabinan, cellulose, casein, arabinoxylan and xylan and degradation of the substrate indicates amylase, arabinase, cellulase, protease or xylanase activity respectively. Such an assay is performed as described in Example 6.
- Enzymatic activity under anaerobic conditions: The term “enzymatic activity under anaerobic conditions” means activity of enzymes produced by a Bacillus strain during growth under anaerobic conditions as described in Example 6.
- European Production Efficacy Factor (EPEF): The European Production Efficacy Factor is a way of comparing the live-bird performance of flocks. This single-figure facilitates comparison of performance within and among farms and can be used to assess environmental, climatic and managemental variables. The EPEF is calculated as [(liveability (%)×Liveweight (kg))/(Age at depletion (days)×FCR)]×100, wherein livability is the percentage of birds alive at slaughter, Liveweight is the average weight of the birds at slaughter, age of depletion is the age of the birds at slaughter and FCR is the feed conversion ratio at slaughter.
- Effective amount/concentration/dosage: The terms “effective amount”, “effective concentration”, or “effective dosage” are defined as the amount, concentration, or dosage of the bacterial strain(s) sufficient to improve the digestion or yield of an animal. The actual effective dosage in absolute numbers depends on factors including: the state of health of the animal in question, other ingredients present. The “effective amount”, “effective concentration”, or “effective dosage” of the bacterial strains may be determined by routine assays known to those skilled in the art. An example of an effective amount for poultry is given in Example 7.
- FCR (Feed Conversion Rate): FCR is a measure of an animal's efficiency in converting feed mass into increases of the desired output. Animals raised for meat—such as swine, poultry and fish—the output is the mass gained by the animal. Specifically FCR is the mass of the food eaten divided by the output, all over a specified period. FCR can be determined as described in Example 8. Improvement in FCR means reduction of the FCR value. A FCR improvement of 2% means that the FCR was reduced by 2%.
- Feeding an animal: The terms “feeding an animal” or “fed to an animal” means that the composition of the present invention is administered orally to the animal one or more times in an effective amount. The oral administration is typically repeated, e.g., one or more times daily over a specified time period such as several days, one week, several weeks, one months or several months. Feeding of poultry can, e.g., be performed as described in Example 7. Accordingly, the terms “feeding” or “fed” mean any type of oral administration such as administration via an animal feed or via drinking water.
- Forage: The term “forage” as defined herein also includes roughage. Forage is fresh plant material such as hay and silage from forage plants, grass and other forage plants, seaweed, sprouted grains and legumes, or any combination thereof. Examples of forage plants are Alfalfa (lucerne), birdsfoot trefoil, brassica (e.g., kale, rapeseed (canola), rutabaga (swede), turnip), clover (e.g., alsike clover, red clover, subterranean clover, white clover), grass (e.g., Bermuda grass, brome, false oat grass, fescue, heath grass, meadow grasses, orchard grass, ryegrass, Timothy-grass), corn (maize), millet, barley, oats, rye, sorghum, soybeans and wheat and vegetables such as beets. Forage further includes crop residues from grain production (such as corn stover; straw from wheat, barley, oat, rye and other grains); residues from vegetables like beet tops; residues from oilseed production like stems and leaves form soy beans, rapeseed and other legumes; and fractions from the refining of grains for animal or human consumption or from fuel production or other industries.
- Isolated: The term “isolated” means that the one or more bacterial strains described herein are in a form or environment which does not occur in nature, that is, the one or more bacterial strains are at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature.
- Non-hemolytic: Hemolysis is the breakdown of red blood cells. The ability of bacterial colonies to induce hemolysis when grown on blood agar is used to classify bacterial strains into hemolytic and non-hemolytic strains. In this context hemolysis is defined as described in EFSA Journal 2011; 9(11):2445, using Bacillus subtilis 168 (BGSC-1A1, Bacillus Genetic Stock Center) as a negative control. A Bacillus strain can be classified as non-hemolytic using the assay described in Example 1.
- Pellet: The terms “pellet” and/or “pelleting” refer to solid rounded, spherical and/or cylindrical tablets or pellets and the processes for forming such solid shapes, particularly feed pellets and solid extruded animal feed. As used herein, the terms “extrusion” or “extruding” are terms well known in the art and refer to a process of forcing a composition, as described herein, through an orifice under pressure.
- Poultry: The term “poultry” means domesticated birds kept by humans for the eggs they produce and/or their meat and/or their feathers. Poultry includes broilers and layers. Poultry include members of the superorder Galloanserae (fowl), especially the order Galliformes (which includes chickens, Guineafowls, quails and turkeys) and the family Anatidae, in order Anseriformes, commonly known as “waterfowl” and including domestic ducks and domestic geese. Poultry also includes other birds that are killed for their meat, such as the young of pigeons. Examples of poultry include chickens (including layers, broilers and chicks), ducks, geese, pigeons, turkeys and quail.
- Prevent C. perfringens infections and/or necrotic enteritis: The term “prevent C. perfringens infections and/or necrotic enteritis” means a method and/or composition that prevents development of a C. perfringens infection and/or necrotic enteritis in an animal.
- Roughage: The term “roughage” means dry plant material with high levels of fiber, such as fiber, bran, husks from seeds and grains and crop residues (such as stover, copra, straw, chaff, sugar beet waste).
- Sensitive to antibiotics: The term “sensitive to antibiotics” means the phenotypic property of a bacterial strain, that growth of said bacterial strain is inhibited under conditions where the bacterial strain would otherwise grow. In this context sensitivity to antibiotics is tested after the CLSI guidelines (M07-A9 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; 2012). The S. aureus ATCC 29213 is used as reference strain, which means that it should be included in the test, and that the results are only valid if S. aureus ATCC 29213 show results in compliance with the breakpoints of the CLSI guideline (see example Table 5.5) (M100-S24 Performance Standards for Antimicrobial Susceptibility Testing; informational Supplement, 2014). A strain of Bacillus is considered sensitive if the growth is detected at or below the breakpoint concentration of the appropriate antibiotic specified in EFSA journal 2012; 10(6):2740.
- Silage: The term “silage” means fermented, high-moisture stored fodder which can be fed to ruminants (cud-chewing animals such as cattle and sheep) or used as a biofuel feedstock for anaerobic digesters. It is fermented and stored in a process called ensilage, ensiling or silaging, and is usually made from grass or cereal crops (e.g., maize, sorghum, oats, rye, timothy etc. forage grass plants),) or legume crops like clovers/trefoils, alfalfa, vetches, using the entire green plant (not just the grain). Silage can be made from many field crops, and special terms may be used depending on type (oatlage for oats, haylage for alfalfa). Silage is made either by placing cut green vegetation in a silo, by piling it in a large heap covered with plastic sheet, or by wrapping large bales in plastic film.
- Spore: The terms “spore” and “endospore” are interchangeable and have their normal meaning which is well known and understood by those of skill in the art. As used herein, the term spore refers to a microorganism in its dormant, protected state.
- Stable: The term “stable” is a term that is known in the art, and in a preferred aspect, stable is intended to mean the ability of the microorganism to remain in a spore form until it is administered to an animal to improve the health of the animal.
- Swine: The term “swine” or “pigs” means domesticated pigs kept by humans for food, such as their meat. Swine includes members of the genus Sus, such as Sus scrofa domesticus or Sus domesticus and include piglets, growing pigs, and sows.
- Vegetable protein: The term “vegetable protein” refers to any compound, preparation or mixture that includes at least one protein derived from or originating from a vegetable, including modified proteins and protein-derivatives.
- It has been surprisingly found that the addition of direct fed microbes (DFM) from Bacillus species to animal feed can be used to prevent and/or control C. perfringens infections and/or necrotic enteritis in mono-gastric animal such as pigs and/or poultry and at the same time improve the body weight gain and/or feed conversion rate (e.g., in both C. perfringens challenged and unchallenged chickens).
- The invention relates to an animal feed or an animal feed additive comprising spores of one or more Bacillus strains according to invention. The spores are preferable stable spores. More specifically the invention relates to the following aspects with respect to an animal feed or an animal feed additive comprising Bacillus strains:
- Aspect 1: An animal feed or an animal feed additive comprising spores of a Bacillus strain characterized in that:
-
- i) the Bacillus strain is non-hemolytic,
- ii) the Bacillus strain has antimicrobial activity against Clostridium perfringens and
- iii) the Bacillus strain improves body weight gain and/or feed conversion rate in chickens fed with the Bacillus strain.
- Aspect 2: An animal feed or an animal feed additive comprising spores of a Bacillus strain characterized in that:
-
- i) the Bacillus strain is non-hemolytic,
- ii) the Bacillus strain has antimicrobial activity against Clostridium perfringens and
- iii) the Bacillus strain is sensitive to at least seven such as eight of the antibiotics selected from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline.
- Aspect 3: An animal feed or an animal feed additive comprising spores of a Bacillus strain characterized in that:
-
- i) the Bacillus strain is non-hemolytic,
- ii) the Bacillus strain is sensitive to at least seven such as eight of the antibiotics selected from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline and
- iii) the Bacillus strain improves body weight gain and/or feed conversion rate in chickens fed with the Bacillus strain.
- Aspect 4: An animal feed or an animal feed additive comprising spores of a Bacillus strain characterized in that:
-
- i) the Bacillus strain is non-hemolytic,
- ii) the Bacillus strain has antimicrobial activity against Clostridium perfringens and
- iii) the Bacillus strain has enzymatic activity under aerobic and/or anaerobic conditions that hydrolyzes one or more of the substrates selected from the group consisting of Amylose, Arabinan, Arabinoxylan, Casein and Xylan.
- Sensitivity to Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline can, e.g., be determined as described in Example 5.
- Enzymatic activity under aerobic and/or anaerobic conditions that hydrolyzes one or more of the substrates selected from the group consisting of Amylose, Arabinan, Arabinoxylan, Casein and Xylan can, e.g., be determined as described in Example 6.
- In one embodiment the Bacillus strain of the animal feed or the animal feed additive according to
Aspect - In one embodiment the Bacillus strain of the animal feed or the animal feed additive according to
Aspect 1 or 3 improves one or more performance parameters in poultry selected from the list consisting of body weight gain, European Production Efficacy Factor and feed conversion rate in chickens fed with the Bacillus strain. - In one embodiment the Bacillus strain of the animal feed or the animal feed additive according to
Aspect 4 has antimicrobial activity against Clostridium perfringens. - In one embodiment the Bacillus strain of the animal feed or the animal feed additive according to Aspect 1 or 2 is sensitive to at least seven such as eight of the antibiotics selected from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline.
- In one embodiment the improvement of feed conversion rate (FCR) for the animal feed or the animal feed additive of
Aspect Aspect - In one embodiment the improvement in body weight gain for the animal feed or the animal feed additive of
Aspect Aspect - In one embodiment the Bacillus strain of the animal feed or the animal feed additive according to
Aspect - In one embodiment the Bacillus strain of the animal feed or the animal feed additive according to
Aspect - In one embodiment the Bacillus strain of the animal feed or the animal feed additive according to
Aspect - In one embodiment the Bacillus strain of the animal feed or the animal feed additive according to
Aspect - In one embodiment the Bacillus strain of the animal feed or the animal feed additive according to
Aspect Aspect Aspect Aspect - The invention relates in one embodiment to an animal feed or an animal feed additive comprising a Bacillus having deposit accession number DSM 29869 or a strain having all of the identifying characteristics of Bacillus DSM 29869 or a mutant thereof. The invention relates in another embodiment to an animal feed or an animal feed additive comprising a Bacillus having deposit accession number DSM 29870 or a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof. The invention relates in another embodiment to an animal feed or an animal feed additive comprising a Bacillus having deposit accession number DSM 29871 or a strain having all of the identifying characteristics of Bacillus DSM 29871 or a mutant thereof. The invention relates in a further embodiment to an animal feed or an animal feed additive comprising a Bacillus having deposit accession number DSM 29872 or a strain having all of the identifying characteristics of Bacillus DSM 29872 or a mutant thereof.
- In one embodiment of
Aspect Aspect - In one embodiment of
Aspect - In one embodiment the animal feed or the animal feed additive comprises one or more coccidiostats wherein the composition is, e.g., a premix.
- In a preferred embodiment the animal feed or the animal feed additive according to
Aspect - In one embodiment the animal feed or animal feed additive is characterized in that at least 70% (such as at least 80% or at least 90%) of the Bacillus spores survive gastric stability in a mono-gastric animal such as chickens.
- The animal feed or the animal feed additive according to
Aspect - The animal feed according to
Aspect - In a preferred embodiment, the bacterial count of each of the bacterial strains in the animal feed additive is between 1×107 and 1×1018 CFU/kg of composition, preferably between 1×109 and 1×1016 CFU/kg of composition, more preferably between 1×1019 and 1×1015 CFU/kg of composition and most preferably between 1×1011 and 1×1014 CFU/kg of dry matter.
- In a preferred embodiment the animal feed or the animal feed additive according to
Aspect - The animal feed or the animal feed additive according to
Aspect - In a preferred embodiment the animal feed or the animal feed additive according to
Aspect - In a preferred embodiment the animal feed or the animal feed additive according to
Aspect - In a further embodiment, the animal feed or animal feed additive comprises one or more bacterial strains such as at least two of the above strains up to and including all of the strains in the group consisting of DSM 29869, DSM 29870, DSM 29871 and DSM 29872.
- In an embodiment to any of the aforementioned embodiments the Bacillus spore kills/inhibits at least 40% (such as at least 45%, at least 50%, at least 60%, at least 70% or at least 80%) of Clostridium perfringens after, e.g., 24 hours, e.g., determined as described in Example 2.
- In another embodiment of the invention the animal feed or animal feed additive further comprises concentrate. In another embodiment of the invention the animal feed or animal feed additive further comprises forage. In another embodiment of the invention the animal feed or animal feed additive such as the animal feed further comprises one or more additional microbes. In another embodiment of the invention the animal feed or animal feed additive further comprises one or more enzymes. In another embodiment of the invention the animal feed or animal feed additive further comprises one or more vitamins. In another embodiment of the invention the animal feed or animal feed additive further comprises one or more minerals. In another embodiment of the invention the animal feed or animal feed additive such as the animal feed further comprises one or more amino acids. In another embodiment of the invention the animal feed or animal feed additive further comprises one or more other feed ingredients.
- In an embodiment to any of the aforementioned embodiments, the animal feed or the animal feed additive also improves the health of the mono-gastric animal when fed to said animal. In another embodiment to any of the aforementioned embodiments, the the animal feed or the animal feed additive also increases the egg yield of poultry when fed said poultry. In an embodiment to any of the aforementioned embodiments, the animal feed or the animal feed additive increases the meat yield of the mono-gastric animal when fed to said animal.
- In a preferred embodiment, the animal feed comprises one or more bacterial strains described herein, wherein the bacterial count of each of the bacterial strains is between 1×104 and 1×1014 CFU/kg of animal feed, preferably between 1×106 and 1×1012 CFU/kg of animal feed, and more preferably between 1×107 and 1×1011 CFU/kg of animal feed. In a more preferred embodiment the bacterial count of each of the bacterial strains described herein is between 1×108 and 1×1010 CFU/kg of animal feed.
- In a preferred embodiment, the animal feed additive comprises one or more bacterial strains described herein, wherein the bacterial count of each of the bacterial strains is between 1×107 and 1×1018 CFU/kg of animal feed additive, preferably between 1×109 and 1×1016 CFU/kg of animal feed additive, and more preferably between 1×1010 and 1×1015 CFU/kg of animal feed additive. In a more preferred embodiment the bacterial count of each of the bacterial strains described herein is between 1×1011 and 1×1014 CFU/kg of animal feed additive.
- In a preferred embodiment, the bacterial count of each of the bacterial strains in the animal feed is between 1×104 and 1×1014 CFU/kg of dry matter, preferably between 1×106 and 1×1012 CFU/kg of dry matter, and more preferably between 1×107 and 1×1011 CFU/kg of dry matter. In a more preferred embodiment the bacterial count of each of the bacterial strains in the animal feed is between 1×108 and 1×1010 CFU/kg of dry matter.
- In a more preferred embodiment the animal feed or the animal feed additive according to
Aspect - In still yet another embodiment of the invention, the one or more bacterial strains are present in the animal feed or the animal feed additive in form of a spore such as a stable spore. In still a further embodiment of the invention, the stable spore will germinate in the intestine and/or stomach of the mono-gastric animal.
- In one embodiment, the one or more bacterial strains are stable when subjected to pressures applied/achieved during an extrusion process for pelleting. In a particular embodiment, the one or more bacterial strains are stable at pressures ranging from 1 bar to bar, particularly 10 bar to 40 bar, more particularly 15 bar to 40 bar, even more particularly bar to 40 bar, still even more particularly 35 bar to 37 bar, even still more particularly 36 bar.
- In a particular embodiment, the one or more bacterial strains are stable at high temperatures. In particular, the bacterial strains are stable when they are subjected to temperatures achieved during an extrusion process for pelleting. In an even more particular embodiment, the one or more bacterial strains are stable at temperatures ranging from 80° C. to 120° C., particularly temperatures ranging from, 90° C. to 120° C., even more particularly temperatures ranging from 95° C. to 120° C.
- In another aspect, the invention relates to an animal feed or animal feed additive comprising a carrier, such as forage, and one or more of the bacteria cultures having characteristics substantially identical to that of a strain selected from the group consisting of:
-
- the strain having the deposit accession number DSM 29869;
- the strain having the deposit accession number DSM 29870;
- the strain having the deposit accession number DSM 29871; and
- the strain having the deposit accession number DSM 29872; or
- any combination thereof.
- In an embodiment, the animal feed or animal feed additive comprises a carrier and the strain having the deposit accession number DSM 29869, or a strain having all of the identifying characteristics of Bacillus DSM 29869 or a mutant thereof.
- In an embodiment, the animal feed or animal feed additive comprises a carrier and the strain having the deposit accession number DSM 29870, or a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof.
- In an embodiment, the animal feed or animal feed additive comprises a carrier and the strain having the deposit accession number DSM 29871, or a strain having all of the identifying characteristics of Bacillus DSM 29871 or a mutant thereof.
- In an embodiment, the animal feed or animal feed additive comprises a carrier and the strain having the deposit accession number DSM 29872, or a strain having all of the identifying characteristics of Bacillus DSM 29872 or a mutant thereof.
- In another embodiment, the animal feed or animal feed additive is for feeding to a mono-gastric animal. Mono-gastric animals include, but are not limited to, pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chicken (including but not limited to broiler chicks, layers); horses (including but not limited to hotbloods, coldbloods and warm bloods) and fish (including but not limited to salmon, trout, tilapia, catfish and carps; and crustaceans (including but not limited to shrimps and prawns). Pigs and/or poultry are preferred mono-gastric animals.
- In an embodiment, the animal feed or animal feed additive further comprises one or more additional microbes. In a particular embodiment, the animal feed further comprises a bacterium from one or more of the following genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium and Megasphaera or any combination thereof.
- In a particular embodiment, animal feed or animal feed additive further comprises a bacterium from one or more of the following strains of Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus pumilus, Bacillus polymyxa, Bacillus licheniformis, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, or any combination thereof.
- In a particular embodiment, the animal feed or animal feed additive further comprises one or more types of yeast. The one or more types of yeast can be selected from the group consisting of Saccharomycetaceae, Saccharomyces (such as S. cerevisiae and/or S. boulardii), Kluyveromyces (such as K. marxianus and K. lactis), Candida (such as C. utilis, also called Torula yeast), Pichia (such as P. pastoris), Torulaspora (such as T. delbrueckii), Phaffia yeasts and Basidiomycota.
- In an embodiment to any of the aforementioned embodiments the animal feed or the animal feed additive further comprises a carrier. The carrier can comprise one or more of the following compounds: water, glycerol, ethylene glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose, sucrose, sorbitol, lactose, wheat flour, wheat bran, corn gluten meal, starch and cellulose.
- In one aspect, the animal feed comprises the Bacillus strain according to
Aspect - Forage as defined herein also includes roughage. Forage is fresh plant material such as hay and silage from forage plants, grass and other forage plants, grass and other forage plants, seaweed, sprouted grains and legumes, or any combination thereof. Examples of forage plants are Alfalfa (lucerne), birdsfoot trefoil, brassica (e.g., kale, rapeseed (canola), rutabaga (swede), turnip), clover (e.g., alsike clover, red clover, subterranean clover, white clover), grass (e.g., Bermuda grass, brome, false oat grass, fescue, heath grass, meadow grasses, orchard grass, ryegrass, Timothy-grass), corn (maize), millet, barley, oats, rye, sorghum, soybeans and wheat and vegetables such as beets. Crops suitable for ensilage are the ordinary grasses, clovers, alfalfa, vetches, oats, rye and maize. Forage further includes crop residues from grain production (such as corn stover; straw from wheat, barley, oat, rye and other grains); residues from vegetables like beet tops; residues from oilseed production like stems and leaves form soy beans, rapeseed and other legumes; and fractions from the refining of grains for animal or human consumption or from fuel production or other industries.
- Roughage is generally dry plant material with high levels of fiber, such as fiber, bran, husks from seeds and grains and crop residues (such as stover, copra, straw, chaff, sugar beet waste).
- Examples of concentrates are feed with high protein and energy concentrations, such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (either whole or prepared by crushing, milling, etc. from, e.g., corn, oats, rye, barley, wheat), oilseed press cake (e.g., from cottonseed, safflower, sunflower, soybean (such as soybean meal), rapeseed/canola, peanut or groundnut), palm kernel cake, yeast derived material and distillers grains (such as wet distillers grains (WDS) and dried distillers grains with solubles (DDGS)).
- In one embodiment, the forage and one or more microbes are mixed with a concentrate. In another embodiment, the forage and one or more microbes are mixed with a premix. In a further embodiment, the forage and one or more microbes are mixed with vitamins and/or minerals. In a further embodiment, the forage and one or more microbes are mixed with one or more enzymes. In a further embodiment, the forage and one or more microbes are mixed with other feed ingredients, such as colouring agents, stabilisers, growth improving additives and aroma compounds/flavorings, polyunsaturated fatty acids (PUFAs); reactive oxygen generating species, anti-microbial peptides, anti-fungal polypeptides and amino acids.
- In particular embodiments, the animal feed may comprise Bacillus stain DSM 29869 and/or DSM 29870 and/or DSM 29871 and/or DSM 29872 or a strain having all of the identifying characteristics of Bacillus DSM 29869 and/or DSM 29870 and/or DSM 29871 and/or DSM 29872 or a mutant of DSM 29869 and/or DSM 29870 and/or DSM 29871 and/or DSM 29872 and 0-80% maize; and/or 0-80% sorghum; and/or 0-70% wheat; and/or 0-70% barley; and/or 0-30% oats; and/or 0-40% soybean meal; and/or 0-10% fish meal; and/or 0-20% whey.
- The animal feed may comprise Bacillus stain DSM 29869 and/or DSM 29870 and/or DSM 29871 and/or DSM 29872 or a strain having all of the identifying characteristics of Bacillus DSM 29869 and/or DSM 29870 and/or DSM 29871 and/or DSM 29872 or a mutant of DSM 29869 and/or DSM 29870 and/or DSM 29871 and/or DSM 29872 and vegetable proteins. In particular embodiments, the protein content of the vegetable proteins is at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% (w/w). Vegetable proteins may be derived from vegetable protein sources, such as legumes and cereals, for example, materials from plants of the families Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, and Poaceae, such as soy bean meal, lupin meal, rapeseed meal, and combinations thereof.
- In a particular embodiment, the vegetable protein source is material from one or more plants of the family Fabaceae, e.g., soybean, lupine, pea, or bean. In another particular embodiment, the vegetable protein source is material from one or more plants of the family Chenopodiaceae, e.g., beet, sugar beet, spinach or quinoa. Other examples of vegetable protein sources are rapeseed, and cabbage. In another particular embodiment, soybean is a preferred vegetable protein source. Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oat, maize (corn), rice, and sorghum.
- In a particular embodiment the animal feed consists of or comprises milk (e.g., from sow), e.g., for feeding of piglets. In another particular embodiment the animal feed consists of or comprises milk replacement, e.g., for feeding of piglets.
- In an embodiment, the animal feed may include a premix, comprising, e.g., vitamins, minerals, enzymes, preservatives, antibiotics, other feed ingredients or any combination thereof which are mixed into the animal feed.
- In one aspect, the invention related to a premix for feeding an animal infected by Clostridium perfringens, the premix comprising at least the Bacillus strain DSM 29869 described above. In a further embodiment, the premix may comprise one or more bacterial strains, in addition to DSM 29869.
- In one aspect, the invention related to a premix for feeding an animal infected by Clostridium perfringens, the premix comprising at least the Bacillus strain DSM 29870 described above. In a further embodiment, the premix may comprise one or more bacterial strains, in addition to DSM 29870.
- In one aspect, the invention related to a premix for feeding an animal infected by Clostridium perfringens, the premix comprising at least the Bacillus strain DSM 29871 described above. In a further embodiment, the premix may comprise one or more bacterial strains, in addition to DSM 29871.
- In one aspect, the invention related to a premix for feeding an animal infected by Clostridium perfringens, the premix comprising at least the Bacillus strain DSM 29872 described above. In a further embodiment, the premix may comprise one or more bacterial strains, in addition to DSM 29872.
- In another embodiment, the animal feed may include one or more vitamins, such as one or more fat-soluble vitamins and/or one or more water-soluble vitamins. In another embodiment, the animal feed may optionally include one or more minerals, such as one or more trace minerals and/or one or more macro minerals.
- Usually fat- and water-soluble vitamins, as well as trace minerals form part of a so-called premix intended for addition to the feed, whereas macro minerals are usually separately added to the feed.
- Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, e.g., vitamin K3.
- Non-limiting examples of water-soluble vitamins include vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g., Ca-D-panthothenate.
- Non-limiting examples of trace minerals include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium and zinc.
- Non-limiting examples of macro minerals include calcium, magnesium, potassium and sodium.
- In another embodiment, the animal feed or animal feed additive described herein optionally include one or more enzymes. Enzymes can be classified on the basis of the handbook Enzyme Nomenclature from NC-IUBMB, 1992), see also the ENZYME site at the internet: expasy.ch/enzyme/. ENZYME is a repository of information relative to the nomenclature of enzymes. It is primarily based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUB-MB), Academic Press, Inc., 1992, and it describes each type of characterized enzyme for which an EC (Enzyme Commission) number has been provided (Bairoch, 2000, The ENZYME database, Nucleic Acids Res. 28:304-305). This IUB-MB Enzyme nomenclature is based on their substrate specificity and occasionally on their molecular mechanism; such a classification does not reflect the structural features of these enzymes.
- Another classification of certain glycoside hydrolase enzymes, such as endoglucanase, xylanase, galactanase, mannanase, dextranase and alpha-galactosidase, in families based on amino acid sequence similarities has been proposed a few years ago. They currently fall into 90 different families: See the CAZy(ModO) internet site (Coutinho, P. M. & Henrissat, B. (1999) Carbohydrate-Active Enzymes server at URL: afmb.cnrs-mrs.fr/˜cazy/CAZY/index.html (corresponding papers: Coutinho, P. M. & Henrissat, B. (1999) Carbohydrate-active enzymes: an integrated database approach. In “Recent Advances in Carbohydrate Bioengineering”, H. J. Gilbert, G. Davies, B. Henrissat and B. Svensson eds., The Royal Society of Chemistry, Cambridge, pp. 3-12; Coutinho, P. M. & Henrissat, B. (1999) The modular structure of cellulases and other carbohydrate-active enzymes: an integrated database approach. In “Genetics, Biochemistry and Ecology of Cellulose Degradation”, K. Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, S. Karita and T. Kimura eds., Uni Publishers Co., Tokyo, pp. 15-23).
- Thus the animal feed or animal feed additive of the invention may also comprise at least one other enzyme selected from the group comprising of phytase (EC 3.1.3.8 or 3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC 3.2.1.22); protease (EC 3.4); phospholipase A1 (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1.1.5); phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4); amylase such as, for example, alpha-amylase (EC 3.2.1.1); lysozyme (EC 3.2.1.17); and beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6), or any mixture thereof.
- In a particular embodiment, the animal feed or animal feed additive of the invention comprises a phytase (EC 3.1.3.8 or 3.1.3.26). 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), the Phyzyme® XP (Verenium/DuPont) and Axtra® PHY (DuPont). Other preferred phytases include those described in, e.g., WO 98/28408, WO 00/43503, and WO 03/066847.
- In a particular embodiment, the animal feed or animal feed additive of the invention comprises a xylanase (EC 3.2.1.8). 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/beta-glucanase, DuPont).
- In a particular embodiment, the animal feed or animal feed additive of the invention comprises a protease (EC 3.4). Examples of commercially available proteases include Ronozyme® ProAct (DSM Nutritional Products).
- The animal feed or animal feed additive of the invention may further comprise one or more amino acids. Examples of amino acids which are used in animal feed are lysine, alanine, beta-alanine, threonine, methionine and tryptophan.
- The animal feed or animal feed additive of the invention may further comprise colouring agents, stabilisers, growth improving additives and aroma compounds/flavorings, polyunsaturated fatty acids (PUFAs); reactive oxygen generating species, anti-microbial peptides and anti-fungal polypeptides.
- Examples of colouring agents are carotenoids such as beta-carotene, astaxanthin, and lutein.
- Examples of aroma compounds/flavorings are creosol, anethol, deca-, undeca- and/or dodeca-lactones, ionones, irone, gingerol, piperidine, propylidene phatalide, butylidene phatalide, capsaicin and tannin.
- Examples of antimicrobial peptides (AMP's) are CAP18, Leucocin A, Tritrpticin, Protegrin-1, Thanatin, Defensin, Lactoferrin, Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000), Plectasins, and Statins, including the compounds and polypeptides disclosed in WO 03/044049 and WO 03/048148, as well as variants or fragments of the above that retain antimicrobial activity.
- Examples of antifungal polypeptides (AFP's) are the Aspergillus giganteus, and Aspergillus niger peptides, as well as variants and fragments thereof which retain antifungal activity, as disclosed in WO 94/01459 and WO 02/090384.
- Examples of polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid.
- Examples of reactive oxygen generating species are chemicals such as perborate, persulphate, or percarbonate; and enzymes such as an oxidase, an oxygenase or a syntethase.
- The animal feed or animal feed additive of the invention may further comprise at least one amino acid. Examples of amino acids which are used in animal feed are lysine, alanine, beta-alanine, threonine, methionine and tryptophan.
- Animal diets can, e.g., be manufactured as mash feed (non-pelleted) or pelleted feed. Typically, the milled feed-stuffs are mixed and sufficient amounts of essential vitamins and minerals are added according to the specifications for the species in question. The bacteria cultures and optionally enzymes can be added as solid or liquid formulations. For example, for mash feed a solid or liquid culture formulation may be added before or during the ingredient mixing step. For pelleted feed the (liquid or solid) culture preparation may also be added before or during the feed ingredient step. Typically a liquid culture preparation comprises the culture of the invention optionally with a polyol, such as glycerol, ethylene glycol or propylene glycol, and is added after the pelleting step, such as by spraying the liquid formulation onto the pellets. The enzyme may also be incorporated in a feed additive or premix.
- The enzyme may be added to the feed mix as a granule, which is optionally pelleted or extruded. The granule typically comprises a core particle and one or more coatings, which typically are salt and/or wax coatings. The core particle can either be a homogeneous blend of an active compound optionally together with salts (e.g., organic or inorganic zinc or calcium salt) or an inert particle with an active compound applied onto it. The active compound is the culture of the invention optionally combined with one or more enzymes. The inert particle may be water soluble or water insoluble, e.g., starch, a sugar (such as sucrose or lactose), or a salt (such as NaCl, Na2SO4). The salt coating is typically at least 1 μm thick and can either be one particular salt or a mixture of salts, such as Na2SO4, K2SO4, MgSO4 and/or sodium citrate. Other examples are those described in, e.g., WO 2008/017659, WO 2006/034710, WO 97/05245, WO 98/54980, WO 98/55599, WO 2000/70034 or polymer coating such as described in WO 2001/00042.
- Methods of Treatment of C. perfringens Infections and/or Necrotic Enteritis
- In one embodiment, the invention relates to a method for treatment of C. perfringens infections and/or necrotic enteritis in an animal such as a mono-gastric animal including poultry using the animal feed or animal feed additive according to
Aspect 1, 2, 3 or 4 (in an effective amount). In a specific embodiment the invention relates to a method for treatment of C. perfringens infections and/or necrotic enteritis in an animal such as an mono-gastric animal including poultry using an animal feed or animal feed additive comprising the Bacillus strain having deposit accession number DSM 29869 and/or DSM 29870 and/or DSM 29871 and/or DSM 29872, or a strain having all of the identifying characteristics of Bacillus DSM 29869 and/or DSM 29870 and/or DSM 29871 and/or DSM 29872 or a mutant of DSM 29869 and/or DSM 29870 and/or DSM 29871 and/or DSM 29872 (in an effective amount). - In one embodiment the animal is not a human being. In a further embodiment, the animal feed is fed to a mono-gastric animal. Mono-gastric animals include, but are not limited to, pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chicken (including but not limited to broiler chicks, layers); horses (including but not limited to hotbloods, coldbloods and warm bloods) and fish (including but not limited to salmon, trout, tilapia, catfish and carps; and crustaceans (including but not limited to shrimps and prawns). Pigs and/or poultry are preferred mono-gastric animals.
- The animal feed can further comprise one or more components selected from the list consisting of concentrate; forage; one or more enzymes; one or more additional microbes; one or more vitamins; one or more minerals; one or more amino acids; and one or more other feed ingredients.
- In a further embodiment, the method comprises administering to the animal feed one or more bacterial strains such as at least two of the above strains, at least three of the above strains, or up to and including all of the above strains.
- In one embodiment, the method comprises administering to the animal feed the Bacillus strains having the deposit accession number DSM 29869 or a strain having all the identifying characteristics of Bacillus DSM 29869 or a mutant thereof.
- In one embodiment, the method comprises administering to the animal feed the Bacillus strains having the deposit accession number DSM 29870 or a strain having all the identifying characteristics of Bacillus DSM 29870 or a mutant thereof.
- In one embodiment, the method comprises administering to the animal feed the Bacillus strains having the deposit accession number DSM 29871 or a strain having all the identifying characteristics of Bacillus DSM 29871 or a mutant thereof.
- In one embodiment, the method comprises administering to the animal feed the Bacillus strains having the deposit accession number DSM 29872 or a strain having all the identifying characteristics of Bacillus DSM 29872 or a mutant thereof.
- In another embodiment of the method, the animal feed further comprises concentrate. In another embodiment of the method, the animal feed further comprises forage. In another embodiment of the method, the animal feed further comprises one or more additional microbes. In another embodiment of the method, the animal feed further comprises one or more enzymes. In another embodiment of the method, the animal feed further comprises one or more vitamins. In another embodiment of the method, the animal feed further comprises one or more minerals. In another embodiment of the method, the animal feed further comprises one or more amino acids. In another embodiment of the method, the animal feed further comprises one or more other feed ingredients.
- In an embodiment to any of the aforementioned embodiments, the method also improves the health of the mono-gastric animal feed. In another embodiment to any of the aforementioned embodiments, the method also increases the egg yield of poultry. In an embodiment to any of the aforementioned embodiments, the method also increases the meat yield of the mono-gastric animal.
- In a preferred embodiment, the method comprises administering to a mono-gastric animal one or more bacterial strains described herein, wherein the bacterial count of each of the bacterial strains is between 1×104 and 1×1014 CFU/kg of forage, preferably between 1×106 and 1×1012 CFU/kg of forage, and more preferably between 1×107 and 1×1011 CFU/kg of animal feed. In a more preferred embodiment the bacterial count of each of the bacterial strains described herein is between 1×108 and 1×1016 CFU/kg of animal feed.
- In a preferred embodiment, the method comprises administering to a mono-gastric animal one or more bacterial strains described herein, wherein the bacterial count of each of the bacterial strains is between 1×105 and 1×1015 CFU/animal/day, preferably between 1×107 and 1×1013 CFU/animal/day, and more preferably between 1×108 and 1×1012 CFU/animal/day. In a more preferred embodiment the bacterial count of each of the bacterial strains described herein is between 1×109 and 1×1011 CFU/animal/day.
- In another aspect, the invention covers the method for treatment of C. perfringens infections comprising:
-
- (a) feeding a mono-gastric animal a feed; and
- (b) administering to an animal one or more Bacillus strains selected from the group consisting of:
- (i) the strain having the deposit accession number DSM 29869; or a strain having all the identifying characteristics of Bacillus DSM 29869 or a mutant thereof;
- (ii) the strain having the deposit accession number DSM 29870; or a strain having all the identifying characteristics of Bacillus DSM 29870 or a mutant thereof;
- (iii) the strain having the deposit accession number DSM 29871; or a strain having all the identifying characteristics of Bacillus DSM 29871 or a mutant thereof; and/or
- (iv) the strain having the deposit accession number DSM 29872; or a strain having all the identifying characteristics of Bacillus DSM 29872 or a mutant thereof or any combination thereof,
- wherein the Bacillus strain antimicrobial activity against Clostridium perfringens and/or E. coli, and wherein step (a) occurs before, after, or simultaneously with step (b).
- In a further embodiment, the method comprises administering to the animal feed one or more bacterial strains such as at least two of the above strains, at least three of the above strains, or up to and including all of the above strains.
- In a preferred embodiment of the method, the animal feed is fed to a mono-gastric animal. In an embodiment of the method, the mono-gastric animal is, e.g., pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chicken (including but not limited to broiler chicks, layers); horses (including but not limited to hotbloods, coldbloods and warm bloods), or fish (including but not limited to salmon, trout, tilapia, catfish and carps; and crustaceans (including but not limited to shrimps and prawns).
- In another embodiment of the method, the animal feed further comprises one or more components selected from the list consisting of concentrate; forage; one or more enzymes; one or more additional microbes; one or more vitamins; one or more minerals; one or more amino acids; and one or more other feed ingredients.
- In another embodiment of the method, the animal feed further comprises concentrate. In another embodiment of the method, the animal feed further comprises forage. In another embodiment of the method, the animal feed further comprises one or more additional microbes. In another embodiment of the method, the animal feed further comprises one or more enzymes. In another embodiment of the method, the animal feed further comprises one or more vitamins. In another embodiment of the method, the animal feed further comprises one or more minerals. In another embodiment of the method, the animal feed further comprises one or more amino acids. In another embodiment of the method, the animal feed further comprises one or more other feed ingredients.
- In still yet another embodiment of the method, the one or more bacterial strains are present in the form of a stable spore. In still a further embodiment of the method, the stable spore will germinate in the intestine and/or stomach of the mono-gastric animal.
- In a preferred embodiment, the method comprises administering to a mono-gastric animal one or more bacterial strains described herein, wherein the bacterial count of each of the bacterial strains is between 1×104 and 1×1014 CFU/kg of forage, preferably between 1×106 and 1×1012 CFU/kg of forage, and more preferably between 1×107 and 1×1011 CFU/kg of forage. In a more preferred embodiment the bacterial count of each of the bacterial strains described herein is between 1×108 and 1×1010 CFU/kg of forage.
- In a preferred embodiment, the method comprises administering to a mono-gastric animal one or more bacterial strains described herein, wherein the bacterial count of each of the bacterial strains is between 1×105 and 1×1015 CFU/animal/day, preferably between 1×107 and 1×1013 CFU/animal/day, and more preferably between 1×108 and 1×1012 CFU/animal/day. In a more preferred embodiment the bacterial count of each of the bacterial strains described herein is between 1×109 and 1×1011 CFU/animal/day.
- In a preferred embodiment, the method comprises administering to a mono-gastric animal one or more bacterial strains described herein, wherein the bacterial count of each Bacillus spore is between 1×105 and 1×1015 CFU/animal/day, preferably between 1×107 and 1×1013 CFU/animal/day, and more preferably between 1×108 and 1×1012 CFU/animal/day. The invention relates in a further embodiment to use of the animal feed or animal feed additive to improve the performance of an animal, such as improving the feed conversion rate and/or improving the European Production Efficacy Factor and/or improving the body weight gain and/or improving the feed efficiency and/or improving the health.
- In one embodiment the improvement of feed conversion rate (FCR) for
Aspect Aspect - In one embodiment the improvement in body weight gain for
Aspect Aspect - The invention relates to the use of a feed or feed premix comprising at least the spores of a Bacillus subspecies to improve the performance of an animal infected by C. perfringens. More specifically the feed or feed premix described within the present comprise a Bacillus DSM 29870 (hereafter DSM 29870). This strain is characterized in that is non-hemolytic, has antimicrobial activity, e.g., against Clostridium perfringens and/or E. coli, is sensitive to Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline, improves body weight gain and/or feed conversion ratio in animal, hydrolyzes one or more of the substrates selected from the group consisting of amylase, arabinan, arabinoxylan, casein and xylan.
- In an embodiment the present invention relates to the use of a feed or feed premix comprising at least the DSM 29870 as described above to improve the performance of an animal infected by C. perfringens. Within the meaning of the invention, “animal performance” may be determined by the body weight gain of the animal and/or by the feed conversion ratio and/or by the feed efficiency and/or by the digestibility of a nutrient in a feed (e.g., amino acid digestibility) and/or digestible energy or metabolizable energy in a feed and/or by nitrogen retention.
- In a preferred embodiment of the invention “animal performance” is determined by the body weight gain of the animal and/or by the feed conversion ratio. By “improved animal performance” it is meant that there is increased body weight gain and/or reduced feed conversion ratio and/or improved digestibility of nutrients or energy in a feed and/or by improved nitrogen retention and/or increased feed efficiency resulting from the use of feed or feed premix of the present invention in feed in comparison to feed which does not comprise said feed or feed premix. Preferably, by “improved animal performance” it is meant that there is increased body weight gain and/or reduced feed conversion ratio.
- In a preferred aspect of the invention, the animal feed or animal feed premix comprises at least the DSM 29870 bacterial strain described herein is used, wherein the bacterial count of the bacterial strains is between 1×104 and 1×1014 CFU/kg of animal feed or feed premix, preferably between 1×106 and 1×1012 CFU/kg of feed or feed premix, and more preferably between 1×107 and 1×1011 CFU/kg of feed or feed premix. In a more preferred embodiment the bacterial count of the bacterial strains described herein is between 1×108 and 1×1010 CFU/kg of feed or feed premix.
- As used herein, the term “feed conversion ratio” refers to the amount of feed fed to an animal to increase the weight of the animal by a specified amount. Specifically FCR is the mass of the food eaten divided by the output, all over a specified period. FCR can be determined as described in the Examples. An improved feed conversion ratio means a lower feed conversion ratio. By “lower feed conversion ratio” or “improved feed conversion ratio” it is meant that the use of a feed additive composition in feed results in a lower amount of feed being required to be fed to an animal to increase the weight of the animal by a specified amount compared to the amount of feed required to increase the weight of the animal by the same amount when the feed does not comprise said feed additive composition. In one embodiment the improvement of feed conversion rate (FCR) for Aspect 5, 6, 7 or 8 results in a FCR of −2.5% or less than −2.5%, such as less than −2.6%, such as less than −2.7%, such as less than −2.8%, such less than −2.9%, such as less than −3.0%. In a preferred embodiment the improvement of FCR results in a FCR of from −5% to −2% such as a FCR from −4% to −2%, such as a FCR of from −3.5% to −2.5%. In a specific embodiment the improvement of FCR for
Aspect - An “increased body weight gain” refers to an animal having increased body weight on being fed feed comprising a feed composition compared with an animal being fed a feed without said feed composition of the invention. Specifically, the Body Weight Gain of an animal is the increase of body weight of the animal over a specified time period. In one embodiment, the improvement in body weight gain if of at least 0.5% such at least 1%, such at least 2%, such at least 2,3%, such at least 3%, such at least 4%, such at least 5%, such at least 6%, such at least 6.5%, such at least 7%, such at least 8%, such at least 8%, such at least 10%. In one embodiment the improvement in body weight gain for Aspect 5, 6, 7 or 8 results in a body weight gain of at least 0.5%, such as at least 0.8%, such as at least 1.5%, such as at least 1.8%, such as at least 2.0%, such as at least 2.3%, such as at least %, such as at least 4.2%, such as at least 5.2%, such as at least 6.5%, such as at least 7.3%. In a preferred embodiment the improvement in body weight gain for
Aspect - As used herein, the term “feed efficiency” refers to the amount of weight gain in an animal that occurs when the animal is fed ad-libitum or a specified amount of food during a period of time. By “increased feed efficiency” it is meant that the use of the feed or the feed premix according the present invention in feed results in an increased weight gain per unit of feed intake compared with an animal fed without said feed or feed premix being present.
- Nutrient digestibility as used herein means the fraction of a nutrient that disappears from the gastro-intestinal tract or a specified segment of the gastro-intestinal tract, e.g., the small intestine. Nutrient digestibility may be measured as the difference between what is administered to the subject and what comes out in the faeces of the subject, or between what is administered to the subject and what remains in the digesta on a specified segment of the gastro intestinal tract, e.g., the ileum. Nutrient digestibility as used herein may be measured by the difference between the intake of a nutrient and the excreted nutrient by means of the total collection of excreta during a period of time; or with the use of an inert marker that is not absorbed by the animal, and allows the researcher calculating the amount of nutrient that disappeared in the entire gastro-intestinal tract or a segment of the gastro-intestinal tract. Such an inert marker may be titanium dioxide, chromic oxide or acid insoluble ash. Digestibility may be expressed as a percentage of the nutrient in the feed, or as mass units of digestible nutrient per mass units of nutrient in the feed. Nutrient digestibility as used herein encompasses starch digestibility, fat digestibility, protein digestibility, mineral digestibility and amino acid digestibility.
- Energy digestibility as used herein means the gross energy of the feed consumed minus the gross energy of the faeces or the gross energy of the feed consumed minus the gross energy of the remaining digesta on a specified segment of the gastro-intestinal tract of the animal, e.g., the ileum. Metabolizable energy as used herein refers to apparent metabolizable energy and means the gross energy of the feed consumed minus the gross energy contained in the faeces, urine, and gaseous products of digestion. Energy digestibility and metabolizable energy may be measured as the difference between the intake of gross energy and the gross energy excreted in the faeces or the digesta present in specified segment of the gastro-intestinal tract using the same methods to measure the digestibility of nutrients, with appropriate corrections for nitrogen excretion to calculate metabolizable energy of feed.
- Nitrogen retention as used herein means a subject's ability to retain nitrogen from the diet as body mass. A negative nitrogen balance occurs when the excretion of nitrogen exceeds the daily intake and is often seen when the muscle is being lost. A positive nitrogen balance is often associated with muscle growth, particularly in growing animals. Nitrogen retention may be measured as the difference between the intake of nitrogen and the excreted nitrogen by means of the total collection of excreta and urine during a period of time. It is understood that excreted nitrogen includes undigested protein from the feed, endogenous proteinaceous secretions, microbial protein, and urinary nitrogen.
- In another aspect the present invention relates to a method for improving the performance of an animal infected by C. perfringens comprising the step of administering to said animal a feed or feed premix comprising at least the Bacillus strain DSM 29870.
- In a preferred aspect of the invention, the feed or feed premix comprises at least the DSMZ 29870 bacterial strain, wherein the bacterial count of each of the bacterial strains is between 1×104 and 1×1014 CFU/kg of feed, preferably between 1×106 and 1×1012 CFU/kg of feed, and more preferably between 1×107 and 1×1011 CFU/kg of feed. In a more preferred embodiment the bacterial count of the bacterial strains described herein is between 1×108 and 1×1010 CFU/kg of feed. Within the meaning of the invention, the terms “improving animal performance” are defined as above in the paragraph “use of the composition”. In a specific embodiment, the invention deals with a method for improving the body weight gain and/or the feed conversion ratio of an animal infected by C. perfringens comprising the step of administering to said animal a feed composition comprising at least the Bacillus strain DSM 29870. As indicated above the animal is selected from the group consisting of swine and poultry. More specifically, the animal is selected from the group comprising chickens, broilers, layers, quails and turkey, domestic ducks or domestic geese or the young of pigeons, pigs, swine, piglets, growing pigs, or sows
- In another aspect the present invention relates to a method for feeding an animal infected by C. perfringens comprising administering a feed or feed premix comprising at least the Bacillus strain DSM29870 to said animal optionally in conjunction with other animal feed ingredients.
- Within the meaning of the invention, the terms “improving animal performance” are defined as above. As indicated above the animal is selected from the group consisting of swine and poultry. More specifically, the animal is selected from the group comprising chickens, broilers, layers, quails and turkey, domestic ducks or domestic geese or the young of pigeons, pigs, swine, piglets, growing pigs, or sows. In a preferred aspect of the invention, the composition such as a feed comprises at least the DSMZ 29870 bacterial strain, wherein the bacterial count of each of the bacterial strains is between 1×104 and 1×1014 CFU/kg of feed, preferably between 1×106 and 1×1012 CFU/kg of feed, and more preferably between 1×107 and 1×1011 CFU/kg of feed. In a more preferred embodiment the bacterial count of the bacterial strains described herein is between 1×108 and 1×1010 CFU/kg of feed. The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.
- Preferred embodiments of the invention are described in the set of items herein below (cf. ITEM SET I, ITEM SET II and ITEM SET III).
-
- Item Set I:
- 1. An animal feed or an animal feed additive comprising a Bacillus strain characterized in that:
- i) the Bacillus strain is non-hemolytic,
- ii) the Bacillus strain has antimicrobial activity against Clostridium perfringens and
- iii) the Bacillus strain improves body weight gain and/or feed conversion rate in chickens fed with the Bacillus strain.
- 2. An animal feed or an animal feed additive comprising a Bacillus strain characterized in that:
- i) the Bacillus strain is non-hemolytic,
- ii) the Bacillus strain has antimicrobial activity against Clostridium perfringens and
- iii) the Bacillus strain is sensitive to at least seven such as eight of the antibiotics selected from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline.
- 3. An animal feed or an animal feed additive comprising a Bacillus strain characterized in that:
- i) the Bacillus strain is non-hemolytic,
- ii) the Bacillus strain is sensitive to at least seven such as eight of the antibiotics selected from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline and
- iii) the Bacillus strain improves body weight gain and/or feed conversion rate in chickens fed with the Bacillus strain.
- 4. An animal feed or an animal feed additive comprising a Bacillus strain characterized in that:
- i) the Bacillus strain is non-hemolytic,
- ii) the Bacillus strain has antimicrobial activity against Clostridium perfringens and
- iii) the Bacillus strain has enzymatic activity under aerobic and/or anaerobic conditions that hydrolyzes one or more of the substrates selected from the group consisting of Amylose, Arabinan, Arabinoxylan, Casein and Xylan.
- 5. The animal feed or the animal feed additive according to any of items 1 to 3, wherein the Bacillus strain has enzymatic activity under aerobic and/or anaerobic conditions that hydrolyzes one or more of the substrates selected from the group consisting of Amylose, Arabinan, Arabinoxylan, Casein and Xylan, e.g., as determined in Example 6.
- 6. The animal feed or the animal feed additive according to any of
items 1 and 4, wherein the Bacillus strain improves one or more performance parameters in poultry selected from the list consisting of body weight gain, European Production Efficacy Factor and feed conversion rate in poultry fed with the Bacillus strain. - 7. The animal feed or the animal feed additive according to
item 3, wherein the Bacillus strain has antimicrobial activity against Clostridium perfringens, e.g., as determined in Example 2. - 8. The animal feed or the animal feed additive according to any of
items 1 and 4, wherein the Bacillus strain is sensitive to at least seven such as eight of the antibiotics selected from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline, e.g., as determined in Example 5. - 9. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus strain comprises 16S rDNA that has more than 98% sequence identity to SEQ ID NO: 1 and/or more than 98% sequence identity to SEQ ID NO: 2 and/or more than 98% sequence identity to SEQ ID NO: 3 and/or more than 98% sequence identity to SEQ ID NO: 4.
- 10. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus strain is a Bacillus subtilis strain or a Bacillus amyloliquefaciens strain.
- 11. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus strain has antimicrobial effect against E. coli, e.g., as determined in Example 4.
- 12. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus strain is the Bacillus strain having deposit accession number DSM 29869, or a strain having all of the identifying characteristics of Bacillus DSM 29869 or a mutant thereof that has antimicrobial activity against Clostridium perfringens.
- 13. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus strain is the Bacillus strain having deposit accession number DSM 29870, or a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof that has antimicrobial activity against Clostridium perfringens.
- 14. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus strain is the Bacillus strain having deposit accession number DSM 29871, or a strain having all of the identifying characteristics of Bacillus DSM 29871 or a mutant thereof that has antimicrobial activity against Clostridium perfringens.
- 15. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus strain is the Bacillus strain having deposit accession number DSM 29872, or a strain having all of the identifying characteristics of Bacillus DSM 29872 or a mutant thereof that has antimicrobial activity against Clostridium perfringens.
- 16. The animal feed or the animal feed additive according to any of items 1 to 15, wherein the Bacillus spores of the animal feed or the animal feed additive are present as dried spores.
- 17. The animal feed or the animal feed additive according to any of items 1 to 16 which further comprises a carrier.
- 18. The animal feed or the animal feed additive according to item 17, wherein the carrier comprises one or more of the following compounds: water, glycerol, ethylene glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose, sucrose, sorbitol, lactose, wheat flour, wheat bran, corn gluten meal, starch and cellulose.
- 19. The animal feed or the animal feed additive according to any of items 1 to 18, wherein the animal feed or animal feed additive comprises from 105 to 1012 CFU/g of isolated Bacillus spores.
- 20. The animal feed or animal feed additive of any of items 1 to 19, wherein at least 70% (such as at least 80% or at least 90%) of the Bacillus spores survive gastric stability in a mono-gastric animal such as chickens.
- 21. The animal feed or animal feed additive according to any of items 1 to 20 which further comprises one or more components selected from the list consisting of:
- one or more enzymes;
- one or more additional microbes;
- one or more vitamins;
- one or more minerals;
- one or more amino acids; and
- one or more other feed ingredients.
- 22. The animal feed or animal feed additive according to any of items 1 to 21, wherein the bacterial count of each Bacillus spore is 1×104 and 1×1014 CFU/kg of animal feed or animal feed additive, preferably between 1×106 and 1×1012 CFU/kg of animal feed or animal feed additive, and more preferably between 1×107 and 1×1011 CFU/kg of animal feed or animal feed additive.
- 23. The animal feed or animal feed additive according to any of items 1 to 22, wherein the animal feed or animal feed additive is a mono-gastric animal feed or animal feed additive.
- 24. The animal feed or animal feed additive according to any of items 1 to 23, wherein the Bacillus strain improves gut health of chickens with infection of Clostridium perfringens by having antimicrobial activity against Clostridium perfringens.
- 25. The mono-gastric animal feed or animal feed additive according to item 24, wherein the mono-gastric animal is selected from the group consisting of pigs, swine, piglets, sows, poultry, turkeys, ducks, chicken, broilers, layers, chicks, fish and crustaceans.
- 26. The animal feed or animal feed additive according to any of items 1 to 25, wherein the Bacillus strain has a high compatibility with monensin such as being compatible with at least 2.0 μg/ml monensin, e.g., as determined in Example 12 (such as at least 2.1 μg/ml monensin, such as at least 2.2 μg/ml monensin, such as at least 2.3 μg/ml monensin, such as at least 2.4 μg/ml monensin, such as at least 2.5 μg/ml monensin, such as at least 2.6 μg/ml monensin or such as at least 2.7 μg/ml monensin as determined in Example 12).
- 27. An animal feed or animal feed additive according to any of items 1 to 26 for the treatment of necrotic enteritis or the treatment of a Clostridium perfringens infection.
- 28. The animal feed or animal feed additive according to item 27 for treatment of mono-gastric animals.
- 29. A method of treating a Clostridium perfringens infection or for treating necrotic enteritis in an animal comprising administrating the animal feed or the animal feed additive according to any of items 1 to 28 to the animal.
- 30. Use of the animal feed or the animal feed additive according to any of items 1 to 28 to treat a Clostridium perfringens infection or for treating necrotic enteritis in an animal.
- 31. Use of the animal feed or the animal feed additive according to any of items 1 to 28 to improve one or more performance parameters of an animal selected from the group consisting of: improving the feed conversion ratio, improving the body weight gain, improving the European Production Efficacy Factor, improving the feed efficiency and improving the health.
- 32. A method of improving one or more performance parameters of an animal selected from the group consisting of: improving the feed conversion ratio, improving the body weight gain, improving the European Production Efficacy Factor, improving the feed efficiency and improving the health, comprising administering the animal feed or animal feed additive according to any of items 1 to 28 to the animal.
- Item Set II:
- 1. Use of an animal feed or an animal feed premix comprising at least the Bacillus strain DSM 29870 for improving the performance of an animal.
- 2. The use of the animal feed or the animal feed premix comprising at least the Bacillus strain DSM 29870 according to item 1 for improving the feed conversion ratio and/or for improving the body weight gain and/or for improving the feed efficiency, and/or the weight, and/or the feed intake.
- 3. The use of the animal feed or the animal feed premix comprising at least the Bacillus strain DSM 29870 according to item 1 or 2 wherein the animal is selected from the group consisting of poultry and swine.
- 4. The use of the animal feed or the animal feed premix comprising at least the Bacillus strain DSM 29870 according to
item 3 wherein the animal is selected from chickens, broilers, layers, quails and turkey, domestic ducks or domestic geese or the young of pigeons, pigs, swine, piglets, growing pigs, or sows. - 5. Use of the animal feed or the animal feed additive according to anyone of items 1 to 4 wherein the composition is a feed composition such an animal feed or a premix.
- 6. Use of the animal feed according to any of item 1 to 4 wherein the feed comprises at least the DSM 29870 Bacillus strain and wherein the bacterial count of each bacterial strain is between 1×104 and 1×1014 CFU/kg of animal feed, preferably between 1×106 and 1×1012 CFU/kg of animal feed, and more preferably between 1×107 and 1×1011 CFU/kg of animal feed and even more preferably between 1×108 and 1×1010 CFU/kg of animal feed.
- 7. A method for improving the performance of an animal comprising administering to said animal an animal feed or an animal feed premix comprising at least the Bacillus strain DSM29870.
- 8. The method for improving the performance of an animal according to item 7 such as improving the feed conversion ratio and/or improving the body weight gain and/or improving the feed efficiency, and/or the weight, and/or improving feed intake.
- 9. The method for improving the performance of an animal according to item 7 or 8 wherein the animal is selected from the group consisting of poultry and swine.
- 10. The method for improving the performance of an animal according to item 9 wherein the animal is selected from the group comprising chickens, broilers, layers, quails and turkey, domestic ducks or domestic geese or the young of pigeons, pigs, swine, piglets, growing pigs, or sows.
- 11. A method for improving the performance of an animal according to anyone of the items 7 to 10 wherein the bacterial count of each bacterial strain is between 1×104 and 1×1014 CFU/kg of animal feed, preferably between 1×106 and 1×1012 CFU/kg of animal feed, and more preferably between 1×107 and 1×1011 CFU/kg of animal feed and even more preferably between 1×108 and 1×1010 CFU/kg of animal feed.
- 12. A method for feeding an animal comprising administering an animal feed or an animal feed premix comprising at least the Bacillus strain DSM 29870 to said animal optionally in conjunction with other animal feed ingredients.
- 13. The method for feeding an animal according to item 12 wherein the animal is selected from the group consisting of poultry and swine.
- 14. The method for feeding an animal according to item 13 wherein the animal is selected from the group comprising chickens, broilers, layers, quails and turkey, domestic ducks or domestic geese or the young of pigeons, pigs, swine, piglets, growing pigs, or sows.
- 15. A method for feeding an animal according to anyone of items 12 to 14 wherein the bacterial count of each bacterial strain is between 1×104 and 1×1014 CFU/kg of animal feed, preferably between 1×106 and 1×1012 CFU/kg of animal feed, and more preferably between 1×107 and 1×1011 CFU/kg of animal feed and even more preferably between 1×108 and 1×1010 CFU/kg of animal feed.
- 16. A feed or a feed premix for improving the performance of poultries and swine comprising at least the Bacillus strain DSM 29870.
- 17. The feed or feed premix according to item 16 further comprising one or more enzymes; one or more additional microbes; one or more vitamins; one or more minerals; one or more amino acids; and one or more other feed or feed premix ingredients.
- 18. The feed according to item 16 or 17 wherein the bacterial count of each bacterial strain is between 1×104 and 1×1014 CFU/kg of animal feed, preferably between 1×106 and 1×1012 CFU/kg of animal feed, and more preferably between 1×107 and 1×1011 CFU/kg of animal feed and even more preferably between 1×108 and 1×1010 CFU/kg of animal feed.
- Item Set III:
- 1. An animal feed or an animal feed additive comprising a Bacillus strain selected from the group consisting of
- a Bacillus strain having deposit accession number DSM 29870, or a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof that has antimicrobial activity against Clostridium perfringens;
- a Bacillus strain having deposit accession number DSM 29869, or a strain having all of the identifying characteristics of Bacillus DSM 29869 or a mutant thereof that has antimicrobial activity against Clostridium perfringens;
- a Bacillus strain having deposit accession number DSM 29871, or a strain having all of the identifying characteristics of Bacillus DSM 29871 or a mutant thereof that has antimicrobial activity against Clostridium perfringens; and
- a Bacillus strain having deposit accession number DSM 29872, or a strain having all of the identifying characteristics of Bacillus DSM 29872 or a mutant thereof that has antimicrobial activity against Clostridium perfringens.
- 2. The animal feed or the animal feed additive according to item 1, wherein the Bacillus strain comprises 16S rDNA that has more than 98% sequence identity to SEQ ID NO: 1 and/or more than 98% sequence identity to SEQ ID NO: 2 and/or more than 98% sequence identity to SEQ ID NO: 3 and/or more than 98% sequence identity to SEQ ID NO: 4.
- 3. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus strain is characterized in that:
- i) the Bacillus strain is non-hemolytic and/or,
- ii) the Bacillus strain has antimicrobial activity against Clostridium perfringens, and/or
- iii) the Bacillus strain improves body weight gain and/or feed conversion rate in chickens fed with the Bacillus strain.
- 4. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus strain is characterized in that the Bacillus strain is sensitive to at least seven such as eight of the antibiotics selected from the group consisting of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline, e.g., as determined in Example 5.
- 5. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus strain is characterized in that the Bacillus strain is non-hemolytic
- 6. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus strain is characterized in that the Bacillus strain improves body weight gain and/or feed conversion rate in chickens fed with the Bacillus strain.
- 7. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus strain is characterized in that the Bacillus strain has antimicrobial activity against Clostridium perfringens, e.g., as determined in Example 2.
- 8. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus strain is characterized in that the Bacillus strain has enzymatic activity under aerobic and/or anaerobic conditions that hydrolyzes one or more of the substrates selected from the group consisting of Amylose, Arabinan, Arabinoxylan, Casein and Xylan, e.g., as determined in Example 6.
- 9. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus strain is characterized in that the Bacillus strain improves one or more performance parameters in poultry selected from the list consisting of body weight gain, European Production Efficacy Factor and feed conversion rate in poultry fed with the Bacillus strain.
- 10. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus strain comprises 16S rDNA that has more than 99% sequence identity to SEQ ID NO: 1 and/or more than 99% sequence identity to SEQ ID NO: 2 and/or more than 99% sequence identity to SEQ ID NO: 3 and/or more than 99% sequence identity to SEQ ID NO: 4.
- 11. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus strain is a Bacillus subtilis strain or a Bacillus amyloliquefaciens strain.
- 12. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus strain has antimicrobial effect against E. coli, e.g., as determined in Example 4.
- 13. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus strain is the Bacillus strain having deposit accession number DSM 29869, or a strain having all of the identifying characteristics of Bacillus DSM 29869 or a mutant thereof that has antimicrobial activity against Clostridium perfringens.
- 14. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus strain is the Bacillus strain having deposit accession number DSM 29870, or a strain having all of the identifying characteristics of Bacillus DSM 29870 or a mutant thereof that has antimicrobial activity against Clostridium perfringens.
- 15. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus strain is the Bacillus strain having deposit accession number DSM 29871, or a strain having all of the identifying characteristics of Bacillus DSM 29871 or a mutant thereof that has antimicrobial activity against Clostridium perfringens.
- 16. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus strain is the Bacillus strain having deposit accession number DSM 29872, or a strain having all of the identifying characteristics of Bacillus DSM 29872 or a mutant thereof that has antimicrobial activity against Clostridium perfringens.
- 17. The animal feed or the animal feed additive according to any of the previous items, wherein the Bacillus spores of the animal feed or the animal feed additive are present as dried spores such as spray dried spores.
- 18. The animal feed or the animal feed additive according to any of the previous items, wherein the animal feed or animal feed additive comprises from 105 to 1012 CFU/g of isolated Bacillus spores.
- 19. The animal feed or animal feed additive of any of the previous items, wherein at least 70% (such as at least 80% or at least 90%) of the Bacillus spores survive gastric stability in a mono-gastric animal such as chickens.
- 20. The animal feed or animal feed additive according to any of the previous items which further comprises one or more components selected from the list consisting of:
- one or more enzymes;
- one or more additional microbes;
- one or more vitamins;
- one or more minerals;
- one or more amino acids; and
- one or more other feed ingredients.
- 21. The animal feed or animal feed additive according to any of the previous items, wherein the bacterial count of each Bacillus spore is 1×104 and 1×1014 CFU/kg of animal feed or animal feed additive, preferably between 1×106 and 1×1012 CFU/kg of animal feed or animal feed additive, and more preferably between 1×107 and 1×1011 CFU/kg of animal feed or animal feed additive.
- 22. The animal feed or animal feed additive according to any of the previous items, wherein the animal feed or animal feed additive is a mono-gastric animal feed or mono-gastric animal feed additive.
- 23. The animal feed or animal feed additive according to any of the previous items, wherein the Bacillus strain improves gut health of chickens with infection of Clostridium perfringens by having antimicrobial activity against Clostridium perfringens.
- 24. The mono-gastric animal feed or animal feed additive according to item 22 or 23, wherein the mono-gastric animal is selected from the group consisting of pigs, swine, piglets, sows, poultry, turkeys, ducks, chicken, broilers, layers, chicks, fish and crustaceans.
- 25. The animal feed or animal feed additive according to any of the previous items, wherein the Bacillus strain has a high compatibility with monensin such as being compatible with at least 2.0 μg/ml monensin, e.g., as determined in Example 12 (such as at least 2.1 μg/ml monensin, such as at least 2.2 μg/ml monensin, such as at least 2.3 μg/ml monensin, such as at least 2.4 μg/ml monensin, such as at least 2.5 μg/ml monensin, such as at least 2.6 μg/ml monensin or such as at least 2.7 μg/ml monensin as determined in Example 12).
- 26. The animal feed or the animal feed additive according to any of the previous items which further comprises a carrier.
- 27. The animal feed or the animal feed additive according to item 26, wherein the carrier comprises one or more of the following compounds: water, glycerol, ethylene glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose, sucrose, sorbitol, lactose, wheat flour, wheat bran, corn gluten meal, starch and cellulose.
- 28. The animal feed or animal feed additive according to any of the previous items for the treatment of necrotic enteritis and/or the treatment of a Clostridium perfringens infection.
- 29. The animal feed or animal feed additive according to any of the previous items comprising at least the Bacillus strain DSM 29870.
- 30. The animal feed or animal feed additive according to any of the previous items comprising at least the Bacillus strain DSM 29869.
- 31. The animal feed or animal feed additive according to any of the previous items comprising at least the Bacillus strain DSM 29871.
- 32. The animal feed or animal feed additive according to any of the previous items comprising at least the Bacillus strain DSM 29872.
- 33. The animal feed or animal feed additive according to any of the previous items for treatment of mono-gastric animals.
- 34. A method of treating a Clostridium perfringens infection or for treating necrotic enteritis in an animal comprising administrating the animal feed or the animal feed additive according to any of items 1 to 33 to the animal.
- 35. Use of the animal feed or the animal feed additive according to any of items 1 to 33 to treat a Clostridium perfringens infection or for treating necrotic enteritis in an animal.
- 36. Use of the animal feed or the animal feed additive according to any of items 1 to 33 to improve one or more performance parameters of an animal selected from the group consisting of: improving the feed conversion ratio, improving the body weight gain, improving the European Production Efficacy Factor, improving the feed efficiency and improving the health.
- 37. The use according to item 35 or 36, wherein the animal feed or the animal feed additive comprises the Bacillus strain DSM 29870.
- 38. A method of improving one or more performance parameters of an animal selected from the group consisting of: improving the feed conversion ratio, improving the body weight gain, improving the European Production Efficacy Factor, improving the feed efficiency and improving the health, comprising administering the animal feed or animal feed additive according to any of items 1 to 33 to the animal.
- 39. The method according to item 38, wherein the animal feed or the animal feed additive comprises the Bacillus strain DSM 29870.
- 40. A method for feeding an animal comprising administering an animal feed or an animal feed premix comprising at least the Bacillus strain DSM 29870 to said animal optionally in conjunction with other animal feed ingredients.
- 41. The method for feeding an animal according to
item 40 wherein the animal is selected from the group consisting of poultry and swine. - 42. A feed or a feed premix for improving the performance of poultries and swine comprising at least the Bacillus strain DSM 29870.
- 43. The feed, feed additive or feed premix according to any of items 1 to 33 and item 42 wherein the bacterial count of each bacterial strain is between 1×104 and 1×1014 CFU/kg of animal feed (or per kg of feed additive or per kg of feed premix), preferably between 1×106 and 1×1012 CFU/kg of animal feed (or per kg of feed additive or per kg of feed premix), and more preferably between 1×107 and 1×1011 CFU/kg of animal feed (or per kg of feed additive or per kg of feed premix) and even more preferably between 1×108 and 1×1010 CFU/kg of animal feed (or per kg of feed additive or per kg of feed premix).
- 44. The feed according to any of items 1 to 33 and item 42 wherein the bacterial count of each bacterial strain is between 1×104 and 1×1014 CFU/kg of animal feed, preferably between 1×106 and 1×1012 CFU/kg of animal feed, and more preferably between 1×107 and 1×1011 CFU/kg of animal feed and even more preferably between 1×108 and 1×1010 CFU/kg of animal feed.
- 45. The feed additive or feed premix according to any of items 1 to 33 and item 42 wherein the bacterial count of each bacterial strain is between 1×107 and 1×1019 CFU/kg of animal feed additive or kg of feed premix, preferably between 1×109 and 1×1017 CFU/kg of animal feed additive or kg of feed premix, and more preferably between 1×1010 and 1×1016 CFU/kg of animal feed additive or kg of feed premix and even more preferably between 1×1011 and 1×1015 CFU/kg of animal feed additive or kg of feed premix.
- Hemolysis was tested according to the Technical Guidance on the assessment of the toxigenic potential of Bacillus species used in animal nutrition, EFSA Journal 2011; 9(11):2445.
- Sheep blood agar plates were purchased as ready to use (Becton Dickenson art 254053 or 254087). Alternatively, the agar plates can be prepared by adding 5% defibrinated sheep blood (obtained from Statens Serum Institute, Denmark) to TS-agar (Oxoid CM 131). Agar should be autoclaved at 121° C. for 20 minutes and cooled down to about 40° C. before adding the blood immediately before pouring the plates.
- The Bacillus strains were taken from the preservation at −80° C. and streaked on TS-agar plate, which was incubated at 30° C. overnight or until growth appeared. From a single colony as little as possible of the material was used to streak a line on ¼ of an agar plate. The plate was incubated at 30° C. for 72 hours. Hemolysis/clearing zones of the Bacillus strains to be tested were compared with the positive and negative control. As positive control Bacillus subtilis ATCC 21332 was used. As negative control Bacillus subtilis 168 (BGSC-1A1, Bacillus Genetic Stock Center) was used.
- In a screening of 599 independent isolates of Bacillus 223 strains (37%) were hemolysis negative. In another screening 21 of 65 independent isolates of Bacillus (32%) were hemolysis negative. Both screenings exclusively comprised strains that based on identification by 16S rDNA sequencing belong to Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus or Bacillus amyloliquefaciens or close relatives to these four species.
- The non-Hemolytic Bacillus strains therefore seem to be common and fairly abundant in nature, but only comprising a minority of the natural strains. The non-hemolytic strains seem to be more abundant in Bacillus licheniformis, while most Bacillus amyloliquefaciens strains appear hemolytic. All the non-hemolytic strains were tested for inhibition of growth of Clostridium perfringens. The following strains from screening were all hemolysis negative and among those selected for further studies: DSM 29869, DSM 29870, DSM 29871 and DSM 29872. Clostat (Bacillus PB6) was determined to be hemolytic.
- All the non-hemolytic strains were tested for inhibition of growth of Clostridium perfringens strains 23 and 48 (both are netB positive) [Gholamiandekhordi et al., 2006, Molecular and phenotypical characterization of Clostridium perfringens isolates from poultry flocks with different diseasestatus, Vet. Microbiol. 113:143-152], were grown overnight in tryptic soy broth (BD part 211822) supplemented with 0.6% yeast extract (BD part 212750) at 35° C. under static anaerobic conditions. 250 μL of the overnight culture of Clostridium perfringens was added to 250 mL of tryptic soy agar supplemented with 0.6% yeast extract at 40° C. and poured into rectangular petri plates (Nunc part 267060). The inoculated agar was allowed to cool at room temperature after which an 8 mm diameter well was made in the agar. Plates were stored in absence of oxygen until use.
- The Bacillus strain DSM 29869, DSM 29870, DSM 29871, or DSM 29872 was grown overnight in tryptic soy broth at 35° C. under aerobic conditions. 1000 μL of Bacillus culture was collected and fractionated into cell-free supernatant and cells by centrifugation. 20 μL of cell-free supernatant or 100×diluted cells in phosphate buffered saline were added directly to the wells in the Clostridium perfringens inoculated agar plates. A control well contained 20 μL of phosphate buffer saline. The plates were incubated for 18 hours at 35° C. under anaerobic conditions.
- Inhibition of the Clostridium perfringens strain was noted by a circular clearing zone around the well of interest. The phosphate buffer saline well was considered a negative control based on lack of clearing zone around the well.
- Cell-free supernatant and 100×diluted cells of Bacillus strains DSM 29869, DSM 29870, DSM 29871, and DSM 29872 were able to consistently inhibit growth of C. perfringens strains 23 and 48 in vitro. Inhibition was also seen by competitor strain “CloSTAT”, for both supernatant and cells, but was not seen with competitor strain GalliproTect. “CloSTAT” is a strain of Bacillus amyloliquefaciens that was isolated from the commercial DFM product CloSTAT, Kemin. “GalliproTect” is a strain of Bacillus licheniformis that was isolated from the commercial product Gallipro Tect, Chr Hansen.
- The following biological materials were deposited under the terms of the Budapest Treaty at Leibniz-Institut DSMZ-Deutsche Sammlung von Mikro-organismen and Zellkulturen GmbH, Inhoffenstraße 7 B, 38124 Braunschweig Germany, and given the following accession numbers:
-
TABLE 3.1 Deposit of Biological Material Identification Accession Number Date of Deposit Bacillus amyloliquefaciens DSM 29869 Jan. 12, 2015 Bacillus subtilis DSM 29870 Jan. 12, 2015 Bacillus subtilis DSM 29871 Jan. 12, 2015 Bacillus amyloliquefaciens DSM 29872 Jan. 12, 2015 - The strains have been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by foreign patent laws to be entitled thereto. The deposits represent a substantially pure culture of the deposited strain. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
- Sequencing of 16S rDNA Genes
- DNA was extracted from a culture of DSM 29869, DSM 29870, DSM 29871 and DSM 29872 using QiaAmp DNA Blood Mini Kit (Qiagen art 51106). The kit was used as recommended for extraction of DNA from gram positive bacteria.
- 16S rDNA was amplified in a total volume of 50 μl by mixing: 10 pmol of each of Primer 16S F and 16S R, 0.2 mM of each nucleotide, 2.5 units Ampli taq, 1× Ampli taq buffer, 5 μl DNA template and by using the following PCR program: 94° C. 2 min (94° C. 30 s, 52° C. 30 S, 72° C. 1 min)×35, 72° C. 10 min on a Perkin Elmer PCR machine. The PCR product was sequenced by Novozymes DNA sequencing facility using primer 530R, 357F, 1390R and 1100F.
-
TABLE 3.2 Primers: Primer Sequence SEQ ID NO 16S-F 5′-GAGTTTGATCCTGGCTCAG-3′ SEQ ID NO: 8 16S-R 5′-AGAAAGGAGGTGATCCAGCC-3′ SEQ ID NO: 9 794-R 5′-ATCTAATCCTGTTTGCTCCCC-3′ SEQ ID NO: 10 357-F 5′-TACGGGAGGCAGCAG-3′ SEQ ID NO: 11 1390-R 5′-CGGTGTGTRCAAGGCCC-3′ SEQ ID NO: 12 1000-F 5′-CAACGAGCGCAACCCT′, SEQ ID NO: 13 - Degeneration of primer 1390-R: R is A or G. The 16 S rDNA sequences from DSM 29869, DSM 29870, DSM 29871 and DSM 29872 are shown as SEQ ID NO: 1-4 in the sequence listing respectively. The 16 S rDNA sequences from DSM 29869, DSM 29870, DSM 29871 and DSM 29872 were analyzed by BLAST against EMBL database and showed identity to 16 S rDNA sequences of Bacillus subtilis (SEQ ID NO: 2 and SEQ ID NO: 3) and to Bacillus amyloliquefaciens (SEQ ID NO: 1 and SEQ ID NO: 4).
- In order to study the phylogenetic affiliation of SEQ ID NO: 1 to SEQ ID NO: 4 the sequences were analyzed by a ClustalW alignment in MegAlign (DNASTAR) using SEQ ID NO: 5 to SEQ ID NO: 7 as benchmark. These sequences are reference 16S rDNA sequences of the type strains of Bacillus vallismortis taken from AB021198 (SEQ ID NO: 5), Bacillus subtilis taken from AJ276351 (SEQ ID NO: 6) and Bacillus amyloliquefaciens taken from AB255669 (SEQ ID NO: 7).
- The ClustalW alignment of SEQ ID NO: 1 to SEQ ID NO: 7 (
FIG. 1 ) shows 7 nucleotide positions where 2 or more sequences have a nucleotide that deviates from the other. For numbering the positions in SEQ ID NO: 6 are used. At position 152 SEQ ID NO: 2 is identical to Bacillus amyloliquefaciens and Bacillus vallismortis, but different from the rest that are identical to each other. At position 174 SEQ ID NO: 1 and SEQ ID NO: 4 are identical to Bacillus amyloliquefaciens, but different from the rest that are identical to each other. At position 257 SEQ ID NO: 4 is identical to Bacillus amyloliquefaciens, but different from the rest that are identical to each other. At position 437, 444 and 455 SEQ ID NO: 2 and SEQ ID NO: 3 are identical to Bacillus subtilis, but different from the rest that are identical to each other. At position 1223 SEQ ID NO: 1 is identical to Bacillus amyloliquefaciens, but different from the rest that are identical to each other. The variation in the 16S rDNA genes support the species affiliation seen in the BLAST report. - Bacillus amyloliquefaciens DSM 29869 was isolated by Novozymes (Novo Nordisk) from an environmental sample collected at Jamaica in 1990. The strain was identified as Bacillus amyloliquefaciens based on 16S rDNA sequencing
- Bacillus subtilis DSM 29870 was isolated by Novozymes (Novo Nordisk) from an environmental sample collected at Jamaica in 1990. The strain was identified as Bacillus subtilis based on 16S rDNA sequencing.
- Bacillus subtilis DSM 29871 was isolated for Novozymes by a Danish high-school student at Brønderslev Gymnasium from a soil sample from private property in Denmark in 2008. The strain was identified as Bacillus subtilis based on 16S rDNA sequencing.
- Bacillus amyloliquefaciens DSM 29872 was isolated from an environmental sample from the USA in 2008. The strain was identified as Bacillus amyloliquefaciens based on 16S rDNA sequencing.
- Escherichia coli strains, ATCC10536 or ATCC25922, were grown overnight in tryptic soy broth (BD part 211822) supplemented with 0.6% yeast extract (BD part 212750) at 35° C. under static anaerobic conditions. 100 μL of the overnight culture of Escherichia coli was added to 250 mL of tryptic soy agar supplemented with 0.6% yeast extract at 40° C. and poured into rectangular petri plates (Nunc part 267060). The inoculated agar was then allowed to cool at room temperature after which an 8 mm diameter well was made in the agar.
- The Bacillus strain DSM 29869, DSM 29870, DSM 29871, or DSM 29872 was grown overnight in tryptic soy broth at 35° C. under aerobic conditions. 1000 μL of Bacillus subtilis culture was collected and fractionated into cell-free supernatant and cells by centrifugation. 20 μL of cell-free supernatant or 100× diluted cells in phosphate buffer saline were added directly to the wells in the Escherichia coli inoculated agar plates. A control well contained 20 μL of phosphate buffer saline. The plates were incubated for 18 hours at 30° C. under aerobic conditions.
- Inhibition of the Escherichia coli strain was noted by a circular clearing zone around the well of interest. The phosphate buffer saline well was considered a negative control based on lack of clearing zone around the well.
- Cell-free supernatant and 100×diluted cells of Bacillus strains DSM 29869, DSM 29870, DSM 29871, and DSM 29872 were able to consistently inhibit growth of the E. coli strains ATCC10535 and ATCC25922 in vitro. Inhibition was also seen by competitor strain CloSTAT, for both supernatant and cells.
- The minimal inhibitory concentrations (MIC) of eight antibiotics against Bacillus strains DSM 29869, DSM 29870, DSM 29871, and DSM 29872 were determined using broth micro dilution essentially as described in the CLSI guidelines (M07-A9 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; 2012). Only modification was that volumes were changed; 90 μl Mueller Hinton Broth 2 with bacteria was added to 10 μl antibiotics dilutions, cfu's of bacteria and concentration of antibiotics were changed so final concentrations and final cfu's matched the guideline. The plates were incubated for 20-24 h instead of 16-20 h. The control strain recommended in the CLSI standard Staphylococcus aureus ATCC 29213 was used as control strain.
-
-
- Bacillus amyloliquefaciens DSM 29869
- Bacillus subtilis DSM 29870
- Bacillus subtilis DSM 29871
- Bacillus amyloliquefaciens DSM 29872
- S. aureus ATCC 29213
-
-
- Chloramphenicol (Sigma C1919, 10 mg/ml, solubilized in 96% ethanol)
- Clindamycin (Sigma PHR1159, 10 mg/ml, solubilized in water)
- Erythromycin (ABBOTICIN from
Amdipharm 40501, 50 mg/ml, solubilized in 96% ethanol) - Gentamycin (Biomedicals inc., 190057, 10 mg/ml, solubilized in water)
- Kanamycin (Sigma, CAS no. 25389-94-0, 50 mg/ml, solubilized in water)
- Streptomycin (Sigma, S1277, 10 mg/ml, solubilized in water)
- Tetracycline (Sigma, T3383, 10 mg/ml, solubilized in water)
- Vancomycin (Sigma, V-8138, 10 mg/ml, solubilized in water)
- Mueller Hinton Broth 2 (Sigma/Fluka 90922)
- 0.9% NaCl (Sigma/RdH 31434/Merck 106404)
- Tryptone soya agar plates (Oxoid CM 131)
- Microtiter plates: Costar plate, polypropylene, round bottom, Corning 3879
- A few colonies of Bacillus spp. (<1 day old) were inoculated into Mueller Hinton Broth 2 (MHB) and incubated for around 4 hours at 37° C. OD600 (BioPhotometer plus, Eppendorf) was measured and adjusted to 0.25 (equivalent to McFarland 0.5) in MHB. For the control strain direct colony suspension was used. A few colonies of S. aureus ATCC 29213 (<1 day old) were suspended in MHB and OD600 (BioPhotometer plus, Eppendorf) was measured and adjusted to 0.10-0.12 (equivalent to McFarland 0.5) in MHB. The bacterial suspensions were diluted 200× in MHB.
- Antibiotics were diluted to the concentration of 640 μg/mL in MHB. A two fold dilution series was prepared in MHB down to the concentration 0.625 μg/ml. 10 μl of each dilution and of each antibiotic was pipetted into a micro-titre plate. Later, when the antibiotics were mixed with the suspension of bacteria, the samples were diluted 10× (10 μL sample in a total volume of 100 μl). This resulted in the final test range of 0.06-64 μg/ml.
- If the plates were not used right away the plates were stored in the freezer at −20° C. until usage.
- 90 μl of the bacterial suspensions were added to the assay plates. The assay plates were incubated in a plastic bag with at wet cloth at 37° C. for 20-24 h. The MIC was determined as the lowest concentration of antibiotic that completely inhibited growth of bacteria as detected by the unaided eye.
- A 10-fold dilution series in 0.9% NaCl was made to the 10−3 of the cultures inoculated into the micro-titre plate. 2×100 ul from the 10−3 dilution were plated onto two TSA plates. The plates were incubated overnight at 37° C. Number of CFU/ml was counted.
- Three biological replicates of the assay were performed for Bacillus subtilis DSM 29871, Bacillus amyloliquefaciens DSM 29869 and Bacillus subtilis DSM 29870 (MIC 1-3), while four biological replicates were performed for Bacillus amyloliquefaciens DSM 29872 (MIC 2-5).
- The MIC values obtained for B. subtilis DSM 29870 showed that the breakpoint values were equal to or below the breakpoint values given in the EFSA guideline (EFSA Journal 2012; 10(6):2740). For B. subtilis DSM 29869 the MIC values obtained in two out of the three biological replicates showed MIC values equal to or below the breakpoint. In MIC 1 the value for Tetracycline was 16. However, due to analytical variance of the method MIC results of one dilution above the breakpoint is in general accepted and the strain may be regarded as being sensitive. Thus, according to EFSA these two strains are regarded as sensitive to all the 8 antibiotics included in the test (Tables 5.2 and 5.3).
- For the strains B. subtilis DSM 29871 and B. amyloliquefaciens DSM 29872 the MIC values showed that the two strains were sensitive to seven out of the eight antibiotics (Tables 5.1 and 5.4). An increased tolerance was observed towards Streptomycin and according to EFSA the strains are classified as resistant to Streptomycin.
- As a control S. aureus ATCC 29213 was tested in parallel and had MIC values within the ranges given by the CLSI standard (M 100-S24 Performance Standards for Antimicrobial Susceptibility Testing; informational Supplement, 2014) (Table 5.5).
- The amount of bacteria inoculated into the assay plates was measured (CFU/ml). In general the CFU/ml was very close to the target value of 5*105 CFU/ml. However, the CFU/ml for Bacillus strains may be associated with some uncertainty, since the bacteria tend to aggregate and once aggregated will only result in one colony forming unit (Tables 5.1 and 5.2).
-
TABLE 5.1 MIC results for B. subtilis DSM 29871 MIC 1 MIC 2 MIC 3EFSA* breakpoints Antibiotic μg/ml μg/ml μg/ml μg/ml Chloramphenicol 8 4 8 8 Clindamycin 0.5 0.25 0.5 4 Erythromycin 0.125 0.125 0.125 4 Gentamycin 0.25 0.125 0.25 4 Kanamycin 2 1 2 8 Streptomycin 32 16 32 8 Tetracycline 2 4 4 8 Vancomycin 0.25 0.25 0.25 4 CFU/ml 3.6*105 n.r. n.r. *EFSA Journal 2012; 10(6): 2740 n.r. not registered -
TABLE 5.2 MIC results for B. amyloliquefaciens DSM 29869 MIC 1 MIC 2 MIC 3EFSA* breakpoints Antibiotic μg/ml μg/ml μg/ml μg/ ml Chloramphenicol 4 4 4 8 Clindamycin 1 0.5 1 4 Erythromycin 0.5 0.06 0.06 4 Gentamycin 0.5 0.125 0.06 4 Kanamycin 2 0.5 0.5 8 Streptomycin 2 4 4 8 Tetracycline 16 8 8 8 Vancomycin 0.25 0.25 0.25 4 CFU/ml 3.9*105 2.7*105 1.2*105 *EFSA Journal 2012; 10(6): 2740 -
TABLE 5.3 MIC results for B. subtilis DSM 29870 MIC 1 MIC 2 MIC 3EFSA* breakpoints Antibiotic μg/ml μg/ml μg/ml μg/ ml Chloramphenicol 4 8 4 8 Clindamycin 0.25 0.25 0.25 4 Erythromycin 0.125 0.125 0.125 4 Gentamycin 0.25 0.125 0.25 4 Kanamycin 2 1 2 8 Streptomycin 4 4 8 8 Tetracycline 0.25 0.25 0.25 8 Vancomycin 0.25 0.25 0.25 4 CFU/ml 4.2*105 2.1*105 1.4*105 *EFSA Journal 2012; 10(6): 2740 -
TABLE 5.4 MIC results for B. amyloliquefaciens DSM 29872 EFSA* break- MIC 2 MIC 3MIC 4MIC 5 points Antibiotic μg/ml μg/ml μg/ml μg/ml μg/ ml Chloramphenicol 4 4 4 4 8 Clindamycin 0.5 0.5 0.5 0.5 4 Erythromycin 0.06 0.125 0.06 0.06 4 Gentamycin 0.06 0.06 0.125 0.125 4 Kanamycin 1 0.5 2 1 8 Streptomycin 8 (32) 8 32 >64 8 Tetracycline 0.25 0.25 0.125 0.125 8 Vancomycin 0.25 0.25 0.125 0.125 4 CFU/ml 2.7*105 1.5*105 4.7*105 6.3*105 *EFSA Journal 2012; 10(6): 2740 -
TABLE 5.5 MIC results for S. aureus ATCC 29213 MIC 1 MIC 2 MIC 3MIC 4MIC 5 CLSI* breakpoints Antibiotic μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml Chloramphenicol 8-16 16 8 16 16 2-16 Clindamycin 0.125 0.25 0.125 0.06 0.06 0.06-0.25 Erythromycin 0.5 0.5 0.5 0.5 0.5 0.25-1 Gentamycin 0.5 0.5 0.5 1 1 0.12-1 Kanamycin 4 4 4 4 4 1-4 Streptomycin 8 8 8 16 8 No information Tetracycline 0.5 1 1 0.5 0.5 0.12-1 Vancomycin 0.5 1 1 1 1 0.5-2 CFU/ml 5.2*105 7.0*105 7.0*105 7.3*105 4.3*105 *M100-S24 Performance Standards for Antimicrobial Susceptibility Testing; informational Supplement, 2014 - Overnight cultivation was made in a 96-deep well plate with 1.25 mL of LB broth (Difco; BD #244610) per well. Each strain was tested in duplicate and inoculated with a single colony from a pure Standard Methods Agar plate (SMA plates, Smith River Biologicals #11-00450). Before incubation, a single sterile Aeraseal breathable seal (Fisher #50-212-463) was placed on top of the plate, and the plate was incubated with shaking at 35° C. overnight under aerobic conditions.
- SMA plates were streaked from the overnight cultures using a 10 μL inoculating loop. Up to four strains were streaked per plate, into separate quadrants.
- Plates were incubated at 39° C. in an AnaeroJar (Thermo Scientific Oxoid, Fisher #OXAG0025A) along with an AnaeroGen sachet (Thermo Scientific Oxoid, Fisher #OXAN0025A) to maintain anaerobic conditions (˜0.1% oxygen). Work was performed under aseptic conditions in biological hood, using sterile materials.
- Preparation of media with AZCL substrates which can be autoclaved AZCL-cellulose (AZCL-HE-Cellulose, I-AZCEL, Megazyme), arabinoxylan (AZCL-Arabinoxylan, wheat, I-AZWAX, Megazyme), arabinan (AZCL-Arabinan, debranched, I-AZDAR, Megazyme):
- Into two (2) 500 mL beakers, 300 mL of 0.1× and 1× LB agar were prepared for each beaker, with a magnetic stir bars added to each. While stirring, 100 mL of LB condition from the beakers were added to three (3) 250 mL Wheaton bottles, thus giving 3 bottles with 0.1× LB and 3 bottles with 1× LB. AZCL-cellulose, arabinan, and arabinoxylan were then added separately to each of a 1× and a 0.1× bottle with stirring. A total of 0.05 grams of substrate is added per 100 mL of media. If more than 100 mL of media is being used, the amount of substrate added is adjusted by using the 0.05 g/100 mL ratio. The media were autoclaved to sterilize. Note: Only these three AZCL substrates can be autoclaved without obvious dye release indicating substrate instability. After autoclaving, media is kept at 50° C. until ready to pour plates. The pH was checked, and if necessary adjusted to pH 7 using 10% HCl or 2N NaOH, maintaining sterility.
- Preparation of Media with AZCL Substrates which Can't be Autoclaved
- These substrates were: AZCL-amylose (I-AZAMY, Megazyme), AZCL-casein (I-AZCAS, Megazyme), AZCL-xylan (Xylan, birchwood (I-AZXBW, Megazyme)
- Into two (2) 1000 mL Wheaton bottles, 400 mL of 0.1×LB and 1×LB was prepared and a magnetic stir bar was added to each. The media was autoclaved to sterilize, prior to adding AZCL reagent. After autoclaving, media was kept at 50-55° C. until ready to add AZCL substrate and pour plates. The pH was checked, and if necessary adjusted to pH 7 using 10% HCl or 2N NaOH, maintaining sterility.
- Work was performed under aseptic conditions in biological hood, using sterile materials. These steps were done relatively quickly, so that the agar did not solidify before transferring into the 96-well plate.
- For substrates which could be autoclaved (AZCL-cellulose, AZCL-arabinoxylan, AZCL-arabinan): The warm (˜50° C.), autoclaved liquid agar media was magnetically stirred in order to suspend the insoluble AZCL substrate. An aliquot was transferred to a sterile solution basin (Periodic mixing of the material in the solution basin was necessary to keep the substrate suspended.) A repeating, multi-channel pipette (e.g.,
Matrix 1250 μL pipet along with the corresponding 1250 μL sterile pipet tips) was used to dispense 180 μL into each well of a sterile 96-well plate. A set of tips would typically last for up to six columns per plate. Once the plate was filled, a sterile lid was added, and it was allowed to solidify. One plate was made for each substrate (AZCL-cellulose, AZCL-arabinoxylan, AZCL-arabinan) and condition (0.1× and 1× LB). - For the substrates that could not be autoclaved (AZCL-amylose, AZCL-casein, AZCL-xylan): Into a sterile 250 mL beaker with magnetic stir-bar was added 100 mL of warm, sterile 0.1× or 1× LB media (no AZCL substrate added). With stirring, add 0.05 mg of AZCL substrate, and continue stirring until re-suspended. It may help to add the substrate slowly at first. Once substrate was re-suspended, it was poured into solution basin and added to plates as described above. One plate was made for each substrate (AZCL-cellulose, AZCL-arabinoxylan, AZCL-arabinan) and condition (0.1× and 1× LB).
- Work was performed under aseptic conditions in biological hood, using sterile materials. The AZCL substrate agar plates were inoculated by applying 2 μL of overnight culture that was dispensed on top of the agar. The added liquid should be visible on top of the agar. When moving to the next plate, be sure that there are no drops left at the end of the pipet tips to ensure that each well has been inoculated. Repeat for all AZCL substrate plates. Control enzyme dilutions were prepared, each at 1/100, diluting in sterile phosphate dilution buffer: 10 μL enzyme preparation+990 μL buffer. Control enzyme wells were then inoculated with 10 μL of the appropriate enzyme dilution for each substrate.
-
TABLE 6.1 Enzymes and respective substrates Enzyme Substrate Amylase Amylose Arabinase Arabinan Cellulase Cellulose Protease Casein Xylanase Arabinoxylan, Xylan -
TABLE 6.2 Enzyme production under aerobic conditions 30° C. Aerobic, 24 h organism ID Amylose Arabinan Arabinoxylan Casein Cellulose Xylan DSM 29869 + + + + − + DSM 29870 + + + + − + DSM 29871 + + + + + + DSM 29872 + + + + − + -
TABLE 6.3 Enzyme production under anaerobic conditions, 24 hours 30° C. Anaerobic, 24 h organism ID Amylose Arabinan Arabinoxylan Casein Cellulose Xylan DSM 29869 + + + − − + DSM 29870 + + +/− + − + DSM 29871 − + + − + + DSM 29872 + + + − − + -
TABLE 6.4 Enzyme production under anaerobic conditions, 48 hours 30° C. Anaerobic, 48 h organism ID Amylose Arabinan Arabinoxylan Casein Cellulose Xylan DSM 29869 + + + + − + DSM 29870 + + +/− + − + DSM 29871 − + + − + + DSM 29872 + + + − − + - Three independent battery studies have been conducted to evaluate the influence of the strain DSM 29870 on the development of an induced necrotic enteritis.
- In each experiment, a total of 256 one day-old male broiler chickens Cobb×Cobb were allocated to Petersime battery cages (8 birds/cage). Cages were used in factorial and completely randomized design with 8 cages per treatment (i.e., 64 animals/treatment).
- An unmedicated corn/soybean meal-based commercial broiler starter ration was formulated (Table 7.1). Feed and water were available ad libitum throughout the trials.
-
TABLE 7.1 Composition of the basal experimental diet4 (starter d 1-d 28) Starter (0-28) Ingredients Corn, yellow, ground 56.12 Soybean meal (48) 37.50 Fat, poultry 3.00 Dicalcium phosphate 1.75 Limestone 0.80 Salt 0.30 Vitamin premix1 0.25 DL-Methionine 0.20 Trace mineral premix2 0.08 Calculated Nutritional Content ME, kcal/kg 3.096 Protein, % 22.30 Lysine, % 1.180 Methionine, % 0.530 Met + Cys, %3 0.890 1Vitamin mix provided the following (per kg of diet): thiamin•mononitrate, 2.4 mg; nicotinic acid, 44 mg; riboflavin, 4.4 mg; D-Ca pantothenate, 12 mg; vitamin B12 (cobalamin), 12.0 μg; pyridoxine•HCL, 4.7 mg; D-biotin, 0.11 mg; folic acid, 5.5 mg; menadione sodium bisulfite complex, 3.34 mg; choline chloride, 220 mg; cholecalciferol, 27.5 ug; trans-retinyl acetate, 1,892 ug; all-rac α tocopheryl acetate, 11 mg; ethoxyquin, 125 mg. 2Trace mineral mix provided the following (per kg of diet): manganese (MnSO4•H2O), 60 mg; iron (FeSO4•7H2O), 30 mg; zinc (ZnO), 50 mg; copper (CuSO4•5H2O), 5 mg; iodine (ethylene diamine dihydroiodide), 0.15 mg; selenium (NaSe03), 0.3 mg. 3Met = methionine; Cys = cysteine. 4The basal feed will not contain any probiotic/prebiotic feed additives, NSPases, coccidiostats or antibiotic growth promoter. - The 4 experimental groups consisted in (Table 7.2): T1, non-infected and non-treated animals; T2, C. perfringens infected and non-treated animals; T3, C. perfringens infected animals fed with the basal diet containing bacitracin (included at 50 ppm, from d1 to d28); T4, C. perfringens infected animals fed with the basal diet containing the DSM 29870 strain (included at 1.109 CFU/kg of feed in trial 1 and 5.108 CFU/kg of feed in
trials 2 and 3, from d1 to d28). -
TABLE 7.2 Experimental treatments Treatments T1 Non-infected, non-treated T2 Infected, non-treated T3 Infected, bacitracin T4 Infected, DSM 29870 - On day 14, all birds were orally inoculated with a coccidial inoculum containing approximately 5,000 oocysts of Eimeria maxima per bird.
- Once daily on d19, d20 and d21, all birds, except T1, were administered (by oral gavage) 1 mL of a fresh broth culture of C. perfringens.
- On d21, three birds from each cage were selected, sacrificed, weighed, and examined for the degree of presence of necrotic enteritis lesions. The scoring was based on a 0 to 3 score, with 0 being normal and 3 being the most severe.
- The live body weight and the feed were recorded at the start (d1) and the end of the experiment (d28), on a cage basis, to calculate performance:
-
Body Weight Gain (BWG)=(Live Body Weight)d28−(Live Body Weight)d1 -
Feed Conversion Ratio (FCR)=ratio between feed consumed and weight gained - Mortality was recorded daily and the FCR was adjusted accordingly.
- Necrotic enteritis lesion scores, total mortality and % of dead birds with necrotic enteritis lesions were also calculated
- Data (n=32) were subjected to an ANOVA, with complete randomized block design using the ANOVA procedure of XLSTAT (Addinsoft 1995-2014) to establish differences between diets. Pen was considered as the experimental unit. The model included diets (n=4) and block as fixed effect. Results are reported as least square means. LS means were assumed to be different at P 0.05.
- The results obtained on performance, necrotic enteritis lesion scores, mortality and % of dead birds with necrotic enteritis lesions parameters are shown in Tables 7.3.
-
TABLES 7.3 Effect of dietary supplementation of the strain DSM 29870 on the negative impact on production parameters due to induced necrotic enteritis in three independent trials. Dead birds Total with necrotic BWG5 Lesion mortality enteritis lesions (g/bird) FCR5 score6 (%) (%) Trial 1 (d 1-d 28) (d 1-d 28) (d 21) (d 1-d 28) (d 1-d 28) T11 720a 1.666c 0.0a 7.5b 0.0b T22 549b 2.071a 0.2a 27.5a 17.2a T33 633ab 1.813bc 0.2a 5.0b 1.6b T44 628ab 1.843bc 0.3a 5.0b 3.1b 1Non-infected, non-treated group fed with the basal diet. 2Infected, non-treated group fed with the basal diet. 3Infected group fed with the basal diet supplemented with bacitracin (50 ppm). 4Infected group fed with the basal diet supplemented with DSM 29870 (1.109 CFU/kg of feed). 5Means from 8 replicates of 8 birds per cage for each group. Raw data (n = 32) were analyzed using variance analysis with block (n = 8) and treatments (n = 4) as fixed effect. 6Means from 8 replicates of 3 birds per cage for each group. Raw data (n = 32) were analyzed using variance analysis with block (n = 8) and treatments (n = 4) as fixed effect. a, b, cValues with different superscripts are significantly (P < 0.05) different from each other. Dead birds Total with necrotic BWG5 Lesion mortality enteritis lesions (g/bird) FCR5 score6 (%) (%) Trial 2 (d 1-d 28) (d 1-d 28) (d 21) (d 1-d 28) (d 1-d 28) T1 685a 1.692b 0.0d 2.5c 0.0c T2 435c 2.404a 1.2a 32.5a 20.3a T3 665ab 1.759b 0.6b 12.5bc 6.3c T4 654ab 1.768b 0.4bc 25.0ab 9.4bc 1 Non-infected, non-treated group fed with the basal diet. 2 Infected, non-treated group fed with the basal diet. 3 Infected group fed with the basal diet supplemented with bacitracin (50 ppm). 4 Infected group fed with the basal diet supplemented with DSM 29870 (5.108 CFU/kg of feed). 5Means from 8 replicates of 8 birds per cage for each group. Raw data (n = 32) were analyzed using variance analysis with block (n = 8) and treatments (n = 4) as fixed effect. 6Means from 8 replicates of 3 birds per cage for each group. Raw data (n = 32) were analyzed using variance analysis with block (n = 8) and treatments (n = 4) as fixed effect. a, b, cValues with different superscripts are significantly (P < 0.05) different from each other. Dead birds Total with necrotic BWG5 Lesion mortality enteritis lesions (g/bird) FCR5 score6 (%) (%) Trial 3 (d 1-d 28) (d 1-d 28) (d 21) (d 1-d 28) (d 1-d 28) T1 667a 1.752c 0.0b 10.0a 0.0c T2 521b 1.988a 0.5a 25.0a 15.6a T3 645a 1.786bc 0.6a 15.0a 4.7bc T4 638a 1.851b 0.4a 10.0a 4.7bc 1 Non-infected, non-treated group fed with the basal diet. 2 Infected, non-treated group fed with the basal diet. 3 Infected group fed with the basal diet supplemented with bacitracin (50 ppm). 4 Infected group fed with the basal diet supplemented with DSM 29870 (5.108 CFU/kg of feed). 5Means from 8 replicates of 8 birds per cage for each group. Raw data (n = 32) were analyzed using variance analysis with block (n = 8) and treatments (n = 4) as fixed effect. 6Means from 8 replicates of 3 birds per cage for each group. Raw data (n = 32) were analyzed using variance analysis with block (n = 8) and treatments (n = 4) as fixed effect. a, b, cValues with different superscripts are significantly (P < 0.05) different from each other. - The data obtained from the three challenge studies showed that the strain DSM 29870 can counteract the negative impact of an induced necrotic enteritis on the production parameters with a significant effect in all challenge trials on the FCR and the mortality due to necrotic enteritis, relative to the positive control group (non-infected, non-treated animals).
- Furthermore, there was no significant difference between the DSM 29870 fed group and the bacitracin fed group with regards to all parameters measured in these trials.
- The challenge studies described above showed that the administration of the DSM 29870 strain could prevent necrotic enteritis in broiler chickens.
- A total of 2000 one day-old male
broiler chickens Cobb 500 were individually weighed (body weight 42 g±1 g) and allocated in floor pens (50 birds/pen). Pens were used in factorial and completely randomized design with 8 pens per treatment (i.e., 400 animals/treatment). The 5 experimental treatments consisted in (Table 8.1): T1, negative control basal diets; T2, basal diets containing the strain DSM 29870 (included at 5.108 CFU/kg of feed, from d1 to d35); T3, basal diets containing the strain DSM 29871 (included at 5.108 CFU/kg of feed, from d1 to d35); T4 basal diets containing the strain DSM 29872 (included at 5.108 CFU/kg of feed, from d1 to d35); T5, basal diet containing a marketed probiotic, GalliPro Max (included at the commercial dose, 8.108 CFU/kg of feed, from d1 to d35). - Basal diets consisted in 3 phases feeding program: starter phase (from 1 to day 12), grower phase (from 13 to day 28) and finisher phase (from 29 to day 35). Every phase was formulated to meet or exceed animal requirement and agree with standard commercial US corn-soybean meal-based broiler diets. The dietary and raw material composition of these diets is given in Table 8.2. All experimental diets include phytase (Ronozyme P, 250 FYT/kg) and did not contain any coccidiostat, NSPase or growth promoting substance. Feed and water were available ad libitum throughout the trial.
-
TABLE 8.1 Experimental treatments Treatments T1 Control T2 T1 + DSM 29870 T3 T1 + DSM 29871 T4 T1 + DSM 29872 T5 T1 + GalliPro Max -
TABLE 8.2 Composition of the basal experimental diets Starter Grower Finisher (0-12 d) (13-28 d) (29-35 d) % Ingredients Corn, yellow, grain 64.673 66.460 68.491 Soybean meal, dehulled, solvent 29.020 26.662 24.677 Ampro 55 (animal by-product 55% 2.500 3.000 3.000 protein) Calcium carbonate 0.886 0.735 0.684 Fat, vegetable 0.883 1.485 1.702 Dicalcium phosphate 0.706 0.612 0.500 Salt, plain (NaCl) 0.439 0.435 0.436 Methionine MHA 0.358 0.259 0.221 L-Lysine 0.273 0.208 0.145 L-Threonine 98.5 0.103 0.000 0.000 Trace Mineral1 0.075 0.075 0.075 Vitamin premix 2 0.065 0.050 0.050 Ronozyme P-(ct) 0.019 0.019 0.019 Calculated Nutritional Content ME (kcal/kg) 3,067 3,130 3,165 Crude protein (%) 20.96 20.03 19.16 Dig. Lysine (%) 1.20 1.10 1.00 Dig. Methionine (%) 0.61 0.52 0.48 Dig. TSAA (%) 0.90 0.80 0.75 Dig. Threonine (%) 0.81 0.68 0.65 Calcium (%) 0.90 0.85 0.8 Avail. phosphorus (%) 0.42 0.42 0.4 1Vitamin mix will provide the following (per kg of diet): thiamin•mononitrate, 2.4 mg; nicotinic acid, 44 mg; riboflavin, 4.4 mg; D-Ca pantothenate, 12 mg; vitamin B12 (cobalamin), 12.0 μg; pyridoxine•HCL, 4.7 mg; D-biotin, 0.11 mg; folic acid, 5.5 mg; menadione sodium bisulfite complex, 3.34 mg; choline chloride, 220 mg; cholecalciferol, 27.5 ug; trans-retinyl acetate, 1,892 ug; all-rac α tocopheryl acetate, 11 mg; ethoxyquin, 125 mg. 2 Trace mineral mix provided the following (per kg of diet): manganese (MnSO4•H2O), 60 mg; iron (FeSO4•7H2O), 30 mg; zinc (ZnO), 50 mg; copper (CuSO4•5H2O), 5 mg; iodine (ethylene diamine dihydroiodide), 0.15 mg; selenium (NaSe03), 0.3 mg. - The live body weight and the feed were recorded at the start (d1) and the end of the experiment (d35), on a pen basis, to calculate performance:
-
Feed Intake (FI)=(Remaining Feed)d35−(Issued Feed)d1 -
Body Weight Gain (BWG)=(Live Body Weight)d35−(Live Body Weight)d1 -
Feed Conversion Ratio (FCR)=ratio between feed consumed and weight gained - Mortality was recorded daily and the FCR was adjusted accordingly.
- Data (n=40) were subjected to an ANOVA, with complete randomized block design using the ANOVA procedure of XLSTAT (Addinsoft 1995-2014) to establish differences between diets. Pen was considered as the experimental unit. The model included diets (n=5) and block as fixed effect. Results are reported as least square means. LS means were assumed to be different at P≥0.05.
- Similar calculations were realized in the example 9 and 10.
- The results obtained on performance parameters for the whole experimental period are shown in Table 8.3.
-
TABLE 8.3 Effect of dietary supplementation of the strains DSM 29870, DSM 29871 and DSM 29872 on broiler performance DSM 298702 DSM 298712 DSM 298722 GalliPro Max3 supplemented supplemented supplemented supplemented Parameters4 Control1 group group group group FI (g/bird) 3245a 3256a 3221a 3263a 3203a Relative to the control — +0.3% −0.7% +0.6% −1.3% BWG (g/bird) 1987ab 2026ab 2002ab 2038a 1973b Relative to the control — +2.0% +0.8% +2.6% −0.7% FCR 1.633a 1.607b 1.609b 1.602b 1.624ab Relative to the control — −1.6% −1.5% −2.0% −0.6% Mortality (%) 3.25a 3.50a 5.25a 4.5a 6.25a 1Group fed with the basal diet. 2DSM 29870/29871/29872 supplemented group fed with the basal diet including strain DSM 29870/29871/29872 at 5.108 CFU/kg of feed. 3GalliPro Max supplemented group fed with the basal diet including GalliPro Max at 8.108 CFU/kg of feed. 4Means from 8 replicates of 50 birds per pen for each group. Raw data (n = 24) were analyzed using variance analysis with block (n = 8) and treatments (n = 3) as fixed effect. a, b, cValues with different superscripts are significantly (P < 0.05) different from each other. - Feed additives did not significantly affect animal feed intake relative to the control. Body weight was slightly increased using the NZB strains whereas GalliPro Max tended to decrease this parameter.
- The DSM 29870, DSM 29871 and DSM 29872 strains also improved significantly the feed conversion ratio by 1.6%, 1.5% and 1.9%, respectively, with no significant differences between the groups fed with NZB strains. GalliPro Max had only a slight and non-significant effect on the feed conversion ratio.
- None of the probiotic tested here had a significant effect on the mortality level.
- A total of 900 one day-old male broiler chickens Ross 708 were individually weighed (body weight 47.1 g±1.1 g) and allocated in floor pens (15 birds/pens). Pens were used in factorial and completely randomized design with 12 pens per treatment (i.e., 180 animals/treatment). The 5 experimental treatments consisted in (Table 9.1): T1, negative control basal diets; T2, basal diets containing the strain DSM 29870 (included at 5.108 CFU/kg of feed, from d1 to d35); T3, basal diets containing the strain DSM 29871 (included at 5.108 CFU/kg of feed, from d1 to d35); T4 basal diets containing the strain DSM 29872 (included at 5.108 CFU/kg of feed, from d1 to d35); T5, basal diet containing a marketed probiotic, GalliPro Max (included at the commercial dose, 8.108 CFU/kg of feed, from d1 to d35).
- Basal diets consisted in 3 phases feeding program: starter phase (from 1 to day 14), grower phase (from 15 to day 21) and finisher phase (from 22 to day 35). Every phases were formulated to meet or exceed animal requirement and agree with standard commercial US corn-soybean meal-based broiler diets. The dietary and raw material composition of these diets is given in Table 9.2. All experimental diets included vitamin and mineral premixes, but did not contain any coccidiostat, NSPase, phytase or growth promoting substance. Feed and water were available ad libitum throughout the trial.
-
TABLE 9.1 Experimental treatments Treatments T1 Control T2 T1 + DSM 29870 T3 T1 + DSM 29871 T4 T1 + DSM 29872 T5 T1 + GalliPro Max -
TABLE 9.2 Composition of the basal experimental diets Starter Grower Finisher (0-14 d) (15-21 d) (22-35 d) % Ingredients Corn 57.59 59.20 62.30 Wheat Flour 1.00 1.00 1.00 Soybean Meal (46% CP) 30.00 28.00 24.50 Poultry Meal 5.00 5.00 5.00 Poultry Fat 2.10 2.86 3.52 Limestone (36% Ca) 1.05 1.02 0.98 Dicalcium Phosphate (18% P) 1.76 1.52 1.42 Salt 0.35 0.32 0.29 Sodium Bicarbonate 0.16 0.14 0.11 L-Lysine HCL (78%) 0.23 0.22 0.21 DL-Methionine (99%) 0.36 0.33 0.29 L-Threonine (98%) 0.05 0.04 0.03 Selenium premix 0.05 0.05 0.05 Vitamin Mix 0.15 0.15 0.15 Mineral Mix 0.15 0.15 0.15 Calculated Nutritional Content ME (kcal/kg) 2968 3032 3096 Crude Protein (%) 23.3 22.4 20.78 Lysine (%) 1.35 1.29 1.18 Methionine (%) 0.69 0.65 0.59 Met + Cys (%) 1.05 1.00 0.92 Threonine (%) 0.86 0.81 0.75 Calcium (%) 1.03 0.96 0.91 Phosphorus Avail. (%) 0.46 0.41 0.39 1Vitamin mix will provide the following (per kg of diet): vitamin A, 13 227 513 IU; vitamin D3, 3 968 254 IU; vitamin E, 66 138 IU; vitamin B12, 40 mg; biotin, 254 mg; menadione, 3 968 mg; thiamine, 3 968 mg; riboflavin, 13 228 mg; d-Pantothenic Acid, 22 046 mg; pyridoxine, 7 937 mg; niacin, 110 229 mg; folic acid, 2 205 mg; Selenium Premix: selenium, 600 ppm + calcium, 36% 2Trace mineral mix provided the following (per kg of diet): calcium, Min: 15.7% and Max: 18.7%; manganese (Mn), 6.0%; zinc (Zn), 6.0%; iron (Fe), 4.0%; copper (Cu), 5000 ppm; iodine (I), 1250 ppm; cobalt (Co), 500 ppm - The results obtained on performance parameters for the whole experimental period are shown in Table 9.3.
-
TABLE 9.3 Effect of dietary supplementation of the strains DSM 29870, DSM 29871 and DSM 29872 on broiler performance DSM 298702 DSM 298712 DSM 298722 GalliPro Max3 supplemented supplemented supplemented supplemented Parameters4 Control1 group group group group FI (g/bird) 3573a 3542a 3606a 3515a 3541a Relative to the control — −0.9% +0.9% −1.0% −0.9% BWG (g/bird) 2361a 2416b 2401ab 2424b 2398ab Relative to the control — +2.3% +1.7% +2.6% +1.5% FCR 1.513a 1.467b 1.501bc 1.451a 1.477ab Relative to the control — −3.6% −0.8% −4.2% −2.4% Mortality (%) 15.6ab 7.8a 11.7ab 19.4b 10.0a 1Group fed with the basal diet 2DSM 29870/29871/29872 supplemented group fed with the basal diet including strain DSM 29870/29871/29872 at 5.108 CFU/kg of feed. 3GalliPro Max supplemented group fed with the basal diet including GalliPro Max at 8.108 CFU/kg of feed. 4Means from 12 replicates of 15 birds per pen for each group. Raw data (n = 36) were analyzed using variance analysis with block (n = 12) and treatments (n = 3) as fixed effect. a, b, cValues with different superscripts are significantly (P < 0.05) different from each other - Also in this experiment, the administration of the strains DSM 29870, DSM 29871, DSM 29872 and GalliPro Max did not significantly reduce the feed intake, but improved the body weight gain by 2.3%, 1.7%, 2.6% and 1.5%, respectively. Therefore, difference relatively to control was significant for DSM 29870 and DSM 29872.
- The strains DSM 29870 and DSM 29872 improved significantly the feed conversion ratio by 3.6% and 4.2%, respectively. DSM 29871 and GalliPro Max also improved (slightly for DSM 29871) the feed conversion ratio in this experiment, but not significantly.
- The strain DSM 29870 allowed a decrease of the mortality level in a numerically higher manner than GalliPro Max.
- A total of 1080 one day-old male broiler chickens Ross PM3 were individually weighed (body weight 42±3.5 g) and allocated in floor pens (18 birds/pens). Pens were used in factorial and completely randomized design with 12 pens per treatment (i.e., 216 animals/treatment). The 3 experimental treatments consisted in (Table 10.1): T1, negative control basal diets; T2, basal diets containing the strain DSM 29870 (included at 5.108 CFU/kg of feed, from d1 to d35); T3, basal diets containing the strain DSM 29871 (included at 5.108 CFU/kg of feed, from d1 to d35); T4 basal diets containing the strain DSM 29872 (included at 5.108 CFU/kg of feed, from d1 to d35); T5, basal diet containing a marketed probiotic, GalliPro Max (included at the commercial dose, 8.108 CFU/kg of feed, from d1 to d35).
- Basal diets consisted in 3 phases feeding program: starter phase (from 1 to day 21) and grower phase (from 22 to day 35). Every phase was formulated to meet or exceed animal requirement and agree with standard commercial EU corn-soybean meal-based broiler diets. The dietary and raw material composition of these diets is given in Table 10.2. All experimental diets included vitamin and mineral premixes, but did not contain any coccidiostat, NSPase, phytase or growth promoting substance. Feed and water were available ad libitum throughout the trial.
-
TABLE 10.1 Experimental treatments Treatments T1 Control T2 T1 + DSM 29870 T3 T1 + DSM 29871 T4 T1 + DSM 29872 T5 T1 + GalliPro Max -
TABLE 10.2 Composition of the basal experimental diets Starter Grower (0-21 d) (22-35 d) % Ingredients Maize 51.68 55.58 Soybean Meal (48% CP) 39.03 34.55 Soybean oil 4.27 5.18 DL-Methionine 0.22 0.17 Calcium carbonate 1.05 1.05 Dicalcium Phosphate 1.78 1.5 Salt 0.37 0.37 Wheat middlings 1.00 1.00 Premix1 0.6 0.6 Calculated Nutritional Content ME (kcal/kg) 3000 3100 Crude Protein (%) 22.25 20.5 Fat (%) 6.76 7.7 Cellulose (%) 3.15 3 Minerals (%) 5.74 5.3 Lysine (%) 1.25 1.13 Methionine (%) 0.55 0.48 Met + Cys (%) 0.92 0.83 Threonine (%) 0.87 0.8 Calcium (%) 0.93 0.85 Total phosphorus 0.7 0.63 Phosphorus Avail. (%) 0.38 0.33 1The premix will provide the following (per kg of diet): vitamin A, 12 000 IU; vitamin D3, 3 000 IU;, vitamin E = 300 IU;, vitamin K3, 3 mg; vitamin B, 2 mg; vitamin B2, 8 mg; vitamin B6, 3 mg; vitamin B12, 0.02 mg; folic acid, 1 mg; biotin, 0.2 mg; calcium pantothenate, 15 mg; nicotinic acid, 40 mg; manganese (Mn), 80 mg; zinc (Zn), 60 mg; iodine (I), 1 mg, iron (Fe), 80 mg; copper (Cu), 15 mg; cobalt (Co), 0.4 mg; selenium (Se), 0.2 mg; magnesium (Mg), 5 mg; Etoxyquin, 0.5 mg; BHA, 0.5 mg, citric acid, 5 mg; phosphoric acid, 5 mg. - The results obtained on performance parameters for the whole experimental period are shown in Table 10.3.
-
TABLE 10.3 Effect of dietary supplementation of the strains DSM 29870, DSM 29871 and DSM 29872 on broiler performance DSM 298702 DSM 298712 DSM 298722 GalliPro Max3 supplemented supplemented supplemented supplemented Parameters4 Control1 group group group group FI (g/bird) 3217a 3295a 3281a 3230a 3246a Relative to the control — +2.4% +2.0% +0.4% +0.9% BWG (g/bird) 2019b 2149a 2167a 2124a 2103ab Relative to the control — +6.5% +7.3% +5.2% +4.2% FCR 1.598a 1.534b 1.513b 1.521b 1.544b Relative to the control — −4.0% −5.3% −4.8% −3.4% Mortality (%) 11.1a 9.7a 8.3a 6.5a 7.4a 1Group fed with the basal diet. 2DSM 29870/29871/29872 supplemented group fed with the basal diet including strain DSM 29870/29871/29872 at 5.108 CFU/kg of feed. 3GalliPro Max supplemented group fed with the basal diet including GalliPro Max at 8.108 CFU/kg of feed. 4Means from 12 replicates of 18 birds per pen for each group. Raw data (n = 36) were analyzed using variance analysis with block (n = 12) and treatments (n = 3) as fixed effect. a, b, cValues with different superscripts are significantly (P < 0.05) different from each other. - In this experiment, the administration of the strains DSM 29870, DSM 29871, DSM 29872 (as GalliPro Max) had not significant effect on the feed intake, but led to the significant improvement of the body weight gain by 6.5%, 7.3% and 5.2%, respectively. GalliPro Max improved also the body weight gain (+4.2%), but not significantly.
- All NZB strains improved significantly the feed conversion ratio by 4.0%, 5.3% and 4.8% (DSM 29870, DSM 29871 and DSM 29872, respectively) as GalliPro Max which reached 3.4% of improvement.
- Regarding the mortality level, there were no significant differences between the treatments.
- The results of the three performance experiments described above are summarized in Table 11.1.
-
TABLE 11.1 Results of the individual studies Relative Relative Relative FI to the BWG to the to the Trial Treatments (g/bird) control (g/bird) control FCR control 1 Control 3245a — 1987ab — 1.633a — DSM 29870 3256a +0.3% 2026ab +2.0% 1.607b −1.6% DSM 29871 3221a −0.7% 2002ab +0.8% 1.609b −1.5% DSM 29872 3263a +0.6% 2038a +2.6% 1.602b −2.0% GalliPro Max 3203a −1.3% 1973b −0.7% 1.624ab −0.6% 2 Control 3573a — 2361a — 1.513a — DSM 29870 3542a −0.9% 2416b +2.3% 1.467b −3.6% DSM 29871 3606a +0.9% 2401ab +1.7% 1.501bc −0.8% DSM 29872 3515a −1.0% 2424b +2.6% 1.451a −4.2% GalliPro Max 3541a −0.9% 2398ab +1.5% 1.477ab −2.4% 3 Control 3217a — 2019b — 1.598a — DSM 29870 3295a +2.4% 2149a +6.5% 1.534b −4.0% DSM 29871 3281a +2.0% 2167a +7.3% 1.513b −5.3% DSM 29872 3230a +0.4% 2124a +5.2% 1.521b −4.8% GalliPro Max 3246a +0.9% 2103ab +4.2% 1.544b −3.4% a, b, c Values with different superscripts are significantly (P < 0.05) different from each other. - These trials having a similar experimental design, all the data obtained were pooled and combined in a meta-analysis (after having been tested for homogeneity). This meta-analysis involved 3 980 broilers in 160 replicates (32 replicates and 796 animals for each experimental group). The results are shown in Table 11.
- Data (n=160) were subjected to a mixed model ANOVA, using the GLIMMIX procedure of SAS (SAS Institute, 2002-2012) to establish differences between diets. Pen was considered as the experimental unit. The model included diets (n=5) as fixed effect and experiment (n=3) as random effect. Results are reported as least square means. LS means were assumed to be different at P≥0.05.
-
TABLE 11.2 meta-analysis of three experiments showing the effect of dietary supplementation of the strain DSM 29870, DSM 29871 and DSM 29872 on broiler performance DSM 298702 DSM 298712 DSM 298722 GalliPro Max3 supplemented supplemented supplemented supplemented Parameters4 Control1 group group group group FI (g/bird) 3343a 3360a 3361a 3356a 3347a Relative to the control — +0.5% +0.5% +0.4% +0.1% BWG (g/bird) 2145b 2208a 2202ab 2211a 2171ab Relative to the control — +2.9% +2.6% +3.0% +1.2% FCR 1.560a 1.530b 1.53bc 1.52c 1.550ab Relative to the control — −2.4% −2.2% −2.7% −1.1% 1Group fed with the basal diet 2DSM 29870/29871/29872 supplemented group fed with the basal diet including strain DSM 29870/29871/29872 at 5.108 CFU/kg of feed. 3GalliPro Max supplemented group fed with the basal diet including GalliPro Max at 8.108 CFU/kg of feed. 4Means from 32 observations for each experimental group (796 animals/group). a, b, cValues with different superscripts are significantly (P < 0.05) different from each other. - This meta-analysis showed that the use of the strain DSM 29870, DSM 29871 or DSM 29872 in a corn/soybean meal-based diet significantly improved in average the body weight gain (+2.9%, +2.6% and +3.0%, respectively) and the feed conversion ratio (−2.4%, −2.2% and −2.7%, respectively) with no effect on the feed intake.
- In the working examples above, positive effects of strain DSM 29870 have been demonstrated on broiler performances (i.e., body weight gain and feed conversion ratio) fed a corn/soybean meal-based diet.
- All the data obtained from the experiments described in the above examples also showed that the effect of strain DSM 29870 on the feed conversion ratio is due to its effect on the body weight. These effects might be associated with either health effect or metabolism improvement.
- GalliPro Max showed no significant effect on the body weight gain in any of the working examples above and showed significant and positive effect on feed conversion ratio in one third of the experiments.
- In the experiments described in the above examples, the better effect of strain DSM 29870 compared to GalliPro Max on broilers performance has been demonstrated. The effect observed using DSM 29870 were in average twice those observed for GalliPro Max.
- Monensin compatibility of Bacillus strains DSM 29869, DSM 29870, DSM 29871, and DSM 29872 was determined using a modified broth micro dilution similar to the method described in the Example 5. Briefly, a single colony of Bacillus spp. (from overnight tryptic soy agar plates) was inoculated into Mueller Hinton Broth (MHB) and cultured overnight. Sterile media was the inoculated with the overnight culture and allowed to grow for 4 hours to test bacteria in log growth phase. Cultures were then diluted once more 1:200 into fresh MHB and 90 μL of this inoculated broth was added to the diluted monensin at the indicated concentrations. Prior art strains were also tested for comparison: NN019785, NN062266 (NRRL B-50013), NN062267 (NRRL B-50104), NN062278 (PTA-6507), NN062319 (FERM BP-1096), NN062440, NN062441 (DSM 17236), NN062439.
-
-
- Bacillus amyloliquefaciens DSM 29869
- Bacillus subtilis DSM 29870
- Bacillus subtilis DSM 29871
- Bacillus amyloliquefaciens DSM 29872
- Bacillus licheniformis NN019785
- Bacillus amyloliquefaciens NN062266 (NRRL B-50013)
- Bacillus subtilis NN062267 (NRRL B-50104)
- Bacillus subtilis NN062278 (PTA-6507)
- Bacillus amyloliquefaciens NN062319 (FERM BP-1096)
- Bacillus subtilis NN062440
- Bacillus licheniformis NN062441 (DSM 17236)
- Bacillus amyloliquefaciens NN062439
-
-
- Monensin sodium salt (Sigma, CAS no. 22373-78-0, solubilized in 96% ethanol)
- Mueller Hinton Broth (Becton, Dickinson and Company, 275730)
- Tryptic soy agar (Becton, Dickinson and Company, 236920)
- Micro titer plates: Costar plate, polypropylene, flat bottom, Corning, 3628
- Borosilicate glass tubes: Kimbale, 16×125 mm, 73500-16125
- Adhesive gas permeable seals: Thermo Scientific, AB-0718
- Bacillus spp. were grown overnight on tryptic soy agar plates (40 g/L) at 37° C. Mueller Hinton broth (21 g/L) was dissolved in water and autoclaved in glass tubes containing 5 mL of broth each. A single colony of Bacillus spp. (from overnight plates) was inoculated into Mueller Hinton Broth (MHB) and incubated overnight at 37° C. shaking at 200 rpm. A 5 mL glass tube of fresh, sterile media was then inoculated with 25 mL of overnight culture and allowed to grow for 4 hours at 37° C. Cultures were then diluted once more 1:200 into fresh MHB. 90 μL of this inoculated broth was then added to the diluted antibiotic at the indicated concentrations.
- Monensin was diluted into 96% ethanol to a concentration of 800 μg/mL. This solution was then diluted 10-fold into sterile phosphate buffer to a concentration of 80 μg/mL. A two fold dilution series was prepared in MHB down to the concentration 2.5 μg/mL. 10 μl of each dilution and of each antibiotic was pipetted into a micro titer plate. Later, when the antibiotics were mixed with the suspension of bacteria, the samples were diluted 10× (10 μL sample in a total volume of 100 μl). This resulted in the final test range of 0.25-8 μg/ml.
- 90 μl of the bacterial suspensions were added to the assay plates. The assay plates were then covered with an adhesive glass permeable seal and incubated overnight at 37° C. shaking at 200 rpm. The maximum compatible concentration was determined similar to a MIC analysis as the concentration above that which inhibited 80% of bacteria as detected by the unaided eye.
- A potential challenge of delivering Bacillus spp. in feed is the common use of antibiotics as growth promoters in feed. Therefore it is necessary to determine the compatibility of strains with commonly-used feed antibiotics in order to identify any potential conflicts with use as a direct fed microbial. Therefore, the monensin compatibility of Bacillus strains DSM 29869, DSM 29870, DSM 29871, and DSM 29872 were determined along with prior art strains. Bacillus strains DSM 29869, DSM 29870, DSM 29871, and DSM 29872 indicated a higher level of compatibility with monensin than most of the prior art strains included herein: NN019785, NN062266 (NRRL B-50013), NN062267 (NRRL B-50104), NN062278 (PTA-6507), NN062319 (FERM BP-1096), NN062440, NN062441 (DSM 17236), NN062439.
-
TABLE 12.1 Monensin compatibility results Monensin Species Product Name (μg/mL) NN062677 DSM29870 Bacillus subtilis 2.7 NN062673 DSM29871 Bacillus subtilis 2.0 NN062683 DSM29872 Bacillus amyloliquefaciens 4.0 NN062676 DSM29869 Bacillus amyloliquefaciens 2.0 NN019785 Bacillus licheniformis BioPlus 2B (Chr. Hansen) 0.8 NN062266 NRRL B- Bacillus amyloliquefaciens Eviva Pro (Dupont) 1.1 50013 NN062267 NRRL B- Bacillus subtilis Eviva Pro (Dupont) 1.1 50104 NN062278 PTA-6507 Bacillus subtilis Eviva Pro (Dupont) 1.4 NN062319 FERM BP- Bacillus amyloliquefaciens Calsporin (Calpis) 2.2 1096 NN062440 Bacillus subtilis GalliPro Max (Chr. Hansen) 0.4 NN062441 DSM17236 Bacillus licheniformis GalliPro Tect (Chr. Hansen) 0.9 NN062439 Bacillus amyloliquefaciens Clostat (Kemin) 2.1
Claims (20)
1. An animal feed or animal feed additive comprising a carrier, a flowability agent and a Bacillus strain having a 16S rDNA with more than 98% sequence identity to SEQ ID NO: 2.
2. The animal feed or animal feed additive of claim 1 , wherein the Bacillus strain has antimicrobial activity against Clostridium perfringens.
3. The animal feed or animal feed additive of claim 1 , wherein the Bacillus strain has antimicrobial activity against Escherichia coli.
4. The animal feed or animal feed additive of claim 1 , wherein the Bacillus strain is sensitive to at least seven of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline.
5. The animal feed or animal feed additive of claim 1 , wherein the Bacillus strain is non-hemolytic.
6. The animal feed or animal feed additive of claim 1 , wherein the Bacillus strain is present in said animal feed or animal feed additive as dried spores.
7. The animal feed or animal feed additive of claim 1 , wherein the Bacillus strain is present in said animal feed or animal feed additive at rate of between 1×104 and 1×1018 colony forming units per kilogram of animal feed or animal feed additive.
8. The animal feed or animal feed additive of claim 1 , wherein the carrier comprises one or more of the following: glycerol, ethylene glycol, 1, 2-propylene glycol or 1, 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose, sucrose, sorbitol, lactose, wheat flour, wheat bran, corn gluten meal, starch, farigel, cassava cores, Sipernat 50S, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 4000, carbopol, and cellulose.
9. The animal feed or animal feed additive of claim 1 , wherein the carrier comprises calcium carbonate.
10. The animal feed or animal feed additive of claim 1 , wherein the flowability agent comprises sodium aluminum silicate and/or colloidal amorphous silica.
11. The animal feed or animal feed additive of claim 1 , further comprising one or more additional components selected from the group consisting of enzymes, microbes, vitamins, minerals, amino acids and other feed ingredients.
12. A method comprising administering the animal feed or animal feed additive of claim 1 to an animal.
13. The method of claim 12 , wherein said animal feed or animal feed additive is administered to said animal in an amount effective to improve one or more performance parameters of said animal.
14. The method of claim 12 , wherein said animal is monogastric.
15. The method of claim 12 , wherein said animal is selected from the group consisting of poultry and swine.
16. An animal feed or animal feed additive comprising a carrier, a flowability agent and dried spores of a Bacillus strain, wherein said Bacillus strain:
has a 16S rDNA with more than 98% sequence identity to SEQ ID NO: 2;
has antimicrobial activity against Clostridium perfringens and/or Escherichia coli;
is sensitive to at least seven of Vancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline; and
is non-hemolytic.
17. A method comprising administering the animal feed or animal feed additive of claim 16 to an animal.
18. The method of claim 17 , wherein said animal feed or animal feed additive is administered to said animal in an amount effective to improve one or more performance parameters of said animal.
19. The method of claim 17 , wherein said animal is monogastric.
20. The method of claim 17 , wherein said animal is selected from the group consisting of poultry and swine.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/746,731 US20230372413A1 (en) | 2015-01-23 | 2022-05-17 | Bacillus strains improving health and performance of production animals |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201562106869P | 2015-01-23 | 2015-01-23 | |
US201562109210P | 2015-01-29 | 2015-01-29 | |
US201562260892P | 2015-11-30 | 2015-11-30 | |
PCT/US2016/014492 WO2016118840A1 (en) | 2015-01-23 | 2016-01-22 | Bacillus strains improving health and performance of production animals |
US201715543663A | 2017-07-14 | 2017-07-14 | |
US17/746,731 US20230372413A1 (en) | 2015-01-23 | 2022-05-17 | Bacillus strains improving health and performance of production animals |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/543,663 Division US11331351B2 (en) | 2015-01-23 | 2016-01-22 | Bacillus strains improving health and performance of production animals |
PCT/US2016/014492 Division WO2016118840A1 (en) | 2015-01-23 | 2016-01-22 | Bacillus strains improving health and performance of production animals |
Publications (1)
Publication Number | Publication Date |
---|---|
US20230372413A1 true US20230372413A1 (en) | 2023-11-23 |
Family
ID=55646836
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/543,663 Active US11331351B2 (en) | 2015-01-23 | 2016-01-22 | Bacillus strains improving health and performance of production animals |
US17/746,731 Pending US20230372413A1 (en) | 2015-01-23 | 2022-05-17 | Bacillus strains improving health and performance of production animals |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/543,663 Active US11331351B2 (en) | 2015-01-23 | 2016-01-22 | Bacillus strains improving health and performance of production animals |
Country Status (3)
Country | Link |
---|---|
US (2) | US11331351B2 (en) |
BR (1) | BR112017015623B1 (en) |
WO (1) | WO2016118840A1 (en) |
Families Citing this family (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016118864A1 (en) | 2015-01-23 | 2016-07-28 | Novozymes A/S | Bacillus strains improving health and performance of production animals |
US10201574B1 (en) | 2015-09-16 | 2019-02-12 | Church & Dwight Co., Inc. | Methods of microbial treatment of poultry |
US11298383B2 (en) | 2016-05-20 | 2022-04-12 | Church & Dwight Co., Inc. | Lactobacillus and bacillus based direct fed microbial treatment for poultry and method of use |
BR112018074133A2 (en) | 2016-05-25 | 2019-03-06 | Church & Dwight Co., Inc. | Bacillus compositions and methods of use with ruminants |
CN106509372A (en) * | 2016-10-09 | 2017-03-22 | 杨松杞 | New water-soluble carrier and preparation method thereof |
MX2019010805A (en) | 2017-03-14 | 2019-12-16 | Chr Hansen As | Bacillus subtilis strains improving animal performance parameters. |
US11622569B2 (en) | 2017-07-24 | 2023-04-11 | Church & Dwight Co., Inc. | Bacillus microbial terroir for pathogen control in swine |
AR113371A1 (en) | 2017-10-18 | 2020-04-22 | Ascus Biosciences Inc | INCREASE IN POULTRY PRODUCTION THROUGH THE ADMINISTRATION OF A SYNTHETIC BIOASSEMBLY OF MICROBES OR PURIFIED STRAINS OF THEM |
EP3740594A1 (en) * | 2018-01-19 | 2020-11-25 | Chr. Hansen A/S | Bacillus subtilis for animal feed |
JP7387095B2 (en) * | 2018-03-16 | 2023-11-28 | 味の素株式会社 | Feed additives and feed |
US20190289878A1 (en) * | 2018-03-23 | 2019-09-26 | Purina Animal Nutrition Llc | Methods of feeding animals feed products containing direct-fed microbials |
US20220117264A1 (en) * | 2019-01-10 | 2022-04-21 | Evonik Operations Gmbh | Fermentation broths and their use |
CN111587954B (en) * | 2020-06-17 | 2023-01-31 | 湖南华佑生物科技有限公司 | Feed particle for improving beef quality and preparation method thereof |
CN112481163A (en) * | 2020-12-01 | 2021-03-12 | 华东师范大学 | Bacterial strain for aquatic feed addition and application thereof |
Family Cites Families (33)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS63209580A (en) | 1987-02-25 | 1988-08-31 | Karupisu Shokuhin Kogyo Kk | Bacillus subtilis c-3102 |
DK91192D0 (en) | 1992-07-10 | 1992-07-10 | Novo Nordisk As | PROTEIN |
US5733355A (en) * | 1994-09-29 | 1998-03-31 | Susumu Hibino | Bacterial Preparation for agricultural use |
CZ87497A3 (en) | 1995-07-28 | 1998-03-18 | Gist-Brocades B. V. | Enzyme preparation stabilized by means of salts |
JP3504672B2 (en) | 1996-12-20 | 2004-03-08 | ノボザイムス アクティーゼルスカブ | Peniophora phytase |
GB2341077B (en) | 1997-06-04 | 2001-06-13 | Dsm Nv | Carbohydrate-based enzyme granulates |
CA2356642A1 (en) | 1999-01-22 | 2000-07-27 | Novozymes A/S | Improved phytases |
DE19922753A1 (en) | 1999-05-18 | 2000-11-23 | Basf Ag | New instant enzyme formulation, useful as animal feed supplement, made by agglomerating a water-soluble powdered carrier by spraying on a solution of an enzyme preparation or a binder |
DE19929257A1 (en) | 1999-06-25 | 2000-12-28 | Basf Ag | Production of polymer-coated granulated animal feed additive, useful in production of pelletized animal feed, involves granulating mixture of carrier and enzyme and coating with suitable organic polymer |
US7319087B2 (en) | 2001-05-04 | 2008-01-15 | Novozymes A/S | Antimicrobial polypeptide from Aspergillus niger |
DE60215534T2 (en) | 2001-11-20 | 2007-09-20 | Novozymes A/S | ANTIMICROBIAL POLYPEPTIDES FROM PSEUDOPLECTANIA NIGRELLA |
WO2003048148A2 (en) | 2001-12-03 | 2003-06-12 | Novozymes A/S | Statin-like compounds |
EP1474508B1 (en) | 2002-02-08 | 2011-11-16 | Novozymes A/S | Phytase variants |
US7247299B2 (en) | 2002-11-27 | 2007-07-24 | Kemin Industries, Inc. | Antimicrobial compounds from Bacillus subtilis for use against animal and human pathogens |
EP2143338A1 (en) | 2004-09-27 | 2010-01-13 | Novozymes A/S | Enzyme Granules |
US20080057047A1 (en) | 2005-11-29 | 2008-03-06 | Benedikt Sas | Use of bacillus amyloliquefaciens PB6 for the prophylaxis or treatment of gastrointestinal and immuno-related diseases |
EP3072399B1 (en) | 2006-08-07 | 2018-12-19 | Novozymes A/S | Enzyme granules for animal feed |
US8021654B2 (en) | 2008-03-14 | 2011-09-20 | Danisco A/S | Methods of treating pigs with Bacillus strains |
US20090318292A1 (en) | 2008-06-23 | 2009-12-24 | Novozymes A/S | Bacillus Subtilis Strain |
US8540981B1 (en) | 2008-07-07 | 2013-09-24 | Dupont Nutrition Biosciences Aps | Bacillus strains useful against calf pathogens and scours |
CA2736885C (en) * | 2008-09-17 | 2016-12-06 | Agraquest, Inc. | Method for using a bacillus subtilis strain to enhance animal health |
US20110318289A1 (en) | 2010-06-24 | 2011-12-29 | Novozymes Biologicals, Inc. | Methods and Compositions for Reducing Body Odor |
WO2012009712A2 (en) | 2010-07-16 | 2012-01-19 | The Board Of Trustees Of The University Of Arkansas | Methods and compositions including spore-forming bacteria for increasing the health of animals |
KR101242821B1 (en) * | 2011-01-31 | 2013-03-12 | 씨제이제일제당 (주) | Probiotics Agent Against Vibrio sp. |
GB201102865D0 (en) * | 2011-02-18 | 2011-04-06 | Danisco | Feed additive composition |
GB201102857D0 (en) | 2011-02-18 | 2011-04-06 | Danisco | Feed additive composition |
PL2748300T3 (en) * | 2011-08-24 | 2019-05-31 | Dupont Nutrition Biosci Aps | Enzyme producing bacillus strains |
CN104768391A (en) | 2011-12-19 | 2015-07-08 | 诺维信公司 | Processes and compositions for increasing the digestibility of cellulosic materials |
WO2013153159A1 (en) * | 2012-04-13 | 2013-10-17 | Chr. Hansen A/S | Antibiotic sensitive bacillus strains having antimicrobial effect against e. coli and clostridium perfringens and having high sporulation capacity |
GB201213801D0 (en) | 2012-08-03 | 2012-09-12 | Dupont Nutrition Biosci Aps | Feed additive composition |
US8906668B2 (en) * | 2012-11-23 | 2014-12-09 | Seres Health, Inc. | Synergistic bacterial compositions and methods of production and use thereof |
CN103882097A (en) | 2012-12-21 | 2014-06-25 | 青岛中仁药业有限公司 | Identification method of bacillus subtilis with high protease output |
US11883443B2 (en) | 2013-04-09 | 2024-01-30 | Novozymes A/S | Compositions and methods for improving the health of aquatic animals |
-
2016
- 2016-01-22 WO PCT/US2016/014492 patent/WO2016118840A1/en active Application Filing
- 2016-01-22 BR BR112017015623-7A patent/BR112017015623B1/en active IP Right Grant
- 2016-01-22 US US15/543,663 patent/US11331351B2/en active Active
-
2022
- 2022-05-17 US US17/746,731 patent/US20230372413A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
BR112017015623B1 (en) | 2022-05-31 |
BR112017015623A2 (en) | 2018-03-13 |
US11331351B2 (en) | 2022-05-17 |
WO2016118840A1 (en) | 2016-07-28 |
US20180264052A1 (en) | 2018-09-20 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20230372413A1 (en) | Bacillus strains improving health and performance of production animals | |
US11801272B2 (en) | Bacillus strains improving health and performance of production animals | |
US11291695B2 (en) | Bacillus subtilis strains improving animal performance parameters | |
CA3004522C (en) | Feed additive composition | |
US20170246222A1 (en) | Bacillus Strains with Fast Germination and Antimicrobial Activity against Clostridium | |
US20230287329A1 (en) | Bacillus subtilis subspecies | |
JP2019520064A (en) | Bacillus subtilis strain exhibiting probiotic activity | |
US11819525B2 (en) | Management of pathogenic lawsonia | |
EA043066B1 (en) | COMPOSITION FOR THE PREVENTION OR CONTROL OF BACTERIAL COLONIZATION OR INFECTION IN THE INTESTINE OF POULTRY |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |