US20180087117A1 - Anti-microbial agent from paenibacillus sp. and methods and uses thereof - Google Patents
Anti-microbial agent from paenibacillus sp. and methods and uses thereof Download PDFInfo
- Publication number
- US20180087117A1 US20180087117A1 US15/727,184 US201715727184A US2018087117A1 US 20180087117 A1 US20180087117 A1 US 20180087117A1 US 201715727184 A US201715727184 A US 201715727184A US 2018087117 A1 US2018087117 A1 US 2018087117A1
- Authority
- US
- United States
- Prior art keywords
- bacterium
- cell culture
- microbial agent
- paenibacillus polymyxa
- polymyxa
- 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.)
- Abandoned
Links
- 239000004599 antimicrobial Substances 0.000 title claims abstract description 159
- 238000000034 method Methods 0.000 title claims abstract description 58
- 241000592795 Paenibacillus sp. Species 0.000 title abstract description 6
- 241000194105 Paenibacillus polymyxa Species 0.000 claims abstract description 156
- 241000894006 Bacteria Species 0.000 claims abstract description 97
- 238000004113 cell culture Methods 0.000 claims abstract description 62
- 239000012228 culture supernatant Substances 0.000 claims abstract description 59
- 239000000203 mixture Substances 0.000 claims abstract description 29
- 230000000845 anti-microbial effect Effects 0.000 claims description 70
- 241000588724 Escherichia coli Species 0.000 claims description 55
- 230000012010 growth Effects 0.000 claims description 52
- 108090000623 proteins and genes Proteins 0.000 claims description 50
- 238000010186 staining Methods 0.000 claims description 41
- 239000000126 substance Substances 0.000 claims description 38
- XDJYMJULXQKGMM-UHFFFAOYSA-N polymyxin E1 Natural products CCC(C)CCCCC(=O)NC(CCN)C(=O)NC(C(C)O)C(=O)NC(CCN)C(=O)NC1CCNC(=O)C(C(C)O)NC(=O)C(CCN)NC(=O)C(CCN)NC(=O)C(CC(C)C)NC(=O)C(CC(C)C)NC(=O)C(CCN)NC1=O XDJYMJULXQKGMM-UHFFFAOYSA-N 0.000 claims description 33
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 32
- 230000002401 inhibitory effect Effects 0.000 claims description 32
- KNIWPHSUTGNZST-UHFFFAOYSA-N polymyxin E2 Natural products CC(C)CCCCC(=O)NC(CCN)C(=O)NC(C(C)O)C(=O)NC(CCN)C(=O)NC1CCNC(=O)C(C(C)O)NC(=O)C(CCN)NC(=O)C(CCN)NC(=O)C(CC(C)C)NC(=O)C(CC(C)C)NC(=O)C(CCN)NC1=O KNIWPHSUTGNZST-UHFFFAOYSA-N 0.000 claims description 26
- 108010040201 Polymyxins Proteins 0.000 claims description 21
- 239000006143 cell culture medium Substances 0.000 claims description 19
- 235000013305 food Nutrition 0.000 claims description 17
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 17
- 241001465754 Metazoa Species 0.000 claims description 16
- XDJYMJULXQKGMM-RVYUQJQSSA-N colistin A Chemical compound CC[C@@H](C)CCCCC(=O)N[C@@H](CCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O XDJYMJULXQKGMM-RVYUQJQSSA-N 0.000 claims description 15
- 239000000047 product Substances 0.000 claims description 15
- 241001646719 Escherichia coli O157:H7 Species 0.000 claims description 13
- KNIWPHSUTGNZST-SSWRVQTPSA-N colistin B Chemical compound CC(C)CCCCC(=O)N[C@@H](CCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O KNIWPHSUTGNZST-SSWRVQTPSA-N 0.000 claims description 13
- 241000605900 Butyrivibrio fibrisolvens Species 0.000 claims description 11
- 241000589540 Pseudomonas fluorescens Species 0.000 claims description 11
- 230000000529 probiotic effect Effects 0.000 claims description 11
- 241000605896 Fibrobacter succinogenes Species 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 10
- 239000006041 probiotic Substances 0.000 claims description 10
- 235000018291 probiotics Nutrition 0.000 claims description 10
- 241000588912 Pantoea agglomerans Species 0.000 claims description 9
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims description 9
- 238000004519 manufacturing process Methods 0.000 claims description 9
- 239000002537 cosmetic Substances 0.000 claims description 8
- 241000282414 Homo sapiens Species 0.000 claims description 6
- 241000589774 Pseudomonas sp. Species 0.000 claims description 6
- 241001135245 Butyrivibrio sp. Species 0.000 claims description 5
- 241000488157 Escherichia sp. Species 0.000 claims description 5
- 241001495179 Fibrobacter sp. Species 0.000 claims description 5
- 241000590008 Helicobacter sp. Species 0.000 claims description 5
- 241000983368 Pantoea sp. Species 0.000 claims description 5
- 241000607149 Salmonella sp. Species 0.000 claims description 5
- 241000607758 Shigella sp. Species 0.000 claims description 5
- 239000005022 packaging material Substances 0.000 claims description 5
- 229930193868 fusaricidin Natural products 0.000 claims description 4
- 108010001478 Bacitracin Proteins 0.000 claims description 3
- 241000589994 Campylobacter sp. Species 0.000 claims description 3
- 229960003071 bacitracin Drugs 0.000 claims description 3
- 229930184125 bacitracin Natural products 0.000 claims description 3
- 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 claims description 3
- 235000013406 prebiotics Nutrition 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 58
- 230000000694 effects Effects 0.000 description 45
- 238000011282 treatment Methods 0.000 description 34
- 239000001974 tryptic soy broth Substances 0.000 description 34
- 108010050327 trypticase-soy broth Proteins 0.000 description 34
- 230000005764 inhibitory process Effects 0.000 description 33
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 30
- 244000005700 microbiome Species 0.000 description 27
- 239000002609 medium Substances 0.000 description 22
- 108010078777 Colistin Proteins 0.000 description 19
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 19
- JORAUNFTUVJTNG-BSTBCYLQSA-N n-[(2s)-4-amino-1-[[(2s,3r)-1-[[(2s)-4-amino-1-oxo-1-[[(3s,6s,9s,12s,15r,18s,21s)-6,9,18-tris(2-aminoethyl)-3-[(1r)-1-hydroxyethyl]-12,15-bis(2-methylpropyl)-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptazacyclotricos-21-yl]amino]butan-2-yl]amino]-3-h Polymers CC(C)CCCCC(=O)N[C@@H](CCN)C(=O)N[C@H]([C@@H](C)O)CN[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O.CCC(C)CCCCC(=O)N[C@@H](CCN)C(=O)N[C@H]([C@@H](C)O)CN[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O JORAUNFTUVJTNG-BSTBCYLQSA-N 0.000 description 18
- 229920001817 Agar Polymers 0.000 description 16
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 16
- 150000001875 compounds Chemical class 0.000 description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 15
- 239000008272 agar Substances 0.000 description 15
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 15
- 108020004414 DNA Proteins 0.000 description 14
- 108010093965 Polymyxin B Proteins 0.000 description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 239000000654 additive Substances 0.000 description 14
- 230000001580 bacterial effect Effects 0.000 description 14
- 229920000024 polymyxin B Polymers 0.000 description 14
- 229960005266 polymyxin b Drugs 0.000 description 14
- 239000006228 supernatant Substances 0.000 description 14
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 13
- 102000016943 Muramidase Human genes 0.000 description 13
- 108010014251 Muramidase Proteins 0.000 description 13
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 13
- 230000000844 anti-bacterial effect Effects 0.000 description 13
- 229960003346 colistin Drugs 0.000 description 13
- 239000000499 gel Substances 0.000 description 13
- 235000010335 lysozyme Nutrition 0.000 description 13
- 239000004325 lysozyme Substances 0.000 description 13
- 229960000274 lysozyme Drugs 0.000 description 13
- 238000003556 assay Methods 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 11
- 235000018102 proteins Nutrition 0.000 description 11
- 238000012360 testing method Methods 0.000 description 11
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 10
- 241001360526 Escherichia coli ATCC 25922 Species 0.000 description 10
- 238000010790 dilution Methods 0.000 description 10
- 239000012895 dilution Substances 0.000 description 10
- DCAYPVUWAIABOU-UHFFFAOYSA-N hexadecane Chemical compound CCCCCCCCCCCCCCCC DCAYPVUWAIABOU-UHFFFAOYSA-N 0.000 description 10
- 238000000746 purification Methods 0.000 description 10
- 230000035945 sensitivity Effects 0.000 description 10
- 235000002639 sodium chloride Nutrition 0.000 description 10
- 230000004083 survival effect Effects 0.000 description 10
- 230000001225 therapeutic effect Effects 0.000 description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 108090000364 Ligases Proteins 0.000 description 9
- 108090000631 Trypsin Proteins 0.000 description 9
- 102000004142 Trypsin Human genes 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 241000894007 species Species 0.000 description 9
- 239000012588 trypsin Substances 0.000 description 9
- 108010062877 Bacteriocins Proteins 0.000 description 8
- 241000287828 Gallus gallus Species 0.000 description 8
- 102000003960 Ligases Human genes 0.000 description 8
- 244000052616 bacterial pathogen Species 0.000 description 8
- 239000004202 carbamide Substances 0.000 description 8
- 235000015872 dietary supplement Nutrition 0.000 description 8
- 150000004665 fatty acids Chemical group 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- -1 6-methyloctanoyl Chemical group 0.000 description 7
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 7
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 7
- 241000193830 Bacillus <bacterium> Species 0.000 description 7
- 108010067770 Endopeptidase K Proteins 0.000 description 7
- 108090001060 Lipase Proteins 0.000 description 7
- 239000004367 Lipase Substances 0.000 description 7
- 102000004882 Lipase Human genes 0.000 description 7
- 241000179039 Paenibacillus Species 0.000 description 7
- 238000005119 centrifugation Methods 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 7
- 239000000284 extract Substances 0.000 description 7
- 238000009472 formulation Methods 0.000 description 7
- 238000011534 incubation Methods 0.000 description 7
- 238000002955 isolation Methods 0.000 description 7
- 235000019421 lipase Nutrition 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 230000004060 metabolic process Effects 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 7
- 238000001228 spectrum Methods 0.000 description 7
- 239000013589 supplement Substances 0.000 description 7
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 239000006137 Luria-Bertani broth Substances 0.000 description 6
- 244000057717 Streptococcus lactis Species 0.000 description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 6
- 239000002253 acid Substances 0.000 description 6
- 239000003242 anti bacterial agent Substances 0.000 description 6
- 229920001429 chelating resin Polymers 0.000 description 6
- DIOQZVSQGTUSAI-UHFFFAOYSA-N decane Chemical compound CCCCCCCCCC DIOQZVSQGTUSAI-UHFFFAOYSA-N 0.000 description 6
- 239000002778 food additive Substances 0.000 description 6
- 230000002496 gastric effect Effects 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 230000036541 health Effects 0.000 description 6
- AGBQKNBQESQNJD-UHFFFAOYSA-N lipoic acid Chemical compound OC(=O)CCCCC1CCSS1 AGBQKNBQESQNJD-UHFFFAOYSA-N 0.000 description 6
- 230000014759 maintenance of location Effects 0.000 description 6
- 230000017066 negative regulation of growth Effects 0.000 description 6
- 229940041153 polymyxins Drugs 0.000 description 6
- 230000000069 prophylactic effect Effects 0.000 description 6
- 230000002829 reductive effect Effects 0.000 description 6
- 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 5
- 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 5
- 241000283690 Bos taurus Species 0.000 description 5
- 108090000317 Chymotrypsin Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 5
- 238000002768 Kirby-Bauer method Methods 0.000 description 5
- 241000018243 Paenibacillus polymyxa M1 Species 0.000 description 5
- 235000014897 Streptococcus lactis Nutrition 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 239000003833 bile salt Substances 0.000 description 5
- 229940093761 bile salts Drugs 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 230000025938 carbohydrate utilization Effects 0.000 description 5
- 238000012512 characterization method Methods 0.000 description 5
- 229960002376 chymotrypsin Drugs 0.000 description 5
- 235000014113 dietary fatty acids Nutrition 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000000194 fatty acid Substances 0.000 description 5
- 229930195729 fatty acid Natural products 0.000 description 5
- 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 5
- 235000013373 food additive Nutrition 0.000 description 5
- 238000013467 fragmentation Methods 0.000 description 5
- 238000006062 fragmentation reaction Methods 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 150000002500 ions Chemical class 0.000 description 5
- 239000013586 microbial product Substances 0.000 description 5
- YKQOSKADJPQZHB-YNWHQGOSSA-N n-[(2s)-4-amino-1-[[(2s,3r)-1-[[(2s)-4-amino-1-oxo-1-[[(3s,6s,9s,12s,15r,18s,21s)-6,9,18-tris(2-aminoethyl)-3-[(1s)-1-hydroxyethyl]-12,15-bis(2-methylpropyl)-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptazacyclotricos-21-yl]amino]butan-2-yl]amino]-3-h Polymers CCC(C)CCCC(=O)N[C@@H](CCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O YKQOSKADJPQZHB-YNWHQGOSSA-N 0.000 description 5
- 235000021590 normal diet Nutrition 0.000 description 5
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- 229920001223 polyethylene glycol Polymers 0.000 description 5
- 102000004196 processed proteins & peptides Human genes 0.000 description 5
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 241000589876 Campylobacter Species 0.000 description 4
- 241000589875 Campylobacter jejuni Species 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 241000590002 Helicobacter pylori Species 0.000 description 4
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- 241000186805 Listeria innocua Species 0.000 description 4
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- 241001354013 Salmonella enterica subsp. enterica serovar Enteritidis Species 0.000 description 4
- 241000293871 Salmonella enterica subsp. enterica serovar Typhi Species 0.000 description 4
- 241000607764 Shigella dysenteriae Species 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 230000001464 adherent effect Effects 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 239000003674 animal food additive Substances 0.000 description 4
- 210000000941 bile Anatomy 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 235000013330 chicken meat Nutrition 0.000 description 4
- 239000000287 crude extract Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000003113 dilution method Methods 0.000 description 4
- 238000000855 fermentation Methods 0.000 description 4
- 230000004151 fermentation Effects 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 210000004051 gastric juice Anatomy 0.000 description 4
- 210000001035 gastrointestinal tract Anatomy 0.000 description 4
- 229940037467 helicobacter pylori Drugs 0.000 description 4
- 230000002209 hydrophobic effect Effects 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 239000003960 organic solvent Substances 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 229940007046 shigella dysenteriae Drugs 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 241000193752 Bacillus circulans Species 0.000 description 3
- 244000063299 Bacillus subtilis Species 0.000 description 3
- 235000014469 Bacillus subtilis Nutrition 0.000 description 3
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 3
- 241000588881 Chromobacterium Species 0.000 description 3
- 241000520134 Enterococcus mundtii Species 0.000 description 3
- 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 description 3
- 108010028921 Lipopeptides Proteins 0.000 description 3
- 241000186779 Listeria monocytogenes Species 0.000 description 3
- 108010053775 Nisin Proteins 0.000 description 3
- NVNLLIYOARQCIX-MSHCCFNRSA-N Nisin Chemical compound N1C(=O)[C@@H](CC(C)C)NC(=O)C(=C)NC(=O)[C@@H]([C@H](C)CC)NC(=O)[C@@H](NC(=O)C(=C/C)/NC(=O)[C@H](N)[C@H](C)CC)CSC[C@@H]1C(=O)N[C@@H]1C(=O)N2CCC[C@@H]2C(=O)NCC(=O)N[C@@H](C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(NCC(=O)N[C@H](C)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCSC)C(=O)NCC(=O)N[C@H](CS[C@@H]2C)C(=O)N[C@H](CC(N)=O)C(=O)N[C@H](CCSC)C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(N[C@H](C)C(=O)N[C@@H]3C(=O)N[C@@H](C(N[C@H](CC=4NC=NC=4)C(=O)N[C@H](CS[C@@H]3C)C(=O)N[C@H](CO)C(=O)N[C@H]([C@H](C)CC)C(=O)N[C@H](CC=3NC=NC=3)C(=O)N[C@H](C(C)C)C(=O)NC(=C)C(=O)N[C@H](CCCCN)C(O)=O)=O)CS[C@@H]2C)=O)=O)CS[C@@H]1C NVNLLIYOARQCIX-MSHCCFNRSA-N 0.000 description 3
- 241001494479 Pecora Species 0.000 description 3
- 241000191998 Pediococcus acidilactici Species 0.000 description 3
- 102000035195 Peptidases Human genes 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 241000589516 Pseudomonas Species 0.000 description 3
- 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 3
- 241000607142 Salmonella Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 241000191967 Staphylococcus aureus Species 0.000 description 3
- 239000012505 Superdex™ Substances 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 230000000996 additive effect Effects 0.000 description 3
- 239000000443 aerosol 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
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 230000008485 antagonism Effects 0.000 description 3
- 108010009943 bacitracin synthetase Proteins 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 229940098773 bovine serum albumin Drugs 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
- 239000002775 capsule Substances 0.000 description 3
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 3
- 125000004122 cyclic group Chemical group 0.000 description 3
- 238000009792 diffusion process Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 235000019152 folic acid Nutrition 0.000 description 3
- 239000011724 folic acid Substances 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 229930195733 hydrocarbon Natural products 0.000 description 3
- 150000002430 hydrocarbons Chemical class 0.000 description 3
- 238000004811 liquid chromatography Methods 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- XRBFWBMDMQIAQF-ZHFMXAAZSA-N n-[(2s)-4-amino-1-[[(2s,3r)-1-[[(2s)-4-amino-1-oxo-1-[[(3s,6s,9s,12s,15r,18s,21s)-6,9,18-tris(2-aminoethyl)-3,12-bis[(1r)-1-hydroxyethyl]-15-(2-methylpropyl)-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptazacyclotricos-21-yl]amino]butan-2-yl]amino]-3-h Chemical compound CCC(C)CCCCC(=O)N[C@@H](CCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O XRBFWBMDMQIAQF-ZHFMXAAZSA-N 0.000 description 3
- 229960003512 nicotinic acid Drugs 0.000 description 3
- 239000004309 nisin Substances 0.000 description 3
- 235000010297 nisin Nutrition 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 108010000785 non-ribosomal peptide synthase Proteins 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 235000020939 nutritional additive Nutrition 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 3
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000010561 standard procedure Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000004885 tandem mass spectrometry Methods 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 239000006150 trypticase soy agar Substances 0.000 description 3
- 230000035899 viability Effects 0.000 description 3
- 229940088594 vitamin Drugs 0.000 description 3
- 229930003231 vitamin Natural products 0.000 description 3
- 235000013343 vitamin Nutrition 0.000 description 3
- 239000011782 vitamin Substances 0.000 description 3
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 2
- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 2
- 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 2
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 2
- 101710126481 ATP-dependent lipid A-core flippase Proteins 0.000 description 2
- 241000588626 Acinetobacter baumannii Species 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 2
- 241000272525 Anas platyrhynchos Species 0.000 description 2
- 241000272814 Anser sp. Species 0.000 description 2
- 241000193755 Bacillus cereus Species 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 2
- 241000606161 Chlamydia Species 0.000 description 2
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 2
- 241000238424 Crustacea Species 0.000 description 2
- 108010069514 Cyclic Peptides Proteins 0.000 description 2
- 102000001189 Cyclic Peptides Human genes 0.000 description 2
- 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 2
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- 108010000912 Egg Proteins Proteins 0.000 description 2
- 241000283073 Equus caballus Species 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 229920002527 Glycogen Polymers 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 239000011786 L-ascorbyl-6-palmitate Substances 0.000 description 2
- QAQJMLQRFWZOBN-LAUBAEHRSA-N L-ascorbyl-6-palmitate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](O)[C@H]1OC(=O)C(O)=C1O QAQJMLQRFWZOBN-LAUBAEHRSA-N 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 241000186781 Listeria Species 0.000 description 2
- 241000186780 Listeria ivanovii Species 0.000 description 2
- 235000010654 Melissa officinalis Nutrition 0.000 description 2
- 244000062730 Melissa officinalis Species 0.000 description 2
- AFVFQIVMOAPDHO-UHFFFAOYSA-M Methanesulfonate Chemical compound CS([O-])(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-M 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 241000193411 Paenibacillus amylolyticus Species 0.000 description 2
- 241000611788 Paenibacillus kobensis Species 0.000 description 2
- 241000178960 Paenibacillus macerans Species 0.000 description 2
- 241001146929 Paenibacillus polymyxa CR1 Species 0.000 description 2
- 241001452311 Paenibacillus polymyxa OSY-DF Species 0.000 description 2
- 241000192001 Pediococcus Species 0.000 description 2
- 102000057297 Pepsin A Human genes 0.000 description 2
- 108090000284 Pepsin A Proteins 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- VYGQUTWHTHXGQB-FFHKNEKCSA-N Retinol Palmitate Chemical compound CCCCCCCCCCCCCCCC(=O)OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C VYGQUTWHTHXGQB-FFHKNEKCSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 2
- 241000607768 Shigella Species 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 241000191940 Staphylococcus Species 0.000 description 2
- 241000194017 Streptococcus Species 0.000 description 2
- 241000194049 Streptococcus equinus Species 0.000 description 2
- 241000282898 Sus scrofa Species 0.000 description 2
- 241000607598 Vibrio Species 0.000 description 2
- 229930003268 Vitamin C Natural products 0.000 description 2
- MECHNRXZTMCUDQ-UHFFFAOYSA-N Vitamin D2 Natural products C1CCC2(C)C(C(C)C=CC(C)C(C)C)CCC2C1=CC=C1CC(O)CCC1=C MECHNRXZTMCUDQ-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 229930003427 Vitamin E Natural products 0.000 description 2
- 239000003463 adsorbent Substances 0.000 description 2
- 238000012459 agar diffusion assay Methods 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 125000000217 alkyl group Chemical group 0.000 description 2
- ANVAOWXLWRTKGA-XHGAXZNDSA-N all-trans-alpha-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=CC=C(C)C=CC1C(C)=CCCC1(C)C ANVAOWXLWRTKGA-XHGAXZNDSA-N 0.000 description 2
- 108010027597 alpha-chymotrypsin Proteins 0.000 description 2
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 description 2
- 239000001166 ammonium sulphate Substances 0.000 description 2
- 235000019730 animal feed additive Nutrition 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 235000006708 antioxidants Nutrition 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Natural products CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 description 2
- 235000010385 ascorbyl palmitate Nutrition 0.000 description 2
- 239000012298 atmosphere Substances 0.000 description 2
- 208000022362 bacterial infectious disease Diseases 0.000 description 2
- 238000002869 basic local alignment search tool Methods 0.000 description 2
- 235000021028 berry Nutrition 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- FUFJGUQYACFECW-UHFFFAOYSA-L calcium hydrogenphosphate Chemical compound [Ca+2].OP([O-])([O-])=O FUFJGUQYACFECW-UHFFFAOYSA-L 0.000 description 2
- 229960002079 calcium pantothenate Drugs 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000022131 cell cycle Effects 0.000 description 2
- 239000013553 cell monolayer Substances 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229910052804 chromium Inorganic materials 0.000 description 2
- 239000011651 chromium Substances 0.000 description 2
- QSWDMMVNRMROPK-UHFFFAOYSA-K chromium(3+) trichloride Chemical compound [Cl-].[Cl-].[Cl-].[Cr+3] QSWDMMVNRMROPK-UHFFFAOYSA-K 0.000 description 2
- 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 2
- 229920002770 condensed tannin Polymers 0.000 description 2
- 239000007799 cork Substances 0.000 description 2
- 239000006071 cream Substances 0.000 description 2
- RMRCNWBMXRMIRW-BYFNXCQMSA-M cyanocobalamin Chemical compound N#C[Co+]N([C@]1([H])[C@H](CC(N)=O)[C@]\2(CCC(=O)NC[C@H](C)OP(O)(=O)OC3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)C)C/2=C(C)\C([C@H](C/2(C)C)CCC(N)=O)=N\C\2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O RMRCNWBMXRMIRW-BYFNXCQMSA-M 0.000 description 2
- 235000013365 dairy product Nutrition 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 230000001627 detrimental effect Effects 0.000 description 2
- 239000010432 diamond Substances 0.000 description 2
- 235000019700 dicalcium phosphate Nutrition 0.000 description 2
- 229940095079 dicalcium phosphate anhydrous Drugs 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 239000002270 dispersing agent Substances 0.000 description 2
- 210000000969 egg white Anatomy 0.000 description 2
- 235000014103 egg white Nutrition 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 229960002061 ergocalciferol Drugs 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 230000001747 exhibiting effect Effects 0.000 description 2
- 235000021050 feed intake Nutrition 0.000 description 2
- 239000006052 feed supplement Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000010200 folin Substances 0.000 description 2
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 210000004211 gastric acid Anatomy 0.000 description 2
- 238000001641 gel filtration chromatography Methods 0.000 description 2
- 108091008053 gene clusters Proteins 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 229960002743 glutamine Drugs 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 229940096919 glycogen Drugs 0.000 description 2
- 244000005709 gut microbiome Species 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 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 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- JJTUDXZGHPGLLC-UHFFFAOYSA-N lactide Chemical compound CC1OC(=O)C(C)OC1=O JJTUDXZGHPGLLC-UHFFFAOYSA-N 0.000 description 2
- 239000000787 lecithin Substances 0.000 description 2
- 229940067606 lecithin Drugs 0.000 description 2
- 235000010445 lecithin Nutrition 0.000 description 2
- 231100000518 lethal Toxicity 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 239000000865 liniment Substances 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000006210 lotion Substances 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000000074 matrix-assisted laser desorption--ionisation tandem time-of-flight detection Methods 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 244000000010 microbial pathogen Species 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 230000004899 motility Effects 0.000 description 2
- 239000007923 nasal drop Substances 0.000 description 2
- 229940100662 nasal drops Drugs 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 235000005152 nicotinamide Nutrition 0.000 description 2
- 239000011570 nicotinamide Substances 0.000 description 2
- DFPAKSUCGFBDDF-UHFFFAOYSA-N nicotinic acid amide Natural products NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 229940111202 pepsin Drugs 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- MBWXNTAXLNYFJB-NKFFZRIASA-N phylloquinone Chemical compound C1=CC=C2C(=O)C(C/C=C(C)/CCC[C@H](C)CCC[C@H](C)CCCC(C)C)=C(C)C(=O)C2=C1 MBWXNTAXLNYFJB-NKFFZRIASA-N 0.000 description 2
- 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 2
- 235000019175 phylloquinone Nutrition 0.000 description 2
- 239000011772 phylloquinone Substances 0.000 description 2
- 229960001898 phytomenadione Drugs 0.000 description 2
- 229940106587 pine bark extract Drugs 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 229940068968 polysorbate 80 Drugs 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 229960003975 potassium Drugs 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 235000013594 poultry meat Nutrition 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- NHZMQXZHNVQTQA-UHFFFAOYSA-N pyridoxamine Chemical compound CC1=NC=C(CO)C(CN)=C1O NHZMQXZHNVQTQA-UHFFFAOYSA-N 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 229960002477 riboflavin Drugs 0.000 description 2
- 229940092258 rosemary extract Drugs 0.000 description 2
- 235000020748 rosemary extract Nutrition 0.000 description 2
- 239000001233 rosmarinus officinalis l. extract Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- XPMYTBIJWHMOIJ-UHFFFAOYSA-N sattabacin Chemical compound CC(C)CC(=O)C(O)CC1=CC=CC=C1 XPMYTBIJWHMOIJ-UHFFFAOYSA-N 0.000 description 2
- AYJJYONYYXAVTL-UHFFFAOYSA-N sattazolin Chemical compound C1=CC=C2C(CC(O)C(=O)CC(C)C)=CNC2=C1 AYJJYONYYXAVTL-UHFFFAOYSA-N 0.000 description 2
- 229910052711 selenium Inorganic materials 0.000 description 2
- 239000011669 selenium Substances 0.000 description 2
- 229940091258 selenium supplement Drugs 0.000 description 2
- 239000002356 single layer Substances 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 description 2
- JZRWCGZRTZMZEH-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 230000000699 topical effect Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 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 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- 235000019154 vitamin C Nutrition 0.000 description 2
- 239000011718 vitamin C Substances 0.000 description 2
- 235000001892 vitamin D2 Nutrition 0.000 description 2
- 239000011653 vitamin D2 Substances 0.000 description 2
- MECHNRXZTMCUDQ-RKHKHRCZSA-N vitamin D2 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)/C=C/[C@H](C)C(C)C)=C\C=C1\C[C@@H](O)CCC1=C MECHNRXZTMCUDQ-RKHKHRCZSA-N 0.000 description 2
- 229940046009 vitamin E Drugs 0.000 description 2
- 235000019165 vitamin E Nutrition 0.000 description 2
- 239000011709 vitamin E Substances 0.000 description 2
- 239000011728 vitamin K2 Substances 0.000 description 2
- 229940011671 vitamin b6 Drugs 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- 230000004584 weight gain Effects 0.000 description 2
- 235000019786 weight gain Nutrition 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 description 1
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- MJYQFWSXKFLTAY-OVEQLNGDSA-N (2r,3r)-2,3-bis[(4-hydroxy-3-methoxyphenyl)methyl]butane-1,4-diol;(2r,3r,4s,5s,6r)-6-(hydroxymethyl)oxane-2,3,4,5-tetrol Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O.C1=C(O)C(OC)=CC(C[C@@H](CO)[C@H](CO)CC=2C=C(OC)C(O)=CC=2)=C1 MJYQFWSXKFLTAY-OVEQLNGDSA-N 0.000 description 1
- FGNPLIQZJCYWLE-BTVCFUMJSA-N (2r,3r,4s,5r)-2-amino-3,4,5,6-tetrahydroxyhexanal;sulfuric acid Chemical compound OS(O)(=O)=O.O=C[C@H](N)[C@@H](O)[C@H](O)[C@H](O)CO FGNPLIQZJCYWLE-BTVCFUMJSA-N 0.000 description 1
- IHPFAGHGAJFPOT-ZDUSSCGKSA-N (2s)-2-hydroxy-1-(4-hydroxyphenyl)-5-methylhexan-3-one Chemical compound CC(C)CC(=O)[C@@H](O)CC1=CC=C(O)C=C1 IHPFAGHGAJFPOT-ZDUSSCGKSA-N 0.000 description 1
- DMASLKHVQRHNES-UPOGUZCLSA-N (3R)-beta,beta-caroten-3-ol 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=CC=C(C)C=CC1=C(C)CCCC1(C)C DMASLKHVQRHNES-UPOGUZCLSA-N 0.000 description 1
- JKQXZKUSFCKOGQ-JLGXGRJMSA-N (3R,3'R)-beta,beta-carotene-3,3'-diol 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=CC=C(C)C=CC1=C(C)C[C@@H](O)CC1(C)C JKQXZKUSFCKOGQ-JLGXGRJMSA-N 0.000 description 1
- PHIQHXFUZVPYII-ZCFIWIBFSA-O (R)-carnitinium Chemical compound C[N+](C)(C)C[C@H](O)CC(O)=O PHIQHXFUZVPYII-ZCFIWIBFSA-O 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- HYPYXGZDOYTYDR-HAJWAVTHSA-N 2-methyl-3-[(2e,6e,10e,14e)-3,7,11,15,19-pentamethylicosa-2,6,10,14,18-pentaenyl]naphthalene-1,4-dione Chemical compound C1=CC=C2C(=O)C(C/C=C(C)/CC/C=C(C)/CC/C=C(C)/CC/C=C(C)/CCC=C(C)C)=C(C)C(=O)C2=C1 HYPYXGZDOYTYDR-HAJWAVTHSA-N 0.000 description 1
- DBTMGCOVALSLOR-UHFFFAOYSA-N 32-alpha-galactosyl-3-alpha-galactosyl-galactose Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(OC2C(C(CO)OC(O)C2O)O)OC(CO)C1O DBTMGCOVALSLOR-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- PVXPPJIGRGXGCY-DJHAAKORSA-N 6-O-alpha-D-glucopyranosyl-alpha-D-fructofuranose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@](O)(CO)O1 PVXPPJIGRGXGCY-DJHAAKORSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 240000002234 Allium sativum Species 0.000 description 1
- 108091093088 Amplicon Proteins 0.000 description 1
- 244000061520 Angelica archangelica Species 0.000 description 1
- 241000382455 Angelica sinensis Species 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 241000186063 Arthrobacter Species 0.000 description 1
- 235000016425 Arthrospira platensis Nutrition 0.000 description 1
- 240000002900 Arthrospira platensis Species 0.000 description 1
- 241000512259 Ascophyllum nodosum Species 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- BHELIUBJHYAEDK-OAIUPTLZSA-N Aspoxicillin Chemical compound C1([C@H](C(=O)N[C@@H]2C(N3[C@H](C(C)(C)S[C@@H]32)C(O)=O)=O)NC(=O)[C@H](N)CC(=O)NC)=CC=C(O)C=C1 BHELIUBJHYAEDK-OAIUPTLZSA-N 0.000 description 1
- 241000416162 Astragalus gummifer Species 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 241001112741 Bacillaceae Species 0.000 description 1
- 208000004429 Bacillary Dysentery Diseases 0.000 description 1
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 1
- 208000031729 Bacteremia Diseases 0.000 description 1
- 201000001178 Bacterial Pneumonia Diseases 0.000 description 1
- 206010004054 Bacterial upper respiratory tract infections Diseases 0.000 description 1
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000282461 Canis lupus Species 0.000 description 1
- 235000002567 Capsicum annuum Nutrition 0.000 description 1
- 240000004160 Capsicum annuum Species 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 241000251730 Chondrichthyes Species 0.000 description 1
- 229910021555 Chromium Chloride Inorganic materials 0.000 description 1
- 229910021556 Chromium(III) chloride Inorganic materials 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- OCUCCJIRFHNWBP-IYEMJOQQSA-L Copper gluconate Chemical compound [Cu+2].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O OCUCCJIRFHNWBP-IYEMJOQQSA-L 0.000 description 1
- 239000004212 Cryptoxanthin Substances 0.000 description 1
- 235000009854 Cucurbita moschata Nutrition 0.000 description 1
- 240000004244 Cucurbita moschata Species 0.000 description 1
- 244000007835 Cyamopsis tetragonoloba Species 0.000 description 1
- 239000011740 D-alpha-tocopherylacetate Substances 0.000 description 1
- 235000002414 D-alpha-tocopherylacetate Nutrition 0.000 description 1
- ZAKOWWREFLAJOT-CEFNRUSXSA-N D-alpha-tocopherylacetate Chemical compound CC(=O)OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C ZAKOWWREFLAJOT-CEFNRUSXSA-N 0.000 description 1
- 150000008574 D-amino acids Chemical class 0.000 description 1
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 description 1
- GZIFEOYASATJEH-UHFFFAOYSA-N D-delta tocopherol Natural products OC1=CC(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1 GZIFEOYASATJEH-UHFFFAOYSA-N 0.000 description 1
- RXVWSYJTUUKTEA-UHFFFAOYSA-N D-maltotriose Natural products OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1OC1C(O)C(O)C(O)C(CO)O1 RXVWSYJTUUKTEA-UHFFFAOYSA-N 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- QWIZNVHXZXRPDR-UHFFFAOYSA-N D-melezitose Natural products O1C(CO)C(O)C(O)C(O)C1OC1C(O)C(CO)OC1(CO)OC1OC(CO)C(O)C(O)C1O QWIZNVHXZXRPDR-UHFFFAOYSA-N 0.000 description 1
- SNPLKNRPJHDVJA-ZETCQYMHSA-N D-panthenol Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCCO SNPLKNRPJHDVJA-ZETCQYMHSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 241000164287 Enterococcus faecalis ATCC 19433 Species 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- PMVSDNDAUGGCCE-TYYBGVCCSA-L Ferrous fumarate Chemical compound [Fe+2].[O-]C(=O)\C=C\C([O-])=O PMVSDNDAUGGCCE-TYYBGVCCSA-L 0.000 description 1
- 241000608038 Fibrobacter succinogenes subsp. succinogenes S85 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
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 1
- 240000006927 Foeniculum vulgare Species 0.000 description 1
- 235000004204 Foeniculum vulgare Nutrition 0.000 description 1
- 108700032487 GAP-43-3 Proteins 0.000 description 1
- 206010017943 Gastrointestinal conditions Diseases 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108010007979 Glycocholic Acid Proteins 0.000 description 1
- 240000004670 Glycyrrhiza echinata Species 0.000 description 1
- 235000001453 Glycyrrhiza echinata Nutrition 0.000 description 1
- 235000006200 Glycyrrhiza glabra Nutrition 0.000 description 1
- 235000017382 Glycyrrhiza lepidota Nutrition 0.000 description 1
- 206010018612 Gonorrhoea Diseases 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 235000001287 Guettarda speciosa Nutrition 0.000 description 1
- 241000606790 Haemophilus Species 0.000 description 1
- 241000205062 Halobacterium Species 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- 241000700588 Human alphaherpesvirus 1 Species 0.000 description 1
- 241000701074 Human alphaherpesvirus 2 Species 0.000 description 1
- 241000735432 Hydrastis canadensis Species 0.000 description 1
- 241000282596 Hylobatidae Species 0.000 description 1
- 235000003368 Ilex paraguariensis Nutrition 0.000 description 1
- 244000188472 Ilex paraguariensis 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
- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- RHGKLRLOHDJJDR-BYPYZUCNSA-N L-citrulline Chemical compound NC(=O)NCCC[C@H]([NH3+])C([O-])=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- 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 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 241000186605 Lactobacillus paracasei Species 0.000 description 1
- 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 description 1
- 208000004023 Legionellosis Diseases 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 241000192132 Leuconostoc Species 0.000 description 1
- 229920000161 Locust bean gum Polymers 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- 239000006391 Luria-Bertani Medium Substances 0.000 description 1
- 208000016604 Lyme disease Diseases 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 108050003365 Malonyl CoA-acyl carrier protein transacylases Proteins 0.000 description 1
- 102100027329 Malonyl-CoA-acyl carrier protein transacylase, mitochondrial Human genes 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 1
- 240000004658 Medicago sativa Species 0.000 description 1
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 1
- 108090000301 Membrane transport proteins Proteins 0.000 description 1
- 102000003939 Membrane transport proteins Human genes 0.000 description 1
- ABSPRNADVQNDOU-UHFFFAOYSA-N Menaquinone 1 Natural products C1=CC=C2C(=O)C(CC=C(C)C)=C(C)C(=O)C2=C1 ABSPRNADVQNDOU-UHFFFAOYSA-N 0.000 description 1
- 206010027202 Meningitis bacterial Diseases 0.000 description 1
- 244000246386 Mentha pulegium Species 0.000 description 1
- 235000016257 Mentha pulegium Nutrition 0.000 description 1
- 235000004357 Mentha x piperita Nutrition 0.000 description 1
- 241001467578 Microbacterium Species 0.000 description 1
- 241000192041 Micrococcus Species 0.000 description 1
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- FFDGPVCHZBVARC-UHFFFAOYSA-N N,N-dimethylglycine Chemical compound CN(C)CC(O)=O FFDGPVCHZBVARC-UHFFFAOYSA-N 0.000 description 1
- 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 1
- RHGKLRLOHDJJDR-UHFFFAOYSA-N Ndelta-carbamoyl-DL-ornithine Natural products OC(=O)C(N)CCCNC(N)=O RHGKLRLOHDJJDR-UHFFFAOYSA-N 0.000 description 1
- 241000588653 Neisseria Species 0.000 description 1
- 235000010679 Nepeta cataria Nutrition 0.000 description 1
- 240000009215 Nepeta cataria Species 0.000 description 1
- 206010029155 Nephropathy toxic Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000219925 Oenothera Species 0.000 description 1
- 235000004496 Oenothera biennis Nutrition 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 206010033078 Otitis media Diseases 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 241000768412 Paenibacillus polymyxa E681 Species 0.000 description 1
- 241000708561 Paenibacillus polymyxa SQR-21 Species 0.000 description 1
- 240000004371 Panax ginseng Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 241001523956 Parengyodontium album Species 0.000 description 1
- 241000198694 Passiflora pallida Species 0.000 description 1
- 241000487050 Pediococcus pentosaceus ATCC 25745 Species 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- 201000005702 Pertussis Diseases 0.000 description 1
- 244000062780 Petroselinum sativum Species 0.000 description 1
- ZYFVNVRFVHJEIU-UHFFFAOYSA-N PicoGreen Chemical compound CN(C)CCCN(CCCN(C)C)C1=CC(=CC2=[N+](C3=CC=CC=C3S2)C)C2=CC=CC=C2N1C1=CC=CC=C1 ZYFVNVRFVHJEIU-UHFFFAOYSA-N 0.000 description 1
- 241001236212 Pinus pinaster Species 0.000 description 1
- 235000005105 Pinus pinaster Nutrition 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 241000132152 Polymyxa Species 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- HLCFGWHYROZGBI-JJKGCWMISA-M Potassium gluconate Chemical compound [K+].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O HLCFGWHYROZGBI-JJKGCWMISA-M 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 206010037151 Psittacosis Diseases 0.000 description 1
- 241000340987 Ptychopetalum olacoides Species 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 1
- NCYCYZXNIZJOKI-OVSJKPMPSA-N Retinaldehyde Chemical compound O=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-OVSJKPMPSA-N 0.000 description 1
- 241000235527 Rhizopus Species 0.000 description 1
- 240000001890 Ribes hudsonianum Species 0.000 description 1
- 235000016954 Ribes hudsonianum Nutrition 0.000 description 1
- 235000001466 Ribes nigrum Nutrition 0.000 description 1
- 241000606701 Rickettsia Species 0.000 description 1
- 235000015422 Rumex crispus ssp. crispus Nutrition 0.000 description 1
- 235000015426 Rumex crispus ssp. fauriei Nutrition 0.000 description 1
- 244000207667 Rumex vesicarius Species 0.000 description 1
- 241000282849 Ruminantia Species 0.000 description 1
- 108010019477 S-adenosyl-L-methionine-dependent N-methyltransferase Proteins 0.000 description 1
- 241000899950 Salix glauca Species 0.000 description 1
- 206010039438 Salmonella Infections Diseases 0.000 description 1
- 241001138501 Salmonella enterica Species 0.000 description 1
- 241000607151 Salmonella enterica subsp. enterica serovar Rubislaw Species 0.000 description 1
- 206010039587 Scarlet Fever Diseases 0.000 description 1
- 206010040070 Septic Shock Diseases 0.000 description 1
- 240000006661 Serenoa repens Species 0.000 description 1
- 235000005318 Serenoa repens Nutrition 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- 206010040550 Shigella infections Diseases 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 239000004115 Sodium Silicate Substances 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 241001180364 Spirochaetes Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241001238546 Streptococcus equinus ATCC 33317 Species 0.000 description 1
- 241000193996 Streptococcus pyogenes Species 0.000 description 1
- 229920006328 Styrofoam Polymers 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 235000019486 Sunflower oil Nutrition 0.000 description 1
- 235000005865 Symphytum officinale Nutrition 0.000 description 1
- 240000002299 Symphytum officinale Species 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 206010044248 Toxic shock syndrome Diseases 0.000 description 1
- 231100000650 Toxic shock syndrome Toxicity 0.000 description 1
- 229920001615 Tragacanth Polymers 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 235000013832 Valeriana officinalis Nutrition 0.000 description 1
- 244000126014 Valeriana officinalis Species 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 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 1
- XWCYDHJOKKGVHC-UHFFFAOYSA-N Vitamin A2 Chemical compound OCC=C(C)C=CC=C(C)C=CC1=C(C)C=CCC1(C)C XWCYDHJOKKGVHC-UHFFFAOYSA-N 0.000 description 1
- 229930003451 Vitamin B1 Natural products 0.000 description 1
- 229930003779 Vitamin B12 Natural products 0.000 description 1
- 229930003471 Vitamin B2 Natural products 0.000 description 1
- 229930003537 Vitamin B3 Natural products 0.000 description 1
- 229930003571 Vitamin B5 Natural products 0.000 description 1
- 229930003756 Vitamin B7 Natural products 0.000 description 1
- 229930003316 Vitamin D Natural products 0.000 description 1
- 229930003448 Vitamin K Natural products 0.000 description 1
- 235000001667 Vitex agnus castus Nutrition 0.000 description 1
- 244000063464 Vitex agnus-castus Species 0.000 description 1
- 241000589634 Xanthomonas Species 0.000 description 1
- 241000607734 Yersinia <bacteria> Species 0.000 description 1
- JKQXZKUSFCKOGQ-LQFQNGICSA-N Z-zeaxanthin Natural products C([C@H](O)CC=1C)C(C)(C)C=1C=CC(C)=CC=CC(C)=CC=CC=C(C)C=CC=C(C)C=CC1=C(C)C[C@@H](O)CC1(C)C JKQXZKUSFCKOGQ-LQFQNGICSA-N 0.000 description 1
- QOPRSMDTRDMBNK-RNUUUQFGSA-N Zeaxanthin Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CCC(O)C1(C)C)C=CC=C(/C)C=CC2=C(C)CC(O)CC2(C)C QOPRSMDTRDMBNK-RNUUUQFGSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- WHMDKBIGKVEYHS-IYEMJOQQSA-L Zinc gluconate Chemical compound [Zn+2].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O WHMDKBIGKVEYHS-IYEMJOQQSA-L 0.000 description 1
- 235000006886 Zingiber officinale Nutrition 0.000 description 1
- 244000273928 Zingiber officinale Species 0.000 description 1
- ZOIORXHNWRGPMV-UHFFFAOYSA-N acetic acid;zinc Chemical compound [Zn].CC(O)=O.CC(O)=O ZOIORXHNWRGPMV-UHFFFAOYSA-N 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000002814 agar dilution Methods 0.000 description 1
- 238000013272 agar well diffusion method Methods 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 229960003767 alanine Drugs 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N aldehydo-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- PNNNRSAQSRJVSB-BXKVDMCESA-N aldehydo-L-rhamnose Chemical compound C[C@H](O)[C@H](O)[C@@H](O)[C@@H](O)C=O PNNNRSAQSRJVSB-BXKVDMCESA-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
- JKQXZKUSFCKOGQ-LOFNIBRQSA-N all-trans-Zeaxanthin 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=CC2=C(C)CC(O)CC2(C)C JKQXZKUSFCKOGQ-LOFNIBRQSA-N 0.000 description 1
- 239000011795 alpha-carotene Substances 0.000 description 1
- 235000003903 alpha-carotene Nutrition 0.000 description 1
- ANVAOWXLWRTKGA-HLLMEWEMSA-N alpha-carotene Natural products C(=C\C=C\C=C(/C=C/C=C(\C=C\C=1C(C)(C)CCCC=1C)/C)\C)(\C=C\C=C(/C=C/[C@H]1C(C)=CCCC1(C)C)\C)/C ANVAOWXLWRTKGA-HLLMEWEMSA-N 0.000 description 1
- 235000020661 alpha-linolenic acid Nutrition 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 235000019728 animal nutrition Nutrition 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 238000011482 antibacterial activity assay Methods 0.000 description 1
- 238000011203 antimicrobial therapy Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 229940114079 arachidonic acid Drugs 0.000 description 1
- 235000021342 arachidonic acid Nutrition 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 229960003121 arginine Drugs 0.000 description 1
- 235000009697 arginine Nutrition 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960005261 aspartic acid Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 108010026917 bacillibactin Proteins 0.000 description 1
- RCQTVEFBFUNTGM-UHFFFAOYSA-N bacillibactin Natural products CC1OC(=O)C(NC(=O)CNC(=O)C=2C(=C(O)C=CC=2)O)C(C)OC(=O)C(NC(=O)CNC(=O)C=2C(=C(O)C=CC=2)O)C(C)OC(=O)C1NC(=O)CNC(=O)C1=CC=CC(O)=C1O RCQTVEFBFUNTGM-UHFFFAOYSA-N 0.000 description 1
- 230000010065 bacterial adhesion Effects 0.000 description 1
- 201000009904 bacterial meningitis Diseases 0.000 description 1
- 201000005008 bacterial sepsis Diseases 0.000 description 1
- 208000033847 bacterial urinary tract infection Diseases 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 229940098396 barley grain Drugs 0.000 description 1
- 229940038481 bee pollen Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 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 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 235000013734 beta-carotene Nutrition 0.000 description 1
- 239000011648 beta-carotene Substances 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
- 235000002360 beta-cryptoxanthin Nutrition 0.000 description 1
- DMASLKHVQRHNES-ITUXNECMSA-N beta-cryptoxanthin 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=CC2=C(C)CCCC2(C)C DMASLKHVQRHNES-ITUXNECMSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 229960002747 betacarotene Drugs 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 1
- 235000021324 borage oil Nutrition 0.000 description 1
- 229910052796 boron Inorganic materials 0.000 description 1
- 229960005069 calcium Drugs 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 235000010376 calcium ascorbate Nutrition 0.000 description 1
- 229940047036 calcium ascorbate Drugs 0.000 description 1
- 239000011692 calcium ascorbate Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 108010033929 calcium caseinate Proteins 0.000 description 1
- FNAQSUUGMSOBHW-UHFFFAOYSA-H calcium citrate Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O FNAQSUUGMSOBHW-UHFFFAOYSA-H 0.000 description 1
- 239000001354 calcium citrate Substances 0.000 description 1
- 229960004256 calcium citrate Drugs 0.000 description 1
- WUKWITHWXAAZEY-UHFFFAOYSA-L calcium difluoride Chemical compound [F-].[F-].[Ca+2] WUKWITHWXAAZEY-UHFFFAOYSA-L 0.000 description 1
- 229910001634 calcium fluoride Inorganic materials 0.000 description 1
- 239000004227 calcium gluconate Substances 0.000 description 1
- 235000013927 calcium gluconate Nutrition 0.000 description 1
- 229960004494 calcium gluconate Drugs 0.000 description 1
- MKJXYGKVIBWPFZ-UHFFFAOYSA-L calcium lactate Chemical compound [Ca+2].CC(O)C([O-])=O.CC(O)C([O-])=O MKJXYGKVIBWPFZ-UHFFFAOYSA-L 0.000 description 1
- 239000001527 calcium lactate Substances 0.000 description 1
- 235000011086 calcium lactate Nutrition 0.000 description 1
- 229960002401 calcium lactate Drugs 0.000 description 1
- BLORRZQTHNGFTI-ZZMNMWMASA-L calcium-L-ascorbate Chemical compound [Ca+2].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] BLORRZQTHNGFTI-ZZMNMWMASA-L 0.000 description 1
- NEEHYRZPVYRGPP-UHFFFAOYSA-L calcium;2,3,4,5,6-pentahydroxyhexanoate Chemical compound [Ca+2].OCC(O)C(O)C(O)C(O)C([O-])=O.OCC(O)C(O)C(O)C(O)C([O-])=O NEEHYRZPVYRGPP-UHFFFAOYSA-L 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- HOPSCVCBEOCPJZ-UHFFFAOYSA-N carboxymethyl(trimethyl)azanium;chloride Chemical compound [Cl-].C[N+](C)(C)CC(O)=O HOPSCVCBEOCPJZ-UHFFFAOYSA-N 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 229960004203 carnitine Drugs 0.000 description 1
- 235000021466 carotenoid Nutrition 0.000 description 1
- 150000001747 carotenoids Chemical class 0.000 description 1
- 235000010418 carrageenan Nutrition 0.000 description 1
- 239000000679 carrageenan Substances 0.000 description 1
- 229920001525 carrageenan Polymers 0.000 description 1
- 229940113118 carrageenan Drugs 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 229920006184 cellulose methylcellulose Polymers 0.000 description 1
- 230000003196 chaotropic effect Effects 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 230000035605 chemotaxis Effects 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
- QWJSAWXRUVVRLH-UHFFFAOYSA-M choline bitartrate Chemical compound C[N+](C)(C)CCO.OC(=O)C(O)C(O)C([O-])=O QWJSAWXRUVVRLH-UHFFFAOYSA-M 0.000 description 1
- 229960004874 choline bitartrate Drugs 0.000 description 1
- 229960000359 chromic chloride Drugs 0.000 description 1
- 229940046374 chromium picolinate Drugs 0.000 description 1
- WYYQVWLEPYFFLP-UHFFFAOYSA-K chromium(3+);triacetate Chemical compound [Cr+3].CC([O-])=O.CC([O-])=O.CC([O-])=O WYYQVWLEPYFFLP-UHFFFAOYSA-K 0.000 description 1
- GJYSUGXFENSLOO-UHFFFAOYSA-N chromium;pyridine-2-carboxylic acid Chemical compound [Cr].OC(=O)C1=CC=CC=N1.OC(=O)C1=CC=CC=N1.OC(=O)C1=CC=CC=N1 GJYSUGXFENSLOO-UHFFFAOYSA-N 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 229960002173 citrulline Drugs 0.000 description 1
- 235000013477 citrulline Nutrition 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000003240 coconut oil Substances 0.000 description 1
- 235000019864 coconut oil Nutrition 0.000 description 1
- 235000012716 cod liver oil Nutrition 0.000 description 1
- 239000003026 cod liver oil Substances 0.000 description 1
- 229960001127 colistin sulfate Drugs 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 229940108925 copper gluconate Drugs 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 235000019244 cryptoxanthin Nutrition 0.000 description 1
- 235000000639 cyanocobalamin Nutrition 0.000 description 1
- 239000011666 cyanocobalamin Substances 0.000 description 1
- 229960002104 cyanocobalamin Drugs 0.000 description 1
- 229960002433 cysteine Drugs 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
- 229960003067 cystine Drugs 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- ZAKOWWREFLAJOT-UHFFFAOYSA-N d-alpha-Tocopheryl acetate Natural products CC(=O)OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C ZAKOWWREFLAJOT-UHFFFAOYSA-N 0.000 description 1
- 229940039770 d-alpha-tocopheryl acetate Drugs 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 229940009976 deoxycholate Drugs 0.000 description 1
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 235000019621 digestibility Nutrition 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000013024 dilution buffer Substances 0.000 description 1
- 108700003601 dimethylglycine Proteins 0.000 description 1
- 206010013023 diphtheria Diseases 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
- YKZPPPNXRZHVGX-PXYKVGKMSA-L dipotassium;(2s)-2-aminobutanedioate;hydron;hydrate Chemical compound [H+].[H+].O.[K+].[K+].[O-]C(=O)[C@@H](N)CC([O-])=O.[O-]C(=O)[C@@H](N)CC([O-])=O YKZPPPNXRZHVGX-PXYKVGKMSA-L 0.000 description 1
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 1
- CDMADVZSLOHIFP-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane;decahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 CDMADVZSLOHIFP-UHFFFAOYSA-N 0.000 description 1
- 239000009588 dong quai Substances 0.000 description 1
- 230000005059 dormancy Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 210000001842 enterocyte Anatomy 0.000 description 1
- ZDKZHVNKFOXMND-UHFFFAOYSA-N epinepetalactone Chemical compound O=C1OC=C(C)C2C1C(C)CC2 ZDKZHVNKFOXMND-UHFFFAOYSA-N 0.000 description 1
- 125000004494 ethyl ester group Chemical group 0.000 description 1
- 239000005038 ethylene vinyl acetate Substances 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 239000011773 ferrous fumarate Substances 0.000 description 1
- 235000002332 ferrous fumarate Nutrition 0.000 description 1
- 229960000225 ferrous fumarate Drugs 0.000 description 1
- 235000013924 ferrous gluconate Nutrition 0.000 description 1
- 239000004222 ferrous gluconate Substances 0.000 description 1
- 229960001645 ferrous gluconate Drugs 0.000 description 1
- 229960001781 ferrous sulfate Drugs 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 229940050549 fiber Drugs 0.000 description 1
- 239000010408 film Substances 0.000 description 1
- 125000004387 flavanoid group Chemical group 0.000 description 1
- 229930003949 flavanone Natural products 0.000 description 1
- 150000002208 flavanones Chemical class 0.000 description 1
- 235000011981 flavanones Nutrition 0.000 description 1
- 229930003944 flavone Natural products 0.000 description 1
- 150000002213 flavones Chemical class 0.000 description 1
- 235000011949 flavones Nutrition 0.000 description 1
- HVQAJTFOCKOKIN-UHFFFAOYSA-N flavonol Natural products O1C2=CC=CC=C2C(=O)C(O)=C1C1=CC=CC=C1 HVQAJTFOCKOKIN-UHFFFAOYSA-N 0.000 description 1
- 150000002216 flavonol derivatives Chemical class 0.000 description 1
- 235000011957 flavonols Nutrition 0.000 description 1
- 235000004426 flaxseed Nutrition 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 229940064302 folacin Drugs 0.000 description 1
- 229940014144 folate Drugs 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 235000019249 food preservative Nutrition 0.000 description 1
- 239000005452 food preservative Substances 0.000 description 1
- 244000078673 foodborn pathogen Species 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 235000021588 free fatty acids Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 229960003692 gamma aminobutyric acid Drugs 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
- 235000004611 garlic Nutrition 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 235000008397 ginger Nutrition 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 229960002449 glycine Drugs 0.000 description 1
- RFDAIACWWDREDC-FRVQLJSFSA-N glycocholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 RFDAIACWWDREDC-FRVQLJSFSA-N 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 235000005679 goldenseal Nutrition 0.000 description 1
- 208000001786 gonorrhea Diseases 0.000 description 1
- 244000000058 gram-negative pathogen Species 0.000 description 1
- 229940038487 grape extract Drugs 0.000 description 1
- 229940087603 grape seed extract Drugs 0.000 description 1
- 235000002532 grape seed extract Nutrition 0.000 description 1
- 235000020688 green tea extract Nutrition 0.000 description 1
- 235000008216 herbs Nutrition 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 229960002885 histidine Drugs 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 235000001050 hortel pimenta Nutrition 0.000 description 1
- 150000001261 hydroxy acids Chemical class 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 208000011140 intestinal infectious disease Diseases 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000007914 intraventricular administration Methods 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000005040 ion trap Methods 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- VRIVJOXICYMTAG-IYEMJOQQSA-L iron(ii) gluconate Chemical compound [Fe+2].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O VRIVJOXICYMTAG-IYEMJOQQSA-L 0.000 description 1
- CJWQYWQDLBZGPD-UHFFFAOYSA-N isoflavone Natural products C1=C(OC)C(OC)=CC(OC)=C1C1=COC2=C(C=CC(C)(C)O3)C3=C(OC)C=C2C1=O CJWQYWQDLBZGPD-UHFFFAOYSA-N 0.000 description 1
- 150000002515 isoflavone derivatives Chemical class 0.000 description 1
- 235000008696 isoflavones Nutrition 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 229960003136 leucine Drugs 0.000 description 1
- 229940010454 licorice Drugs 0.000 description 1
- 229960004488 linolenic acid Drugs 0.000 description 1
- 235000019136 lipoic acid Nutrition 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000012669 liquid formulation Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 235000010420 locust bean gum Nutrition 0.000 description 1
- 239000000711 locust bean gum Substances 0.000 description 1
- 235000012680 lutein Nutrition 0.000 description 1
- 229960005375 lutein Drugs 0.000 description 1
- 239000001656 lutein Substances 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
- 229960003646 lysine Drugs 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 1
- 239000001095 magnesium carbonate Substances 0.000 description 1
- 229910000021 magnesium carbonate Inorganic materials 0.000 description 1
- VTHJTEIRLNZDEV-UHFFFAOYSA-L magnesium dihydroxide Chemical compound [OH-].[OH-].[Mg+2] VTHJTEIRLNZDEV-UHFFFAOYSA-L 0.000 description 1
- 239000001755 magnesium gluconate Substances 0.000 description 1
- 235000015778 magnesium gluconate Nutrition 0.000 description 1
- 229960003035 magnesium gluconate Drugs 0.000 description 1
- 239000000347 magnesium hydroxide Substances 0.000 description 1
- 229910001862 magnesium hydroxide Inorganic materials 0.000 description 1
- 239000000395 magnesium oxide Substances 0.000 description 1
- CPLXHLVBOLITMK-UHFFFAOYSA-N magnesium oxide Inorganic materials [Mg]=O CPLXHLVBOLITMK-UHFFFAOYSA-N 0.000 description 1
- 229940091250 magnesium supplement Drugs 0.000 description 1
- IAKLPCRFBAZVRW-XRDLMGPZSA-L magnesium;(2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanoate;hydrate Chemical compound O.[Mg+2].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O IAKLPCRFBAZVRW-XRDLMGPZSA-L 0.000 description 1
- AXZKOIWUVFPNLO-UHFFFAOYSA-N magnesium;oxygen(2-) Chemical compound [O-2].[Mg+2] AXZKOIWUVFPNLO-UHFFFAOYSA-N 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 239000011572 manganese Substances 0.000 description 1
- 239000011683 manganese gluconate Substances 0.000 description 1
- 235000014012 manganese gluconate Nutrition 0.000 description 1
- 229940072543 manganese gluconate Drugs 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- OXHQNTSSPHKCPB-IYEMJOQQSA-L manganese(2+);(2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanoate Chemical compound [Mn+2].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O OXHQNTSSPHKCPB-IYEMJOQQSA-L 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- FYGDTMLNYKFZSV-UHFFFAOYSA-N mannotriose Natural products OC1C(O)C(O)C(CO)OC1OC1C(CO)OC(OC2C(OC(O)C(O)C2O)CO)C(O)C1O FYGDTMLNYKFZSV-UHFFFAOYSA-N 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013622 meat product Nutrition 0.000 description 1
- QWIZNVHXZXRPDR-WSCXOGSTSA-N melezitose Chemical compound O([C@@]1(O[C@@H]([C@H]([C@@H]1O[C@@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O)CO)CO)[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O QWIZNVHXZXRPDR-WSCXOGSTSA-N 0.000 description 1
- 230000009061 membrane transport Effects 0.000 description 1
- DKHGMERMDICWDU-GHDNBGIDSA-N menaquinone-4 Chemical compound C1=CC=C2C(=O)C(C/C=C(C)/CC/C=C(C)/CC/C=C(C)/CCC=C(C)C)=C(C)C(=O)C2=C1 DKHGMERMDICWDU-GHDNBGIDSA-N 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 229960004452 methionine Drugs 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 230000003641 microbiacidal effect Effects 0.000 description 1
- 244000005706 microflora Species 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 229910052750 molybdenum Inorganic materials 0.000 description 1
- 239000011733 molybdenum Substances 0.000 description 1
- JPSLIQUWHBPNBM-NBKAJXASSA-N n-[(2s)-4-amino-1-[[(2s,3r)-1-[[(2s)-4-amino-1-oxo-1-[[(3s,6s,9s,12s,15r,18s,21s)-6,9,18-tris(2-aminoethyl)-3-[(1r)-1-hydroxyethyl]-12,15-bis(2-methylpropyl)-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptazacyclotricos-21-yl]amino]butan-2-yl]amino]-3-h Chemical compound CS(O)(=O)=O.CCC(C)CCCC(=O)N[C@@H](CCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O JPSLIQUWHBPNBM-NBKAJXASSA-N 0.000 description 1
- ZESIAEVDVPWEKB-ORCFLVBFSA-N n-[(2s)-4-amino-1-[[(2s,3r)-1-[[(2s)-4-amino-1-oxo-1-[[(3s,6s,9s,12s,15r,18s,21s)-6,9,18-tris(2-aminoethyl)-3-[(1r)-1-hydroxyethyl]-12,15-bis(2-methylpropyl)-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptazacyclotricos-21-yl]amino]butan-2-yl]amino]-3-h Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.CC(C)CCCCC(=O)N[C@@H](CCN)C(=O)N[C@H]([C@@H](C)O)CN[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O.CCC(C)CCCCC(=O)N[C@@H](CCN)C(=O)N[C@H]([C@@H](C)O)CN[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O ZESIAEVDVPWEKB-ORCFLVBFSA-N 0.000 description 1
- 230000007694 nephrotoxicity Effects 0.000 description 1
- 231100000417 nephrotoxicity Toxicity 0.000 description 1
- 229960003966 nicotinamide Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 238000002414 normal-phase solid-phase extraction Methods 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
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 235000020660 omega-3 fatty acid Nutrition 0.000 description 1
- 229940012843 omega-3 fatty acid Drugs 0.000 description 1
- 239000006014 omega-3 oil Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 201000000901 ornithosis Diseases 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 229940055726 pantothenic acid Drugs 0.000 description 1
- 239000011713 pantothenic acid Substances 0.000 description 1
- 235000019161 pantothenic acid Nutrition 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000006072 paste Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 229960000292 pectin Drugs 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 235000011197 perejil Nutrition 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 229960005190 phenylalanine Drugs 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 235000020741 pine bark extract Nutrition 0.000 description 1
- 239000000419 plant extract Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920001200 poly(ethylene-vinyl acetate) Polymers 0.000 description 1
- 229920001515 polyalkylene glycol Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920002503 polyoxyethylene-polyoxypropylene Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229940068988 potassium aspartate Drugs 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 229960002816 potassium chloride Drugs 0.000 description 1
- 239000001508 potassium citrate Substances 0.000 description 1
- 229960002635 potassium citrate Drugs 0.000 description 1
- QEEAPRPFLLJWCF-UHFFFAOYSA-K potassium citrate (anhydrous) Chemical compound [K+].[K+].[K+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O QEEAPRPFLLJWCF-UHFFFAOYSA-K 0.000 description 1
- 235000011082 potassium citrates Nutrition 0.000 description 1
- 239000004224 potassium gluconate Substances 0.000 description 1
- 235000013926 potassium gluconate Nutrition 0.000 description 1
- 229960003189 potassium gluconate Drugs 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000002540 product ion scan Methods 0.000 description 1
- 229960002429 proline Drugs 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 230000022558 protein metabolic process Effects 0.000 description 1
- 239000008171 pumpkin seed oil Substances 0.000 description 1
- 229960003581 pyridoxal Drugs 0.000 description 1
- 235000008164 pyridoxal Nutrition 0.000 description 1
- 239000011674 pyridoxal Substances 0.000 description 1
- 235000008151 pyridoxamine Nutrition 0.000 description 1
- 239000011699 pyridoxamine Substances 0.000 description 1
- 235000008160 pyridoxine Nutrition 0.000 description 1
- 239000011677 pyridoxine Substances 0.000 description 1
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 description 1
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 description 1
- 239000011764 pyridoxine hydrochloride Substances 0.000 description 1
- 229960004172 pyridoxine hydrochloride Drugs 0.000 description 1
- 229940076788 pyruvate Drugs 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 239000013557 residual solvent Substances 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 230000003938 response to stress Effects 0.000 description 1
- 235000020945 retinal Nutrition 0.000 description 1
- 239000011604 retinal Substances 0.000 description 1
- 229960003471 retinol Drugs 0.000 description 1
- 235000020944 retinol Nutrition 0.000 description 1
- 239000011607 retinol Substances 0.000 description 1
- 229960000342 retinol acetate Drugs 0.000 description 1
- 235000019173 retinyl acetate Nutrition 0.000 description 1
- 239000011770 retinyl acetate Substances 0.000 description 1
- 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 1
- 229940108325 retinyl palmitate Drugs 0.000 description 1
- 235000019172 retinyl palmitate Nutrition 0.000 description 1
- 239000011769 retinyl palmitate Substances 0.000 description 1
- 239000002151 riboflavin Substances 0.000 description 1
- 235000019192 riboflavin Nutrition 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 239000003813 safflower oil Substances 0.000 description 1
- 235000005713 safflower oil Nutrition 0.000 description 1
- 206010039447 salmonellosis Diseases 0.000 description 1
- 239000010018 saw palmetto extract Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 230000024053 secondary metabolic process Effects 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 229960001153 serine Drugs 0.000 description 1
- 230000009528 severe injury Effects 0.000 description 1
- 239000002453 shampoo Substances 0.000 description 1
- 201000005113 shigellosis Diseases 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000004460 silage Substances 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 235000010378 sodium ascorbate Nutrition 0.000 description 1
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 description 1
- 229960005055 sodium ascorbate Drugs 0.000 description 1
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 description 1
- 235000010234 sodium benzoate Nutrition 0.000 description 1
- 239000004299 sodium benzoate Substances 0.000 description 1
- 235000019795 sodium metasilicate Nutrition 0.000 description 1
- 239000011684 sodium molybdate Substances 0.000 description 1
- 235000015393 sodium molybdate Nutrition 0.000 description 1
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 239000011781 sodium selenite Substances 0.000 description 1
- 235000015921 sodium selenite Nutrition 0.000 description 1
- 229960001471 sodium selenite Drugs 0.000 description 1
- NTHWMYGWWRZVTN-UHFFFAOYSA-N sodium silicate Chemical compound [Na+].[Na+].[O-][Si]([O-])=O NTHWMYGWWRZVTN-UHFFFAOYSA-N 0.000 description 1
- 229910052911 sodium silicate Inorganic materials 0.000 description 1
- 235000010265 sodium sulphite Nutrition 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 description 1
- 230000003381 solubilizing effect Effects 0.000 description 1
- 229940082787 spirulina Drugs 0.000 description 1
- 230000028070 sporulation Effects 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229910052712 strontium Inorganic materials 0.000 description 1
- CIOAGBVUUVVLOB-UHFFFAOYSA-N strontium atom Chemical compound [Sr] CIOAGBVUUVVLOB-UHFFFAOYSA-N 0.000 description 1
- 239000008261 styrofoam Substances 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 239000002600 sunflower oil Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 208000006379 syphilis Diseases 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 101150075675 tatC gene Proteins 0.000 description 1
- 229960003080 taurine Drugs 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 231100001274 therapeutic index Toxicity 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- UIERGBJEBXXIGO-UHFFFAOYSA-N thiamine mononitrate Chemical compound [O-][N+]([O-])=O.CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N UIERGBJEBXXIGO-UHFFFAOYSA-N 0.000 description 1
- 229960002663 thioctic acid Drugs 0.000 description 1
- 229960002898 threonine Drugs 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 238000001269 time-of-flight mass spectrometry Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 229960000984 tocofersolan Drugs 0.000 description 1
- 239000012049 topical pharmaceutical composition Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 235000010487 tragacanth Nutrition 0.000 description 1
- 239000000196 tragacanth Substances 0.000 description 1
- 229940116362 tragacanth Drugs 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 235000013337 tricalcium citrate Nutrition 0.000 description 1
- 235000019731 tricalcium phosphate Nutrition 0.000 description 1
- 125000005457 triglyceride group Chemical group 0.000 description 1
- WGIWBXUNRXCYRA-UHFFFAOYSA-H trizinc;2-hydroxypropane-1,2,3-tricarboxylate Chemical compound [Zn+2].[Zn+2].[Zn+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O WGIWBXUNRXCYRA-UHFFFAOYSA-H 0.000 description 1
- 239000007195 tryptone soya broth Substances 0.000 description 1
- 229960004799 tryptophan Drugs 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 229960004441 tyrosine Drugs 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 235000016788 valerian Nutrition 0.000 description 1
- 229960004295 valine Drugs 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 229910052720 vanadium Inorganic materials 0.000 description 1
- LEONUFNNVUYDNQ-UHFFFAOYSA-N vanadium atom Chemical compound [V] LEONUFNNVUYDNQ-UHFFFAOYSA-N 0.000 description 1
- VLOPEOIIELCUML-UHFFFAOYSA-L vanadium(2+);sulfate Chemical compound [V+2].[O-]S([O-])(=O)=O VLOPEOIIELCUML-UHFFFAOYSA-L 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 235000019155 vitamin A Nutrition 0.000 description 1
- 239000011719 vitamin A Substances 0.000 description 1
- NCYCYZXNIZJOKI-UHFFFAOYSA-N vitamin A aldehyde Natural products O=CC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-UHFFFAOYSA-N 0.000 description 1
- 235000010374 vitamin B1 Nutrition 0.000 description 1
- 239000011691 vitamin B1 Substances 0.000 description 1
- 235000019163 vitamin B12 Nutrition 0.000 description 1
- 239000011715 vitamin B12 Substances 0.000 description 1
- 235000019164 vitamin B2 Nutrition 0.000 description 1
- 239000011716 vitamin B2 Substances 0.000 description 1
- 235000019160 vitamin B3 Nutrition 0.000 description 1
- 239000011708 vitamin B3 Substances 0.000 description 1
- 235000009492 vitamin B5 Nutrition 0.000 description 1
- 239000011675 vitamin B5 Substances 0.000 description 1
- 235000019158 vitamin B6 Nutrition 0.000 description 1
- 239000011726 vitamin B6 Substances 0.000 description 1
- 235000011912 vitamin B7 Nutrition 0.000 description 1
- 239000011735 vitamin B7 Substances 0.000 description 1
- 235000019166 vitamin D Nutrition 0.000 description 1
- 239000011710 vitamin D Substances 0.000 description 1
- 150000003710 vitamin D derivatives Chemical class 0.000 description 1
- 235000005282 vitamin D3 Nutrition 0.000 description 1
- 239000011647 vitamin D3 Substances 0.000 description 1
- 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 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
- 235000019143 vitamin K2 Nutrition 0.000 description 1
- 229940045997 vitamin a Drugs 0.000 description 1
- 229940046008 vitamin d Drugs 0.000 description 1
- 229940021056 vitamin d3 Drugs 0.000 description 1
- 229940046010 vitamin k Drugs 0.000 description 1
- 229940041603 vitamin k 3 Drugs 0.000 description 1
- 239000001717 vitis vinifera seed extract Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000010497 wheat germ oil Substances 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
- 235000010930 zeaxanthin Nutrition 0.000 description 1
- 239000001775 zeaxanthin Substances 0.000 description 1
- 229940043269 zeaxanthin Drugs 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000004246 zinc acetate Substances 0.000 description 1
- 229960000314 zinc acetate Drugs 0.000 description 1
- 235000013904 zinc acetate Nutrition 0.000 description 1
- 239000011746 zinc citrate Substances 0.000 description 1
- 235000006076 zinc citrate Nutrition 0.000 description 1
- 229940068475 zinc citrate Drugs 0.000 description 1
- 239000011670 zinc gluconate Substances 0.000 description 1
- 235000011478 zinc gluconate Nutrition 0.000 description 1
- 229960000306 zinc gluconate Drugs 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 1
- AFVLVVWMAFSXCK-UHFFFAOYSA-N α-cyano-4-hydroxycinnamic acid Chemical compound OC(=O)C(C#N)=CC1=CC=C(O)C=C1 AFVLVVWMAFSXCK-UHFFFAOYSA-N 0.000 description 1
- 239000002076 α-tocopherol Substances 0.000 description 1
- 235000004835 α-tocopherol Nutrition 0.000 description 1
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 1
- FYGDTMLNYKFZSV-BYLHFPJWSA-N β-1,4-galactotrioside 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]2[C@@H](O[C@@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-BYLHFPJWSA-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
- WGVKWNUPNGFDFJ-DQCZWYHMSA-N β-tocopherol Chemical compound OC1=CC(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C WGVKWNUPNGFDFJ-DQCZWYHMSA-N 0.000 description 1
- QUEDXNHFTDJVIY-DQCZWYHMSA-N γ-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1 QUEDXNHFTDJVIY-DQCZWYHMSA-N 0.000 description 1
- GZIFEOYASATJEH-VHFRWLAGSA-N δ-tocopherol Chemical compound OC1=CC(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1 GZIFEOYASATJEH-VHFRWLAGSA-N 0.000 description 1
Images
Classifications
-
- 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
-
- C12R1/01—
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B4/00—General methods for preserving meat, sausages, fish or fish products
- A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
- A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
- A23B4/20—Organic compounds; Microorganisms; Enzymes
- A23B4/22—Microorganisms; Enzymes; Antibiotics
-
- 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
-
- 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
- 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
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3571—Microorganisms; Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- 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
-
- 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
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/005—Antimicrobial preparations
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
- C07K7/60—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation occurring through the 4-amino group of 2,4-diamino-butanoic acid
- C07K7/62—Polymyxins; Related peptides
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- 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
Definitions
- the invention is in the field of anti-microbial agents. More specifically, the invention relates to anti-microbial agents derived from Paenibacillus.
- Bacteriocins are natural proteinaceous antimicrobial compounds produced by bacteria and active against taxonomically related bacteria (Klaenhammer, 1993). Species that produce bacteriocins have been studied extensively in the hope of finding safe and efficient means of inhibiting the growth of pathogenic bacteria, especially in foods (Cleveland et al. 2001). Bacteriocins produced by Gram-positive staining bacteria, such as lactic acid bacteria, have become a focus of interest as alternatives to conventional antibiotics (Nes et al. 1996).
- Nisin the first bacteriocin ever isolated and now widely used as a food additive, was approved by the World Health Organization for use as a food preservative in 1973.
- This peptide is generally inactive against Gram-negative staining bacteria, imposing a limitation on its effectiveness when major food-borne pathogens such as Escherichia coli, Salmonella and Yersinia are involved (Du and Shen 1999; Zheng et al. 1999).
- Davies et al. (1998) reported that nisin produced by Lactococcus lactis was thermostable and remained active after treatment at 121° C. for 15 min at pH 3.
- Nisin is about 4.4 kDa and is stabilized by disulfide bonds.
- Polymyxins a class of antimicrobial agents, are synthesized by a non-ribosomal process.
- the peptide-synthase directed condensation reactions by which polymyxins are formed in the cell cytoplasm have been reviewed (Marahiel et al. 1997) and their biosynthesis in a cell-free enzyme system reported ( Komura et al. 1985).
- DeCrescenzo et al. (2007) isolated a new Paenibacillus species ( P. amylolyticus C27) that produces polymyxins E1 and E2 (colistin A and B).
- the new antimicrobial peptides were reported to be effective against Gram-negative staining bacteria such as E. coli, Pseudomonas, Salmonella , and Shigella .
- DeCrescenzo et al. (2007) also reported that polymyxin E produced by P.
- amylolyticus C27 inhibited Gram-positive staining bacteria such as Staphylococus aureus ATCC 6538, Enterococcus faecalis ATCC 19433 and Streptococcus pyogenes ATCC 19165.
- Zengguo et al. (2007) reported the co-production of polymyxin and lantibiotic by natural isolates of P. polymyxa .
- the two antimicrobial peptides were reported to display potent activity against many Gram-negative staining bacteria, including E. coli, Pseudomonas aeruginosa and Acinetobacter baumannii , and against Gram-positive food-borne pathogenic bacteria.
- Zengguo et al. (2007) also reported that polymyxin produced by P. polymyxa OSY-DF is stable from pH 2.0 to 9.0 and retained its activity after a short autoclaving.
- Colistin is a polymyxin antibiotic discovered in the late 1940s for the treatment of Gram-negative infections. After several years of clinical use, colistin was associated with significant nephrotoxicity and neurotoxicty (Lim et al. 2010), rendering its use questionable. Colistin has a bactericidal effect against Gram-negative bacteria and acts as a detergent-like molecule (Landman et al. 2008).
- polymyxin E1 The coproduction of polymyxin E1 and a lantibiotic has been reported for P. polymyxa OSY-DF; a strain isolated from food (He et al. 2007). Polymyxin E1 was active against Gram-negative bacteria, whilst paenibacillin, a proteinaceous compound, exhibited activity against Gram-positive bacteria (He et al. 2007 & 2008). P. kobensis M isolated from soil produced mattacin (polymixin M) (Nathaniel et al. 2003) and Bacillus sp.
- strain B-60 produced various inhibitory molecules named sattabacin, hydroxysattabacin, sattazolin and methylsattazolin, with antiviral activity against herpes simplex viruses types 1 and 2 (HSV1 and HSV2) (Lampis et al. 1995).
- Strains of P. polymyxa have been isolated from different ecological niches including food matrices such as butternut squash, potatoes, rice, and wheat flour (Fangio et al. 2010).
- E. coli is regarded as an inoffensive and common bacterium of the gastrointestinal tract of ruminants.
- O157:H7 The emergence of the enterohemorrhagic strain O157:H7 has become a major public concern worldwide. Indeed this pathogen is able to produce potent endotoxins with severe damage to the lining of the intestine, in particular in humans, allowing the strain to invade the body and infect organs such as the kidneys, sometimes with fatal consequences.
- Spore-forming species such as Paenibacillus or other probiotic bacteria are known in the art. Andersson et al. (1997) showed that Bacillus cereus produced spores able to adhere to Caco-2 cell monolayers and to germinate in an environment similar to that of the small intestine and noted that the hydrophobicity of the spore is a contributing factor to adhesion.
- Angioi et al. (1995) evaluated the capacity of Bacillus subtilis strains to colonise the surface of Caco-2 cells, testing the bacteria both in the spore state and following stimulation of germination by exposure to low pH (as under gastric conditions) or to high temperatures. The degree of adhesion was found to vary in relation to the different physiological phases of the bacterial cells.
- the present invention provides, in part, an isolated Paenibacillus sp. bacterium comprising SEQ ID NO: 1.
- the invention provides an isolated Paenibacillus polymyxa (Strain JB05-01-1) bacterium deposited at the ATCC®) under the terms of the Budapest Treaty and designated Accession Number PTA-10436, or a strain comprising the identifying characteristics thereof.
- the bacterium may be isolated from a direct-fed microbial product. For example, JB05-01-1 was found while examining a mixed culture preparation of RE3TM.
- the bacterium may include an anti-microbial activity, such as an anti-bacterial activity.
- the anti-bacterial activity may include inhibiting the growth of a Gram-negative staining bacterium, such as one or more of Escherichia sp. (e.g., Escherichia coli such as Escherichia coli RR1, Escherichia coli TB1, or Escherichia coli O157:H7), Pantoea sp. (e.g., Pantoea agglomerans BC1), Pseudomonas sp. (e.g., Pseudomonas fluorescens R73), Butyrivibrio sp.
- Escherichia sp. e.g., Escherichia coli such as Escherichia coli RR1, Escherichia coli TB1, or Escherichia coli O157:H7
- Pantoea sp. e.g
- Fibrobacter sp. e.g., Fibrobacter succinogenes
- Salmonella sp. e.g., Salmonella enteritidis or Salmonella typhi
- Shigella sp. e.g., Shigella dysenteriae
- Helicobacter sp. e.g., Helicobacter pylori
- Campylobacter sp e.g., Campylobacter jejuni ).
- the anti-bacterial activity may include inhibiting the growth of a Gram-negative staining bacterium, such as one or more of Escherichia coli RR1, Escherichia coli TB1, and Escherichia coli O157:H7, Pantoea agglomerans BC1, Pseudomonas fluorescens R73, Butyrivibrio fibrisolvens OR85 , Fibrobacter succinogenes and Pseudomonas aeruginosa.
- a Gram-negative staining bacterium such as one or more of Escherichia coli RR1, Escherichia coli TB1, and Escherichia coli O157:H7
- Pantoea agglomerans BC1 Pseudomonas fluorescens R73
- Butyrivibrio fibrisolvens OR85 Fibrobacter succinogenes and Pseudomonas aeruginosa.
- the bacterium may not inhibit the growth of a Gram-positive staining bacterium, such as one or more of a Listeria innocua, Listeria monocytogenes, Pediococcus acidilactici, Paenibacillus polymyxa, Paenibacillus macerans, Bacillus lecheniformis, Bacillus subtilis, Bacillus circulans 9E2, Streptococcus bovis, Enterococcus mundtii, Staphylococcus aureus , or Lactococcus lactis.
- a Gram-positive staining bacterium such as one or more of a Listeria innocua, Listeria monocytogenes, Pediococcus acidilactici, Paenibacillus polymyxa, Paenibacillus macerans, Bacillus lecheniformis, Bacillus subtilis, Bacillus circulans 9E2, Streptococcus bovis, Enterococcus mundtii,
- the anti-microbial activity may be sensitive to an enzyme selected from the group consisting of one or more of proteinase K, trypsin, chymotrypsin or lipase; or may be sensitive to sodium dodecyl sulphate (SDS) or urea; or may be sensitive to a temperature in excess of about 90° C. for about 30 minutes; or may be sensitive to a temperature of about 100° C. for about 10 minutes; or may be insensitive to a temperature up to about 80° C.
- SDS sodium dodecyl sulphate
- the invention provides a cell culture including a bacterium as described herein.
- the cell culture may be a starter culture.
- the invention provides a cell culture supernatant derived from growing a bacterium as described herein in a cell culture medium.
- the supernatant may include an anti-microbial activity, such as an anti-bacterial activity.
- the invention provides an anti-microbial agent isolated from a bacterium, cell culture, or cell culture supernatant as described herein.
- the anti-microbial agent may include a peptide, such as a lipopeptide.
- the anti-microbial agent may include a molecular weight between about 1000 daltons to about 2500 daltons.
- the anti-microbial agent may be a polymyxin.
- the anti-microbial agent may be colistin A and/or colistin B.
- the invention provides a bacterium, cell culture, cell culture supernatant, or anti-microbial agent as described herein.
- the invention provides a pharmaceutical, veterinary, cosmetic or hygiene composition including a bacterium, cell culture, cell culture supernatant, or anti-microbial agent as described herein and a suitable carrier.
- the invention provides a food or feed additive comprising a bacterium, cell culture, cell culture supernatant, or anti-microbial agent as described herein.
- the invention provides a packaging material comprising a bacterium, cell culture, cell culture supernatant, or anti-microbial agent as described herein.
- the invention provides a kit comprising a bacterium, cell culture, cell culture supernatant, or anti-microbial agent as described herein together with instructions for use in inhibiting growth of a micro-organism.
- the invention provides a method of producing an anti-microbial agent, by providing a live Paenibacillus sp. bacterium comprising SEQ ID NO: 1; and culturing the live Paenibacillus sp. bacterium in a cell culture medium, under conditions suitable for production of the anti-microbial agent.
- the invention provides a method of producing an anti-microbial agent, by providing a live Paenibacillus polymyxa (Strain JB05-01-1) bacterium or a strain comprising the identifying characteristics thereof; and culturing the live Paenibacillus polymyxa (Strain JB05-01-1) bacterium or a strain comprising the identifying characteristics thereof in a cell culture medium, under conditions suitable for production of the anti-microbial agent.
- the methods may further include isolating the anti-microbial agent from the bacterium.
- the culturing may be performed under conditions suitable for secretion of the anti-microbial agent into the cell culture medium.
- the methods may further include separating the bacterium from the cell culture medium to provide a cell culture supernatant comprising the anti-microbial agent.
- the methods may further include isolating the anti-microbial agent from the cell culture supernatant.
- the invention provides an anti-microbial agent produced by the methods as described herein.
- the anti-microbial agent may include a peptide, such as a lipopeptide.
- the anti-microbial agent may include a molecular weight between about 1000 daltons to about 2500 daltons.
- the anti-microbial agent may be a polymyxin.
- the anti-microbial agent may be colistin A and/or colistin B.
- the anti-microbial agent may include an anti-microbial activity selected from one or more of: sensitivity to proteinase K, trypsin, chymotrypsin and lipase, sodium dodecyl sulphate (SDS), urea, acetonitrile, or hexane; insensitivity to chloroform, propanol, methanol, ethanol or toluene; insensitivity to pH; sensitivity to a temperature in excess of about 90° C. for about 30 minutes; sensitivity to a temperature of about 100° C. for about 10 minutes; insensitivity to a temperature up to about 80° C. for about 30 minutes; inhibition of growth of a Gram-negative staining bacterium; or no inhibition of growth of a Gram-positive staining bacterium.
- SDS sodium dodecyl sulphate
- the invention provides a method of inhibiting the growth of a microorganism in a subject or substance in need thereof by administering or applying an effective amount of the bacterium, cell culture, cell culture supernatant or anti-microbial agent, as described herein, to the subject or substance.
- the inhibition of growth may be selective.
- the microorganism may be a bacterium, such as a Gram-negative staining bacterium, such as one or more of one or more of Escherichia sp. (e.g., Escherichia coli such as Escherichia coli RR1, Escherichia coli TB1, or Escherichia coli O157:H7), Pantoea sp. (e.g., Pantoea agglomerans BC1), Pseudomonas sp. (e.g., Pseudomonas fluorescens R73 or Pseudomonas aeruginosa ), Butyrivibrio sp.
- Escherichia sp. e.g., Escherichia coli such as Escherichia coli RR1, Escherichia coli TB1, or Escherichia coli O157:H7
- Pantoea sp. e.g
- Fibrobacter sp. e.g., Fibrobacter succinogenes
- Salmonella sp. e.g., Salmonella enteritidis or Salmonella typhi
- Shigella sp. e.g., Shigella dysenteriae
- Helicobacter sp. e.g., Helicobacter pylori
- Campylobacter sp e.g., Campylobacter jejuni ).
- the Gram-negative staining bacterium may be one or more of Escherichia coli RR1, Escherichia coli TB1, and Escherichia coli O157:H7, Pantoea agglomerans BC1, Pseudomonas fluorescens R73, Butyrivibrio fibrisolvens OR85 , Fibrobacter succinogenes and Pseudomonas aeruginosa.
- the bacterium may be a pathogenic bacterium, such as a food-borne pathogenic bacterium.
- the microorganism may be a food-borne pathogenic micro-organism or a food-spoilage micro-organism.
- the subject may be an animal, such as a human, a pet, or an agricultural animal (e.g., cow, horse, pig, sheep, goat, chicken, turkey, duck, goose, fish, or crustacean).
- the substance may be a cosmetic, hygiene, feed or food product (e.g., dairy or meat product) or packaging material thereof.
- the invention provides an isolated nucleic acid molecule including SEQ ID NO: 1.
- the invention provides the use of an effective amount of a bacterium, cell culture, cell culture supernatant or anti-microbial agent, as described herein for inhibiting the growth of a microorganism in a subject or substance in need thereof.
- a method of inhibiting growth of a Gram-negative staining bacterium in a subject or substance in need thereof comprising administering or applying an effective amount of an anti-microbial composition to the subject or substance, wherein the anti-microbial composition comprises: (a) an isolated Paenibacillus polymyxa bacterium which has a 16S ribosomal RNA (16SrRNA) gene containing a DNA comprising the base sequence of SEQ ID NO: 1; (b) an isolated Paenibacillus polymyxa bacterium deposited at the ATCC® under the terms of the Budapest Treaty and designated Accession Number PTA-10436; (c) a cell culture comprising the Paenibacillus polymyxa bacterium; (d) a cell culture supernatant derived from growing the Paenibacillus polymyxa bacterium in a cell culture medium; or (e) an anti-microbial agent isolated from the Paenibacillus polymyxa bacter
- a method of producing an anti-microbial agent comprising growing or culturing an isolated Paenibacillus polymyxa bacterium which has a 16S ribosomal RNA (16SrRNA) gene containing a DNA comprising the base sequence of SEQ ID NO: 1, or an isolated Paenibacillus polymyxa bacterium deposited at the ATCC® under the terms of the Budapest Treaty and designated Accession Number PTA-10436, in a cell culture medium to produce a cell culture supernatant comprising the anti-microbial agent.
- 16SrRNA 16S ribosomal RNA
- a method of producing an anti-microbial agent comprising growing or culturing an isolated Paenibacillus polymyxa bacterium which has a 16S ribosomal RNA (16SrRNA) gene containing a DNA comprising the base sequence of SEQ ID NO: 1, or an isolated Paenibacillus polymyxa bacterium deposited at the ATCC® under the terms of the Budapest Treaty and designated Accession Number PTA-10436, in a cell culture medium to produce a cell culture comprising the anti-microbial agent.
- 16SrRNA 16S ribosomal RNA
- a method of reducing mortality or morbidity of an animal comprising administering an effective amount of an anti-microbial composition to the animal, wherein the anti-microbial composition comprises: (a) an isolated Paenibacillus polymyxa bacterium which has a 16S ribosomal RNA (16SrRNA) gene containing a DNA comprising the base sequence of SEQ ID NO: 1; (b) an isolated Paenibacillus polymyxa bacterium deposited at the ATCC® under the terms of the Budapest Treaty and designated Accession Number PTA-10436; (c) a cell culture comprising the Paenibacillus polymyxa bacterium; (d) a cell culture supernatant derived from growing the Paenibacillus polymyxa bacterium in a cell culture medium; or (e) an anti-microbial agent isolated from the Paenibacillus polymyxa bacterium, the cell culture or the cell culture supernatant.
- a method of identifying a bacterium useful in producing an antimicrobial agent a bacterium useful for inhibiting growth of a Gram-negative staining bacterium in a subject or substance in need thereof; or a bacterium useful in reducing mortality or morbidity of an animal.
- the method may comprise assessing active mixed cultures for the presence of a bacterium comprising a 16S ribosomal RNA (16SrRNA) gene containing a DNA comprising the base sequence of SEQ ID NO: 1; isolating the bacterium that comprises said sequence, and culturing the bacterium as a pure culture.
- the active mixed culture may be a direct-fed microbial mixed culture.
- the method may further comprise identifying cultures using the phenotypic or genetic traits of JB05-01-1 disclosed herein.
- FIG. 1 is a graph showing Kinetics of P. polymyxa JB05-01-1 growth and production of “JB05-01-1” during stirred batch culture in Luria-Bertani broth at 30° C. Filled diamond is culture optical density; open triangle is inhibitory activity expressed as AU/ml, determined by micro-dilution assay
- FIG. 2 is a pair of graphs showing the antimicrobial activity against E. coli RR1.
- DiVerent amounts of crude P. polymyxa culture supernatant (1 mg/ml) containing antimicrobial substance(s) “JB05-01-1” at 0 (open diamond), 32 (open square), and 96 (open triangle) AU/ml or polymyxin B (0.1 ⁇ g/ml) (open circle) were added to exponentially growing culture of Escherichia coli RR1 in tryptic soy broth at 30° C., wherein graph A is optical density at 600 nm and graph B is viable cell counts (CFU, colony forming unit). Arrows show crude antimicrobials peptides addition.
- FIG. 3 is a photograph showing SDS-PAGE profiles of partially purified antimicrobial substance produced by Paenibacillus polymyxa JB05-01-1, wherein the gel in part A was stained with Coomassie Brilliant Blue staining R-250 and the gel in part B was recovered with TSBA pre-overlaid with E. coli RR1.
- Lane 1 contains the molecular weight markers (in KD);
- Lane 2 contains partly purified antimicrobial substance JB05-01-1. Arrows in gel of parts A and B show a band and a zone of inhibition, respectively.
- FIG. 4 is a partial sequence of P. polymyxa JB05-01-1 16S rRNA gene (GenBank Accession Number GQ184435; SEQ ID NO: 1).
- FIG. 5 is a Fast Protein Liquid Chromatography (FPLC) of purified antimicrobials peptides produced by P. polymyxa JB05-01-1 eluted at 30% acetonitrile (CH 3 CN), 1% TFA.
- FPLC Fast Protein Liquid Chromatography
- FIG. 6A is a mass spectrometry analysis of purified Colistin A produced by P. polymyxa JB05-01-1, and structure determination.
- FIG. 6B is a mass spectrometry analysis of purified Colistin B produced by P. polymyxa JB05-01-1, and structure determination.
- Molecular weights were determined using MS analysis (Colistin A: 1169 Da; Colistin B: 1155 Da).
- FIG. 6C is a chemical structure of fatty acid (FA): Colistin A, 6-methyloctanoyl; Colistin B, 6-methylhepatanoyl.
- FA fatty acid
- FIG. 7B is a paper disc spot test showing inhibition of E. coli ATCC 25922 by P. polymyxa JB05-01-1.
- FIG. 8 shows the affinity of P. polymyxa JB05-01-1, P. polymyxa ATCC 43865 and P. polymyxa ATCC 7070 strains for hexadecane (White) and decane (grey). Values presented are the means of three independent measurements.
- FIG. 9 shows the tolerance (Log CFU per ml) of selected P. polymyxa JB05-01-1 ( ⁇ ), P. polymyxa ATCC 43865 ( ) and P. polymyxa ATCC 7070 ( ) strains to a simulated gastric juice (pH 2.0) over time (mins) Results are means of two independent experiments, vertical bars represent standard deviations.
- the present invention provides, in part, a Paenibacillus sp. isolate, designated Paenibacillus polymyxa JB05-01-1 (deposited on Oct. 21, 2009, with the American Type Culture Collection (ATCC®) under the terms of the Budapest Treaty and assigned Accession Number PTA-10436) obtained from an animal feed additive and identified by amplification and sequencing of the 16S rRNA gene. Characterization of the physical properties and anti-microbial activities of a substance secreted into the culture supernatant of a Paenibacillus polymyxa JB05-01-1 cell culture resulted in the identification of at least one anti-microbial agent.
- ATC® American Type Culture Collection
- an “anti-microbial agent,” as used herein, refers to an agent that exhibits one or more “anti-microbial activity” i.e., any activity that inhibits the growth of a micro-organism.
- inhibit By “inhibit,” “inhibition” or “inhibiting” is meant to destroy, prevent, control, decrease, slow or otherwise interfere with the growth or survival of a micro-organism by at least about 10% to at least about 100%, or any value therebetween for example about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% when compared to the growth or survival of the micro-organism in the absence of the anti-microbial agent.
- inhibitor is meant to destroy, prevent, control, decrease, slow or otherwise interfere with the growth or survival of a micro-organism by at least about 1-fold or more, for example, about 1.5-fold to about 100-fold, or any value therebetween for example about 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95-fold when compared to the growth or survival of the micro-organism in the absence of the anti-microbial agent.
- the “inhibition” may be more than 100-fold. In alternative embodiments, the “inhibition” may be substantially complete inhibition of growth i.e., the growth rate may be reduced to about zero in the presence of the anti-microbial agent, and the anti-microbial agent may cause death of a micro-organism, when compared to the growth or survival of the micro-organism in the absence of the anti-microbial agent. Accordingly, an anti-microbial agent may be microbicidal or may be microbistatic.
- the anti-microbial agent may be an anti-bacterial agent i.e., an agent that exhibits one or more “anti-bacterial activity” i.e., any activity that inhibits the growth of a bacterium.
- the anti-bacterial agent may be bactericidal or bacteriostatic.
- an anti-bacterial agent according to the invention may selectively inhibit the growth of a Gram-negative staining bacterium.
- selective inhibit By “selectively inhibit” “selective inhibition” or “selectively inhibiting” is meant to destroy, prevent, control, decrease, slow or otherwise interfere with the growth or survival of a Gram-negative staining bacterium by at least about 10% to at least about 100%, or any value therebetween for example about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% when compared to the growth or survival of a Gram-positive staining bacterium.
- “selectively inhibit” “selective inhibition” or “selectively inhibiting” is meant to destroy, prevent, control, decrease, slow or otherwise interfere with the growth or survival of a Gram-negative staining bacterium by at least about 1-fold or more, for example, about 1.5-fold to about 100-fold, or any value therebetween for example about 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95-fold when compared to the growth or survival of a Gram-positive staining bacterium.
- the “selective inhibition” may be more than 100-fold. In alternative embodiments, the “selective inhibition” may be substantially complete inhibition of growth of a Gram-negative staining bacterium i.e., the growth rate could be reduced to about zero and the anti-bacterial agent may cause death of a Gram-negative staining bacterium when compared to the growth or survival of Gram-positive staining bacterium.
- Gram-negative staining bacteria include without limitation Escherichia sp., Pantoea sp., Pseudomonas sp., Salmonella sp., Shigella sp., Pseudomonas sp., Helicobacter sp., Butyrivibrio sp., Fibrobacter sp. or Campylobacter sp.
- Gram-negative staining bacteria species include without limitation Escherichia coli (e.g., Escherichia coli RR1, Escherichia coli TB1, Escherichia coli O157:H7), Pantoea agglomerans, Pseudomonas fluorescens, Pseudomonas aeruginosa, Salmonella enteritidis, Salmonella typhi, Shigella dysenteriae, Helicobacter pylori, Butyrivibrio fibrisolvens, Fibrobacter succinogenes or Campylobacter jejuni.
- Escherichia coli e.g., Escherichia coli RR1, Escherichia coli TB1, Escherichia coli O157:H7
- Pantoea agglomerans Pseudomonas fluorescens, Pseudomonas aeruginosa
- Salmonella enteritidis Salmon
- an anti-microbial agent according to the invention does not substantially inhibit the growth of Gram-positive staining bacteria, such as one or more of Listeria innocua, Listeria monocytogenes, Pediococcus acidilactici, Paenibacillus polymyxa, Paenibacillus macerans, Bacillus lecheniformis, Bacillus subtilis, Bacillus circulans 9E2, Streptococcus bovis, Enterococcus mundtii, Staphylococcus aureus , or Lactococcus lactis.
- Gram-positive staining bacteria such as one or more of Listeria innocua, Listeria monocytogenes, Pediococcus acidilactici, Paenibacillus polymyxa, Paenibacillus macerans, Bacillus lecheniformis, Bacillus subtilis, Bacillus circulans 9E2, Streptococcus bovis, Enterococcus mundtii, Staphylococc
- an anti-microbial agent according to the invention may be sensitive or insensitive to various treatments.
- sensitive or “sensitivity” is meant loss or reduction of anti-microbial activity by at least about 10% to at least about 100%, or any value therebetween for example about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% when an anti-microbial agent is subjected to a particular treatment, when compared to anti-microbial activity in the absence of the treatment.
- sensitivity loss or reduction of anti-microbial activity by at least about 1-fold or more, for example, about 1.5-fold to about 100-fold, or any value therebetween for example about 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95-fold when an anti-microbial agent is subjected to a particular treatment, when compared to anti-microbial activity in the absence of the treatment.
- the “sensitivity” may include loss or reduction of anti-microbial activity of more than 100-fold.
- sensitivity may vary with the time of treatment.
- the time of treatment may range from a few minutes to many hours.
- the time of treatment may be about 5 minutes to over 25 hours, such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 minutes or any value therebetween, or such as 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0 hours, or any value therebetween.
- Anti-microbial activity may be tested by standard methods such as agar diffusion tests and micro-dilution assay as described herein, or by other standard methods such as disk diffusion, agar dilution, or through the use of automated instrumental testing systems (see, for example, Manual of Clinical Microbiology. 1995. P. M. Murray (ed). ASM Press, Washington, D.C.).
- insensitive or “insensitivity” is meant no substantial observable effect or sensitivity when an anti-microbial agent is subjected to a particular treatment, when compared to anti-microbial activity in the absence of the treatment.
- insensitive or “insensitivity” is meant an observable effect of less than about 10%, for example about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% when an anti-microbial agent is subjected to a particular treatment, when compared to anti-microbial activity in the absence of the treatment.
- an anti-microbial agent according to the invention may be sensitive to treatment with proteases such as proteinase K, trypsin, chymotrypsin or with a lipase.
- proteases such as proteinase K, trypsin, chymotrypsin or with a lipase.
- an anti-microbial agent according to the invention may be sensitive to treatment with surfactants such as sodium dodecyl sulphate (SDS), chaotropics agents such as urea, or solvents such as acetonitrile or hexane.
- surfactants such as sodium dodecyl sulphate (SDS), chaotropics agents such as urea, or solvents such as acetonitrile or hexane.
- an anti-microbial agent according to the invention may be insensitive to an organic solvent such as chloroform, propanol, methanol, ethanol or toluene.
- an anti-microbial agent according to the invention may be insensitive to pH, for example, pH ranging from about 2 to about 9.
- an anti-microbial agent according to the invention may be sensitive to a temperature in excess of about 80° C. In some embodiments, an anti-microbial agent according to the invention may be sensitive to a temperature in excess of about 90° C. Accordingly, in some embodiments, an anti-microbial agent according to the invention may be sensitive to a temperature in excess of about 90° C. when exposed for at least about 30 minutes. In alternative embodiments, an anti-microbial agent according to the invention may be sensitive to a temperature of about 100° C. when exposed for at least about 10 minutes.
- an anti-microbial agent according to the invention may be insensitive to a temperature up to about 80° C. when exposed for about 30 minutes.
- sensitivity of an anti-microbial agent according to the invention may include: the loss (about 100%) of anti-microbial activity after treatment of a composition including the anti-microbial agent with proteinase K for 10 minutes at 100° C.; the reduction of anti-microbial activity to about 83% or about 75% by trypsin and chymotrypsin, respectively, or to about 62% by lipase; the reduction of anti-microbial activity to about 66% after SDS treatment and about 58% after Urea treatment.
- the molecular weight of an anti-microbial agent according to the invention may be about 1,000 Da to about 2,500 Da.
- an anti-microbial agent according to the invention may include more than one molecule having a molecular weight in the range of about 1,000 Da to about 2,500 Da.
- the agent may be a peptide, for example, a lipopeptide.
- an anti-microbial agent according to the invention may be a peptidic compound including for example a nonproteinaceous amino acid, such as a D-amino acid or a hydroxy acid and/or may be modified for example by N methylation, acylation, glycosylation, or heterocyclic ring formation.
- an anti-microbial agent according to the invention may be a polymyxin.
- polymyxin is meant a peptide having anti-microbial activity.
- the structure of a polymyxin may include a cyclic peptide e.g., a cyclic decapeptide, with a terminal fatty acid moiety, that is capable of inhibiting the growth of a micro-organism such as a Gram-negative staining bacterium.
- an anti-microbial agent according to the invention may be one or both of colistin A and colistin B.
- colistin A/B is meant a polymyxin having anti-microbial activity.
- colistin A has a molecular weight of about 1169 Da and includes fatty acid moiety 6-methyloctanoyl.
- Colistin B generally has a molecular weight of 1155 Da and includes fatty acid moiety 6-methylhepatanoyl.
- An anti-microbial agent according to the invention may include an anti-microbial agent produced by Paenibacillus polymyxa JB05-01-1, ATCC® Accession Number PTA-10436, or by a naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:1.
- an anti-microbial agent may include one or more compounds.
- An anti-microbial agent may be present in a cell, cell membrane, or crude extract, cell culture, or cell culture supernatant thereof.
- the cell may be a Paenibacillus polymyxa JB05-01-1 cell or a naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:1.
- Anti-microbial agent(s) may be obtained from Paenibacillus polymyxa JB05-01-1 or from other sources.
- RE3 Basic Environmental Systems & Technology Inc. Edmonton, AB, Canada
- RE3 is a direct-fed microbial product used to improve in vitro ruminal fermentation of barley grain/barley silage-based diets and includes a non-sterile liquid formulation containing L. paracasei and L. lactis cultures and their fermentation products.
- Paenibacillus polymyxa JB05-01-1 was obtained by culturing a sample of RE3TM.
- Other anti-microbial agents may similarly be found by routine screening for isolates that include the 16S rRNA sequence of SEQ ID NO:1 as described herein or known in the art.
- Anti-microbial agent(s) may be produced by growing or culturing Paenibacillus polymyxa JB05-01-1 or a bacterium that includes the 16S rRNA sequence of SEQ ID NO:1 in an appropriate cell culture medium under conditions suitable for production of anti-microbial agent(s) as described herein or known in the art.
- Paenibacillus polymyxa JB05-01-1 or a bacterium that includes the 16S rRNA sequence of SEQ ID NO:1 may be grown in an appropriate cell culture medium under conditions suitable for secretion of anti-microbial agent(s) into the cell culture supernatant as described herein or known in the art.
- the cell culture medium may be a minimal medium or a complete medium.
- the cell culture medium may be LB medium (Luria-Bertani medium).
- the medium may be a liquid medium or may be a solid or semi-solid medium, such as nutrient broth or agar, or tryptic soy broth or agar.
- the cell culture medium includes a carbon/energy source, NH 4 —N, and biotin.
- the cell culture conditions e.g., temperature, time, etc.
- the cell culture conditions may be varied as appropriate to optimize growth and/or production of the anti-microbial agent(s).
- the temperature may range from about 5° C. to about 40° C., such as 10° C., 15° C., 20° C., 25° C., 30° C., or 35° C., or any value therebetween. In alternative embodiments, the temperature may be about 30° C.
- the time may range from about 5 hours to about 48 hours or any value therebetween. In alternative embodiments, the time may be greater than 48 hours. In alternative embodiments, the time may be about 20 hours.
- the cell culture conditions may be aerobic or anaerobic.
- Standard separation processes may be used to obtain a substantially pure preparation of an anti-microbial agent.
- An agent or compound is “substantially pure” or “isolated” when it is separated from the components that naturally accompany it.
- an anti-microbial agent or compound is substantially pure when it is at least 10%, 20%, 30%, 40%, 50%, or 60%, more generally 70%, 75%, 80%, or 85%, or over 90%, 95%, or 99% by weight, of the total material in a sample.
- a substantially pure preparation or culture of a cell expressing an anti-microbial agent such as a Paenibacillus polymyxa JB05-01-1 cell or a naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:1
- a substantially pure Paenibacillus polymyxa JB05-01-1 cell or a substantially pure naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:1 is a preparation of cells or “cell culture” that contains 100% of such cells.
- an anti-microbial agent that is isolated by known purification techniques, or isolated as described herein, will be generally be substantially free from its naturally associated components.
- a substantially pure anti-microbial agent can be obtained, for example, by extraction from a natural source such as a Paenibacillus polymyxa JB05-01-1 cell or a naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:1.
- an anti-microbial agent according to the invention will form part of a composition, for example, a crude extract containing other substances.
- an anti-microbial agent may be present in a crude extract of a Paenibacillus polymyxa JB05-01-1 cell or a naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:1 that may also contain the other naturally occurring components found in such a cell.
- a crude extract of a Paenibacillus polymyxa JB05-01-1 cell or a naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:1 may be prepared by routine procedures, for example, disruption of the cells using standard mechanical or non-mechanical techniques such as freeze-thaw techniques, osmotic shock, enzyme (e.g., lysozyme) treatment, ultrasonication, liquid extrusion, etc., which may be followed by removal of the cell debris by for example centrifugation.
- standard mechanical or non-mechanical techniques such as freeze-thaw techniques, osmotic shock, enzyme (e.g., lysozyme) treatment, ultrasonication, liquid extrusion, etc.
- an anti-microbial agent may be present in a cell culture supernatant, such as a supernatant obtained from growing a Paenibacillus polymyxa JB05-01-1 cell or a naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:1 in a suitable cell culture medium under conditions suitable for secretion of the anti-microbial agent into the supernatant.
- culture supernatant refers to the liquid broth remaining when cells grown in a medium are separated from the culture medium by for example centrifugation, filtration, sedimentation, or other means well known in the art.
- a salt such as ammonium sulphate may be used at various concentrations, initially. Residual ammonium sulphate may then be removed by dialysis against water. The suspended precipitate containing one or more than one antimicrobial compound may be chromatographed on a column such as an ion exchanger, and the various compounds in the culture supernatant may be separated by monitoring absorbance at 280 nm. Active fractions can be determined from among the compounds thus separated, and selected on the basis of the efficacy with which aliquots thereof kill or inhibit the growth of microbes such as bacterial cells, i.e.
- the indicator strain known to be sensitive to the anti-microbial agent(s). Active fractions may then be pooled. Further purification may be carried out by high performance liquid chromatography (HPLC) based on the charge of the compound. The various peaks obtained by monitoring absorbance at 280 nm may be separated and again tested for activity against the indicator strain. Purity can be measured using any appropriate method such as column chromatography, gel electrophoresis, HPLC, etc.
- the anti-microbial agent may be isolated from the cell culture supernatant using solid phase extraction with columns such as Amberlite XAD-16 column and Sep-Pak C18 Column.
- the active fraction eluted from the column(s) may be further purified based on molecular weight separation using a Fast Protein Liquid Chromatography (FPLC) system.
- FPLC Fast Protein Liquid Chromatography
- Typical methods include, without limitation, size exclusion or ion exchange chromatography, ammonium sulfate, alcohol, or chloroform extraction, or centrifugation with size filters.
- anti-microbial activity of an anti-microbial agent may be determined by routine methods or as described herein.
- anti-microbial activity may be detected by agar diffusion tests or micro-dilution assay.
- Anti-microbial agent(s) according to the invention may be used in a variety of applications in which inhibition of growth of a micro-organism, such as a bacterium, is desirable. Such applications include, without limitation, pharmaceutical and veterinary applications (e.g., for the treatment of a microbial infection), nutritional supplements and animal feed, personal care (cosmetic or hygiene) applications, etc. In alternative embodiments, anti-microbial agent(s) according to the invention may be used to inhibit the growth of a microorganism (e.g., a bacterium) involved in the spoilage of food or other products.
- a microorganism e.g., a bacterium
- Food spoilage micro-organisms include without limitation one or more species of Clostridium, Pseudomonas, Porteus, Chromobacterium, Chromobacterium, Lactobacillus, Penicillium, Aspergillus, Rhizopus, Micrococcus, Bacillus, Streptococcus, Pediococcus, Leuconostoc, Chromobacterium, Halobacterium, Alcaigenes, Xanthomonas, Botryitis, Aerobacter, Cornebacterium, Arthrobacter, Microbacterium, Serratia , etc.
- the micro-organism may be a pathogenic micro-organism.
- the bacterium may a pathogenic bacterium, such as food-borne pathogenic bacterium.
- Food-borne pathogenic bacteria include without limitation one or more species of Staphylococcus, Escherichia, Listeria, Salmonella, Streptococcus, Vibrio, Campylobacter, Enterobacter, Shigella , etc.
- the bacterium may include a Gram-negative staining bacterium.
- Gram-negative staining bacteria include without limitation one or more species of Escherichia sp., Pantoea sp., Pseudomonas sp., Salmonella sp., Shigella sp., Helicobacter sp., Campylobacter sp. or Butyrivibrio sp., and Fibrobacter sp.
- Gram-negative bacteria species include without limitation Escherichia coli (e.g., Escherichia coli RR1, Escherichia coli TB1, Escherichia coli O157:H7), Pantoea agglomerans, Pseudomonas fluorescens, Pseudomonas aeruginosa, Salmonella enteritidis, Salmonella typhi, Shigella dysenteriae, Helicobacter pylori, Campylobacter jejuni, Butyrivibrio fibrisolvens , or Fibrobacter succinogenes.
- Escherichia coli e.g., Escherichia coli RR1, Escherichia coli TB1, Escherichia coli O157:H7
- Pantoea agglomerans Pseudomonas fluorescens, Pseudomonas aeruginosa
- Salmonella enteritidis Salmonella
- bacteria include without limitation Gram-negative rods such as enteric Gram-negative staining rods, curved Gram-negative staining rods, parvobacteria and Haemophilus , Gram-negative staining cocci such as Neisseria , non-sporing anaerobes, and bacteria such as spirochaetes, rickettsia and chlamydia.
- Gram-negative rods such as enteric Gram-negative staining rods, curved Gram-negative staining rods, parvobacteria and Haemophilus
- Gram-negative staining cocci such as Neisseria
- non-sporing anaerobes non-sporing anaerobes
- bacteria such as spirochaetes, rickettsia and chlamydia.
- microial infections such as bacterial infections
- bacterial infections include without limitation chlamydia , gonorrhea, salmonellosis , shigellosis, tuberculosis, syphilis, bacterial pneumonia, bacterial sepsis (bacteremia), bacterial urinary tract infections, vaginosis, bacterial upper respiratory tract infections, bacterial meningitis, bacterial enteritis, diphtheria, legionellosis, pertussis, scarlet fever, toxic shock syndrome, psittacosis, otitis media, lyme disease, etc.
- Anti-microbial agents of the invention can be provided alone or in combination with other compounds, in the presence of any pharmaceutically, veterinary or cosmetically acceptable carrier, diluent, and/or excipient in a form suitable for administration to animals, for example, humans, cattle, sheep, pigs, poultry, etc. If desired, administration or application of an anti-microbial agent according to the invention may be combined with more traditional and existing anti-microbial therapies, treatments, supplements, or additives, or with other desirable therapies, treatments, supplements, or additives.
- Anti-microbial agents according to the invention may be provided chronically or intermittently.
- Chronic administration refers to administration of the anti-microbial agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time.
- Intermittent administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.
- Suitable formulations or compositions to administer the anti-microbial agent(s) to subjects suffering from or presymptomatic for a microbial infection.
- Any appropriate route of administration may be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracisternal, intraperitoneal, intranasal, intra-anal, intravaginal, aerosol, topical, or oral administration.
- Therapeutic formulations may be in the form of liquid solutions, syrups, or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols; and topical formulations may come in the form of balms, creams, and lotions.
- Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
- Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
- parenteral delivery systems for include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
- Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
- the anti-microbial agent(s) are administered to a subject in an amount sufficient to inhibit the growth of a micro-organism.
- an “effective amount” of an anti-microbial agent(s) according to the invention includes a therapeutically effective amount or a prophylactically effective amount.
- a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as inhibition of the growth of a micro-organism.
- a therapeutically effective amount of an anti-microbial agent(s) may vary according to factors such as the disease state, age, sex, and weight of the individual or subject, and the ability of the anti-microbial agent(s) to elicit a desired response in the individual or subject. Dosage regimens may be adjusted to provide the optimum therapeutic or prophylactic response.
- a therapeutically effective amount is also one in which any toxic or detrimental effects of the anti-microbial agent(s) are outweighed by the therapeutically beneficial effects.
- a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as such as inhibition of the growth of a micro-organism. Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount.
- An exemplary range for therapeutically or prophylactically effective amounts of an anti-microbial agent(s) may be any value from about 0.1 nM to about 0.1M, for example about 0.1 nM to about 0.05M, about 0.05 nM to about 15 ⁇ M or about 0.01 nM to about.
- dosage values may vary with the severity of the condition to be alleviated.
- specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the anti-microbial agent(s).
- Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical or veterinary practitioners.
- the amount of active anti-microbial agent(s) in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic or prophylactic response.
- a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
- a subject may be a mammal, an agricultural (e.g., farm) or domestic animal, an experimental animal or any animal that may benefit from the anti-microbial agents as described herein.
- a subject may include a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, chicken, turkey, duck, goose, dog, cat, fish, crustacean, etc.
- anti-microbial agent(s) of the invention should be used without causing substantial toxicity.
- Toxicity of the anti-microbial agent(s) of the invention can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD50 (the dose lethal to 50% of the population) and the LD100 (the dose lethal to 100% of the population). In some circumstances however, such as in severe disease conditions, it may be necessary to administer substantial excesses of the anti-microbial agent(s).
- an “effective amount” of an anti-microbial agent according to the invention includes an amount effective to inhibit the growth of a micro-organism, such as a bacterium. It is to be understood that such amounts need not be therapeutic or prophylactic amounts, as long as the amount of the anti-microbial agent is capable of inhibiting the growth of a micro-organism, such as a bacterium, in the context in which it is administered or applied, for example, for prevention of food spoilage, etc.
- an anti-microbial agent according to the invention may be provided in a cell, for example a substantially pure Paenibacillus polymyxa JB05-01-1 cell or a substantially pure naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:1, or a cell culture thereof.
- the cell may be provided in a liquid, or may be frozen or dried, e.g., freeze-dried.
- the cell culture may be concentrated.
- the cell culture may be a “starter” culture for example for a dairy product (e.g., milk, cheese, etc.), or for selective media in a laboratory.
- the anti-microbial agent may be provided in a therapeutic, veterinary, hygiene, cosmetic, food, drink or feed product.
- the anti-microbial agent may be provided in the packaging material for, for example, a therapeutic, veterinary, hygiene, cosmetic, food, drink or feed product.
- the packaging material may include without limitation, plastic, film, Styrofoam, etc.
- an anti-microbial agent according to the invention may be provided in a kit that may optionally include additional anti-microbial agents or desirable therapies, treatments, supplements, or additives, optionally with instructions for use thereof.
- an anti-microbial agent according to the invention may be provided as a nutritional or food additive, or feed supplement or additive.
- a “nutritional additive” or “food additive” refers to a substance that is added to food, generally to affect the characteristics of the food, such as spoilage.
- a food additive may be “direct” in that it is directly added to food for example to inhibit growth of a micro-organism.
- a food additive may be considered “indirect” when it is exposed to food during processing, packaging, or storage but is not present in the final food product.
- feed additive or “feed supplement” refers to products used in animal nutrition for purposes of improving the quality of feed, or to improve the animals' performance and health, e.g. providing enhanced digestibility of the feed materials or inhibiting the growth of micro-organisms.
- an animal feed additive is a direct-fed microbial product which refers to a mono or mixed culture of live micro-organisms, which when applied to a host affects beneficially the host by improving the properties of the indigenous microflora.
- a non-limiting example of a direct-fed microbial product is RE3TM from Basic Environmental Systems & Technology Inc. Edmonton, AB, Canada.
- RE3TM may be specifically excluded from a feed additive according to the invention.
- a probiotic candidate should display the ability to survive under gastrointestinal tract conditions, namely resistance to salivary lysozyme, gastric acid (HCl) and bile.
- the “candidate” should also be able to establish itself among the intestinal microbiota and adherence to the intestinal epithelium is considered a major parameter determining candidate's ability to colonize the intestinal tract and thus be effective as a probiotic.
- P. polymyxa JB05-01-1 exhibited probiotic characteristics such as production of antimicrobials ( FIGS. 7A and 7B ), resistance to gastrointestinal conditions (e.g. lysozyme and acidic pH—Table 5) and tolerance of hydrogen peroxide (Table 5), as well as adhesion to Caco-2 cells (Table 7). Therefore in alternative embodiments, the bacterium, cell culture, supernatant, or the anti-microbial agent according to the invention may be used as a probiotic.
- anti-microbial agents of the invention can be provided in combination with other feed or nutritional supplements or additives.
- at least one supplement or additive, such as listed herein can be included for consumption with the anti-microbial agent of the invention and may have, for example, antioxidant, dispersant, antimicrobial, or solubilizing properties.
- a suitable antioxidant is, for example, vitamin C, vitamin E or rosemary extract.
- a suitable dispersant is, for example, lecithin, an alkyl polyglycoside, polysorbate 80 or sodium lauryl sulfate.
- a suitable antimicrobial is, for example, sodium sulfite or sodium benzoate.
- a suitable solubilizing agent is, for example, a vegetable oil such as sunflower oil, coconut oil, and the like, or mono-, di- or tri-glycerides.
- Additives include vitamins such as vitamin A (retinol, retinyl palmitate or retinol acetate), vitamin B1 (thiamin, thiamin hydrochloride or thiamin mononitrate), vitamin B2 (riboflavin), vitamin B3 (niacin, nicotinic acid or niacinamide), vitamin B5 (pantothenic acid, calcium pantothenate, d-panthenol or d-calcium pantothenate), vitamin B6 (pyridoxine, pyridoxal, pyridoxamine or pyridoxine hydrochloride), vitamin B12 (cobalamin or cyanocobalamin), folic acid, folate, folacin, vitamin H (biotin), vitamin C (ascorbic acid, sodium ascorbate, calcium ascorbate or ascorbyl palmitate), vitamin D (cholecalciferol, calciferol or ergocalciferol), vitamin E (d-alpha-to
- additives include minerals such as boron (sodium tetraborate decahydrate), calcium (calcium carbonate, calcium caseinate, calcium citrate, calcium gluconate, calcium lactate, calcium phosphate, dibasic calcium phosphate or tribasic calcium phosphate), chromium (GTF chromium from yeast, chromium acetate, chromium chloride, chromium trichloride and chromium picolinate) copper (copper gluconate or copper sulfate), fluorine (fluoride and calcium fluoride), iodine (potassium iodide), iron (ferrous fumarate, ferrous gluconate or ferrous sulfate), magnesium (magnesium carbonate, magnesium gluconate, magnesium hydroxide or magnesium oxide), manganese (manganese gluconate and manganese sulfate), molybdenum (sodium molybdate), phosphorus (dibasic calcium phosphate
- additives include amino acids, peptides, and related molecules such as alanine, arginine, asparagine, aspartic acid, carnitine, citrulline, cysteine, cystine, dimethylglycine, gamma-aminobutyric acid, glutamic acid, glutamine, glutathione, glycine, histidine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, serine, taurine, threonine, tryptophan, tyrosine and valine.
- amino acids such as alanine, arginine, asparagine, aspartic acid, carnitine, citrulline, cysteine, cystine, dimethylglycine, gamma-aminobutyric acid, glutamic acid, glutamine, glutathione, glycine, histidine, isoleucine, leucine, lysine, me
- additives include animal extracts such as cod liver oil, marine lipids, shark cartilage, oyster shell, bee pollen and d-glucosamine sulfate.
- animal extracts such as cod liver oil, marine lipids, shark cartilage, oyster shell, bee pollen and d-glucosamine sulfate.
- Other additives include unsaturated free fatty acids such as ⁇ -linoleic, arachidonic and ⁇ -linolenic acid, which may be in an ester (e.g. ethyl ester or triglyceride) form.
- ester e.g. ethyl ester or triglyceride
- herb and plant extracts such as kelp, pectin, Spirulina , fiber, lecithin, wheat germ oil, safflower seed oil, flax seed, evening primrose, borage oil, blackcurrant, pumpkin seed oil, grape extract, grape seed extract, bark extract, pine bark extract, French maritime pine bark extract, muira puama extract, fennel seed extract, dong quai extract, chaste tree berry extract, alfalfa, saw palmetto berry extract, green tea extracts, angelica , catnip, cayenne, comfrey, garlic, ginger, ginseng , goldenseal, juniper berries, licorice, olive oil, parsley, peppermint, rosemary extract, valerian, white willow, yellow dock and yerba mate.
- herbs and plant extracts such as kelp, pectin, Spirulina , fiber, lecithin, wheat germ oil, safflower seed oil, fla
- miscellaneous substances such as menaquinone, choline (choline bitartrate), inositol, carotenoids (beta-carotene, alpha-carotene, zeaxanthin, cryptoxanthin or lutein), para-aminobenzoic acid, betaine HCl, free omega-3 fatty acids and their esters, thiotic acid (alpha-lipoic acid), 1,2-dithiolane-3-pentanoic acid, 1,2-dithiolane-3-valeric acid, alkyl polyglycosides, polysorbate 80, sodium lauryl sulfate, flavanoids, flavanones, flavones, flavonols, isoflavones, proanthocyanidins, oligomeric proanthocyanidins, vitamin A aldehyde, a mixture of the components of vitamin A 2 , the D Vitamins (D 1 , D 2 , D 3 and D 4 ) which
- the supplement or additive may be packaged for consumption in softgel, capsule, tablet or liquid form. It can be supplied in edible polysaccharide gums, for example carrageenan, locust bean gum, guar, tragacanth, cellulose and carboxymethylcellulose.
- Cosmetic or hygiene supplements may be provided in for example, shampoos, conditioners, creams, pastes, lotions, lipsticks, lip balms, etc.
- the bacterial indicator strains used are listed in Table 1. All were maintained at ⁇ 80° C. in appropriate media containing 10% glycerol (w/v).
- P. polymyxa and all indicator strains except Butyrivibrio fibrisolvens and Fibrobacter succinogenes were propagated aerobically at 30° C. in their respective culture media as indicated in Table 1.
- the media used were: Tryptic soy broth (TSB) (Difco Laboratories, Sparks, Md., USA), de Man, Rogosa and Sharpe broth (MRS) (Rosell Institute, Montreal, PQ, Canada) (de Man et al. 1960) and Luria-Bertani (LB) broth.
- TLB Tryptic soy broth
- MFS Rogosa and Sharpe broth
- LB Luria-Bertani
- Liquid or solid (1.2% w/v agar) anaerobic L-10 medium containing glucose, maltose and soluble starch as carbon sources were used for the growth of B. fibrisolvens and F. succinogenes (Caldwell and Bryant 1966). Their growth was carried out at 39° C. in a CO 2 :H 2 atmosphere (95:5 v/v). Before starting the experiments, all strains were sub-cultured at least three times at 24-h intervals using 1% volume transfers.
- the antimicrobial agent producer P. polymyxa JB05-01-1 was isolated from a direct-fed microbial product (RE3TM Basic Environmental Systems & Technology Inc. Edmonton, AB, Canada).
- RE3TM was screened for bacteria producing compounds inhibiting the growth of E. coli by a deferred antagonism plating procedure as described by Tagg et al. (1976). Briefly, 100 ⁇ l of 10 3 -10 4 dilutions of RE3TM in L-10 or TSB media were spread on L-10 or TSB plates and incubated overnight at 39° C. and 37° C.
- the plates were replicated, and then the bacterial colonies on the original plates were washed from the agar surface and the plates were surface sterilized under Ultraviolet (UV) light at 254 nm for 20 minutes.
- the plates were then overlaid with 5 ml of melted LB (0.5% agar) containing 50 ⁇ l of an overnight culture of E. coli RR1 and incubated overnight at 37° C. Colonies producing clearing zones were identified and picked from the replica plates for testing for activity against E. coli 0157:H7.
- Gram staining, motility, catalase and oxidase tests were conducted as a preliminary step in the characterization of the selected colonies. Tentative identifications were confirmed by amplification and sequencing of 16S ribosomal RNA genes.
- the Paenibacillus strain (JB05-01-1) was grown in 3 ml of TSB at 30° C. overnight. The cells were harvested by centrifugation at 5000 ⁇ g for 5 min. DNA was extracted using a Power Soil DNA Kit (MoBio Laboratories Inc., Carlsbad, Calif., USA) according to the manufacturer's instructions. The DNA concentration was measured using the PicoGreen dsDNA quantitation kit (Molecular Probes, Invitrogen, Eugene, Oreg., USA) in a Multi Detection Microplate Reader (Model SIAFRM, BioTek Instruments, Winooski, Vt., USA) using calf thymus DNA (Sigma-Aldrich, St. Louis, Mo., USA) as the standard.
- the PCR amplification targeted the approximately 1500 bp of the 16S rRNA gene.
- the PCR reaction contained 10 ng of template DNA, 2.5 ml of 10 ⁇ dilution buffer, 10 pmol of each primer and 1 U of Taq polymerase (Takara Shuzo, Japan) in a final volume of 25 ml.
- the primers used were the universal bacterial primers 8-27 F (5′-AGA GTT TGA TCC TGG CTC)AGA ⁇ 3° (Liu et al., 1997) and 1492R (5′-TAC CTT GTT ACG ACT T-3′) (Kane et al., 1993).
- the amplification conditions involved denaturation at 95° C. for 1 min, followed by 25 cycles of 95° C. for 30 s, 55° C. for 30 s and 72° C. for 1.5 min.
- the nomenclature of the primers used was based on E. coli numbering system.
- Amplicons from the PCR reaction were electrophoresed on 1% agarose gel and purified by excising the correct sized bands.
- the DNA was extracted from the gel using QIAquick PCR purification Kit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions.
- the purified DNA was cloned into TOPO vector (Invitrogen, Carlsbad, Calif.) and further used to transform electrocompetent E. coli (DH5- ⁇ cells) by electroporation.
- the cells were then plated on LB/Kanamycin (50 mg/L) agar plates and incubated overnight at 37° C. Three clones, verified for correct inserts, were grown overnight in LB/Kanamycin (100 mg/L).
- LB medium was inoculated with 10 ml of a fresh, overnight culture of P. polymyxa JB05-01-1 and incubated at 30° C. with agitation at 200 rpm.
- the culture optical density at 600 nm was measured every two hours using a Multi-detection micro-plate reader (Bio-Teck instrument Inc., Winooski, Vt., USA), and 1 mL of culture was centrifuged (8,000 rpm, 10 min, 4° C.) to remove the cells.
- the supernatant was heated at 70° C. for 10 min to inactivate any protease activity, as described by Martin et al. (2003).
- the agar diffusion assay and micro-dilution method were used to test the heated supernatants for antimicrobial activity as described herein.
- soluble protein was done using the Folin phenol reagent method as described by Lowry et al. (1951) with bovine serum albumin as standard.
- Polymyxin E, Polymyxin B and Nisin A were used as positive control for antimicrobial activity.
- Nisin A stock solutions were prepared from pure Nisin obtained from Aplin and Barrett (Beaminster, UK) in the form of NisaplinTM, which contains 2.5% (w/w) Nisin A.
- Polymyxin E and Polymyxin B were purchased from Sigma-Aldrich (Oakville, ON, Canada).
- the secretion of the antimicrobial agent was shown to start in the exponential phase and reach its maximum in the early stationary phase ( FIG. 1 ). Production thus appeared to be growth associated and activity levels remained stable through 48 h.
- Inhibition zone diameters at 48 h were approximately 8 ⁇ 1 mm and activity was 96 AU ml ⁇ 1 .
- the qualitative antimicrobial spectrum of P. polymyxa culture supernatant was determined using the agar well diffusion method (Wolf and Gibbons 1996). Briefly, a 25-ml volume of molten tryptic soy agar (0.75% agar w/v) was cooled to 47° C. and seeded with 1% (v/v) overnight TSB culture of an indicator strain. The seeded agar was then poured into a sterile Petri plate and allowed to solidify at room temperature. Wells (7 mm) were cut in the solidified agar using a sterile metal cork borer and filled with 80 of supernatant. The plates were left at 5° C. for 2 h to allow diffusion of the tested aliquot and then incubated aerobically for 18 h at 30° C. Absence or presence of inhibition zones as well as their diameters were recorded.
- the antimicrobial activity was also determined by the micro-dilution method described by Daba et al. (1994). Activity was expressed in arbitrary units per milliliter (AU ml ⁇ 1 ) using the formula (1000/125) x (1/D), where D was the highest dilution causing inhibition of the indicator strains.
- the minimum inhibitory concentration (MIC) of P. polymyxa JB05-01-1 culture supernatant and pure polymyxin E or polymyxin B against E. coli RR1 was determined using a MicrotestTM polystyrene micro-plate assay (96-well, Becton Dickinson Labware, Lincoln Park, N.J.) as described by Kheadr et al. (2004).
- the inhibitory spectrum of the culture supernatant is presented in Table 1.
- Gram-negative staining bacteria E. coli RR1, Pantoea agglomerans BC1, Pseudomonas fluorescens R73 , B. fibrisolvens OR85 and F. succinogenes S85
- P. polymyxa JB05-01-1 The spectrum of activity of the antimicrobial agent produced by P. polymyxa JB05-01-1 was different from that of Nisin A but similar to polymyxin E and polymyxin B used as a positive control (Table 1).
- E. coli RR1 cells were cultivated in the presence of concentrated P. polymyxa culture supernatant containing JB05-01-1 at final total activity of 0, 32, and 96 AU/ml.
- the OD 600 nm of culture was determined every two hours using a multi-detection microplate reader (Bio-Teck instrument Inc, Winooski, Vt., USA).
- the corresponding final cell concentration expressed as CFU/ml was determined as described by Naghmouchi et al. (2008).
- the sensitivities of the antimicrobial agent to proteases was tested by treating P. polymyxa JB05-01-1 culture supernatant with 2 mg ml ⁇ 1 final concentration of proteinase K (Tritirachium album), ⁇ -chymotrypsin (bovine pancreas), lipase (Sigma-Aldrich, Oakville, ON), trypsin (porcine pancreas), urea (Sigma-Aldrich, St. Louis Mo.), sodium dodecyl sulfate (SDS) (Sigma-Aldrich, St. Louis Mo.) for 1 h at 37° C.
- the antimicrobial activity remained unchanged after heating at 80° C. for 30 min. Loss of activity of about 60% was observed after heating at 90° C. for 30 min. Heating to 100° C. for 10 min completely eliminated the antimicrobial activity.
- Organic solvents such as chloroform, propanol, methanol, ethanol and toluene did not affect the activity of the antimicrobial peptide.
- Activity also remained stable after a two-hour incubation at pH ranging from 2 to 9.
- P. polymyxa JB05-01-1 culture supernatant was analysed in duplicate using a NuPAGE 12% Bis-Tris gel kit (Invitrogen, Burlington, ON, Canada) as per manufacturer's instructions at 200 V (constant) for 40 min.
- the 2.5-200 kDa molecular weight marker kit from Invitrogen was used as a molecular weight standard.
- After electrophoresis, one gel was stained with Coomassie Brilliant Blue R250 (Invitrogen).
- a duplicate gel was used for the plate overlay assay to estimate the molecular weight of the antimicrobial compounds as described by Bhunia et al. (1987).
- a SDS-PAGE gel prewashed with sterile water was placed in a Petri dish and overlaid with 10 ml tryptic soy agar containing growing cells of E. coli RR1 at about 10 5 CFU ml ⁇ 1 .
- the agar was allowed to solidify, held at 4° C. for 60 min, and then incubated for 18 h at 30° C.
- the formation of an inhibition zone indicated the position and size of the active antimicrobial peptide in the gel.
- Coomassie Brilliant Blue staining of SDS-PAGE gels of P. polymyxa JB05-01-1 culture supernatant revealed no distinct protein bands.
- inhibitory activity was detected as a clearly defined zone of inhibition in the region corresponding to a molecular mass of ⁇ 2.5 kDa ( ⁇ 2500 Da) after gels were overlaid with E. coli RR1-seeded agar ( FIG. 3 ). No inhibitory activity was detected when the SDS gels were overlaid with Listeria innocua.
- the bacterial strains used and their culture media are listed in Table 4. All strains were stored previously at ⁇ 80° C. in media containing 20% glycerol (w/v) and were sub-cultured twice in the appropriate media prior to being used in experiments. Fresh cultures were prepared by inoculation of 10 ml of the corresponding medium with 100 ⁇ l of the frozen stock followed by incubation for 18-24 h at the corresponding temperature.
- P. polymyxa JB05-01-1 was grown aerobically at 30° C. in Luria-Bertani (LB) broth (Difco Laboratories, Detroit, Mich., USA) for 16 h.
- Colistin-sensitive strain Escherichia coli RR1 E. coli RR1
- TLB tryptic soy broth
- P. polymyxa JB05-01-1 culture was used to inoculate 500 ml of Luria-Bertani (LB) broth (1% inoculum) in a 1-liter flask and incubated at 30° C. for 20 h. Cells in the fermentation broth were separated by centrifugation (12,000 g, 20 min, 4° C.). The resulting cell-free supernatant was heated (100° C.
- the eluted active fraction was then evaporated to 30% aqueous volume (approximately 150 ml) using a rotary evaporator (Laborota 4001 Efficient; Krackeler Scientific, Inc., Albany, N.Y., U.S.A.) at 30° C.
- the Amberlite XAD-16 column was washed with 500 ml of HPLC grade water for future use.
- the evaporated active fraction was passed through a Waters Sep-Pak Vac 35 cc C18-10 g column (Waters, Milford, Mass., USA) via Gilson MiniPlus 2 Pump at a flow rate of 2.5 ml/min.
- the column was washed with 30 ml of 20% acetonitrile (CH 3 CN) 0.5 M HCl and the active fraction eluted with 50 ml of 50% acetonitrile 0.5 M HCl (Seim et al. 2005).
- the active fraction eluted from the Sep-Pak C18 column was evaporated under similar conditions as hereinbefore described to 20 ml and further concentrated to 5 ml using Centricon YM-3 centrifugal filter unit (Millipore: Bedford, Mass., USA) at 3000 g for 120 min at 4° C.
- the evaporated active fraction eluted from the Sep-Pak C18 column was designated as Fraction E.
- Fraction E was applied to Superdex 75 HR 16/60 gel filtration chromatography to obtain the active fraction based on molecular weight separation.
- the Superdex 75 HR 16/60 column was equilibrated with 100 mM sodium phosphate extraction buffer (pH 7.4) containing 150 mM NaCl. This step was carried out at 4° C. with a flow rate of 0.5 ml/min using a Fast Protein Liquid Chromatography (FPLC) system (GE Healthcare Canada Inc.).
- FPLC Fast Protein Liquid Chromatography
- the mobile phase consisted of 30% CH 3 CN/70% HPLC grade water, 0.1% trifluoroacetic acid (TFA) single buffer. Elution was monitored at wavelength detection of 218 nm during a total running time of about 600 min. Fractions exhibiting antibacterial activity, at a given retention time, were collected from different FPLC runs, pooled and lyophilized. The resulting powder was dissolved in 0.1 ml HPLC grade water and checked again for activity against E. coli RR 1 and the other bacteria listed in Table 4.
- TFA trifluoroacetic acid
- Antibacterial activity of the fractions obtained at each stage of the isolation process was determined by the micro-dilution method (Daba et al. 1994). Total activity was expressed in arbitrary units per milliliter (AU/ml), using the formula (1000/125) ⁇ (1/D), where D was the highest dilution causing complete inhibition of E. coli RR1, the indicator strain (Naghmouchi et al. 2010).
- soluble protein concentration was performed in triplicate with the Folin phenol reagent method (Lowry et al. 1951).
- Bovine serum albumin (BSA) was used as internal standard.
- Polymyxin E solution standard was obtained from Sigma-Aldrich (Oakville, ON, Canada).
- MIC Minimum inhibitory concentration for FPLC purified peptide peak (retention time of 361.55 min) and that of polymyxin (colistin—standard) were determined by the micro-dilution assay (Naghmouchi et al. 2010).
- MSMS MSMS fragmentation
- the antimicrobial substance(s) was/were subsequently applied to Sep-Pack C18 column and the most active fraction purified with 50% acetonitrile and 0.5 M HCl.
- the recovery from the Sep-Pack C18 step was 13.3% with the specific activity of 2,032 AU/mg of protein.
- the antimicrobial fraction obtained from the Sep-Pack C18 recovery step was separated with the FPLC system connected to Hiload 16/60 Superdex 75 Prep grade size exclusion.
- RT peak retention time
- FIG. 5 fractions with a peak retention time (RT) of 361.55 min were identified ( FIG. 5 ). These fractions were active against E. coli RR1.
- Approximately 1.1% of the culture supernatant was recovered as antimicrobial substance(s) through the FPLC filtration process.
- the procedure increased the specific activity (per mg of protein) of the antimicrobial substance(s) 190-fold.
- Total purified antimicrobial substance(s) obtained from 500 ml of the fermented broth were approximately 0.05 mg.
- the MIC of FPLC purified peptide peak (retention times of 361.55 min) against E. coli RR1 was approximately 0.13 ⁇ g/ml.
- Tandem mass spectrometry MS analysis was performed for a single active peak obtained from the final FPLC purification (361.55 min) step. Data analysis revealed the presence of two compounds with the following molecular weights 1169.7 Da ( FIG. 6A ) and 1155.7 Da ( FIG. 6B ), which are equivalent to those of colistin A and colistin B. Further confirmation of this structure (colistin) was performed with MS/MS analysis on TOF and ESI. Fragmentation of peak 585.3 Da (A) in the ion trap permitted the identification in the spectrum ( FIG.
- FIG. 6C shows the chemical structure of fatty acid colistin A and colistin B.
- purified colistin (A/B) produced by P. polymyxa JB05-01-1 had inhibitory activity against Gram-negative bacteria including E. coli O157:H7, E. coli RR1, Pseudomonas fluorescens R73 and Pseudomonas aeruginosa .
- the MIC values of the purified peptides were about 0.13 and 0.162 ⁇ g/ml for E. coli O157:H7 and P. fluorescens LRC R73, respectively. Similar MIC values were obtained with the colistin standard.
- Example 7 Determination of the Potential of P. polymyxa JB05-01-1 as a Probiotic
- Paenibacillus polymyxa JB05-01-1 and Paenibacillus polymyxa reference strains ATCC (43865 and 7070) were aerobically grown for 18 h at 30° C. with agitation in Tryptic Soy Broth (TSB) (Difco Laboratories, Detroit, Mich., USA) containing 1% of yeast extract leading to a medium named TSBYE.
- Escherichia coli generic ATCC 25922 E. coli ATCC 25922
- the four strains were stored at ⁇ 80° C. in the corresponding medium containing 20% glycerol (w/v).
- Frozen stocks were activated by re-suspending 100 ⁇ l of each conserved strain in 10 ml of growth medium, followed by incubation for 18-24 h at the corresponding temperature.
- P. polymyxa JB05-01-1 culture (10 ml) was centrifuged and the cells were resuspended in 50 ⁇ l of LB broth. 20 ⁇ l, of the suspension were deposited into a 5-mm paper disc. The paper discs impregnated with bacterial cell suspension were placed on the surface of TSBYE seeded with overnight culture of E. coli ATCC 25922 (0.25 ml in 25 ml), solidified with 0.75% agar and poured at 47° C. into Petri plates. The plates were incubated aerobically at 30° C. for 18 h and checked for zones of inhibition (clear agar) surrounding the paper discs (Lenni and Mauliya, 2010).
- bovine bile salts (Sigma) were added to the medium at different concentrations (0.1%, 0.2%, 0.3%, and 0.4% w/v).
- different concentrations (0, 2.5, 5, 10, 20 and 40 ⁇ g/ml) of H 2 O 2 purchased from Merck (Darmstadt, Germany) was added.
- Wells (in duplicate) containing 200 ⁇ l of tested broth were inoculated with 20 ⁇ l of overnight culture of P. polymyxa diluted 1/100 in TSBYE. Microplates were incubated under anaerobic conditions at 30° C. for 18 h.
- Optical densities were read at 630 nm using a Thermomax microplate reader (Zeus, scientific Inc, Raritan, N.J.). Resistance to H 2 O 2 and lysozyme (minimum inhibitory concentration) was expressed as the lowest concentration that maintained the OD under 50% of the OD in the “0” concentration wells. Acid resistance was expressed as the lowest pH at which the tested strain had an OD at least 40% of its level at pH 6.5.
- a simulated gastric juice was prepared by suspending pepsin (3 mg/ml) in sterile saline (0.5% w/v) and adjusting the pH to 2.0 with concentrated HCl. Overnight 2 ml cultures of the three P. polymyxa variants were subjected to centrifugation in an Eppendorf centrifuge at 5000 g for 5 min and washed two times in quarter-strength Ringer's solution at pH 7.
- 0.3 mL of the washed suspension was added to 1.5 mL of simulated gastric juice (pH 2.0) in a 2.0 mL Eppendorf tube and vortexed.
- simulated gastric juice was replaced by 1.5 mL quarter-strength Ringer's solution and these samples were used for determination of the initial cell counts.
- Aliquots of 0.2 mL were removed after 15, 30, and 90 min at 37° C. and viable counts were determined by plating serial 10-fold dilutions on LB agar and counting the colony numbers after anaerobic incubation at 37° C. for 48 h. Experiments were carried out in duplicate and were repeated two times. Results are expressed as the mean and standard deviation of three determinations.
- P. polymyxa JB05-01-1 and other P. polymyxa strains (ATCC 43865, ATCC 7070) to utilize sugars, glycogen and pyruvate, to grow in the presence of 6.5% NaCl and finally to tolerate polymyxin B were determined using the Vitek 2 colorimetric GN-BCL card for the identification of Gram-positive spore-forming microorganisms of the family Bacillaceae (BioMerieux, Inc. Hazelwood, Mo., USA) according to the manufacturer's instructions (Scheldeman et al. 2004). Results were reported as negative or positive. The carbohydrate utilization profiles were compared and the homology was determined using the Vitek 2 compact system (BioMerieux, Inc. Hazelwood, Mo., USA).
- P. polymyxa JB05-01-1 and reference strains ATCC 43865 and ATCC 7070 were grown in TSBYE media at 30° C. to the logarithmic growth phase, harvested by centrifugation (3000 g, 10 min), washed twice, and resuspended at a concentration of 10 8 CFU ml 1 in 0.15 mol l ⁇ 1 NaCl solution. Hexadecane or decane (0.4 ml) was vortexed for 60 sec and the suspension was allowed to stand for 30 min (Doyle and Rosenberg, 1995). Absorbance by the aqueous phase at 400 nm was measured using a spectrophotometer (Molecular Devices Corp., Sunnyvale, Calif., USA). The percentage of cells extracted into each hydrocarbon liquid was determined by the following formula: 100 ⁇ [1 ⁇ (A/A 0 )], where A 0 and A are absorbance by the aqueous phase prior to and after mixing.
- Enterocyte-like Caco-2 cells obtained from the American Type Culture Collection (Rockville, Md.) were cultured routinely under a 5% (v/v) CO 2 atmosphere at 37° C. as monolayer in Dulbecco-modified Eagle's minimal essential medium (DMEM; Gibco) containing 25 mM glucose, 4 mM glutamine and 1 mM sodium pyruvate (Gagnon et al. 2004), and then in medium supplemented with 20% (v/v) fetal calf serum (Hyclone Laboratories, Logan, Utah, USA). Wells in Multiwell tissue culture plates (Falcon, Becton-Dickinson) were seeded each with 10 4 cells.
- DMEM Dulbecco-modified Eagle's minimal essential medium
- the culture medium was replaced every 48 h and confluent monolayers obtained after the tenth sub-culture was used for the adhesion assay. Trypan Blue was used for viable staining and cells were counted by hemocytometer (Hausser scientific, Horsham, Pa., USA).
- Adhesion to Caco-2 cells was evaluated at 15, 30 and 60 min by washing twice with sterile PBS followed by addition of 0.25 ml of trypsin-EDTA (0.05% trypsin, 0.53 mM Na 4 -EDTA; Gibco). After 15 min of incubation at 37° C., 0.25 ml of DMEM supplemented with 20% FCS was added to each well to stop the trypsin reaction and the Caco-2 layer was detached by pipetting. Serial 10-fold dilutions were then plated on Bacillus cereus agar (Difco Laboratories), followed by incubation under aerobic conditions for 24 h at 37° C.
- the antimicrobial substance(s) produced by P. polymyxa JB0501 was confirmed qualitatively using the agar diffusion test ( FIG. 7A ) against E. coli ATCC 25922 as the indicator strain.
- P. polymyxa JB05-01-1 was found to be antagonistic towards E. coli ATCC 25922 ( FIG. 7B ). Paper discs loaded with P. polymyxa JB05-01-1 produced clear zones of inhibition about 8 mm in diameter. TSB medium was tested as negative control.
- ATCC 43865 and ATCC 7070 isolates were able to tolerate higher concentrations of 31.25-62.5 ⁇ g/ml. All three strains of P. polymyxa grew at pH 4.75 or lower (Table 5). Indeed JB05-01-1 and ATCC 43865 were able to grow at a lower pH (4.5). Acidity is believed to be the most detrimental factor affecting the growth and viability of probiotic bacteria, growth being slowed down significantly below pH 4.5 (Lankaputhra et al. 1995). P. polymyxa JB05-01-1 should therefore survive in fermented products and perhaps contribute to prolonging product shelf life. No growth was observed for any of the strains in the presence of 0.2% bile salts. The maximum H 2 O 2 concentration tolerated by P. polymyxa JB05-01-1 was between 7.3 to 14.6 ⁇ g/ml.
- Table 6 shows the carbohydrate utilization profiles, the tolerance of salt or polymyxin B, and the calculated homology based on these 14 traits for the three variants of P. polymyxa .
- Strain JB05-01-1 displayed a homology of approximately 98%, confirming the high probability that it does indeed belong to the genus Paenibacillus .
- adhesion ranged from about 0.35% to about 6.5% of the initial numbers contacted with the enterocytes; the proportion being decreased at the higher initial load suggesting that saturation of the adhesion sites was approached at the higher bacterial load tested.
- Contact time with the Caco-2 monolayer had little effect on the number of adherent bacterial cells counted.
- a preparation of JB05-01-1 supernatant (labeled P3) was produced as a dietary supplement for a field trial.
- a TSB medium with 0.4% yeast extract was prepared for a 1 liter fermentor which was in turn inoculated with the test culture. The culture was grown at 30° C. for 18 hours. The resulting culture was centrifuged at 12,000 g for 15 minutes at 4° C. The centrifuged cultures were then heated to between 80 and 100° C. in a water bath allowing the supernatant to be separated from the pellet. The supernatant was then stored at 4° C. for use in the trial. 500 day-old broiler chicks were divided into five treatment groups, each group containing 4 cohorts of 25 birds. The five different treatment groups of broiler chickens were given different amounts of dietary supplements for a four week period. The treatment regimes were as follows:
- T3 Normal Diet+0.5 ml P3 per kg feed
- T4 Normal Diet+1 ml P3 per kg feed
- JB05-01-1 genome The genome of Paenibacillus polymyxa JB05-01-1 (“JB05-01-1 genome”) was sequenced by MiSeq Illumina sequencing (Eurofins MWG, Germany). The size of the genome was 5.9 Mb, with a 45.7% G/C content.
- the JB05-01-1 genome sequence was compared with available sequenced genomes of five Paenibacillus polymyxa strains (E681, SC2, M1, CR1 et SQR21). The contigs were reordered based on the reference strain E681 with ACT and WebACT. The JB05-01-1 genome was annotated using the RAST server (automated annotation), which identified 5489 coding sequences and 127 RNAs.
- the JB05-01-1 genome contains genes coding for several antibacterial peptides, for example, polymyxin synthesis gene cluster, bacitracin and fusaricidin synthetases.
- the JB05-01-1 genome also contains multiple antibiotic resistance genes.
- Genes coding for bacitracin synthetases (annotated as bacitracin synthetase 3 BA3) were identified as follows:
- the 2613 bp lantionine synthetase LanL (PEG 2150, contig 48 697422-700034) was also identified.
- two other genes coding for non-ribosomal peptide synthetases were identified: a 1407 bp gene (PEG1966, contig 48, 468244-469650: 1407 bp) and a 2025 bp gene (PEG1971, contig 48, 484672-486696).
- the JB05-01-1 genome also contains a polymyxin synthesis gene cluster consisting of five genes, sharing high homology with the pmxABCDE genes (95-99% identities).
- the genes coding for PmxB, PmxC and PmxD located on contig 63 were identified as:
- pmxA and pmxE genes in the JB05-01-1 genome were not identified by automated annotation.
- the pmxA (15 030 bp) and the pmxE (18 940 bp) genes of P. polymyxa M1 were used as references to search the JB05-01-1 genome. Fragments of pmxA were found on contigs 62 (bases 1 to 1619), 60 (1796-2688), 66 (3132-5604), 67 (6075-8553) and 68 (9252-13125, 13659-15033).
- the pmxE gene appeared to be located on contigs 63 and 61.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Polymers & Plastics (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Food Science & Technology (AREA)
- Organic Chemistry (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Birds (AREA)
- Gastroenterology & Hepatology (AREA)
- Animal Husbandry (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Physiology (AREA)
- Biophysics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Nutrition Science (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dermatology (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides, in part, a Paenibacillus sp. isolate, designated Paenibacillus polymyxa JB05-01-1, as well as an anti-microbial agent obtained from the bacterium or cell culture supernatant thereof. Compositions, methods and uses are also provided.
Description
- The present application is a continuation of U.S. patent application Ser. No. 14/642,158, filed Mar. 9, 2015, which is a continuation-in-part of U.S. patent application Ser. No. 13/611,160, filed Sep. 12, 2012, which is a continuation-in-part of U.S. patent application Ser. No. 13/296,063, which was filed on Nov. 14, 2011, the contents of each are all incorporated by reference herein in their entireties. U.S. patent application Ser. No. 13/296,063 is a continuation-in-part of PCT/CA2009/001808, filed on Dec. 9, 2009, and published as WO 2011/069227, the contents of which are incorporated by reference herein in their entireties.
- The following application contains a sequence listing in computer readable format (CRF), submitted as a text file in ASCII format entitled “Sequence_Listing,” created on Oct. 5, 2017, as 6 KB. The content of the CRF is hereby incorporated by reference.
- The invention is in the field of anti-microbial agents. More specifically, the invention relates to anti-microbial agents derived from Paenibacillus.
- In response to the increasing prevalence of antibiotic resistance in pathogenic bacteria, the pharmacokinetic properties and safety profiles of many novel antimicrobial peptides have been investigated. Bacteriocins are natural proteinaceous antimicrobial compounds produced by bacteria and active against taxonomically related bacteria (Klaenhammer, 1993). Species that produce bacteriocins have been studied extensively in the hope of finding safe and efficient means of inhibiting the growth of pathogenic bacteria, especially in foods (Cleveland et al. 2001). Bacteriocins produced by Gram-positive staining bacteria, such as lactic acid bacteria, have become a focus of interest as alternatives to conventional antibiotics (Nes et al. 1996). Nisin, the first bacteriocin ever isolated and now widely used as a food additive, was approved by the World Health Organization for use as a food preservative in 1973. This peptide is generally inactive against Gram-negative staining bacteria, imposing a limitation on its effectiveness when major food-borne pathogens such as Escherichia coli, Salmonella and Yersinia are involved (Du and Shen 1999; Zheng et al. 1999). Davies et al. (1998) reported that nisin produced by Lactococcus lactis was thermostable and remained active after treatment at 121° C. for 15 min at
pH 3. Nisin is about 4.4 kDa and is stabilized by disulfide bonds. - Polymyxins, a class of antimicrobial agents, are synthesized by a non-ribosomal process. The peptide-synthase directed condensation reactions by which polymyxins are formed in the cell cytoplasm have been reviewed (Marahiel et al. 1997) and their biosynthesis in a cell-free enzyme system reported (Komura et al. 1985).
- Many species within the genus Paenibacillus produce variants of polymyxins, which are generally composed of a cyclic decapeptide with a terminal fatty acid moiety (Martin et al. 2003). Five chemically distinct compounds, polymyxins A to E, differing in amino acid and fatty acid composition have been identified to date. Martin et al. (2003) reported that mattacin activity (800 AU ml−1) produced by P. kobensis M was maximal at 12 h of fermentation. Martin et al. (2003) also reported that mattacin and polymyxin B inhibited all Gram-negative staining species tested including E. coli O157:H7, Salmonella enterica serovar Rubislaw and Vibrio parahemeolyticus G1-166 but both failed to inhibit strains of Listeria and Bacillus.
- DeCrescenzo et al. (2007) isolated a new Paenibacillus species (P. amylolyticus C27) that produces polymyxins E1 and E2 (colistin A and B). The new antimicrobial peptides were reported to be effective against Gram-negative staining bacteria such as E. coli, Pseudomonas, Salmonella, and Shigella. DeCrescenzo et al. (2007) also reported that polymyxin E produced by P. amylolyticus C27 inhibited Gram-positive staining bacteria such as Staphylococus aureus ATCC 6538, Enterococcus faecalis ATCC 19433 and Streptococcus pyogenes ATCC 19165.
- Zengguo et al. (2007) reported the co-production of polymyxin and lantibiotic by natural isolates of P. polymyxa. The two antimicrobial peptides were reported to display potent activity against many Gram-negative staining bacteria, including E. coli, Pseudomonas aeruginosa and Acinetobacter baumannii, and against Gram-positive food-borne pathogenic bacteria. Zengguo et al. (2007) also reported that polymyxin produced by P. polymyxa OSY-DF is stable from pH 2.0 to 9.0 and retained its activity after a short autoclaving.
- Svetoch et al. (2005) reported the isolation of a new class IIa bacteriocin from P. polymyxa NRRL-B-30509, which has been used for the control of Campylobacter in poultry.
- Among antimicrobial substances produced by Bacillus polymyxa are polymyxins, which are cyclic peptides with a long hydrophobic tail. Colistin is a polymyxin antibiotic discovered in the late 1940s for the treatment of Gram-negative infections. After several years of clinical use, colistin was associated with significant nephrotoxicity and neurotoxicty (Lim et al. 2010), rendering its use questionable. Colistin has a bactericidal effect against Gram-negative bacteria and acts as a detergent-like molecule (Landman et al. 2008). Recently, its application has returned as the last resort against multidrug-resistant organisms including Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae (Falagas and Kasiakou 2005).
- The need for antibiotics with activity against these multidrug-resistant Gram-negative pathogens is urgent and in the absence of viable alternatives, clinicians now recommend colistin treatment when confronted with some multidrug resistant bacterial infections (Lim et al. 2010). This has led to the development of less toxic colistin molecules (Jian Li et al. 2004; Falagas et al., 2006). Colistin fomulations available for clinical uses are colistin sulfate (for topical use) and colistin methane sulfonate for parenteral and aerosol therapy (Jian Li et al. 2006).
- The coproduction of polymyxin E1 and a lantibiotic has been reported for P. polymyxa OSY-DF; a strain isolated from food (He et al. 2007). Polymyxin E1 was active against Gram-negative bacteria, whilst paenibacillin, a proteinaceous compound, exhibited activity against Gram-positive bacteria (He et al. 2007 & 2008). P. kobensis M isolated from soil produced mattacin (polymixin M) (Nathaniel et al. 2003) and Bacillus sp. strain B-60 produced various inhibitory molecules named sattabacin, hydroxysattabacin, sattazolin and methylsattazolin, with antiviral activity against herpes
simplex viruses types 1 and 2 (HSV1 and HSV2) (Lampis et al. 1995). Strains of P. polymyxa have been isolated from different ecological niches including food matrices such as butternut squash, potatoes, rice, and wheat flour (Fangio et al. 2010). - Usually, E. coli is regarded as an inoffensive and common bacterium of the gastrointestinal tract of ruminants. The emergence of the enterohemorrhagic strain O157:H7 has become a major public concern worldwide. Indeed this pathogen is able to produce potent endotoxins with severe damage to the lining of the intestine, in particular in humans, allowing the strain to invade the body and infect organs such as the kidneys, sometimes with fatal consequences.
- Spore-forming species such as Paenibacillus or other probiotic bacteria are known in the art. Andersson et al. (1997) showed that Bacillus cereus produced spores able to adhere to Caco-2 cell monolayers and to germinate in an environment similar to that of the small intestine and noted that the hydrophobicity of the spore is a contributing factor to adhesion. Angioi et al. (1995) evaluated the capacity of Bacillus subtilis strains to colonise the surface of Caco-2 cells, testing the bacteria both in the spore state and following stimulation of germination by exposure to low pH (as under gastric conditions) or to high temperatures. The degree of adhesion was found to vary in relation to the different physiological phases of the bacterial cells. Doyle et al. (1984) showed that spores or cells from several Bacillus species displayed a strong affinity for hexadecane and other hydrophobic solvents. Treatment of Bacillus spore suspensions with strong denaturants promoted their adherence to hexadecane (Koshikawa et al. 1989).
- The present invention provides, in part, an isolated Paenibacillus sp. bacterium comprising SEQ ID NO: 1.
- In alternative embodiments, the invention provides an isolated Paenibacillus polymyxa (Strain JB05-01-1) bacterium deposited at the ATCC®) under the terms of the Budapest Treaty and designated Accession Number PTA-10436, or a strain comprising the identifying characteristics thereof. The bacterium may be isolated from a direct-fed microbial product. For example, JB05-01-1 was found while examining a mixed culture preparation of RE3™.
- The bacterium may include an anti-microbial activity, such as an anti-bacterial activity. The anti-bacterial activity may include inhibiting the growth of a Gram-negative staining bacterium, such as one or more of Escherichia sp. (e.g., Escherichia coli such as Escherichia coli RR1, Escherichia coli TB1, or Escherichia coli O157:H7), Pantoea sp. (e.g., Pantoea agglomerans BC1), Pseudomonas sp. (e.g., Pseudomonas fluorescens R73), Butyrivibrio sp. (e.g., Butyrivibrio fibrisolvens OR85), Fibrobacter sp. (e.g., Fibrobacter succinogenes), Salmonella sp. (e.g., Salmonella enteritidis or Salmonella typhi), Shigella sp. (e.g., Shigella dysenteriae), Helicobacter sp. (e.g., Helicobacter pylori), or Campylobacter sp (e.g., Campylobacter jejuni).
- The anti-bacterial activity may include inhibiting the growth of a Gram-negative staining bacterium, such as one or more of Escherichia coli RR1, Escherichia coli TB1, and Escherichia coli O157:H7, Pantoea agglomerans BC1, Pseudomonas fluorescens R73, Butyrivibrio fibrisolvens OR85, Fibrobacter succinogenes and Pseudomonas aeruginosa.
- In alternative embodiments, the bacterium may not inhibit the growth of a Gram-positive staining bacterium, such as one or more of a Listeria innocua, Listeria monocytogenes, Pediococcus acidilactici, Paenibacillus polymyxa, Paenibacillus macerans, Bacillus lecheniformis, Bacillus subtilis, Bacillus circulans 9E2, Streptococcus bovis, Enterococcus mundtii, Staphylococcus aureus, or Lactococcus lactis.
- In alternative embodiments, the anti-microbial activity may be sensitive to an enzyme selected from the group consisting of one or more of proteinase K, trypsin, chymotrypsin or lipase; or may be sensitive to sodium dodecyl sulphate (SDS) or urea; or may be sensitive to a temperature in excess of about 90° C. for about 30 minutes; or may be sensitive to a temperature of about 100° C. for about 10 minutes; or may be insensitive to a temperature up to about 80° C. for about 30 minutes; or may be sensitive to acetonitrile and hexane; or may be insensitive to an organic solvent selected from the group consisting of chloroform, propanol, methanol, ethanol and toluene; or may be insensitive to pH, for example, pH ranging from about 2 to about 9.
- In alternative embodiments, the invention provides a cell culture including a bacterium as described herein. The cell culture may be a starter culture.
- In alternative embodiments, the invention provides a cell culture supernatant derived from growing a bacterium as described herein in a cell culture medium. The supernatant may include an anti-microbial activity, such as an anti-bacterial activity.
- In alternative embodiments, the invention provides an anti-microbial agent isolated from a bacterium, cell culture, or cell culture supernatant as described herein. The anti-microbial agent may include a peptide, such as a lipopeptide. The anti-microbial agent may include a molecular weight between about 1000 daltons to about 2500 daltons. The anti-microbial agent may be a polymyxin. The anti-microbial agent may be colistin A and/or colistin B.
- In alternative embodiments, the invention provides a bacterium, cell culture, cell culture supernatant, or anti-microbial agent as described herein.
- In alternative embodiments, the invention provides a pharmaceutical, veterinary, cosmetic or hygiene composition including a bacterium, cell culture, cell culture supernatant, or anti-microbial agent as described herein and a suitable carrier.
- In alternative embodiments, the invention provides a food or feed additive comprising a bacterium, cell culture, cell culture supernatant, or anti-microbial agent as described herein.
- In alternative embodiments, the invention provides a packaging material comprising a bacterium, cell culture, cell culture supernatant, or anti-microbial agent as described herein.
- In alternative embodiments, the invention provides a kit comprising a bacterium, cell culture, cell culture supernatant, or anti-microbial agent as described herein together with instructions for use in inhibiting growth of a micro-organism.
- In alternative embodiments, the invention provides a method of producing an anti-microbial agent, by providing a live Paenibacillus sp. bacterium comprising SEQ ID NO: 1; and culturing the live Paenibacillus sp. bacterium in a cell culture medium, under conditions suitable for production of the anti-microbial agent.
- In alternative embodiments, the invention provides a method of producing an anti-microbial agent, by providing a live Paenibacillus polymyxa (Strain JB05-01-1) bacterium or a strain comprising the identifying characteristics thereof; and culturing the live Paenibacillus polymyxa (Strain JB05-01-1) bacterium or a strain comprising the identifying characteristics thereof in a cell culture medium, under conditions suitable for production of the anti-microbial agent.
- The methods may further include isolating the anti-microbial agent from the bacterium. The culturing may be performed under conditions suitable for secretion of the anti-microbial agent into the cell culture medium. The methods may further include separating the bacterium from the cell culture medium to provide a cell culture supernatant comprising the anti-microbial agent. The methods may further include isolating the anti-microbial agent from the cell culture supernatant.
- In alternative embodiments, the invention provides an anti-microbial agent produced by the methods as described herein. The anti-microbial agent may include a peptide, such as a lipopeptide. The anti-microbial agent may include a molecular weight between about 1000 daltons to about 2500 daltons. The anti-microbial agent may be a polymyxin. The anti-microbial agent may be colistin A and/or colistin B.
- The anti-microbial agent may include an anti-microbial activity selected from one or more of: sensitivity to proteinase K, trypsin, chymotrypsin and lipase, sodium dodecyl sulphate (SDS), urea, acetonitrile, or hexane; insensitivity to chloroform, propanol, methanol, ethanol or toluene; insensitivity to pH; sensitivity to a temperature in excess of about 90° C. for about 30 minutes; sensitivity to a temperature of about 100° C. for about 10 minutes; insensitivity to a temperature up to about 80° C. for about 30 minutes; inhibition of growth of a Gram-negative staining bacterium; or no inhibition of growth of a Gram-positive staining bacterium.
- In alternative embodiments, the invention provides a method of inhibiting the growth of a microorganism in a subject or substance in need thereof by administering or applying an effective amount of the bacterium, cell culture, cell culture supernatant or anti-microbial agent, as described herein, to the subject or substance. The inhibition of growth may be selective.
- The microorganism may be a bacterium, such as a Gram-negative staining bacterium, such as one or more of one or more of Escherichia sp. (e.g., Escherichia coli such as Escherichia coli RR1, Escherichia coli TB1, or Escherichia coli O157:H7), Pantoea sp. (e.g., Pantoea agglomerans BC1), Pseudomonas sp. (e.g., Pseudomonas fluorescens R73 or Pseudomonas aeruginosa), Butyrivibrio sp. (e.g., Butyrivibrio fibrisolvens OR85), Fibrobacter sp. (e.g., Fibrobacter succinogenes), Salmonella sp. (e.g., Salmonella enteritidis or Salmonella typhi), Shigella sp. (e.g., Shigella dysenteriae), Helicobacter sp. (e.g., Helicobacter pylori), or Campylobacter sp (e.g., Campylobacter jejuni).
- The Gram-negative staining bacterium, may be one or more of Escherichia coli RR1, Escherichia coli TB1, and Escherichia coli O157:H7, Pantoea agglomerans BC1, Pseudomonas fluorescens R73, Butyrivibrio fibrisolvens OR85, Fibrobacter succinogenes and Pseudomonas aeruginosa.
- The bacterium may be a pathogenic bacterium, such as a food-borne pathogenic bacterium. The microorganism may be a food-borne pathogenic micro-organism or a food-spoilage micro-organism.
- The subject may be an animal, such as a human, a pet, or an agricultural animal (e.g., cow, horse, pig, sheep, goat, chicken, turkey, duck, goose, fish, or crustacean). The substance may be a cosmetic, hygiene, feed or food product (e.g., dairy or meat product) or packaging material thereof.
- In alternative embodiments, the invention provides an isolated nucleic acid molecule including SEQ ID NO: 1.
- In alternative embodiments, the invention provides the use of an effective amount of a bacterium, cell culture, cell culture supernatant or anti-microbial agent, as described herein for inhibiting the growth of a microorganism in a subject or substance in need thereof.
- In alternative embodiments, there is provided a method of inhibiting growth of a Gram-negative staining bacterium in a subject or substance in need thereof, the method comprising administering or applying an effective amount of an anti-microbial composition to the subject or substance, wherein the anti-microbial composition comprises: (a) an isolated Paenibacillus polymyxa bacterium which has a 16S ribosomal RNA (16SrRNA) gene containing a DNA comprising the base sequence of SEQ ID NO: 1; (b) an isolated Paenibacillus polymyxa bacterium deposited at the ATCC® under the terms of the Budapest Treaty and designated Accession Number PTA-10436; (c) a cell culture comprising the Paenibacillus polymyxa bacterium; (d) a cell culture supernatant derived from growing the Paenibacillus polymyxa bacterium in a cell culture medium; or (e) an anti-microbial agent isolated from the Paenibacillus polymyxa bacterium, the cell culture or the cell culture supernatant.
- In alternative embodiments, there is provided a method of producing an anti-microbial agent comprising growing or culturing an isolated Paenibacillus polymyxa bacterium which has a 16S ribosomal RNA (16SrRNA) gene containing a DNA comprising the base sequence of SEQ ID NO: 1, or an isolated Paenibacillus polymyxa bacterium deposited at the ATCC® under the terms of the Budapest Treaty and designated Accession Number PTA-10436, in a cell culture medium to produce a cell culture supernatant comprising the anti-microbial agent.
- In alternative embodiments, there is provided a method of producing an anti-microbial agent comprising growing or culturing an isolated Paenibacillus polymyxa bacterium which has a 16S ribosomal RNA (16SrRNA) gene containing a DNA comprising the base sequence of SEQ ID NO: 1, or an isolated Paenibacillus polymyxa bacterium deposited at the ATCC® under the terms of the Budapest Treaty and designated Accession Number PTA-10436, in a cell culture medium to produce a cell culture comprising the anti-microbial agent.
- In alternative embodiments, there is provided a method of reducing mortality or morbidity of an animal comprising administering an effective amount of an anti-microbial composition to the animal, wherein the anti-microbial composition comprises: (a) an isolated Paenibacillus polymyxa bacterium which has a 16S ribosomal RNA (16SrRNA) gene containing a DNA comprising the base sequence of SEQ ID NO: 1; (b) an isolated Paenibacillus polymyxa bacterium deposited at the ATCC® under the terms of the Budapest Treaty and designated Accession Number PTA-10436; (c) a cell culture comprising the Paenibacillus polymyxa bacterium; (d) a cell culture supernatant derived from growing the Paenibacillus polymyxa bacterium in a cell culture medium; or (e) an anti-microbial agent isolated from the Paenibacillus polymyxa bacterium, the cell culture or the cell culture supernatant.
- In alternative embodiments, there is provided a method of identifying a bacterium useful in producing an antimicrobial agent; a bacterium useful for inhibiting growth of a Gram-negative staining bacterium in a subject or substance in need thereof; or a bacterium useful in reducing mortality or morbidity of an animal. The method may comprise assessing active mixed cultures for the presence of a bacterium comprising a 16S ribosomal RNA (16SrRNA) gene containing a DNA comprising the base sequence of SEQ ID NO: 1; isolating the bacterium that comprises said sequence, and culturing the bacterium as a pure culture. The active mixed culture may be a direct-fed microbial mixed culture. The method may further comprise identifying cultures using the phenotypic or genetic traits of JB05-01-1 disclosed herein.
- This summary of the invention does not necessarily describe all features of the invention.
- These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:
-
FIG. 1 is a graph showing Kinetics of P. polymyxa JB05-01-1 growth and production of “JB05-01-1” during stirred batch culture in Luria-Bertani broth at 30° C. Filled diamond is culture optical density; open triangle is inhibitory activity expressed as AU/ml, determined by micro-dilution assay -
FIG. 2 is a pair of graphs showing the antimicrobial activity against E. coli RR1. DiVerent amounts of crude P. polymyxa culture supernatant (1 mg/ml) containing antimicrobial substance(s) “JB05-01-1” at 0 (open diamond), 32 (open square), and 96 (open triangle) AU/ml or polymyxin B (0.1 μg/ml) (open circle) were added to exponentially growing culture of Escherichia coli RR1 in tryptic soy broth at 30° C., wherein graph A is optical density at 600 nm and graph B is viable cell counts (CFU, colony forming unit). Arrows show crude antimicrobials peptides addition. -
FIG. 3 is a photograph showing SDS-PAGE profiles of partially purified antimicrobial substance produced by Paenibacillus polymyxa JB05-01-1, wherein the gel in part A was stained with Coomassie Brilliant Blue staining R-250 and the gel in part B was recovered with TSBA pre-overlaid with E. coli RR1.Lane 1 contains the molecular weight markers (in KD);Lane 2 contains partly purified antimicrobial substance JB05-01-1. Arrows in gel of parts A and B show a band and a zone of inhibition, respectively. -
FIG. 4 is a partial sequence of P. polymyxa JB05-01-1 16S rRNA gene (GenBank Accession Number GQ184435; SEQ ID NO: 1). -
FIG. 5 is a Fast Protein Liquid Chromatography (FPLC) of purified antimicrobials peptides produced by P. polymyxa JB05-01-1 eluted at 30% acetonitrile (CH3CN), 1% TFA. -
FIG. 6A is a mass spectrometry analysis of purified Colistin A produced by P. polymyxa JB05-01-1, and structure determination.FIG. 6B is a mass spectrometry analysis of purified Colistin B produced by P. polymyxa JB05-01-1, and structure determination. Molecular weights were determined using MS analysis (Colistin A: 1169 Da; Colistin B: 1155 Da). MS/MS spectrums relevant to the species at m/z 585.3 and 578.4 observed in the chromatograms ofFIG. 6A andFIG. 6B , respectively. The assignments of the different peaks are shown for the corresponding peptide sequence.FIG. 6C is a chemical structure of fatty acid (FA): Colistin A, 6-methyloctanoyl; Colistin B, 6-methylhepatanoyl. -
FIG. 7A is an agar diffusion test showing inhibition of E. coli ATCC 25922 by supernatant of 20 h culture of Paenibacillus polymyxa JB05-01-1 (right); left=negative control (Tryptic soy broth) -
FIG. 7B is a paper disc spot test showing inhibition of E. coli ATCC 25922 by P. polymyxa JB05-01-1. -
FIG. 8 shows the affinity of P. polymyxa JB05-01-1,P. polymyxa ATCC 43865 andP. polymyxa ATCC 7070 strains for hexadecane (White) and decane (grey). Values presented are the means of three independent measurements. -
- The present invention provides, in part, a Paenibacillus sp. isolate, designated Paenibacillus polymyxa JB05-01-1 (deposited on Oct. 21, 2009, with the American Type Culture Collection (ATCC®) under the terms of the Budapest Treaty and assigned Accession Number PTA-10436) obtained from an animal feed additive and identified by amplification and sequencing of the 16S rRNA gene. Characterization of the physical properties and anti-microbial activities of a substance secreted into the culture supernatant of a Paenibacillus polymyxa JB05-01-1 cell culture resulted in the identification of at least one anti-microbial agent.
- An “anti-microbial agent,” as used herein, refers to an agent that exhibits one or more “anti-microbial activity” i.e., any activity that inhibits the growth of a micro-organism. By “inhibit,” “inhibition” or “inhibiting” is meant to destroy, prevent, control, decrease, slow or otherwise interfere with the growth or survival of a micro-organism by at least about 10% to at least about 100%, or any value therebetween for example about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% when compared to the growth or survival of the micro-organism in the absence of the anti-microbial agent. In alternative embodiments, by “inhibit” “inhibition” or “inhibiting” is meant to destroy, prevent, control, decrease, slow or otherwise interfere with the growth or survival of a micro-organism by at least about 1-fold or more, for example, about 1.5-fold to about 100-fold, or any value therebetween for example about 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95-fold when compared to the growth or survival of the micro-organism in the absence of the anti-microbial agent. In alternative embodiments, the “inhibition” may be more than 100-fold. In alternative embodiments, the “inhibition” may be substantially complete inhibition of growth i.e., the growth rate may be reduced to about zero in the presence of the anti-microbial agent, and the anti-microbial agent may cause death of a micro-organism, when compared to the growth or survival of the micro-organism in the absence of the anti-microbial agent. Accordingly, an anti-microbial agent may be microbicidal or may be microbistatic.
- In some embodiments, the anti-microbial agent may be an anti-bacterial agent i.e., an agent that exhibits one or more “anti-bacterial activity” i.e., any activity that inhibits the growth of a bacterium. In alternative embodiments, the anti-bacterial agent may be bactericidal or bacteriostatic.
- In some embodiments, an anti-bacterial agent according to the invention may selectively inhibit the growth of a Gram-negative staining bacterium.
- By “selectively inhibit” “selective inhibition” or “selectively inhibiting” is meant to destroy, prevent, control, decrease, slow or otherwise interfere with the growth or survival of a Gram-negative staining bacterium by at least about 10% to at least about 100%, or any value therebetween for example about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% when compared to the growth or survival of a Gram-positive staining bacterium. In alternative embodiments, by “selectively inhibit” “selective inhibition” or “selectively inhibiting” is meant to destroy, prevent, control, decrease, slow or otherwise interfere with the growth or survival of a Gram-negative staining bacterium by at least about 1-fold or more, for example, about 1.5-fold to about 100-fold, or any value therebetween for example about 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95-fold when compared to the growth or survival of a Gram-positive staining bacterium. In alternative embodiments, the “selective inhibition” may be more than 100-fold. In alternative embodiments, the “selective inhibition” may be substantially complete inhibition of growth of a Gram-negative staining bacterium i.e., the growth rate could be reduced to about zero and the anti-bacterial agent may cause death of a Gram-negative staining bacterium when compared to the growth or survival of Gram-positive staining bacterium.
- Gram-negative staining bacteria include without limitation Escherichia sp., Pantoea sp., Pseudomonas sp., Salmonella sp., Shigella sp., Pseudomonas sp., Helicobacter sp., Butyrivibrio sp., Fibrobacter sp. or Campylobacter sp. Examples of Gram-negative staining bacteria species include without limitation Escherichia coli (e.g., Escherichia coli RR1, Escherichia coli TB1, Escherichia coli O157:H7), Pantoea agglomerans, Pseudomonas fluorescens, Pseudomonas aeruginosa, Salmonella enteritidis, Salmonella typhi, Shigella dysenteriae, Helicobacter pylori, Butyrivibrio fibrisolvens, Fibrobacter succinogenes or Campylobacter jejuni.
- In alternative embodiments, an anti-microbial agent according to the invention does not substantially inhibit the growth of Gram-positive staining bacteria, such as one or more of Listeria innocua, Listeria monocytogenes, Pediococcus acidilactici, Paenibacillus polymyxa, Paenibacillus macerans, Bacillus lecheniformis, Bacillus subtilis, Bacillus circulans 9E2, Streptococcus bovis, Enterococcus mundtii, Staphylococcus aureus, or Lactococcus lactis.
- In some embodiments, an anti-microbial agent according to the invention may be sensitive or insensitive to various treatments. By “sensitive” or “sensitivity” is meant loss or reduction of anti-microbial activity by at least about 10% to at least about 100%, or any value therebetween for example about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% when an anti-microbial agent is subjected to a particular treatment, when compared to anti-microbial activity in the absence of the treatment. In alternative embodiments, by “sensitive” or “sensitivity” is meant loss or reduction of anti-microbial activity by at least about 1-fold or more, for example, about 1.5-fold to about 100-fold, or any value therebetween for example about 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95-fold when an anti-microbial agent is subjected to a particular treatment, when compared to anti-microbial activity in the absence of the treatment. In alternative embodiments, the “sensitivity” may include loss or reduction of anti-microbial activity of more than 100-fold.
- It is to be understood that sensitivity may vary with the time of treatment. In alternative embodiments, the time of treatment may range from a few minutes to many hours. For example, the time of treatment may be about 5 minutes to over 25 hours, such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 minutes or any value therebetween, or such as 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0 hours, or any value therebetween. Anti-microbial activity may be tested by standard methods such as agar diffusion tests and micro-dilution assay as described herein, or by other standard methods such as disk diffusion, agar dilution, or through the use of automated instrumental testing systems (see, for example, Manual of Clinical Microbiology. 1995. P. M. Murray (ed). ASM Press, Washington, D.C.).
- By “insensitive” or “insensitivity” is meant no substantial observable effect or sensitivity when an anti-microbial agent is subjected to a particular treatment, when compared to anti-microbial activity in the absence of the treatment. In alternative embodiments, by “insensitive” or “insensitivity” is meant an observable effect of less than about 10%, for example about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% when an anti-microbial agent is subjected to a particular treatment, when compared to anti-microbial activity in the absence of the treatment.
- In some embodiments, an anti-microbial agent according to the invention may be sensitive to treatment with proteases such as proteinase K, trypsin, chymotrypsin or with a lipase.
- In some embodiments, an anti-microbial agent according to the invention may be sensitive to treatment with surfactants such as sodium dodecyl sulphate (SDS), chaotropics agents such as urea, or solvents such as acetonitrile or hexane.
- In some embodiments, an anti-microbial agent according to the invention may be insensitive to an organic solvent such as chloroform, propanol, methanol, ethanol or toluene.
- In some embodiments, an anti-microbial agent according to the invention may be insensitive to pH, for example, pH ranging from about 2 to about 9.
- In some embodiments, an anti-microbial agent according to the invention may be sensitive to a temperature in excess of about 80° C. In some embodiments, an anti-microbial agent according to the invention may be sensitive to a temperature in excess of about 90° C. Accordingly, in some embodiments, an anti-microbial agent according to the invention may be sensitive to a temperature in excess of about 90° C. when exposed for at least about 30 minutes. In alternative embodiments, an anti-microbial agent according to the invention may be sensitive to a temperature of about 100° C. when exposed for at least about 10 minutes.
- In some embodiments, an anti-microbial agent according to the invention may be insensitive to a temperature up to about 80° C. when exposed for about 30 minutes.
- In particular embodiments, sensitivity of an anti-microbial agent according to the invention may include: the loss (about 100%) of anti-microbial activity after treatment of a composition including the anti-microbial agent with proteinase K for 10 minutes at 100° C.; the reduction of anti-microbial activity to about 83% or about 75% by trypsin and chymotrypsin, respectively, or to about 62% by lipase; the reduction of anti-microbial activity to about 66% after SDS treatment and about 58% after Urea treatment.
- In some embodiments, the molecular weight of an anti-microbial agent according to the invention may be about 1,000 Da to about 2,500 Da. In some embodiments, an anti-microbial agent according to the invention may include more than one molecule having a molecular weight in the range of about 1,000 Da to about 2,500 Da. The agent may be a peptide, for example, a lipopeptide.
- In some embodiments, an anti-microbial agent according to the invention may be a peptidic compound including for example a nonproteinaceous amino acid, such as a D-amino acid or a hydroxy acid and/or may be modified for example by N methylation, acylation, glycosylation, or heterocyclic ring formation.
- In some embodiments, an anti-microbial agent according to the invention may be a polymyxin. By “polymyxin” is meant a peptide having anti-microbial activity. In general, the structure of a polymyxin may include a cyclic peptide e.g., a cyclic decapeptide, with a terminal fatty acid moiety, that is capable of inhibiting the growth of a micro-organism such as a Gram-negative staining bacterium.
- In some embodiments, an anti-microbial agent according to the invention may be one or both of colistin A and colistin B. By “colistin A/B” is meant a polymyxin having anti-microbial activity. In general, colistin A has a molecular weight of about 1169 Da and includes fatty acid moiety 6-methyloctanoyl. Colistin B generally has a molecular weight of 1155 Da and includes fatty acid moiety 6-methylhepatanoyl.
- An anti-microbial agent according to the invention may include an anti-microbial agent produced by Paenibacillus polymyxa JB05-01-1, ATCC® Accession Number PTA-10436, or by a naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:1.
- In some embodiments, an anti-microbial agent may include one or more compounds.
- An anti-microbial agent may be present in a cell, cell membrane, or crude extract, cell culture, or cell culture supernatant thereof. The cell may be a Paenibacillus polymyxa JB05-01-1 cell or a naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:1.
- Anti-microbial agent(s) may be obtained from Paenibacillus polymyxa JB05-01-1 or from other sources. For example, RE3 (Basic Environmental Systems & Technology Inc. Edmonton, AB, Canada) is a direct-fed microbial product used to improve in vitro ruminal fermentation of barley grain/barley silage-based diets and includes a non-sterile liquid formulation containing L. paracasei and L. lactis cultures and their fermentation products. Paenibacillus polymyxa JB05-01-1 was obtained by culturing a sample of RE3™. Other anti-microbial agents may similarly be found by routine screening for isolates that include the 16S rRNA sequence of SEQ ID NO:1 as described herein or known in the art.
- Anti-microbial agent(s) may be produced by growing or culturing Paenibacillus polymyxa JB05-01-1 or a bacterium that includes the 16S rRNA sequence of SEQ ID NO:1 in an appropriate cell culture medium under conditions suitable for production of anti-microbial agent(s) as described herein or known in the art. In alternative embodiments, Paenibacillus polymyxa JB05-01-1 or a bacterium that includes the 16S rRNA sequence of SEQ ID NO:1 may be grown in an appropriate cell culture medium under conditions suitable for secretion of anti-microbial agent(s) into the cell culture supernatant as described herein or known in the art.
- The cell culture medium may be a minimal medium or a complete medium. In some embodiments, the cell culture medium may be LB medium (Luria-Bertani medium). The medium may be a liquid medium or may be a solid or semi-solid medium, such as nutrient broth or agar, or tryptic soy broth or agar. In general, the cell culture medium includes a carbon/energy source, NH4—N, and biotin.
- The cell culture conditions (e.g., temperature, time, etc.) may be varied as appropriate to optimize growth and/or production of the anti-microbial agent(s).
- In some embodiments, the temperature may range from about 5° C. to about 40° C., such as 10° C., 15° C., 20° C., 25° C., 30° C., or 35° C., or any value therebetween. In alternative embodiments, the temperature may be about 30° C.
- In some embodiments, the time may range from about 5 hours to about 48 hours or any value therebetween. In alternative embodiments, the time may be greater than 48 hours. In alternative embodiments, the time may be about 20 hours.
- The cell culture conditions may be aerobic or anaerobic.
- Standard separation processes may be used to obtain a substantially pure preparation of an anti-microbial agent. An agent or compound is “substantially pure” or “isolated” when it is separated from the components that naturally accompany it. Typically, an anti-microbial agent or compound is substantially pure when it is at least 10%, 20%, 30%, 40%, 50%, or 60%, more generally 70%, 75%, 80%, or 85%, or over 90%, 95%, or 99% by weight, of the total material in a sample. Thus, for example, a substantially pure preparation or culture of a cell expressing an anti-microbial agent, such as a Paenibacillus polymyxa JB05-01-1 cell or a naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:1, is a preparation of cells or “cell culture” in which contaminating cells that are not a Paenibacillus polymyxa JB05-01-1 cell, or do not have the desired 16S rRNA sequence of SEQ ID NO:1, or do not express an anti-microbial agent as described herein, constitute less than 1%, 5%, 10%, 20%, 30%, 40%, or 50%, of the total number of cells in the preparation. In some embodiments, a substantially pure Paenibacillus polymyxa JB05-01-1 cell or a substantially pure naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:1, is a preparation of cells or “cell culture” that contains 100% of such cells.
- In some embodiments, an anti-microbial agent that is isolated by known purification techniques, or isolated as described herein, will be generally be substantially free from its naturally associated components. A substantially pure anti-microbial agent can be obtained, for example, by extraction from a natural source such as a Paenibacillus polymyxa JB05-01-1 cell or a naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:1.
- In some instances, an anti-microbial agent according to the invention will form part of a composition, for example, a crude extract containing other substances. For example, an anti-microbial agent may be present in a crude extract of a Paenibacillus polymyxa JB05-01-1 cell or a naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:1 that may also contain the other naturally occurring components found in such a cell. A crude extract of a Paenibacillus polymyxa JB05-01-1 cell or a naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:1 may be prepared by routine procedures, for example, disruption of the cells using standard mechanical or non-mechanical techniques such as freeze-thaw techniques, osmotic shock, enzyme (e.g., lysozyme) treatment, ultrasonication, liquid extrusion, etc., which may be followed by removal of the cell debris by for example centrifugation.
- In alternative embodiments, an anti-microbial agent may be present in a cell culture supernatant, such as a supernatant obtained from growing a Paenibacillus polymyxa JB05-01-1 cell or a naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:1 in a suitable cell culture medium under conditions suitable for secretion of the anti-microbial agent into the supernatant. The term “culture supernatant” refers to the liquid broth remaining when cells grown in a medium are separated from the culture medium by for example centrifugation, filtration, sedimentation, or other means well known in the art. As an example, if the anti-microbial agent(s) is to be isolated from cell culture supernatant, a salt such as ammonium sulphate may be used at various concentrations, initially. Residual ammonium sulphate may then be removed by dialysis against water. The suspended precipitate containing one or more than one antimicrobial compound may be chromatographed on a column such as an ion exchanger, and the various compounds in the culture supernatant may be separated by monitoring absorbance at 280 nm. Active fractions can be determined from among the compounds thus separated, and selected on the basis of the efficacy with which aliquots thereof kill or inhibit the growth of microbes such as bacterial cells, i.e. the indicator strain, known to be sensitive to the anti-microbial agent(s). Active fractions may then be pooled. Further purification may be carried out by high performance liquid chromatography (HPLC) based on the charge of the compound. The various peaks obtained by monitoring absorbance at 280 nm may be separated and again tested for activity against the indicator strain. Purity can be measured using any appropriate method such as column chromatography, gel electrophoresis, HPLC, etc.
- The anti-microbial agent may be isolated from the cell culture supernatant using solid phase extraction with columns such as Amberlite XAD-16 column and Sep-Pak C18 Column. The active fraction eluted from the column(s) may be further purified based on molecular weight separation using a Fast Protein Liquid Chromatography (FPLC) system.
- A person skilled in the art would understand that other conventional concentration, purification or fractionation methods may be used to obtain one or more isolated or substantially purified anti-microbial agent(s) or partially purified fractions exhibiting an anti-microbial activity from whole or lysed cells or from cell culture supernatant. Typical methods include, without limitation, size exclusion or ion exchange chromatography, ammonium sulfate, alcohol, or chloroform extraction, or centrifugation with size filters.
- The anti-microbial activity of an anti-microbial agent may be determined by routine methods or as described herein. For example, anti-microbial activity may be detected by agar diffusion tests or micro-dilution assay.
- Anti-microbial agent(s) according to the invention may be used in a variety of applications in which inhibition of growth of a micro-organism, such as a bacterium, is desirable. Such applications include, without limitation, pharmaceutical and veterinary applications (e.g., for the treatment of a microbial infection), nutritional supplements and animal feed, personal care (cosmetic or hygiene) applications, etc. In alternative embodiments, anti-microbial agent(s) according to the invention may be used to inhibit the growth of a microorganism (e.g., a bacterium) involved in the spoilage of food or other products.
- Food spoilage micro-organisms include without limitation one or more species of Clostridium, Pseudomonas, Porteus, Chromobacterium, Chromobacterium, Lactobacillus, Penicillium, Aspergillus, Rhizopus, Micrococcus, Bacillus, Streptococcus, Pediococcus, Leuconostoc, Chromobacterium, Halobacterium, Alcaigenes, Xanthomonas, Botryitis, Aerobacter, Cornebacterium, Arthrobacter, Microbacterium, Serratia, etc.
- The micro-organism may be a pathogenic micro-organism. The bacterium may a pathogenic bacterium, such as food-borne pathogenic bacterium. Food-borne pathogenic bacteria include without limitation one or more species of Staphylococcus, Escherichia, Listeria, Salmonella, Streptococcus, Vibrio, Campylobacter, Enterobacter, Shigella, etc.
- The bacterium may include a Gram-negative staining bacterium. Gram-negative staining bacteria include without limitation one or more species of Escherichia sp., Pantoea sp., Pseudomonas sp., Salmonella sp., Shigella sp., Helicobacter sp., Campylobacter sp. or Butyrivibrio sp., and Fibrobacter sp. Examples of Gram-negative bacteria species include without limitation Escherichia coli (e.g., Escherichia coli RR1, Escherichia coli TB1, Escherichia coli O157:H7), Pantoea agglomerans, Pseudomonas fluorescens, Pseudomonas aeruginosa, Salmonella enteritidis, Salmonella typhi, Shigella dysenteriae, Helicobacter pylori, Campylobacter jejuni, Butyrivibrio fibrisolvens, or Fibrobacter succinogenes.
- Other examples of bacteria include without limitation Gram-negative rods such as enteric Gram-negative staining rods, curved Gram-negative staining rods, parvobacteria and Haemophilus, Gram-negative staining cocci such as Neisseria, non-sporing anaerobes, and bacteria such as spirochaetes, rickettsia and chlamydia.
- Examples of microial infections, such as bacterial infections, include without limitation chlamydia, gonorrhea, salmonellosis, shigellosis, tuberculosis, syphilis, bacterial pneumonia, bacterial sepsis (bacteremia), bacterial urinary tract infections, vaginosis, bacterial upper respiratory tract infections, bacterial meningitis, bacterial enteritis, diphtheria, legionellosis, pertussis, scarlet fever, toxic shock syndrome, psittacosis, otitis media, lyme disease, etc.
- Anti-microbial agents of the invention can be provided alone or in combination with other compounds, in the presence of any pharmaceutically, veterinary or cosmetically acceptable carrier, diluent, and/or excipient in a form suitable for administration to animals, for example, humans, cattle, sheep, pigs, poultry, etc. If desired, administration or application of an anti-microbial agent according to the invention may be combined with more traditional and existing anti-microbial therapies, treatments, supplements, or additives, or with other desirable therapies, treatments, supplements, or additives.
- Anti-microbial agents according to the invention may be provided chronically or intermittently. “Chronic” administration refers to administration of the anti-microbial agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. “Intermittent” administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.
- Conventional pharmaceutical or veterinary practice may be employed to provide suitable formulations or compositions to administer the anti-microbial agent(s) to subjects suffering from or presymptomatic for a microbial infection. Any appropriate route of administration may be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracisternal, intraperitoneal, intranasal, intra-anal, intravaginal, aerosol, topical, or oral administration. Therapeutic formulations may be in the form of liquid solutions, syrups, or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols; and topical formulations may come in the form of balms, creams, and lotions.
- Methods well known in the art for making formulations are found in, for example, “Remington's Pharmaceutical Sciences” (19th edition), ed. A. Gennaro, 1995, Mack Publishing Company, Easton, Pa. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel. For therapeutic or prophylactic compositions, the anti-microbial agent(s) are administered to a subject in an amount sufficient to inhibit the growth of a micro-organism.
- An “effective amount” of an anti-microbial agent(s) according to the invention includes a therapeutically effective amount or a prophylactically effective amount. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as inhibition of the growth of a micro-organism. A therapeutically effective amount of an anti-microbial agent(s) may vary according to factors such as the disease state, age, sex, and weight of the individual or subject, and the ability of the anti-microbial agent(s) to elicit a desired response in the individual or subject. Dosage regimens may be adjusted to provide the optimum therapeutic or prophylactic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the anti-microbial agent(s) are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as such as inhibition of the growth of a micro-organism. Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount. An exemplary range for therapeutically or prophylactically effective amounts of an anti-microbial agent(s) may be any value from about 0.1 nM to about 0.1M, for example about 0.1 nM to about 0.05M, about 0.05 nM to about 15 μM or about 0.01 nM to about.
- It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the anti-microbial agent(s). Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical or veterinary practitioners. The amount of active anti-microbial agent(s) in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic or prophylactic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, a subject may be a mammal, an agricultural (e.g., farm) or domestic animal, an experimental animal or any animal that may benefit from the anti-microbial agents as described herein. For example, a subject may include a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, chicken, turkey, duck, goose, dog, cat, fish, crustacean, etc.
- In general, anti-microbial agent(s) of the invention should be used without causing substantial toxicity. Toxicity of the anti-microbial agent(s) of the invention can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD50 (the dose lethal to 50% of the population) and the LD100 (the dose lethal to 100% of the population). In some circumstances however, such as in severe disease conditions, it may be necessary to administer substantial excesses of the anti-microbial agent(s).
- In alternative embodiments, an “effective amount” of an anti-microbial agent according to the invention includes an amount effective to inhibit the growth of a micro-organism, such as a bacterium. It is to be understood that such amounts need not be therapeutic or prophylactic amounts, as long as the amount of the anti-microbial agent is capable of inhibiting the growth of a micro-organism, such as a bacterium, in the context in which it is administered or applied, for example, for prevention of food spoilage, etc.
- In alternative embodiments, an anti-microbial agent according to the invention may be provided in a cell, for example a substantially pure Paenibacillus polymyxa JB05-01-1 cell or a substantially pure naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:1, or a cell culture thereof. The cell may be provided in a liquid, or may be frozen or dried, e.g., freeze-dried. The cell culture may be concentrated. The cell culture may be a “starter” culture for example for a dairy product (e.g., milk, cheese, etc.), or for selective media in a laboratory.
- The anti-microbial agent may be provided in a therapeutic, veterinary, hygiene, cosmetic, food, drink or feed product. In alternative embodiments, the anti-microbial agent may be provided in the packaging material for, for example, a therapeutic, veterinary, hygiene, cosmetic, food, drink or feed product. The packaging material may include without limitation, plastic, film, Styrofoam, etc.
- In alternative embodiments, an anti-microbial agent according to the invention may be provided in a kit that may optionally include additional anti-microbial agents or desirable therapies, treatments, supplements, or additives, optionally with instructions for use thereof.
- In alternative embodiments, an anti-microbial agent according to the invention may be provided as a nutritional or food additive, or feed supplement or additive.
- A “nutritional additive” or “food additive” refers to a substance that is added to food, generally to affect the characteristics of the food, such as spoilage. A food additive may be “direct” in that it is directly added to food for example to inhibit growth of a micro-organism. A food additive may be considered “indirect” when it is exposed to food during processing, packaging, or storage but is not present in the final food product. The term “feed additive” or “feed supplement” refers to products used in animal nutrition for purposes of improving the quality of feed, or to improve the animals' performance and health, e.g. providing enhanced digestibility of the feed materials or inhibiting the growth of micro-organisms. An example of an animal feed additive is a direct-fed microbial product which refers to a mono or mixed culture of live micro-organisms, which when applied to a host affects beneficially the host by improving the properties of the indigenous microflora. A non-limiting example of a direct-fed microbial product is RE3™ from Basic Environmental Systems & Technology Inc. Edmonton, AB, Canada. In some embodiments, RE3™ may be specifically excluded from a feed additive according to the invention.
- A probiotic candidate should display the ability to survive under gastrointestinal tract conditions, namely resistance to salivary lysozyme, gastric acid (HCl) and bile. The “candidate” should also be able to establish itself among the intestinal microbiota and adherence to the intestinal epithelium is considered a major parameter determining candidate's ability to colonize the intestinal tract and thus be effective as a probiotic. As shown in the Examples, P. polymyxa JB05-01-1 exhibited probiotic characteristics such as production of antimicrobials (
FIGS. 7A and 7B ), resistance to gastrointestinal conditions (e.g. lysozyme and acidic pH—Table 5) and tolerance of hydrogen peroxide (Table 5), as well as adhesion to Caco-2 cells (Table 7). Therefore in alternative embodiments, the bacterium, cell culture, supernatant, or the anti-microbial agent according to the invention may be used as a probiotic. - In alternative embodiments, anti-microbial agents of the invention can be provided in combination with other feed or nutritional supplements or additives. For example, at least one supplement or additive, such as listed herein, can be included for consumption with the anti-microbial agent of the invention and may have, for example, antioxidant, dispersant, antimicrobial, or solubilizing properties.
- A suitable antioxidant is, for example, vitamin C, vitamin E or rosemary extract. A suitable dispersant is, for example, lecithin, an alkyl polyglycoside,
polysorbate 80 or sodium lauryl sulfate. A suitable antimicrobial is, for example, sodium sulfite or sodium benzoate. A suitable solubilizing agent is, for example, a vegetable oil such as sunflower oil, coconut oil, and the like, or mono-, di- or tri-glycerides. Additives include vitamins such as vitamin A (retinol, retinyl palmitate or retinol acetate), vitamin B1 (thiamin, thiamin hydrochloride or thiamin mononitrate), vitamin B2 (riboflavin), vitamin B3 (niacin, nicotinic acid or niacinamide), vitamin B5 (pantothenic acid, calcium pantothenate, d-panthenol or d-calcium pantothenate), vitamin B6 (pyridoxine, pyridoxal, pyridoxamine or pyridoxine hydrochloride), vitamin B12 (cobalamin or cyanocobalamin), folic acid, folate, folacin, vitamin H (biotin), vitamin C (ascorbic acid, sodium ascorbate, calcium ascorbate or ascorbyl palmitate), vitamin D (cholecalciferol, calciferol or ergocalciferol), vitamin E (d-alpha-tocopherol, d-beta-tocopherol, d-gamma-tocopherol, d-delta-tocopherol or d-alpha-tocopheryl acetate) and vitamin K (phylloquinone or phytonadione). Other additives include minerals such as boron (sodium tetraborate decahydrate), calcium (calcium carbonate, calcium caseinate, calcium citrate, calcium gluconate, calcium lactate, calcium phosphate, dibasic calcium phosphate or tribasic calcium phosphate), chromium (GTF chromium from yeast, chromium acetate, chromium chloride, chromium trichloride and chromium picolinate) copper (copper gluconate or copper sulfate), fluorine (fluoride and calcium fluoride), iodine (potassium iodide), iron (ferrous fumarate, ferrous gluconate or ferrous sulfate), magnesium (magnesium carbonate, magnesium gluconate, magnesium hydroxide or magnesium oxide), manganese (manganese gluconate and manganese sulfate), molybdenum (sodium molybdate), phosphorus (dibasic calcium phosphate, sodium phosphate), potassium (potassium aspartate, potassium citrate, potassium chloride or potassium gluconate), selenium (sodium selenite or selenium from yeast), silicon (sodium metasilicate), sodium (sodium chloride), strontium, vanadium (vanadium sulfate) and zinc (zinc acetate, zinc citrate, zinc gluconate or zinc sulfate). Other additives include amino acids, peptides, and related molecules such as alanine, arginine, asparagine, aspartic acid, carnitine, citrulline, cysteine, cystine, dimethylglycine, gamma-aminobutyric acid, glutamic acid, glutamine, glutathione, glycine, histidine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, serine, taurine, threonine, tryptophan, tyrosine and valine. Other additives include animal extracts such as cod liver oil, marine lipids, shark cartilage, oyster shell, bee pollen and d-glucosamine sulfate. Other additives include unsaturated free fatty acids such as γ-linoleic, arachidonic and α-linolenic acid, which may be in an ester (e.g. ethyl ester or triglyceride) form. Other additives include herbs and plant extracts such as kelp, pectin, Spirulina, fiber, lecithin, wheat germ oil, safflower seed oil, flax seed, evening primrose, borage oil, blackcurrant, pumpkin seed oil, grape extract, grape seed extract, bark extract, pine bark extract, French maritime pine bark extract, muira puama extract, fennel seed extract, dong quai extract, chaste tree berry extract, alfalfa, saw palmetto berry extract, green tea extracts, angelica, catnip, cayenne, comfrey, garlic, ginger, ginseng, goldenseal, juniper berries, licorice, olive oil, parsley, peppermint, rosemary extract, valerian, white willow, yellow dock and yerba mate. Other additives include miscellaneous substances such as menaquinone, choline (choline bitartrate), inositol, carotenoids (beta-carotene, alpha-carotene, zeaxanthin, cryptoxanthin or lutein), para-aminobenzoic acid, betaine HCl, free omega-3 fatty acids and their esters, thiotic acid (alpha-lipoic acid), 1,2-dithiolane-3-pentanoic acid, 1,2-dithiolane-3-valeric acid, alkyl polyglycosides,polysorbate 80, sodium lauryl sulfate, flavanoids, flavanones, flavones, flavonols, isoflavones, proanthocyanidins, oligomeric proanthocyanidins, vitamin A aldehyde, a mixture of the components of vitamin A2, the D Vitamins (D1, D2, D3 and D4) which can be treated as a mixture, ascorbyl palmitate and vitamin K2. - The supplement or additive may be packaged for consumption in softgel, capsule, tablet or liquid form. It can be supplied in edible polysaccharide gums, for example carrageenan, locust bean gum, guar, tragacanth, cellulose and carboxymethylcellulose. Cosmetic or hygiene supplements may be provided in for example, shampoos, conditioners, creams, pastes, lotions, lipsticks, lip balms, etc.
- The present invention will be further illustrated in the following examples.
- The following examples are intended to illustrate embodiments of the invention and should not be construed as limiting.
- The bacterial indicator strains used are listed in Table 1. All were maintained at −80° C. in appropriate media containing 10% glycerol (w/v). P. polymyxa and all indicator strains except Butyrivibrio fibrisolvens and Fibrobacter succinogenes were propagated aerobically at 30° C. in their respective culture media as indicated in Table 1. The media used were: Tryptic soy broth (TSB) (Difco Laboratories, Sparks, Md., USA), de Man, Rogosa and Sharpe broth (MRS) (Rosell Institute, Montreal, PQ, Canada) (de Man et al. 1960) and Luria-Bertani (LB) broth. Liquid or solid (1.2% w/v agar) anaerobic L-10 medium containing glucose, maltose and soluble starch as carbon sources (each at 0.1% w/w) was used for the growth of B. fibrisolvens and F. succinogenes (Caldwell and Bryant 1966). Their growth was carried out at 39° C. in a CO2:H2 atmosphere (95:5 v/v). Before starting the experiments, all strains were sub-cultured at least three times at 24-h intervals using 1% volume transfers.
- The antimicrobial agent producer P. polymyxa JB05-01-1 was isolated from a direct-fed microbial product (RE3™ Basic Environmental Systems & Technology Inc. Edmonton, AB, Canada). RE3™ was screened for bacteria producing compounds inhibiting the growth of E. coli by a deferred antagonism plating procedure as described by Tagg et al. (1976). Briefly, 100 μl of 103-104 dilutions of RE3™ in L-10 or TSB media were spread on L-10 or TSB plates and incubated overnight at 39° C. and 37° C. The plates were replicated, and then the bacterial colonies on the original plates were washed from the agar surface and the plates were surface sterilized under Ultraviolet (UV) light at 254 nm for 20 minutes. The plates were then overlaid with 5 ml of melted LB (0.5% agar) containing 50 μl of an overnight culture of E. coli RR1 and incubated overnight at 37° C. Colonies producing clearing zones were identified and picked from the replica plates for testing for activity against E. coli 0157:H7.
- Gram staining, motility, catalase and oxidase tests were conducted as a preliminary step in the characterization of the selected colonies. Tentative identifications were confirmed by amplification and sequencing of 16S ribosomal RNA genes.
- The Paenibacillus strain (JB05-01-1) was grown in 3 ml of TSB at 30° C. overnight. The cells were harvested by centrifugation at 5000×g for 5 min. DNA was extracted using a Power Soil DNA Kit (MoBio Laboratories Inc., Carlsbad, Calif., USA) according to the manufacturer's instructions. The DNA concentration was measured using the PicoGreen dsDNA quantitation kit (Molecular Probes, Invitrogen, Eugene, Oreg., USA) in a Multi Detection Microplate Reader (Model SIAFRM, BioTek Instruments, Winooski, Vt., USA) using calf thymus DNA (Sigma-Aldrich, St. Louis, Mo., USA) as the standard.
- Polymerase Chain Reaction Amplification of 16S rRNA Genes
- The PCR amplification targeted the approximately 1500 bp of the 16S rRNA gene. The PCR reaction contained 10 ng of template DNA, 2.5 ml of 10× dilution buffer, 10 pmol of each primer and 1 U of Taq polymerase (Takara Shuzo, Japan) in a final volume of 25 ml. The primers used were the universal bacterial primers 8-27 F (5′-AGA GTT TGA TCC TGG CTC)AGA−3° (Liu et al., 1997) and 1492R (5′-TAC CTT GTT ACG ACT T-3′) (Kane et al., 1993). The amplification conditions involved denaturation at 95° C. for 1 min, followed by 25 cycles of 95° C. for 30 s, 55° C. for 30 s and 72° C. for 1.5 min. The nomenclature of the primers used was based on E. coli numbering system.
- Cloning and Sequencing of 16S rRNA Genes
- Amplicons from the PCR reaction were electrophoresed on 1% agarose gel and purified by excising the correct sized bands. The DNA was extracted from the gel using QIAquick PCR purification Kit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions. The purified DNA was cloned into TOPO vector (Invitrogen, Carlsbad, Calif.) and further used to transform electrocompetent E. coli (DH5-α cells) by electroporation. The cells were then plated on LB/Kanamycin (50 mg/L) agar plates and incubated overnight at 37° C. Three clones, verified for correct inserts, were grown overnight in LB/Kanamycin (100 mg/L). All clones were sequenced by the University of Calgary Core DNA Services, Calgary, AB, Canada. The 16S rRNA sequence was a consensus from three clones. The 16S rDNA sequences of the isolates were compared with DNA sequences from the National Center for Biotechnology Information (NCBI) database using the standard nucleotide-nucleotide homology search Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990).
- Sequencing of the 16S rDNA PCR products from the isolate showing the highest activity against E. coli O157:H7 identified an organism that shared 99% homology with Paenibacillus polymyxa. This isolate was designated as Paenibacillus polymyxa JB05-01-1. The 16S rRNA gene partial sequence of P. polymyxa JB05-01-1 was deposited with GenBank and has been assigned Accession Number GQ184435 (
FIG. 4 , SEQ ID NO: 1). -
TABLE 1 Antimicrobial spectrum of inhibitory substance produced by Paenibacillus polymyxa JB05-01-1 culture supernatant (CS-JB05-01-1) and polymyxin B, used as positive control. Inhibition* Polymyxin B Indicator strains Source Medium CS-JB05-01-1 (0.1 μg ml−1) Bacillus circulans LRC 9E2 LB − − Bacillus lecheniformis LRC 8422 LB − − Butyrivibrio fibrisolvens LRC 0R85 L-10 + + Escherichia coli LRC RR1 TSB +++ +++ Escherichia coli LRC TB1 TSB ++ +++ Escherichia coli LRC SA1650 TSB ++ ++ Enterococcus mundtii LRC 8369 LB − − Fibrobacter succinogenes ATCC 19169 L-10 + + Lactococcus lactis ATCC 7962 MRS − − Listeria innocua HPB 13 TSB − − Listeria ivanovii HPB 28 TSB − − Pantoea agglomerans LRC BC1 TSB + + Pseudomonas fluorescens LRC R73 TSB ++ ++ Paenibacillus polymyxa ATCC 43865 LB − − Paenibacillus polymyxa ATCC 7070 LB − − Paenibacillus polymyxa JB05-01A This study LB − − Pediococcus acidilactici ATCC 25740 MRS − − Pediococcus pentosaceus ATCC 25745 LB − − Streptococcus bovis ATCC 33317 LB − − *Determined from two individual assays − No inhibition at concentrations up of 0.1 μg ml−1. + Diameter of the inhibition zone 10 ± 2 mm++ Diameter of the inhibition zone 16 ± 2 mm+++ Diameter of the inhibition zone 28 ± 2 mm ATCC: American Type Culture Collection, LRC: Lethbridge Research Center HPB: Health Product Branch, (Health and Welfare Canada, Ottawa, ON, Canada) - One litre of LB medium was inoculated with 10 ml of a fresh, overnight culture of P. polymyxa JB05-01-1 and incubated at 30° C. with agitation at 200 rpm. The culture optical density at 600 nm was measured every two hours using a Multi-detection micro-plate reader (Bio-Teck instrument Inc., Winooski, Vt., USA), and 1 mL of culture was centrifuged (8,000 rpm, 10 min, 4° C.) to remove the cells. The supernatant was heated at 70° C. for 10 min to inactivate any protease activity, as described by Martin et al. (2003). The agar diffusion assay and micro-dilution method were used to test the heated supernatants for antimicrobial activity as described herein.
- The determination of soluble protein was done using the Folin phenol reagent method as described by Lowry et al. (1951) with bovine serum albumin as standard. Polymyxin E, Polymyxin B and Nisin A were used as positive control for antimicrobial activity. Nisin A stock solutions were prepared from pure Nisin obtained from Aplin and Barrett (Beaminster, UK) in the form of Nisaplin™, which contains 2.5% (w/w) Nisin A. Polymyxin E and Polymyxin B were purchased from Sigma-Aldrich (Oakville, ON, Canada).
- Inhibition of Escherichia coli RR1 by supernatant of a batch culture grown in Luria-Bertani broth, measured by the micro-dilution method, was maximal at 20 h and remained so through 48 h. Thus, based on agar diffusion and micro-dilution tests, the secretion of the antimicrobial agent was shown to start in the exponential phase and reach its maximum in the early stationary phase (
FIG. 1 ). Production thus appeared to be growth associated and activity levels remained stable through 48 h. Inhibition zone diameters at 48 h were approximately 8±1 mm and activity was 96 AU ml−1. - The qualitative antimicrobial spectrum of P. polymyxa culture supernatant was determined using the agar well diffusion method (Wolf and Gibbons 1996). Briefly, a 25-ml volume of molten tryptic soy agar (0.75% agar w/v) was cooled to 47° C. and seeded with 1% (v/v) overnight TSB culture of an indicator strain. The seeded agar was then poured into a sterile Petri plate and allowed to solidify at room temperature. Wells (7 mm) were cut in the solidified agar using a sterile metal cork borer and filled with 80 of supernatant. The plates were left at 5° C. for 2 h to allow diffusion of the tested aliquot and then incubated aerobically for 18 h at 30° C. Absence or presence of inhibition zones as well as their diameters were recorded.
- The antimicrobial activity was also determined by the micro-dilution method described by Daba et al. (1994). Activity was expressed in arbitrary units per milliliter (AU ml−1) using the formula (1000/125)x(1/D), where D was the highest dilution causing inhibition of the indicator strains.
- The minimum inhibitory concentration (MIC) of P. polymyxa JB05-01-1 culture supernatant and pure polymyxin E or polymyxin B against E. coli RR1 was determined using a Microtest™ polystyrene micro-plate assay (96-well, Becton Dickinson Labware, Lincoln Park, N.J.) as described by Kheadr et al. (2004).
- The inhibitory spectrum of the culture supernatant is presented in Table 1. Gram-negative staining bacteria (E. coli RR1, Pantoea agglomerans BC1, Pseudomonas fluorescens R73, B. fibrisolvens OR85 and F. succinogenes S85) were inhibited while no activity was detected against Gram-positive staining bacteria. The spectrum of activity of the antimicrobial agent produced by P. polymyxa JB05-01-1 was different from that of Nisin A but similar to polymyxin E and polymyxin B used as a positive control (Table 1).
- To determine the effect of P. polymyxa JB05-01-1 culture supernatant, E. coli RR1 cells were cultivated in the presence of concentrated P. polymyxa culture supernatant containing JB05-01-1 at final total activity of 0, 32, and 96 AU/ml. The OD600 nm of culture was determined every two hours using a multi-detection microplate reader (Bio-Teck instrument Inc, Winooski, Vt., USA). The corresponding final cell concentration expressed as CFU/ml was determined as described by Naghmouchi et al. (2008). The inhibitory activity (I.A.) of culture supernatant containing JB05-01-1 was calculated as a percentage, I.A.=100−100[OD600(x)/OD600(i)], where (x) is culture supernatant containing JB05-01-1 at the corresponded total activity and (i) is the control culture. Data was reported as means of duplicate analyses.
- To determine the mode of action of culture supernatant containing JB05-01-1, the viability of E. coli RR1 cells was examined (
FIG. 2 ) upon their contact with culture supernatant containing JB05-01-1. Thus, addition of culture supernatant containing JB05-01-1 at total activity of 32 or 96 AU/ml reduced the final cells concentrations of E. coli RR1 by about 2.7 and 3.4 log10 CFU/ml after 2 h of exposure, respectively (graph B). The corresponding inhibitory activities were estimated to 47.1% and 51%, respectively. The OD600 nm of the cell suspension was constant through the experiments (graph A). The data obtained indicates that P. polymyxa JB05-01-1 has bactericidal action like polymyxin B, which was used as a positive control. - The sensitivities of the antimicrobial agent to proteases (all from Sigma-Aldrich, Oakville, ON) or other agents was tested by treating P. polymyxa JB05-01-1 culture supernatant with 2 mg ml−1 final concentration of proteinase K (Tritirachium album), α-chymotrypsin (bovine pancreas), lipase (Sigma-Aldrich, Oakville, ON), trypsin (porcine pancreas), urea (Sigma-Aldrich, St. Louis Mo.), sodium dodecyl sulfate (SDS) (Sigma-Aldrich, St. Louis Mo.) for 1 h at 37° C. (Motta et al. 2007). Thermal stability of the antimicrobial activity was determined by holding aliquots (1000 μl) of 20 h culture supernatant at temperatures ranging from 50° C. to 90° C. for 30 min or at 100° C. for 10 min. The effect of pH was determined by adjusting the pH of P. polymyxa JB05-01-1 culture supernatant from 2 to 9 using 5 M HCl or NaOH. The activity of each sample was compared with the activity of untreated P. polymyxa JB05-01 culture supernatant at pH 6.8.
- The effect of several organic solvents was evaluated by stirring 20 h culture supernatant for 2 h with 10% (v/v) acetonitrile, hexane, propanol, ethanol, toluene, acetone, butanol or methanol (all solvents were obtained from Sigma-Aldrich, St. Louis Mo.). Residual antimicrobial activities were tested using the agar diffusion assay against E. coli RR1 as described herein, with controls for effects of residual solvent.
- The effect of enzymes, detergents and other compounds on the anti-E. coli activity of the P. polymyxa JB05-01-1 culture supernatant is shown in Table 2.
-
TABLE 2 Inhibition of Escherichia coli RR1 by Paenibacillus polymyxa JB05-01-1 culture supernatant (CS-JB05-01-1) or polymyxin B treated with enzymes, detergents or urea, as determined by the agar diffusion test. % of residual activity* Treatment Polymyxin B agent CS-JB05-01-1 (0.1 μg ml−1) None 100 100 Proteinase K 0 4.5 Trypsin 83.3 62.5 Chymotrypsin 75 79.5 Lipase 62.5 58.3 SDS 66 70.1 Urea 58 62.5 *Antimicrobial activities of culture supernatant without additives are 100%. Results are means of two individual assays with an SD less than 5% about the mean. - Activity was eliminated after proteinase K treatments. Lipase, trypsin, α-chymotrypsin, sodium dodecyl sulphate (SDS) and urea reduced the antimicrobial activity by 38%, 17%, 25%, 34% and 42% respectively when compared to untreated activity.
- The antimicrobial activity remained unchanged after heating at 80° C. for 30 min. Loss of activity of about 60% was observed after heating at 90° C. for 30 min. Heating to 100° C. for 10 min completely eliminated the antimicrobial activity.
- Organic solvents such as chloroform, propanol, methanol, ethanol and toluene did not affect the activity of the antimicrobial peptide. Acetonitrile or hexane treatment at the same concentration (10%, v/v) reduced the antimicrobial activity by about 5% and 20%, respectively. Activity also remained stable after a two-hour incubation at pH ranging from 2 to 9.
- P. polymyxa JB05-01-1 culture supernatant was analysed in duplicate using a NuPAGE 12% Bis-Tris gel kit (Invitrogen, Burlington, ON, Canada) as per manufacturer's instructions at 200 V (constant) for 40 min. The 2.5-200 kDa molecular weight marker kit from Invitrogen was used as a molecular weight standard. After electrophoresis, one gel was stained with Coomassie Brilliant Blue R250 (Invitrogen). A duplicate gel was used for the plate overlay assay to estimate the molecular weight of the antimicrobial compounds as described by Bhunia et al. (1987). Briefly, a SDS-PAGE gel prewashed with sterile water was placed in a Petri dish and overlaid with 10 ml tryptic soy agar containing growing cells of E. coli RR1 at about 105 CFU ml−1. The agar was allowed to solidify, held at 4° C. for 60 min, and then incubated for 18 h at 30° C. The formation of an inhibition zone indicated the position and size of the active antimicrobial peptide in the gel.
- Coomassie Brilliant Blue staining of SDS-PAGE gels of P. polymyxa JB05-01-1 culture supernatant revealed no distinct protein bands. However, inhibitory activity was detected as a clearly defined zone of inhibition in the region corresponding to a molecular mass of <2.5 kDa (<2500 Da) after gels were overlaid with E. coli RR1-seeded agar (
FIG. 3 ). No inhibitory activity was detected when the SDS gels were overlaid with Listeria innocua. - The bacterial strains used and their culture media are listed in Table 4. All strains were stored previously at −80° C. in media containing 20% glycerol (w/v) and were sub-cultured twice in the appropriate media prior to being used in experiments. Fresh cultures were prepared by inoculation of 10 ml of the corresponding medium with 100 μl of the frozen stock followed by incubation for 18-24 h at the corresponding temperature.
- P. polymyxa JB05-01-1 was grown aerobically at 30° C. in Luria-Bertani (LB) broth (Difco Laboratories, Detroit, Mich., USA) for 16 h. Colistin-sensitive strain Escherichia coli RR1 (E. coli RR1), was obtained from the Lethbridge Research Center culture collection. E. coli RR1 was grown aerobically for 18 h at 30° C. in tryptic soy broth (TSB, Difco Laboratories, USA).
- P. polymyxa JB05-01-1 culture was used to inoculate 500 ml of Luria-Bertani (LB) broth (1% inoculum) in a 1-liter flask and incubated at 30° C. for 20 h. Cells in the fermentation broth were separated by centrifugation (12,000 g, 20 min, 4° C.). The resulting cell-free supernatant was heated (100° C. for 10 min) and passed through a 5×50 cm column (Amersham Pharmacia Biotech) packed with 60 g Amberlite XAD-16 resin (Sigma-Aldrich, Oakville, ON, Canada) via a
Gilson MiniPlus 2 Pump at a flow rate of 5 ml/min to remove hydrophobic components from the medium as described by Martin et al. (2003). The Amberlite XAD-16 column was washed with 300 ml of 30% ethanol after which the active fraction was eluted with 500 ml of 70% isopropanol (pH 2; achieved through the addition of 1 M HCl) at a flow rate of 5 ml/min. The eluted active fraction was then evaporated to 30% aqueous volume (approximately 150 ml) using a rotary evaporator (Laborota 4001 Efficient; Krackeler Scientific, Inc., Albany, N.Y., U.S.A.) at 30° C. The Amberlite XAD-16 column was washed with 500 ml of HPLC grade water for future use. - The evaporated active fraction was passed through a Waters Sep-Pak Vac 35 cc C18-10 g column (Waters, Milford, Mass., USA) via
Gilson MiniPlus 2 Pump at a flow rate of 2.5 ml/min. The column was washed with 30 ml of 20% acetonitrile (CH3CN) 0.5 M HCl and the active fraction eluted with 50 ml of 50% acetonitrile 0.5 M HCl (Seim et al. 2005). The active fraction eluted from the Sep-Pak C18 column was evaporated under similar conditions as hereinbefore described to 20 ml and further concentrated to 5 ml using Centricon YM-3 centrifugal filter unit (Millipore: Bedford, Mass., USA) at 3000 g for 120 min at 4° C. The evaporated active fraction eluted from the Sep-Pak C18 column was designated as Fraction E. - All fractions obtained at each stage of the isolation process were assessed for antibacterial activity using the micro-dilution method as described below.
- Fraction E was applied to Superdex 75
HR 16/60 gel filtration chromatography to obtain the active fraction based on molecular weight separation. The Superdex 75HR 16/60 column was equilibrated with 100 mM sodium phosphate extraction buffer (pH 7.4) containing 150 mM NaCl. This step was carried out at 4° C. with a flow rate of 0.5 ml/min using a Fast Protein Liquid Chromatography (FPLC) system (GE Healthcare Canada Inc.). One ml of Fraction E was applied onto the column and fractions of 1 ml were collected during the elution process using an isocratic gradient. The mobile phase consisted of 30% CH3CN/70% HPLC grade water, 0.1% trifluoroacetic acid (TFA) single buffer. Elution was monitored at wavelength detection of 218 nm during a total running time of about 600 min. Fractions exhibiting antibacterial activity, at a given retention time, were collected from different FPLC runs, pooled and lyophilized. The resulting powder was dissolved in 0.1 ml HPLC grade water and checked again for activity againstE. coli RR 1 and the other bacteria listed in Table 4. - Antibacterial activity of the fractions obtained at each stage of the isolation process was determined by the micro-dilution method (Daba et al. 1994). Total activity was expressed in arbitrary units per milliliter (AU/ml), using the formula (1000/125)×(1/D), where D was the highest dilution causing complete inhibition of E. coli RR1, the indicator strain (Naghmouchi et al. 2010).
- The determination of soluble protein concentration was performed in triplicate with the Folin phenol reagent method (Lowry et al. 1951). Bovine serum albumin (BSA) was used as internal standard. Polymyxin E solution standard was obtained from Sigma-Aldrich (Oakville, ON, Canada).
- Minimum inhibitory concentration (MIC) for FPLC purified peptide peak (retention time of 361.55 min) and that of polymyxin (colistin—standard) were determined by the micro-dilution assay (Naghmouchi et al. 2010).
- Two microlitres of the peak sample obtained from final FPLC purification step (retention time 361.55 min) was analysed directly by over-spotting with 1 μL α-Cyano-4-hydroxycinnamic acid (3 mg/mL containing 1.8 mg/mL ammonium citrate dissolved in 50% ACN/0.1% TFA). In addition, 5 μL of each sample was diluted with 50 μl, 0.1% TFA/water C18 Ziptip desalted and eluted with matrix. The samples were air dried and then analysed on an Applied Biosystems MDS Sciex 4800 TOF/TOF Mass Analyser over mass range 600-4000 m/z collecting 500 laser shots. Ions of highest intensity were manually selected for MSMS fragmentation for 1000 laser shots and data acquired.
- Four microliters of the peak sample obtained from final FPLC purification step (retention time 361.55 min) was placed into a silica Au/Pd coated nanoES spray tip (Proxeon, Odense. Denmark). Static manual nanospray was performed on an Applied Biosystems/MDS Sciex QStar Pulsar I fitted with a Protana/Proxeon Nanospray source. Spray was established by applying a tip voltage of 1200V and collecting a TOFMS survey scan 300-1200 m/z consisting of 50 accumulated 1 sec scans. Mass spectrometer parameters used were as follows: Declustering potential setting of 55, Focusing Potential setting of 265, Curtain gas setting of 25 and CAD gas setting of 6 with Nitrogen in the collision cell. The most intense double charged ions were selected for MSMS fragmentation. Product ion scans were acquired over the mass range 100-1500 m/z range and collision energy was manually increased to provide the best fragmentation during the 50 accumulated 1 second scans. Mass spectrometer parameters used for MSMS were as follows: Declustering potential setting of 50, Focusing Potential setting of 220, Curtain gas setting of 25 and CAD gas setting of 7 with Nitrogen in the collision cell.
- Isolation, Purification and Characterization of Antimicrobial Substance(s) Produced by P. polymyxa JB05-01
- The characterization of antimicrobial substance(s) from Luria-Bertani broth cultured with P. polymyxa JB05-01-1 is summarized in Table 3.
-
TABLE 3 Antimicrobial substance(s) recovery at different stages of purification. Total and partial purified substance(s) were quantified by the micro-dilution test using E. coli RR1 as indicator strain. Increase in Total Total Specific Specific Re- Volume protein activity1 activity2 activity covery Step/fraction (ml) (mg) (AU) (AU/mg) (fold) (%) Supernatant 500 1750 48000 27.42 1 100 Amberlate 200 196 25600 130.61 4.8 53 XAD-16 Sep- Pack C 1850 3.15 6400 2031.7 74.09 13.33 eluate FPLC- system 1 0.05 256 5120 189.62 1.06 1Activity (AU/ml) determined by micro-titer plate assay using E. coli RR1 as indicator strain and multiplied by the volume in milliliters. 2Activity (AU/ml) divided by total protein (mg). - Extraction of the antimicrobial substance(s) was achieved using Amberlite XAD-16-adsorbent, a non-ionic macro-reticular resin that adsorbs and releases ionic species through hydrophobic and polar interactions (Zengguo et al. 2007). By applying)(AD-16 resin to clarified cell-free culture supernatant, the antimicrobial substance(s) was/were selectively adsorbed, whilst other water-soluble components remained in the liquid phase. The antimicrobial substance(s) was/were eluted from)(AD-16 by 70% acid and isopropyl alcohol, and the resulting fraction was evaporated to 30% aqueous volume which retained most of the antimicrobial activity. The specific activity obtained after Amberlite XAD-16-adsorbent was approximately 130.61 AU/mg of protein.
- The antimicrobial substance(s) was/were subsequently applied to Sep-Pack C18 column and the most active fraction purified with 50% acetonitrile and 0.5 M HCl. The recovery from the Sep-Pack C18 step was 13.3% with the specific activity of 2,032 AU/mg of protein.
- Finally, the antimicrobial fraction obtained from the Sep-Pack C18 recovery step was separated with the FPLC system connected to
Hiload 16/60 Superdex 75 Prep grade size exclusion. In the FPLC profile, fractions with a peak retention time (RT) of 361.55 min were identified (FIG. 5 ). These fractions were active against E. coli RR1. Approximately 1.1% of the culture supernatant was recovered as antimicrobial substance(s) through the FPLC filtration process. The procedure increased the specific activity (per mg of protein) of the antimicrobial substance(s) 190-fold. Total purified antimicrobial substance(s) obtained from 500 ml of the fermented broth were approximately 0.05 mg. - The MIC of FPLC purified peptide peak (retention times of 361.55 min) against E. coli RR1 was approximately 0.13 μg/ml.
- The chemical nature of the antibacterial substance(s) produced by P. polymyxa JB05-01-1 was elucidated by MALDI TOF/TOF analysis and Nanospray analysis (
FIGS. 6A and 6B ) as described below. - Tandem mass spectrometry MS analysis was performed for a single active peak obtained from the final FPLC purification (361.55 min) step. Data analysis revealed the presence of two compounds with the following molecular weights 1169.7 Da (
FIG. 6A ) and 1155.7 Da (FIG. 6B ), which are equivalent to those of colistin A and colistin B. Further confirmation of this structure (colistin) was performed with MS/MS analysis on TOF and ESI. Fragmentation of peak 585.3 Da (A) in the ion trap permitted the identification in the spectrum (FIG. 6A ) of the first series of product ions with m/z 829.5, 728.5, 628.4, 527.3, 427.3, 327.2, 227.2, which are formed by subsequent loss of amino acid moieties. Similar results were observed for peak 578.4 Da (FIG. 6B ). The m/z ions obtained in the second series were identical to fragmentation patterns reported in the literature (Govaerts et al. 2002; De Crescenzo et al. 2007). -
FIG. 6C shows the chemical structure of fatty acid colistin A and colistin B. Thus it was concluded that the active antimicrobial substance purified from P. polymyxa JB0501 are colistin A and colistin B. - As shown in Table 4, purified colistin (A/B) produced by P. polymyxa JB05-01-1 had inhibitory activity against Gram-negative bacteria including E. coli O157:H7, E. coli RR1, Pseudomonas fluorescens R73 and Pseudomonas aeruginosa. The MIC values of the purified peptides were about 0.13 and 0.162 μg/ml for E. coli O157:H7 and P. fluorescens LRC R73, respectively. Similar MIC values were obtained with the colistin standard.
-
TABLE 4 Antimicrobial spectrum and minimal inhibitory concentration (MIC) of purified peptide (colistins A and B) produced by Paenibacillus polymyxa JB05-01-1 Inhibition zone * attributed to Purified Growth peptide MIC Indicator strains Source Medium at (5 μg/ml) (μg/ml) Listeria ivanovii HPB28 TSB − 2.5-5 L. monocytogenes LSD530 TSB − 5-10 Escherichia coli MC4100 TSB ++ 0.13-0.26 E. coli O157:H7 ATCC TSB ++ 0.13 35150 E. coli ATCC TSB ++ 0.13 25922 E. coli RR1 TSB +++ 0.13 Pseudomonas ATCC TSB +++ 0.52 aeruginosa 19442 P. fluorescens LRC R73 TSB +++ 0.162 Lactococcus lactis UL719 MRS − − Paenibacillus ATCC MRS − − polymyxa 43865 Pediococcus UL5 MRS − − acidilactici Salmonella enterica UL TSB + 1.04-2.08 Staphylococcus Scott A3 TSB − − aureus * Determined from two individual assays − No inhibition at concentrations up of 5 μg ml−1 + Diameter of the inhibition zone 10 ± 2 mm++ Diameter of the inhibition zone 16 ± 2 mm+++ Diameter of the inhibition zone 28 ± 2 mm ATCC: American Type Culture Collection; UL: University Laval, Quebec, Canada LRC: Lethbridge Research Center, Alberta, Canada; LSD: Laboratory Services Division, Ottawa, ON, Canada; HPB: Health Product Branch, (Health and Welfare Canada, Ottawa, ON, Canada). MRS: de Man Rogosa and Sharpe medium TSB: Tryptone Soya Broth medium - Paenibacillus polymyxa JB05-01-1 and Paenibacillus polymyxa reference strains ATCC (43865 and 7070) were aerobically grown for 18 h at 30° C. with agitation in Tryptic Soy Broth (TSB) (Difco Laboratories, Detroit, Mich., USA) containing 1% of yeast extract leading to a medium named TSBYE. Escherichia coli generic ATCC 25922 (E. coli ATCC 25922) was grown for 16 h at 37° C. in TSB (Difco). The four strains were stored at −80° C. in the corresponding medium containing 20% glycerol (w/v). Frozen stocks were activated by re-suspending 100 μl of each conserved strain in 10 ml of growth medium, followed by incubation for 18-24 h at the corresponding temperature.
- Antimicrobial Activity of Paenibacillus polymyxa JB05-01-1 Culture Supernatant
- To demonstrate that inhibition of E. coli ATCC 25922 by P. polymyxa JB05-01-1 was attributed in part to secreted substances, culture supernatant was tested using a qualitative agar well diffusion method (Wolf and Gibbons, 1996). Briefly, TSBYE containing 0.75% (w/v) agar was cooled to 47° C., seeded with overnight culture of E. coli ATCC 25922 (1% vol), and poured into sterile Petri plates. Wells (7 mm) were cut in the solidified agar using a sterile metal cork borer and filled with 80 μl of P. polymyxa culture supernatant. The plates were incubated at 5° C. for 2 h to allow diffusion of the supernatant; the incubation continued aerobically for 18 h at 30° C. After this period, the plates were inspected for the presence or absence of zones of inhibition.
- Antagonism Between P. polymyxa JB05-01-1 and E. coli ATCC 25922
- P. polymyxa JB05-01-1 culture (10 ml) was centrifuged and the cells were resuspended in 50 μl of LB broth. 20 μl, of the suspension were deposited into a 5-mm paper disc. The paper discs impregnated with bacterial cell suspension were placed on the surface of TSBYE seeded with overnight culture of E. coli ATCC 25922 (0.25 ml in 25 ml), solidified with 0.75% agar and poured at 47° C. into Petri plates. The plates were incubated aerobically at 30° C. for 18 h and checked for zones of inhibition (clear agar) surrounding the paper discs (Lenni and Mauliya, 2010).
- Tolerance of P. polymyxa JB05-01-1 to Lysozyme, Acid, Bile and Hydrogen Peroxide (H2O2)
- Tolerance of the three P. polymyxa strains to lysozyme, acidic conditions, bile salts and hydrogen peroxide was tested using sterile flat-bottom 96-well microtiter plates (Falcon, Becton Dickinson and Company, Frankin Lakes, N.J., USA; Gagnon et al. 2004). Egg-white lysozyme (Sigma, 48,000 U/mg protein) was added at different concentrations (0, 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, 500, 1000 μg/ml) to TSBYE broth. The medium was adjusted to different pH values (7.0, 6.5, 6. 5.5, 5.0, 4.75, 4.5, 4.25, 4.0) using 1M HCl. For another purpose, bovine bile salts (Sigma) were added to the medium at different concentrations (0.1%, 0.2%, 0.3%, and 0.4% w/v). Similarly, to study the effect of H2O2, different concentrations (0, 2.5, 5, 10, 20 and 40 μg/ml) of H2O2 purchased from Merck (Darmstadt, Germany) was added. Wells (in duplicate) containing 200 μl of tested broth were inoculated with 20 μl of overnight culture of P. polymyxa diluted 1/100 in TSBYE. Microplates were incubated under anaerobic conditions at 30° C. for 18 h. Optical densities (OD) were read at 630 nm using a Thermomax microplate reader (Zeus, scientific Inc, Raritan, N.J.). Resistance to H2O2 and lysozyme (minimum inhibitory concentration) was expressed as the lowest concentration that maintained the OD under 50% of the OD in the “0” concentration wells. Acid resistance was expressed as the lowest pH at which the tested strain had an OD at least 40% of its level at pH 6.5.
- Determination of the tolerance to a simulated gastric transit was based on the procedure described by Charteris et al. (1998). A simulated gastric juice was prepared by suspending pepsin (3 mg/ml) in sterile saline (0.5% w/v) and adjusting the pH to 2.0 with concentrated HCl. Overnight 2 ml cultures of the three P. polymyxa variants were subjected to centrifugation in an Eppendorf centrifuge at 5000 g for 5 min and washed two times in quarter-strength Ringer's solution at pH 7. Then, 0.3 mL of the washed suspension was added to 1.5 mL of simulated gastric juice (pH 2.0) in a 2.0 mL Eppendorf tube and vortexed. In the controls, simulated gastric juice was replaced by 1.5 mL quarter-strength Ringer's solution and these samples were used for determination of the initial cell counts. Aliquots of 0.2 mL were removed after 15, 30, and 90 min at 37° C. and viable counts were determined by plating serial 10-fold dilutions on LB agar and counting the colony numbers after anaerobic incubation at 37° C. for 48 h. Experiments were carried out in duplicate and were repeated two times. Results are expressed as the mean and standard deviation of three determinations.
- The abilities of P. polymyxa JB05-01-1 and other P. polymyxa strains (
ATCC 43865, ATCC 7070) to utilize sugars, glycogen and pyruvate, to grow in the presence of 6.5% NaCl and finally to tolerate polymyxin B were determined using theVitek 2 colorimetric GN-BCL card for the identification of Gram-positive spore-forming microorganisms of the family Bacillaceae (BioMerieux, Inc. Hazelwood, Mo., USA) according to the manufacturer's instructions (Scheldeman et al. 2004). Results were reported as negative or positive. The carbohydrate utilization profiles were compared and the homology was determined using theVitek 2 compact system (BioMerieux, Inc. Hazelwood, Mo., USA). - P. polymyxa JB05-01-1 and
reference strains ATCC 43865 andATCC 7070 were grown in TSBYE media at 30° C. to the logarithmic growth phase, harvested by centrifugation (3000 g, 10 min), washed twice, and resuspended at a concentration of 108 CFU ml1 in 0.15 mol l−1 NaCl solution. Hexadecane or decane (0.4 ml) was vortexed for 60 sec and the suspension was allowed to stand for 30 min (Doyle and Rosenberg, 1995). Absorbance by the aqueous phase at 400 nm was measured using a spectrophotometer (Molecular Devices Corp., Sunnyvale, Calif., USA). The percentage of cells extracted into each hydrocarbon liquid was determined by the following formula: 100×[1−(A/A0)], where A0 and A are absorbance by the aqueous phase prior to and after mixing. - Enterocyte-like Caco-2 cells (ATCC HTB-37) obtained from the American Type Culture Collection (Rockville, Md.) were cultured routinely under a 5% (v/v) CO2 atmosphere at 37° C. as monolayer in Dulbecco-modified Eagle's minimal essential medium (DMEM; Gibco) containing 25 mM glucose, 4 mM glutamine and 1 mM sodium pyruvate (Gagnon et al. 2004), and then in medium supplemented with 20% (v/v) fetal calf serum (Hyclone Laboratories, Logan, Utah, USA). Wells in Multiwell tissue culture plates (Falcon, Becton-Dickinson) were seeded each with 104 cells. The culture medium was replaced every 48 h and confluent monolayers obtained after the tenth sub-culture was used for the adhesion assay. Trypan Blue was used for viable staining and cells were counted by hemocytometer (Hausser scientific, Horsham, Pa., USA).
- The procedure of Cepeljnik et al. (2007) was followed. Caco-2 cell monolayers were washed twice with sterile phosphate-buffered saline (PBS, 100 mM, pH 7.3) and submerged in DMEM for 18 h, followed by one wash with PBS. Overnight culture of P. polymyxa in TSBYE broth was centrifuged and the bacterial cells were washed twice with PBS, suspended in DMEM at approximately 4×104 CFU/ml or 4×106 CFU/ml and 2 ml were added to the wells. Adhesion to Caco-2 cells was evaluated at 15, 30 and 60 min by washing twice with sterile PBS followed by addition of 0.25 ml of trypsin-EDTA (0.05% trypsin, 0.53 mM Na4-EDTA; Gibco). After 15 min of incubation at 37° C., 0.25 ml of DMEM supplemented with 20% FCS was added to each well to stop the trypsin reaction and the Caco-2 layer was detached by pipetting. Serial 10-fold dilutions were then plated on Bacillus cereus agar (Difco Laboratories), followed by incubation under aerobic conditions for 24 h at 37° C.
- Antimicrobial Activity of Paenibacillus polymyxa JB05-01-1 Culture Supernatant
- The antimicrobial substance(s) produced by P. polymyxa JB0501 was confirmed qualitatively using the agar diffusion test (
FIG. 7A ) against E. coli ATCC 25922 as the indicator strain. - Antagonism Between Paenibacillus polymyxa JB05-01-1 and E. coli ATCC 25922
- Using the spot test, P. polymyxa JB05-01-1 was found to be antagonistic towards E. coli ATCC 25922 (
FIG. 7B ). Paper discs loaded with P. polymyxa JB05-01-1 produced clear zones of inhibition about 8 mm in diameter. TSB medium was tested as negative control. - Lysozyme, Acid, Bile and Hydrogen Peroxide Tolerance by Paenibacillus polymyxa JB05-01-1
- For a probiotic candidate, it must display the ability to survive under gastrointestinal tract conditions. This means to begin with, resistance to salivary lysozyme, gastric acid (HCl) and bile. The “candidate” should also be able to establish itself among the intestinal microbiota. The ability of P. polymyxa JB05-01-1 to grow in the presence of lysozyme, acidic pH, bile salts and H2O2 is shown in Table 5. P. polymyxa JB05-01-1 was able to grow in the presence of egg-white lysozyme concentrations up to 16.5 μg/ml which is supposed to be more than the physiological concentration of intestinal lysozyme (Suskovic et al. 1997).
ATCC 43865 andATCC 7070 isolates were able to tolerate higher concentrations of 31.25-62.5 μg/ml. All three strains of P. polymyxa grew at pH 4.75 or lower (Table 5). Indeed JB05-01-1 andATCC 43865 were able to grow at a lower pH (4.5). Acidity is believed to be the most detrimental factor affecting the growth and viability of probiotic bacteria, growth being slowed down significantly below pH 4.5 (Lankaputhra et al. 1995). P. polymyxa JB05-01-1 should therefore survive in fermented products and perhaps contribute to prolonging product shelf life. No growth was observed for any of the strains in the presence of 0.2% bile salts. The maximum H2O2 concentration tolerated by P. polymyxa JB05-01-1 was between 7.3 to 14.6 μg/ml. -
TABLE 5 Tolerance of gastrointestinal factors by Paenibacillus polymyxa Substance Lysozyme Acid Bile salts H2O2 Variant or strain (μg/ml)1 (HCl)2 (0.2%) (μg/ml)1 P. polymyxa JB05-01-1 16.25 4.5 — 3 7.3-14.6 P. polymyxa ATCC 4386531.25-62.5 4.5 — 14.6 P. polymyxa ATCC 707031.25-62.5 4.75 — 7.3-14.6 1Minimum inhibitory concentration (MIC) 2Minimum growth pH 3 —No growth - Exposure of P. polymyxa JB05-01-1,
P. polymyxa ATCC 43865 andP. polymyxa ATCC 7070 strains to a simulated gastric buffer containing pepsin at pH 2.0 resulted in a rapid loss of viability (FIG. 9 ). Viable counts of variant P. polymyxa JB05-01-1 strains decreased from about 6.93 to 1.61 Log CFU per ml within 15 min. P. polymyxa JB05-01-1 displayed higher affinity for both hexadecane and decane than the P. polymyxaATCC 43865 and P. polymyxa ATCC 7070 (FIG. 8 ). - Table 6 shows the carbohydrate utilization profiles, the tolerance of salt or polymyxin B, and the calculated homology based on these 14 traits for the three variants of P. polymyxa. Strain JB05-01-1 displayed a homology of approximately 98%, confirming the high probability that it does indeed belong to the genus Paenibacillus.
-
TABLE 6 Carbohydrate utilization profile, growth in 6.5% NaCl, polymyxin B resistance and homology of three strains of Paenibacillus polymyxa Bacterial strain ATCC ATCC Physiological test JB05-01-1 43865 7070 D-Galactose + + + Maltotriose + + + D-Mannose + + + D-Melezitose + − − D-Tagatose − − − D-Trehalose + + + Palatinose + + + L-Rhamnose − − − D-Glucose + + + d-Ribose + + + Glycogen + + + Pyruvate − + + Growth in 6.5% NaCl − + + Tolerance of polymyxin B + + + Homology (%) 98 91 88
Adhesion of Paenibacillus polymyxa to Caco-2 Cells - The quantitative adhesion of P. polymyxa JB05-01-1 and
P. polymyxa ATCC 43865 andATCC 7070 to monolayers of enterocyte-like cells is depicted in Table 7. Starting from initial numbers of about 8.7×106 and 6.8×106 CFU/well, the number of adherent bacterial cells after 1 h of contact was about 3.4±0.16×104 CFU/well and 4.8±0.26×104 CFU/well respectively for P. polymyxa JB05-01-1 andP. polymyxa ATCC 43865. Starting frominitial numbers 100 times smaller, the numbers of adherent bacterial cells were about ten times smaller. In both P. polymyxa JB05-01-1 andP. polymyxa ATCC 43865, adhesion ranged from about 0.35% to about 6.5% of the initial numbers contacted with the enterocytes; the proportion being decreased at the higher initial load suggesting that saturation of the adhesion sites was approached at the higher bacterial load tested. Contact time with the Caco-2 monolayer had little effect on the number of adherent bacterial cells counted. -
TABLE 7 Adhesion of Paenibacillus polymyxa JB05-01-1, Paenibacillus polymyxa ATCC 43865 andPaenibacillus polymyxa ATCC 7070 to Caco-2 cellsAdherent bacteria (CFU/well)* Initial load 15 min 30 min 60 min P. polymyxa 8.7 ± 0.53 × 106 3.1 ± 0.1 × 104 3.1 ± 0.31 × 104 3.4 ± 0.16 × 104 JB05-01-1 8.7 ± 0.45 × 104 5.3 ± 0.22 × 103 4.4 ± 0.68 × 103 4.8 ± 0.42 × 103 P. polymyxa 6.8 ± 0.45 × 106 3.2 ± 0.21 × 104 2.8 ± 0.53 × 104 4.8 ± 0.26 × 104 ATCC 438656.8 ± 0.37 × 104 4.4 ± 0.33 × 103 2.4 ± 0.22 × 103 3.7 ± 0.32 × 103 P. polymyxa 7.1 ± 0.65 × 106 3.8 ± 0.21 × 104 3.3 ± 0.13 × 104 4.8 ± 0.46 × 104 ATCC 70707.1 ± 0.22 × 104 2.4 ± 0.33 × 102 5.5 ± 0.58 × 102 4.1 ± 0.51 × 102 *Mean S.E. of duplicate analyses - A preparation of JB05-01-1 supernatant (labeled P3) was produced as a dietary supplement for a field trial. A TSB medium with 0.4% yeast extract was prepared for a 1 liter fermentor which was in turn inoculated with the test culture. The culture was grown at 30° C. for 18 hours. The resulting culture was centrifuged at 12,000 g for 15 minutes at 4° C. The centrifuged cultures were then heated to between 80 and 100° C. in a water bath allowing the supernatant to be separated from the pellet. The supernatant was then stored at 4° C. for use in the trial. 500 day-old broiler chicks were divided into five treatment groups, each group containing 4 cohorts of 25 birds. The five different treatment groups of broiler chickens were given different amounts of dietary supplements for a four week period. The treatment regimes were as follows:
- T1=Normal Diet (control)
- T2=Normal Diet+1.5 ml RE3™ per kg feed
- T3=Normal Diet+0.5 ml P3 per kg feed
- T4=Normal Diet+1 ml P3 per kg feed
- T5=Normal Diet=1.5 ml P3 per kg feed
- Summary of results at the end of
week 4 are shown in Table 8. -
TABLE 8 The effect of varying levels of P3 on the growth performance of broiler chickens Dietary treatment T2 T3 T4 T5 1.5 ml 0.5 ml 1 ml 1.5 ml T1 RE3/kg P3/kg P3/kg P3/kg Parameter Control feed feed feed feed Initial weight/bird (g) 47 47 47 47 47 Final weight/bird (g) 1041 1006 1058 1025 1036 Feed intake/bird (g) 1780 1673 1729 1711 1731 Weight gain/bird (g) 994 959 1011 978 989 FCR (intake/gain) 1.79 1.75 1.71 1.75 1.75 Mortality (%) 7 5 5 1 1 - Addition of dietary supplements RE3 or P3 to chicken feed resulted in a slightly better feed conversion rate (FCR) of the feed than when no dietary supplement is added (control) indicating that addition of these dietary supplements results in a higher weight gain in chickens for equivalent feed intake. Provision of dietary supplements RE3 or P3 to feed also resulted in fewer mortalities, with supplement P3 added at levels of 1 ml or 1.5 ml per kg of feed having the lowest mortality rate.
- The genome of Paenibacillus polymyxa JB05-01-1 (“JB05-01-1 genome”) was sequenced by MiSeq Illumina sequencing (Eurofins MWG, Germany). The size of the genome was 5.9 Mb, with a 45.7% G/C content.
-
TABLE 9 Features of the assembled JB05-01-1 genome Features of the assembled JB05-01-1 genome Contig/Scaffold Overview Sample: PPJB005 All Large Large Contigs Contigs Scaffolds Scaffolds Total Entries: 70 30 42 5 Total Length: 5961671 5949216 5993163 5981904 Min Length: 173 1029 173 1029 Max Length: 1668666 1668666 5976888 5976888 Mean Length: 85166 198307 142694 1196380 N50: 512780 512780 5976888 5976888 N90: 173989 173989 1561 1561 GC Content: 45.7 45.7 45.7 45.7 #A: 1618632 1615645 1618632 1615858 #T: 1619418 1616533 1619418 1616700 #G: 1358695 1355323 1358695 1355642 #C: 1364926 1361715 1364926 1362143 #N: 0 0 31492 31492 Sample: PPJB005 Scaffold Length k-mer coverage scf_77 5976888 30.694033 scf_12 1561 87.979660 scf_10 1292 133.208954 scf_41 1134 56.627861 scf_7 1029 373.888641
The JB05-01-1 genome consists of 70 contigs, ranging from 173 bp to 1 658 666 bp in size, assembled in 5 large scaffolds. - The JB05-01-1 genome sequence was compared with available sequenced genomes of five Paenibacillus polymyxa strains (E681, SC2, M1, CR1 et SQR21). The contigs were reordered based on the reference strain E681 with ACT and WebACT. The JB05-01-1 genome was annotated using the RAST server (automated annotation), which identified 5489 coding sequences and 127 RNAs.
-
TABLE 10 Overview of annotated JB05-01-1 genome: gene subsystems and functions Feature Percentage Subsystems Count % Cofactors, Vitamins, Prosthetic 235 7.94 Groups Pigments Cell Wall and Capsule 154 5.20 Virulence, Disease, and Defense 95 3.21 Potassium metabolism 9 0.30 Photosynthesis 0 0.0 Miscellaneous 24 0.81 Phages, Prophages, Transposable 16 0.54 elements, Plasmids Membrane Transport 92 3.11 Iron acquisition and metabolism 35 1.18 RNA Metabolism 161 5.44 Nucleosides and Nucleotides 115 3.88 Protein Metabolism 223 7.53 Cell Division and Cell Cycle 27 0.91 Motility and Chemotaxis 69 2.33 Regulation and Cell signaling 59 1.99 Secondary Metabolism 125 4.22 Fatty acids, Lipids, and Isoprenoids 131 4.42 Nitrogen Metabolism 31 1.05 Dormancy and Sporulation 97 3.28 Respiration 53 1.79 Stress Response 85 2.87 Metabolism & Aromatic compounds 7 0.24 Amino Acids & derivatives 344 11.62 Sulfur Metabolism 51 1.72 Phosphorus Metabolism 67 2.26 Carbohydrates 656 22.15 Total 2961 100.00 - The JB05-01-1 genome contains genes coding for several antibacterial peptides, for example, polymyxin synthesis gene cluster, bacitracin and fusaricidin synthetases. The JB05-01-1 genome also contains multiple antibiotic resistance genes. Genes coding for bacitracin synthetases (annotated as
bacitracin synthetase 3 BA3) were identified as follows: -
- A 23727 bp synthetase (PEG 83, contig 43b 88285-112011), which is 97% identical to the P. Polymyxa
E681 bacitracin synthetase 3 - A 7767 bp synthetase (PEG 1749, contig 48 209298-217064), which shares 95% identity with the P. polymyxa M1 non-ribosomal peptide synthase NrsB
- A 12543 bp synthetase (PEG1750, contig 48 217075-229617), which is 92% identical to P. polymyxa M1 non-ribosomal peptide synthase NrsC
- A 4494 bp gene (PEG1962, contig 48, 460576-456083), which shares 99% identities with a gene annotated as a peptide synthetase in P. polymyxa CR1.
- A 3870 bp gene (PEG1977, contig 48, 512190-516059).
- A 23727 bp synthetase (PEG 83, contig 43b 88285-112011), which is 97% identical to the P. Polymyxa
- Another gene of interest was found (PEG 3093, contig 52 6122-17335) coding for a 11214 bp Malonyl CoA-acyl carrier protein transacylase which shares 99% identity with the P. polymyxa CR1 fusaricidin synthetase gene.
-
TABLE 11 Primers sequences for the bacitracin and fusaricidin synthetases identified in the JB05-01-1 genome G/C con- Length tent Tm Sequence (5′ to 3′) (bp) (%) (° C.) PEG83_F ATGAATGACATGCAGTTATA 20 30 44 (SEQ ID NO: 2) PEG83_R TTAGGACAAAATGGGCTTGT 20 40 48 (SEQ ID NO: 3) PEG1749_F ATGAGAAACGCACTCATTCA 20 40 48 (SEQ ID NO: 4) PEG1749_R CTAGGAAGATGTATTATTTT 25 24 48 TATAG (SEQ ID NO: 5) PEG1750_F ATGAGTAAAGATCTGCAAAT 23 31 48 TCA (SEQ ID NO: 6) PEG1750_R TTACAGAACCGCTGCCCAGC 20 60 56 (SEQ ID NO: 7) PEG1962_F ATGTTCTATGCATTAACGCA 21 33 47 T (SEQ ID NO: 8) PEG1962_R TTATTCAAAAAGCAATTCAA 24 21 45 TATC (SEQ ID NO: 9) PEG1977_F ATGCATTATGAAGGATATGA 20 30 44 (SEQ ID NO: 10) PEG1977_R TTACCTCAAAAATGTATTCA 20 25 42 (SEQ ID NO: 11) PEG3093_F TTGAAAGCCTTATTTGAGAA 21 33 47 G (SEQ ID NO: 12) PEG3093_R TCAAGATTTATGATGTGCCA 20 35 46 (SEQ ID NO: 13) - The 2613 bp lantionine synthetase LanL (PEG 2150, contig 48 697422-700034) was also identified. In addition, two other genes coding for non-ribosomal peptide synthetases were identified: a 1407 bp gene (PEG1966, contig 48, 468244-469650: 1407 bp) and a 2025 bp gene (PEG1971, contig 48, 484672-486696).
- The JB05-01-1 genome also contains a polymyxin synthesis gene cluster consisting of five genes, sharing high homology with the pmxABCDE genes (95-99% identities). The genes coding for PmxB, PmxC and PmxD located on contig 63 were identified as:
-
- pmxB: PEG5404 (1899 bp, contig 63 10624-8726) annotated as a bacillibactin synthetase component F (97% identical to the pmxB gene in Paenibacillus polymyxa SQR-21)
- pmxC: PEG5403 (1827 bp, contig 63 8736-6910) annotated as lipid A export ATP-binding/permease protein MsbA (96% identical to the pmxC gene in P. polymyxa M1
- pmxD: PEG5402 (1734 bp, contig 63 6913-5180) annotated as lipid A export ATP-binding/permease protein MsbA (98% identical to the pmxD gene in P. polymyxa M1).
-
TABLE 12 Primer sequences for the polymyxin synthesis genes identified in the JB05-01-1 genome G/C Length content Tm Sequence (5′ to 3′) (bp) (%) (° C.) PEG5402_F TTGAAAAAGGGCGGATGGC 23 52 57 (6913) TCTC (SEQ ID NO: 14) PEG5402_R CTAGCCGTACAGCCGGGC 18 72 57 (5180) (SEQ ID NO: 15) PEG5403_F ATGGAAGCTGACCGGCAG 18 61 53 (8736) (SEQ ID NO: 16) PEG5403_R TCAAGTGTACGCCACCTCC 19 58 53 (6910) (SEQ ID NO: 17) PEG5404_F ATGTACGGAGCGCTGCTGT 19 58 53 (10624) (SEQ ID NO: 18) PEG5404_R TCAGCTTCCATGCAGTACC 19 53 51 (8726) (SEQ ID NO: 19) - The pmxA and pmxE genes in the JB05-01-1 genome were not identified by automated annotation. The pmxA (15 030 bp) and the pmxE (18 940 bp) genes of P. polymyxa M1 were used as references to search the JB05-01-1 genome. Fragments of pmxA were found on contigs 62 (
bases 1 to 1619), 60 (1796-2688), 66 (3132-5604), 67 (6075-8553) and 68 (9252-13125, 13659-15033). The pmxE gene appeared to be located oncontigs 63 and 61. -
- Andersson, A., Granumb, P. E., Ronnera, U. (1998). The adhesion of Bacillus cereus spores to epithelial cells might be an additional virulence mechanism International Journal of Food Microbiology 39, 93-99.
- Altschul S F, Gish W, Miller W, Myers E W, Lipman D J. Basic local alignment search tool. J Mol Biol 1990; 215:403-410.
- Angioi, A., Zanetyi, S., Sanna, A., Delogu, G., Fadda, G. (1995) Adhesiveness of Bacillus subtilis Strains to Epithelial Cells Cultured in vitro. Microbial Ecology in Health and Disease. 8: 71-77
- Bhunia A K, Johnson M C, Ray, B. Direct detection of an antibacterial peptide of Pediococcus acidilactici in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J Ind Microbiol 1987; 2:319-322.
- Caldwell D R, Bryant M P. Medium without rumen fluid for non selective enumeration and isolation of rumen bacteria. Appl Microbiol 1966; 14:794-801.
- Cepeljnik, T., Lah, B., Narat, M., Marinsek-Logar, R. (2007) Adaptation of adhesion test using Caco-2 cells for anaerobic bacterium Pseudobutyrivibrio xylanivorans, a probiotic candidate. Folia Microbiol (Praha). 52:367-73.
- Charteris, W. P., Kelly, P. M., Morelli, L., and Collins, J. K. (1998). Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract. Journal of Applied Microbiology, 84, 759-768.
- Cleveland J, Montville T J, Nes I F, Chikindas M L. Bacteriocins: Safe, natural antibacterials for food preservation. hit. J. Food Microbiol, 2001; 71:11-20.
- Daba H, Lacroix C, Huang J, Simard R E, Lemieux L. Simple method of purification and sequencing of a bacteriocin produced by Pediococcus acidilactici UL5. J Appl Bacteriol 1994; 77:682-688.
- Davies E A, Bevis H E, Potter R, Harris J, Williams G C, Delves-Broughton J. The effect of pH on the stability of nisin solution during autoclaving. Lett Appl Microbiol 1998; 27:186-187.
- de Man J C, Rogosa M, Sharpe M E. A medium for the cultivation of lactobacilli. J Appl Bacteriol 1960; 23:130-135.
- DeCrescenzo H E, Phillips D R, Doran Peterson J B. Polymyxin E production by P. amylolyticus. Letters Appl Microbiol 2007; 45:491-496.
- Doyle, R. J., Nedjat-Haiem F. Singh J. S. (1984). Hydrophobic characteristics of Bacillus spores. Current Microbiology. 10: 329-333.
- Doyle, R. J., Rosenberg, M. (1995). Measurement of microbial adhesion to hydrophobic substrata. Methods in Enzymology, 253, 542-550.
- Du L, Shen B. Identification and characterization of a type II peptidyl carrier protein from the bleomycin producer Streptomyces verticillus ATCC 15003. Chem Biol. 1999; 6:507-517.
- Falagas M E, and Kasiakou S K (2005). Colistin: the revival ofpolymyxins for the management of multidrug-resistant gram-negative bacterial infections. Clin Infect Dis. 40:1333-1341.
- Falagas M E, Kasiakou S J (2006) Toxicity of polymyxins: a systematic review of the evidence from old and recent studies. Crit Care. 10:R27.
- Fangio M F, Roura S I, Fritz R (2010) Isolation and identification of Bacillus spp. and related genera from different starchy foods. J Food Sci. 75:218-221.
- Gagnon, M., Kheadr, E., Le Blay G., Fliss, I. (2004) vitro inhibition of Escherichia coli O157:H7 by bifidobacterial strains of human origin. International Journal of Food Microbiology 92, 69-78.
- Govaerts C, Orwa J, Van Schepdael A, Roets E, Hoogmartens J (2002) Characterization of polypeptide antibiotics of the polymyxin series by liquid chromatography electrospray ionization ion trap tandem mass spectrometry. J. Pept. Sci. 8:45-55.
- He Z, Kisla D, Zhang L, Yuan C, Green-Church K B, YousefAE (2007) Isolation and identification of a Paenibacillus polymyxa strain that coproduces a novel lantibiotic and polymyxin. Appl Env Microbiol. 73:168-178.
- He Z, Yuan C, Zhang L, Yousef A E (2008) N-terminal acetylation in paenibacillin, a novel lantibiotic. FEBS Lett. 582:2787-2792.
- Jian Li, Milnel R W, Nation R L, Turnidge J D, Smeaton T C, Coulthard K (2004) Pharmacokinetics of colistin methanesulphonate and colistin in rats following an intravenous dose of colistin methanesulphonate J Antimicrobial Chemother. 53: 837-840.
- Kane M D, Paulsen L K, Stahl D A. Monitoring the enrichment and isolation of sulfate-reducing bacteria by using oligonucleotide hybridization probes designed from environmentally derived 16S rRNA sequences. Appl Environ Microbiol 1993; 59:682-686.
- Kheadr E, Bernoussi N, Lacroix C, Fliss I. Comparison of the sensitivity of commercial strains and infant isolates of bifidobacteria to antibiotics and bacteriocins, Int Dairy J 2004; 14; 273-285.
- Klaenhammer T R. Genetics of bacteriocins produced by lactic bacteria. FEMS Microbiol Rev 1993; 12:39-86.
- Komura S, Kurahashi K. Biosynthesis of polymyxin E by a cell-free enzyme system. J Biochem 1985; 97:1409-1417.
- Koshikawa, T., Yamazaki, M., Yoshimi, M., Ogawa, S., Yamada, A., Watabe, K., Tori, A., (1989) Surface Hydrophobicity of Spores of Bacillus spp. Journal of General Microbiology 135, 2717-2722.
- Lampis G, Deidda D, Maullu C, Madeddu M A, Pompei R, Delle Monachie F, Satta G (1995) Sattabacins and sattazolins: new biologically active compounds with antiviral properties extracted from a Bacillus sp. J Antibiot (Tokyo). 48:967-72.
- Landman D, Georgescu C, Martin D A, Quale J (2008) Polymyxins Revisited Clinical. Microbiol Rev. 21, 449-465.
- Lankaputhra, E. V., Shah, N. P., 1995. Survival of Lactobacillus acidophilus and Bifidobacterium spp. in the presence of acid and bile salts. Cult. Dairy Prod. J. 30, 2-7.
- Lenni Fitri and Betty Mauliya Bustam (2010) Screening of antimicrobial producing strains isolated from the soil of grassland rhizosphere in Pocut Meurah Intan Forest Park, Seulawah, Aceh Besar Biodiversitas, 11 129-132.
- Lim L M, Ly N, Anderson D, Yang J C, Macander L, Jarkowski A 3rd, Forrest A, Bulitta J B, Tsuji B T (2010) Resurgence of colistin: a review of resistance, toxicity, pharmacodynamics, and dosing. Pharmacotherapy. 30:1279-91.
- Liu W T, Marsh T L, Cheng H, Forney L J. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl Environ Microbiol 1997; 63:4516-4522.
- Lowry O H, Rosebrough N J, Farr A L, Randall R J. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:267-275.
- Marahiel M A, Stachelhaus T, Mootz H D. Modular peptide synthetases involved in nonribosomal peptide synthesis. Chem Rev 1997; 97:2651-2673.
- Martin N I, Hu H, Moake M M, Churey J J, Whittal R, Worobo R W, Vederas J C. Isolation, structural characterization, and properties of mattacin (polymyxin M), a cyclic peptide antibiotic produced by Paenibacillus kobensis M. J Biol. Chem 2003; 278:13124-13132.
- Motta A S, Lorenzini D M, Brandelli A. Purification and partial characterization of an antibacterial peptide produced by a novel Bacillus sp. Isolated from the amazon basin. Curr Microbiol 2007; 54:282-286.
- Naghmouchi K, Drider D, Baah J, Teather R (2010) Nisin A and Polymyxin B as Synergistic Inhibitors of Gram-positive and Gram-negative Bacteria. Prob Ant Prot. 2: 98-103.
- Naghmouchi K, Drider D, Fliss I. Action of divergicin M35, a class IIa bacteriocin, on liposomes and Listeria. J Appl Microbiol. 2007; 102:1508-17.
- Naghmouchi K, Drider D, Hammami R, Fliss I. (2008) Effect of Antimicrobial Peptides Divergicin M35 and Nisin A on Listeria monocytogenes LSD530 Potassium Channels. Current Microbiol 56:609-612.
- Nathaniel I M, Hu H, Moake M M, Churey J J, Whittal R, Worobo R W, and Vederas J C (2003) Isolation, Structural Characterization, and Properties of Mattacin (Polymyxin M), a Cyclic Peptide Antibiotic Produced by Paenibacillus kobensis M. J Biol Chem. 278: 13124-13132.
- Nes S F, Diep D B, Havarstein L S, Bruberg M B, Eijsink V G, Holo H. Biosynthesis of bacteriocins in lactic acid bacteria. Antonie Leeuwenhock 1996; 70:113-128.
- Scheldeman, P. Goossens, K., Rodriguez-Diaz, M, Pil, A., Goris, J., Herman, L., De Vos, P., Logan, N A., Heyndrickx M (2004). Paenibacillus lactis sp. nov., isolated from raw and heat-treated milk. International journal of systematic and evolutionary microbiology 54, 885-891.
- Selim S, Negrel J, Govaerts C, Gianinazzi S, van Tuinen D (2005) Isolation and Partial Characterization of Antagonistic Peptides Produced by Paenibacillus sp. Strain B2 Isolated from the Sorghum Mycorrhizosphere. Appl Environ Microbiol. 71: 501-6507.
- Suskovic, J., Brkic, B., Matosic, S., Maric, V., 1997. Lactobacillus acidophilus M92 as potential probiotic strain. Milchwissenschaft 52, 430-435.
- Svetoch E A, Stern N J, Eruslanov B V, Kovalev Y N, Volodina L I, Perelygin V V, Mitsevich E V, Mitsevich I P, Pokhilenko V D, Borzenkov V N, Levchuk V P, Svetoch O E, Kudriavtseva T Y. Isolation of Bacillus circulans and Paenibacillus polymyxa strains inhibitory to Campylobacter jejuni and characterization of associated bacteriocins. J Food Prot 2005; 68:11-17.
- Tagg J R, Dajani A S, Wannamaker L W. Bacteriocins of gram-positive bacteria Bacteriol Rev 1976; 40:722-56.
- Wolf C E, Gibbons W R. Improved method for quantification of the bacteriocin nisin. J Appl Bacteriol 1996; 80:453-457.
- Zengguo He, Kisla D, Zhang L, Yuan C, Green-Church K B, Yousef A. E. Isolation and identification of a Paenibacillus polymyxa strain that coproduces a novel lantibiotic and polymyxin. Appl Environ Microbiol 2007; 73:168-178.
- Zheng G. Yan L Z, Vederas J C, Zuber P. Genes of the sbo-alb locus of Bacillus subtilis are required for production of the antilisterial bacteriocin subtilosin. J Bacteriol 1999; 181:7346-355.
- The present invention has been described with regard to one or more embodiments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims. Accordingly, although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the spirit and scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range, and of sub-ranges encompassed therein. As used herein, the terms “comprising”, “comprises”, “having” or “has” are used as an open-ended terms, substantially equivalent to the phrase “including, but not limited to”. Terms such as “the,” “a,” and “an” are to be construed as indicating either the singular or plural. Citation of references herein shall not be construed as an admission that such references are prior art to the present invention. All publications are incorporated herein by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings.
Claims (20)
1. A method of inhibiting growth of a Gram-negative staining bacterium in a subject or substance in need thereof, the method comprising administering or applying an effective amount of an anti-microbial composition to the subject or substance, wherein the anti-microbial composition comprises:
(a) an isolated Paenibacillus polymyxa bacterium which has a 16S ribosomal RNA (16SrRNA) gene containing a DNA comprising the base sequence of SEQ ID NO: 1;
(b) an isolated Paenibacillus polymyxa bacterium deposited at the ATCC® under the terms of the Budapest Treaty and designated Accession Number PTA-10436;
(c) a cell culture comprising the Paenibacillus polymyxa bacterium;
(d) a cell culture supernatant derived from growing the Paenibacillus polymyxa bacterium in a cell culture medium; or
(e) an anti-microbial agent isolated from the Paenibacillus polymyxa bacterium, the cell culture or the cell culture supernatant.
2. The method of claim 1 , wherein the Gram-negative staining bacterium is selected from the group consisting of Escherichia sp., Pantoea sp., Pseudomonas sp., Butyrivibrio sp., Fibrobacter sp., Salmonella sp., Shigella sp., Helicobacter sp., and Campylobacter sp.
3. The method of claim 1 , wherein the Gram-negative staining bacterium is selected from the group consisting of Escherichia coli RR1, Escherichia coli TB1, Escherichia coli O157:H7, Pantoea agglomerans BC1, Pseudomonas fluorescens R73, Butyrivibrio fibrisolvens OR85, Fibrobacter succinogenes and Pseudomonas aeruginosa.
4. The method of claim 1 , which does not inhibit the growth of a Gram-positive staining bacterium.
5. The method of claim 1 , wherein the anti-microbial agent comprises a peptide.
6. The method of claim 1 , wherein the isolated Paenibacillus polymyxa bacterium DNA comprises at least one gene needed for the production of a bacitracin, polymyxin, or fusaricidin.
7. The method of claim 1 , wherein the anti-microbial agent has a molecular weight between about 1000 daltons to about 2500 daltons.
8. The method of claim 1 , wherein the anti-microbial agent comprises a polymyxin.
9. The method of claim 1 , wherein the anti-microbial agent comprises at least one of colistin A and colistin B.
10. The method of claim 1 , wherein the subject is a human, a pet or an agricultural animal.
11. The method of claim 1 , wherein the anti-microbial composition is used as a probiotic.
12. The method of claim 1 , wherein the substance is selected from the group consisting of a cosmetic product, a hygiene product, a feed product, a food product and packaging material thereof.
13. The method of claim 12 wherein the food product is a dry feed product.
14. A method of producing an anti-microbial agent comprising growing or culturing an isolated Paenibacillus polymyxa bacterium which has a 16S ribosomal RNA (16SrRNA) gene containing a DNA comprising the base sequence of SEQ ID NO: 1, or an isolated Paenibacillus polymyxa bacterium deposited at the ATCC® under the terms of the Budapest Treaty and designated Accession Number PTA-10436, in a cell culture medium to produce a cell culture supernatant comprising the anti-microbial agent.
15. The method of claim 14 , further comprising isolating or purifying the anti-microbial agent from the cell culture supernatant.
16. A method of producing an anti-microbial agent comprising growing or culturing an isolated Paenibacillus polymyxa bacterium which has a 16S ribosomal RNA (16SrRNA) gene containing a DNA comprising the base sequence of SEQ ID NO: 1, or an isolated Paenibacillus polymyxa bacterium deposited at the ATCC® under the terms of the Budapest Treaty and designated Accession Number PTA-10436, in a cell culture medium to produce a cell culture comprising the anti-microbial agent.
17. The method of claim 16 , further comprising isolating or purifying the anti-microbial agent from the cell culture.
18. A method of reducing mortality or morbidity of an animal comprising administering an effective amount of an anti-microbial composition to the animal, wherein the anti-microbial composition comprises:
(a) an isolated Paenibacillus polymyxa bacterium which has a 16S ribosomal RNA (16SrRNA) gene containing a DNA comprising the base sequence of SEQ ID NO: 1;
(b) an isolated Paenibacillus polymyxa bacterium deposited at the ATCC® under the terms of the Budapest Treaty and designated Accession Number PTA-10436;
(c) a cell culture comprising the Paenibacillus polymyxa bacterium;
(d) a cell culture supernatant derived from growing the Paenibacillus polymyxa bacterium in a cell culture medium; or
(e) an anti-microbial agent isolated from the Paenibacillus polymyxa bacterium, the cell culture or the cell culture supernatant.
19. The method of claim 18 , wherein the animal is a human, a pet or an agricultural animal.
20. The method of claim 18 , wherein the anti-microbial composition is used as a probiotic or a prebiotic.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US15/727,184 US20180087117A1 (en) | 2009-12-09 | 2017-10-06 | Anti-microbial agent from paenibacillus sp. and methods and uses thereof |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/CA2009/001808 WO2011069227A1 (en) | 2009-12-09 | 2009-12-09 | Anti-microbial agent from paenibacillus sp. and methods and uses thereof |
US13/296,063 US20120121543A1 (en) | 2009-12-09 | 2011-11-14 | Anti-microbial agent from paenibacillus sp. and methods and uses thereof |
US13/611,160 US20130101559A1 (en) | 2009-12-09 | 2012-09-12 | Anti-microbial agent from paenibacillus sp. and methods and uses thereof |
US14/642,158 US9809865B2 (en) | 2009-12-09 | 2015-03-09 | Anti-microbial agent from Paenibacillus sp. and methods and uses thereof |
US15/727,184 US20180087117A1 (en) | 2009-12-09 | 2017-10-06 | Anti-microbial agent from paenibacillus sp. and methods and uses thereof |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/642,158 Continuation US9809865B2 (en) | 2009-12-09 | 2015-03-09 | Anti-microbial agent from Paenibacillus sp. and methods and uses thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
US20180087117A1 true US20180087117A1 (en) | 2018-03-29 |
Family
ID=54366868
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/642,158 Expired - Fee Related US9809865B2 (en) | 2009-12-09 | 2015-03-09 | Anti-microbial agent from Paenibacillus sp. and methods and uses thereof |
US15/727,184 Abandoned US20180087117A1 (en) | 2009-12-09 | 2017-10-06 | Anti-microbial agent from paenibacillus sp. and methods and uses thereof |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/642,158 Expired - Fee Related US9809865B2 (en) | 2009-12-09 | 2015-03-09 | Anti-microbial agent from Paenibacillus sp. and methods and uses thereof |
Country Status (1)
Country | Link |
---|---|
US (2) | US9809865B2 (en) |
Families Citing this family (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107995925B (en) | 2015-03-26 | 2022-04-29 | 拜耳作物科学有限合伙公司 | Novel Paenibacillus strains, antifungal compounds, and methods of use thereof |
MX2019006817A (en) * | 2016-12-16 | 2019-08-26 | Dupont Nutrition Biosci Aps | Bacillus-based components for inhibiting or delaying the growth of enterococcus spp. in animals. |
CN110055188A (en) * | 2019-03-07 | 2019-07-26 | 南京师范大学 | One plant of Paenibacillus polymyxa XW4 for producing bacteriostatic peptide and its separation screening and application |
CN112501071B (en) * | 2020-12-15 | 2022-04-22 | 河北省科学院生物研究所 | Paenibacillus polymyxa SWGC4112 and culture method and application thereof |
CN112940982B (en) * | 2021-03-24 | 2021-09-17 | 中国水产科学研究院珠江水产研究所 | Snakehead source Bacillus belezii and application thereof |
CN113621536B (en) * | 2021-07-20 | 2023-04-25 | 华南农业大学 | Paenibacillus polymyxa SP1 and application thereof |
CN113729109B (en) * | 2021-07-20 | 2023-04-28 | 华南农业大学 | Fermented feed rich in antibacterial substances and preparation method thereof |
US20230080731A1 (en) * | 2021-08-31 | 2023-03-16 | The Board Of Trustees Of The University Of Arkansas | Bactericidal protein for control of campylobacter and listeria |
CN116240123B (en) * | 2022-08-02 | 2024-01-23 | 岳阳渔美康生物科技有限公司 | Paenibacillus polymyxa, microecological preparation and preparation method thereof |
CN116585449B (en) * | 2023-05-08 | 2024-03-15 | 郑州大学 | Nanometer antibacterial drug delivery system based on probiotics spores and preparation method and application thereof |
CN117402784B (en) * | 2023-10-19 | 2024-04-12 | 甘肃省科学院生物研究所 | Paenibacillus polymyxa Mxdg-1, microbial inoculum and application thereof |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103146614B (en) * | 2013-03-14 | 2014-07-23 | 中国科学院成都生物研究所 | Camptotheca endophytic bacterium LY214 for producing camptothecin and application thereof |
-
2015
- 2015-03-09 US US14/642,158 patent/US9809865B2/en not_active Expired - Fee Related
-
2017
- 2017-10-06 US US15/727,184 patent/US20180087117A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
US9809865B2 (en) | 2017-11-07 |
US20150320830A1 (en) | 2015-11-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US9809865B2 (en) | Anti-microbial agent from Paenibacillus sp. and methods and uses thereof | |
US20120121543A1 (en) | Anti-microbial agent from paenibacillus sp. and methods and uses thereof | |
Guo et al. | Identification and characterization of Bacillus subtilis from grass carp (Ctenopharynodon idellus) for use as probiotic additives in aquatic feed | |
Cheng et al. | Isolation and characterization of antimicrobial peptides derived from Bacillus subtilis E20-fermented soybean meal and its use for preventing Vibrio infection in shrimp aquaculture | |
Khochamit et al. | Antibacterial activity and genotypic–phenotypic characteristics of bacteriocin-producing Bacillus subtilis KKU213: potential as a probiotic strain | |
JP5185996B2 (en) | Broad spectrum antibacterial and antifungal activity of Lactobacillus johnsonii D115 | |
Strompfová et al. | In vitro study on bacteriocin production of Enterococci associated with chickens | |
Strompfová et al. | Enterococcus faecium EK13—an enterocin A-producing strain with probiotic character and its effect in piglets | |
Kaktcham et al. | Quantitative analyses of the bacterial microbiota of rearing environment, tilapia and common carp cultured in earthen ponds and inhibitory activity of its lactic acid bacteria on fish spoilage and pathogenic bacteria | |
Shin et al. | Isolation and characterization of bacteriocin‐producing bacteria from the gastrointestinal tract of broiler chickens for probiotic use | |
Urdaci et al. | Antimicrobial activity of Bacillus probiotics | |
US11857596B2 (en) | Animal feed additive and animal feed | |
CA2923982A1 (en) | Antiviral methods and compositions comprising probiotic bacterial molecules | |
Dec et al. | Antimicrobial activity of Lactobacillus strains of chicken origin against bacterial pathogens | |
WO2009030040A1 (en) | Antimicrobial activity of bacteriocin-producing lactic acid bacteria | |
Xiang et al. | Purification, characterization, and antibacterial and antibiofilm activity of a novel bacteriocin against Salmonella Enteritidis | |
Garcés et al. | Antimicrobial activity of bacteriocin‐producing Carnobacterium spp. isolated from healthy Patagonian trout and their potential for use in aquaculture | |
US20130101559A1 (en) | Anti-microbial agent from paenibacillus sp. and methods and uses thereof | |
Daba et al. | Evaluation of Enterococcus strains newly isolated from Egyptian sources for bacteriocin production and probiotic potential | |
Ansari | Bacteriocin from LAB for medical and health applications | |
Class et al. | Patent application title: ANTI-MICROBIAL AGENT FROM PAENIBACILLUS SP. AND METHODS AND USES THEREOF Inventors: Ronald Teather (Lethbridge, CA) John Baah (Lethbridge, CA) Karim Naghmouchi (Tunis, TN) James Gibbs Watson (Edmonton, CA) Djamel Drider (Villeneuve D'Ascq Cedex, FR) | |
KR20090129019A (en) | Novel bacteriocin-producing lactic acid bacteria and mixed microbial composition using it for broiler chickens | |
Meidong et al. | Mixed culture of Bacillus aerius B81e and Lactiplantibacillus paraplantarum L34b-2 derived from in vivo screening using hybrid catfish exhibits high probiotic effects on Pangasius bocourti | |
Zhang | Bacterial-derived antimicrobials as potential alternatives for antibiotic-free broiler production | |
Arul | Isolation and probiotic characterization of lactic acid bacteria enterococcus faecalis from country chicken, and duck and its in vivo attributes in broiler chicken |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: BEST ENVIRONMENTAL TECHNOLOGIES, INC., BARBADOS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:TEATHER, RONALD;BAAH, JOHN;WATSON, JAMES GIBBS;AND OTHERS;SIGNING DATES FROM 20161208 TO 20170223;REEL/FRAME:044148/0465 |
|
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 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |