JP2006081536A - Medium for lactobacillus - Google Patents
Medium for lactobacillus Download PDFInfo
- Publication number
- JP2006081536A JP2006081536A JP2005034089A JP2005034089A JP2006081536A JP 2006081536 A JP2006081536 A JP 2006081536A JP 2005034089 A JP2005034089 A JP 2005034089A JP 2005034089 A JP2005034089 A JP 2005034089A JP 2006081536 A JP2006081536 A JP 2006081536A
- Authority
- JP
- Japan
- Prior art keywords
- lactic acid
- growth
- acid bacteria
- medium according
- concentration
- 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.)
- Granted
Links
- 241000186660 Lactobacillus Species 0.000 title abstract description 8
- 229940039696 lactobacillus Drugs 0.000 title description 2
- 241000894006 Bacteria Species 0.000 claims abstract description 159
- 229940121375 antifungal agent Drugs 0.000 claims abstract description 37
- 239000003429 antifungal agent Substances 0.000 claims abstract description 37
- 229910052784 alkaline earth metal Inorganic materials 0.000 claims abstract description 29
- 229910052783 alkali metal Inorganic materials 0.000 claims abstract description 27
- 150000001340 alkali metals Chemical class 0.000 claims abstract description 26
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims abstract description 12
- 150000001540 azides Chemical class 0.000 claims abstract description 10
- 150000001342 alkaline earth metals Chemical class 0.000 claims abstract description 7
- 238000012258 culturing Methods 0.000 claims abstract description 7
- 229910000019 calcium carbonate Inorganic materials 0.000 claims abstract description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 5
- XJRPTMORGOIMMI-UHFFFAOYSA-N ethyl 2-amino-4-(trifluoromethyl)-1,3-thiazole-5-carboxylate Chemical compound CCOC(=O)C=1SC(N)=NC=1C(F)(F)F XJRPTMORGOIMMI-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000008103 glucose Substances 0.000 claims abstract description 5
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 230
- 235000014655 lactic acid Nutrition 0.000 claims description 115
- 239000004310 lactic acid Substances 0.000 claims description 115
- 239000002609 medium Substances 0.000 claims description 82
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical group [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 38
- 239000003910 polypeptide antibiotic agent Substances 0.000 claims description 33
- -1 alkaline earth metal azide Chemical class 0.000 claims description 26
- 108010093965 Polymyxin B Proteins 0.000 claims description 19
- 229920000024 polymyxin B Polymers 0.000 claims description 19
- 229960005266 polymyxin b Drugs 0.000 claims description 19
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 18
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 18
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 13
- 244000063299 Bacillus subtilis Species 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- YPHMISFOHDHNIV-FSZOTQKASA-N cycloheximide Chemical compound C1[C@@H](C)C[C@H](C)C(=O)[C@@H]1[C@H](O)CC1CC(=O)NC(=O)C1 YPHMISFOHDHNIV-FSZOTQKASA-N 0.000 claims description 10
- 239000001963 growth medium Substances 0.000 claims description 10
- 241000193755 Bacillus cereus Species 0.000 claims description 9
- 238000000855 fermentation Methods 0.000 claims description 9
- 230000004151 fermentation Effects 0.000 claims description 9
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims description 8
- 229960004884 fluconazole Drugs 0.000 claims description 8
- RFHAOTPXVQNOHP-UHFFFAOYSA-N fluconazole Chemical compound C1=NC=NN1CC(C=1C(=CC(F)=CC=1)F)(O)CN1C=NC=N1 RFHAOTPXVQNOHP-UHFFFAOYSA-N 0.000 claims description 8
- 239000002459 polyene antibiotic agent Substances 0.000 claims description 8
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 claims description 7
- 241000588724 Escherichia coli Species 0.000 claims description 7
- 241000607142 Salmonella Species 0.000 claims description 7
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 claims description 7
- 229960003942 amphotericin b Drugs 0.000 claims description 7
- 108010021062 Micafungin Proteins 0.000 claims description 5
- BYBLEWFAAKGYCD-UHFFFAOYSA-N Miconazole Chemical compound ClC1=CC(Cl)=CC=C1COC(C=1C(=CC(Cl)=CC=1)Cl)CN1C=NC=C1 BYBLEWFAAKGYCD-UHFFFAOYSA-N 0.000 claims description 5
- 229960002159 micafungin Drugs 0.000 claims description 5
- PIEUQSKUWLMALL-YABMTYFHSA-N micafungin Chemical compound C1=CC(OCCCCC)=CC=C1C1=CC(C=2C=CC(=CC=2)C(=O)N[C@@H]2C(N[C@H](C(=O)N3C[C@H](O)C[C@H]3C(=O)N[C@H](C(=O)N[C@H](C(=O)N3C[C@H](C)[C@H](O)[C@H]3C(=O)N[C@H](O)[C@H](O)C2)[C@H](O)CC(N)=O)[C@H](O)[C@@H](O)C=2C=C(OS(O)(=O)=O)C(O)=CC=2)[C@@H](C)O)=O)=NO1 PIEUQSKUWLMALL-YABMTYFHSA-N 0.000 claims description 5
- 229960002509 miconazole Drugs 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 4
- YKSVGLFNJPQDJE-YDMQLZBCSA-N (19E,21E,23E,25E,27E,29E,31E)-33-[(2R,3S,4R,5S,6R)-4-amino-3,5-dihydroxy-6-methyloxan-2-yl]oxy-17-[7-(4-aminophenyl)-5-hydroxy-4-methyl-7-oxoheptan-2-yl]-1,3,5,7,37-pentahydroxy-18-methyl-9,13,15-trioxo-16,39-dioxabicyclo[33.3.1]nonatriaconta-19,21,23,25,27,29,31-heptaene-36-carboxylic acid Chemical compound CC(CC(C)C1OC(=O)CC(=O)CCCC(=O)CC(O)CC(O)CC(O)CC2(O)CC(O)C(C(CC(O[C@@H]3O[C@H](C)[C@@H](O)[C@@H](N)[C@@H]3O)\C=C\C=C\C=C\C=C\C=C\C=C\C=C\C1C)O2)C(O)=O)C(O)CC(=O)C1=CC=C(N)C=C1 YKSVGLFNJPQDJE-YDMQLZBCSA-N 0.000 claims description 3
- 108010001478 Bacitracin Proteins 0.000 claims description 3
- 108010078777 Colistin Proteins 0.000 claims description 3
- IIUZTXTZRGLYTI-UHFFFAOYSA-N Dihydrogriseofulvin Natural products COC1CC(=O)CC(C)C11C(=O)C(C(OC)=CC(OC)=C2Cl)=C2O1 IIUZTXTZRGLYTI-UHFFFAOYSA-N 0.000 claims description 3
- AGJUUQSLGVCRQA-SWOUQTJZSA-N Fungichromin Chemical compound CCCCC[C@@H](O)[C@@H]1[C@@H](O)C[C@@H](O)C[C@@H](O)C[C@@H](O)C[C@@H](O)C[C@@H](O)[C@@H](O)[C@H](O)\C(C)=C\C=C\C=C\C=C\C=C\[C@H](O)[C@@H](C)OC1=O AGJUUQSLGVCRQA-SWOUQTJZSA-N 0.000 claims description 3
- UXWOXTQWVMFRSE-UHFFFAOYSA-N Griseoviridin Natural products O=C1OC(C)CC=C(C(NCC=CC=CC(O)CC(O)C2)=O)SCC1NC(=O)C1=COC2=N1 UXWOXTQWVMFRSE-UHFFFAOYSA-N 0.000 claims description 3
- 229930195098 Hamycin Natural products 0.000 claims description 3
- DDUHZTYCFQRHIY-UHFFFAOYSA-N Negwer: 6874 Natural products COC1=CC(=O)CC(C)C11C(=O)C(C(OC)=CC(OC)=C2Cl)=C2O1 DDUHZTYCFQRHIY-UHFFFAOYSA-N 0.000 claims description 3
- AGJUUQSLGVCRQA-UHFFFAOYSA-N Pentamycin Natural products CCCCCC(O)C1C(O)CC(O)CC(O)CC(O)CC(O)CC(O)C(O)C(O)C(C)=CC=CC=CC=CC=CC(O)C(C)OC1=O AGJUUQSLGVCRQA-UHFFFAOYSA-N 0.000 claims description 3
- NCXMLFZGDNKEPB-UHFFFAOYSA-N Pimaricin Natural products OC1C(N)C(O)C(C)OC1OC1C=CC=CC=CC=CCC(C)OC(=O)C=CC2OC2CC(O)CC(O)(CC(O)C2C(O)=O)OC2C1 NCXMLFZGDNKEPB-UHFFFAOYSA-N 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
- 229960004348 candicidin Drugs 0.000 claims description 3
- 229960003346 colistin Drugs 0.000 claims description 3
- DDUHZTYCFQRHIY-RBHXEPJQSA-N griseofulvin Chemical compound COC1=CC(=O)C[C@@H](C)[C@@]11C(=O)C(C(OC)=CC(OC)=C2Cl)=C2O1 DDUHZTYCFQRHIY-RBHXEPJQSA-N 0.000 claims description 3
- 229960002867 griseofulvin Drugs 0.000 claims description 3
- 229950006942 hamycin Drugs 0.000 claims description 3
- 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 Chemical compound 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 claims description 3
- 229960003255 natamycin Drugs 0.000 claims description 3
- NCXMLFZGDNKEPB-FFPOYIOWSA-N natamycin Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C[C@@H](C)OC(=O)/C=C/[C@H]2O[C@@H]2C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 NCXMLFZGDNKEPB-FFPOYIOWSA-N 0.000 claims description 3
- 229960000988 nystatin Drugs 0.000 claims description 3
- VQOXZBDYSJBXMA-NQTDYLQESA-N nystatin A1 Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/CC/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 VQOXZBDYSJBXMA-NQTDYLQESA-N 0.000 claims description 3
- 229960000339 pentamycin Drugs 0.000 claims description 3
- 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 3
- 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 3
- 238000001514 detection method Methods 0.000 abstract description 10
- 239000003242 anti bacterial agent Substances 0.000 abstract description 7
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- 230000002401 inhibitory effect Effects 0.000 description 10
- 241000233866 Fungi Species 0.000 description 9
- 239000001967 plate count agar Substances 0.000 description 9
- 244000005700 microbiome Species 0.000 description 7
- ABIUHPWEYMSGSR-UHFFFAOYSA-N bromocresol purple Chemical compound BrC1=C(O)C(C)=CC(C2(C3=CC=CC=C3S(=O)(=O)O2)C=2C=C(Br)C(O)=C(C)C=2)=C1 ABIUHPWEYMSGSR-UHFFFAOYSA-N 0.000 description 6
- 235000000346 sugar Nutrition 0.000 description 6
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- 240000001046 Lactobacillus acidophilus Species 0.000 description 3
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- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
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- 241000194033 Enterococcus Species 0.000 description 1
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- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
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- 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
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- CUBVWSWVTSEQON-UHFFFAOYSA-N [N-]=[N+]=[N-].[Fr+] Chemical compound [N-]=[N+]=[N-].[Fr+] CUBVWSWVTSEQON-UHFFFAOYSA-N 0.000 description 1
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- 239000007640 basal medium Substances 0.000 description 1
- UETLMBWMVIQIGU-UHFFFAOYSA-N calcium azide Chemical compound [Ca+2].[N-]=[N+]=[N-].[N-]=[N+]=[N-] UETLMBWMVIQIGU-UHFFFAOYSA-N 0.000 description 1
- AYTVLULEEPNWAX-UHFFFAOYSA-N cesium;azide Chemical compound [Cs+].[N-]=[N+]=[N-] AYTVLULEEPNWAX-UHFFFAOYSA-N 0.000 description 1
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- 229960004130 itraconazole Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- GUWHRJQTTVADPB-UHFFFAOYSA-N lithium azide Chemical compound [Li+].[N-]=[N+]=[N-] GUWHRJQTTVADPB-UHFFFAOYSA-N 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
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- 238000004519 manufacturing process Methods 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- TZLVRPLSVNESQC-UHFFFAOYSA-N potassium azide Chemical compound [K+].[N-]=[N+]=[N-] TZLVRPLSVNESQC-UHFFFAOYSA-N 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
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- GEWQYWRSUXOTOL-UHFFFAOYSA-N rubidium(1+);azide Chemical compound [Rb+].[N-]=[N+]=[N-] GEWQYWRSUXOTOL-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
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Abstract
Description
本発明は、乳酸菌以外の菌の発育を抑制または軽減させることで、乳酸菌と乳酸菌以外の菌を含む試料中から乳酸菌の選択的な検出を可能とする簡易検出培地を提供するものである。 The present invention provides a simple detection medium that enables selective detection of lactic acid bacteria from a sample containing lactic acid bacteria and bacteria other than lactic acid bacteria by suppressing or reducing the growth of bacteria other than lactic acid bacteria.
乳酸菌は、グラム陽性球菌または桿菌であり、消費したブドウ糖に対し50%以上の乳酸を産生し、カタラーゼ試験陰性・内性胞子形成をせず、運動性はまれに示すものがある細菌である（非特許文献１参照）。具体的な菌種としてはラクトバシルス属、ペディオコッカス属、テトラジェノコッカス属、キャルノバクテリウム属、ヴァゴコッカス属、ロイコノストック属、ワイセラ属、オエノコッカス属、アトポビウム属、ストレプトコッカス属、エンテロコッカス属、ラクトコッカス属細菌が含まれ、従来の乳酸菌検査ではBCP加プレートカウント寒天培地、MRS培地、APT培地、トマトジュース培地等による培養が行われている。しかしながら、これらの培地は、発酵食品の乳酸菌数検査など、乳酸菌以外の雑菌が存在しない条件下での使用が主であり、検体中に乳酸菌以外の菌を含む検体からの乳酸菌検出については、他の菌が乳酸菌の発育を阻害、または発育性状指示薬の反応を阻害することがあり、乳酸菌の検出が困難な状況に遭遇することがしばしばあった。例えば、食品衛生管理上、製造工程環境から拭き取り法などによって検体を採取し、乳酸菌を検出する場合、従来用いられている乳酸菌用培地により培養した場合には、バチルス属細菌や、酵母、グラム陰性桿菌が乳酸菌と同時に発育することにより、乳酸菌の発育が阻害されたり、培地中に含まれる乳酸菌鑑別指示薬等の反応が阻害され、結果として正確な乳酸菌の検出ができないという状況に陥る。 Lactic acid bacteria are Gram-positive cocci or gonorrhea, which produce 50% or more of lactic acid with respect to the consumed glucose, are not catalase test negative, do not form internal spores, and have motility rarely ( Non-patent document 1). Specific bacterial species include Lactobacillus genus, Pediococcus genus, Tetragenococcus genus, Carnobacterium genus, Vagococcus genus, Leuconostoc genus, Weisella genus, Onococcus genus, Atopobium genus, Streptococcus genus, Enterococcus genus, Lactococcus The bacterium belonging to the genus Coccus is included, and in the conventional lactic acid bacteria test, the culture is performed using a BCP-added plate count agar medium, an MRS medium, an APT medium, a tomato juice medium, and the like. However, these media are mainly used under conditions where there are no bacteria other than lactic acid bacteria, such as the number of lactic acid bacteria in fermented foods. For the detection of lactic acid bacteria from specimens containing bacteria other than lactic acid bacteria, In many cases, it was difficult to detect a lactic acid bacterium because the bacterium could inhibit the growth of a lactic acid bacterium or the reaction of a growth property indicator. For example, in food hygiene management, when a sample is collected from the manufacturing process environment by a wiping method to detect lactic acid bacteria, when cultured in a conventionally used medium for lactic acid bacteria, Bacillus bacteria, yeast, gram negative When the koji mold grows at the same time as the lactic acid bacterium, the growth of the lactic acid bacterium is inhibited, or the reaction of the lactic acid bacteria identification indicator contained in the medium is inhibited, resulting in a situation where the lactic acid bacterium cannot be detected accurately.
これを阻止するために、例えば、BCP加プレートカウント寒天培地にアジ化ナトリウムを10ppm添加する方法（非特許文献２参照）などが文献等で開示されているが、一部のグラム陰性菌やバチルス属細菌は発育が抑制されないため、乳酸菌と乳酸菌以外の菌を含む試料からの乳酸菌の検出方法としては最適な方法ではなかった。また、阻害薬を用いて標的微生物以外の微生物の増殖を阻害して標的微生物を検出する方法についても報告されていたが、乳酸菌のみを選択的に検出する方法については報告されていなかった（特許文献１参照）。 In order to prevent this, for example, a method of adding 10 ppm of sodium azide to a BCP-added plate count agar medium (see Non-Patent Document 2) and the like have been disclosed in the literature, but some gram-negative bacteria and Bacillus Since the growth of the genus bacteria is not suppressed, it was not an optimal method for detecting lactic acid bacteria from samples containing lactic acid bacteria and bacteria other than lactic acid bacteria. In addition, a method for detecting the target microorganism by inhibiting the growth of microorganisms other than the target microorganism using an inhibitor has been reported, but a method for selectively detecting only lactic acid bacteria has not been reported (patent) Reference 1).
本発明の目的は、乳酸菌以外の菌の発育を抑制または軽減させることで、乳酸菌と乳酸菌以外の菌を含む試料中から乳酸菌の検出を可能とする簡易検出培地を提供することである。 An object of the present invention is to provide a simple detection medium that enables detection of lactic acid bacteria from a sample containing lactic acid bacteria and bacteria other than lactic acid bacteria by suppressing or reducing the growth of bacteria other than lactic acid bacteria.
本願発明者らは、鋭意研究の結果、アルカリ金属またはアルカリ土類金属のアジ化物とポリペプチド系抗生物質を同時添加することにより、衛生管理上の乳酸菌検出試験において試料中にしばしば存在するバチルス属細菌、グラム陰性桿菌などの発育が抑制されることを見出し、さらに、ポリエン系抗生物質、シクロヘキシミド、カビサイジン、ミコナゾール、フルコナゾール、ミカファンギン等の抗真菌剤を添加することにより酵母・真菌類の発育を抑制させ、また、乳酸発酵の指標として従来用いられてきたブロムクレゾールパープルと糖を添加した培地にこれらの抗生物質・抗菌剤を添加することで乳酸菌を効果的に検出することが可能となる本発明を完成した。 As a result of diligent research, the inventors of the present invention have found that by simultaneously adding an azide of an alkali metal or an alkaline earth metal and a polypeptide antibiotic, a bacterium belonging to the genus Bacillus often present in a sample in a lactic acid bacteria detection test for hygiene management. We found that the growth of bacteria, Gram-negative bacilli, etc. was suppressed, and further, the growth of yeasts and fungi was suppressed by adding antifungal agents such as polyene antibiotics, cycloheximide, cavicidin, miconazole, fluconazole, and micafungin In addition, the present invention makes it possible to effectively detect lactic acid bacteria by adding these antibiotics and antibacterial agents to a medium supplemented with bromcresol purple and sugar, which has been conventionally used as an indicator of lactic acid fermentation. Was completed.
すなわち、本発明は、一定の濃度のアルカリ金属またはアルカリ土類金属のアジ化物とポリペプチド系抗生物質を培地中に同時に添加することで、該濃度のアルカリ金属またはアルカリ土類金属のアジ化物のみ、または、該濃度のポリペプチド系抗生物質のみでは抑制されなかった枯草菌の発育を抑制する事を可能とし、かつ、該濃度のアルカリ金属またはアルカリ土類金属のアジ化物のみでは抑制されなかった緑膿菌等のグラム陰性細菌や、該濃度のポリペプチド系抗生物質のみでは抑制されなかったセレウス菌に対してもその発育抑制を可能としたことを特徴する。 That is, the present invention can add only a certain concentration of an alkali metal or alkaline earth metal azide and a polypeptide antibiotic to the medium at the same time, so that only the alkali metal or alkaline earth metal azide at that concentration is added. Or the growth of Bacillus subtilis which was not suppressed only by the polypeptide antibiotics at the concentration, and was not suppressed only by the alkali metal or alkaline earth metal azide at the concentration It is characterized in that its growth can be suppressed even against Gram-negative bacteria such as Pseudomonas aeruginosa and Bacillus cereus that were not suppressed only by the polypeptide antibiotics at this concentration.
また、酵母・真菌類に関しては、これらの菌類は、前記濃度のアルカリ金属またはアルカリ土類金属のアジ化物のみの添加では十分な抑制効果が得られない為に、ポリエン系抗生物質、シクロヘキシミド、カビサイジン、ミコナゾール、フルコナゾール、ミカファンギン等の抗真菌剤を添加することにより、これらの菌類の発育抑制を可能とし、更に乳酸菌以外の微生物に対して十分な抑制を可能とした。 In addition, regarding yeasts and fungi, these fungi cannot be sufficiently inhibited by adding only the alkali metal or alkaline earth metal azide at the above-mentioned concentration, so that polyene antibiotics, cycloheximide, and cavicidin By adding antifungal agents such as miconazole, fluconazole, and micafungin, it was possible to suppress the growth of these fungi and to sufficiently suppress microorganisms other than lactic acid bacteria.
本発明は、これら、ポリペプチド系抗生物質、アルカリ金属またはアルカリ土類金属のアジ化物、抗真菌剤を添加することで相乗的および特異的に乳酸菌以外の雑菌の発育を抑制する事を可能とする。本発明は更に、発酵を検出するためのブロムクレゾールパープルと糖を添加した培地を提供する。 The present invention makes it possible to synergistically and specifically suppress the growth of various bacteria other than lactic acid bacteria by adding these polypeptide antibiotics, alkali metal or alkaline earth metal azides, and antifungal agents. To do. The present invention further provides a medium supplemented with bromcresol purple and sugar for detecting fermentation.
本発明の態様は、以下のとおりである。
[１] ポリペプチド系抗生物質およびアルカリ金属もしくはアルカリ土類金属のアジ化物を含む、乳酸菌を選択的に培養するための培地。
[２] さらに、抗真菌剤を含む、[１]の培地、
[３] アルカリ金属またはアルカリ土類金属のアジ化物がアジ化ナトリウムである[１]または[２]の培地、
[４] ポリペプチド系抗生物質がポリミキシンB、コリスチンおよびバシトラシンからなる群から選択される少なくとも１種類である[１]から[３]のいずれかの培地、
[５] 抗真菌剤がポリエン系抗生物質、シクロヘキシミド、カビサイジン、ミコナゾール、フルコナゾールおよびミカファンギンからなる群から選択される少なくとも１種類である[２]から[４]のいずれかの培地、
[６] ポリエン系抗生物質がアムホテリシンB、ナイスタチン、ピマリシン、トリコマイシン、グリセオフルビン、ペンタマイシン、カンディシジン、ハマイシンおよびクロミンからなる群から選択される少なくとも１種類である[５]の培地、
[７] アルカリ金属もしくはアルカリ土類金属のアジ化物ならびにポリペプチド系抗生物質を両者を混合することによりバチルス属細菌およびグラム陰性菌の発育を阻止するが、乳酸菌の発育を阻止しない濃度で含む、[１]から[６]のいずれかの培地、
[８] アルカリ金属もしくはアルカリ土類金属のアジ化物ならびにポリペプチド系抗生物質を単独では大腸菌、サルモネラ菌、緑膿菌、セレウス菌、枯草菌の一部の菌の発育を阻止しないが、両者を混合することにより大腸菌、サルモネラ菌、緑膿菌、セレウス菌、枯草菌の発育を阻止し、なおかつ乳酸菌の発育を阻止しない濃度で含む、[７]の培地、
[９] さらに、抗真菌剤を酵母の発育を阻止する濃度で含む、[７]または[８]の培地、
[１０] アルカリ金属またはアルカリ土類金属のアジ化物の濃度が５〜200μg/mLであり、ポリペプチド系抗生物質の濃度が１〜100μg/mLである[１]から[９]のいずれかの培地、
[１１] アルカリ金属またはアルカリ土類金属のアジ化物の濃度が50〜100μg/mLであり、ポリペプチド系抗生物質の濃度が１〜16μg/mLである[１０]の培地、
[１２] 抗真菌剤の濃度が１〜200μg/mLである[２]から[１１]のいずれかの培地、
[１３] 抗真菌剤の濃度が２〜80μg/mlである[１２]の培地、
[１４] ５〜200μg/mLのアジ化ナトリウムおよび１〜100μg/mLのポリミキシンBを含む[１]から[１０]のいずれかの培地、
[１５] さらに、１〜200μg/mLの抗真菌剤を含む[１４]の培地、
[１６] 培地のpHが6.0〜7.5である[１]から[１５]のいずれかの培地、
[１７] さらに、少なくともブドウ糖ならびにブロモクレゾールパープルもしくは炭酸カルシウム粉末を含む培地であって、特異的に培養した乳酸菌による乳酸発酵を検出することにより乳酸菌の存在を検出し得る[１]から[１６]のいずれかの培地、
[１８] [１]から[１７]のいずれかの培地を用いて、乳酸菌を選択的に培養する方法、ならびに
[１９] [１７]の培地を用いて、乳酸菌を選択的に培養し、乳酸菌の存在を検出する方法。
Aspects of the present invention are as follows.
[1] A medium for selectively culturing lactic acid bacteria, comprising a polypeptide antibiotic and an azide of an alkali metal or alkaline earth metal.
[2] The medium according to [1], further comprising an antifungal agent,
[3] The medium according to [1] or [2], wherein the alkali metal or alkaline earth metal azide is sodium azide,
[4] The medium according to any one of [1] to [3], wherein the polypeptide antibiotic is at least one selected from the group consisting of polymyxin B, colistin and bacitracin,
[5] The medium according to any one of [2] to [4], wherein the antifungal agent is at least one selected from the group consisting of polyene antibiotics, cycloheximide, cavicidin, miconazole, fluconazole, and Micafungin,
[6] The medium according to [5], wherein the polyene antibiotic is at least one selected from the group consisting of amphotericin B, nystatin, pimaricin, tricomycin, griseofulvin, pentamycin, candicidin, hamycin and chromin.
[7] Alkaline metal or alkaline earth metal azide and polypeptide antibiotics are mixed to inhibit the growth of Bacillus and Gram-negative bacteria, but at a concentration that does not prevent the growth of lactic acid bacteria. The medium of any one of [1] to [6],
[8] Alkali metal or alkaline earth metal azide and polypeptide antibiotics alone do not prevent the growth of some Escherichia coli, Salmonella, Pseudomonas aeruginosa, Bacillus cereus, Bacillus subtilis, but a mixture of both The medium according to [7], containing a concentration that inhibits the growth of Escherichia coli, Salmonella, Pseudomonas aeruginosa, Bacillus cereus, Bacillus subtilis and does not inhibit the growth of lactic acid bacteria,
[9] The medium according to [7] or [8], further comprising an antifungal agent at a concentration that inhibits yeast growth,
[10] Any one of [1] to [9], wherein the concentration of alkali metal or alkaline earth metal azide is 5 to 200 μg / mL and the concentration of polypeptide antibiotic is 1 to 100 μg / mL Culture medium,
[11] The medium according to [10], wherein the alkali metal or alkaline earth metal azide concentration is 50 to 100 μg / mL, and the polypeptide antibiotic concentration is 1 to 16 μg / mL,
[12] The medium according to any one of [2] to [11], wherein the concentration of the antifungal agent is 1 to 200 μg / mL,
[13] The medium according to [12], wherein the concentration of the antifungal agent is 2 to 80 μg / ml,
[14] The medium according to any one of [1] to [10], containing 5 to 200 μg / mL sodium azide and 1 to 100 μg / mL polymyxin B;
[15] The medium according to [14], further comprising 1 to 200 μg / mL of an antifungal agent,
[16] The medium according to any one of [1] to [15], wherein the pH of the medium is 6.0 to 7.5,
[17] Furthermore, it is a medium containing at least glucose and bromocresol purple or calcium carbonate powder, and the presence of lactic acid bacteria can be detected by detecting lactic acid fermentation by specifically cultured lactic acid bacteria [1] to [16] Any medium,
[18] A method for selectively cultivating lactic acid bacteria using the medium according to any one of [1] to [17], and
[19] A method for detecting the presence of lactic acid bacteria by selectively culturing lactic acid bacteria using the medium of [17].
本発明の方法により、バチルス属細菌、酵母・真菌類、グラム陰性細菌の発育を抑制し、環境中から乳酸菌を効果的に検出する培地・方法を確立することができた。 By the method of the present invention, the growth of Bacillus bacteria, yeasts / fungi, and Gram-negative bacteria was suppressed, and a medium / method for effectively detecting lactic acid bacteria from the environment could be established.
本発明の乳酸菌と乳酸菌以外の菌を含む試料中から乳酸菌の検出を可能とする培地は、アルカリ金属またはアルカリ土類金属のアジ化物とポリペプチド系抗生物質、抗真菌剤を含むことを特徴とする。また、本発明の方法は、アルカリ金属またはアルカリ土類金属のアジ化物とポリペプチド系抗生物質、抗真菌剤を、糖の発酵を検出することのできる培地に添加することを特徴とする。 A medium capable of detecting lactic acid bacteria from a sample containing lactic acid bacteria and bacteria other than lactic acid bacteria according to the present invention comprises an azide of an alkali metal or alkaline earth metal, a polypeptide antibiotic, and an antifungal agent. To do. The method of the present invention is characterized by adding an azide of an alkali metal or alkaline earth metal, a polypeptide antibiotic, or an antifungal agent to a medium capable of detecting sugar fermentation.
本発明の培地の一形態として、一定濃度のアルカリ金属またはアルカリ土類金属のアジ化物とポリペプチド系抗生物質を含む培地が挙げられ、該培地を用いることにより、セレウス菌や枯草菌を含むバチルス属細菌、グラム陰性桿菌等の細菌の発育を阻止することができる。また、一定濃度の抗真菌剤をさらに含む培地が挙げられ、該培地を用いることにより、酵母、真菌の発育をも阻止することができる。 One embodiment of the medium of the present invention includes a medium containing a constant concentration of an alkali metal or alkaline earth metal azide and a polypeptide antibiotic, and by using this medium, Bacillus containing Bacillus cereus and Bacillus subtilis. The growth of bacteria such as genus bacteria and gram-negative bacilli can be prevented. Moreover, the culture medium which further contains an antifungal agent of a fixed density | concentration is mentioned, By using this culture medium, growth of yeast and fungi can also be inhibited.
本発明において、乳酸菌以外の細菌の発育を阻止するとは、他の菌が増殖することによって起こり得る、乳酸菌の発育の抑制や発育性状指示薬の反応の阻害が起きない程度に乳酸菌以外の細菌の発育を阻止することをいう。従って、乳酸菌以外の細菌の発育を阻止するとは、該細菌を完全に殺菌することをいう場合もあれば、細菌が生存しているが増殖せず、培養開始時と培養終了時の細菌の個数が変化しないように処理することをいう場合もある。ここで、乳酸菌の発育性状指示薬とは、例えば、乳酸発酵により低下した培地のpHを測定し得るpH指示薬をいう。 In the present invention, the prevention of the growth of bacteria other than lactic acid bacteria means that the growth of bacteria other than lactic acid bacteria can be caused by the growth of other bacteria to the extent that inhibition of the growth of lactic acid bacteria and inhibition of the reaction of the growth property indicator do not occur. It means to prevent. Therefore, preventing the growth of bacteria other than lactic acid bacteria may refer to the complete sterilization of the bacteria, or the bacteria are alive but not proliferating, and the number of bacteria at the start and end of the culture. In some cases, processing is performed so as not to change. Here, the growth property indicator of lactic acid bacteria refers to, for example, a pH indicator capable of measuring the pH of a medium lowered by lactic acid fermentation.
アルカリ金属またはアルカリ土類金属のアジ化物は、高濃度では乳酸菌の発育を阻止するが、乳酸菌の発育を阻止しない濃度範囲で、バチルス属細菌、グラム陰性菌や酵母の発育を阻止する。ポリペプチド系抗生物質は高濃度では乳酸菌の発育を阻止するが、乳酸菌の発育を阻止しない濃度範囲で、グラム陰性菌の発育を阻止する。単独では一部のバチルス属細菌およびグラム陰性細菌の発育を阻止しない濃度のアルカリ金属もしくはアルカリ土類金属のアジ化物およびポリペプチド系抗生物質を混合して用いた場合、単独では発育が阻止されないバチルス属細菌およびグラム陰性細菌の発育を広く阻止するようになる。一方、バチルス属細菌およびグラム陰性細菌の発育を広く阻止する濃度で両者を混合して用いても、乳酸菌の発育を阻止しない場合がある。本発明においては、このようにバチルス属細菌およびグラム陰性細菌の発育を広く阻止するが、乳酸菌の発育を阻止しないような濃度でアルカリ金属もしくはアルカリ土類金属のアジ化物およびポリペプチド系抗生物質を混合して用いればよい。すなわち、本発明の培地は、アルカリ金属またはアルカリ土類金属のアジ化物とポリペプチド系抗生物質が相乗的に作用して、バチルス属細菌およびグラム陰性細菌の発育を広く阻止する。さらに、本発明においては、酵母、真菌類の発育を阻止するために、抗真菌剤が用いられる。抗真菌剤も酵母、真菌類の発育を阻止するが乳酸菌の発育を阻止しない濃度範囲で用いられる。 Alkali metal or alkaline earth metal azide inhibits the growth of lactic acid bacteria at high concentrations, but inhibits the growth of Bacillus bacteria, Gram-negative bacteria and yeasts in a concentration range that does not inhibit the growth of lactic acid bacteria. Polypeptide antibiotics inhibit the growth of lactic acid bacteria at high concentrations, but inhibit the growth of gram-negative bacteria in a concentration range that does not inhibit the growth of lactic acid bacteria. Bacillus that does not inhibit growth when used alone in combination with alkali metal or alkaline earth metal azides and polypeptide antibiotics that do not inhibit the growth of some Bacillus and Gram-negative bacteria. It broadly inhibits the growth of genus bacteria and gram-negative bacteria. On the other hand, there is a case where the growth of lactic acid bacteria is not inhibited even when both are mixed and used at a concentration that widely inhibits the growth of Bacillus bacteria and Gram-negative bacteria. In the present invention, an alkali metal or alkaline earth metal azide and a polypeptide antibiotic are used in such concentrations that broadly inhibit the growth of Bacillus bacteria and Gram-negative bacteria but do not inhibit the growth of lactic acid bacteria. What is necessary is just to mix and use. That is, in the culture medium of the present invention, alkali metal or alkaline earth metal azide and polypeptide antibiotics act synergistically to broadly inhibit the growth of Bacillus and Gram-negative bacteria. Furthermore, in the present invention, an antifungal agent is used to inhibit the growth of yeast and fungi. Antifungal agents are also used in a concentration range that inhibits the growth of yeast and fungi but does not inhibit the growth of lactic acid bacteria.
アルカリ金属のアジ化物として、アジ化リチウム、アジ化ナトリウム、アジ化カリウム、アジ化ルビジウム、アジ化セシウム、アジ化フランシウム等が挙げられ、アルカリ土類金属のアジ化物として、アジ化カルシウム、アジ化バリウム等が挙げられる。ポリペプチド系抗生物質として、ポリミキシンB、コリスチン、バシトラシン等が挙げられ、これらの抗生物質の１つ以上を添加すればよい。抗真菌剤として、ポリエン系抗生物質、アゾール系抗真菌剤、キャンディン系抗真菌剤、シクロヘキシミド、カビサイジン等が挙げられ、これらの中から１つ以上を添加すればよい。ポリエン系抗生物質としてアンフォテリシンB、ナイスタチン、ピマリシン、トリコマイシン、グリセオフルビン、ペンタマイシン、カンディシジン、ハマイシン、クロミン等が、アゾール系抗真菌剤としてミコナゾール、フルコナゾール、イトラコナゾール等が、キャンディン系抗真菌剤としてミカファンギン等が挙げられる。また、カビサイジンは、Streptomyces gougerotiiに属する菌培養液から抽出した抗生物質であるが、その他の微生物由来の真菌類に対する抗生物質も用いることができる。 Examples of alkali metal azides include lithium azide, sodium azide, potassium azide, rubidium azide, cesium azide, and francium azide. Alkaline earth metal azides include calcium azide and azide. Barium etc. are mentioned. Polymyxin B, colistin, bacitracin and the like can be mentioned as polypeptide antibiotics, and one or more of these antibiotics may be added. Examples of antifungal agents include polyene antibiotics, azole antifungal agents, candin antifungal agents, cycloheximide, and cavicidin. One or more of these may be added. Amphotericin B, nystatin, pimaricin, tricomycin, griseofulvin, pentamycin, candicidin, hamycin, chromin etc. as polyene antibiotics, miconazole, fluconazole, itraconazole etc. as azole antifungal agents, Micafungin etc. as candin antifungal agents, etc. Is mentioned. In addition, moldycin is an antibiotic extracted from a bacterial culture solution belonging to Streptomyces gougerotii , but antibiotics against fungi derived from other microorganisms can also be used.
それぞれの培地に添加する濃度は、乳酸菌以外の発育を阻止するが乳酸菌の発育を阻止しない濃度であり、微生物を用いた発育阻止試験を行うことにより容易に決定することができる。例えば、乳酸菌以外の発育を阻止しようとする細菌に対する最小発育阻止濃度（MIC）以上の濃度で用いればよい。また、乳酸菌の発育が阻止されない濃度である限り、乳酸菌以外の細菌を死滅させる濃度である最小殺菌濃度（MFC）以上の濃度で用いてもよい。例えば、アルカリ金属またはアルカリ土類金属のアジ化物が５〜200μg/mL、好ましくは50〜100μg/mL、ポリペプチド系抗生物質が１〜100μg/mL、さらに好ましくは１〜50μg/mL、さらに好ましくは１〜20μg/mL、さらに好ましくは１〜16μg/mL、抗真菌剤が１〜200μg/mL、好ましくは１〜100μg/mL、さらに好ましくは２〜80μg/mL、さらに好ましくは２〜40μg/mL、さらに好ましくは２〜20μg/mLである。ポリペプチド系抗生物質や抗真菌剤の有効濃度は、種類や組合わせによって異なってくることがある。それぞれの種類における有効濃度、組合わせにおける有効濃度は、乳酸菌と他の菌を混合培養する際にポリペプチド系抗生物質および抗真菌剤を添加し培養して、乳酸菌以外の発育を阻止するが乳酸菌の発育を阻止しない濃度を決定することができる。用いるポリペプチド系抗生物質の種類や他の薬剤との組合わせによっては、ポリペプチド系抗生物質の濃度は、１〜８μg/mL、好ましくは１〜４μg/ｍL、さらに好ましくは１〜２μg/mLあるいは２〜４μg/mLであり、用いる抗真菌剤の種類や他の薬剤との組合わせによっては、抗真菌剤の濃度は、１〜８μg/mL、好ましくは１〜４μg/ｍL、さらに好ましくは１〜２μg/mLあるいは２〜４μg/mLである。ポリペプチド系抗生物質を高濃度で、例えば４μg/mL以上添加した場合、乳酸菌の発育が若干弱くなることがあるが、発育が阻止されない限り、乳酸菌の検出は可能であり、ポリペプチド系抗生物質は、例え、乳酸菌の増殖に影響を与えたとしても乳酸菌の検出が可能な濃度範囲で用いることができる。 The concentration added to each medium is a concentration that inhibits the growth of other than lactic acid bacteria but does not inhibit the growth of lactic acid bacteria, and can be easily determined by performing a growth inhibition test using microorganisms. For example, it may be used at a concentration equal to or higher than the minimum growth inhibitory concentration (MIC) for bacteria other than lactic acid bacteria that are trying to inhibit growth. Moreover, as long as the concentration is such that the growth of lactic acid bacteria is not inhibited, it may be used at a concentration equal to or higher than the minimum bactericidal concentration (MFC), which is a concentration that kills bacteria other than lactic acid bacteria. For example, alkali metal or alkaline earth metal azide is 5 to 200 μg / mL, preferably 50 to 100 μg / mL, polypeptide antibiotic is 1 to 100 μg / mL, more preferably 1 to 50 μg / mL, more preferably 1-20 μg / mL, more preferably 1-16 μg / mL, antifungal agent 1-200 μg / mL, preferably 1-100 μg / mL, more preferably 2-80 μg / mL, more preferably 2-40 μg / mL mL, more preferably 2 to 20 μg / mL. Effective concentrations of polypeptide antibiotics and antifungal agents may vary depending on the type and combination. The effective concentration in each type and the effective concentration in the combination is that when lactic acid bacteria and other bacteria are mixed and cultured, polypeptide antibiotics and antifungal agents are added and cultured to prevent growth other than lactic acid bacteria. The concentration that does not prevent the growth of can be determined. Depending on the type of polypeptide antibiotic used and the combination with other drugs, the concentration of polypeptide antibiotic is 1 to 8 μg / mL, preferably 1 to 4 μg / mL, more preferably 1 to 2 μg / mL. Alternatively, it is 2 to 4 μg / mL, and depending on the type of antifungal agent used and the combination with other drugs, the concentration of the antifungal agent is 1 to 8 μg / mL, preferably 1 to 4 μg / mL, more preferably It is 1-2 μg / mL or 2-4 μg / mL. When a polypeptide antibiotic is added at a high concentration, for example, 4 μg / mL or more, the growth of lactic acid bacteria may be slightly weakened. However, as long as the growth is not blocked, detection of lactic acid bacteria is possible. Can be used in a concentration range in which lactic acid bacteria can be detected even if they affect the growth of lactic acid bacteria.
培地としては、BCP加プレートカウント寒天培地、MRS培地、APT培地、トマトジュース培地、GYP白亜寒天培地等の乳酸菌検査に用いられる培地を用いることができ、これらの培地に上記のアルカリ金属またはアルカリ土類金属のアジ化物とポリペプチド系抗生物質、抗真菌剤等を添加すればよい。また、pHは、6.0〜7.5、好ましくは6.5〜6.9である。 As the medium, a medium used for lactic acid bacteria testing such as BCP-added plate count agar medium, MRS medium, APT medium, tomato juice medium, GYP chalk agar medium, etc. can be used. Metal azides, polypeptide antibiotics, antifungal agents and the like may be added. Moreover, pH is 6.0-7.5, Preferably it is 6.5-6.9.
好ましくは、50〜100μg/mLアジ化ナトリウム、１〜100μg/mLポリミキシンB、１〜200μg/mL抗真菌剤、あるいは50〜100μg/mLアジ化ナトリウム、１〜16μg/mLポリミキシンB、２〜20μg/mL抗真菌剤をBCP加プレートカウント寒天培地に添加し、pHを6.7±0.2に調製した形態である。 Preferably, 50-100 μg / mL sodium azide, 1-100 μg / mL polymyxin B, 1-200 μg / mL antifungal agent, or 50-100 μg / mL sodium azide, 1-16 μg / mL polymyxin B, 2-20 μg / mL antifungal agent was added to BCP-added plate count agar medium, and the pH was adjusted to 6.7 ± 0.2.
尚、培地pHの測定は、半導体センサpHメーター（例えば、新電元社 pHBOY-P2）を用い、培地が固化したのちに、細分化した培地片をセンサ部に圧着させることで計測する。 The medium pH is measured by using a semiconductor sensor pH meter (for example, Shindengen pHBOY-P2), and after the medium is solidified, the fragmented medium piece is pressure-bonded to the sensor unit.
本発明の培地は、バチルス属細菌に対する発育阻止濃度範囲と乳酸菌に対する発育支持濃度範囲とが互いに重複し、かつ、一部のバチルス属細菌・酵母に対する発育阻止濃度範囲と乳酸菌に対する発育阻止濃度範囲とが一部重複するアルカリ金属またはアルカリ土類金属のアジ化物と、グラム陰性細菌に対する発育阻止濃度範囲と乳酸菌に対する発育支持濃度範囲とが互いに重複し、かつ、一部のバチルス属細菌に対する発育阻止濃度範囲と乳酸菌に対する発育阻止濃度範囲とが一部重複するポリペプチド系抗生物質と、酵母に対する発育阻止濃度範囲と乳酸菌に対する発育支持濃度範囲とが互いに重複する抗真菌剤とを、酵母、バチルス属細菌、グラム陰性細菌の発育を実質的に阻止するが、乳酸菌の発育を支持するような関係力価濃度を以って含有してなる乳酸菌簡易検出用培地である。 In the medium of the present invention, the growth inhibitory concentration range for Bacillus bacteria and the growth support concentration range for lactic acid bacteria overlap each other, and the growth inhibitory concentration range for some Bacillus bacteria and yeast and the growth inhibitory concentration range for lactic acid bacteria Alkaline metal or alkaline earth metal azide partially overlapping, growth inhibitory concentration range for Gram-negative bacteria and growth support concentration range for lactic acid bacteria overlap, and growth inhibitory concentrations for some Bacillus bacteria A polypeptide antibiotic having a partial overlap of the range and the growth inhibitory concentration range for lactic acid bacteria, and an antifungal agent having a growth inhibitory concentration range for yeast and the growth support concentration range for lactic acid bacteria overlapping each other. , Which effectively inhibits the growth of gram-negative bacteria, but has a relevant titer concentration that supports the growth of lactic acid bacteria. Is a lactic acid bacterium simple detection medium, which comprises me.
さらに、本発明の培地は、乳酸菌の存在を示す乳酸発酵を検出する試薬を含んでいてもよい。このような試薬として、ブドウ糖、ショ糖、乳糖、マンニット、果糖等の糖、ブロムクレゾールパープル等のpH指示薬が挙げられ、糖は0.1〜40g/mL、好ましくは１g/mLで、ブロムクレゾールパープルは0.002〜１g/Lの濃度で添加すればよい。ブロムクレゾールパープルの代わりに、乳酸菌の発酵により産生された酸によるpHの低下を測定し得る他のpH指示薬を用いることもできる。ブロムクレゾールパープルを含む培地としては、BCP加プレートカウント寒天培地が挙げられる。また、炭酸カルシウムの粉末を加えた培地も用いることができる。このような培地としては、例えば、GYP白亜寒天培地が挙げられる。 Furthermore, the culture medium of the present invention may contain a reagent for detecting lactic acid fermentation indicating the presence of lactic acid bacteria. Examples of such reagents include glucose, sucrose, lactose, mannitol, fructose and other sugars, and pH indicators such as bromocresol purple. The sugar is 0.1 to 40 g / mL, preferably 1 g / mL, and bromocresol purple. May be added at a concentration of 0.002 to 1 g / L. Instead of bromcresol purple, other pH indicators that can measure the decrease in pH due to the acid produced by fermentation of lactic acid bacteria can also be used. Examples of the medium containing bromcresol purple include BCP-added plate count agar medium. A medium to which calcium carbonate powder is added can also be used. An example of such a medium is GYP chalk agar medium.
本発明の培地に、乳酸菌が存在し得る試料を添加し、37℃で48〜72時間培養することにより、乳酸菌以外の微生物は発育できないので、死滅する。培地に糖及びブロムクレゾールパープルを添加しておいた場合、発酵により産生される酸により培地のpHが下がりpH指示薬であるブロムクレゾールパープルの色が紫から黄色に変色することにより、乳酸菌の存在を検出することができる。また、培地に炭酸カルシウムの粉末を加えた場合、不溶性の炭酸カルシウムの白濁が乳酸菌のコロニーの周りでは、生成した乳酸によって溶かされ、きれいなクリヤー（透明）ゾーンを生じるので、乳酸菌の増殖を検出することができる。 By adding a sample in which lactic acid bacteria can be present to the medium of the present invention and culturing at 37 ° C. for 48 to 72 hours, microorganisms other than lactic acid bacteria cannot grow and die. When sugar and bromcresol purple are added to the medium, the pH of the medium is lowered by the acid produced by fermentation, and the color of bromcresol purple, which is a pH indicator, changes from purple to yellow. Can be detected. In addition, when calcium carbonate powder is added to the medium, the insoluble calcium carbonate cloudiness is dissolved by the lactic acid produced around the colonies of lactic acid bacteria, resulting in a clear (clear) zone. be able to.
以下、実施例を挙げて更に詳細に説明するが、本発明の本質はアジ化物と抗生物質の相乗効果を利用した乳酸菌検出培地であり、以下の内容は単に本発明を実施するための最良の組成、添加量であり、本発明は以下の実施例だけに限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples, but the essence of the present invention is a lactic acid bacteria detection medium utilizing the synergistic effect of azide and antibiotics, and the following content is simply the best for practicing the present invention. It is a composition and an addition amount, and the present invention is not limited only to the following examples.
〔実施例１〕
アジ化ナトリウムのみを培地に添加した場合、ポリミキシンBのみを培地に添加した場合、双方を同時に添加した場合の菌の発育を比較した。従来乳酸菌の菌数測定で用いられているBCP加プレートカウント寒天培地に、アジ化ナトリウムとポリミキシンBを添加し、細菌の発育を観察した。
[Example 1]
When only sodium azide was added to the medium, polymyxin B alone was added to the medium, and the growth of the bacteria when both were added simultaneously was compared. Sodium azide and polymyxin B were added to a BCP-added plate count agar medium conventionally used for counting the number of lactic acid bacteria, and bacterial growth was observed.
ポリミキシンBのみを添加した場合には枯草菌の発育を抑制するためには４μg/mLの添加量が必要である。また、４μg/mLのポリミキシンBを添加した場合には、乳酸菌ds-01株の発育にも影響がでる。 When only polymyxin B is added, an addition amount of 4 μg / mL is necessary to suppress the growth of Bacillus subtilis. In addition, the addition of 4 μg / mL polymyxin B also affects the growth of the lactic acid bacteria ds-01 strain.
アジ化ナトリウムのみを添加した場合には枯草菌の発育を抑制するためには200μg/mLの添加量が必要である。また、200μg/mLのアジ化ナトリウムを添加した場合には、乳酸菌の発育にも影響がでる。 When only sodium azide is added, an addition amount of 200 μg / mL is necessary to suppress the growth of Bacillus subtilis. In addition, the addition of 200 μg / mL sodium azide also affects the growth of lactic acid bacteria.
アジ化ナトリウムとポリミキシンBを組み合わせることで乳酸菌への影響がない状態で、枯草菌の発育を抑制することが可能となった。また、アジ化ナトリウム、ポリミキシンBそれぞれ単独では抑制されなかったセレウス菌やその他のグラム陰性桿菌についても完全に抑制することが可能となった。 By combining sodium azide and polymyxin B, it was possible to suppress the growth of Bacillus subtilis without affecting lactic acid bacteria. In addition, it became possible to completely suppress Bacillus cereus and other Gram-negative rods that were not suppressed by sodium azide and polymyxin B alone.
〔実施例２〕
従来乳酸菌の菌数測定で用いられているBCP加プレートカウント寒天培地に、アジ化ナトリウムとポリミキシンB、アムホテリシンBを添加し、細菌の発育を観察した。
[Example 2]
Sodium azide, polymyxin B, and amphotericin B were added to a BCP-added plate count agar medium that has been used in the conventional count of lactic acid bacteria, and bacterial growth was observed.
50μg/mLアジ化ナトリウムと２μg/mLポリミキシンB、４μg/mLアムホテリシンBを添加した培地に、ラクトバシルス・カゼイ（乳酸菌１）、ラクトバシルス・アシドフィルス（乳酸菌２）、大腸菌、サルモネラ、緑膿菌、バシルス・セレウス、枯草菌、酵母をそれぞれ1×106cfu/mLに希釈した菌液を1μLずつ接種し、37℃、72時間培養した場合の発育状況を比較した。 Lactobacillus casei (Lactic acid bacteria 1), Lactobacillus acidophilus (Lactic acid bacteria 2), E. coli, Salmonella, Pseudomonas aeruginosa, Bacillus, Bacillus, to a medium supplemented with 50 μg / mL sodium azide and 2 μg / mL polymyxin B Growth conditions were compared when inoculating 1 μL each of B. subtilis, Bacillus subtilis, and yeast diluted to 1 × 10 6 cfu / mL and culturing at 37 ° C. for 72 hours.
乳酸菌以外の菌については完全に発育を抑制し、乳酸菌のみが発育し、培地を黄変させた。
また、実施例１にて発育が抑制されていなかった酵母についても、アムホテリシンBを添加したことによりその発育が抑制された。
For bacteria other than lactic acid bacteria, growth was completely suppressed, only lactic acid bacteria were grown, and the medium was yellowed.
Moreover, also about the yeast whose growth was not suppressed in Example 1, the growth was suppressed by adding amphotericin B.
〔実施例３〕
従来乳酸菌の菌数測定で用いられているBCP加プレートカウント寒天培地に
アジ化ナトリウム（NaN3）：50μg/mL
ポリミキシンB ：4μg/mL
を添加し、さらに、各濃度の抗真菌剤（フルコナゾール）を添加し、各種細菌の生育を比較した。
Example 3
Sodium azide (NaN 3 ): 50 μg / mL in a BCP-added plate count agar medium that has been used in the conventional count of lactic acid bacteria
Polymyxin B: 4μg / mL
Furthermore, antifungal agent (fluconazole) of each concentration was added, and the growth of various bacteria was compared.
ラクトバシルス・カゼイ（乳酸菌1）、ラクトバシルス・アシドフィルス（乳酸菌２）、大腸菌、サルモネラ、緑膿菌、バチルス・セレウス、枯草菌、酵母1、酵母2をそれぞれ1×106cfu/mLに希釈した菌液を1μLずつ接種し、37℃、72時間培養した場合の発育状況を比較した。 Lactobacillus casei (lactic acid bacteria 1), Lactobacillus acidophilus (lactic acid bacteria 2), Escherichia coli, Salmonella, Pseudomonas aeruginosa, Bacillus cereus, Bacillus subtilis, yeast 1 and yeast 2 diluted to 1 x 10 6 cfu / mL 1 μL each was inoculated and the growth status was compared when cultured at 37 ° C. for 72 hours.
表３に示すように、フルコナゾール濃度が10〜80μg/mLの範囲で乳酸菌以外の菌の増殖を抑制しつつ、乳酸菌を培養することが可能であった。特にフルコナゾール濃度が20μg/mL以上では、乳酸菌以外の菌の増殖は完全に抑えられた。 As shown in Table 3, it was possible to culture lactic acid bacteria while suppressing the growth of bacteria other than lactic acid bacteria when the fluconazole concentration was in the range of 10 to 80 μg / mL. In particular, when the fluconazole concentration was 20 μg / mL or more, the growth of bacteria other than lactic acid bacteria was completely suppressed.
〔実施例４〕
基礎培地：自社調製（BCP加プレートカウント寒天培地組成からL-システインを抜いた）培地に、
アジ化ナトリウム（NaN3） ：50μg/mL
アムホテリシンB（AMPH-B） ：４μg/mL
を添加し、さらに、各濃度の抗真菌剤（ポリミキシンB）を添加し、各種細菌の生育を比較した。
Example 4
Basal medium: In-house prepared (excluding L-cysteine from BCP-added plate count agar composition)
Sodium azide (NaN3): 50μg / mL
Amphotericin B (AMPH-B): 4μg / mL
Was added, and each concentration of antifungal agent (polymyxin B) was added, and the growth of various bacteria was compared.
ラクトバシルス・カゼイ（乳酸菌1）、ラクトバシルス・アシドフィルス（乳酸菌２）、大腸菌、サルモネラ、緑膿菌、バチルス・セレウス、枯草菌、酵母1、酵母2をそれぞれ1×106cfu/mLに希釈した菌液を1μLずつ接種し、37℃、72時間培養した場合の発育状況を比較した。 Lactobacillus casei (lactic acid bacteria 1), Lactobacillus acidophilus (lactic acid bacteria 2), Escherichia coli, Salmonella, Pseudomonas aeruginosa, Bacillus cereus, Bacillus subtilis, yeast 1 and yeast 2 diluted to 1 x 10 6 cfu / mL 1 μL each was inoculated and the growth status was compared when cultured at 37 ° C. for 72 hours.
表４に示すように、ポリミキシンＢ濃度が16μg/mLで乳酸菌以外の菌の増殖を完全に抑制しつつ、乳酸菌を培養することが可能であった。すなわち、ポリミキシンBの濃度が16μg/mLで本発明が実施可能であった。 As shown in Table 4, it was possible to culture lactic acid bacteria while completely inhibiting the growth of bacteria other than lactic acid bacteria at a polymyxin B concentration of 16 μg / mL. That is, the present invention could be practiced with a polymyxin B concentration of 16 μg / mL.
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| JP2008131897A (en) * | 2006-11-28 | 2008-06-12 | Nissui Pharm Co Ltd | Culture medium for detecting enterococcus |
| CN104483363A (en) * | 2015-01-05 | 2015-04-01 | 天津商业大学 | Method for manufacturing electrochemical biosensor for measuring antibiotic |
| CN108641969A (en) * | 2018-06-21 | 2018-10-12 | 赤峰制药股份有限公司 | A kind of griseofulvin production bacterium culture medium |
| WO2021005813A1 (en) * | 2019-07-05 | 2021-01-14 | Jnc株式会社 | Culture medium for selectively separating lactic acid bacterium |
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| WO1996015435A2 (en) * | 1994-11-04 | 1996-05-23 | Idexx Laboratories, Inc. | Medium for detecting enterococci in a sample |
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| JP2008131897A (en) * | 2006-11-28 | 2008-06-12 | Nissui Pharm Co Ltd | Culture medium for detecting enterococcus |
| CN104483363A (en) * | 2015-01-05 | 2015-04-01 | 天津商业大学 | Method for manufacturing electrochemical biosensor for measuring antibiotic |
| CN108641969A (en) * | 2018-06-21 | 2018-10-12 | 赤峰制药股份有限公司 | A kind of griseofulvin production bacterium culture medium |
| WO2021005813A1 (en) * | 2019-07-05 | 2021-01-14 | Jnc株式会社 | Culture medium for selectively separating lactic acid bacterium |
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