MD1106Z - Nutrient medium for cultivation of Bacillus subtilis CNMN-BB-09 strain - Google Patents
Nutrient medium for cultivation of Bacillus subtilis CNMN-BB-09 strain Download PDFInfo
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- MD1106Z MD1106Z MDS20160087A MDS20160087A MD1106Z MD 1106 Z MD1106 Z MD 1106Z MD S20160087 A MDS20160087 A MD S20160087A MD S20160087 A MDS20160087 A MD S20160087A MD 1106 Z MD1106 Z MD 1106Z
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- nutrient medium
- bacillus subtilis
- cnmn
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- strain
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- 235000015097 nutrients Nutrition 0.000 title claims abstract description 16
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 14
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 15
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 7
- 239000001509 sodium citrate Substances 0.000 claims abstract description 7
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims abstract description 7
- 239000012137 tryptone Substances 0.000 claims abstract description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 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
- 229940061607 dibasic sodium phosphate Drugs 0.000 claims abstract description 5
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims abstract description 5
- 239000008103 glucose Substances 0.000 claims abstract description 5
- 229940111688 monobasic potassium phosphate Drugs 0.000 claims abstract description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 5
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 3
- 239000011780 sodium chloride Substances 0.000 claims abstract description 3
- 235000011187 glycerol Nutrition 0.000 claims description 6
- 230000000843 anti-fungal effect Effects 0.000 abstract description 10
- 230000001580 bacterial effect Effects 0.000 abstract description 8
- 239000000243 solution Substances 0.000 description 6
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 244000052769 pathogen Species 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 2
- 235000019270 ammonium chloride Nutrition 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 241000223602 Alternaria alternata Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Invenţia se referă la microbiologie, şi anume la un mediu nutritiv pentru cultivarea tulpinii Bacillus subtilis CNMN-BB-09. The invention relates to microbiology, namely to a nutrient medium for cultivating the Bacillus subtilis strain CNMN-BB-09.
Invenţia se referă la domeniul microbiologiei, în particular la elaborarea unui mediu nutritiv pentru cultivarea microorganismelor care pot fi utilizate pentru protecţia biologică a plantelor. The invention relates to the field of microbiology, in particular to the development of a nutrient medium for cultivating microorganisms that can be used for biological plant protection.
Este cunoscut mediul nutritiv M9 cu compoziţia (g/L): clorură de amoniu - 1,0, fosfat monobazic de potasiu - 3,0, fosfat bibazic de sodiu - 6,0, sulfat de magneziu - 0,25, glucoză - 2,0, apă - restul [1]. The M9 nutrient medium is known with the composition (g/L): ammonium chloride - 1.0, monobasic potassium phosphate - 3.0, dibasic sodium phosphate - 6.0, magnesium sulfate - 0.25, glucose - 2.0, water - the rest [1].
Dezavantajul acestui mediu nutritiv constă în creşterea lentă a boimasei, calitatea şi activitatea antifungică redusă a bacteriilor cultivate. The disadvantage of this nutrient medium is the slow growth of biomass, the quality and reduced antifungal activity of the cultured bacteria.
Problema pe care o rezolvă prezenta invenţie constă în elaborarea unui mediu nutritiv care să accelereze creşterea biomasei şi să stimuleze activitatea antifungică a culturii. The problem solved by the present invention consists in developing a nutrient medium that accelerates biomass growth and stimulates the antifungal activity of the culture.
Mediul nutritiv, conform invenţiei, conţine, g/L: sulfat de amoniu - 1,4, fosfat de potasiu monobazic - 1,5, fosfat de sodiu dibazic - 2,5, clorură de sodiu - 0,5, glucoză - 2,0, drojdii uscate - 4,0, citrat de sodiu - 0,25, triptonă - 0,25, glicerină - 2,0 ml, apă - restul, având un pH de 5,5…6,0. The nutrient medium, according to the invention, contains, g/L: ammonium sulfate - 1.4, monobasic potassium phosphate - 1.5, dibasic sodium phosphate - 2.5, sodium chloride - 0.5, glucose - 2.0, dry yeast - 4.0, sodium citrate - 0.25, tryptone - 0.25, glycerin - 2.0 ml, water - the rest, having a pH of 5.5...6.0.
Rezultatul invenţiei constă în sporirea creşterii şi a activităţii antifungice a culturii bacteriene. The result of the invention consists in increasing the growth and antifungal activity of the bacterial culture.
Exemplu de realizare a invenţiei Example of embodiment of the invention
Obiectul cercetării a fost o tulpină activă a bacteriei Bacillus subtilis CNMN-BB-09, izolată din rizosfera plantelor de tomate. În prima fază a cultivării tulpinii a fost utilizat mediul M9 cu următoarea compoziţie, în g/L: clorură de amoniu - 1,0, fosfat monobazic de potasiu - 3,0, fosfat bibazic de sodiu - 6,0, sulfat de magneziu - 0,25, glucoză - 2,0, apa - restul. Pentru a îmbunătăţi proprietăţile de creştere ale mediului nutritiv şi pentru sporirea activităţii antifungice a culturii bacteriene Bacillus subtilis CNMN-BB-09 în mediul nutritiv suplimentar au fost introduse următoarele componente, în g/L: drojdii uscate - 4,0, citrat de sodiu - 0,25, triptonă - 0,25, glicerină - 2,0. The object of the research was an active strain of the bacterium Bacillus subtilis CNMN-BB-09, isolated from the rhizosphere of tomato plants. In the first phase of the cultivation of the strain, M9 medium was used with the following composition, in g/L: ammonium chloride - 1.0, monobasic potassium phosphate - 3.0, dibasic sodium phosphate - 6.0, magnesium sulfate - 0.25, glucose - 2.0, water - the rest. In order to improve the growth properties of the nutrient medium and to enhance the antifungal activity of the bacterial culture Bacillus subtilis CNMN-BB-09, the following components were introduced into the additional nutrient medium, in g/L: dry yeast - 4.0, sodium citrate - 0.25, tryptone - 0.25, glycerin - 2.0.
Bacteriile tulpinii Bacillus subtilis CNMN-BB-09 au fost cultivate în flacoane agitate conţinând 250 ml de medii nutritive (mediul optimizat şi mediul conform celei mai apropiate soluţii). Cultivarea a fost realizată la temperatura de 28°C, pe parcursul a 4 zile. Probele au fost prelevate la 2, 3 şi 4 zile de cultivare. Determinarea titrului culturii bacteriene a fost realizată prin diluţii limitatoare [Егоров Н.С. Руководство к практическим занятиям по микробиологии. Москва, 1995, 224 p]. Datele privind titrul culturii Bacillus subtilis CNM-BB-09 sunt prezentate în tabelul 1. Pentru a determina activitatea antifungică în calitate de cultură de testare a fost aleasă Alternaria alternata utilizând metoda de disc de hârtie [Егоров Н.С. Основы учения об антибиотиках. Москва, 2004, 528 p]. The bacteria of the Bacillus subtilis strain CNMN-BB-09 were cultivated in shaken flasks containing 250 ml of nutrient media (optimized medium and medium according to the closest solution). Cultivation was carried out at a temperature of 28°C, during 4 days. Samples were taken on 2, 3 and 4 days of cultivation. Determination of the titer of the bacterial culture was carried out by limiting dilutions [Егоров Н.С. Руководство к практическим занятиям по микробиологии. Москва, 1995, 224 p]. Data on the titer of the Bacillus subtilis CNM-BB-09 culture are presented in Table 1. To determine the antifungal activity as a test culture, Alternaria alternata was chosen using the paper disk method [Егоров Н.С. Основы учения об антибиотиках. Москва, 2004, 528 p].
Tabelul 1 Table 1
Numărul de unităţi formatoare ale tulpinii Bacillus subtilis CNMN-BB-09 după 2, 3, 4 zile de cultivare Number of forming units of the Bacillus subtilis strain CNMN-BB-09 after 2, 3, 4 days of cultivation
Mediul nutritiv Durata de cultivare, zile 2 3 4 М9 (cea mai apropiată soluţie) 1,3 × 108 8,8 × 108 2,6 × 109 М9+ citrat de sodiu 1,3 × 109 2,3 × 109 4,8 × 109 М9+ drojdii uscate 5,9 × 109 4,9 × 109 4,1 × 109 М9+ triptonă 7,1 × 108 1,7 × 109 5,7 × 109 М9+ glicerină 1,3 × 108 9,2 × 108 5,7 × 109 Mediul propus (invenţie) 7,5 × 109 8,9 × 109 1,7 × 1010 Nutrient medium Cultivation duration, days 2 3 4 М9 (closest solution) 1.3 × 108 8.8 × 108 2.6 × 109 М9+ sodium citrate 1.3 × 109 2.3 × 109 4.8 × 109 М9+ dry yeast 5.9 × 109 4.9 × 109 4.1 × 109 М9+ tryptone 7.1 × 108 1.7 × 109 5.7 × 109 М9+ glycerin 1.3 × 108 9.2 × 108 5.7 × 109 Proposed medium (invention) 7.5 × 109 8.9 × 109 1.7 × 1010
Datele privind activitatea antifungică a tulpinii Bacillus subtilis CNMN-BB-09 sunt prezentate în tabelul 2 şi 3. Data on the antifungal activity of the Bacillus subtilis strain CNMN-BB-09 are presented in Tables 2 and 3.
Tabelul 2 Table 2
Activitatea antifungică a tulpinii Bacillus subtilis CNMN-BB-09 după 2 zile de cultivare Antifungal activity of Bacillus subtilis strain CNMN-BB-09 after 2 days of cultivation
Mediul nutritiv Concentraţiile de suspensie bacteriană, diametrul zonei de inhibiţie, mm 5,0% 3,0% 1,0% 0,5% 0,2% 0,1% М9(cea mai apropiată soluţie) 5(0)* 0(3) 0(0) 0(0) 0(0) 0(0) М9+ citrat de sodiu 10,3(2,6) 10(3) 8(2) 8(2) 7(3) 7(3) М9+ drojdii uscate 15(6) 14(7) 15(6) 12(5) 10(5) 8(3) М9+ triptonă 16,3(5) 15(5) 15(10) 13(10) 11(7) 10(5) М9+ glicerină 11,6(9) 11(5) 9(5) 9(3) 8,5(3,5) 5,5(3) Mediul propus (invenţie) 18,2(5,6) 17,7(8,8) 15(10) 15(10) 15(2) 12(2);De exemplu: ;5(0)*, - 5 (cifra din faţa parantezei) diametrul zonei de creştere a agentului patogen complet suprimată, Nutrient medium Bacterial suspension concentrations, diameter of the zone of inhibition, mm 5.0% 3.0% 1.0% 0.5% 0.2% 0.1% M9(closest solution) 5(0)* 0(3) 0(0) 0(0) 0(0) 0(0) M9+ sodium citrate 10.3(2.6) 10(3) 8(2) 8(2) 7(3) 7(3) M9+ dry yeast 15(6) 14(7) 15(6) 12(5) 10(5) 8(3) M9+ tryptone 16.3(5) 15(5) 15(10) 13(10) 11(7) 10(5) M9+ glycerin 11.6(9) 11(5) 9(5) 9(3) 8.5(3.5) 5.5(3) Proposed medium (invention) 18.2(5.6) 17.7(8.8) 15(10) 15(10) 15(2) 12(2);For example: ;5(0)*, - 5 (the number in front of the parenthesis) diameter of the pathogen growth zone completely suppressed,
- (0) (cifra din paranteză) diametrul zonei de liză a miceliului agentului patogen - (0) (number in parentheses) diameter of the lysis zone of the pathogen's mycelium
Tabelul 3 Table 3
Activitatea antifungică a tulpinii bacteriene Bacillus subtilis CNMN-BB-09 după 4 zile de cultivare Antifungal activity of the bacterial strain Bacillus subtilis CNMN-BB-09 after 4 days of cultivation
Mediul nutritiv Concentraţiile de suspensie bacteriană 3,0% 1,0% 0,5% 0,2% 0,1% М9(cea mai apropiată soluţie) 5(1,2) 3(0) 0(0) 0(0) 0(0) М9+ citrat de sodiu 12(5) 9(4.5) 10(4) 8(3) 8(3) М9+ drojdii uscate 15(5) 15(5) 12(2) 9(5) 8(3) М9+ triptonă 15(5) 10(5) 10(4) 8(3) 8(3) М9+ glicerină 15(4) 9(3) 9(3) 7(3) 6,5(3) Mediul propus (invenţie) 18,3(4,8) 16(5,1) 16(4,2) 14(2,1) 10(3)Nutrient medium Bacterial suspension concentrations 3.0% 1.0% 0.5% 0.2% 0.1% M9 (closest solution) 5(1.2) 3(0) 0(0) 0(0) 0(0) M9+ sodium citrate 12(5) 9(4.5) 10(4) 8(3) 8(3) M9+ dry yeast 15(5) 15(5) 12(2) 9(5) 8(3) M9+ tryptone 15(5) 10(5) 10(4) 8(3) 8(3) M9+ glycerin 15(4) 9(3) 9(3) 7(3) 6.5(3) Proposed medium (invention) 18.3(4.8) 16(5.1) 16(4.2) 14(2,1) 10(3)
Titrul maxim al tulpinii Bacillus subtilis CNMN-BB-09 pe mediul cunoscut a fost observat în ziua a 4-a de cultivare atingând valoarea de 2,6 x 109, în timp ce pe mediul din invenţia propusă, după două zile de cultivare titrul culturii a atins valoarea de 7,5 x 109, iar în ziua a patra de 1,7 x 1010, ceea ce depăşeşte de 6,5 ori titrul obţinut pe mediul din cea mai apropiată soluţie. The maximum titer of the Bacillus subtilis strain CNMN-BB-09 on the known medium was observed on the 4th day of cultivation, reaching the value of 2.6 x 109, while on the medium of the proposed invention, after two days of cultivation, the culture titer reached the value of 7.5 x 109, and on the fourth day of 1.7 x 1010, which exceeds by 6.5 times the titer obtained on the medium from the closest solution.
Astfel, în zonele de inhibare a creşterii miceliului format de patogen activitatea antifungică a culturii bacteriene a depăşit-o pe cea a culturii obţinute pe mediul din cea mai apropiată soluţie în toate concentraţiile testate. Thus, in the areas of inhibition of the growth of the mycelium formed by the pathogen, the antifungal activity of the bacterial culture exceeded that of the culture obtained on the medium from the closest solution in all tested concentrations.
1. Маслак Д.В., Феклистова И.Н., Гринева И.А. и др. Активность целлюлолитического комплекса индуцированных мутантов Bacillus subtilis. Труды БГУ, 2015, том 10, часть 1, Микробиология, р. 83-89 1. Maslak Д.В., Феклистова И.Н., Гринева И.А. and others Активность целлюлотического комплекса дуцизированных мутантов Bacillus subtilis. Труды БГУ, 2015, том 10, часть 1, Микробиология, р. 83-89
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MD727G2 (en) * | 1995-02-23 | 1998-01-31 | Crudu Valeriu | Medium for cultivation of the tuberculosis mycrobacteries |
| MD4304B1 (en) * | 2013-08-13 | 2014-09-30 | Institutul De Zoologie Al Academiei De Ştiinţe A Moldovei | Strain of Bacillus thuringiensis subsp. kurstaki bacteria - bioinsecticide for the control of Lymantria genus lepidopterans |
| RU2571945C1 (en) * | 2014-11-17 | 2015-12-27 | Федеральное государственное бюджетное учреждение "Всероссийский научно-исследовательский и испытательный институт медицинской техники" (ФГБУ "ВНИИИМТ" Росздравнадзора) | Nutrient medium to cultivate bacteria bacillus subtilis bn-135- producent of pectate lyase |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MD727G2 (en) * | 1995-02-23 | 1998-01-31 | Crudu Valeriu | Medium for cultivation of the tuberculosis mycrobacteries |
| MD4304B1 (en) * | 2013-08-13 | 2014-09-30 | Institutul De Zoologie Al Academiei De Ştiinţe A Moldovei | Strain of Bacillus thuringiensis subsp. kurstaki bacteria - bioinsecticide for the control of Lymantria genus lepidopterans |
| RU2571945C1 (en) * | 2014-11-17 | 2015-12-27 | Федеральное государственное бюджетное учреждение "Всероссийский научно-исследовательский и испытательный институт медицинской техники" (ФГБУ "ВНИИИМТ" Росздравнадзора) | Nutrient medium to cultivate bacteria bacillus subtilis bn-135- producent of pectate lyase |
Non-Patent Citations (1)
| Title |
|---|
| Маслак Д.В., Феклистова И.Н., Гринева И.А. и др. Активность целлюлолитического комплекса индуцированных мутантов Bacillus subtilis. Труды БГУ, 2015, том 10, часть 1, Микробиология, р. 83-89 * |
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