Method for submerged cultivation of bacillus brevis for producing gramicidin s

FIELD: chemistry.

SUBSTANCE: disclosed is a method of cultivating Bacillus brevis strain 101 for producing gramicidin S. Submerged cultivation of a culture is carried out on a synthetic culture medium. The medium contains yeast autolysate and casein hydrolysate in concentration of 0.1 g/l and 0.2 g/l on amine nitrogen, glycerine in concentration of 20 ml/l, edible 40% lactic acid 2.0-4.0 ml/l, ammonium phosphate-chloride 0-3.4 g/l, di-substituted ammonium phosphate 0.85-4.5 g/l, mono-substituted potassium phosphate 0-1.0 g/l, magnesium sulphate heptahydrate 0.9 g/l, sodium citrate 1.0 g/l. Content of amine nitrogen in the initial medium is equal to 1.3-1.6 g/l. When concentration of amine nitrogen falls to less than 1.4 g/l, a concentrated culture solution is added to the medium until achieving concentration of 1.75 g/l. The concentrated culture solution contains glycerine, edible 40% lactic acid, di-substituted ammonium phosphate and chloride and magnesium sulphate with ratio of concentration of glycerin, lactic acid, nitrogen, phosphorus and magnesium equal to 1:(0.008-0.032):(0.027-0.036):(0-0.008):(0.002-0.008). During growth, the rate of stirring is increased from 200 to 500 rpm. pH is kept at 6.5-6.8 by adding potassium and sodium hydroxide. The process is stopped 2-6 after the onset of a stationary phase.

EFFECT: method enables reproducible production of a large amount of gramicidin S.

10 tbl, 5 ex

 

Usage: the Invention relates to biotechnology, in particular to the microbiological industry, and can be used to obtain gramicidin a highly effective microbiological method

The inventive method includes periodic cultivation producer of gramicidin From Bacillus brevis strain 101 in a balanced composition environment and additional recharge concentrate the nutrient solution in the growing process, the input of which is produced when the values amine (restored) of nitrogen in the culture liquid (QL) below 1.4 g/l to achieve a value of no more than about 1.75 g/l under oxygen limitation in terms of providing developing cultures of oxygen due to the gradual increase in mixing intensity on the rate of mass transfer of oxygen from 0.25·10-3up to 1·10-3mol O2/l·min and the stirring speed from 200 to 500 rpm while maintaining the partial content of dissolved oxygen in the culture, is close to zero (0-10%), maintaining the pH level of 6.5-6.8 and when you stop growing process through 2-6 h after the beginning of the stationary phase, as evidenced by changes in the concentration of biomass (increase or decrease) of optical density by a value not more than 1 g/L.

The invention relates to microbiological and medical industry the items and can be used for high-performance production of gramicidin S.

Known methods of obtaining of gramicidin a in submerged cultivation producer Bacillus brevis synthetic environments [1-6].

Existing periodic process With gramicidin when growing the most productive strains of Bacillus brevis, selected as a prototype [8], which is used for synthesis of the product under cultivation of Bacillus brevis strain 101 [7], characterized by the fact that is disposable loading of the nutrient medium in the fermenter, inoculation seeds, cultivation under aeration and stirring for 27 hours to achieve a pH of 6.0 and below.

The analysis of this method showed that the fermentation medium contains the entire stock of nutrient substrates with the time of sowing. Therefore, previously under cultivation of Bacillus brevis for growth and biosynthesis of gramicidin a is not used feed concentrates nutrient solutions optimized composition, as they were used in [9]. The nutrient environment of the prototype is not optimal, because the lack of individual nutrients (phosphorus and magnesium), and at the same time contains in excess of other (glycerin and oxalic ammonium). As a result of high inhibitory concentration of glycerol and amino nitrogen (2,0-3,0 g/l) in the environment of growth of producer slowed down. Due to lack of mineral components, the concentration of biomass was usegoogle 10 g/l on average and no more than 15 g/L. A high concentration of lactic acid (10.5 g/l) contributes to the accumulation of clones, not producing gramicidin S. When growing producer in this medium pH was not regulated, and it decreased to values below 6.0. Low rate of mass transfer for oxygen (1,28·10-2g O2/l·min) during aeration of 0.66 l/l·min and stirring (259-303 rpm) also was not conducive to obtaining a culture with a high concentration of biomass and product. For this reason, the content of gramicidin in the culture at best amounted to about 2 g/L.

The aim of the invention is to increase the output of gramicidin a in the periodic cultivation of Bacillus brevis.

Using the proposed method allows to obtain more than 3.0 g/l of gramicidin S. the essence of the invention lies in the fact that in order to increase the output of gramicidin a in submerged cultivation producer Bacillus brevis synthetic environment growing produce in the medium of the following composition:

- glycerin - 20 ml/l,

- food 40% lactic acid - 2-4 ml/l;

the autolysate of yeast - 0.1 g/l (on the amine nitrogen);

- hydrolyzed casein - 0.2 g/l (on the amine nitrogen);

- ammonium chloride, NH4Cl - 0-3,4 g/l;

- disubstituted ammonium phosphate, (NH4)2HPO4(DAP) - 0,85-4.5 g/liter;

- potassium postorogenic one-deputizing, KH2PO4- 0-1,0 g/l;

- magni is sulfate, 7-water, MgSO4·7H2O - 0.9 g/l;

- sodium citrate, dnovotny - 1.0 g/L.

The content in the environment amine (restored) nitrogen - 1,3-1,6 g/l

The method includes the additional recharge concentrated nutrient solution, which consists of glycerin, food 40%lactic acid, chloride and disubstituted phosphate ammonium sulfate and magnesium at a ratio of concentrations of glycerol, lactic acid, nitrogen, phosphorus and magnesium 1: (0,008-0,032):(0,027-0,036):(0-0,008):(0,002-0,008), the input which is produced when the values of amine nitrogen in the culture fluid below 1.4 g/l to achieve a concentration of no more than about 1.75 g/l, while limiting the growth by the lack of oxygen in the conditions smooth increasing mixing intensity on the rate of mass transfer of oxygen from 0.25·10-3to 1.0·10-3mol O2/l·min and the stirring speed from 200 to 500 rpm and maintaining the partial content of dissolved oxygen in the culture, is close to zero (0-10%), maintaining the pH level of 6.5-6.8 and when you stop growing process through 2-6 h after the beginning of the stationary phase, as evidenced by changes in the concentration of biomass (increase or decrease) of optical density by a value not more than 1 g/L.

Example 1.

Cultivation of Bacillus brevis strain 101 in the fermenter with a capacity of 10 litres

Characteristicsin material:

- volume of 0.5 l,

- typical morphology,

the biomass of 2.5-3.5 g/l,

a pH of 6.4 to 6.7.

to portirovali prior to sterilization of the medium to pH
Table 1
The composition of the medium for cultivation in a fermenter
Components1 l medium
Yeast extract (on the amine nitrogen, g/l0.1
Casein hydrolysate (amino nitrogen), g/l0.2
Sodium citrate, g/l1,0
KN2RHO4, g/l1,0
(NH4)2NRA4, g/l4,5
MgSO4·7H2O, g/l0,9
Lactic acid food, 40%aq, ml/l2,0
Glycerin, ml/l20,0
Water, lto 1.0
Antifoam (labral), ml1,0
KOH (15% solution)
6,7-6,8
to portirovali after sterilization of the medium to pH6,7-6,8
HCl (10% solution), if necessary, after sterilization, to pH6,7-6,8

The working volume of the fermenter 6 l, so bookmark components 6 l environment (including input 0.5 l of inoculum) was carried out according to table 1. In the composition of the concentrated nutrient solution (concentrate) was composed of glycerin, food 40%lactic acid, ammonium chloride and 7-water sulfate magnesium at a ratio of glycerol, lactic acid, nitrogen and magnesium 1,0:0,032:0,027:0,002. To maintain the pH at 6.6 and 6.7 have used a 15% solution of caustic potash. If necessary and to prevent active pricing added antifoam Laprol, which is not a component of the nutrient medium to 1 ml/l of culture fluid. Wednesday, concentrated nutrient solution and the alkaline solution was sterilized at a temperature of 120°C for 30 min Feed concentrate during fermentation was carried out by lowering the concentration of amine nitrogen below 1.4 g/l Volume of injected concentrate was determined, taking into account that the submission of 10 ml concentrate 6 l caused an increase in amino nitrogen in QOL 3.6·10 -2g/L. In the growth of the mixing rate gradually increased from 202 rpm to 461 rpm, maintaining Rho2at the level of 0-10%.

Table 2
Indicators of culturing in the fermenter with a capacity of 10 l
τ, hn, R/minKLa, n·10-3mol O2/l·minRho2, %pHAmine nitrogen, g/lBiomass, g/lGramicidin S, g/lImpurities, % of gramicidin C
02020,251006,91,380,43n/an/a
32300,291,46,91,380,78n/an/a
6 2530,321,26,91,411,25n/an/a
92700,341,36,81,312,30n/an/a
122810,361,46,91,383,340,412of 5.4
153000,390,96,71,544,390,6555,6
183480,493,06,71,686,901,0855,8
213540,501,76,71,40to 9.571,4655,3
243930,601,56,61,4012,542,3466,0
273930,600,96,60,9118,102,7005,8
304050,640,86,60,8422,603,7228,4
334500,71the 9.76,70,7027,104,287,9
364610,8310,06,60,4929,304,7106,4
384610,834,56,60,4928,204,9446,4

The data of table 2 show that the cultivation of a producer in a 10-liter fermenter made of 38 hours in the conditions of limited growth on oxygen. In the process of growth of the mixing rate gradually increased from 202 to 461 rpm under the control of dissolved oxygen, holding Rho2at the level of 0.9-10%. The rate of mass transfer was increased from 0.25·10-3mol O2/l·min to 0.83·10-3mol O2/l·min In the growth process, starting with the 7th hour culture was introduced concentrate. Feed rate appropriate to the needs of culture, as evidenced by the low values of Rho20-10% and the concentration of amino nitrogen in the culture of not higher than 1.68 g/L. This suggests that managed to avoid growth inhibition by high concentrations and growth restrictions under taccom power.

The concentration of hydrogen ions in QOL supported the introduction of a 15%aqueous solution of caustic potassium. The values of biomass concentration, which was determined by optical density, the culture entered the stationary growth phase at the 33rd hour. The cultivation was stopped after 4-5 hours after cessation of growth. The result was obtained culture containing 28-29 g/l biomass and 4,944 g/l of gramicidin a with the impurity content of 6.4% of gramicidin S.

Example 2.

Cultivation of Bacillus brevis strain 101 in the fermenter with a capacity of 10 litres

Characteristics of seed:

- volume of 0.5 l,

- typical morphology,

the biomass of 2.5-3.5 g/l,

a pH of 6.4 to 6.7.

Table 3
The composition of the medium for cultivation in a fermenter
Components1 l medium
Yeast extract (on the amine nitrogen, g/l0.1
Casein hydrolysate (amino nitrogen), g/l0.2
Glycerin, m/l20,0
NH4Cl, g/l3,4
(NH4)2NRA4, g/l 0,85
MgSO4·7H2O, g/l0,9
Lactic acid food 40%R-R, ml/l4,0
Sodium citrate, ml/l1,0
Water, lto 1.0
Antifoam (labral), ml1,0
KOH (15% solution)
to portirovali prior to sterilization of the medium to pH6,7-6,8
to portirovali after sterilization of the medium to pH6,7-6,8
HCl (10% solution),
necessary, after sterilization, to pH
6,7-6,8

The working volume of the fermenter 6 l, so bookmark components 6 l environment (including input inoculum) was carried out according to table 3. Used a concentrated nutrient solution (concentrate), composed of glycerin, food 40%lactic acid, chloride and disubstituted phosphoric acid ammonium sulfate and magnesium at a ratio of concentrations of glycerol, lactic acid, nitrogen, phosphorus and magnesium 1:0,008:0,036:0,005:0,00. To maintain the pH at 6.6 and 6.7 have used a 15% solution of caustic potash. If necessary and to prevent active pricing added antifoam Laprol, which is not a component of the nutrient medium to 1 ml/l of culture fluid. Wednesday, the concentrate and the alkaline solution was sterilized at a temperature of 120°C for 30 minutes

Feed concentrate was carried out by lowering the concentration of amine nitrogen below 1.4 g/l Volume of injected concentrate was determined, taking into account that the submission of 10 ml concentrate 6 l causes an increase in amino nitrogen in QOL on 5,27·10-2g/L. In the growth of the mixing rate gradually increased from 202 rpm to 500 rpm, maintaining Rho2at the level of 0-10%.

0
Table 4
Indicators of culturing in the fermenter with a capacity of 10 l
τ, hn, R/minKLa, n·10-3mol O2/l·minRho2, %pHAmine nitrogen, g/lBiomass, g/lGramicidin S, g/lImpurities, % of gramicidin C
2020,251006,71,470,37n/an/a
32020,252,46,81,400,68n/an/a
62250,282,26,61,262,04n/an/a
92470,313,36,71,333,45n/an/a
122700,342,26,61,264,810,576,22
152980,391,56,61,40to 7.32is 0.9986,10
183200,431,76,60,91for 9.641,639of 9.30
213430,481,86,60,8413,492,2978,90
243930,602,06,60,8416,943,1638,90
273930,602,06,60,8421,34 3,3608,28
304500,782,26,60,7726,154,0838,60
335000,992,86,60,9828,244,1628,30
365000,992,46,60,7733,894,5539,10
395000,992,46,61,0537,684,7728,90
425000,9963,06,81,05 38,015,2828,44

The data of table 4 show that the cultivation of a producer in a 10-liter fermenter produced within 42 hours under conditions limiting growth on oxygen. In the process of growth of the mixing rate gradually increased from 202 to 500 rpm under the control of dissolved oxygen, holding pO2at the level of 1.5-10%. Mass transfer rates were increased from 0.25·10-3mol O2/l·min to 0.99·10-3mol O2/l·min In the growth process, starting with the 7th hour culture was introduced concentrate environment. Feed rate appropriate to the needs of culture, as evidenced by the low values of Rho20-10% and the concentration of amino nitrogen in the culture of not higher than 1.47 g/L. This suggests that managed to avoid growth inhibition by high concentrations and growth constraints lack power.

The concentration of hydrogen ions in QOL supported the introduction of a 15%aqueous solution of caustic potassium. The values of biomass concentration, which was determined by optical density, the culture entered the stationary growth phase at the 39th hour growth. The cultivation was stopped after 3 hours after cessation of growth. The result was obtained culture containing 38 g/l of biomass and 5,282 g/l of gramicidin a with the content of impurities 8,44% of gramicidin S.

Example 3.

Growing acillus brevis strain 101 in the fermenter with a capacity of 100 liters Seed material was prepared in a 10-liter fermenter.

Characteristics of seed:

volume 5.2 l,

- typical morphology,

the biomass of 2.93 g/l,

- pH 6.4.

Table 5
The composition of the medium for cultivation in a fermenter "BioR 0,1"
Components1 l medium
Yeast extract (on the amine nitrogen, g/l0.1
Casein hydrolysate (amino nitrogen), g/l0.2
Sodium citrate, g/l1,0
KN2RHO4, g/l1,0
(NH4)2NRA4, g/l4,5
MgSO4·7H2O, g/l0,9
Lactic acid food 40%R-R, ml/l2,0
Glycerin, ml/l20,0
Water, lto 1.0
Antifoam (labral), ml 1,0
KOH (15% solution)
to portirovali prior to sterilization of the medium to pH6,7-6,8
to portirovali after sterilization of the medium to pH6,7-6,8
HCl (10% solution), if necessary, after sterilization, to pH6,7-6,8

The working volume of the fermenter 60 l, so bookmark component 60 l environment (including input 5.2 l of inoculum) was carried out according to table 5.

In the composition of the concentrated nutrient solution (concentrate) was composed of glycerin, food 40%lactic acid, ammonium chloride and 7-water sulfate magnesium at a ratio of glycerol, lactic acid, nitrogen and magnesium 1,0:0,032:0,030:0,002. To maintain the pH at 6.6 and 6.7 used a 15%solution of caustic potash. If necessary and to prevent active foaming added antifoam Laprol, which is not a component of the nutrient medium to 1 ml/l of culture fluid. Wednesday, the concentrate and the alkaline solution was sterilized at a temperature of 120°C for 30 min Feed concentrate was carried out by lowering the concentration of amine nitrogen below 1.4 g/l Volume of injected concentrate was determined, taking into account that under the cha 100 ml concentrate 60 l causes an increase in amino nitrogen in QOL on the 4.29·10 -2g/l

In the process of growth of the mixing rate gradually increased from 202 rpm to 502 rpm, maintaining Rho2at the level of 0-10%.

Table 6
Indicators of culturing in the fermenter with a capacity of 100 l
τ, hn, R/minKLa,n·10-3mol O2/l·minRho2, %pHAmine nitrogen, g/lBiomass, g/lGramicidin S, g/lImpurities, % of gramicidin C
02030,371006,81,400,25n/an/a
32030,370,26,81,400,68n/an/a
6 2760,470,46,81,401,26n/an/a
93210,550,56,71,402,09n/an/a
123500,610,66,61,473,030,4745,9
153890,691,06,61,40a 3.870,632the 5.7
184000,723,26,61,265,021,0135,8
214310,801,06,61,19to 7.321,5038,1
244680,911,06,61,1910,041,8888,6
275001,017,16,60,1211,942,6548,8
305021,020,86,60,1215,902,8927,7
335021,020,86,60,9117,803,211,2
365021,0214,86,60,6321,703,4987,9
395021,020,96,70,5622,563,8077,6

The data of table 6 show that the cultivation of a producer in a 100-liter fermenter produced within 39 hours in the conditions of limited growth on oxygen. In the process of growth of the mixing rate gradually increased from 202 to 502 rpm under the control of dissolved oxygen, holding Rho2at the level of 0.2-15%. The rate of mass transfer was increased from 0.37·10-3mol O2/l·min to 1.02·10-3mol O2/l·min In the growth process, starting with the 7th hour culture was introduced concentrate environment. Feed rate appropriate to the needs of culture, as evidenced by the low values of Rho2(0-10%) and the concentration of amino nitrogen in the culture of not higher than 1.4 g/L. This suggests that managed to avoid growth inhibition by high concentrations and limitations grew the and lack power.

The concentration of hydrogen ions in QOL supported the introduction of a 15%aqueous solution of caustic potassium. The values of biomass concentration, which was determined by optical density, the culture entered the stationary growth phase at the 36th hour of growth. The cultivation was stopped after 3 hours after cessation of growth. In a culture containing a 22.5 g/l biomass and 3,807 g/l of gramicidin a with the impurity content of 7.6% of gramicidin S.

Example 4.

Cultivation of Bacillus brevis strain 101 in the fermenter with a capacity of 10 L. the Characteristic seed:

- volume of 0.5 l,

- typical morphology,

the biomass of 2.5-3.5 g/l,

a pH of 6.4 to 6.7.

0,85
Table 7
The composition of the medium for cultivation in a fermenter
Components1 l medium
Yeast extract (on the amine nitrogen, g/l0.1
Casein hydrolysate (amino nitrogen), g/l0.2
Glycerin, m/l20,0
NH4Cl, g/l3,4
(NH4)2NRA4, g/l
MgSO4·7H2O, g/l0,9
Lactic acid food, 40%aq, ml/l4,0
Sodium citrate, ml/l1,0
Water, lto 1.0
Antifoam (labral), ml1,0
Paon (15% solution)
to portirovali prior to sterilization of the medium to pH6,7-6,8
to portirovali after sterilization of the medium to pH6,7-6,8
HCl (10% solution), if necessary, after sterilization, to pH6,7-6,8

The working volume of the fermenter 6 l, so bookmark components 6 l environment (including input inoculum) was carried out according to table 7. Used a concentrated nutrient solution, composed of glycerin, food 40%lactic acid, chloride and disubstituted phosphate ammonium sulfate and magnesium at a ratio of concentrations of glycerol, lactic acid, nitrogen, phosphorus and magnesium 1:0,009:0,038:0,008:0,008. To maintain a pH level of 6.6-6.7 COI is litovali 15% solution of sodium hydroxide. If necessary and to prevent active foaming added antifoam Laprol, which is not a component of the nutrient medium to 1 ml/l of culture fluid. Wednesday, the concentrate and the alkaline solution was sterilized at a temperature of 120°C for 30 minutes

Feed concentrate was carried out by lowering the concentration of amine nitrogen below 1.4 g/l Volume of injected concentrate was determined, taking into account that the submission of 10 ml concentrate 6 l causes an increase in amino nitrogen in QOL by 4.37·10-2g/L. In the growth of the mixing rate gradually increased from 202 rpm to 472 rpm, maintaining Rho2at the level of 0-10%.

Table 8
Indicators of culturing in the fermenter with a capacity of 10 l
τ, hn, R/minKLa, n·10-3mol O2/l·minRho2, %pHAmine nitrogen, g/lBiomass, g/lGramicidin S, g/lImpurities, % of gramicidin C
0202 0,251006,71,400,40n/an/a
32020,250,46,81,120,84n/an/a
62530,320,76,71,052,09n/an/a
92920,382,16,90,913,45n/an/a
123030,404,16,61,195,440,8885,9
15 3320,461,06,70,49to 7.321,42511,3
183540,500,56,70,638,162,19914,6
213540,500,66,70,9111,132,6679,1
243990,620,46,70,9814,432,84710,3
274270,7110,06,70,7717,123,33711,1
304780,891,26,70,8419,683,79311,4
334720,872,96,70,7023,44RUB 3.67411,4
364720,876,56,70,6327,203,87012,3
394720,873,76,70,7728,804,64311,1
424100,651,16,70,9929,305,366 the 11.6

The data of table 8 show that the cultivation of a producer in a 10-liter fermenter produced within 42 hours under conditions limiting growth on oxygen. In the process of growth of the mixing rate gradually increased from 202 to 472 rpm under the control of dissolved oxygen, holding Rho2at the level of 1.5-10%. Mass transfer rates were increased from 0.25·10-3mol O2/l·min to 0.87·10-3mol O2/l·min In the growth process, starting with the 7th hour culture was introduced concentrate environment. Feed rate appropriate to the needs of culture, as evidenced by the low values of Rho20-10% and the concentration of amino nitrogen in the culture of not higher than 1.4 g/L. This suggests that managed to avoid growth inhibition by high concentrations and growth constraints lack power.

The concentration of hydrogen ions in QOL supported the introduction of a 15%aqueous solution of sodium hydroxide. The values of biomass concentration, which was determined by optical density, the culture entered the stationary growth phase at the 39th hour growth. The cultivation was stopped after 3 hours after cessation of growth. The result was obtained culture containing 29 g/l of biomass and 5,366 g/l of gramicidin a with the impurity content of 11.6% of the content of gramicidin S.

Example 5.

Cultivation of Bacillus brevis strain 101 in the fermenter with a capacity of 100 liters

Characteristics of inoculum for 100-liter unit:

- volume of 5.0 l,

- the absence of extraneous microflora,

the typical morphology of vegetative cells

- biomass 2,82 g/l,

- rn.

Table 9
The composition of the medium for cultivation in a fermenter "BioR 0,1"
Components1 l medium
Yeast extract (on the amine nitrogen, g/l0.1
Casein hydrolysate (amino nitrogen), g/l0.2
Sodium citrate, g/l1
KN2RHO4, g/l1
(NH4)2NRA4, g/l4,5
MgSO4·7H2O, g/l0,9
Lactic acid food, 40%aq, ml/l2,0
Glycerin, ml/l20,0
Water, lto 1.0
Pinag the Khabibullina (labral), ml1,0
KOH (15% solution)
to portirovali prior to sterilization of the medium to pH6,7-6,8
to portirovali after sterilization of the medium to pH6,7-6,8
HCl (10% solution), if necessary, after sterilization, to pH6,7-6,8

The working volume of the fermenter 60 l, so bookmark component 60 l environment (with the input of 5.0 l of inoculum produced on table.

In the composition of the concentrated nutrient solution (concentrate) was composed of glycerin, food 40%lactic acid, ammonium chloride and 7-water sulfate magnesium at a ratio of glycerol, lactic acid, nitrogen and magnesium 1,0:0,032:0,030:0,002. To maintain the pH at 6.6 and 6.7 have used a 15% solution of caustic potash. If necessary and to prevent active foaming added antifoam Laprol, which is not a component of the nutrient medium to 1 ml/l of culture fluid. Wednesday, the concentrate and the alkaline solution was sterilized at a temperature of 120°C for 30 minutes

Feed concentrate was carried out by lowering the concentration of amine nitrogen below 1.4 g/l Volume of injected concentrate was determined,whereas, that supply 100 ml concentrate 60 l causes an increase in amino nitrogen in QOL on the 4.29·10-2g/l

In the process of growth of the mixing rate gradually increased from 202 rpm to 502 rpm and maintaining pO2at the level of 0-10%.

Table 10
Indicators of culturing in the fermenter with a capacity of 100 l
τ, hn, R/minKLa, n·10-3mol O2/l·minRho2, %pHAmine nitrogen, g/lBiomass, g/lGramicidin S, g/lImpurities, % of gramicidin C
02030,371007,11,540,33n/ODAn/a
32030,371,27,11,470,42 n/ODAn/a
63020,521,16,91,750,78n/ODAn/a
93210,551,46,91,751,62n/ODAn/a
123500,611,06,81,752,620,4549,3
154000,721,06,81,753,660,6118,4
184500,851,26,61,75 5,640,8818,4
215021,021,56,61,61to 7.321,437the 9.7
244850,961,46,61,6110,001,84410,0
274910,981,36,61,5413,102,3529,4
305021,021,16,61,5415,692,7478,0
335021,021,16,6 1,4019,663,1368,1
365021,021,26,61,2623,013,5328,6
394940,991,16,60,9127,204,1738,3
425021,0226,36,60,5627,604,2678,0

The data of table 10 show that the cultivation of a producer in a 100-liter fermenter produced within 41 hours in the conditions of limited growth on oxygen. In the process of growth of the mixing rate gradually increased from 202 to 502 rpm under the control of dissolved oxygen, holding Rho2at the level of 1.0 to 1.5. In the process of raising the rate of mass transfer was increased from 0.37·10-3mol O2/l·min is about 1,02·10 -3mol O2/l·minutes since 8 hours, the culture was injected concentrate environment. Feed rate appropriate to the needs of culture, as evidenced by the low values of Rho2(0-10%) and the concentration of amino nitrogen in the culture of not higher than about 1.75 g/L. This suggests that managed to avoid growth inhibition by high concentrations and growth constraints lack power.

The concentration of hydrogen ions in QOL supported the introduction of a 15%aqueous solution of caustic potassium. The values of biomass concentration, which was determined by optical density, the culture entered the stationary growth phase at the 39th hour growth. The cultivation was stopped after 2 hours after cessation of growth. In a culture containing a 27.6 g/l biomass and 4,267 g/l of gramicidin s With impurities content of 8.0%.

Sources of information

1. Kites CENTURIES Physiology of metabolism of Bacillus brevis subsp. G.-B. in connection with the biosynthesis of gramicidin S. - Candles. Diss., 1962, M, MSU.

2. Korshunov V., Egorov NS Synthetic environment for the development of Bacillus brevis and education gramedia. Microbiology, 1962; 31:3, 515-519.

3. Shaposhnikov, V.N., Rabotnov I.L., Silaev A.B., etc. a method of obtaining a crystal of gramicidin S. - Copyright certificate for invention №187243, 1963.

4. Kupletskaya could BE the Influence of aeration on the development and education of gramedia culture of Bacillus brevis vr. G.-B. Microbiology, 1965; 34:5, 905-909.

5. Zharikov, the Natural variability of spore-forming bacteria and biosynthesis of polypeptide antibiotics. - Prof. Diss., 1972), Moscow state University.

6. Berezovskaya A.I., Wypych A.N., Egorov NS and other Strain of Bacillus brevis 101 - producer of gramicidin S. - Copyright certificate for invention №686463, 1978.

7. Yudin OTHER Physiological and biochemical characteristics of the producers of gramedia Bacillus brevis subsp. G.-B. and their variants. - Kida. Diss., 2002, M., Moscow state University.

8. Udalova OTHER Features of the growth and development of Bacillus brevis var. G.-B in connection with the biosynthesis of gramicidin S. - Candles. Diss., 1979, M, MSU.

9. Derbyshev CENTURIES, deaf N.N., Canines S. p., Nabokov A.P., Shcherbakov GY Method of producing biomass aerobiotic microorganisms. - RF patent №2111246, 1992.

The method of cultivation of Bacillus brevis strain 101 for receiving gramicidin S, including deep cultivation of culture on synthetic nutrient medium containing yeast autolysate and casein hydrolysate in a concentration of 0.1 g/l and 0.2 g/l of amine nitrogen, respectively, characterized in that the cultivation of produce on the environment in which the concentration of glycerin, food 40%lactic acid, chloride and disubstituted phosphoric acid ammonium phosphoric acid one-deputizing potassium sulfate 7-water magnesium and citric acid sodium is 20 ml/l; 2,0-4,0 ml/l; 0-3,4 g/l; 0,85-4.5 g/l; 0-1,0 g/l; 0.9 g/l and 1.0 g/is, respectively, and when the content of amine nitrogen in the original environment of 1.3-1.6 g/l with water a concentrated nutrient solution, which consists of glycerin, food 40%lactic acid, chloride and disubstituted phosphoric acid ammonium sulfate and magnesium at a ratio of concentrations of glycerol, lactic acid, nitrogen, phosphorus and magnesium 1÷(0,008-0,032)÷(0,027-0,036)÷(0-0,008)÷(0,002-0,008), supplied in a fermentation medium when the values of amine nitrogen in the culture fluid below 1.4 g/l to achieve a concentration of no more than about 1.75 g/l, while limiting the growth by the lack of oxygen at high mixing intensity on the rate of mass transfer of oxygen from 0.25·10-3mol O2/(l·min) to 1.0·10-3mol O2/(l·min), achieved by increasing the stirring speed from 200 to 500 rpm, while maintaining the pH in the range of about 6.5-6.8 introduction of sterile solutions of caustic potassium or sodium, and when you stop growing process through 2-6 h after the onset of stationary phase.



 

Same patents:

FIELD: food industry.

SUBSTANCE: method for production of Lactococcus lactis strain version producing, under standard fermentation conditions, a quantity of vitamin K2 exceeding that produced by the stock/parent bacterial strain inoculated under the same conditions approximately 1.2 times, the said method including, at least: a) inoculation of the sock bacterial strain under standard fermentation conditions in a cultural medium causing a change in the oxidation-reduction state of a cell containing bacitracin or a peroxide and b) selection of the strain version if producing a quantity of vitamin K2 exceeding that produced by the stock/parent bacterial strain inoculated under the same conditions approximately 1.2 times. Lactococcus lactis subsp.cremoris 1-3557 strain deposited in Collection CNCN of Pasteur Institute on 20.01.2006 and Lactococcus lactis subsp.cremoris 1-3558 strain deposited in Collection CNCM of Pasteur Institute on 20.01.2006 produce at least 1.2 times more vitamin K2 than the stock/parent bacterial strain inoculated under the same conditions. Additionally, the invention deals with a lactic acid starter containing at least one of the above strains and to the method for preparation of a cultured milk product containing the above strain and/or the lactic acid starter.

EFFECT: invention enables increase of vitamin K2 content in the product.

11 cl, 5 tbl

FIELD: food industry.

SUBSTANCE: invention deal with a method for production of Lactobacillus delbrueckii subsp. bulgaricus strain with reduced lactic acid postproduction. The method involves exposure of Lactobacillus delbrueckii subsp. bulgaricus, mother strain possessing high texturising properties and high acid postproduction to the impact of a weak mutagen represented by ethyl methanesulfonate (EMS) and subsequent selection of strains with preset characteristics. Characterisation of selected Lactobacillus delbrueckii subsp. bulgaricus texturising strains (versions) is as follows: a) they have pH (measured by standard method) within the range of 4.25-4.55 after 14 days of storage; b) they have texturising properties (measured using viscosity determination analysis) no less than 25 Pa or a) hey reduce pH by at least 0.20 or 0.30 pH units after 14 days of storage at a temperature of 8°C; b) they have texturising properties (measured using viscosity determination analysis) no less than 25 Pa. Invention relates to application of the selected strains for production of fermented dairy products and dairy products containing Lactobacillus delbrueckii subsp. bulgaricus strain with the above characteristics.

EFFECT: invention enables production of a dairy product having more expressed texturising properties with less acid postproduction.

12 cl, 5 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: Saccharomyces cerevisiae yeast strain, which is isolated from fermented milk whey and can be cultured on dairy cheese whey, is deposited in the Russian National Collection of Industrial Microorganisms under number VKPM Y-3414 and is a producer of ethyl alcohol.

EFFECT: invention enables to obtain a microbial strain which is adapted to culturing on dairy cheese whey and solid media and increases output of ethyl alcohol.

1 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a Saccharomyces cerevisiae VKPM Y-3415 yeas strain which produces ethyl alcohol. The strain is adapted to culturing on dairy cheese whey.

EFFECT: strain is capable of producing ethyl alcohol with high output and is antagonistic to accompanying microflora.

1 ex

FIELD: agriculture.

SUBSTANCE: strain Streptomyces cellulosae WH9, deposited in CGMCC under the number NO.2167 and used to produce a microbial fertiliser. The strain Aspergillus versicolor WH13, deposited in CGMCC under the number NO.2171 and used to produce a microbial fertiliser. The microbial phosphate fertiliser contains a product of fermentation of a microbial composition containing the following four microorganisms: a strain Bacillus subtilis WH2 (CGMCC NO.0395.2), a strain Bacillus licheniformis WH4 (CGMCC NO.0395.4), a strain Streptomyces cellulosae WH9 and a strain Aspergillus versicolor WH13. Also the method is provided to manufacture the specified microbial phosphate fertiliser, where production of the specified microbial phosphate fertiliser may include using the ground phosphate rock containing 8%-12% P2O5.

EFFECT: improved properties of the strain.

8 cl, 3 tbl, 8 ex

FIELD: agriculture.

SUBSTANCE: strain Streptomyces cellulosae WH9, deposited in CGMCC under the number NO.2167 and used to produce a microbial fertiliser. The strain Aspergillus versicolor WH13, deposited in CGMCC under the number NO.2171 and used to produce a microbial fertiliser. The microbial phosphate fertiliser contains a product of fermentation of a microbial composition containing the following four microorganisms: a strain Bacillus subtilis WH2 (CGMCC NO.0395.2), a strain Bacillus licheniformis WH4 (CGMCC NO.0395.4), a strain Streptomyces cellulosae WH9 and a strain Aspergillus versicolor WH13. Also the method is provided to manufacture the specified microbial phosphate fertiliser, where production of the specified microbial phosphate fertiliser may include using the ground phosphate rock containing 8%-12% P2O5.

EFFECT: improved properties of the strain.

8 cl, 3 tbl, 8 ex

FIELD: agriculture.

SUBSTANCE: strain Streptomyces cellulosae WH9, deposited in CGMCC under the number NO.2167 and used to produce a microbial fertiliser. The strain Aspergillus versicolor WH13, deposited in CGMCC under the number NO.2171 and used to produce a microbial fertiliser. The microbial phosphate fertiliser contains a product of fermentation of a microbial composition containing the following four microorganisms: a strain Bacillus subtilis WH2 (CGMCC NO.0395.2), a strain Bacillus licheniformis WH4 (CGMCC NO.0395.4), a strain Streptomyces cellulosae WH9 and a strain Aspergillus versicolor WH13. Also the method is provided to manufacture the specified microbial phosphate fertiliser, where production of the specified microbial phosphate fertiliser may include using the ground phosphate rock containing 8%-12% P2O5.

EFFECT: improved properties of the strain.

8 cl, 3 tbl, 8 ex

FIELD: agriculture.

SUBSTANCE: strain Streptomyces cellulosae WH9, deposited in CGMCC under the number NO.2167 and used to produce a microbial fertiliser. The strain Aspergillus versicolor WH13, deposited in CGMCC under the number NO.2171 and used to produce a microbial fertiliser. The microbial phosphate fertiliser contains a product of fermentation of a microbial composition containing the following four microorganisms: a strain Bacillus subtilis WH2 (CGMCC NO.0395.2), a strain Bacillus licheniformis WH4 (CGMCC NO.0395.4), a strain Streptomyces cellulosae WH9 and a strain Aspergillus versicolor WH13. Also the method is provided to manufacture the specified microbial phosphate fertiliser, where production of the specified microbial phosphate fertiliser may include using the ground phosphate rock containing 8%-12% P2O5.

EFFECT: improved properties of the strain.

8 cl, 3 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: recombinant method is used to produce a microorganism of Mycobacterium tuberculosis complex which involves phoP gene inactivation or deletion and fadD26 gene inactivation or deletion. The produced microorganism is used for preventing tuberculosis in humans and animals.

EFFECT: invention allows producing the vaccine microorganism showing the properties of high attenuation and immune protection against tuberculous infection.

7 cl, 27 dwg, 9 ex

FIELD: agriculture.

SUBSTANCE: invention represents reference strains Francisella tularensis of tularensis subtype, including two genetic groups AI and AII, of holartica subtype, including biovars japonica, erythromycin-sensitive and erythromycin-resistant, Erys and EryR, and of mediasiatica subtype. Strains are deposited in the State Collection of Pathogenic Microbes "Microbe". All strains are natural, apart from the strain of holartica subtype, biovar japonica, which is extracted from a human being. Strains are produced by selection of clones that stably preserve phenotypic properties and containing genes, nucleotide sequence of which is specified for the type and subtype. The set of reference DNA preparations is created on the basis of these strains and may be used for genetic and immunological research.

EFFECT: using the invention will make it possible to increase efficiency of diagnostic research, to standardise and to increase quality of diagnostic preparations at production stages.

4 cl, 4 dwg, 1 tbl, 10 ex

FIELD: food industry.

SUBSTANCE: method for production of Lactococcus lactis strain version producing, under standard fermentation conditions, a quantity of vitamin K2 exceeding that produced by the stock/parent bacterial strain inoculated under the same conditions approximately 1.2 times, the said method including, at least: a) inoculation of the sock bacterial strain under standard fermentation conditions in a cultural medium causing a change in the oxidation-reduction state of a cell containing bacitracin or a peroxide and b) selection of the strain version if producing a quantity of vitamin K2 exceeding that produced by the stock/parent bacterial strain inoculated under the same conditions approximately 1.2 times. Lactococcus lactis subsp.cremoris 1-3557 strain deposited in Collection CNCN of Pasteur Institute on 20.01.2006 and Lactococcus lactis subsp.cremoris 1-3558 strain deposited in Collection CNCM of Pasteur Institute on 20.01.2006 produce at least 1.2 times more vitamin K2 than the stock/parent bacterial strain inoculated under the same conditions. Additionally, the invention deals with a lactic acid starter containing at least one of the above strains and to the method for preparation of a cultured milk product containing the above strain and/or the lactic acid starter.

EFFECT: invention enables increase of vitamin K2 content in the product.

11 cl, 5 tbl

FIELD: chemistry.

SUBSTANCE: invention discloses acylamidase enzyme AA37 from Rhodococcus erythropolis 37 All-Russian collection of industrial microorganisms Ac-1793, with a sequence given in the description. A nucleotide sequence which codes this enzyme is defined. The invention describes a method for synthesis of N-substituted acrylamides in aqueous medium from acrylamide and amines in the presence of an acylamidase biocatalyst in isolated state or in a composition with E.coli cells.

EFFECT: invention enables to obtain N-substituted aliphatic acrylamides from acrylamide and primary aliphatic amines in an aqueous medium.

3 cl, 14 ex

FIELD: medicine.

SUBSTANCE: method for producing an Escherichia coli cell fraction showing protease inhibiting activity provides bacteria cells preservation in the presence of buffered 80-90% glycerine, processing by 3% triton X-100 for removing a cell membrane. Produced cytoplasm proteins are extracted by the increasing salt concentration, namely 0.14 M, 0.35 M; 2 M NaCl, 6 M guanidine hydrochloride with 0.1% β-mercaptoethanole. It is followed by affine sepharose 4B chromatography with immobilised trypsin and estimation of protease inhibiting activity in eluates.

EFFECT: invention can be used in analysis of molecular-genetic mechanisms of procaryote cell structure formation and roles of protein components in their organisation, and also genome remodelling nature that is necessary for disclosing of regulation pathways of mechanisms of macro-and microorganism action, and also search of new drug targets and development of ecologically safe new drugs.

3 tbl, 4 dwg

FIELD: medicine.

SUBSTANCE: method of bacterial cell immobilisation provides introduction of bacterial cells Erwinia rhapontici B-9292 in the concentration 10 % in a poly-N-vinylpyrrolidone solution of molecular weight 500 kDa in the concentration 1 mg/ml in an acetate buffer pH=6.0. The received reaction mixture is kept for 3 hours at temperature 22-25°C, and then the immobilised bacterial cells are centrifuged. The isomaltulose yield is 92-95%.

EFFECT: method allows to intensifying the saccharose transformation process and providing higher isomaltulose yield.

1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: Brevibacillus laterosporus bacteria strain, Russian National Collection of Industrial Microorganisms No. B-10531 is produced by multistage selection from natural Brevibacillus laterosporus 16-336 strain. An algicide activity of the strain makes 10.5-52.5 % of residual optical density in 24-h incubation. A diameter of a zone of growth retardation of plant pathogenic fungi makes 3-14 mm. A diameter of a zone of growth retardation of pathogenic bacteria makes 5-12 mm.

EFFECT: strain produces a wide spectrum of biologically active compounds for control of microscopic algae of various taxonomic types, plant pathogenic fungi and pathogenic bacteria.

5 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, namely to preparing physiologically active compounds, and concerns a bacteriorhodopsin producer strain. The Halobacterium salinarum ST 2 strain is produced by multistage selection of a Halobacterium salinarum halophilic bacterial strain, Russian National Collection of Industrial Microorganisms No. B-9025, without chemical pretreatment and is deposited in Russian National Collection of Industrial Microorganisms No. B-10425.

EFFECT: Halobacterium salinarum strain Russian National Collection of Industrial Microorganisms No B-10425 exceeds both a prototype, and a parent strain in efficiency.

2 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: method provides recovery of a diagnostic anthrax allergen directly from a concentrated suspension of vegetative cells of a vaccine strain Bac. anthracis STI-1. The strain is grown on a nutrient medium based on a muriatic hydrolyzate of a fish flour by deep cultivation. The produced bacterial mass is washed in a separator to produce the concentrated suspension of vegetative cells. An allergenic protein fraction is recovered by alkaline hydrolysis, and the prepared hydrolyzate is fractioned. A protein allergen fraction is recovered from a supernatant of the recovered fraction by fractioning with an acetic acid solution; further the precipitation containing an end product is dissolved, dialyzed to produce a purified anthrax protein allergen. The preparation is presented in liquid and lyophilised forms.

EFFECT: method allows higher safety of producing the preparation and reduced length of a technological process, with preserving long-storage activity, effectiveness both in evaluating the anti-anthrax immunity stress, and diagnosing the infection.

4 cl, 4 tbl

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically to obtaining proteolytic fractions from procariotic cells, and can be used in analysis of molecular-genetic mechanisms of formation of procaryote cell structures and the role of protein components in their organisation, which is needed to obtain additional information in design and construction of computer models of organisation of gene and epigene control networks. Escherichia coli cells are preserved in the presence of buffered 80-90% glycerin and then treated with 3% triton X-100 in order to remove the cell membrane. Further, the obtained cytoplasmic proteins are extracted with increasing concentrations of salts: 0.14 M, 0.35 M; 2 M NaCl, 6 M guanidine hydrochloride with 0.1% β-mercaptoethanol. Affinity chromatography on sepharose 4B with immobilised trypsin inhibitor is carried out and proteolytic activity is evaluated.

EFFECT: invention enables to obtain a fraction from procaryote cells.

4 dwg, 3 tbl

FIELD: medicine.

SUBSTANCE: method for preparing isotope-marked secondary metabolic products of fungi provides immobilised fungi growing on an inert carrier with an artificial fluid culture medium added. All carbon, nitrogen and/or sulphur atoms in the specified medium are substituted by stable isotopes chosen from the group including 13C, 15N, 33S and 34S. Also, an isotope-marked secondary metabolic product made of fungi is presented.

EFFECT: high-purity end product preparation.

13 cl, 2 dwg, 9 ex

FIELD: microbiology.

SUBSTANCE: antibacterial substance DM0507 is produced by cultivation of bacteria strain Bacillus subtilis DB9011 (FERM BP-3418), collection of supernatant of produced culture, collection of sediment produced by means of pH regulation up to 3 and less, with further extraction of sediment with ethanol. Antibacterial substance DM0507 is characterised with infrared spectrum and NMR spectrum, which are specified in dwg 1 and dwg 2 accordingly. Antibacterial substance DM0507 has antimicrobial activity against wide spectrum of microorganisms.

EFFECT: antimicrobial agent, food product or drink are produced.

5 cl, 2 dwg, 2 tbl, 10 ex

FIELD: food industry.

SUBSTANCE: invention deal with a method for production of Lactobacillus delbrueckii subsp. bulgaricus strain with reduced lactic acid postproduction. The method involves exposure of Lactobacillus delbrueckii subsp. bulgaricus, mother strain possessing high texturising properties and high acid postproduction to the impact of a weak mutagen represented by ethyl methanesulfonate (EMS) and subsequent selection of strains with preset characteristics. Characterisation of selected Lactobacillus delbrueckii subsp. bulgaricus texturising strains (versions) is as follows: a) they have pH (measured by standard method) within the range of 4.25-4.55 after 14 days of storage; b) they have texturising properties (measured using viscosity determination analysis) no less than 25 Pa or a) hey reduce pH by at least 0.20 or 0.30 pH units after 14 days of storage at a temperature of 8°C; b) they have texturising properties (measured using viscosity determination analysis) no less than 25 Pa. Invention relates to application of the selected strains for production of fermented dairy products and dairy products containing Lactobacillus delbrueckii subsp. bulgaricus strain with the above characteristics.

EFFECT: invention enables production of a dairy product having more expressed texturising properties with less acid postproduction.

12 cl, 5 dwg, 2 ex

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