Liquid nutrient medium for submerged cultivation of bacteria plesimonas shigelloides strain no. 16

 

(57) Abstract:

The invention relates to biotechnology and can be used for microbial protein. Nutrient medium as the basis contains the enzymatic hydrolysate of casein with the content of amino nitrogen 138 mg %, potassium disubstituted phosphate, potassium phosphate one-deputizing, magnesium sulfate, sodium Chernovetskiy, initial concentration of hydrogen ions pH of 7.2. When growing bacteria Plesiomonas shigelloides strain No. 16 - producer Loubressac protein, the latter allows to increase the concentration of protein at the level 6,82·109C.n.·cm-3. The environment can be used for the accumulation of bacterial mass of a wide range of microorganisms. 4 table.

The invention relates to the field of Microbiology, and can be used to produce microbial protein, initiating the ice.

For submerged cultivation of bacteria Plesiomonas shigelloides strain No. 16 using known liquid nutrient medium, for example enzymatic hydrolysate of bovine meat, enzymatic hydrolysate of carcasses of fur-bearing animals, acid hydrolysate of casein, enzymatic hydrolysate of casein, this is ralista carcasses of fur-bearing animals ("Method for obtaining basics of nutrient media for cultivation of microorganisms. IPC612 N 1/20, 12 N 01/04, No. 2061038), having in its composition the following ratio of components, g· l-1:

WITH6H12ABOUT620

NaCl 3,0

KCl And 0.2

Na2HPO46,0

the content of amine nitrogen in the environment (70... 120) mg %;

initial concentration of hydrogen ions (7,20,1) pH units

In common with the claimed solution is the content of potassium: potassium disubstituted phosphate, potassium phosphate one-deputizing, potassium chloride. Sodium salt: sodium Chernovetskiy, sodium chloride, in the following ratio of components (g· l)-1:

KN2RHO42,2

TO2NRA41,7

PA2S2O30,6

Na2HPO46,0

KCl And 0.2

The disadvantages of this environment should include the instability and low level results in accumulation of concentrations Loubressac protein. In addition, this environment is complicated to manufacture and to obtain use of scarce and expensive raw materials-meat, fur-bearing animals, as well as an expensive enzyme of the pancreas - Pancreatin.

Known liquid nutrient medium on the basis of acid gidrolizom composed of the following ratio of components, g· l-1:

KN2RHO42,2

TO2NRA41,7

MgSO40,6

Na2S2O30,6

the content of amine nitrogen in the environment 138 mg %;

initial concentration of hydrogen ions in the medium 7,15 pH unit.

In common with the claimed solution is the presence of potassium salts: potassium disubstituted phosphate, potassium phosphate one-deputizing, salts of sodium and magnesium: magnesium sulfate and sodium Chernovetskiy in the following ratio of components, g· l-1:

KN2RHO42,2

TO2NRA41,7

MgSO40,6

PA2S2O30,6

The disadvantages of this environment should include not high enough level results in accumulation of concentrations Loubressac protein. In addition, this environment is complicated to manufacture and to obtain use of expensive reagents and raw materials, in particular hydrochloric acid, and milk protein - casein.

Known liquid synthetic culture medium (instructions for the preparation and control of vaccine associated inactivated against colibacillosis, salmonellosis, Klebsiella and proteaceae infection of young animals villages of the components, g· l-1:

TO2NRA42

KN2RHO45

MgSO4×7H2O 0,55

FeSO4×3H2O 0,025

In common with the claimed solution is the presence of potassium salts: potassium disubstituted phosphate, potassium phosphate one-deputizing, magnesium salt is magnesium sulfate as, in the following ratio of components, g· l-1:

K2HPO42

KN2RHO40,5

gSO4×7H2ABOUT 0,55

The disadvantages of this environment include high consumption of 50% glucose solution in the process of deep cultivation and low biomass yield. In QOL (culture liquid) as a consequence, low centers of nucleation. In addition, when the manufacturing environment using scarce reagents, in particular magnesium sulfate as, iron (II) sulfate trihydrate.

The closest is a liquid nutrient medium on the basis of enzymatic hydrolysate of bovine animals (Regulation of production of the vaccine plague live dry No. 377-97, enacted in 1997), which has in its composition the following component ratio is>P CLASS="ptx2">PA2S2ABOUT30,6

the content of amine nitrogen in the environment 140 mg %;

initial concentration of hydrogen ions in the medium - 7,15 pH

In common with the claimed solution is the presence of potassium salts: potassium disubstituted phosphate, potassium phosphate one-deputizing, salts of sodium and magnesium: magnesium sulfate and sodium Chernovetskiy in the following ratio of components, g· l-1:

KN2RHO42,2

TO2NRA41,7

MgSO40,6

Na2S2CO30,6

the content of amine nitrogen in the environment 140 mg %;

initial concentration of hydrogen ions in the medium pH 7,05 (Regulation of production of the vaccine plague live dry No. 377-97, enacted in 1997).

The disadvantages of this environment should include the instability and low level results in accumulation of biomass and concentration Loubressac protein. In addition, this environment is complicated to manufacture and to obtain use of scarce and expensive raw materials, in particular enzymatic hydrolysate meat.

The enzymatic hydrolysate meat is a source of nitrogen and a native protein, prevents dissol the hydrogen ions, the presence of magnesium sulfate and sodium servational ensures the presence in the environment MD ions and prevents the destruction of cells.

The invention consists in that the liquid medium on the basis of enzymatic hydrolysate of casein for submerged cultivation of bacteria Plesiomonas shigelloides strain No. 16, containing appropriate protein basis, the optimum salt composition and concentration of amino nitrogen and the initial concentration of hydrogen ions in the environment allows to increase the concentration Loubressac protein level:

4,65× 1010C.n.× cm-3;

4,74× 109C.n.× cm-3;

4,62× 1010C.n.× cm-3.

This technical result in the implementation of the invention is achieved due to the fact that the liquid nutrient medium for submerged cultivation of bacteria Plesiomonas shigelloides strain No. 16, containing salts: potassium disubstituted phosphate, potassium phosphate one-deputizing, magnesium sulfate, sodium Chernovetskiy, as the basis of the nutrient medium contains an enzymatic hydrolysate of casein with the content of amino nitrogen 138 mg % and the initial concentration of hydrogen ions 7>1,7

MgSO40,6

Na2S2O30,6

enzymatic

casein hydrolysate else

The cheapening of the environment is achieved by replacing costly enzymatic hydrolysate meat and use instead of cheaper raw materials - enzymatic hydrolysate of casein. Comparative cost of 1 DM3ready environment are shown in table 1.

Maintaining the growth properties of the nutrient medium due to the presence of nutrients through a balanced composition of carbohydrates and mineral salts.

Cultivation of bacteria of the species Plesiomonas shigelloides strain No. 16 is carried out in a biological reactor, in a continuous mixing and aeration. The proposed environment contains readily available raw materials and reagents domestic production, easy to manufacture and provides high output concentration of microbial protein active LOA (Loubressac activity). Under the influence of the ingredients of the nutrient medium improves the structure of cell membranes, increases the consumption of nutrients from the nutrient solution, improves metabolism through direct effects on the enzyme systems of the cell, acilitators stages of cultivation, increasing resistance of bacterial cells to sharp fluctuations of the initial concentration of hydrogen ions in the environment. Ingredients medium capable of binding bacterial toxins, products of cellular metabolism, the breakdown products of dying cells. Selected protein nutritional Foundation has much more nutritional value for this species of bacteria and easier to digest. In addition, the components of the nutrient medium in their optimal ratio prevents the formation of a dense precipitate during long-term storage of finished products, including the NC (native culture) during its deposition. All the above allows to obtain NC (native culture) with high enough, results in the accumulation of growth concentration Loubressac protein, compared with the prototype and with a higher content of living cells, with the outer membrane active protein component.

The choice of nutrient medium, the concentration of amino nitrogen, the concentration of hydrogen ions, the duration of cultivation determined empirically on the basis of the results of cultivation in flasks with a capacity of 250 cm3on shuttel-apparatus and growing in paratabulation preparation of the working culture), (IRC-production culture), dry libfilesystem culture in ampoules and vials, when grown in flasks with a capacity of 250 cm3and in the apparatus with a capacity of 0,007 m3detection and selection of colonies was used standard bacteriological technique, consisting in the germination of colonies studied strain on the solid nutrient medium (base - enzymatic hydrolysate of bovine meat), followed by subculture in liquid nutrient medium in the vials (base - enzymatic hydrolysate of bovine meat), and then beveled dense nutrient medium in mattresses (base - enzymatic hydrolysate of bovine meat). The obtained agar culture were washed in a certain solution, the stabilizing liquid and protective environment depending on the purpose of the preparation and use of seeds. To get at certain stages crops the content of extraneous microflora is not allowed.

As a result of the planned experiments on the cultivation of bacteria Plesiomonas shigelloides strain No. 16 in flasks with a capacity of 250 cm3on shuttel machine, the following data were obtained. Was installed m the de amine nitrogen level 138 mg% and growing culture within (18... 30) hours regardless of the initial concentration of hydrogen ions in a liquid nutrient medium in the interval from (6,8 7,7...) pH unit. In the process of growing in aseptic conditions was carried out sampling in certain plan of experiment hours of growth.

In the samples was determined such key indicators as:

OK (optical concentration billion MIC.CL.× cm-1);

BQh(biological concentration on Petri dishes billion live MIC.CL.HSM-1);

LOA (Loubressac activity in C. n.× cm-3);

pH (concentration of hydrogen ions, pH);

the presence of extraneous microflora (presence or absence).

Based on the analysis of experimental results the highest values on LOA (Loubressac activity) were obtained under cultivation in flasks (nutrient base - enzymatic hydrolysate of casein - Nam- 138 mg %; pH of 7.0 units) 6,82× 109C.n.× cm-3in comparison with the liquid nutrient medium on the basis of enzymatic hydrolysate meat and acid hydrolysate of casein.

Indicators Loubressac activity for these environments were: nutrient base - enzymatic hydrolysate m the test basis acid hydrolyzed casein (Loubressac activity - 3,79 kg.n.× cm4; Nam- 146 mg %; pH of 7.0 units).

When reducing the duration of cultivation in liquid nutrient medium on the basis of enzymatic hydrolysate of casein (Nam- 138 mg %, pH - 7,15%) were obtained indicators Loubressac activity - 5,50× 109C.N. × cm-31.25× 109C.n.× cm-3.

In comparison with the liquid nutrient medium on the basis of enzymatic hydrolysate meat (Nam140 mg %, pH of 7.7 units) figure Loubressac activity amounted to 1.38× 106and of 5.29× 107C.n.× cm-3). OK (optical concentration billion MIC.CL.× cm-1) was determined by diluting the sample culture of 0.9% sodium chloride solution and comparing the result with the optical standard 10 units turbidity gisk named after. L. A. Lytvyn. BQh(biological concentration billion live MIC.CL.× cm-1) were determined using standard bacteriological methods by plating on Petri dishes with nutrient dense environment (base enzymatic hydrolysate meat). The presence of extraneous microflora were recorded using a Gram-stained smear, which is used to assess the purity of the bacterial mass. The reaction is itawamba by conducting analysis on minciotti MK-70 with adjustable temperature.

Example. Liquid nutrient medium for culturing bacteria Plesiomonas shigelloides strain No. 16 was prepared as follows. In the digester pour the calculated amount of distilled water and hydrolyzed to the amine content of nitrogen (14515) mg %. Components are mixed, boiled until complete disappearance of flocculent sludge and select the sample for the control and adjustment of the magnitude of the amine nitrogen (1382) mg %. If necessary, add water or hydrolyzed, the analysis is repeated. Add a portion of salt and boil until dissolved salts, then select the sample for the control and adjustment of the concentration of hydrogen ions in the environment within (7,10,1) pH unit. The pH adjustment spend 10% solution of Hcl or NaOH.

A hot medium is filtered through a cardboard mark "T" on the suction filter f-52, Packed in vials of 0.1 DM3according to (505) cm3and bottles with a capacity of 20 DM3(100,5) DM3. After filtering, they control the environment. The bottles are closed with a cotton-gauze plugs. Into each bottle with a capacity of 20 DM3with the environment enter the antifoam VM-5 (division No. 5, regulation of production of the vaccine plague live dry No. 377-97, enacted in 1997) is based on 1 cm3&) PM

The control environment. Liquid nutrient medium in appearance is a liquid dark yellow color, does not contain extraneous inclusions, slight precipitate. According to its physico-chemical properties it must satisfy the following requirements:

the content of amino nitrogen, mg % - 12010 (determined at the preparation, operation TP-5-1, regulation of the production of the vaccine plague live dry No. 377-97, enacted in 1997);

the concentration of hydrogen ions, pH - 7,10,1.

Control methods described in section 13 (Regulation of production of the vaccine plague live dry No. 377-97, enacted in 1997).

When performing control should be guided by the requirements set forth in the Compilation of safety instructions". Normalized operation time is 0.5 h (Regulation of production of the vaccine plague live dry No. 377-97, enacted in 1997).

Operation TP-5-4. Sterilization (Regulation of production of the vaccine plague live dry No. 377-97, enacted in 1997).

Vials and bottles with the medium is sterilized in an autoclave according to the mode: temperature (120... 125)° C, exposure time (30... 35) minutes of Sterile nutrient medium may acii environment should be guided by the requirements set forth in the "Handbook on security". Normalized operation time is 3 regulated 0.5 h (Regulation of production of the vaccine plague live dry No. 377-97, enacted in 1997).

Sterile nutrient medium is evaluated only in appearance, which must meet the following requirements: liquid dark yellow light when the antifoam, does not contain extraneous inclusions, slight precipitate. Appearance assessed by visual examination of youporon with sterile nutrient medium. Normalized operation time is 0.5 hours

Upon receipt of the crop of bacteria Plesiomonas shigelloides strain No. 16 for submerged cultivation in vehicles with a capacity of 0,007 m3use of bacteriological methods described above for flasks with a capacity of 250 cm3. To get at certain stages crops the presence of extraneous microflora is not allowed. Received sowing culture on solid medium (nutrient base - enzymatic hydrolysate meat) in Petri dishes must have optical concentration, after the end of the incubation period when flushing with 0.9% solution of chlorite is storona cells. Sowing culture obtained in the liquid nutrient medium in vials (nutrient base - enzymatic hydrolysate meat) must have optical concentration after an incubation period of not less than 1× 109billion MIC.CL.× cm-1in the field of view of the smear should not contain any extraneous cells. Sowing culture obtained by flushing with beveled dense nutrient medium in mattresses (nutrient base - enzymatic hydrolysate meat) must have optical concentration after an incubation period of not less than 1× 1011billion MIC.CL.HSM-1in the field of view of the smear should not contain any extraneous cells. The culture fluid fulfill the above requirements, used as seed material for the fermenter MD-400, with a capacity of 0,007 m3. The cultivation is carried out in the environment, prepared on the basis of enzymatic hydrolysate of casein with Nam. - 138 mg % and the initial pH of the medium was 7.2% After administration of glucose during the entire process of raising the pH adjustment is carried out by adding 20% aqueous solution of ammonia and 43% glucose solution. The cultivation is carried out at constant mechanical AC is a mini sample and determine the main biological indicators. The list of defined indicators and methods of their determination are described above under cultivation of bacteria Plesiomonas shigelloides strain No. 16 on the flasks with a capacity of 250 cm3on shuttel machine.

After culturing the obtained NC (native culture) must meet the following requirements:

concentration loopazoid nuclei active at temperatures of minus 9° C, C.n.× cm-3- not less than 1× 109;

optical concentration, (OK) billion MIC.CL.× cm-1not less than 10;

biological concentration when grown on Petri dishes (BCh), a billion lives.a MIC.CL.× cm-1- not less than 3;

the concentration of hydrogen ions, the pH is in the range of 6.6... 7,6;

the content of extraneous microflora is not allowed.

At the end of the growing process they control the purity of the obtained biomass by microscopy of smears stained by Gram, by sowing in a test tube with a beveled dense nutrient medium on the basis of enzymatic hydrolysate of meat, as well as on Petri dishes with nutrient dense environment based on enzymatic hydrolysate meat.

Simultaneously determined such basic biological indicators, as is grown on Petri dishes (BCh) - a billion lives.a MIC.CL.× cm-1;

LOA (Loubressac activity) - TS.n.× cm-3;

the concentration of hydrogen ions is pH;

the presence of extraneous microflora (presence or absence).

Thus, the use of liquid nutrient medium on the basis of enzymatic hydrolysate of casein allows you to get a bacterial mass with a relatively high level of growth concentration Loubressac protein. This environment can be used for culturing bacterial mass of a wide range of microorganisms. The use of the proposed environment can simplify the composition of the medium and to reduce the cost of technology of its receipt by replacing expensive and scarce enzymatic hydrolysate of bovine meat in the enzymatic hydrolysate of casein.

Liquid nutrient medium for submerged cultivation of bacteria Plesiomonas shigelloides strain No. 16, containing a nutrient basis, potassium disubstituted phosphate, potassium phosphate one-deputizing, magnesium sulfate, sodium Chernovetskiy, characterized in that it contains as basic nutritional environment fer is 7,2% the pH when the following component, g/l:

Potassium phosphate disubstituted 2,2

Potassium phosphate one-deputizing 1,7

Magnesium sulfate 0,6

Sodium Chernovetskiy 0,6

Enzymatic hydrolysate of casein

with the content of amino nitrogen 138 mg%

and the initial concentration of hydrogen

ions of 7.2 pH units Rest



 

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FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea.

EFFECT: valuable medicinal properties of strain.

5 cl, 8 dwg, 10 ex

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