RU2239654C2 - Liquid nutrient medium for submerged culturing microorganism plesiomonas shigelloides, strain 16 - Google Patents

Abstract

FIELD: biotechnology, microbiology.

SUBSTANCE: invention can be used for preparing bacterial protein. Nutrient medium comprises casein hydrolyzate with the content of amine nitrogen 138 mg% as a base, potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, sodium thiosulfate in the parent concentration of hydrogen ions 7.2 U pH. In culturing microorganism Plesiomonas shigelloides, strain № 16, as a producer of ice-forming protein the latter provides preparing the gain of protein concentration. Medium can be used also for accumulation of bacterial mass from the broad range of microorganisms.

EFFECT: valuable properties of nutrient.

4 tbl, 1 ex

Description

The invention relates to the field of microbiology and can be used to obtain a microbial protein that initiates ice formation.

For deep cultivation of bacteria Plesiomonas shigelloides strain No. 16, known liquid nutrient media are used, for example, enzymatic hydrolyzate of cattle meat, enzymatic hydrolyzate of carcasses of fur animals, acid hydrolyzate of casein, enzymatic hydrolyzate of casein, synthetic liquid nutrient medium.

Known liquid nutrient medium based on an enzymatic hydrolyzate of carcasses of fur-bearing animals ("A method for producing a base of nutrient media for the cultivation of microorganisms". IPC ⁶ C 12 N 1/20, C 12 N 01/04, No. 2061038), which includes the following ratio of components , g · l ^-1 :

C ₆ H ₁₂ O ₆ 20

NaCl 3.0

KCl 0.2

Na ₂ HPO ₄ 6.0

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

initial concentration of hydrogen ions (7.2 ± 0.1) u

A common with the proposed solution is the content of potassium salts: potassium phosphate disubstituted, potassium phosphate monosubstituted, potassium chloride. Sodium salts: sodium sulfate, sodium chloride, in the following ratio of components (g · l) ^-1 :

KH ₂ PO ₄ 2.2

K ₂ HPO ₄ 1.7

Na ₂ S ₂ O ₃ 0.6

Na ₂ HPO ₄ 6.0

KCl 0.2

The disadvantages of this environment include instability and insufficiently high level of results on the accumulation of the concentration of ice-forming protein. In addition, this medium is difficult to manufacture and to obtain it use scarce and expensive raw materials and meat of fur animals, as well as an expensive pancreatic enzyme - pancreatin.

Known liquid nutrient medium based on acid hydrolyzate of casein (Schedule for the production of plague live dry vaccine No. 377-97, entered into force in 1997), having in its composition the following ratio of components, g · l ^-1 :

KH ₂ PO ₄ 2.2

K ₂ HPO ₄ 1.7

MgSO ₄ 0.6

Na ₂ S ₂ O ₃ 0.6

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

the initial concentration of hydrogen ions in the medium 7.15 u

A common with the proposed solution is the presence of potassium salts: potassium phosphate disubstituted, potassium phosphate monosubstituted, salts of sodium and magnesium: magnesium sulfate and sodium sulfate in the following ratio, g · l ^-1 :

KH ₂ PO ₄ 2.2

K ₂ HPO ₄ 1.7

MgSO ₄ 0.6

Na ₂ S ₂ O ₃ 0.6

The disadvantages of this environment include the insufficiently high level of results on the accumulation of the concentration of ice-forming protein. In addition, this medium is difficult to manufacture and expensive reagents and raw materials, in particular hydrochloric acid and milk protein - casein, are used for its preparation.

Known liquid synthetic nutrient medium (Instructions for the preparation and control of a vaccine associated inactivated against colibacteriosis, salmonellosis, Klebsiella and Proteus infection of young farm animals and fur animals / JSC "Agrovet", 1995), which includes the following ratio of components, g l ^-1 :

K ₂ HPO ₄ 2

KN ₂ PO ₄ 5

MgSO ₄ × 7H ₂ O 0.55

FeSO ₄ × 3H ₂ O 0.025

A common with the proposed solution is the presence of potassium salts: potassium phosphate disubstituted, potassium phosphate monosubstituted, magnesium salts - magnesium sulfate, in the following ratio, g · l ^-1 :

K ₂ HPO ₄ 2

KN ₂ PO ₄ 0.5

MgSO ₄ × 7H ₂ O 0.55

The disadvantages of this environment include the high consumption of 50% glucose solution in the process of deep cultivation and a low level of biomass yield. In QOL (culture fluid), as a result, the low level of nucleation centers. In addition, in the manufacture of this medium, scarce reagents are used, in particular magnesium sulphate heptahydrate, iron (II) sulphate trihydrate.

The closest is a liquid nutrient medium based on an enzymatic hydrolyzate of cattle meat (Schedule for production of plague live dry vaccine No. 377-97, put into effect in 1997), which has the following ratio of components, g · l ^-1 :

KH ₂ PO ₄ 2.2

K ₂ HPO ₄ 1.7

MgSO ₄ 0.6

Na ₂ S ₂ O ₃ 0.6

the content of amino nitrogen in the medium of 140 mg%;

the initial concentration of hydrogen ions in the medium is 7.15 units of pH

A common with the proposed solution is the presence of potassium salts: potassium phosphate disubstituted, potassium phosphate monosubstituted, salts of sodium and magnesium: magnesium sulfate and sodium sulfate in the following ratio, g · l ^-1 :

KH ₂ PO ₄ 2.2

K ₂ HPO ₄ 1.7

MgSO ₄ 0.6

Na ₂ S ₂ CO ₃ 0.6

the content of amino nitrogen in the medium of 140 mg%;

the initial concentration of hydrogen ions of the medium is 7.05 u.RN (Schedule for the production of plague live dry vaccine No. 377-97, entered into force in 1997).

The disadvantages of this environment include instability and insufficiently high level of results on the accumulation of biomass and concentration of ice-forming protein. In addition, this medium is difficult to manufacture and scarce and expensive raw materials, in particular enzymatic meat hydrolyzate, are used for its production.

Enzymatic meat hydrolyzate is a source of nitrogen and native protein, prevents the dissociation of microorganisms, potassium phosphate one and disubstituted maintain a buffer balance, the concentration of hydrogen ions, the presence of magnesium sulfate and sodium sulfate ensures the presence of Mg ions in the medium and prevents cell destruction.

The essence of the invention lies in the fact that a liquid nutrient medium based on enzymatic hydrolyzate of casein for deep cultivation of bacteria Plesiomonas shigelloides strain No. 16, containing the corresponding protein base, the most optimal salt composition, as well as the concentration of amine nitrogen and the initial concentration of hydrogen ions in the medium allows to obtain an increase concentration of ice-forming protein at:

4.65 × 10 ¹⁰ cps × cm ^-3 ;

4.74 × 10 ⁹ cpx cm ^-3 ;

4.62 × 10 ¹⁰ cpx cm ^-3 .

The specified technical result in the implementation of the invention is achieved due to the fact that the liquid nutrient medium for the deep cultivation of bacteria Plesiomonas shigelloides strain No. 16, containing salts: potassium phosphate disubstituted, potassium phosphate monosubstituted, magnesium sulfate, sodium sulfate, contains enzymatic hydrolysis as the basis of the nutrient medium with an amine nitrogen content of 138 mg% and an initial concentration of hydrogen ions of 7.2 units. pH at the following component content, g / l:

KN ₂ RO ₄ 2.2

K ₂ HPO ₄ 1.7

MgSO ₄ 0.6

Na ₂ S ₂ O ₃ 0.6

enzymatic

casein hydrolyzate rest

Cheaper environment is achieved by replacing an expensive enzymatic meat hydrolyzate and using cheaper raw materials instead - casein enzymatic hydrolyzate. The comparative cost of 1 dm ^{3 of the} finished medium is shown in table 1.

Preservation of the growth properties of the nutrient medium is due to the presence in the nutrient base of a balanced composition of carbohydrates and mineral salts.

The cultivation of bacteria of the species Plesiomonas shigelloides strain No. 16 is carried out in a biological reactor, in the mode of constant mixing and aeration. The proposed environment contains readily available raw materials and reagents of domestic production, is easy to manufacture and provides a high yield of concentration of microbial protein active in LOA (ice-forming activity). Under the influence of the ingredients of the proposed nutrient medium, the structure of cell membranes improves, the consumption of substances from the nutrient solution increases, the metabolism improves due to the direct effect on the enzyme systems of the cells, oxidation-reduction processes, the phenomena of self-agglutination and lysis of bacterial cells at the final stages of cultivation are prevented, the stability of bacterial cells is increased cells to sharp fluctuations in the initial concentration of hydrogen ions in the medium. The ingredients of the medium are capable of binding bacterial toxins, products of cellular metabolism, and decay products of dying cells. The selected protein nutritional base has significantly greater nutritional value for this type of bacteria and is easier to digest. In addition, the components of the nutrient medium in their optimal ratio prevent the formation of a dense precipitate during long-term storage of finished products, in particular obtained NK (native culture) during its deposition. All of the above allows one to obtain NK (native culture) with fairly high results on the accumulation of an increase in the concentration of ice-forming protein, compared with the prototype and with a higher content of living cells having an active protein component on the outer membrane.

The choice of nutrient medium, the concentration of amine nitrogen, the concentration of hydrogen ions, the cultivation time were determined empirically on the basis of the results of cultivation in flasks with a capacity of 250 cm ³ on a shuttle machine and as a result of growing in the apparatus of the company "Marubishi" model MD-400 with a capacity of 0.007 m ³ .

When preparing the seed (SPRK-stabilized preparation of the working culture), (PRK-production working culture), dry lyophilized culture in ampoules and vials, when grown on flasks with a capacity of 250 cm ³ and in apparatus with a capacity of 0.007 m ³ , the detection and selection of colonies was the standard bacteriological technique was used, consisting in the germination of the colonies of the studied strain on a solid nutrient medium (the basis is the enzymatic hydrolyzate of cattle meat), followed by reseeding in liquid nutrient medium into vials (the base is an enzymatic hydrolyzate of cattle meat), and then on a beveled dense nutrient medium in mattresses (the base is an enzymatic hydrolyzate of cattle meat). The resulting agar culture was washed off with a specific solution, a stabilizing liquid and a protective medium, depending on the purpose of preparation and use of seed. In the obtained at certain stages of sowing crops, the content of extraneous microflora is not allowed.

As a result of conducting planned experiments on the cultivation of bacteria Plesiomonas shigelloides strain No. 16 in flasks with a capacity of 250 cm ³ on the shuttle apparatus, the following data were obtained. The maximum growth rate of the concentration of ice-forming protein was established when the content of amine nitrogen in the liquid nutrient medium was 138 mg% and the culture was grown for (18 ... 30) hours regardless of the initial concentration of hydrogen ions in the liquid nutrient medium in the range from (6, 8 ... 7.7) In the process of growing under aseptic conditions, samples were taken at the growth hours determined by the experimental design.

In the selected samples were determined by such key indicators as:

OK (optical concentration billion microcl. × cm ^-1 );

Bq _h (biological concentration in Petri dishes billion live microl.cl.s.cm ^-1 );

LOA (ice-forming activity in c.n. × cm ^-3 );

pH (concentration of hydrogen ions, pH units);

the presence of extraneous microflora (presence or absence).

Based on the study of experimental results, the highest LOA values (ice-forming activity) were obtained: when cultured in flasks (nutrient base — casein enzymatic hydrolyzate — N _am — 138 mg%; pH — 7.0 units) 6.82 × 10 ⁹ cn × cm ^-3 , in comparison with a liquid nutrient medium based on an enzymatic meat hydrolyzate and casein acid hydrolyzate.

Indicators of ice-forming activity for these environments were: the nutrient base is an enzymatic meat hydrolyzate (ice-forming activity - 6.93 × 10 ⁴ tsn. Cm ^-3 ; N _am . - 146 mg%; pH - 7.1 units).

The nutrient base is casein acid hydrolyzate (ice-forming activity - 3.79 c.n. × cm ⁴ ; N _am - 146 mg%; pH - 7.0 units).

With a decrease in the duration of cultivation in a liquid nutrient medium based on an enzymatic hydrolyzate of casein (N _am - 138 mg%, pH of the medium - 7.15 units), ice-forming activity indicators of 5.50 × 10 ⁹ c.n. were obtained. × cm ^-3 and 1.25 × 10 ⁹ tsn × cm ^-3 .

In comparison with a liquid nutrient medium based on an enzymatic meat hydrolyzate (N _am - 140 mg%, pH of the medium - 7.7 units), the ice-forming activity index was 1.38 × 10 ⁶ and 5.29 × 10 ⁷ tsn × cm ^-3 ). OK (optical concentration of billion microcl. × cm ^-1 ) was determined by diluting the culture sample with 0.9% sodium chloride solution and comparing the result with the optical standard for 10 turbidity units GISK them. L.A. Tarasovich. BC _h (biological concentration of billion living microcl. × cm ^-1 ) was determined using standard bacteriological methods by plating on Petri dishes with a dense nutrient medium (the basis of the enzymatic meat hydrolyzate). The presence of extraneous microflora was recorded using Gram-stained smears, which are used to assess the purity of the bacterial mass. The reaction of the culture medium was determined using a universal ionomer. The indicator of ice-forming activity is taken into account by analysis on a temperature-controlled minicryostat MK-70.

Example. A liquid nutrient medium for the cultivation of bacteria Plesiomonas shigelloides strain No. 16 is prepared as follows. The calculated amount of distilled water and hydrolyzate is poured into the digester to the amine nitrogen content (145 ± 15) mg%. The components are mixed, boiled until the flocculent precipitate completely disappears and a sample is taken to control and adjust the amount of amine nitrogen (138 ± 2) mg%. If necessary, add water or hydrolyzate, the analysis is repeated. A portion of salts is added and boiled until the salts are completely dissolved, after which a sample is taken to control and adjust the concentration of hydrogen ions in the medium within (7.1 ± 0.1) p.N. The pH adjustment is carried out with a 10% solution of Hcl or NaOH.

The hot medium is filtered through brand “T” cardboard on an F-52 nutsche filter, packaged in bottles with a capacity of 0.1 dm ³ for (50 ± 5) cm ³ and bottles with a capacity of 20 dm ³ for (10 ± 0.5) dm ³ . After filtration, environmental control is performed. Vials are closed with cotton-gauze plugs. Defoamer VM-5 (prescription No. 5, production schedule for plague live dry vaccine No. 377-97, entered into force in 1997) is introduced into each bottle with a capacity of 20 dm ³ with the medium, at the rate of 1 cm ³ × l ^-1 . The bottle is equipped with appropriate equipment. The normalized operation time is (1.5 ... 2) hours.

Environmental control. The liquid nutrient medium in appearance is a dark yellow liquid that does not contain impurities, a small precipitate is allowed. According to its physical and chemical properties, it must satisfy the following requirements:

the content of amine nitrogen, mg% - 120 ± 10 (determined during the preparation of the broth, operation TP-5-1, production schedule for the plague live dry vaccine No. 377-97, entered into force in 1997);

concentration of hydrogen ions, unit pH - 7.1 ± 0.1.

Control methods are set out in section 13 (Schedule for the production of the plague live dry vaccine No. 377-97, entered into force in 1997).

When performing the control, one should be guided by the requirements set forth in the "Collection of safety instructions". The normalized operation time is 0.5 hours (Regulation for the production of the plague live dry vaccine No. 377-97, entered into force in 1997).

Operation TP-5-4. Sterilization (Schedule for the production of the plague live dry vaccine No. 377-97, entered into force in 1997).

Vials and bottles of medium are sterilized in an autoclave according to the following conditions: temperature (120 ... 125) ° С, exposure (30 ... 35) min. Sterile culture medium can be stored at a temperature of (18 ... 25) ° C for no more than 15 days. At a temperature of (2 ... 6) ° C no more than 30 days. When sterilizing the environment, it is necessary to be guided by the requirements set forth in the "Collection of safety instructions." The normalized operation time is 3, regulated by 0.5 hours (Regulation for the production of plague live dry vaccine No. 377-97, entered into force in 1997).

A sterile nutrient medium is evaluated only by its appearance, which must meet the following requirements: dark yellow liquid with opalescence with an antifoam introduced, which does not contain foreign matter, a small precipitate is allowed. Appearance is assessed by visual inspection of closures with a sterile culture medium. The normalized operation time is 0.5 hours.

When receiving inoculum cultures of bacteria Plesiomonas shigelloides strain No. 16 for deep cultivation in apparatuses with a capacity of 0.007 m ³ use the bacteriological method described above for flasks with a capacity of 250 cm ³ . Extraneous microflora is not allowed in the crops obtained at certain stages of the crop. The resulting seed culture on a solid nutrient medium (the nutrient base is an enzymatic meat hydrolyzate) in Petri dishes should have an optical concentration, after the end of the incubation period when washing with a 0.9% sodium chloride solution, at least 1 × 10 ⁹ billion microl.cl.s.cm ^-1 , no foreign cells should be contained in the field of view of the smear. The seed culture obtained in a liquid nutrient medium in vials (the nutrient base is an enzymatic meat hydrolyzate) should have an optical concentration after the end of the incubation period, at least 1 × 10 ⁹ billion microcl. × cm ^-1 , in the field of view of the smear should not contained no extraneous cells. The sowing culture obtained as a result of washing away from the mowed dense nutrient medium in mattresses (the nutrient base is an enzymatic meat hydrolyzate) should have an optical concentration after the end of the incubation period, at least 1 × 10 ¹¹ billion microl.cl.s.cm ^-1 , in the field of view the smear should not contain a single foreign cell. The culture fluid that meets the above requirements is used as seed for the fermenter MD-400, with a capacity of 0.007 m ³ . The cultivation is carried out in a medium prepared on the basis of an enzymatic hydrolyzate of casein with N _am . - 138 mg% and the initial pH of the medium is 7.2 units. After the introduction of glucose and throughout the growing process, the pH adjustment is carried out by adding 20% aqueous ammonia solution and 43% glucose solution. Cultivation is carried out with constant mechanical stirring and aeration at the rate of 0.1 vol. (rpm) ^-1 .

During the growing process, samples are taken under aseptic conditions and the main biological indicators are determined. The list of defined indicators and methods for their determination are described above during the cultivation of bacteria Plesiomonas shigelloides strain No. 16 on flasks with a capacity of 250 cm ³ on a shuttle apparatus.

At the end of cultivation, the obtained NK (native culture) must meet the following requirements:

ice nuclei concentration, active at minus 9 ° C × ts.n. cm ^-3 - not less than 1 × ¹⁰ September;

optical concentration, (OK) billion micro-cells × cm ^-1 - not less than 10;

biological concentration when grown on Petri dishes (Bq _h ), billion live microl.cl. × cm ^-1 - not less than 3;

concentration of hydrogen ions, pH units - in the range of 6.6 ... 7.6;

the content of extraneous microflora is not allowed.

At the end of the growing process, the purity of the obtained biomass is monitored by microscopy of Gram stained smears by inoculation into test tubes with a beveled solid nutrient medium based on an enzymatic meat hydrolyzate, as well as on Petri dishes with a dense nutrient medium based on an enzymatic meat hydrolyzate.

At the same time, such basic biological indicators were determined as:

optical concentration, (OK) - billion micro-cells × cm ^-1 ;

biological concentration when grown on Petri dishes (Bq _h ) - billion living microl.cl. × cm ^-1 ;

LOA (ice-forming activity) - c.n. × cm ^-3 ;

concentration of hydrogen ions - units rN;

the presence of extraneous microflora (presence or absence).

Thus, the use of a liquid nutrient medium based on an enzymatic casein hydrolyzate allows one to obtain a bacterial mass with a rather high increase in the concentration of ice-forming protein. This medium can be used to cultivate the bacterial mass of a wide range of microorganisms. Using the proposed medium allows to simplify the composition of the medium and reduce the cost of its production technology by replacing the expensive and scarce enzymatic hydrolyzate of cattle meat with an enzymatic casein hydrolyzate.

Claims (1)

Liquid nutrient medium for the deep cultivation of bacteria Plesiomonas shigelloides strain No. 16, containing a nutrient base, disubstituted potassium phosphate, monosubstituted potassium phosphate, magnesium sulfate, sodium sulfate, characterized in that it contains enzymatic nitrogenous hydrolyzate casein 138 as the basis of the nutrient medium mg% and the initial concentration of hydrogen ions of 7.2 units pH at the following component content, g / l: Potassium phosphate disubstituted 2.2 Potassium phosphate monosubstituted 1.7 Magnesium Sulfate 0.6 Sodium sulfate 0.6 Casein Enzymatic Hydrolyzate with an amine nitrogen content of 138 mg% and the initial concentration of hydrogen ions of 7.2 units pH rest