RU2803269C1 - Nutrient medium for the cultivation of pasteurella in industrial bioreactors - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: nutrient medium for cultivating Pasteurella containing CMB pasteurellous digest - 120 l, HCl c.p. - 0.6 l, peptone - 4 kg, NaCl - 0.9 kg, KH₂PO₄ (aq.) - 0.8 kg, Na₂HPO₄ * 12H₂O - 4 kg, cysteine - up to 0.8 kg, yeast extract 25% - 10 l, MgSO₄ * 7H₂O - 400, NaOH, 20% - up to pH 7.8-8.0, water - up to 800 l, providing an output/yield of Pasteurella cells of at least 10 billion cells per 1 ml with a reproducible result in at least four out of every five fermentations, without losing the immunogenic properties of the resulting vaccines.

EFFECT: enhancement of production.

1 cl, 1 tbl, 7 ex

Description

The invention relates to biotechnology and can be used in the industrial production of live and inactivated vaccines against animal pasteurellosis.

The compositions of nutrient media for the cultivation of Pasteurella have been well studied at the laboratory and industrial levels.

The Vaccine against streptococcosis and nutria pasteurellosis is known according to patent No. 2099084, copyright holder Viktor Arsentievich Esepenok et al., A61K 39/116, publ. 12/20/1997, where grown cultures of streptococci and pasteurella are combined in a 1:1 ratio and a 0.3% formaldehyde solution is added. After the inactivation period has expired and the required results of monitoring the purity of growth have been obtained, an adjuvant is added to the bacterial mass - a 3% solution of aluminum hydroxide at the rate of 15% to the volume of the culture. The second adjuvant saponin is added at the rate of 100 mg per 1 liter of biomass. Adjuvants are added with continuous shaking, the culture medium used is glucose 1.0 2.0, formalin 0.3 0.4, aluminum hydroxide 10 15, saponin the rest.

The disadvantages of the known solution are:

The concentrations of microorganisms obtained according to the patent range from ~2.5-3, but below 10-15 billion cells/ml of nutrient medium, which is lower than the concentrations obtained according to the proposed composition of the nutrient medium in the claimed composition.

A known method for producing a vaccine against pasteurellosis, according to patent No. 2129015, All-Russian Research and Technological Institute of Biological Industry, C12N 1/20, publ. 04/20/1999, where the process of growing pasteurella is carried out using a continuous-flow chemostat method, i.e. with the continuous supply of certain quantities of nutrient medium and glucose solution to the fermenter, and also the continuous draining from the apparatus of the same amount of bacterial suspension, which is used at subsequent stages of vaccine production. Cultivation of the Pasteurella vaccine strain in a liquid nutrient medium based on meat hydrolyzate with pH regulation at 7.8 - 8.2 units, and the partial pressure of oxygen dissolved in the culture liquid at 30 - 40%, and the initial concentration of glucose in the nutrient medium is 1 - 5 g/l at a dilution rate of 0.5 - 0.9 1/hour so that the total volume of nutrient medium and glucose solution entering the fermenter in 1 hour is 0.5-0.9 of the volume of suspension in the apparatus, and the concentration of glucose in the incoming nutrient the environment was in the range of 1-5 g/l.

The disadvantages of the known solution are:

a) high probability of contamination (contamination with foreign microflora) of devices (fermenters);

b) a high frequency of mutations in the pathogen, which can lead to a complete loss of beneficial (immunological) properties.

Wherein:

- the available positive data in the short term (1, 2.3 months, as stated in the Patent) do not allow us to draw conclusions about longer periods regarding these properties;

- there are unacceptable reactogenic variants of the resulting vaccine;

c) data on the required equipment regarding the fermenter volume possible for operation and the power of the mixing device is not provided, which may complicate the implementation of this cultivation method and the nutrient medium used for a specific production.

A known method of obtaining a hemophilus influenzae type b conjugate vaccine according to patent No. 2704452 of the copyright holder FSUE "St. Petersburg Research Institute of Vaccines and Serums and an Enterprise for the Production of Bacterial Preparations", C12N 1/20, publ. 10/28/2019, where a liquid nutrient medium of the following composition (g/l) is used: glucose - 5.0; yeast extract - 2.0; peptone - 15.0; NH4Cl - 0.7; Na2HPO4 - 2.5; KCl - 0.5; NaCl - 3.3; L-glutamic acid - 1.2; L-cysteine - 0.015; NAD - 0.02; hemin - 0.04.

The disadvantages of the known solution are:

The concentrations of microorganisms obtained according to the patent range from ~2.5-3, but below 10-15 billion cells/ml of nutrient medium, which is lower than the concentrations obtained according to the proposed composition of the nutrient medium in the claimed composition.

As a prototype, the applicant is considering Author's Certificate No. 1566533, Method for producing a vaccine against avian pasteurellosis, copyright holder - All-Union Scientific Research and Technological Institute of Biological Industry, A61K 39/102, publ. 11/15/1994, where a nutrient medium based on Hottinger’s digest is used to obtain pasteurella bacterial mass. The nutrient medium includes Hottinger's digest, yeast extract, and sodium phosphate. Deep cultivation is carried out for 6 - 8 hours at 37°C, taking into account the phases of the physiological state of the culture.

The disadvantage of the known prototype method is:

a) the prototype does not contain cysteine in the required concentration - as is done in the proposed application - 0.5-1 g/l in the nutrient medium of cysteine) and for this reason the reproducibility of the required cell yield of 10-15 billion cells/ml in four out of every five fermentations, while according to this method, cases of obtaining up to 4 billion cells/ml are known;

b) the volumes of required equipment to obtain reproducible values for the concentration of microorganisms are not shown;

c) the reproducibility of the results in this cultivation method and the PS composition used is not shown;

d) additional equipment is used, namely sensors and instruments for measuring the redox potential of the nutrient medium, which may not always be achievable during industrial cultivation.

The achieved technical result lies in the fact that the claimed nutrient medium for the cultivation of bacteria makes it possible to increase the yield of bacterial biomass, reduce the duration of the process and increase the fermentation rate.

The prototype nutrient medium is modified by adding liver extract 1 liter per 800 liters of PS, Martin's peptone 40 liters per 800 liters of PS and KH ₂ PO ₄ (aq.) 0.8 kg per 800 liters of PS. This environment is further used under the name “regulatory” PS.

The declared result is achieved in a nutrient medium of the following composition, with component consumption for a final volume of 800 liters of PS:

Pasteurellosis CMB digest - 120 l, chemically pure HCl - 0.6 l, peptone - 4 kg, NaCl - 0.9 kg, KH ₂ PO ₄ (aq.) 0.8 kg, Na ₂ HPO ₄ * 12H ₂ O-4 kg, cysteine - up to 0.8 kg, yeast extract 25% -10l, MgSO ₄ *7H ₂ 0-400, NaOH, 20% - up to pH 7.8-8.0, water - up to 800l;

Table 1. The yield of microbial cells for various nutrient media in a bioreactor with a total volume of 1 m ³ . No. date Strain Cell concentration at the end of the process, X,
billion/ml The initial volume in the reactor,
V ₀ , l The final volume in the reactor, calculated from the sum of all additives and seed material,
V _Final , l Percentage, %, increase in yield compared to Control according to the formula: * 1 4.03 656 10 450 571 47 2 20.03 1231 eleven 400 514 45 3 20.03 1231 10 400 515 32 4 24.04 796 15 450 585 126 5 8.05 T-80 12 450 577 78 6 8.05 T-80 10 450 572 47 7 3.07 796 12 450 567 75 8 Regular fermentation control, May-July 2019 -"- Average 6.82 400-450 510-570 -"-

1. TABLE 1. Balance FERMENTATIONS MARCH 4 - JULY 3, 2020

* %=[(X*V _Final - 6.82*570)/(6.82*570)]*100%, where X is the cell biomass yield, billion/ml.

Table 1 shows the following features when conducting the process of cell cultivation and the resulting cell cultures:

• Exclusion of liver extract and Martin's peptone from PS;

• Cultivation time is 7-8 hours.

Wherein:

The average yield of cell concentration in every four out of five fermentations is at least 10.0 billion cells ml;

The percentage, %, increase in yield compared to the Control is in the range of 32-126% for various pasteurella strains;

• Consumption of main nutritional components does not exceed similar indicators of regulatory control processes.

• The immunogenicity of the resulting bacterial mass is not lower than that of the bacterial mass obtained in a routine manner.

Description of the method for obtaining the claimed composition

Example 1. Cultivation of Pasteurella using a regulated method.

To prepare 1 liter of nutrient medium based on CMB for the cultivation of pasteurella, the following components were used per 1 liter of nutrient medium:

1. Hottinger's basic digest - 0.175 l (175 ml)

2. HCl reagent grade - 0.0009 l (0.9 ml)

3. Peptone Martin 0.05 l (50 ml)

4. Peptone - 0.005 kg (5 g)

5. Liver extract (beef) 1:1 - 0.02 l (20 ml)

6. NaHPO ₄ * 12H ₂ O - 0.005 kg (5 g)

7. KH ₂ PO ₄ - 0.001 kg (1 g)

8. NaCl reagent grade - 0.0007 kg (0.7 g)

9. H ₂ O purified up to 1 l

10. NaOH 20% - to pH 7.8 - 8.0.

Preparation of mother broods

Obtaining brood culture of production strain of Pasteurella in vials and cylinders .

The starting materials of microorganisms were stored in a lyophilized state at a temperature not exceeding -40 °C. Cultivation of matte broods occurred without aeration and stirring, in plastic and glass cylinders, for 20-24 hours.

To obtain matte broods, pasteurella from sealed pipettes or ampoules were sown into bottles with a capacity of 0.2-0.5 liters with a nutrient medium intended for the cultivation of pasteurella. The broth culture from the bottles was transferred into matte bottles with a nutrient medium for the cultivation of Pasteurella. At the end of cultivation, the inoculum was checked visually (macroscopically) and by microscopy of Gram-stained smears. Pure inoculum was used to inoculate the nutrient medium in the reactors.

Inoculation of the matrix culture into the bioreactor and cultivation

Three bioreactors were used to cultivate Pasteurella cultures. The strain of each serotype was grown in a separate bioreactor. Seed material was sown in bioreactors at the rate of 5-10% of the volume of the nutrient medium.

Industrial cultivation is carried out in a reactor equipped with a magnetic stirrer. A “Candle” with a diameter of 50 mm is used as a bubbler. No bumpers were used in the fermenters.

The contents of the bioreactor were stirred for 5-7 minutes and incubated for the first two hours without stirring at 36-37°C. Two hours after inoculation, the mixture was continuously stirred with sterile air for aeration until the end of cultivation. The amount of air blown is 1-1.5 volumes per minute.

To intensify the growth of the culture, a sterile 40% glucose solution was added at the rate of 0.2% in terms of dry matter to the volume of the growing culture (1 ml per 1 liter of medium). The glucose solution was fed fractionally, 3-4 times during the cultivation period, the first portion added 2 hours after inoculation.

Pasteurella cultures were cultivated in bioreactors for 10-14 hours at a temperature of 36-37°C. Cultivation was terminated when the concentration did not increase or increased only slightly.

At the end of cultivation, the culture was monitored for purity and the pH and concentration of microbial cells were determined. It was assumed that the concentration should be at least 10 billion/ ^cm3 .

The average concentration of microorganisms (Pasteurella) for various strains with this cultivation method was 6.82 billion cells/ml, with the initial volume of the culture liquid in the bioreactor being 410-450 liters and the final volume of the culture liquid being 510-570 liters, see Table 1. column/line No. 8.

EXAMPLE 2. Cultivation of Pasteurella, strain 656, with a modified nutrient medium.

For the cultivation of strain 656, a modified medium containing pasteurellosis CMB digest-120 l was used,

HCl reagent grade - 0.6 l,

peptone - 4 kg,

NaCl - 0.9 kg,

KH ₂ PO ₄ (aq.) 0.8 kg,

Na ₂ HPO ₄ *12H ₂ O - 4 kg,

cysteine - up to 0.8 kg,

yeast extract 25% – 10 l,

MgSO ₄ *7H ₂ O - 400,

NaOH, 20% - up to pH 7.8-8.0,

Tap water - up to 800 l.

Nutrient medium indicators

No.
p/p Components under study Regulatory Experimental 1 Amine nitrogen 175-200 mg/%. 175-200 mg/%. 2 NaCl 0.3%. 0.3%. 3 Peptone Not less than 1.7 mg/%. Not less than 1.7 mg/%.

Preparation of the components of the nutrient medium, their mixing, preparation of pasteurella inoculum, sowing were carried out according to the conditions given in EXAMPLE 1 , with the exception of additional factors not used for the preparation of the nutrient medium, namely, the addition of cysteine, yeast extract, reducing the concentration of peptone by 20% , excluding Martin's peptone and liver extract.

EXAMPLE 2 shows the following features when conducting the process of cell cultivation and the resulting cell cultures:

• Exclusion of liver extract and Martin's peptone from PS;

• Cultivation time is 7-8 hours.

Wherein:

The cell concentration yield is at least 10.0 billion cells. ml;

The percentage, %, increase in yield compared to the Control is in the range of 32-126% for various pasteurella strains;

• Consumption of main nutritional components does not exceed similar indicators of regulatory control processes.

• The immunogenicity of the resulting bacterial mass is not lower than that of the bacterial mass obtained by the regulatory method, Table 1 , column/line No. 1.

EXAMPLE 3. Cultivation of Pasteurella, strain 1231, with a modified nutrient medium.

For the cultivation of strain 1231, a modified medium containing pasteurellosis CMB digest-120 l was used,

HCl reagent grade - 0.6 l,

peptone - 4 kg,

NaCl - 0.9 kg,

KH ₂ PO ₄ (aq.) 0.8 kg,

Na ₂ HPO ₄ *12H ₂ O - 4 kg,

cysteine - up to 0.8 kg,

yeast extract 25% – 10 l,

MgSO ₄ *7H ₂ O - 400,

NaOH, 20% - up to pH 7.8-8.0,

Tap water - up to 800 l.

Preparation of the components of the nutrient medium, their mixing, preparation of pasteurella inoculum, sowing were carried out according to the conditions given in EXAMPLE 1 , with the exception of additional factors not used for the preparation of the nutrient medium, namely, the addition of cysteine, yeast extract, reducing the concentration of peptone by 20% , excluding Martin's peptone and liver extract.

EXAMPLE 3 shows the following features when conducting the process of cell cultivation and the resulting cell cultures:

• Exclusion of liver extract and Martin's peptone from PS;

• Cultivation time is 7-8 hours.

Wherein:

The cell concentration yield is at least 10.0 billion cells ml;

The percentage, %, increase in yield compared to the Control is in the range of 32-126% for various pasteurella strains;

• Consumption of main nutritional components does not exceed similar indicators of regulatory control processes.

• The immunogenicity of the resulting bacterial mass is not lower than that of the bacterial mass obtained by the regulatory method, Table 1 , column/line No. 2.

EXAMPLE 4. Cultivation of Pasteurella, strain 1231, with a modified nutrient medium.

For the cultivation of strain 1231, a modified medium containing pasteurellosis CMB digest-120 l was used,

HCl reagent grade - 0.6 l,

peptone - 4 kg,

NaCl - 0.9 kg,

KH ₂ PO ₄ (aq.) 0.8 kg,

Na ₂ HPO ₄ *12H ₂ O - 4 kg,

cysteine - up to 0.8 kg,

yeast extract 25% – 10 l,

MgSO ₄ *7H ₂ 0 - 400,

NaOH, 20% - up to pH 7.8-8.0,

Tap water - up to 800 l.

Preparation of the components of the nutrient medium, their mixing, preparation of pasteurella inoculum, sowing were carried out according to the conditions given in EXAMPLE 1 , with the exception of additional factors not used for the preparation of the nutrient medium, namely, the addition of cysteine, yeast extract, reducing the concentration of peptone by 20% , excluding Martin's peptone and liver extract.

EXAMPLE 4 shows the following features when conducting the cell cultivation process and the resulting cell cultures:

• Exclusion of liver extract and Martin's peptone from PS;

• Cultivation time is 7-8 hours.

Wherein:

The cell concentration yield is at least 10.0 billion cells. ml;

The percentage, %, increase in yield compared to the Control is in the range of 32-126% for various pasteurella strains;

• Consumption of main nutritional components does not exceed similar indicators of regulatory control processes.

• The immunogenicity of the resulting bacterial mass is not lower than that of the bacterial mass obtained by the regulatory method, Table 1 , column/line No. 3.

EXAMPLE 5. Cultivation of Pasteurella, strain 796, with a modified nutrient medium.

For the cultivation of strain 796, a modified medium containing pasteurellosis CMB digest-120 l was used,

HCl reagent grade - 0.6 l,

peptone - 4 kg,

NaCl - 0.9 kg,

KH ₂ PO ₄ (aq.) 0.8 kg,

Na ₂ HPO ₄ *12H ₂ O - 4 kg,

cysteine - up to 0.8 kg,

yeast extract 25% – 10 l,

MgSO ₄ *7H ₂ O - 400,

NaOH, 20% - up to pH 7.8-8.0,

Tap water - up to 800 l.

Preparation of the components of the nutrient medium, their mixing, preparation of pasteurella inoculum, sowing were carried out according to the conditions given in EXAMPLE 1 , with the exception of additional factors not used for the preparation of the nutrient medium, namely, the addition of cysteine, yeast extract, reducing the concentration of peptone by 20% , excluding Martin's peptone and liver extract.

EXAMPLE 5 shows the following features when conducting the process of cell cultivation and the resulting cell cultures:

• Exclusion of liver extract and Martin's peptone from PS;

• Cultivation time is 7-8 hours.

Wherein:

The cell concentration yield is at least 10.0 billion cells ml;

The percentage, %, increase in yield compared to the Control is in the range of 32-126% for various pasteurella strains;

• Consumption of main nutritional components does not exceed similar indicators of regulatory control processes.

• The immunogenicity of the resulting bacterial mass is not lower than that of the bacterial mass obtained by the regulatory method, Table 1 , column/line No. 4.

EXAMPLE 5. Cultivation of Pasteurella, strain T-80, with a modified nutrient medium.

For the cultivation of strain T-80, a modified medium containing pasteurellosis CMB digest-120 l was used,

HCl reagent grade - 0.6 l,

peptone - 4 kg,

NaCl - 0.9 kg,

KH ₂ PO ₄ (aq.) 0.8 kg,

Na ₂ HPO ₄ *12H ₂ O - 4 kg,

cysteine - up to 0.8 kg,

yeast extract 25% – 10 l,

MgSO ₄ *7H ₂ O - 400,

NaOH, 20% - up to pH 7.8-8.0,

Tap water - up to 800 l.

Preparation of the components of the nutrient medium, their mixing, preparation of pasteurella inoculum, sowing were carried out according to the conditions given in EXAMPLE 5 (Table 1), with the exception of additional factors not used for the preparation of the nutrient medium, namely, the addition of cysteine, yeast extract, reducing the concentration peptone by 20%, excluding Martin's peptone and liver extract.

EXAMPLE 5 shows the following features when conducting the process of cell cultivation and the resulting cell cultures:

• Exclusion of liver extract and Martin's peptone from PS;

• Cultivation time is 7-8 hours.

Wherein:

The cell concentration yield is at least 10.0 billion cells ml;

The percentage, %, increase in yield compared to the Control is in the range of 32-126% for various pasteurella strains;

• Consumption of main nutritional components does not exceed similar indicators of regulatory control processes.

• The immunogenicity of the resulting bacterial mass is not lower than that of the bacterial mass obtained by the regulatory method, Table 1 , column/line No. 5.

EXAMPLE 6. Cultivation of Pasteurella, strain T-80, with a modified nutrient medium.

For the cultivation of strain T-80, a modified medium containing pasteurellosis CMB digest-120 l was used,

HCl reagent grade - 0.6 l,

peptone - 4 kg,

NaCl - 0.9 kg,

KH ₂ PO ₄ (aq.) 0.8 kg,

Na ₂ HPO ₄ *12H ₂ O - 4 kg,

cysteine - up to 0.8 kg,

yeast extract 25% – 10 l,

MgSO ₄ *7H ₂ O - 400,

NaOH, 20% - up to pH 7.8-8.0,

Tap water - up to 800 l.

Preparation of the components of the nutrient medium, their mixing, preparation of pasteurella inoculum, sowing were carried out according to the conditions given in EXAMPLE 6 (Table 1), with the exception of additional factors not used for the preparation of the nutrient medium, namely, the addition of cysteine, yeast extract, reducing the concentration of peptone by 20%, excluding Martin's peptone and liver extract.

EXAMPLE 6 shows the following features when conducting the process of cell cultivation and the resulting cell cultures:

• Exclusion of liver extract and Martin's peptone from PS;

• Cultivation time is 7-8 hours.

Wherein:

The cell concentration yield is at least 10.0 billion cells. ml;

The percentage, %, increase in yield compared to the Control is in the range of 32-126% for various pasteurella strains;

• Consumption of main nutritional components does not exceed similar indicators of regulatory control processes.

• The immunogenicity of the resulting bacterial mass is not lower than that of the bacterial mass obtained by the regulatory method, Table 1 , column/line No. 6.

EXAMPLE 7. Cultivation of Pasteurella, strain 796, with a modified nutrient medium.

For the cultivation of strain 796, a modified medium containing pasteurellosis CMB digest-120 l was used,

HCl reagent grade - 0.6 l,

peptone - 4 kg,

NaCl - 0.9 kg,

KH ₂ PO ₄ (aq.) 0.8 kg,

Na ₂ HPO ₄ *12H ₂ O - 4 kg,

cysteine - up to 0.8 kg,

yeast extract 25% – 10 l,

MgSO ₄ *7H ₂ O - 400,

NaOH, 20% - up to pH 7.8-8.0,

Tap water - up to 800 l.

Preparation of the components of the nutrient medium, their mixing, preparation of pasteurella inoculum, sowing were carried out according to the conditions given in EXAMPLE 1, with the exception of additional factors not used for the preparation of the nutrient medium, namely, the addition of cysteine, yeast extract, reducing the concentration of peptone by 20% , excluding Martin's peptone and liver extract.

EXAMPLE 7 shows the following features when conducting the process of cell cultivation and the resulting cell cultures:

• Exclusion of liver extract and Martin's peptone from PS;

• Cultivation time is 7-8 hours.

Wherein:

The cell concentration yield is at least 10.0 billion cells ml;

The percentage, %, increase in yield compared to the Control is in the range of 32-126% for various pasteurella strains;

• Consumption of main nutritional components does not exceed similar indicators of regulatory control processes.

• The immunogenicity of the resulting bacterial mass is not lower than that of the bacterial mass obtained by the regulatory method, Table 1 , column/line No. 7.

To obtain a nutrient medium for the cultivation of pasteurella (Pasteurella multocida), industrial bioreactors with a total volume of up to 1 m ³ are used.

Claims (1)

Nutrient medium for the cultivation of pasteurella in industrial bioreactors, containing pasteurellosis CMB digest - 120 l, chemically pure HCl. - 0.6 l, peptone - 4 kg, NaCl - 0.9 kg, KH ₂ PO ₄ (aq.) - 0.8 kg, Na ₂ HPO ₄ *12H ₂ O - 4 kg, cysteine - up to 0.8 kg, yeast extract 25% - 10 l, MgSO ₄ *7H ₂ O - 400, NaOH, 20% - to pH 7.8-8.0, water - up to 800 l.