CN112646747B

CN112646747B - Clostridium tetani culture medium

Info

Publication number: CN112646747B
Application number: CN202110020869.2A
Authority: CN
Inventors: 胡浩; 雷丹
Original assignee: Suzhou Ju Microbiology Technology Co ltd
Current assignee: Suzhou Ju Microbiology Technology Co ltd
Priority date: 2021-01-08
Filing date: 2021-01-08
Publication date: 2024-03-15
Anticipated expiration: 2041-01-08
Also published as: CN112646747A

Abstract

The invention relates to the field of microorganisms, in particular to a clostridium tetani culture medium. The culture medium comprises a strain activation culture medium and a toxigenic culture medium, and the components do not contain animal source nitrogen sources. According to the formula, the proper combination of the components and the content of the animal-origin-free nitrogen source is adopted, the expression level of clostridium tetani toxin is improved on the basis of removing the animal-origin-free components, and the toxin yield can reach 95Lf/ml in a pilot experiment scale.

Description

Clostridium tetani culture medium

Technical Field

The invention relates to the field of microorganisms, in particular to a clostridium tetani culture medium.

Background

Tetanus is a life threatening disease caused by clostridium tetani infection (clostridium tetani). Clostridium tetani causes disease by releasing tetanus neurotoxin, with a minimum lethal dose of only 2.5ng/kg. Tetanus toxoid vaccine has been used for many years to prevent this disease. Tetanus vaccines are based on detoxified toxins produced by clostridium tetani.

The production of toxoids involves the following steps: 1) After one or more steps of strain activation culture, bacteria can keep good growth in a seed activation culture medium; 2) Inoculating the activated strain into a fermentation tank, and fermenting in a toxigenic culture medium to generate tetanus toxin; 3) Finally, the toxin is separated from the culture, and the toxoid is produced after purification and detoxification.

In conventional production methods, clostridium tetani is cultured in a medium containing animal-derived components that meet the nutritional requirements of the bacteria for proteins, peptides, amino acids and essential growth factors. However, animal-derived components of the culture medium may introduce harmful contaminants or impurities such as allergens, viruses, prions or other uncontrollable biological effector substances into the toxoids, causing adverse reactions to the human body.

With the development of the age, the public has higher and higher requirements on the safety of vaccines, and in recent years, development and research on animal-source-free culture media are continuously carried out to reduce adverse reactions of vaccines on human bodies. In recent years, although there are some related inventions and documents concerning animal-origin-free clostridium tetani culture medium, the toxin production is low

Disclosure of Invention

The first aspect of the invention relates to a clostridium tetani species activation medium which, when formulated as a solution, operates at a concentration of a solution comprising:

1.4g/L to 1.6g/L of total nitrogen of non-animal source nitrogen source, 4.5g/L to 5.5g/L of yeast extract powder, 4.5g/L to 5.5g/L of monopotassium phosphate, 0.4g/L to 0.6g/L of L-cysteine hydrochloride, 0.0009g/L to 0.0011g/L of resazurin, 0.0009g/L to 0.0011g/L of vitamin K and 1.4g/L to 1.6g/L of agar powder.

Alternatively, the strain-activating medium as described above, the non-animal-derived nitrogen source comprises at least one of soybean peptone, selective soybean peptone and Hysoy 4D, yeast peptone and rice peptone.

The second aspect of the invention relates to a clostridium tetani toxigenic medium which, when formulated as a solution, has a working concentration of a solution comprising:

1.6g/L to 2g/L of total nitrogen of non-animal nitrogen source, 4.5g/L to 5.5g/L of yeast extract powder, 4.5g/L to 5.5g/L of sodium acetate, 0.9g/L to 1.1g/L of monopotassium phosphate, 0.9g/L to 1.1g/L of disodium hydrogen phosphate, 55ml/L to 65ml/L of glycerin, 2.3g/L to 2.7g/L of glucose, 0.029g/L to 0.031g/L of ferric trichloride hexahydrate, 4.9ml/L to 5.1ml/L of factor I, 1.96ml/L to 2.05ml/L of factor II, 0.95ml/L to 1.05ml/L of factor III and 1.8g/L to 2.2g/L of activated carbon;

the factor I comprises the following components:

thiamine hydrochloride 0.29 g/L-0.31 g/L, riboflavin 0.29 g/L-0.31 g/L, pyridoxine hydrochloride 0.29 g/L-0.31 g/L, calcium pantothenate 1.14 g/L-1.26 g/L, biotin 0.003 g/L-0.005 g/L, and absolute ethyl alcohol 240 ml/L-260 ml/L;

the factor II comprises the following components:

2.4g/L to 2.6g/L of nicotinic acid, 2.4g/L to 2.6g/L of uracil, 74g/L to 76g/L of L-cystine, 9.5g/L to 10.5g/L of zinc sulfate heptahydrate, 37.9g/L to 38.9g/L of magnesium sulfate heptahydrate and 140ml/L to 160ml/L of concentrated hydrochloric acid;

the factor III comprises the following components:

vitamin B12 is 0.004g/L to 0.006g/L.

Alternatively, the non-animal source nitrogen source comprises at least one of soy peptone, selective soy peptone, hysoy 4D, yeast peptone and rice peptone as described above.

Alternatively, the non-animal source nitrogen source comprises two different components, at least one of soy peptone, selective soy peptone, hysoy 4D and rice peptone, respectively, independently of each other, as described above.

Alternatively, the concentration of the two different components of the toxigenic medium as described above is 0.8g/L to 1g/L.

Alternatively, the activated carbon is a type 302 pharmaceutically active carbon, as described above in the toxigenic medium.

A third aspect of the invention relates to a Clostridium tetani culture medium combination comprising a strain activation medium as described above, and a toxigenic medium as described above.

A fourth aspect of the invention relates to a method for culturing Clostridium tetani, comprising:

the culture medium was activated with the species as described above, and as described above, in sequence. The toxigenic medium cultures clostridium tetani.

Alternatively, the culture method as described above, wherein the culture conditions in the strain activation medium are 33℃to 35℃for 24 to 72 hours;

the culture condition in the toxigenic culture medium is 33-35 ℃ for 54-96 hours.

The beneficial effects of the invention are as follows:

the formulation of the invention has proper combination of components and content of animal source free nitrogen source, improves the expression level of clostridium tetani toxin on the basis of removing animal source components, and can reach 95Lf/ml toxin yield in a pilot experiment scale.

Detailed Description

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

The terms "comprising," "including," and "comprising," as used herein, are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, and the endpoints recited.

In the present invention, the culture medium may be packaged in the form of a solution or may be packaged in the form of a dry powder composition, and then dissolved at the time of use. The proportion of each component in the composition or the solution satisfies the predetermined concentration when the composition or the solution is prepared into the working concentration. The "working concentration" is used to define the concentration of the main active ingredient of the medium in the culture of Clostridium tetani, and the concentration of the main active ingredient in the medium may be the working concentration or may be a mother liquor (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50-fold concentrated mother liquor) that can be diluted to that concentration. The solvent of the solution is preferably water.

In the present invention, the non-animal-derived nitrogen source total nitrogen may be of plant origin, microbial origin or synthetic non-animal-derived nitrogen source total nitrogen.

The form of addition of total nitrogen of non-animal origin is, for example, peptone.

Peptone is an organic compound. In this application peptone is a powder which is prepared from raw materials of animal origin, for example, vegetable proteins, casein or gelatin, which are hydrolyzed with acids or proteases and dried to give a pale yellow appearance. Peptones can also be formed after the proteins are decomposed by acids, bases or proteases. One of the preliminary digestion products of proteins in the stomach is peptone. Peptone is rich in organic nitrogen compounds, also contains some vitamins and saccharides, and can provide nutrients such as C source, N source, growth factors and the like for microorganisms.

The plant-derived nitrogen source total nitrogen is preferably of soybean and/or rice origin.

The microbial nitrogen source total nitrogen is preferably yeast powder.

In some embodiments, the non-animal source nitrogen source comprises at least one of soy peptone, selective soy peptone and Hysoy 4D, yeast peptone and rice peptone.

In some embodiments, when the strain activation medium is formulated as a solution, the working concentration is a solution comprising:

1.60g/L of non-animal nitrogen source, 5g/L of yeast extract powder, 5g/L of monopotassium phosphate, 0.5g/L of L-cysteine hydrochloride, 0.001g/L of resazurin, 0.001g/L of vitamin K and 1.5g/L of agar powder.

the factor I comprises the following components:

the factor II comprises the following components:

the factor III comprises the following components:

vitamin B12 is 0.004g/L to 0.006g/L.

The components useful in the media formulations as discussed herein may be in anhydrous or hydrated forms, and many such anhydrous and hydrated forms of the components are known to those of skill in the art. For example, as shown above, in some embodiments, the composition comprises hydrated forms of ferric chloride, zinc sulfate, magnesium sulfate. Each of these components may be replaced with an anhydrous form, and the concentration range may be recalculated based on the molecular weight difference between the hydrated and anhydrous forms. Also, any anhydrous form mentioned in this specification can be replaced with a hydrated form and the concentration ranges recalculated accordingly.

Accordingly, it will be appreciated that for the components of L-cysteine hydrochloride, pyridoxine hydrochloride, etc., the same may be replaced by pure substances of L-cysteine or pyridoxine or other salts, in particular soluble salts.

In addition, the use of amino acid supplements, including glycine, alanine, valine, leucine, isoleucine, arginine, lysine, aspartic acid, cysteine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, asparagine, glutamine, histidine and serine, is not excluded from the present application or is an additional addition of some nutritional supplements, such as microorganisms and amino acids, to the medium of the present application.

The medium of the present application may also contain a buffer, the type of which is not particularly limited and may be of a type well known to those skilled in the art. A "buffer" is a solution that resists changes in pH by the action of its acid-base conjugate components. Various buffers that may be employed depending on, for example, the desired pH of the buffer (as well as the microbial growth and metabolic characteristics, microbial culture system, pH control and the medium used) are described in buffers, aguide for the Preparation and Use of Buffers in Biological Systems, gueffroy, d., edit Calbiochem Corporation (1975). In one embodiment, the buffer has a pH in the range of about 2 to about 9, or about 3 to about 8, or about 4 to about 7, or about 5 to about 7. Non-limiting examples of buffers that will control the pH in this range include MES, MOPS, MOPSO, tris, HEPES, phosphate, acetate, citrate, succinate and ammonium buffers, as well as combinations of these. In some embodiments, the buffer is selected from the group consisting of 3- (N-morpholino) propanesulfonic acid (MOPS) free acid, 3- (N-morpholino) propanesulfonic acid (MOPS) Na, hydroxyethylpiperazine ethanesulfonic acid (HEPES), and sodium bicarbonate. In some embodiments, the formulation comprises a combination of MOPS free acid and MOPS Na.

In some embodiments, formulation aids, such as defoamers, may also be added to the medium. In some embodiments, the defoamer of the medium formulation comprises an ionic or nonionic surfactant.

In some embodiments, the toxigenic medium, when formulated as a solution, has a working concentration of a solution comprising:

hyso 4D total nitrogen 0.90g/L, rice peptone total nitrogen 0.90g/L, yeast extract powder 5.0g/L, sodium acetate 5.0g/L, potassium dihydrogen phosphate 1.00g/L, disodium hydrogen phosphate 1.00g/L, glycerol 60ml/L, glucose 2.5g/L, ferric trichloride hexahydrate 0.030, factor I5.0 ml/L, factor II 2.00ml/L, factor III 1.00ml/L,302 activated carbon 2g/L;

the factor I consists of the following components in percentage by weight:

thiamine hydrochloride 0.30g/L, riboflavin 0.30g/L, pyridoxine hydrochloride 0.30g/L, calcium pantothenate 1.20g/L, biotin 0.004g/L, and absolute ethanol 250ml/L.

The factor II consists of the following components in percentage by weight:

nicotinic acid 2.5g/L, uracil 2.5g/L, L-cystine 75g/L, zinc sulfate heptahydrate 10.0g/L, magnesium sulfate heptahydrate 38.4g/L, concentrated hydrochloric acid 150ml/L.

The factor III consists of the following components in percentage by weight:

vitamin B12 0.005g/L.

In some embodiments, the non-animal source nitrogen source comprises at least one of soy peptone, selective soy peptone, hysoy 4D, yeast peptone, and rice peptone.

In some embodiments, the non-animal source nitrogen source comprises two different components, each independently selecting at least one of soy peptone, selective soy peptone, hysoy 4D and rice peptone.

In some embodiments, the concentration of the two different components is 0.8g/L to 1g/L, or may be 0.9g/L.

In some embodiments, the activated carbon is a type 302 pharmaceutical activated carbon.

clostridium tetani was subsequently cultivated with the strain activation medium as described above and the toxigenic medium as described above.

In some embodiments, the culture conditions in the strain activation medium are 33 ℃ to 35 ℃ for 24 to 72 hours;

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The reagents used in the examples below are all commercially available. Wherein yeast extract, soybean peptone, optionally soybean peptone, etc. are produced by BD company in America, and rice peptone is produced by Ireland Kerry company. The original strain of Clostridium tetani (strain number: CMCC 64008) was purchased from the China Committee for culture Collection of microorganisms. Tetanus antitoxin flocculent reaction standard was purchased from chinese food and drug assay institute. The following examples are not specifically described and are conventional.

Example 1

1. Culture medium

Seed culture medium: 1.60g/L of soybean peptone total nitrogen, 5g/L of yeast extract powder, 5g/L of potassium dihydrogen phosphate, 0.5g/L of L-cysteine hydrochloride, 0.001g/L of resazurin, 0.001g/L of vitamin K, 1.5g/L of agar powder and pH 7.0+/-0.1.

Fermentation medium: soybean peptone total nitrogen 0.90g/L, rice peptone total nitrogen 0.90g/L, yeast extract 4.0g/L, sodium acetate 5.0g/L, potassium dihydrogen phosphate 1.00g/L, disodium hydrogen phosphate 1.00g/L, glycerin 60ml/L, glucose 2.5g/L, ferric trichloride hexahydrate 0.030, factor I5.0 ml/L, factor II 2.00ml/L, factor III 1.00ml/L,302 activated carbon 2g/L, and pH 7.7+ -0.1.

Factor I: thiamine hydrochloride 0.30g/L, riboflavin 0.30g/L, pyridoxine hydrochloride 0.30g/L, calcium pantothenate 1.20g/L, biotin 0.004g/L, and absolute ethanol 250ml/L.

Factor II: nicotinic acid 2.5g/L, uracil 2.5g/L, L-cystine 75g/L, zinc sulfate heptahydrate 10.0g/L, magnesium sulfate heptahydrate 38.4g/L, concentrated hydrochloric acid 150ml/L.

Factor III: vitamin B12 0.005g/L.

2. Bacterial culture

Taking clostridium tetani freeze-dried bacteria, breaking tubes, inoculating the freeze-dried bacteria into a strain activation culture medium, culturing for 24-72 hours at 34+/-1 ℃, inoculating second-generation strains into a toxigenic culture medium after 2-generation strain activation, culturing for 24-48 hours at 34+/-1 ℃, inoculating qualified third-generation strains into the toxigenic culture medium, culturing for 66 hours at 34+/-1 ℃ to form fermentation liquor, sampling the fermentation liquor in the culturing process, and detecting flocculent units of the fermentation liquor.

3. Determination of flocculent unit content of fermentation liquor

Reference 2020 edition pharmacopoeia code 3506 toxoid flocculent unit assay.

The results are shown in Table 2.

Example 2-example 3

Example 2 and example 3 the strain activation medium was the same as example 1 except that the materials listed in table 1 were different from example 1, and the other components, their contents and the preparation operations were the same as example 1, and the toxigenic medium was the same as example 1, and will not be described again. The results are shown in Table 2.

The results of the above 3 examples show that: the formulas of the strain activation culture mediums of the examples 1-3 are all suitable for the growth of clostridium tetani, and the strains prepared by the three formulas are inoculated to a toxigenic culture medium for fermentation, so that the toxin yield is equivalent, and the addition of three components of selective soybean peptone, soybean peptone and Hysoy 4D into the strain activation culture medium is suitable.

Example 4-example 10

Example 4-example 10 the strain activation medium was the same as example 3, and the other components, their contents and formulation and operation were the same as example 3 except that the materials listed in table 3 were different, and no further description was given. The results are shown in Table 4.

The above 8 example results show that: the medium formulation of example 5 was the most suitable formulation for clostridium tetani fermentation, so the duplicate runs were made for example 5, examples 11-13 below.

Example 11-example 13

The culture medium formulations of examples 11-13 are the same as example 5, and the operation is the same as example 1, and the test results are shown in Table 10.

Example 14-example 15

In the embodiment 14 and the embodiment 15, the formulation of the embodiment 5 is used as a reference, the content of each component is changed by the ± variation of the technical scheme of the present invention, and the rest operations are the same as those of the embodiment 1, and are not repeated. The formulations of each of the media in examples 14 and 15 are shown in tables 5, 6, 7, 8 and 9. The test results are shown in Table 10.

TABLE 1 different components and contents of the Strain activation Medium in example 1 and example 2

TABLE 2 flocculent units Lf/ml of fermentation broths in example 1, example 2

Cultivation time	42h	54h	60h	66h
					Example 1	30	40	50	55
Example 2	30	45	55	55
					Example 3	35	45	55	60

TABLE 3 different Components and contents in examples 3-10

TABLE 4 flocculent units Lf/ml of fermentation broths in example 3-example 10

Cultivation time	42h	54h	60h	66h
					Example 3	35	50	65	75
Example 4	25	40	50	55
					Example 5	50	80	90	90
Example 6	25	50	55	55
					Example 7	20	40	45	45
Example 8	25	30	45	55
					Example 9	20	30	45	50
Example 10	45	60	65	60

TABLE 5 culture media formulations for activating strains in example 5, example 14, and example 15

Table 6 factor I formulations in example 5, example 14, example 15

Table 7 factor II formulations of examples 5, 14 and 15

Table 8 factor III formulations in example 5, example 14, example 15

Table 9 the formulation of the toxigenic medium in example 5, example 14, example 15

TABLE 10 flocculent units Lf/ml of fermentation broths in examples 11-15

Cultivation time	42h	54h	60h	66h
					Example 9	40	70	85	90
Example 10	45	75	90	90
					Example 11	50	80	90	85
Example 12	45	80	90	95
					Example 13	40	75	80	85

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. A clostridium tetani toxigenic medium, wherein when formulated as a solution, the working concentration is comprised of a solution of:

hyso 4D 0.9g/L, rice peptone 0.9g/L, yeast extract 4g/L, sodium acetate 5g/L, potassium dihydrogen phosphate 1g/L, disodium hydrogen phosphate 1g/L, glycerol 60ml/L, glucose 2.5g/L, ferric trichloride hexahydrate 0.03g/L, factor I5 ml/L, factor II 2ml/L, factor III 1ml/L, activated carbon 2g/L;

the factor I is the following components:

thiamine hydrochloride 0.3g/L, riboflavin 0.3g/L, pyridoxine hydrochloride 0.3g/L, calcium pantothenate 1.2g/L, biotin 0.004g/L, and absolute ethanol 250ml/L;

the factor II is the following components:

2.5g/L of nicotinic acid, 2.5g/L of uracil, 75g/L of L-cystine, 10g/L of zinc sulfate heptahydrate, 38.4g/L of magnesium sulfate heptahydrate and 150ml/L of concentrated hydrochloric acid;

the factor III is as follows:

vitamin B12 0.005g/L.

2. The toxigenic medium of claim 1, wherein the activated carbon is a type 302 pharmaceutically active carbon.