JPH0375151B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for culturing a microorganism belonging to the genus Bordetella, which is known as a pathogenic bacterium, and a medium used in the culture. Microorganisms belonging to the genus Bordetella include B. pertussis, B. parapertussis, and B. bronchiseptica.B. It is known as the main pathogen of whooping cough, which is a susceptible disease. Therefore, in clinical practice, rapid and reliable detection of Bordetella pertussis is desired, and for this purpose, a culture method or medium that promotes the growth of clinically isolated bacteria is required. In addition, pertussis vaccine (killed whole body vaccine or component vaccine) is used to prevent whooping cough, but in order to efficiently produce the vaccine, culture that promotes the growth of pertussis bacteria is required. A method or medium is preferred. In addition, for example, a culture of pertussis bacteria (culture medium and bacterial cells) can be used to produce insulin secretion-enhancing active substances (I slet
activating protein (hereinafter abbreviated as IAP) and leukocytosis promoting factor-, which is attracting attention as a vaccine component for Bordetella pertussis.
hemagglutinin (hereinafter abbreviated as LPF-HA)
Medically effective biologically active substances such as filamentous-hemagglutinin (hereinafter abbreviated as F-HA) can be obtained, but in order to efficiently produce such biologically active substances, it is necessary to efficiently culture Bordetella pertussis. A method is needed. As a culture medium for the growth of bacteria belonging to the genus Bordetella, especially the pertussis bacteria, Cohen et al. (American Journal of Public Health;
American Journal of Public Health 36 371
~376 (1946)) and Sutherland et al.
of Pathology and Bacteriology;
Journal of Pathology and Bacteriology 82
431-438 (1961)). Culture media containing activated carbon, starch, or ion exchange resin are known. However, activated carbon and ion exchange resins form a heterogeneous system, making them unsuitable for tracking changes in bacterial growth over time.Also, because they have nonspecific adsorption properties, they do not absorb factors that inhibit bacterial growth, such as fatty acids. The removal effect cannot necessarily be said to be selective. The effect of starch on bacterial growth was not sufficient, and there was a desire to develop a new culture medium that would compensate for the above deficiencies. In addition, when clinically isolating Bordetella pertussis as a single colony,
It is necessary to bring the mouth cough spray of a pertussis patient into direct contact with a solid agar medium containing blood, such as Bordet-Jyoung medium (hereinafter abbreviated as BG medium), but these mediums do not require fresh blood. Since it is used as a component, it has poor shelf life and poor reproducibility of colony forming ability. Therefore, from this point of view, there has been a desire to develop a clinical separation medium that has a certain chemical composition and has a long shelf life. Recently, a synthetic medium for large-scale cultivation of Bordetella pertussis was developed by Stainer and Scholte (J. Gen.
Microbiol) vol. 63, pp. 211-220, 1971). This Stenner-Scholte medium (hereinafter abbreviated as SS medium) contains natural products such as blood and polypeptone.
Since the lot difference does not include things that may fluctuate,
Since the culture medium composition of bacteria can be strictly controlled,
Features include being able to culture without causing changes in bacterial properties, and preventing unnecessary contamination of proteins from other species when separating and purifying biologically active substances such as IAP or LPF-HA mentioned above. In recent years, it has been widely used to produce pertussis vaccines and biologically active substances from Bordetella pertussis on an industrial scale. If the inoculation size is not sufficient and the inoculation size is less than 10 ⁷ cells/ml, stable growth characteristics cannot be obtained. Also SS
In an agar medium (hereinafter abbreviated as SSA medium) obtained by adding 1 to 2% agar to the medium and solidifying it, no colony formation was observed when seeding 10 ⁷ cells or less. have a defect. An object of the present invention is to provide a stable and efficient method for culturing microorganisms belonging to the genus Bordetella. Another object of the present invention is to provide a medium for use in such culture. The purpose of the present invention is to use casamino acids when culturing microorganisms belonging to the genus Bordetella.
0.1-20g/, ascorbic acid 0.01-1g/
A method for culturing microorganisms belonging to the genus Bordetella, characterized by using a medium containing 0.1 to 5 g of glutathione and 0.001 to 5 g of methylcyclodextrin, and containing casamino acid, ascorbic acid, glutathione, and methylcyclodextrin. This is achieved by a culture medium. In the present invention, microorganisms belonging to the genus Bordetella include Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica. Bordetella pertussis is preferably used in the present invention, and Bordetella pertussis is particularly preferred. Regarding the mycological properties and culture conditions of microorganisms belonging to the genus Bordetella, please refer to Bergys Manusl of
Determinative Bacteriology, 8th edition, 1974,
Published by The Williams & Willkins Co. and J.Exp
Med. vol. 129, pages 523-550, 1969, or Bacteriology Practice Abstracts, 3rd edition, pages 80 et seq., 1972, published by Maruzen, etc., and are already known. The casamino acids used as a component of the culture medium of the present invention are acid hydrolysates of casein and are available as a dry powder. Furthermore, ascorbic acid is known as vitamin C, and glutathione is a type of peptide that is widely distributed in yeast, animal liver, muscle, and the like. Glutathione may be in an oxidized form, a reduced form, or a mixture thereof. The culture medium of the present invention contains 0.1 to 20 g/, preferably 0.5 to 10 g of casamino acids.
g/, preferably 0.1 to 0.4 g/, and glutathione should be contained in an amount of 0.1 to 5 g/, preferably 0.1 to 1 g/. If it is outside the above range, the object of the present invention will not be fully achieved. In addition to the above-mentioned components, the medium of the present invention contains 0.001 to 5 g/, preferably 0.005 to 5 g/of methylcyclodextrin. Methylcyclodextrin may be added to the medium containing the above-mentioned components as it is and mixed, or it may be added to the clathrate after forming an inclusion compound with glutathione in advance. In the present invention, methylcyclodextrin refers to methyl etherified cyclodextrin, and especially hexakis (2,6-O
-dimethyl) α-cyclodextrin (Me α-
CD) and heptakis (2,6-O-dimethyl)β
-Cyclodextrin (Me β-CD) is preferred. In the present invention, the medium means a conventionally known liquid medium such as bouillon or peptone water, or a conventionally known solid medium made by adding agar, gelatin, egg white, serum, etc. to a liquid medium, but preferred is SS medium (or SSB medium) and SSA that is solidified by adding about 1 to 2% (W/V) agar to it
It is a medium. For example, SS medium usually contains sodium glutamate, l-proline,
Potassium chloride, magnesium chloride, calcium chloride, trishydroxymethylaminomethane, 10.7, 0.24, 2.5, 0.5, 0.2, 0.1, 0.02, respectively.
An aqueous solution containing 1.525 g was adjusted to pH 7.6 with concentrated hydrochloric acid, and then sterilized in an autoclave at 121°C for 15 minutes. L-cystine, ferrous sulfate,
1 ascorbic acid, niacin, glutathione
1.0% (V/
V). In the present invention, the required amounts of casamino acid, ascorbic acid, and glutathione are added to such a medium, and the required amount of methylcyclodextrin is further added. Since the culture medium of the present invention allows bacteria to grow stably and efficiently, it can be preferably used for the growth and detection of clinical isolates of pertussis. In addition, IAP, which is expected to have medical effects as a diabetes treatment drug,
Expected as pertussis vaccine component
It is also an extremely advantageous medium for the production of active substances such as LPF-HA, F-HA, and bacterial vaccines. The method and conditions for cultivating microorganisms belonging to the genus Bordetella using such a medium are not particularly limited, and conventionally known methods and conditions can be employed;
Shaking culture is preferable to static culture, and the culture temperature is 30 to 38°C and the culture time is 10 to 100 hours. The method and means for collecting the produced biologically active substance from the culture (culture medium and bacterial cells) are not particularly limited, and known methods and means can be used. For example, to obtain LPF-HA, pertussis bacteria (Bordetella patassis Higashihama strain) is cultured in the medium of the present invention at 35°C for 48 hours, and the centrifuged supernatant (PH8.3) of the resulting culture solution is Pass through a hydroxyapatite column equilibrated with 0.01M phosphate buffer at pH 8.0. Then, the resulting permeate has a pH of 6.0.
The protein fraction is adsorbed onto a hydroxyapatite column equilibrated with 0.01M phosphate buffer, and eluted with 0.1M phosphate buffer (PH7.0) containing 0.5M sodium chloride to obtain a protein fraction. This protein fraction was adsorbed on affinity chromatography using haptoglobin-Sepharose 4B as a support.
LPF-
You can get HA. In addition, the adsorbed material on a hydroxyapatite column with a pH of 8.0 containing 0.5M sodium chloride
Elute with 0.1M phosphate buffer (PH7.0) to obtain protein fraction. This protein fraction is passed through affinity chromatography using haptoglobin sepharose 4B as a support, and F-HA can be obtained from the passed-through solution. Hereinafter, the present invention will be explained in detail with reference to Examples. Example 1 Regular SSA medium (ascorbic acid 0.02g/
1.5 times the concentration of glutathione in
Furthermore, the concentrations of casamino acids were changed to 0, 0.5, 1.0, 2.5, 5.0, and 10.0 g/, respectively, and the concentrations of Me β-CD were changed to 0, 0.05, and 0.05, respectively.
Approximately 100 pertussis bacteria Higashihama strains were spread on an improved SSA medium adjusted to 0.10, 0.25, 0.50, and 1.0 g/g, and cultured at 35°C for 3 days. Then, we calculated the number of colonies formed under the same conditions.
The number of colonies formed in BG medium was compared. The results are shown in Table 1. Me β−CD 0.50~
It can be seen that by adding 1.0 g/ml, approximately the same number of colonies were formed as in the case of BG medium. The presence of 0.5-5.0 g/casamino acids made the morphology of emerging colonies more obvious.

[Table] Rate *: Colony is small.
Example 2 Add 10% of casamino acids to the basal medium of normal SSB medium.
g/g/, and further glutathione is added.
A replacement fluid adjusted to have 1.5 times ascorbic acid and 20 times ascorbic acid was added at a ratio of 1.0% (V/V). The improved SSB medium thus obtained (casamino acids
Add Me β-CD to 0, 0.05, 0.5, 2.0, and 5.0 g/, ascorbic acid 0.4 g/, and glutathione 0.15 g/), and divide 200 ml of each into Sakaguchi flasks. After the injection, Pertussis cobacterium Higashihama strain was inoculated at 1.5 x 10 ⁹ cells/ml and cultured at 35°C. The result is the first
It is shown in Fig. 2 and Fig. 2. Figure 1 shows the relationship between bacterial concentration (IOU/ml) and culture time, and Figure 2 shows the change over time in LPF-HA activity. It can be seen that when the medium of the present invention is used, the proliferation of Bordetella pertussis and the production of LPF-HA are remarkable. It can also be seen that this effect is promoted by the addition of Me β-CD. In addition, the bacterial concentration and LPF-HA activity were determined by the following method. The bacterial concentration was determined by measuring the turbidity (OD650) of the culture solution. IOU and
Abbreviation for International Opacity Unit, IIOU
= ¹⁰⁹ pieces/ml. LPF-HA activity was determined by collecting LPF-HA produced by the following method and measuring its activity. Centrifuged culture supernatant (PH8.6)
was passed through a hydroxyapatite column equilibrated with 0.01M phosphate buffer at pH 8.0, and the resulting pass-through was adjusted to pH 6.0.
It was adsorbed onto a hydroxyapatite column equilibrated with 0.01M phosphate buffer, and eluted with 0.1M phosphate buffer (PH7.0) containing 0.5M sodium chloride to obtain a protein fraction. This protein fraction was adsorbed onto affinity chromatography using haptoglobin-Sepharose as a support, and 0.5M
Contains NaCl and 3M Potassium Thiocyanide
LPF-HA was obtained by desorption with 0.1M Tris buffer.
LPF-HA activity was determined by enzyme-linked immunosorbent assay (ELISA) by Sato et al.
Law, 28th Toxin Symposium (July 23, 1981 -
The activity unit (u) of LPF-HA is 0.1 per unit volume (ml) of OD400mμ.
It is expressed as the dilution factor of each sample that gives . Example 3 In the same manner as in Example 2 (however, Me β-CD
(without adding), ascorbic acid, glutathione,
Culture media containing various concentrations of casamino acids were prepared, and Bordetella pertussis was inoculated and cultured in the same manner as in Example 2.
LPF-HA activity was measured after hours. The results were as shown in Tables 2, 3, and 4.

【table】

【table】

【table】

[Table] Example 4 (1) Culture 10 g/casamino acid and 2 g/Me β-CD were added to the basal medium of normal SSB medium, and a supplementary solution adjusted to have 20 times the amount of glutathionic acid was added at 1.0 % (V/V). To the thus obtained improved SSB medium 4, 1.0×10 ⁹
pertussis bacteria was inoculated at a concentration of
Culture was performed using a 5.0 incubator. After 48 hours, the bacterial concentration was approximately 10 ^{to 12} cells/ml, and LPF-HA was
The ELISA value was 1250U/ml. Culture finished solution
The supernatant (3.6) obtained by centrifugation at 6000 rpm for 30 minutes was used for subsequent LPF-HA purification. (2) Separation and purification of LPF-HA from culture solution Culture supernatant 3.6 (PH8.3) was heated to 0.01 at around 4℃.
The mixture was passed through a hydroxylapatite (manufactured by BDH Chemicals) column (100 ml) equilibrated with molar phosphate buffer (PH8) at a flow rate of 200 ml/hour. Wash the column with 0.01M phosphate buffer (PH8) 0.4, and wash the resulting effluent 4.0 with concentrated hydrochloric acid.
Adjusted to PH6.0. This solution was passed through a hydroxylapatite column (140 ml) equilibrated with 0.01M phosphate buffer (PH6.0) at a flow rate of 100 ml/hour. 0.01M phosphate buffer (PH6.0) 0.5
The column was then washed with 250 ml of 0.1 molar phosphate buffer (PH7.0), and further eluted and fractionated with 0.1 molar phosphate buffer (PH7.0) containing 0.5 molar salt. Collect the obtained protein fraction (100ml) 0.5
It was passed through a haptoglobin-Sepharose 4B column (30 ml) equilibrated with 0.1 molar phosphate buffer containing molar saline. The adsorbed material was eluted and fractionated with a 0.1 molar phosphate buffer containing 3 molar thiocyanate and 0.5 molar sodium chloride. The obtained protein fractions were collected and dialyzed against 0.1M phosphate buffer (PH7.0) containing 0.5M NaCl to obtain 20.7mg of LPF-HA.
Obtained. This product showed a single band in polyacrylamide gel (polyacrylamide concentration 7.5%, 1N KOH-glacial acetic acid buffer (PH4.3)) disc electrophoresis, and the migration position matched that of existing LPF-HA. Also, the ELISA value of this product is 111.0U/
It was μg.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the relationship between bacterial concentration and culture time when the medium of the present invention is used, and FIG. 2 is a diagram showing the relationship between LPF-HA activity and culture time.

Claims (1)

[Claims] 1. When culturing microorganisms belonging to the genus Bordetella, casamino acids are added at 0.1 to 20 g/,
A method for culturing microorganisms belonging to the genus Bordetella, which comprises using a medium containing 0.01 to 1 g of ascorbic acid, 0.1 to 5 g of glutathione, and 0.001 to 5 g of methylcyclodextrin. 2 A medium for detecting and/or culturing microorganisms belonging to the genus Bordetella, which contains casamino acids.
0.1-20g/, ascorbic acid 0.01-1g/
, 0.1 to 5 g/g of glutathione and 0.001 to 5 g/g of methylcyclodextrin.