JPS6028277B2 - Cultivation method and medium for microorganisms belonging to the genus Bordetella - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the use of Pordetella (B.
A method for culturing microorganisms belonging to the genus Ordetella;
The present invention relates to a medium used in this case and a method for producing biologically active substances using this medium.

Microorganisms belonging to the Bordetella genus include Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica.
It is known as the main pathogen of pertussis, which is an acute infection of the trachea, bronchi, and small bronchi that is accompanied by a characteristic cough that lasts for 100 days. Therefore, from a clinical standpoint, rapid and reliable detection of Bordetella pertussis and the like is desired, and for this purpose, culture methods and media that promote the growth of clinically isolated bacteria are required. In addition, pertussis vaccines (killed whole body vaccines or component vaccines) are used to prevent whooping cough, but in order to efficiently produce vaccines, culture methods that promote the growth of pertussis bacteria or Medium is preferred. In addition, for example, a culture of pertussis 1 phase bacteria (culture medium and bacterial cells) can be used as an insulin secretion-enhancing active substance (Isletac), which is expected to be developed as a diabetes treatment or prevention drug.
tivating, protein (hereinafter abbreviated as lAP) and Les ocytosis promoti, which is attracting attention as a vaccine component for Bordetella pertussis.
ng Nector-hema key lutinin (hereinafter L
(abbreviated as PF-HA) and mamentous-hem
Medically effective biologically active substances such as aggutinin (hereinafter abbreviated as F-day A) can be obtained, but in order to efficiently produce such biologically active substances, an efficient culture method for Bordetella pertussis is required. is necessary. As a culture medium for the growth of bacteria belonging to the genus Rdetella, especially the pertussis 1 phase bacteria, Cohen et al. (American Journal of Public Health;
blicHealth 36 371-376 (194
6)) and Sutherland et al. (Journal of Pathology and Bacteriology; JomM1 of P
athology and bacteriology
82431-4 (1961)), which contains activated carbon, starch, or ion exchange fat, is known.

However, activated carbon and ion exchange resins form a heterogeneous system,
It is not possible to track changes in bacterial growth over time, and because of non-specific adsorption, the effect of removing factors that inhibit bacterial growth, such as fatty acids, 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 isolating clinical B. pertussis as a single colony, oral cough fluid from pertussis patients is directly injected into Bordey-Jyoung medium (
These media require contact with a solid agar medium containing blood, such as BG medium (hereinafter abbreviated as BG medium).
Since fresh blood is an essential component, it has poor storage stability and poor reproducibility of colony formation. Therefore, from this point of view as well, there has been a desire to develop a clinical separation tower having a certain chemical composition and a long shelf life. In recent years, Stainer and Scholte (
A synthetic medium for mass culture of Bordetella pertussis was developed by J. Scholte (J. Gen. Microbiology).
iol) 6 Kasagata, pp. 211-220, 1971).

This Stainer-Sholte medium (hereinafter abbreviated as SS medium) does not contain additives that may vary between lots, such as naturally derived blood and polybeptone, so the bacterial medium composition can be strictly controlled. Therefore, the culture can be carried out without causing any change in the bacterial properties, and the above-mentioned l
During the separation and purification of biologically active substances such as AP or LPF-HA, pertussis vaccines and biologically active substances derived from Bordetella pertussis have been used in recent years because of their ability to prevent the contamination of ominous proteins from other species. Although it is widely used for production on an industrial scale, LPF-HA production is not sufficient under rope-dropping or static liquid culture conditions, and stable growth may occur if the inoculation size is less than 107 cells/I. The disadvantage is that the characteristics cannot be obtained. In addition, agar toji (agar toji) obtained by adding 1 to 2% agar to SS medium and solidifying it
The SSA medium (hereinafter abbreviated as SSA medium) has a major defect in that colony formation is not observed when seeding 107 cells or less. An object of the present invention is to provide a stable and efficient method for culturing and separating microorganisms belonging to the genus Bordetella.

Another object of the present invention is to provide a medium for use in such culture. Another object of the present invention is to provide a method for producing biologically active substances by culturing microorganisms belonging to the genus Poldetella using this medium. The object of the present invention is to use 0.1 to 20% of casamino acid and 0.00% of ascorbic acid when culturing microorganisms belonging to the genus Bordetella.
A method for culturing microorganisms belonging to the genus Poldetella, characterized by using a medium containing 0.1 to 1 night/day and glutathione 0.1 to 5 days/day, a medium containing Kazami acid, ascorbic acid and glutathione, A biological method, which is characterized by culturing microorganisms belonging to the genus Bordetella in a medium containing casamino acids, ascorbic acid, and glutathione, and collecting biologically active substances from the culture (cultivation substrate and bacterial cells). This is achieved by the method of preparation of the active substance.

In the present invention, microorganisms belonging to the genus Bordetella include Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica.

In the present invention, Bordetella pertussis is preferably used, with Bordetella pertussis being particularly preferred. Regarding the mycological properties and culture conditions of microorganisms belonging to the genus Bordetella,
BergySManuS10fDete dishinative
eBacteriology, 8th edition, 1974, T
He Williams & Willkins, Co. Publishing and J. ExpMed. 129 萱, pp. 523-550, 1969 or Bacteriology Practice Summary, 3rd edition, No. 80
The following pages were published by Maruzen in 1972 and are already publicly known. Kazami/acid used as a component of the culture medium of the present invention is an acid hydrolyzate of casein and is 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 medium of the present invention contains 0 casamino acids.
．． 1-20 evenings/evening, preferably 0.5-10 evenings/so, ascorbic acid 0.01-1 evening/evening, preferably 0.1
It is necessary that the amount of glutathione is 0.1 to 5 times/day, preferably 0.1 to 1 night/day. If it is outside the above range, the object of the present invention will not be fully achieved. When the medium of the present invention contains cyclodextrin or a derivative thereof in addition to the above-mentioned components, the object of the present invention can be more advantageously achieved. be done.

Cyclodextrin or its derivative may be added to and mixed with the medium containing the above-mentioned components as is, but it is also possible to form a clathrate compound with glutathione in advance and then add this clathrate compound to the medium. In the present invention, cyclodextrin means Q,8,y cyclodextrin, and cyclodextrin derivatives include, for example, aminated derivatives such as aminocyclodextrin and aminodeoxycyclodextrin, and esters such as acetylcyclodextrin and nitrocyclodextrin. derivatives, methylcyclodextrin, ethylcyclodextrin, propylcyclodextrin, carboxymethylcyclodextrin. Refers to etherified derivatives such as dextrins (etherified cyclodextrins). Preferred in the present invention are etherified cyclodextrins, especially hexakis (
2,610-dimethyl)Q-cyclodextrin (Me
Methyldextrins such as Q-CD) and heptakis(2,610-dimethyl)8-cyclodextrin (MeB-CD) are particularly preferred. In the present invention, the medium refers to b.
It means a conventionally known liquid medium such as agar, beptone water, etc., 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 add 1 agar to it.
This is an SSA medium to which approximately 2% (W/V) was added and solidified.

For example, SS medium typically contains monosodium glutamate, cubrolin, sodium chloride, diphosphoric acid,
Potassium hydrogen, potassium chloride, magnesium chloride, calcium chloride, trishydroxymethylaminomethane,
10.7, 0.24, 2.5, 0.5 0.2 respectively
, 0.1, 0.02, and 1.525 yen to pH 7.6 with concentrated hydrochloric acid, and then sterilized in an autoclave at 121°C for 1 hour. Cystine, ferrous sulfate, ascorbic acid, niacin, and glutathione per serving are 4, 1, 2, 0.4, and 10, respectively.
The replacement solution obtained by sterilizing the solution containing the sterilized water with a millibore filter (0.45 french) is added to the basal medium at 1.0% (V/
V). In the present invention, the required amounts of casamino acids, ascorbic acid, and glutathione are added to the medium, and optionally, the required amount of cyclodextrin or its derivative is 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, lA is expected to have medicinal effects as a diabetes treatment drug.
P, L expected to be a pertussis vaccine component
It is also an extremely advantageous medium for the production of active substances such as PF Ichinichi A, F-HA, and bacterial vaccines. The method and conditions for culturing microorganisms belonging to the genus Bordetella using such a medium are not particularly limited, and conventionally known methods and conditions can be adopted, but shaking culture is preferable to static culture, and culture temperature An appropriate culture time is 30 to 38°C and 10 to 10 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 1 phase bacteria (Bordetella patasis Higashihama strain) is cultured in the medium of the present invention at 35°C for 4 hours, and the resulting culture solution is centrifuged and the supernatant is adjusted to pH 8.
3) is passed through a hydroxyapatite column equilibrated with 0.01M phosphate buffer at pH 8.0. After adjusting the resulting permeate to a pH of 6.0, this time it was adsorbed onto a hydroxyapatite column equilibrated with a 0.01M phosphate buffer with a pH of 6.0, and the pH was adjusted to 0.9.
0.1M phosphate buffer (pH 7) containing M sodium chloride
．． 0) to obtain the protein fraction. This protein fraction was adsorbed on affinity chromatography using habtoglobin-sepharose as a support, and 0.9M NaCI
LPF 1 day A can be obtained by desorbing milk M with a 0.1M phosphate buffer (7.0) containing potassium thiocyanide. Further, the protein fraction adsorbed on the pH 8.0 hydroxyapatite column is eluted with 0.1M phosphate buffer (pH 7.0) containing 0.9M sodium chloride to obtain a protein fraction. This protein fraction is passed through affine chromatography using haptoglobin sepharose crab 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 The concentration of glutathione in normal SSN Akeji (containing ascorbic acid 0.02 times) was increased by 1.5 times, i.e. 0.15
evening/evening, and the concentration of casamino acids was changed to 0, 0, respectively.
5, 1.0, 2.5, 5.0, 10.0, and the concentrations of Me8-CD were 0, 0.05, 0.0, respectively.
Approximately 10 boxes of pertussis 1 phase bacteria Higashihama strain were spread on the improved SSA tower soil adjusted to be 10, 0.25, 0.50, 1.0 evening/even and cultured at 360 for 3 days. .

The number of colonies formed was then compared with the number of colonies formed on BG tower site under the same conditions. The result is the first
Shown in the table. It can be seen that by adding MeB-CD at 0.50 to 1.0 ml, colonies were formed in approximately the same number as in the case of BG medium. 0. of casamino acids.
The presence of 5 to 5.0 Tanok made the morphology of the emergent colony more obvious. Table 1 Example 2 Casamino acids were added to a normal SSB soil basal medium for 10 days/day.
Add glutathione to 1. Fang moss,
1 tangerine juice prepared to have 20 times ascorbic acid
．． It was added at a ratio of 0% (V/V).

Me8-CD was added to the thus obtained improved SSB base (containing casamino acid 10/day, ascorbic acid 0.4/day, and glutathione 0.15/day), respectively.
05, 0.5, 2.0.5.0 evening/evening, and after dividing 200 pieces of each into Sakaguchi flasks, pertussis 1 phase bacteria Higashihama strain was added at 1.5 x 1 pieces/day. The cells were inoculated so that the cells were secreted and cultured at 35 qo. The results are shown in Figures 1 and 2. Figure 1 shows the relationship between the bacterial concentration (10 U/desktop) and culture time, and Figure 2 shows the change over time in LPF daily A activity.

When the culture medium of the present invention is used, the growth of Bordetella pertussis and LPF-H
It can be seen that A production is significant. And the effect of ) is M
It can also be seen that this is promoted by the addition of eB-CD. In addition, the bacterial concentration and LPF-HA activity were determined by the following methods.

The bacterial concentration was determined by measuring the turbidity (OD65o) of the culture solution. 10U and ln rMtional0pacityUn
It is an abbreviation of "it" and corresponds to U of 1 = 1 piece/scream.

LPF daily A activity is calculated using LP produced in the following manner.
F day A was collected and its activity was measured. After centrifuging the culture solution (pH 8.6) and passing it through a hydroxyapatite column equilibrated with 0.01M phosphate buffer at pH 8.0, the resulting effluent was adjusted to a concentration of 6.0. , this time was adsorbed onto a hydroxyapatite column equilibrated with 0.01M phosphate buffer at pH 60, and this was absorbed into a 0.9M phosphate buffer.
0.1M phosphate buffer containing sodium chloride (pH 7.
0) to obtain a protein fraction. This protein fraction was adsorbed on affinity chromatography using habtoglobin-sepharose as a support. Seishu aCI and 3
LPF-HA was obtained by desorption with a 0.1M Tris buffer containing M potassium thiocyanide. LPF-HA activity is
Root 8 was detected by the enzyme antibody method (ELISA method) of Sato et al.
The activity unit (U) of LPF-HA is OD40 h
It was expressed as the dilution factor of each sample giving a volume of 0.1 per unit volume (I). Example 3 In the same manner as in Example 2 (however, without adding Me8-CD), a culture medium containing various concentrations of ascorbic acid, glutathione, and casamino acids was prepared, and in the same manner as in Example 2, pertussis The bacteria were inoculated and cultured, and LPF daily A activity was measured 48 hours later.

The results were as shown in Tables 2, 3, and 4.

Table 2 Reduced glutathione: 0.15 evenings / Casamino acid: 10 evenings / Table 3 Ascorbic acid: 0.4 evenings / Casamino acid: 10 evenings / Table 4 Ascorbic acid: 0.4 evenings / Reducing glutathione: 0.15/Z Example 4 '11 culture To the basal medium of normal SSB medium, add 10/2 casamino acids and 2/2 MeB-CD, and add glutathione to the standard composition of 1/Z. A replacement solution prepared so that the ascorbic acid content was 1.0% (V/V) was added at a ratio of 1.0% (V/V).

Four volumes of the improved SSB medium thus obtained were inoculated with pertussis 1 phase bacteria at a rate of 1.0 x 1 p/p, and cultured using the incubator. After 4 hours, the bacterial concentration was about 1 or 2 bacteria/me, and LPF daily A was 125 in ELISA value.
It became 0U/scream.

The cultured solution was centrifuged at 600 pm for 30 minutes, and the obtained supernatant (3.6 cm) was used for subsequent LPF-HN purification. ■ Isolation and purification of LPF-HA from the culture solution Hydroxylapatite (manufactured by BDH Chemicals) was equilibrated with 0.01 molar phosphate buffer (pH 8) at around 4°C. ) column (Z of 100) at a flow rate of 200'/h.

The column was washed with 0.4 mm of 0.01 M phosphate buffer (pH 8), and the resulting effluent (4.0 mm) was adjusted to pH 6.0 with concentrated hydrochloric acid.

This solution was passed through a hydroxylapatite column (140 column) equilibrated with 0.01M phosphate buffer (pH 6.0) at a flow rate of 100'/hour. Wash the column with 0.5 molar phosphate buffer (pH 6.0), then wash with 250 molar phosphate buffer (pH 7.0), and then wash with 0.5 molar buffer containing 0.5 molar saline. Elution and fractionation were performed with 1 molar phosphate buffer (pH 7.0).

The resulting protein fractions were collected and passed through a haptoglobin-Sepharose membrane (30') equilibrated with 0.1 molar phosphate buffer containing 0.5 molar saline (100'). The adsorbed material was eluted and fractionated with a 0.1 molar phosphate buffer containing 3 molar potassium thiocyanate and 0.5 molar sodium chloride. The resulting protein fractions were collected and dialyzed against a 0.1 molar phosphate buffer (pH 7.0) containing 0.5 molar sodium chloride to obtain an LPF daily A of 20.7.9. This product is a polyacrylamide gel (polyacrylamide concentration 7.5%, IN
KOH-glacial acetate buffer (pH 4.3)) disk electrophoresis showed a single band, and the electrophoresis position was the same as that of the existing LPF-HA.
matched.

Moreover, the ELISA value of this product was 111.1 U/F2.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the relationship between bacterial concentration and culture time when using the medium of the present invention, and FIG. 2 is a diagram showing the relationship between LPF daily A
FIG. 3 is a diagram showing the relationship between activity and culture time. Figure 1 Old Man Z diagram

Claims (1)

[Claims] 1. When culturing microorganisms belonging to the genus Bordetella, casamino acids are added at 0.1 to 20 g/
1. A method for culturing microorganisms belonging to the genus Bordetella, which comprises using a medium containing 0.01 to 1 g/l of ascorbic acid and 0.1 to 5 g/l of glutathione. 2 In addition to casamino acid, ascorbic acid, and glutathione, the medium contains 0.0 cyclodextrin or its derivatives.
2. The method for culturing microorganisms belonging to the genus Bordetella according to claim 1, wherein the microorganism is contained in an amount of 0.01 to 5 g/l. 3 A medium for detecting and/or culturing microorganisms belonging to the genus Bordetella, containing 0.1 to 20 g of casamino acids.
/l, ascorbic acid in an amount of 0.01 to 1 g/l, and glutathione in an amount of 0.1 to 5 g/l. 4 In addition to casamino acid, ascorbic acid, and glutathione, cyclodextrin or its derivatives are added at 0.001 to 5
The culture medium according to claim 3, which contains g/l.