JPS5953833B2 - Culture method and medium - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention utilizes Bordetella (B.
A method for culturing microorganisms belonging to the genus Ordetella;
Regarding the culture medium used at that time.

Microorganisms belonging to the Bordetella genus include Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica.
It is known as the main pathogen of pertussis, 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 a culture medium that promotes the growth of clinically isolated bacteria is required.

Additionally, a pertussis vaccine is used to prevent pertussis, and in order to efficiently produce a vaccine that is usually prepared in the form of a bacterial suspension, a medium that promotes the growth of pertussis bacteria is desirable.

Conventionally, Borna-Jangoo medium (BG medium) and Steiner-Scholte (SS medium) medium have been used for this purpose, but even when such medium is used, Bordetella pertussis and the like can mutate on the medium. The problem is that it is difficult to cultivate stably because it is easily stimulated, and especially when the number of inoculated bacteria is small (on the order of 103 colonies or less), the growth is very unstable.

The present inventors have arrived at the present invention as a result of intensive research into a method for stably and efficiently culturing microorganisms belonging to the genus Bordetella by improving the shortcomings of the prior art.

□ That is, the present invention provides a method for culturing a microorganism belonging to the genus Bordetella, which is characterized by adding cyclodextrin or a derivative thereof to the medium when culturing the microorganism belonging to the genus Bordetella, and a medium used therein. It is.

゛ The microorganisms belonging to the genus Bordetella in the present invention are
This refers to Bordetella pertussis, Bordetella pertussis, Bordetella bronchiseptica, etc.

Regarding the mycological properties and culture conditions of microorganisms belonging to the genus Bordetella, see Bergy's Manual
of Determinative Bacteriol
ogy, 8th edition, 1974, The William
Published by S & Willkins Co0, J, E
xp, Med, Vol. 129, pp. 523-550, 1
It is already publicly known, as it was published in 1969 or Bacteriology Practice Handbook, 3rd edition, pages 80 and below, in 1972, in a round neck.

Cyclodextrin is a starch or starch hydrolyzate that is produced by bacteria such as Bacillus macerans.
It is a crown-shaped molecule obtained by the action of ylase, in which 6 to 10 D-glycopyranose groups are linked in a ring through α-1,4 glucosidic bonds.

The main ones are composed of 6, 7 or 8 D-glycopyranose groups and are respectively called α-5β-5γ-cyclodextrins.

In the present invention, cyclotex I-phosphorus refers to the above-mentioned α,
Refers to cyclodextrins such as β and γ, or mixtures thereof.

Since the Sifurobukis 1he9fu molecule has a large number of primary and secondary hydroxyl groups, various derivatives can be obtained by applying reactions widely used for monosaccharides.

Cyclodextrin derivatives in the present invention refer to derivatives obtained by such methods, such as aminated derivatives such as aminocyclodextrin and aminocyclodextrin, esterified derivatives such as acetylcyclodextrin and nitrocyclodextrin, and methylcyclodextrin. There are etherified derivatives (etherified cyclodextrin) such as , ethyl cyclodextrin, propyl cyclodextrin, and carboxymethyl cyclodextrin.

Preferred in the present invention are etherified cyclodextrin, among which hexakis(2,6-0-
dimethyl) α-cyclodextrin and heptakis (2,
Methyldextrins such as 6-0-dimethyl)β-cyclodextrin are particularly 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 These are an SS medium and an SS medium obtained by adding about 1 to 2% (w/v) agar to this medium and solidifying it.

SS medium contains 11 sodium glutamate, 1
-proline, sodium chloride, potassium dihydrogen phosphate,
10 each of potassium chloride, magnesium chloride, calcium chloride, and trishydroxymethylaminomethane,
7.0, 24.2, 5.0, 5゜0.2.0, 1.0,
After adjusting the aqueous solution containing 1,525 g of 0.02.0.02. to 7.6 g with concentrated hydrochloric acid, sterilize it in an autoclave at 121°C for 15 minutes.
11 ascorbic acid, niacin, and reduced glutathione
4°1, 2.0, 4. A supplementary solution obtained by sterilizing a solution containing Log with a Millipore filter (0.45μ) is 1.0% (V/V) based on the basal medium.
It is obtained by adding in the proportion of .

In the present invention, the amount of cyclodextrin or its derivative added to the medium depends on the amount of bacteria to be inoculated, but for example, if the bacteria to be inoculated are 103 colonies, it is 200 to 5000 μg/ml. .

In the case of 106 colors, 1 to 500 μg/ml is suitable.

The method and conditions for culturing microorganisms belonging to the genus Volteterra using such a medium are not particularly limited, and conventionally known methods and conditions can be employed, but shaking culture is preferable to static culture, and culture temperature The appropriate temperature is around 35°C and the culture time is 10 to 100 hours.

Hereinafter, the present invention will be explained in detail with reference to Examples.

Example 1 Bordetella pertu
ssis ) (Bacillus pertussis) Higashihama strain I freeze-dried cells were suspended in a 1% casamino acid solution, and defibrinated horse blood was added to
Borna Shangu (Bnrdet-Geng) containing %
ou) medium (hereinafter referred to as BG medium) at 35°C for 3 days.

One platinum loopful of the bacteria was scraped off, and the bacteria was refreshed with BG medium for 24 hours and suspended in SS medium to obtain an inoculated bacterial suspension of 5 x 103 colonies/ml.

Prepare in advance a solid SS medium containing cyclodextrin or its derivatives at a concentration of 1.2% agar so that it has a predetermined final concentration (μg/ml), and the number of bacteria per plate is 103 colonies. It was spread like this.

Table 1 shows the bacterial growth status (number of colonies) after culturing at 35°C for 4 days.

The etherified cyclodextrin used was
H, 5chlenk et al.'s method (Ab
str, Pap, Amer, Chem, 5oc0, 14
9, IC11965).

Hereinafter, α-cyclodextrin is α-CD, its methylated product (hexakis(2,6-0-dimethyl)α-cyclodextrin) is Meα-CD, and its ethylated product is Etα.
-CD, β-cyclotexttrin as β-CD, its methylated product as Meβ-CD (heptakis(2,6-0-
dimethyl)β-cyclodextrin), ethylated product as E
It is abbreviated as tβ-CD.

As is clear from Table 1, the growth of Bordetella pertussis is promoted by adding cyclodextrin or its derivative to the medium.

Furthermore, it can be seen that the methylated or ethylated products thereof are better than α-CD or β-CD, and the β-form is better than the α-form.

Example 2 The inoculum suspension obtained in the same manner as in Example 1 was inoculated into 10 ml of SS liquid medium containing a predetermined concentration of Meβ-CD so that the inoculation size was 106 colonies, and an L-shaped test was conducted. The cells were cultured at 35°C with reciprocal shaking in tubes.

The changes over time in the turbidity (OD650 mμ) of the obtained medium were as shown in Table 2.

From Table 2, Meβ-CD was added to the medium at 1 to 500 μg/m.
It can be seen that when added within the range of 1, the turbidity of the medium increases as the amount added increases, that is, the growth of bacteria is promoted.

Claims (1)

[Scope of Claims] 1. A method for culturing a microorganism belonging to the genus Bordetella, which comprises adding cyclodextrin or a derivative thereof to a medium when culturing the microorganism belonging to the genus Bordetella. 2. The culture method according to claim 1, wherein the cyclodextrin derivative is an etherified cyclodextrin. 3. The culture method according to claim 2, wherein the etherified cyclodextrin is methylcyclodextrin. 4. A medium containing cyclodextrin or a derivative thereof for culturing microorganisms belonging to the genus Bordetella. 5 Cyclodextrin or its derivatives from 1 to 5000
The culture medium according to claim 4, which contains the medium in a proportion of μg/ml.