JPH0740919B2 - Growth method and culture medium of Staphylococcus epidermidis - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention relates to Staphylococcus epidermidis (St.
aphylococcus epidermidis) and a culture medium used for the growth method.

(Prior Art) Staphylococcus epidermidis is a kind of bacteria that constitutes the colonization flora of the skin and mucous membranes of humans and warm-blooded animals, and has another name for Staphylococcus epidermidis. Staphylococcus aureus of the same genus
Is a potential pathogen of various infectious diseases and food poisoning, whereas many of these are considered to be obligate or facultative parasites (Kazuo Iwata, "Pathogenic Microbiology, Nanzandou"). .

This Staphylococcus epidermidis has also been reported to produce various enzymes such as lipase, phospholipase, lipoprotein lipase, esterase, lyase and protease ("Bergey's
manual of Determinative Bacteriology 8th Edition ",
Cohen, 1972, "THE STAPHYLOCOCCI", WIEY-INTE-RSCIE
NC etc.).

Lipolytic enzymes produced by microorganisms such as Staphylococcus epidermidis have been conventionally used in various industrial fields. For example, for medical use, Bacillus
Lipase (Triacylglycerol Acylhydrolase) produced by subtilis (Bacillus Subtiiis), Aspergillus niger (Aspegillus Niger) and other bacteria has been put to practical use (Shibata et al., “Natural Natural Substances”, Nanzandou, Ueno / Omura, “Microbial Chemical Chemistry”). Revised second edition, Nanzandou, etc.). In the field of clinical diagnostic tests, Rhizopus japonicus (Rh
The lipoprotein lipase (Triacylglyceroprotein acylhydolase) produced by izopus Japonicus) has been put to practical use for the diagnosis of insulin deficiency diabetes, uremia, nephrotic syndrome, and alcohol poisoning (the second edition of "Microbial Chemistry Chemistry", Nanzan). Etc.). Furthermore, in the field of food processing, Rhizopu Delema
The lipase produced by r) has been put to practical use in the production of flavors and the like (the above-mentioned “Microbial Chemistry” Rev. 2)
Edition, such as Nanzandou).

Industrially useful methods of producing Staphylococcus spp., Such as lipolytic enzymes, have been conventionally propagated, glucose, fructose, maltose, sucrose, glycerin, dextrin, starch, sugar-tight, meat extract,
Medium containing inorganic ions such as peptone, corn steep liquor, soybean meal, casamino acid, NZ amine, yeast extract, Na ⁺ , K ⁺ , Ca ²⁺ , PO ₄ ^3- (JP-B-63-2593), polypeptone It is carried out using a culture medium such as a medium containing 15% and 0.6% of salt, and a medium containing 1% of meat extract, 1% of peptone and 3% of salt (Japanese Patent Publication No. 63-2594). It was

(Problems to be Solved by the Invention) However, in the above-mentioned conventional method using a culture medium, not only the amount of salt or polypeptone added was abnormally large, but also good growth was not always achieved, and the production could not be performed.

The present invention has been made based on the above-mentioned background, and the object thereof is industrially useful, and a proliferation method capable of promoting the proliferation of Staphylococcus epidermidis producing a lipolytic enzyme and a method thereof. The purpose is to provide a culture medium that can be used in the method.

(Means for Solving the Problems) As a result of various research and development for solving the above problems, the inventors have found that oleic acid used together with a solubilizing agent,
It was found that the addition of a sodium salt, potassium salt, or ammonium salt to a medium in a small amount unexpectedly significantly promotes growth, and it was found to be effective in achieving the object of the present invention, and the present invention was completed. Came to do.

That is, the method for growing Staphylococcus epidermidis according to the present invention is characterized in that a small amount of solubilized at least one promoter carried from the group consisting of oleic acid and its sodium salt, potassium salt, and ammonium salt is added to a standard agar medium. With the added culture medium, Staphylococcus
It is characterized in that it comprises multiplying Epidermidis.

In a preferred embodiment of the present invention, as a culture medium, after adding a small amount of a promoter to standard agar medium, 115 to 125 ° C,
It can be autoclaved for 15 to 25 minutes.

Staphylococcus used in the breeding method of the present invention
The culture medium for growing Epidermidices is characterized in that a small amount of at least one solubilized promoter selected from the group consisting of oleic acid, its sodium salt, potassium salt and ammonium salt is added to a standard agar medium. To do.

In a preferred embodiment of the present invention, the addition amount of the promoter oleic acid or a salt thereof is 0.001 to the whole culture medium.
2.0% by weight.

Hereinafter, the present invention will be described more specifically.

The target bacterium grown in the present invention is Staphylococcus epidermidis. This Staphylococcus epidermidis is a known bacterium and can be easily obtained by those skilled in the art. For example, wipe a certain area of human epidermis (preferably epidermis where sebum is secreted) with a sterile cotton swab moistened with sterile water, and wash it in sterile peptone water (1%), Disperse 1 ml of the wash-out liquid on a standard agar medium and incubate at 35 ° C for 24 to 48 hours to give a round or slightly irregular dome-shaped white colony with a smooth or granular surface. Staphylococcus epidermidis can be easily obtained by culturing the obtained colony several times and culturing it purely on a standard agar medium. The fungus thus obtained is
This can be easily confirmed using a reagent for identifying this bacterium (for example, "ID Kit SP-18", manufactured by Nissui Pharmaceutical Co., Ltd.). In addition, the type culture of this bacterium can be found in the microbial accession No. Registered as -03726 and ATCC-31432, and easily available from now on.

The culture medium used in the present invention is obtained by adding a small amount of at least one solubilized promoter selected from the group consisting of oleic acid, its sodium salt, potassium salt and ammonium salt to a standard agar medium. is there.

Examples of the salt of oleic acid added as a promoter include sodium salt, potassium salt and ammonium salt.

The above accelerators can be used alone or in combination of two or more. The combination can be appropriately selected and changed according to the purpose.

The basic medium composition used for the culture medium in this invention is
It is similar to the composition of a standard agar medium (medium for bacterial and fungal tests of the Japanese Pharmacy) and its composition.

In addition to the above-mentioned components, various additives can be added to the culture medium of the present invention depending on the purpose. Such additives include solubilizers, buffers, facilitating aids and the like.

The amount of the promoter added to this culture medium can be appropriately selected and changed according to the culture conditions, but may be small. For example,
When oleic acid or a salt thereof is used as a promoter, the addition amount thereof is 0.001 to 2.0% by weight based on the whole culture medium.

An example of the method for producing the culture medium in the present invention will be described.

First, a predetermined amount of accelerator is mixed with a solubilizer and stirred to dissolve. Next, a predetermined amount of this mixed solubilized product is weighed, and purified water is added to obtain a predetermined total amount.

This solution is added to a predetermined amount of standard agar medium before heat treatment, and autoclaved at a temperature of 115 to 125 ° C. for 15 to 25 minutes, for example. In this way, the culture medium can be produced.

The prepared culture medium can be used to grow Staphylococcus epidermidis using conventional techniques.

(Effect of the invention) As demonstrated by the above description and the following examples,
The proliferation method according to the present invention and the culture medium that can be used in the method have the following effects.

(1) It is industrially useful, and Staphylococcus epidermidis, which produces a lipolytic enzyme, can be grown rapidly and in large amounts.

(2) Lipolytic enzymes derived from microorganisms are applied in various fields such as medical care, clinical diagnostic tests, and food processing.

Conventionally, those derived from fungi or yeasts such as Aspergillus, Penicillium, Muco, Rhizopus, and Candida are mainly used, and bacteria derived from Pseudomonas and Bacillus are known. Many of the lipases produced by these microorganisms mainly act on the α-position of triglyceride, but in particular, Aspergillus niger, Aspergillus flapas, Candida cylindracea, and Staphylococcus vulgaris highly similar to Staphylococcus epidermidis. It is known that the lipase produced by Aureus acts on both α-position and β-position of triglyceride (edited by Eiji Ichishima, “Food Industry and Enzymes”, Asakura Shoten).

In the utilization situation of lipase derived from bacterial cells as described above,
This invention, which can effectively grow Staphylococcus epidermidis, provides a means for efficiently producing a lipolytic enzyme derived from a proliferating bacterium that is applied in various fields such as medical care, clinical diagnostic tests, and food processing. Will be provided.

(Examples) Hereinafter, the present invention will be specifically described based on Examples, but the present invention is not limited to the following examples as long as the gist thereof is not exceeded.

Experimental Example 1 0.5 g of accelerator oleic acid was dissolved with 10 g of solubilizer T-80 by mixing and stirring. Next, 1.05 g of this mixed solubilized product was weighed and purified water was added to make the total amount 105 g.

10 g of this solution was added to 90 g of standard agar medium before heat treatment,
It was autoclaved at a temperature of 121 ° C. for 20 minutes. Thus, the culture medium was prepared. The composition of the obtained culture medium is shown below.

One platinum loop of Staphylococcus epidermidis was smeared on the prepared culture medium and cultured at 35 ° C. for 24 hours, and the colony was observed by a strick.

A photograph showing the state of the colony is shown below the reference photograph 1.

For comparison, the cells were cultured in a control culture medium containing only the standard agar medium to which the growth promoting component of A was not added. A photograph showing the state of the colony is shown above the reference photograph 1.

A growth promoting effect was clearly recognized as compared with the control. The results are shown in Table 3.

Experimental Example 2 10 g of a promoter, sodium oleate, was dissolved in 70 g of purified water, mixed and stirred with 10 g of T-80, a solubilizing agent, and then purified water was added to make a total amount of 100 g.

10 g of this solution was added to 90 g of standard agar medium before heat treatment,
It was autoclaved at a temperature of 121 ° C. for 20 minutes. Thus, the culture medium was prepared. The composition of the obtained culture medium is shown below.

One platinum loop of Staphylococcus epidermidis was smeared on the prepared culture medium and cultured at 35 ° C. for 24 hours, and the colony was observed by a strick.

A photograph showing the state of the colony is shown below the reference photograph 2.

For comparison, the cells were cultured in a control culture medium containing only the standard agar medium to which the growth promoting component of A was not added. A photograph showing the state of the colony is shown above the reference photograph 2.

A growth promoting effect was clearly recognized as compared with the control. The results are shown in Table 3.

Claims (3)

[Claims] 1. A culture medium comprising at least one solubilized promoter selected from the group consisting of oleic acid and salts thereof in a standard agar medium in an amount of 0.001 to 2.0% by weight based on the whole culture medium. A method for growing Staphylococcus epidermidis, which comprises multiplying Philococcus epidermidis. 2. The growth method according to claim 1, wherein the culture medium is obtained by adding a small amount of a promoter to a standard agar medium and then autoclaving at 115 to 125 ° C. for 15 to 25 minutes. 3. A culture medium for growing Staphylococcus epidermidis obtained by adding a small amount of at least one solubilized promoter selected from the group consisting of oleic acid and salts thereof to a standard agar medium.