JP2514174B2 - Gelled solid culture medium - Google Patents

Description

The present invention relates to a solid culture medium for bacteria, filamentous fungi, basidiomycetes, etc., which can be used for planar culture, bacterial species separation, bacterial count measurement, inoculum culture, and the like.

Conventionally, a gelling solid culture medium of this kind of microorganism is used by adding water to various nutrients and a gelling agent (agar or gelatin), sterilizing by heating, cooling and gelling (hereinafter, this is used as an agar medium. Called).

However, various nutrients used here are natural products such as wort, fruit juice and koji juice; sugars such as glucose, maltose and sucrose, which are referred to as synthetic solutions, peptides such as peptone and asparagine, purified products such as amino acids, reagents. Standard P, K,
It is composed of one kind or two or more kinds of inorganic substances such as Fe and ammonia salts, and both are extremely expensive.

Moreover, in order to prepare an agar medium using these nutrients, one or more expensive nutrients must be accurately weighed, respectively, and the operation becomes complicated.

In addition, the agar medium prepared in this manner is required in large amounts when it is practically used for bacterial strain separation, bacterial count measurement, inoculum culture, etc., except when used as a medium for storage of strains, and it is a consumable product. Since it is handled as, there is a drawback that the cost is high.

Further, since the surface of the agar medium is hard and smooth, even if the hyphae of basidiomycetes are inoculated in order to utilize it for inoculum culture of basidiomycetes, the mycelia do not come into good contact with the agar medium surface,
Growth is slow due to poor nutrient and water supply, and inoculum culture cannot be obtained in a short period of time.

Further, the inoculum culture thus obtained has a flat disc shape,
Since it is hard and tough, it must be shredded in advance in an aseptic atmosphere before inoculating and mixing it as an inoculum into an agar scrap medium or the like.

Therefore, as a result of various studies conducted by the present inventor in view of such a current situation, the corn bran contains a large amount of nutrient components necessary for the growth of various microorganisms, and it is added to water in an amount of 5 to 5
It was discovered that when 30% by weight of dispersion is carried out, the mixture is heated to gelatinize and then cooled, it gels like an agar medium and can be used as a solid culture medium for various microorganisms, and the present invention was completed based on this finding.

That is, the present invention is a gelled solid culture medium obtained by dispersing 5 to 30% by weight of corn bran in water, gelatinizing by heating and then cooling.

 The present invention will be described in detail below.

First, corn bran is a by-product when producing corn starch, and its composition is water 12.5%, crude protein 10.0%, crude fat 6.7%, soluble nitrogen-free matter 64.2%, crude fiber 4.1%, crude ash 2.5. % ("Japanese standard feed ingredient table" 1980
The annual edition, Ministry of Agriculture, Forestry and Fisheries (Agricultural, Forestry and Fisheries Technology Conference Secretariat edition)), it contains about 25% more soluble nitrogen-free substances (mainly sugars) than rice bran.

The corn bran can usually be used as it is in the case of a uniform fine powder, but if there is a coarse particle portion, it is preferable to pulverize this before use. Such fine pulverization is preferable because it is easy to disperse in water, and when the solid culture medium is used, the coarse portion does not settle.

Next, in order to prepare the solid culture medium of the present invention, corn bran is dispersed in water in an amount of 5 to 30% by weight, and heat sterilized by a conventional method. When sterilized by heating in this way, the dispersion liquid is completely gelatinized and becomes a translucent colloidal state. At this time, if necessary, the medium may be adjusted to a pH optimum for growth of various microorganisms, and other various nutrients may be added to the medium.

Next, when the heat-sterilized medium is hot, it is dispensed into a culture container such as a petri dish and allowed to stand and cooled, whereby the medium is gelated to obtain the solid culture medium of the present invention. The solid culture medium obtained in this manner contains each nutrient such as a nitrogen source, a carbon source and inorganic salts necessary for the growth of various fungi in a well-balanced manner. Almost no hassle of weighing and blending,
Therefore, the preparation of the medium is very simple.

Further, the solid culture medium of the present invention shows a gelled solid state, does not disintegrate even if the petri dish is erected at a right angle, and has strength enough to retain the shape, but it has a surface area larger than that of the agar medium. Hardness is low. Therefore, when a flat columnar basidiomycete seed piece obtained by a conventional method is placed and inoculated on the center of a conventional agar medium, it becomes a shape placed on a flat surface, and the agar surface and the contact surface are in a small state. When placed and inoculated in the center of the solid culture medium, the seed piece sinks 1/2 place, the number of contact surfaces with the top surface of the medium increases, and water and water-soluble nutrient sources smoothly migrate to the seed piece, and the basidiomycete hyphae grow. Becomes vigorous.

Also, inoculate the solid culture medium of the present invention with basidiomycete seed pieces,
When the basidiomycete hyphae are completely grown on the entire surface of the medium, it is not necessary to cut the pieces into small pieces in advance because they are easily disintegrated and evenly mixed when they are mixed after being added to the sawdust medium.

Also, the medium for flat culture in a petri dish or the like is a thin layer of several mm, but this is gelatinized in a deep container of 30 mm or more and inoculated with inoculum to the inside with a thick medium layer. Alternatively, the culture may be performed anaerobically, or the culture may be shaken to form a sol and aerobically cultured. When culturing the basidiomycete, appropriate wood flour may be added for inducing enzyme production of the basidiomycete.

 Hereinafter, the present invention will be specifically described with reference to examples.

Example 1 100 ml of distilled water was placed in a 300 ml Erlenmeyer flask, and corn bran (“Corn Blanc” manufactured by Toyonen Refinery Co., Ltd.) was weighed and added thereto in such a ratio as shown in Table 1. Then, the mixture was shaken well to obtain a uniform dispersion liquid without lumps.

This was heated on a hot water bath for 5 minutes to carry out a gelatinization reaction to reach a semitransparent state.

Next, it was transferred to an autoclave and saturated water vapor pressure 1k.
G / cm ² (gauge pressure), heat sterilized at 119 ° C for 60 minutes, then 80 ° C
After cooling to a clean bench, dispense about 15 ml each into a petri dish with a diameter of 9 cm, cover with a lid and let it cool to room temperature for gelation, and the degree of fluidity and gel hardness at the time of dispensing. To obtain solid culture media different from each other.

From the results shown in Table 1, when the addition ratio of corn bran to water is 30% or less, it is highly fluid at 80 ° C and can be easily dispensed from an Erlenmeyer flask into a petri dish.
It is understood that if it exceeds 30%, the fluidity becomes poor and dispensing becomes difficult.

Further, it can be seen that when the addition ratio of corn bran is less than 5%, the medium cannot be used as a solid culture medium because it does not gel even if the medium is cooled after dispensing.

Example 2 200 ml of distilled water was placed in a 500 ml Erlenmeyer flask, and 20 ml of this was added.
After adding g (10%) of corn bran (“Corn Blanc” from Hosei Oil & Oil Co., Ltd.) to a silico stopper, shaken, and evenly disperse, heat gelatinize on hot water for 5 minutes to form a semitransparent fluid medium. Obtained.

Next, it was transferred to an autoclave and saturated water vapor pressure 1k.
Heat sterilize for 60 minutes at g / cm ² (gauge) (about 120 ° C) and
After cooling to ℃, it was dispensed into 12 petri dishes, allowed to cool and solidified to obtain the solid culture medium of the present invention (diameter 9 mm, thickness 3 mm).

Next, the above-mentioned solid culture medium is divided into 6 sections into 2 sections, 1 teaspoon of Nameko inoculum (Oga inoculum) manufactured by Kitaken Co., Ltd. in the center of each of the sections, and each of the other sections One inoculation of Shiitake inoculum (flat columnar piece of Koma inoculum) from Meiji Seika Co., Ltd. was inoculated in the central part and cultured in a constant temperature room at 25 ° C. On the 10th day, the mycelium was in a petri dish medium. It spread all over the surface.

From the above, it is understood that according to the present invention, a suitable solid culture medium for basidiomycetes can be obtained only from corn bran without the need for expensive various nutrients and gelling agents such as agar.

Example 3 Instead of using “20 g (10%)” of corn bran in the method for preparing a solid culture medium of Example 2 described above, “30 g (15
%) ”Was used in the same manner as above except that a solid culture medium contained in a petri dish was obtained.

When 6 pieces of this petri dish were removed from the mushroom shavings yard for 5 minutes and examined for airborne bacteria by a conventional method, the occurrence of Trichoderma bacterium and bacteria was observed.

From the above results, it is understood that the solid culture medium of the present invention can be used as a medium for bacterial test.

Example 4 Instead of using “20 g (10%)” of corn bran in the method for preparing a solid culture medium of Example 2 above, “40 g (20%
%) ”, Except that the solid culture medium contained in the petri dish was obtained in exactly the same manner.

One platinum loop of spores of the Aspergillus oryzae "(Koji mold for miso", manufactured by Kojiya Sanzaemon Co., Ltd.) was inoculated into the center of each of the 6 plates of this dish and cultured in a temperature-controlled room at 28 ° C. Also in the medium, the mycelia grew and spread on the entire surface of the medium in the petri dish.

From this result, it is understood that the solid culture medium obtained in the present invention can be used as a plate culture medium for Aspergillus oryzae.

Example 5 Beech wood flour was mixed with rice bran in a weight ratio of 6: 1, and water was added to the mixture to give a water content of 65% by weight, and 500 g of each was added.
It was divided into one bat, covered with a lid, sterilized by heating in an autoclave at a saturated steam pressure of 1 kg / cm ² (gauge) for 60 minutes, and then cooled to room temperature to prepare a basidiomycete culture medium.

Next, instead of using “20 g (10%)” of corn bran in the method for producing a solid culture medium of Example 2 described above, “3
A solid culture medium contained in a petri dish was obtained in exactly the same manner except that 0 g (15%) was used.

For comparison, a Czapek agar medium was obtained by a conventional method using the nutrients and agar having the following compositions.

Czapek agar medium composition NaNO ₃ 3g K ₂ HPO ₄ 1g MgSO / 7H ₂ O 0.5g KCl 0.5g FeSO ₄ / 7H ₂ O 0.01g sucrose 30g agar 15g distilled water 1,000ml 9 solid culture media of the present invention and control 9 Tsapeck agar medium, inoculated with 1 teaspoon of oyster mushroom inoculum in the center of each medium and cultivated in a temperature-controlled room at 25 ° C. The former was on the 10th day and the latter was on the 12th day. The hyphae spread on the entire surface of the medium in the petri dish.

Next, three oyster mushroom cultures (inoculum) petri dishes, each of which was prepared as described above, were inoculated per one bat of the basidiomycete culture medium prepared above.

At this time, the oyster mushroom culture using the solid culture medium of the present invention, when mixed with a basidiomycete culture medium, the shape of the culture was shattered so fine that it could not be confirmed, but the oyster mushroom culture using the control agar medium I was forced to cut it into 1 cm square pieces with a scalpel because it is strong and does not easily collapse.

Next, add the contents of each vat to the wide-mouth cultivation bottle (volume
900ml) Each 3 bottles are filled and sealed in a conventional manner at a temperature of 25
Cultivation was carried out for 28 days in a thermostatic chamber at 70 ° C and 70% humidity.

In the former, the mycelia were completely sprinkled on the surface of the culture medium inside the bottle, whereas in the latter, about 90% of the growth was on the surface of the culture medium.

From the above results, it is understood that the present invention has the following effects.

(1) Conventional agar media are expensive nutrients, agar, etc.
Although various components have to be weighed individually and the operation is complicated, the solid culture medium of the present invention requires only corn bran as a necessary component, and thus the medium preparation is very simple.

(2) In addition, the solid culture medium of the present invention has a stronger growth of microorganisms, particularly basidiomycetes, than the agar medium, so that the desired culture can be obtained in a short period of time.

(3) In addition, since the agar medium is hard and durable and cannot be mixed well with the agar scrap medium, etc., it must be cut into dice with a scalpel, etc. and compared to obtain the desired culture. Although it takes a long time, the solid culture medium of the present invention does not have such inconvenience.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12R 1: 645) C12R 1: 645) (C12N 1/14 (C12N 1/14 C12R 1:69) C12R 1:69) (C12N 1/20 (C12N 1/20 C12R 1:01) C12R 1:01)

Claims (1)

(57) [Claims] 1. A gelled solid culture medium prepared by dispersing 5 to 30% by weight of corn bran in water, gelatinizing by heating and then cooling.