JP4452869B2 - Method for promoting the growth of microbiota - Google Patents

Description

実施例２
実施例１のシクロデキストリン化合物に替えて、表２に示す多糖０．１％をプレートに添加した。
【００２６】
３０℃での培養２日目および１０日目に、プレート上に形成されたコロニーを計数した。
表２から分かるように、グリコーゲン、プルラン、カードラン、セルロースおよびグルコマンナンは、微生物叢の増殖促進効果を示した。
【表２】

実施例３
実施例１のシクロデキストリン化合物に替えて、表３に示す有機酸０．１％をプレートに添加した。
３０℃での培養２日目および１０日目に、プレート上に形成されたコロニーを計数した。
表３から分かるように、乳酸およびα−ケトグルタル酸は、微生物叢の増殖促進効果を示した。
【表３】

実施例４
土壌（殺菌していない）５０ｇにアンスラニル酸１０ｍｇを加え、よく攪拌混合した。
一方、Nutrient Broth８ｇとグルコース２ｇをよく混ぜ合わせ（Ａ）、これにグリコーゲン１ｇ（Ｂ）または２，６−ジ−Ｏ−メチル−β−シクロデキストリン１ｇ（Ｃ）を添加し、再度よく攪拌混合した。
前記土壌１０ｇに、それぞれ０．２ｇのＡ（土壌Ａ）、Ｂ（土壌Ｂ）またはＣ（土壌Ｃ）を加えてよく攪拌混合した。
得られた土壌Ａ、土壌Ｂおよび土壌Ｃを３０℃、湿度８５％の恒温槽内に置き、５日目および１０日目にサンプルを採取し、土壌中の生菌数（５日目および１０日目）とアンスラニル酸の含量（１０日目）を測定した。結果を表４に示す。
【表４】

【発明の効果】
本発明の方法によれば、上に述べたように、複数種の微生物が存在する微生物叢の増殖を促進することができ、それによって活性汚泥処理や土壌中の有害物質の無毒化を効率的に進めることができる。また、微生物叢中の特定の微生物種を効率よく単離することが可能になる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for promoting the growth of a microflora in which a plurality of types of microorganisms are present.
[0002]
[Prior art]
It is known that a plurality of types of microorganisms are involved in the treatment of activated sludge and the detoxification or detoxification treatment of harmful substances released to the outdoor environment. These beneficial microbiota need to actively proliferate the entire microbial community. In addition, when trying to isolate a specific microbial species from a certain microflora, it is often necessary to grow the entire microbial group included in the target microflora. Furthermore, in the natural world, there are many examples in which various microorganisms combine to produce useful substances while affecting each other. Industrial use in this respect is also expected in the future.
[0003]
As a method of promoting the growth of the microflora, considering the fact that the actual microbiota growth environment is an outdoor environment, even in an artificial simulated environment created in the laboratory, the temperature, aerobic conditions ( For example, it was performed by adjusting physical conditions such as oxygen enrichment and pH ("Microbial Engineering Handbook", pages 9-11, (Asakura Shoten) published in 1990). Regarding chemical conditions such as nutrients, inorganic salts were only enriched in the growth environment of the microflora except for the growth of the microflora on the agar complete medium in the laboratory ("Soil Microbial Experiment Method" 397, (Yokendo) published in 1997). More recently, it was reported that the number of colonies increased significantly when sodium pyruvate was added to the medium when enumeration of river bacteria by plate culture (Sugidate et al., Microbes and Enviroments, Vol. 14, 85 -87, (1999)).
[0004]
[Problems to be solved by the invention]
Therefore, for efficient activated sludge treatment and treatment of harmful substances in the outdoor environment, or for the isolation of specific microbial species from certain microbiota, and for more efficient substance production, the growth of the microflora There is still a need for improved methods to promote this. The object of the present invention is therefore to improve the method of promoting the growth of the microflora.
[0005]
[Means for Solving the Problems]
The present inventors have added γ-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, 2,6-di-O-methyl-β-cyclodextrin, to the growth environment of the microflora in which a plurality of types of microorganisms exist. And at least one cyclodextrin compound selected from the group consisting of 2,3,6-tri-O-methyl-β-cyclodextrin, or a polysaccharide selected from the group consisting of glycogen, pullulan, curdlan, cellulose, and glucomannan It has been found that the growth of microflora can be promoted by the presence of at least one of the above.
[0006]
That is, the present invention is an effective amount of γ-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin for promoting the growth of the above-mentioned microflora in the growth environment of the microflora in which a plurality of types of microorganisms exist. At least one cyclodextrin compound selected from the group consisting of 2,6-di-O-methyl-β-cyclodextrin and 2,3,6-tri-O-methyl-β-cyclodextrin, or glycogen, pullulan, curd A method for promoting the growth of a microflora, comprising the presence of at least one polysaccharide selected from the group consisting of orchid, cellulose and glucomannan.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
As for the microflora that needs to be promoted, several types of microorganisms are involved in the treatment of activated sludge and detoxification of harmful substances in the microflora. The purpose of conversion is not achieved. Or it is the microflora when trying to isolate a specific microbial species from a certain microbiota.
[0008]
An example of a microflora that needs to be promoted is an activated sludge-treated microflora. The activated sludge treatment microflora includes, for example, Paracolobacterium aerogenoides, Flavobacterium sp., Bacillus cereus, Escherichia intermidium, Nocardia actinomorpha, Escherichia coli, Pseudomonas ovalis, Alcaligenes faecalis, Bacillus lentus, Zoogloea ramigera, etc. Yes.
[0009]
Other microbiota that need to be promoted include rumen microbiota of ruminants, microorganisms that detoxify harmful chemical substances present in soil, for example, microorganisms that sequentially decompose and detoxify organic chemicals There are microbial fermentation flora such as flora, high concentration industrial waste liquid, microbial flora involved in decolorization and detoxification of cooking oil waste liquid such as olive oil, microbiota detoxifying soil contaminated with diesel oil and the like.
[0010]
Examples of microbiota from which specific microbial species are to be isolated include microorganisms in organisms such as higher animals such as humans, insects, soil protozoa or marine organisms, soil, mud, lake water, river water, and swamps. Microflora collected from mild environments such as water, microflora collected from polar regions such as the deep sea, hot and humid regions, extremely cold regions, volcanic regions, strong acid regions, high-salt regions, and arid regions List microbial flora collected from the natural environment where microbial species are present in large quantities, and microbial flora collected from artificial environments such as air and activated sludge and food processing industry raw materials (flavored juice, etc.) in hospitals and food factories Can do.
[0011]
The outdoor or artificial growth environment is an environment in which multiple types of microorganisms are involved in the treatment of activated sludge, detoxification of harmful substances, and the like in these environments. For example, an outdoor soil environment, an activated sludge treatment environment, a laboratory environment, an artificial decomposition treatment environment with chemical compound microorganisms, and the like are included. It is also a laboratory environment where all the microorganisms in the microflora are grown to isolate specific microbial species.
[0012]
As is well known, the laboratory environment is roughly classified into a solid medium environment and a liquid medium environment, and there are an aerobic environment and an anaerobic environment, respectively. It is well known that the energy source, nitrogen source, etc., temperature and pH in these environments mainly depend on the collection source of the microflora and the microorganism species to be isolated, and vary widely.
[0013]
In these outdoor or artificial growth environments, γ-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, 2,6-di-O-methyl-β-cyclodextrin, and 2,3,6-tri-O-methyl -At least one cyclodextrin compound selected from the group consisting of β-cyclodextrin, or at least one polysaccharide selected from the group consisting of glycogen, pullulan, curdlan, cellulose, and glucomannan are present.
[0014]
Here, “to make it exist” means that, for example, in activated sludge treatment, the cyclodextrin compound or the polysaccharide is introduced into an aeration tank, and in order to detoxify harmful substances present in outdoor soil. , Spraying the cyclodextrin compound or the polysaccharide solution or powder onto the soil. Alternatively, in a laboratory environment, the cyclodextrin or the polysaccharide is added to the growth medium of the microflora.
[0015]
The “effective amount for promoting the growth of the microflora” depends on the characteristics and environment of the microflora and is not uniform, but those skilled in the art can easily determine such an amount. That is, the number of microorganisms in an environment in which different amounts of cyclodextrin compound or polysaccharide are present may be measured. This is within the scope of routine work for those skilled in the art.
[0016]
Better results are often obtained by having at least one of the cyclodextrin compounds and at least one of the polysaccharides present in the growth environment.
[0017]
The presence of at least one organic acid in the growth environment of the microbiota may further promote the growth of the microbiota. Examples of such organic acids include, but are not limited to, lactic acid and α-ketoglutaric acid.
[0018]
As for other growth environment conditions of the microflora, known conditions can be applied in the method of the present invention.
[0019]
Thus, the growth of various microflora can be promoted by the method of the present invention.
[0020]
【Example】
Example 1
10 ml of activated sludge derived from a food factory was filtered with gauze, the filtrate was stirred with a mixer, then sonicated, further added with physiological saline, and stirred with a mixer to disperse the cells. This bacterial cell dispersion was diluted with physiological saline and measured by viable cell staining with viable cell count of 3 × 10 ⁴ cells / ml [6-carboxy-fluorescein diacetate (J. Porter et al., J. Appl. Bacteriol., Vol. 79, 399-408 (1995))].
[0021]
As the medium, Nutrient Broth (Difco) (1: 3 mixture of Bact Beef Extract and Bacto Peptone) added with 0.2% glucose and 2% agar was heat sterilized and solidified in a plate.
[0022]
To this plate, 0.1% by weight of a cyclodextrin compound (preliminarily sterilized) shown in Table 1 was added, and then 0.1 ml of the above microbial dispersion was seeded.
[0023]
Plates were placed at 30 ° C. and the number of colonies on the plates was counted on days 2 and 10.
[0024]
As shown in Table 1, γ-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, 2,6-di-O-methyl-β-cyclodextrin and 2,3,6-tri-O-methyl-β -Cyclodextrin showed an effect of promoting the growth of the microflora.
[0025]
[Table 1]

Example 2
Instead of the cyclodextrin compound of Example 1, 0.1% of the polysaccharide shown in Table 2 was added to the plate.
[0026]
Colonies formed on the plates were counted on the 2nd and 10th days of cultivation at 30 ° C.
As can be seen from Table 2, glycogen, pullulan, curdlan, cellulose and glucomannan showed the effect of promoting the growth of the microflora.
[Table 2]

Example 3
Instead of the cyclodextrin compound of Example 1, the organic acid 0.1% shown in Table 3 was added to the plate.
Colonies formed on the plates were counted on the 2nd and 10th days of cultivation at 30 ° C.
As can be seen from Table 3, lactic acid and α-ketoglutaric acid exhibited a microbiota growth promoting effect.
[Table 3]

Example 4
10 mg of anthranilic acid was added to 50 g of soil (not sterilized) and mixed well with stirring.
On the other hand, 8 g of Nutrient Broth and 2 g of glucose were mixed well (A), 1 g of glycogen (B) or 1 g of 2,6-di-O-methyl-β-cyclodextrin (C) was added thereto, and the mixture was thoroughly stirred again. .
To 10 g of the soil, 0.2 g of A (soil A), B (soil B), or C (soil C) was added and mixed well.
The obtained soil A, soil B, and soil C were placed in a thermostat at 30 ° C. and 85% humidity, and samples were taken on the 5th and 10th days, and the number of viable bacteria in the soil (the 5th and 10th days). Day) and anthranilic acid content (day 10). The results are shown in Table 4.
[Table 4]

【The invention's effect】
According to the method of the present invention, as described above, it is possible to promote the growth of a microflora in which a plurality of types of microorganisms are present, thereby efficiently treating activated sludge and detoxifying harmful substances in soil. Can proceed. In addition, it is possible to efficiently isolate a specific microbial species in the microflora.

Claims (3)

An effective amount of 2,6-di-O-methyl-β-cyclodextrin for promoting the growth of the microflora is present in the growth environment of the microflora in which a plurality of microbe species exist. A method for promoting the growth of activated sludge microbiota. The method according to claim 1 , wherein at least one polysaccharide selected from the group consisting of glycogen, pullulan, curdlan, cellulose and glucomannan is further present in the growth environment. The method according to any one of claims 1 to 2 , wherein α-ketoglutaric acid and / or sodium lactate is further present in the growth environment.