JP4257978B2 - Indigo reducing microorganism and method for reducing indigo using the indigo reducing microorganism - Google Patents

Description

The present invention relates to a novel indigo-reducing microorganism and the like. In particular, the present invention relates to an indigo-reducing alkaliphilic microorganism that can grow on the high alkali side of pH 10 to pH 12 and has indigo reduction ability.

Currently, staining with indigo is (1) a method of fermentation using a natural indigo plant, that is, a spider prepared from plant kyan indigo (Sho Ai Ken), and (2) chemical synthesis in the fermentation building fluid of (1). A method of adding the synthesized indigo (wariken), (3) a method of reducing the scum prepared from indigo and the synthetic indigo with hydrosulfite (hydrobuilding) has been carried out.

  Among these, the product dyed by the method of (1) sukumo derived from natural indigo (2) and fermented building fluids of (1) with synthetic indigo is more elegant and clear due to the action of the microorganisms involved. And has many features such as high water resistance.

  However, the method of (1) using fermentation and the method of adding synthetic indigo to (1) have the disadvantages that fermentation takes a long time, and it is very difficult to manage and cannot be built without considerable experience. is doing.

So far, only the “microbial reduction method of synthetic indigo” (Patent Document 1) by Takahara et al. Has been issued as a patent relating to microbial reduction of indigo. In this connection, Takahara et al. Isolated a microorganism having the ability to reduce indigo in a fermented indigo broth in a research paper (Non-patent Document 2) and developed a new species of the genus Bacillus , namely a Bacillus alkali. We proposed phyllus ( Bacillus alkaliphilus ), but the microorganism is not stored anywhere. Furthermore, this microorganism requires a peptide composed of seven kinds of amino acids contained in indigo (a sphere prepared from spears), and cannot grow on a medium that does not contain these peptides.

On the other hand, in the research paper of 1999 Paden et al. {International Journal of Systematic Bacteriology, Vol. 49, pp. 1025-1031 (1999)} isolated obligate anaerobic bacterium Clostridium Isachijisu with indigo reduction ability from the fermentation solution obtained by fermentation of plant leaves in the vat (Clostridium isatidis). However, there are problems such as that the cultivar is different from the dairy blue and that this microorganism is obligately anaerobic, making it difficult to cultivate it, and the growth of pH 10 and above, which is the normal reducing condition, is not good. Many.

  As described above, microorganisms having the ability to reduce indigo have been isolated so far, but there is no example in which fermentation using these microorganisms is currently performed.

Patent 124188 (issue date: 1959.5.14) "Journal of Fermentation Engineering" Vol. 40, No. 2, pp. 77-80 (1962) International Journal of Systematic Bacteriology, Vol. 49, pp. 1025-1031 (1999)

  At present, indigo staining using a specific microorganism having indigo reduction ability is not industrially performed at all.

  Currently, the fermentation construction method generally used is dyed by dipping the fiber in a fermented indigo broth in which indigo is solubilized from spider as a natural dye, or in which synthetic indigo is added to this fermented construction fluid. However, this method requires a considerable number of days to make the fermented building fluid ready for staining. Therefore, if a new microorganism having excellent indigo reduction ability can be isolated, not only can the number of days be significantly reduced by adding such a microorganism to the fermentation building liquid, but also a clear color, It can be expected to obtain products with deep color and strong water resistance.

  Therefore, an object of the present invention relates to isolation of a microorganism excellent in indigo reduction ability and a method for reducing indigo using the microorganism.

  As a result of intensive studies to achieve the above-mentioned object, the present inventors have found that microorganisms excellent in indigo reduction ability exist in the fermented building liquid prepared from natural silkworms. It came to complete.

The present inventors have succeeded in isolating a microorganism excellent in indigo reducing ability from a fermentation liquid prepared in Date City, Hokkaido, and named the isolated indigo reducing bacterium as IDR2-2 strain. And the characteristics of this microorganism were analyzed, and the knowledge that this microorganism was a new species belonging to the genus Alkalibacterium was obtained.

The gist of the present invention is as follows.
That is, the present invention is (1) a microorganism belonging to the genus Alkalibacterium having a growth pH of pH 10 to pH 12 and having indigo reduction ability.

Further, the present invention is (2) a microorganism belonging to the alkali Agrobacterium (Alkalibacterium) genus, alkali Agrobacterium cycle Lal potash stearothermophilus above (1) a microorganism belonging to the (Alkalibacterium psychralcaliphilus) with indigo reducibility in microorganism according is there.

Further, the present invention is (3) a microorganism belonging to the alkaline Agrobacterium cycle Lal potassium stearothermophilus (Alkalibacterium psychralcaliphilus) is above (2), wherein the alkali Agrobacterium cycle Lal potassium stearothermophilus (Alkalibacterium psychralcaliphilus) IDR2-2 ^T strain microorganism It is.

The present invention also relates to (4) the microorganism according to (2) above, wherein the microorganism belonging to Alkalibacterium psychralcaliphilus is Alkalibacterium psychralcaliphilus FERM P-19648. It is.

  Moreover, this invention is (5) the composition for indigo reduction containing the microorganisms in any one of said (1) thru | or (4).

  Moreover, this invention is (6) the composition for indigo reduction which fix | immobilized the microorganisms in any one of said (1) thru | or (4).

  The present invention also includes (7) contacting indigo with the microorganism described in any one of (1) to (4) above in a fermentation building liquid containing sap prepared from a growth medium or indigo. A method for reducing indigo.

  Moreover, this invention contacts the indigo and the composition for indigo reduction | restoration as described in said (5) thru | or (6) in the fermentation building liquid containing the spice prepared from (8) growth medium or indigo. A method for reducing indigo.

According to the present invention, a novel indigo-reducing microorganism and a method for reducing indigo using the same are provided. The indigo-reducing microorganism of the present invention reduces not only a liquid culture of synthetic indigo alone, but also a liquid culture solution of soot prepared from a plant indigo-containing plant and a culture solution to which synthetic indigo is added. Show performance. In addition, since the indigo-reducing microorganism of the present invention is alkaliphilic, it can reduce indigo on the high alkali side.

  Hereinafter, the present invention will be described in detail.

First, the indigo reducing microorganism of the present invention will be described.
In the present specification, the term “indigo” includes not only synthetic indigo but also plants containing indigo components (such as abalone, large blue, Ryukyu indigo, and indigo). For example, synthetic indigo plant-derived indigo, microorganism-derived indigo, other indigo-containing products, and the like can be mentioned.

The indigo-reducing microorganism of the present invention is a microorganism belonging to Alkalibacterium psychralcaliphilus that can grow at pH 10 to pH 12, for example, Alkalibacterium psychralcaliphilus IDR2 -2 ^T stock. The present microorganism may be an alkaline bacterium Cyclical Caliphyrus variant or mutant strain that can grow at the pH of 10 to 12 and has indigo reduction ability. Furthermore, the present invention also includes all microorganisms having an indigo reduction ability at a growth pH of pH 10 to pH 12 belonging to the genus Alkalibacterium spp.

One example microorganism (IDR2-2 ^T strain) isolated by the present inventors has been deposited under the accession number FERM P-19648. The above-mentioned microorganism is isolated from a fermentation building liquid obtained by fermenting spiders prepared from garnet. For the isolation of IDR2-2 ^T strain, peptone / yeast extract / synthetic indigo / inorganic salts / alkaline (PYA) medium and fortified clostridium agar (RCA) medium were used to produce reduced indigo in PYA medium. Reduction ability was selected as an index.

The microbiological properties of this microorganism are as follows.
[1] Morphological properties (1) Gram staining: positive (2) Spore: None (3) Motility: Existence (4) Fungal form: Aspergillus [2] Growth characteristics in culture medium Color is colorless and colony shape Is circular and the surface of the colony is smooth.
[3] Chemical taxonomic characteristics The G + C molar content of DNA is 40.6%.
[4] Physiological and biochemical characteristics Catalase test: Negative oxidase test: Negative starch degradation: Negative gelatin degradation: Negative Growth at each pH
6:-
7:-
8:-
9:-
10: +
11: +
12: +
Growth at each salt (NaCl) concentration
0%: +
3%: +
5%: +
7%: +
9%: +
10%: +
11%: +
12%: +
13%: +
14%: +
15%: +
16%: +
17%: +
18%:-
20%:-
Growth at each temperature
0 ° C:-
5 ° C: +
10 ° C: +
20 ° C: +
30 ° C: +
40 ° C: +
45 ° C: +
50 ° C:-

The indigo-reducing microorganism of the present invention produces an acid from D-arabinose, D-xylose, D-glucose, and maltose. However, it does not produce acid from raffinose, myo -inositol, mannitol, and sucrose.

The types and fatty acid composition of the cell fatty acids of the indigo-reducing microorganism of the present invention are decanoic acid (C _{10: 0} ; 0.7%), tetradecanoic acid (C _{14: 0} ; 10.9%), hexadecanoic acid (C _{16: 0} ; 37.7%). ), Hexadecenoic acid (C _16: 17c; 2.2%), hexadecenoic acid (C _16: 19c; 34.7%), octadecanoic acid (C _{18: 0} ; 1.5%), and octadecenoic acid (C _18: 19c; 10.3%).

DNA of IDR2-2 ^T strain was extracted, 16S rRNA gene was amplified by PCR method, base sequence of 16S rRNA gene obtained by amplification was analyzed with auto sequencer, and similar base sequence was examined by blast search . Based on the results, multiple alignments with similar base sequences were taken, and a phylogenetic tree was created based on the results obtained by the Neighbor-Joining method. As a result, IDR2-2 ^T strain was genus Alkalibacterium . It was found to be classified as a bacterium. The created phylogenetic tree is shown in FIG. FIG. 1 is a diagram showing systematic positions based on the 16S rRNA gene of Alkalibacterium psychralcaliphilus IDR2-2 ^T strain, and the numbers in the phylogenetic tree indicate that the bootstrap value is 500 or more. Bars indicate 0.1 K _unc units.

Above all, it showed the highest homology with Alkalibacterium olivoaprovliticus .

Regarding the alkaline homology of Alkalibacterium olivoaprovliticus and Marinilactibacillus psychrotolerans having high homology in the base sequence of the 16S rRNA gene, microorganisms were obtained from the preservation agency, and IDR2-2 DNA was extracted from strain ^T strain, to create a probe labeled with DNA of IDR2-2 ^T strains photobiotin, the披検DNA was fixed to the black microplates, homology is calculated by fluorescence measurement. The results showed that IDR2-2 ^T strain had 24.3% and 7.6% homology with Alkalibacterium olivoaprovliticus and Marinilactibacillus psychrotolerans , respectively. In the DNA-DNA homology test, the IDR2-2 ^T strain was shown to be a new species belonging to the genus Alkalibacterium , although it is considered to be the same species as 70% or more.

  The indigo-reducing microorganism of the present invention can directly reduce synthetic indigo alone. In addition, in order to make fermented building fluid ready to be dyed by adding this indigo-reducing microorganism to the fermented building fluid of natural indigo solubilized from indigo as a natural dye and the fermented building fluid in which synthetic indigo is added to this solution. The number of days can be greatly shortened. The microorganism itself may be added directly to a growth medium or fermentation building fluid containing indigo, or to a solid support (for example, a photocurable resin, urethane prepolymer, epoxy resin, agar, chitosan, alginic acid, etc.) It may be used after fixing the body. At this time, the fermentation pH is preferably 10-12. In addition, since the indigo-reducing microorganism of the present invention can grow at a temperature in the range of 5 to 45 ° C., it can be used in a wide temperature range.

  Next, the indigo reduction method of the present invention will be described. The indigo-reducing microorganism of the present invention can be used as it is for reduction of synthetic indigo, and can also reduce indigo contained in the fermentation building liquid from sorghum. Furthermore, you may use as a composition for indigo reduction containing the indigo reduction microorganism of this invention.

Examples of such indigo reducing compositions include liquid preparations and powder preparations.
In order to obtain a liquid preparation, for example, the following method can be used.
Indigo-reducing microorganisms are cultured, the culture is centrifuged, the cells are collected, suspended in an appropriate concentration by adding physiological saline adjusted to pH 10, and used as an indigo-reducing composition. . Moreover, you may add a pH adjuster etc. to the composition for indigo reduction as needed.

In order to obtain a powder formulation, for example, the following method can be used.
Indigo-reducing microorganisms are cultured, and the culture is dried by freeze drying or the like to obtain a powder formulation. Alternatively, the cells may be collected from the culture solution by centrifugation, suspended in a physiological saline adjusted to pH 10 or a fresh medium, and then dried by lyophilization or the like. Further, a pH adjusting agent or the like may be added before lyophilization.

  The indigo reduction composition of the present invention can be produced by fixing the above indigo-reducing microorganism to a solid phase carrier. The solid phase carrier for immobilizing the indigo-reducing microorganism is not particularly limited as long as it can be used for immobilizing microorganisms. For example, alginic acid, activated carbon, diatomaceous earth ceramic porous body and the like can be mentioned. As the shape of such a solid phase carrier, a spherical shape or a cylindrical shape is suitable. In the case of a spherical shape, the particle size is preferably about 0.3 to 2.8 mm, and more preferably about 0.3 to 1.2 mm.

  Next, the method for reducing the indigo component of the present invention will be described. The method for reducing indigo of the present invention is characterized in that the indigo-reducing microorganism of the present invention or the indigo-reducing composition is brought into contact with a medium or a fermentation broth containing an indigo component.

  The method for reducing the indigo component of the present invention can be carried out by administering the indigo-reducing microorganism of the present invention or the indigo-reducing composition to a medium containing the indigo component. There is no particular limitation on the dose of the indigo-reducing microorganism or the indigo-reducing composition, and it may be an amount that can reduce indigo in the fermentation building fluid from the culture medium or scum derived from natural indigo. The pH of the culture medium or fermentation building fluid is preferably in the range of 10 to 12, but may be lower. By using the microorganism of the present invention, fibers can be dyed with reduced indigo obtained by a method of reducing indigo in a fermentation liquid from synthetic indigo or scum derived from natural indigo.

  Hereinafter, the present invention will be described specifically by way of examples. However, the technical scope of the present invention is not limited to these examples. In the following examples, “%” represents “% by mass” unless otherwise specified.

Example 1
A fermented building fluid containing indigo prepared from the natural temple prepared in Date City, Hokkaido, was used as a material for isolating indigo-reducing microorganisms.

For enrichment culture to obtain microorganisms with excellent indigo reduction ability, PYA medium {PY medium (peptone, 8 g; yeast extract, 3 g; indigo 0.1 g; K ₂ HPO ₄ , 1 g; EDTA, 3.5 mg ZnSO ₄ · 7H ₂ O, 3 mg; FeSO ₄ · 7H ₂ O, 10 mg; MnSO ₄ · nH ₂ O, 2 mg; CuSO ₄ · 5H ₂ O, 0.1 mg; Co (NO ₃ ) ₂ · 6H ₂ O, 2 mg; H ₃ BO ₃ , 1 mg; distilled water, 900 ml), and 100 ml of alkaline buffer solution (1 M NaHCO ₃ / Na ₂ CO ₃ ) for adjusting to pH 10 are separately sterilized. , Sterilized and mixed}.

For isolation of indigo-reducing microorganisms obtained by enrichment culture, alkali-RCA agar medium (alkali-enriched clostridial agar medium) {RCA medium (Reinforced Clostridial agar medium: Sigma) 900 ml and pH 10 100 ml of an alkaline buffer (1 M NaHCO ₃ / Na ₂ CO ₃ ) were separately sterilized and mixed, and the resulting mixture was dispensed into a petri dish and solidified to obtain an indigo reducing microorganism isolation medium.

  First, in order to obtain indigo-reducing microorganisms, 5 ml of the fermentation building solution was added to a 100 ml Erlenmeyer flask containing 100 ml of PYA medium, followed by stationary culture at 27 ° C. After confirming that indigo reduction by microorganisms was performed, 5 ml of this culture solution was taken and added to the culture medium for accumulation culture in a 100 ml Erlenmeyer flask. Such an operation was performed 5 times to select microorganisms having excellent indigo reduction ability.

  Regarding the presence or absence of indigo reduction, the indigo to be used was synthesized and was insoluble in water and settled at the bottom of the flask before culturing, but it was precipitated by reduction by microorganisms. Solubilizes and dissolves in the culture medium. As indigo reduction proceeds, the color of the culture changes to dark green or green. It was judged that indigo reduction was occurring due to a change in the color of the culture solution, that is, a change to dark green or green.

  In order to isolate the indigo-reducing microorganism, one platinum loop of a culture solution containing a microorganism having excellent indigo-reducing ability was picked, streaked (inoculated) on the alkali-RCA agar medium, and cultured at 27 ° C. After 48 hours of culture, a single colony was picked and streaked again on an alkaline-RCA agar medium to generate a single colony. This isolation operation was repeated 5 times to isolate a microorganism having excellent indigo reduction ability.

  In order to check the indigo reducing ability of the isolated microorganisms obtained, the microorganisms were separately sterilized with PYGA medium (900 ml of PY medium supplemented with 2 g of glucose and 100 ml of alkaline buffer solution for adjusting pH to 10, respectively) After inoculation into a mixture after sterilization), the culture was performed, and it was observed that the culture broth changed to dark green or green.

The isolated microorganism thus obtained was named IRD2-2 ^T strain. This IDR2-2 ^T strain is deposited under the accession number FERM P-19648 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology. Then, this strain result of studying the bacteriological properties, this bacterium has obtained a finding a new species belonging to the alkali Agrobacterium (Alkalibacterium) genus, was identified as an alkali Agrobacterium cycle Lal potassium stearothermophilus (Alkalibacterium psychralcaliphilus).

Example 2
Alkalibacterium psychralcaliphilus IDR2-2 ^T strain is aseptically inoculated on a plate obtained by dispensing the alkali-RCA agar medium shown in Example 1 into a petri dish and solidifying it, Static culture is performed at 27 ° C. for 5 days. The cells were separated from the obtained culture, and the cells were used as an indigo reduction composition.

Example 3
A slope (slant) of the alkaline-RCA agar medium shown in Example 1 was prepared, and this slant was aseptically inoculated with Alkalibacterium psychralcaliphilus IDR2-2 ^T strain as one white fungus ear, Static culture is performed at 27 ° C. for 5 days. The cells were separated from the obtained culture, and the cells were used as an indigo reduction composition.

Example 4
Alkaline-RCB agar medium {Alkaline-enhanced Clostridium liquid medium, RCB medium (Reinforced Clostridial liquid medium): Sigma} 225 ml was placed in a shoulder flask (500 ml), sterilized by autoclave, and then alkaline buffer (1M NaHCO ₃ / Na ₂ CO ₃ ) is added to adjust the pH to 10, and this medium is aseptically inoculated with Alkalibacterium psychralcaliphilus IDR2-2 ^T strain and inoculated one white fungus ear at 27 ° C. for 1 day (about 24 hours) Shaking culture (12 reciprocations) was performed. The obtained culture broth was used as it was or the culture broth was collected by centrifugation (6,000 rpm, 30 minutes) to obtain a composition for indigo reduction.

Example 5
Alkaline-RCB agar medium {Alkali-enriched Clostridium liquid medium, RCB medium (Reinforced Clostridial liquid medium): 90 ml of Sigma} was placed in an Erlenmeyer flask (100 ml), sterilized by autoclave, and then alkaline buffer (1 M NaHCO ₃ / Na ₂ CO ₃ ) Add 10 ml to adjust the pH to 10, add the indigo reduction composition obtained in Example 2 to this medium, and add 15 ml of liquid sterilized liquid paraffin which is sterilized separately to adjust the medium to be slightly aerobic or anaerobic. Then, static culture was performed at 27 ° C. The color of the culture broth began to change on the first day of culture, and the color of the culture broth changed to dark green on the fifth day, confirming that indigo had been completely reduced.

  Furthermore, after removing the upper layer liquid paraffin from the culture flask, it was transferred to a 200 ml beaker and dyeing of a piece of cloth was attempted in the same manner as a commonly used fermentation building liquid, and similar dyeing results were obtained.

It is a figure which shows the systematic position based on the 16S rRNA gene of Alkalibacterium psychral caliphilus ( Alkalibacterium psychralcaliphilus ) IDR2-2 ^T strain | stump | stock.

Claims (5)

A microorganism whose growth pH is pH 10 to pH 12 and which is Alkalibacterium psychralcaliphilus FERM P-19648 having indigo reduction ability. A composition for indigo reduction, comprising the microorganism according to claim 1 . The composition for indigo reduction which fix | immobilized the microorganisms of Claim 1 . Fermentation Ken'eki in containing Sukumo prepared from the growth medium or indigo, characterized by contacting the microorganism indigo with claim 1, a method of reducing the indigo. A method for reducing indigo, comprising contacting indigo with the indigo-reducing composition according to claim 2 or 3 in a fermentation building liquid containing sorghum prepared from a growth medium or indigo.

Images