JP2007110913A - New indigo-reducing microorganism and method for reducing indigo by using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microorganism capable of proliferating in a high alkaline side and more excellent in indigo-reducing function. <P>SOLUTION: This microorganism of A11 strain belonging to Alkalibacterium polygonumreducens having pH 10-12 growth pH and an indigo reducing function is provided. Also, a method for reducing the indigo is provided by bringing the microorganisms in contact with a growth medium containing a synthetic indigo or a fermentation vat liquid containing fermented indigo leaf which is called as "sukumo", prepared from a natural indigo. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a novel indigo-reducing microorganism and use thereof. More specifically, the present invention relates to a novel indigo-reducing alkaliphilic microorganism capable of growing on the high alkali side of pH 10 to pH 12 and having indigo reduction ability, and use thereof.

  Currently, dyeing with indigo is (i) fermenting natural indigo, that is, fermented liquor (fermented building fluid) containing wheat bran and lye into “sukumo” prepared by fermenting the leaves of plants. (Ii) a method of adding synthetic indigo chemically synthesized to the fermentation building fluid of (i) (split construction), (iii) “sukumo” prepared from indigo and synthetic indigo with hydrosulfite It is carried out by immersing the cloth in a dyeing solution prepared by a method using a chemical building to be reduced (hydro building).

  Of these, products dyed by the method of (i) fermentation building and (ii) the method of adding synthetic indigo to the fermentation building fluid are able to obtain a more elegant and clear color tone due to the action of the microorganisms involved. Compared with the chemical construction method of (iii), it has many advantages such as strong water resistance, no discoloration and fading. However, all of these methods have the drawbacks that fermentation takes a long time and management is very difficult and cannot be built without considerable experience. Therefore, if a microorganism having a superior 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 colors and strong water resistance.

Until now, as a patent relating to microbial reduction of indigo, there is a “microbial reduction method of synthetic indigo” by Takahara et al. (Patent Document 1). In a research paper (Non-patent Document 1), Takahara et al. Isolated a microorganism having the ability to reduce indigo present in an indigo fermentation liquid, and a new species of the genus Bacillus , namely Bacillus alkaliphilus ( Bacillus alkaliphilus). However, at present, this microorganism is not stored anywhere. In addition, this microorganism requires a peptide consisting of seven kinds of amino acids contained in indigo (a spice prepared from dwarf) and cannot grow on a medium that does not contain these peptides. It has been reported.

The 1999 Paden (AN Padden) et al., Large blue (Taisei) obligate anaerobic bacterium Clostridium Isachijisu with indigo reducing ability of the leaves of plants in the Brassicaceae family include plants from the fermentation solution obtained by fermentation in the cask called (Clostridium isatidis ) has been isolated (Non-patent Document 2). However, Daiko is different in variety from Kansai, and this microorganism is obligately anaerobic, making it difficult to cultivate it, and culturing at pH 10 or higher, which is the optimal reduction condition for normal indigo. There are many problems. As described above, microorganisms having indigo reduction ability have been isolated so far, but there is still no example of actual fermentation using these microorganisms.

On the other hand, the present inventors have been able to culture at pH 10 to pH 12 from a fermented building fluid containing “sukumo” prepared from lanthanum, and have the ability to reduce indigo Alkaalibacterium psychralcaliphilus IDR2 -2 ^T strain (FERM P-19648) (patent document 2, non-patent document 3) and Alkalibacterium iburiense M3 ^T , 41A and 41C strains have been found (non-patent document 4).

Patent 124188 (issue date: 1959.5.14) JP 2005-270028 (release date: 2005.10.6) "Journal of Fermentation Engineering" Vol. 40, No. 2, pp. 77-80 (1962) International Journal of Systematic Bacteriology, Vol. 49, pp. 1025-1031 (1999) International Journal of Systematic and Evolutionary Microbiology, 54, pp. 2379-2383 (2004) International Journal of Systematic and Evolutionary Microbiology, Volume 55, pp. 1525–1530 (2005)

  As mentioned above, fermented buildings using “Sukumo” and split buildings using “Sukumo” in combination with synthetic indigo are superior in water resistance and fading resistance to chemical buildings, but at an industrial and practical level. At present, it has not been possible to implement.

  Therefore, an object of the present invention is to isolate a microorganism having a better indigo reduction ability and to use it to efficiently reduce indigo in a shorter time.

  As a result of intensive studies to achieve the above-mentioned problems, the present inventors have found a microorganism excellent in indigo reducing ability from a fermentation liquid prepared in Aizumi-cho, Itano-gun, Tokushima Prefecture, and have completed the present invention. It was.

That is, the present invention includes the following inventions.
(1) A microorganism belonging to Alkalibacterium polygonumreducens having a growth pH of pH 10 to pH 12 and having indigo reduction ability.
(2) The microorganism according to (1), wherein the microorganism belonging to Alkalibacterium polygonumreducens is Alkalibacterium polygonumreducens A11 ^T strain (FERM P-20596).
(3) A composition for indigo reduction, comprising the treated product of the microorganism according to (1) or (2).
(4) A composition for indigo reduction, comprising the microorganism or the treated product of the microorganism according to (1) or (2).
(5) A microorganism according to (1) or (2) is brought into contact with a growth medium containing synthetic indigo or a fermentation building fluid containing `` sukumo '' prepared from natural indigo. Reduction method.
(6) The indigo reduction composition according to (3) or (4) is brought into contact with a growth medium containing synthetic indigo or a fermentation building liquid containing “sukumo” prepared from natural indigo. How to reduce Indigo.
(7) The method according to (5) or (6), wherein the fermented building fluid containing “sukumo” prepared from natural indigo is further added with synthetic indigo.

  The present invention provides a novel indigo-reducing microorganism and a method for reducing indigo using the microorganism. The indigo-reducing microorganism of the present invention has excellent reducing ability not only for synthetic indigo alone but also for a culture solution (fermented building fluid) containing “sukumo” prepared from natural indigo and a culture solution to which synthetic indigo is added. Indicates. Therefore, the use of the indigo-reducing microorganism of the present invention can greatly reduce the number of days for making the fermented building fluid into a state that can be dyed, and the produced reduced indigo can stain fibers with excellent fading resistance. Become. 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.
1. Indigo-reducing microorganisms of the present invention According to the present invention, microorganisms belonging to Alkalibacterium polygonumreducens having a growth pH of pH 10 to pH 12 and having indigo-reducing ability are provided. Examples of such microorganisms include Alkalibacterium polygonumreducens ( Alkalibacterium polygonumreducens ) A11 ^T strain (hereinafter “ A11 ^T strain ”). The indigo-reducing microorganism of the present invention also includes the above-mentioned A11 ^T strain mutants and mutants.

Isolation of the A11 ^T strain uses PYA medium containing peptone / yeast extract / synthetic indigo / inorganic salts / alkali and reinforced Clostridium agar (RCA) medium. Reduced indigo is produced on PYA medium and its reduction ability is indicated. Selected as.

  In the present specification, “indigo” includes not only synthetic indigo chemically synthesized but also natural indigo. Examples of natural indigo include indigo derived from plants containing indigo components (eg, indigo, large blue, Ryukyu indigo, and indigo indigo), and microorganism-derived indigo.

The mycological properties of the A11 ^T strain are as follows.
[1] Morphological properties (1) Gram staining: negative (property positive, negative staining)
(2) Spore: None (3) Motility: Existence (weak)
(4) Fungus shape: Aspergillus [2] Culture characteristics in medium The color tone is cream white, the shape of the colony is circular, and the surface of the colony is smooth.
[3] Chemical taxonomic characteristics The G + C molar content of DNA is 47.1%.
[4] Physiological and biochemical characteristics Catalase test: Negative oxidase test: Negative aminopeptidase test: Negative Culture at each pH
10: +
11: +
12: +
Incubation at each temperature
10 ° C:-
15 ° C: ±
20 ° C: +
30 ° C: +
40 ° C: +
45 ° C:-
Utilization of each carbon source
D-arabinose: +
D-xylose: +
D-glucose: +
D-glucose hexaphosphate:-
D-cellobiose: +
Yeast extract: +

The A11 ^T strain does not produce acid from any of D-arabinose, D-xylose, D-glucose, mannitol and sucrose.

DNA of A11 ^T strain was extracted, 16S rRNA gene was amplified by PCR, the base sequence of 16S rRNA gene obtained by amplification was analyzed with an autosequencer, and similar base sequences were examined by blast search. A similar tree sequence and multiple alignments were taken, and based on the results obtained, a phylogenetic tree was created by the neighbor-joining method. As a result, the A11 ^T strain was classified as an Alkalibacterium genus. There was found. The created phylogenetic tree is shown in FIG. FIG. 1 is a diagram showing a systematic position based on the 16S rRNA gene of Alkalibacterium polygonumreducens A11 ^T strain, and the numbers in the phylogenetic tree indicate a bootstrap value of 500 or more. Bars indicate 0.1 K _unc units.

A11 ^T strains alkali Agrobacterium Oribo APRO retainer capitalize (Alkalibacterium olivoaprovliticus) and 98%, the alkali Agrobacterium cycle Lal potassium stearothermophilus (Alkalibacterium psychralcaliphilus) IDR2-2 ^T strain showed 97% high homology.

Alkalibacterium olivoaprovliticus and Alkalibacterium psychralcaliphilus IDR2-2 ^T strains with high homology in the base sequence of the above 16S rRNA gene, DNAR with them DNA was extracted from A11 ^T strain to compare the homology of A11 ^T strain, and the DNA of A11 ^T strain was labeled with photobiotin to create a probe, the test DNA was immobilized on a black microplate, and the homology was calculated by fluorescence measurement did. As a result, the A11 ^T strain showed 18.9% and 36.3% homology with Alkalibacterium olivoaprovliticus and Alkalibacterium psychralcaliphilus IDR2-2 ^T strain, respectively. In the DNA-DNA homology test, 70% or more is considered to be the same species, but the A11 ^T strain was judged to be a new species belonging to the genus Alkalibacterium . The species name “ polygonumreducens ” is derived from the reduction (rejusense) of polygonum (genus name of polygonum; Greek origin).

The A11 ^T strain was deposited on July 15, 2005 at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (IPOD) (1-1-3 Higashi 1-1-3, Tsukuba, Ibaraki) under the accession number FERM P-20596. Yes.

2. Indigo reduction method The above-mentioned indigo-reducing microorganisms can directly reduce synthetic indigo, and can also reduce indigo in fermented building fluids containing "spider" and a mixture of the indigo and synthetic indigo as well. it can.

  Therefore, the indigo reduction method of the present invention can be carried out by bringing the above indigo-reducing microorganism into contact with a growth medium containing synthetic indigo or a fermentation building fluid containing “sukumo” prepared from natural indigo. . In the case of reducing synthetic indigo alone, it is sufficient to make contact with an appropriate growth medium in which indigo-reducing microorganisms can grow. When doing, it should just contact in fermentation building liquid. Here, “contact” means that indigo-reducing microorganisms are added to the growth medium or fermentation building fluid and cultured for a certain period of time. There is no restriction | limiting in particular in the addition amount to the growth culture medium or fermentation building liquid of an indigo reduction microorganism, What is necessary is just the quantity which can reduce indigo in a growth culture medium or fermentation building liquid. Moreover, what is necessary is just to perform the said culture | cultivation for 24 to 120 hours, for example in the range of 20-40 degreeC. The pH of the growth medium or fermentation building fluid is preferably 10-12. If the pH is lowered within the same range, the growth of the bacteria is promoted and it builds quickly. Therefore, it is not preferable.

  In addition, the indigo-reducing microorganism of the present invention may be used as an indigo-reducing composition containing the treated cell product. As used herein, “treated cell product” refers to an enzyme extracted from a crushed cell material, a culture, or a culture (including cells and culture supernatant), and the form thereof is a liquid preparation or a powder preparation. Is mentioned.

  For example, the indigo-reducing microorganism of the present invention is cultured, the liquid culture is centrifuged, the cells are collected, and the liquid preparation is suspended by adding physiological saline adjusted to pH 10 to obtain an appropriate concentration. Can be prepared. Moreover, you may add a pH adjuster etc. as needed.

  The powder preparation can be prepared, for example, by culturing the indigo-reducing microorganism of the present invention and drying the culture solution by freeze-drying or the like. 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 reducing composition of the present invention can also be produced by fixing the above-mentioned indigo-reducing microorganism cells or treated cells to a solid phase carrier. The solid phase carrier for immobilizing indigo-reducing microorganism cells or treated cells is not particularly limited as long as it can be used for immobilizing microorganisms. Examples thereof include resins, urethane prepolymers, epoxy resins, agar, chitosan, alginic acid, activated carbon, and diatomaceous earth ceramic porous bodies. Further, the shape of such a solid phase carrier is preferably a spherical shape or a cylindrical shape. In the case of a spherical shape, the particle size is preferably about 0.3 to 2.8 mm, more preferably about 0.3 to 1.2 mm.

  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
Fermented building fluid containing indigo prepared from natural temple in Aizumi-cho, Itano-gun, Tokushima Prefecture 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; synthetic 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; 900 ml of distilled water (900 ml) and 100 ml of alkaline buffer solution (1 M NaHCO ₃ / Na ₂ CO ₃ ) for adjusting to pH 10 were sterilized separately and mixed after sterilization]. .

Indigo-reducing microorganisms obtained by enrichment culture were isolated using alkaline-RCA medium [RCA medium (Reinforced Clostridial Agar medium: Sigma) 900 ml and alkaline buffer solution (1 M NaHCO 3) for adjustment to pH 10. ₃ / Na ₂ CO ₃ ) 100 ml separately sterilized and mixed] was dispensed into a petri dish and solidified to obtain an indigo reducing microorganism isolation medium.

  First, in order to obtain indigo-reducing microorganisms, add 5 ml of the above fermented building solution to an Erlenmeyer flask (100 ml volume) containing 100 ml of PYA medium, add 15 ml of liquid paraffin separately sterilized, and perform static culture at 27 ° C. went. After confirming that indigo reduction was performed by microorganisms, 5 ml of this culture solution was taken and added to another Erlenmeyer flask (100 ml volume) containing 100 ml of PYA medium. Such an operation was performed five times to select microorganisms having excellent indigo reduction ability.

  Synthetic indigo is a water-insoluble powder that sinks to the bottom of the flask before culturing, but when it is reduced by microorganisms, the precipitated indigo is solubilized and dissolved in the culture solution, and as the reduction of indigo proceeds The color of the culture medium changes from dark green to green. Therefore, the selection of the microorganism having excellent indigo reduction ability was performed using the color change of the culture solution (change to dark green or green) as an index.

  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 and streaked (inoculated) on the alkali-RCA medium. Further, they were transferred to an anaerobic jar, and the inside of the jar was replaced with argon gas, followed by culturing at 27 ° C. Then, after culturing for 5 days, a single colony was collected, streaked again in an alkaline-RCA medium, and a single colony was generated. 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 obtained isolated microorganism, alkaline-RCB medium containing 0.01% indigo [RCB medium (Reinforced Clostridial Broth medium: Sigma) 90 ml was sterilized by autoclaving, and then alkaline buffer (1M 10 ml of NaHCO ₃ / Na ₂ CO ₃ ) adjusted to pH 10] After inoculating the microbial cells in an Erlenmeyer flask containing 100 ml, add 15 ml of liquid paraffin sterilized separately and perform static culture at 27 ° C. The culture broth was observed to change to dark green or green.

The isolated microorganism thus obtained was designated as A11 ^T strain. This A11 ^T strain has been deposited at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology under the accession number FERM P-20596.

(Example 2)
Alkalibacterium polygonumreducens A11 ^T strain is aseptically inoculated on the plate obtained by dispensing the alkali-RCA medium shown in Example 1 into a petri dish and solidified, and inoculated at 27 ° C. Static culture was performed 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 alkali-RCA medium shown in Example 1 was prepared, and this slant was aseptically inoculated with Alkalibacterium polygonumreducens A11 ^T strain at a temperature of 27 ° C. Static culture was performed 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 medium [RCB medium (Reinforced Clostridial Broth medium: Sigma) 225 ml was placed in a shoulder flask (500 ml volume), sterilized by autoclave, and then added with 25 ml of alkaline buffer (1M NaHCO ₃ / Na ₂ CO ₃ ) pH adjusted to 10], aseptically inoculate one white fungus with Alkalibacterium polygonumreducens A11 ^T strain, and shake culture at 27 ° C for 2 days (about 48 hours) (120 round trips) Went. 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 medium [RCB medium (Reinforced Clostridial Broth medium: Sigma) 90 ml was placed in an Erlenmeyer flask (100 ml volume) and sterilized by autoclaving, then 10 ml of alkaline buffer (1M NaHCO ₃ / Na ₂ CO ₃ ) was added. In addition, the indigo reduction composition obtained in Example 2 was added to the one adjusted to pH 10, and 15 ml of liquid paraffin sterilized separately was added, and the medium was adjusted so as to be slightly aerobic or anaerobic. Static culture was performed at 0 ° C. On the first day of culture, the color of the culture started to change, and on the fifth day, the color of the culture changed to dark green, 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 liquid, and similar dyeing results were obtained.

It is a figure which shows the systematic position based on 16S rRNA gene of alkaline bacterium polygonumreducens ( Alkalibacterium polygonumreducens ) A11 ^T strain | stump | stock. The numerical value on the phylogenetic tree shows that bootstrap values of 1000 or more are over 500. Bars indicate 0.1 K _unc units.

Claims (7)

A microorganism belonging to Alkalibacterium polygonumreducens having a growth pH of pH 10 to pH 12 and having indigo reduction ability. The microorganism according to claim 1, wherein the microorganism belonging to Alkalibacterium polygonumreducens is Alkalibacterium polygonumreducens A11 ^T strain (FERM P-20596).   The composition for indigo reduction containing the microbial cell processed material of Claim 1 or 2.   The composition for indigo reduction containing the fixed body of the microbial cell of microorganisms of Claim 1 or 2, or a microbial cell processed material.   A method for reducing indigo, comprising contacting the microorganism according to claim 1 or 2 with a growth medium containing synthetic indigo or a fermented building fluid containing "sukumo" prepared from natural indigo.   Reduction of indigo, wherein the composition for indigo reduction according to claim 3 or 4 is brought into contact with a growth medium containing synthetic indigo or a fermentation building fluid containing "sukumo" prepared from natural indigo. Method.   The method according to claim 5 or 6, wherein the fermented building fluid containing "sukumo" prepared from natural indigo is further added with synthetic indigo.

Images