KR101513853B1 - The reduction dyeing of indigo using bacteria - Google Patents

Abstract

The present invention relates to a method for reducing and dyeing indigo using Dietzia sp. KDB1, and more particularly, to a method for reducing and staining indigo using Dietzia sp. Strain KDB1, This method can be applied not only to natural indigo but also to synthetic indigo, and can increase the amount of dyeing of the fabric efficiently, and simplifies the difficult reduction process such as complicated and complicated fermentation conditions And can contribute greatly to the field of dyeing using indigo.

Description

The reduction dyeing of indigo using bacteria

The present invention relates to the reduction dyeing of natural and synthetic indigo using bacterial strains, and a more efficient and reproducible eco-friendly reduced dyeing method using the bacteria identified and separated from the fermentation dye of traditional indigo dyes and bacterial strains used therein. About.

Indigo is insoluble in water, but when reduced under alkaline conditions, it turns into a leuco compound and becomes water-soluble, and has affinity for various fibers, and the water-soluble structure is oxidized and changes to an insoluble structure, resulting in color. In Korea, fermentation dyeing using raw juice salt and niram has been mainly performed, and the fermentation dyeing requires a process of reducing (fermenting) neram at the time required, and carbohydrates such as starch syrup based on lye in relation to the reduction Methods of adding ingredients are known. However, these conditions for reducing fermentation are difficult and complex, requiring a lot of time, labor, and detailed techniques that depend on experience.

In addition, the reduction method of insoluble indigo to water-soluble leuco-indigo is similar to the _{chemical reduction method using sodium hyposulfite (Na 2} S ₂ O ₄ ), which is a strong reducing agent widely used commercially, and the traditional dyeing method. It can be broadly divided into biological reduction methods using microorganisms (Bozic et al. 2009, Enzyme and Microbial Technology 45(4):317-323). However, the biological reduction method using microorganisms is a process that is difficult to apply in actual industrial sites as well as taking a lot of time because the specific species or amount of microorganisms have not been identified. Therefore, chemical reduction processes using strong reducing agents such as sodium hyposulfite or sodium sulfide (Na ₂ S) are actually used in industrial sites (Bozic et al. 2010, Applied Microbiology and Biotechnology 85(3):563-571).

However, chemical reduction using a reducing agent such as sodium hyposulfate or sodium sulfide causes many environmental problems and instability of coloration, and has a strong reducing power, causing great harm to the aerobic environment when discharged to the external environment. It has several problems (Aino et al., 2010, FEMS Microbiol Ecol74(1):174-183).

Above all, the process of reduction, which is difficult to standardize, is a major obstacle to the modernization of traditional indigo dyeing.

Republic of Korea Patent Publication 2012-0111714 Japanese Patent Application Publication JP 2005-270028

The present invention isolates a microorganism capable of reducing indigo without using a chemical reducing agent that has a detrimental effect on the environment to determine the physiological characteristics of the microorganism, and a method for reducing indigo using such characteristics, and It is intended to provide microorganisms used for this.

In addition, the present invention overcomes the disadvantages that it takes a long time depending on the external environment when dyeing indigo using bacteria involved in natural fermentation, the success or failure is uncertain, and lack of color reproducibility, and is applied not only to natural indigo but also to synthetic indigo. This is to provide an eco-friendly indigo reduction method with high reproducibility, simple, and good dyeing rate.

In order to solve the above problems, the present invention can provide four kinds of novel microorganisms having indigo reducing ability.

That is, the present invention is indigo (Indigo) having a reducing ability Dietzia species (Dietzia sp.) Can provide KDB1.

The present invention is indigo (Nesterenkonia species) having indigo reducing ability (Nesterenkonia sp.) Can provide KDB2.

The present invention is indigo (Nesterenkonia species) having indigo reducing ability (Nesterenkonia sp.) Can provide KDB3.

The present invention is indigo (Nesterenkonia species) having indigo reducing ability (Nesterenkonia sp.) Can provide KDB4.

The present invention is Dietzia species (Dietzia sp.) KDB1, Nesterenkonia species (Nesterenkonia sp.) KDB2, Nesterenkonia sp. sp.) KDB3 and Nesterenkonia species (Nesterenkonia sp.) It may be a microbial preparation for reducing indigo containing any one of the KDB4 strains or a mixed strain thereof as an active ingredient.

In addition, the present invention is Dietzia species (Dietzia sp.) KDB1, Nesterenkonia sp. sp.) KDB2, Nesterenkonia sp. sp.) KDB3 and Nesterenkonia species (Nesterenkonia sp.) It may be a method of manufacturing a microbial preparation for reducing indigo comprising any one of the KDB4 strains or a mixed strain thereof as an active ingredient.

The present invention is Dietzia species (Dietzia sp.) KDB1, Nesterenkonia sp. sp.) KDB2, Nesterenkonia sp. sp.) KDB3 and Nesterenkonia species (Nesterenkonia sp.) It may be a reduction dyeing method of indigo using any one of the KDB4 strains or a mixed strain thereof.

The reduction dyeing method of indigo according to the present invention may be a reduction dyeing method of indigo performed in an aqueous alkali solution.

Dietzia species according to the present invention (Dietzia sp.) KDB1, Nesterenkonia sp. sp.) KDB2, Nesterenkonia sp. sp.) KDB3 and Nesterenkonia species (Nesterenkonia sp.) The KDB4 strain is a Gram-positive bacteria and may be a reduction staining method for indigo, a strain having activity against nitrogen source decomposing enzymes.

The reduction dyeing of indigo using a bacterial colony according to the present invention is an eco-friendly indigo reduction method that can efficiently increase the dyeing amount of fabrics by efficiently reducing both natural indigo and synthetic indigo using a simple process. to provide.

In addition, in the reduction dyeing of indigo using bacterial strains, there is an effect of increasing the economical efficiency for use of coloring by increasing the reduction rate of indigo.

1 is an exemplary diagram showing four strains isolated from a traditional reducing solution.
Figure 2 is Dietzia sp. It is a diagram of (a) PH change and (b) K/S change according to the elapsed days upon reduction of natural indigo by KDB 1.
3 is Dietzia sp. It is a diagram of (a) PH change and (b) K/S change according to the elapsed days upon reduction of synthetic indigo by KDB 1.
Figure 4 is Nesterenkonia sp. It is a diagram of (a) PH change and (b) K/S change according to the elapsed days upon reduction of natural indigo by KDB 2.
5 is Nesterenkonia sp. It is a diagram of (a) PH change and (b) K/S change according to the elapsed days upon reduction of synthetic indigo by KDB 2.
6 is Nesterenkonia sp. It is a diagram of (a) PH change and (b) K/S change according to the elapsed days upon reduction of natural indigo by KDB 3.
7 is Nesterenkonia sp. It is a diagram of (a) PH change and (b) K/S change according to the elapsed days upon reduction of synthetic indigo by KDB 3.
8 is Nesterenkonia sp. It is a diagram of (a) PH change and (b) K/S change according to the elapsed days upon reduction of natural indigo by KDB 4.
Figure 9 is Nesterenkonia sp. It is a diagram of (a) PH change and (b) K/S change according to the elapsed days upon reduction of synthetic indigo by KDB 4.
Figure 10 is Nesterenkonia sp. It is a diagram of (a) PH change and (b) K/S change according to the elapsed days upon re-reduction of synthetic indigo by KDB 4.

The present invention provides a bacterial strain having indigo reducing ability and an indigo reducing dyeing method using the same.

In addition, the present invention provides a microbial preparation comprising a bacterial strain having an indigo reducing ability or a mixed strain thereof as an active ingredient and a method for producing the same.

The bacterial strain according to the present invention provides an eco-friendly indigo reduction method capable of efficiently increasing the dyeing amount of fabric by efficiently reducing both natural indigo and synthetic indigo using a simple process.

In addition, in the reduction dyeing of indigo using a bacterial strain, there is an effect of increasing the economical efficiency for the use of coloring by increasing the reduction rate of indigo.

Hereinafter, the present invention will be described in more detail in the following examples, but the present invention is only examples limited to the gist. Meanwhile, it is obvious to those skilled in the art that the present invention is not limited to the process conditions presented in the following examples, and can be arbitrarily selected within the range of conditions necessary to achieve the object of the present invention.

The present invention provides a bacterial strain having indigo reducing ability.

More specifically, the bacterial strain having indigo reducing ability according to the present invention is Dietzia spp. sp.) KDB1, Nesterenkonia sp. sp.) KDB2, Nesterenkonia sp. sp.) KDB3, Nesterenkonia sp. sp.) It may be any one of KDB4.

The dietzia species (Dietzia sp.) KDB1 applied for a deposit on May 23, 2013 to the Agricultural Genetic Resource Center of the National Academy of Agricultural Sciences, and was assigned an accession number on June 26, 2013 (accession number KACC91821P).

The Nesterenkonia species (Nesterenkonia sp.) KDB2 applied for a deposit on May 23, 2013 to the Agricultural Genetic Resource Center of the National Academy of Agricultural Sciences, and was assigned an accession number on June 26, 2013 (accession number KACC91822P).

The Nesterenkonia species (Nesterenkonia sp.) KDB3 applied for a deposit on May 23, 2013 to the Agricultural Genetic Resource Center of the National Academy of Agricultural Sciences, and was assigned an accession number on June 26, 2013 (accession number KACC91823P).

The Nesterenkonia species (Nesterenkonia sp.) KDB4 applied for a deposit on May 23, 2013 to the Agricultural Genetic Resource Center of the National Academy of Agricultural Sciences, and was assigned an accession number on June 26, 2013 (accession number KACC91824P).

In addition, the present invention provides a microbial preparation for indigo reduction comprising the strain or a culture solution thereof as an active ingredient.

The microbial preparation for indigo reduction of the present invention is Dietzia species as an active ingredient. sp.) KDB1, Nesterenkonia sp. sp.) KDB2, Nesterenkonia sp. sp.) KDB3, Nesterenkonia sp. sp.) As well as using KDB4 alone, it can be prepared by mixing these strains.

As a non-limiting example, the microorganism preparation for indigo reduction of the present invention is an active ingredient, Dietzia species (Dietzia sp.) KDB1 can be used alone, or Dietzia species (Dietzia sp . ) KDB1 and Nesterenkonia species sp.) KDB2 may be mixed and used, or all four types may be mixed and used, but the present invention is not limited thereto.

When preparing a microbial preparation for reducing indigo by mixing the four strains according to the present invention, the indigo reducing effect can be enhanced.

The microbial preparation for indigo reduction according to the present invention may be prepared as a solution, powder, suspension, dispersion, emulsion, oil dispersion, paste, dust, scattering material or granules, but is not limited thereto. In addition, the microbial preparation for reducing indigo may be used by spraying, spraying, spraying, scattering or pouring. The form of use depends on the intended purpose, which in all cases should be such that the distribution of the microbial preparation according to the invention is as fine and uniform as possible.

Hereinafter, a process and method for selecting indigo reducing microorganisms will be described through an embodiment of the present invention.

Materials and methods

1) DNA extraction for bacterial community analysis of traditional fermentation broth

DNA was extracted from salt solutions made in Naju, Jeollanam-do through traditional fermentation in 2008 and 2010, which dyed well, and from 2009, which dyed poorly. The fermentation broth used in the experiment was stored at -70°C until DNA extraction. After each fermentation broth was centrifuged at 13000 rpm for 10 minutes, the obtained 0.1 to 0.2 g of precipitate was suspended again in 5 ml of sterile distilled water. Of these, 1 ml of genomic DNA was extracted using the Genomic DNA Purifictioin Kit (Promega).

2) 16s rRNA library construction and selection method

A 16s rRNA library was constructed using the forward 27F (5'-AGAGTTTGATCMTGGCTCAG-3'), reverse 1492R (5'-GGTTACCTTGTTACGACTT-3') universal primers. PCR was performed using Taq polymerase for gene amplification. PCR conditions were 10 minutes denaturation at 95°C, 1 minute denaturation at 95°C extension, and 1 minute annealing at 58°C. (annealing), 25 cycles were performed at 72°C for 1.5 minutes under extension conditions. The amplified PCR product was developed by electrophoresis on an agarose gel for 30 minutes. A band of 1.5 to 1.6 kb was cut out on a gel and purified using a PCR Clean-Up kit (manufactured by Qiagen). The purified 16s rRNA DNA was ligated using pGEM®-T vector system (Promega) and T-Blunt vector system (Solgent) and transformed into E. coli XL1-Blue. Subsequently, for selection, colonies having a white color were selected by culturing in Macconkey solid medium containing ampicillin (50 μg/ml).

3) Confirmation of microbial community through FLP (Restriction Fragment Length Polymorphism)

The selected clones were checked for insertion of 16s rRNA through colony cracking, and cultured in a liquid medium of LB ampicillin (50㎍/ml) to obtain a miniprep DNA purification system kit (manufactured by Qiagen) from the induced strain. The plasmid was extracted using. Among the restriction enzymes that recognize and cut the amplified 16S rRNA into a specific sequence part of a gene, a cocktail enzyme solution was prepared and treated with BamH II , EcoR I, and Hind III having six recognition sequences. After analyzing and classifying the pattern of the fragments appearing on the gel, it is the process of re-analyzing the fragments that appeared on the gel by recognizing 4 specific sequences and processing Sau 3AI with a high cleavage frequency. Clones showing different patterns are finally selected and sequenced. Was analyzed. For pattern analysis, an enzyme cocktail solution [(cocktail enzyme solution) ( BamH Ⅱ , EcoR I, Hind Ⅲ)] and Sau 3AI enzyme 0.5-1U were each mixed using 12-25 ng of extracted DNA, and 1 hour 30 minutes to 3 minutes. It was allowed to react for hours. Thereafter, electrophoresis was performed at 100V on a 1.2% agarose gel and an 8% polyacrylamide gel for 30 minutes and 2 hours and 30 minutes, respectively, to compare patterns cut by restriction enzymes. Plasmid DNA with different patterns was PCR performed with forward (27F, 5'-AGAGTTTGATCCTGGCTCAG-3') and reverse (1492R, 5'-GGTTACCTTGTTACGACTT-3') primers and Teq polymerase for 16s rRNA amplification. 25 cycles were performed under conditions of 10 minutes denaturation at 95°C, 1 minute denaturation at 95°C, 1 minute annealing at 58°C, and 1.5 minutes extension at 72°C. The amplified PCR product was developed by electrophoresis on an agarose gel for 30 minutes, and then a band of 1.5 to 1.6 kb was cut out on the gel and purified using a PCR Clean-Up kit (promega). The purified 16s rDNA was sequenced from Genoteck and Macrogen for sequence confirmation. Genotech reads the sequence from both ends of the 16s rDNA using a forward (27F) primer and a reverse (1492R) primer. In Macrogen, sequence analysis was performed by reading the sequence from the middle of 16s rDNA using forward (518F) and reverse (800R) primers. The obtained two results were confirmed to the final sequence using a sequence aligment program (DNAMAN, CLUWTALW). The identified sequence was compared with the 16s rDNA sequence of the strain registered in the GeneBank Database using the Blast program provided by NCBI, and the strain identified in the fermentation broth was identified and the lineage was identified.

<Composition of reaction solution for 16s rRNA gene amplification> Component Μl Colony - 27F primer (5pmol) 0.5 1492R primer (5pmol) 0.5 dNTFs(10mM) One 10X PCR buffer 2.5 DDW 20.2 Taq polymerase 0.3 Final volume 25

<Conditions of reaction solution for 16s rRNA gene amplification> Reaction temperature Reaction time Reaction cycle Initial denatuararion 94℃ 5min 1 cycle Denatuaration 94℃ 1min 25 cycle Annealing 58℃ 1min - Polymerization 72℃ 1.5min -

4) Selection of strains that can be cultured in traditional fermentation solution

Nutrient medium (1% Trypton, 0.5% Yeast extract, 0.3g Beef Extract, 5% NaCl and pH 10) based on the strain identification results using 16s rRNA sequence in order to cultivate a strain that can be isolated among the complex strains of fermented saline. ) Was used. The fermentation broth was spread on a nutrient medium according to the dilution ratio, and cultured at 30° C. for 4 days. In addition, indigo dye precursor indican glycoside (indoxyl-glucose) was used as a carbon source and cultured in M9 minimal nutrient medium under the same conditions to find viable strains. The cultured strain was selected for the final analysis target bacterial strain according to the general phenotypic difference and whether the indican glycoside was used as a carbon source.

5) Identification of strains using Gram-staining

By comparing the 16s rRNA gene sequence, it was confirmed that the most similar bacteria were Gram-positive bacteria. To clearly confirm this, Gram-positive bacteria were determined through gram-staining. Drop the sterilized distilled water on the slide-glass and smear the bacteria. Indirectly, heat is applied to dry it to fix the bacteria. Tilt so as to flow into the dried bacteria from the edge of the slide-glass, add a crystal violet staining reagent, and react for 1 minute. Wash the staining reagent by allowing distilled water to flow through the back of the slide-glass. Add iodine solution and react for 1 minute. After washing in the same way, 95% ethanol is poured into it for bleaching. After washing with distilled water, safranin staining reagent was added and reacted for 1 minute. After washing with distilled water, water was removed, and staining was checked using a microscope.

6) Identification of strains using KOH test

Gram staining is the most basic method for classifying and identifying bacteria. However, some Gram-positive bacteria are sometimes stained as Gram-negative, making it difficult to read. In particular, in anaerobic bacteria, Gram-positive bacteria are not less prone to negatively bleaching than other bacteria, and may be negatively bleached depending on the incubation period. Meanwhile, Mobiluncus species sp.) shows various Gram staining results because it has the same structure as the cell wall of Gram-positive bacteria. The KOH test, first described by Ryu in 1938, is a method that uses differences in the composition of cell membranes of positive and negative bacteria. In other words, the cell membrane of Gram-positive bacteria is relatively stable in a low concentration of KOH solution, but the cell membrane of Gram-negative bacteria is easily destroyed, thereby releasing DNA, and the free DNA increases consistency, resulting in string phenomenon. 20 µl of a 3% KOH solution (3% KOH, 10% glycerin solution, stored at room temperature) was dropped on a glass plate, and 2 to 3 strains were dissociated. Gram negative and gram positive can be judged by observing the increase in viscosity for 30 to 60 seconds. If the viscosity was strong within 0 to 60 seconds, it was judged as positive, and if it progressed more than 60 seconds, it was judged as negative. In general, in the case of Gram-negative bacteria, it can be confirmed that the viscosity increases within 15 seconds, and in the case of Gram-positive bacteria, more time is required or the viscosity is not shown.

7) Identification of strains through analysis of biochemical properties

For the identification of the bacteria, physiological and biochemical characteristics, morphology and culture characteristics were confirmed. The genus can be identified mainly by morphological and cultural characteristics, but physiological and biochemical characterization is required to identify the species. For the identification of the strains, a test was performed to confirm the physiological activity characteristics of each strain.

① This is a method of investigating the ability to hydrolyze protein. It can be confirmed by adding _{mercury chloride (HgCl 2) to the transparent ring formed by bacteria grown on skim milk agar medium.} The bacteria are cultured by adding skim milk to a nutrient plate medium (3g/1L of enzyme extract, 8g/1L of peptone, 10g/1L of agar) so that it becomes 0.5%. _{A 10% mercury chloride (HgCl 2} )/20% hydrochloric acid (HCl) solution was added to the transparent ring formed as the bacteria grew, and the resolution for casein was confirmed.

② To check the resolution of starch, 0.15% soluble starch was added to a nutrient plate medium (yeast extract 3g/1L, peptone 8g/1L, agar 10g/1L) to prepare a test medium. After inoculating and culturing the bacteria in the prepared medium, it was confirmed that a transparent ring was formed by dropping the iodine solution used for gram-staining around the colonies of the grown bacteria.

③ The resolution of each bacteria to gelatin was confirmed. Gelatin medium [12% gelatin, peptone 20g/1L, enzyme extract 10g/1L, dextrose 20g/1L] was solidified in a 15ml test tube. The bacteria were inoculated in the middle of the hardened gelatin medium using platinum and cultured at room temperature (20 to 23°C). By confirming that the gelatin was decomposed and liquefied, it was confirmed whether or not it has gelatin decomposition.

④ Catalase is an enzyme that has heme as a prosthetic group. It is present in aerobic but mainly anaerobic bacteria with cytochrome. In the Catalase test, 3% (v/v) hydrogen peroxide is mixed so as to be 10 Vol% of the bacteria culture drug to check whether bubbles are generated, or by dropping hydrogen peroxide solution on the colonies of the plate medium to check whether bubbles are generated. I can. Each bacteria was inoculated on Nutrient plate medium and cultured at 30°C. 5 µl of 3% hydrogen peroxide solution was added to a single colony of each bacteria to check whether air bubbles were generated around the bacteria.

⑤ Oxidase test is used to distinguish between aerobic bacteria and conditional anaerobic bacteria. This is a method to check the activity of cytochrome oxidase, which catalyzes the oxidation of reduced cytochrome by oxygen, the final electron acceptor of the electron transport system. The bacteria were inoculated on Nutrient plate medium and cultured at 30°C. After adding a-napthol/95% ethanol: 1% dimethyl-p-phenylenediamine oxalate solution = 1:1 solution to a single colony of each bacteria, 10 It was confirmed whether or not the colony showed a dark blue color within to 30 seconds.

⑥ Indole test is a method to check whether tryptophan is used among amino acids. Inoculate the bacteria in peptone water (peptone 10g/1L, sodium chloride 5g/1L) and incubate in an incubator at 30°C, and then Kovac solution [(p-dimethylaminobenzaldehyde) 3g, butanol 75ml, hydrochloric acid [( HCl) 25ml] was added and mixed with 100 µl, and if it is positive, it appears pink or red, and if it is negative, there is no change in color.

8) Identification of biochemical characteristics using API ZYM kit

API ZYM kit (manufactured by BioMerieux) is a kit mainly used to test the enzyme activity of microorganisms and can be applied to almost all kinds of strains. With a small sample amount, the activity of 19 enzymes can be quickly confirmed, and the activity of enzymes in an unpurified mixed sample can be confirmed. Rich plate medium (1% tryptone, 0.5% sodium chloride (NaCl), 0.5% yeast extract, 0.3% beef) Incubated in Bef extract, 1% agar, pH10) and YPD plate medium (1% yeast extract, 2% peptone, 2% dextrose, 1% agar, pH10) One bacterium was dissolved in sterile distilled water or 0.85% sodium chloride (NaCl) solution so that the absorbance was 1 to 1.5 at 550 nm. 150 µl of the bacterial solution was inoculated into each cue of the kit, and incubated for 4 hours in a 30 incubator. After incubation, the ZYM A solution and the ZYM B solution included in the kit were added dropwise in order, reacted for 5 minutes, and the results were analyzed. According to the progress of time, it was confirmed whether or not color development was conducted by reacting for 1 hour and 12 hours at room temperature.

9) Medium Optimization: Test for Carbon Source

In order to create culture conditions suitable for the characteristics of the four strains extracted from the traditional reduced solution, a test was performed on each medium condition. The most important basic component in culture conditions is a carbon source. Therefore, an experiment was conducted to find out whether or not the strain for the carbon source was grown and its preference. 1% carbon source was added to the M9 minimal nutrient medium. 100mM sodium bicarbonate (NaHCO ₃ ) / sodium carbonate (Na ₂ CO ₃ ) solution (pH10) to glucose (Glucose), xylose (xylose), maltose (maltose), sucrose (sucrose), starch (starch) The bacteria were inoculated in M9 minimal nutrient plate medium (agar 1%) containing 0% and 1%, respectively. Incubated for 3 days in a 30 ℃ incubator. The growth of the bacteria was confirmed as the cultivation time elapsed.

<M9 salt composition> Component g Na ₂ HPO ₄ 64 KH ₂ PO ₄ 15 NaCl 2.5 NH ₄ Cl 5 DDW 900 ml

<9M minimum nutrient medium composition> Component ml 5X M9 salt 200 1M MgSO ₄ 2 Nutrient 20 1M CaCl ₂ 0.1 DDW 777.9 Final volume 1000

10) Medium optimization: testing for organic nitrogen sources

Yeast extract, beef extract, peptone, and tryptone were each 0% in 100 mM sodium bicarbonate (NaHCO ₃ )/sodium carbonate (Na ₂ CO _{3) solution (pH 10).} , 0.1% 0.5%, 1%, 5% was added to prepare a flat medium (1% agar). The pH of the medium was adjusted to 9 to 10 depending on the concentration of each nitrogen source component. It was confirmed that the higher the concentration of the nitrogen source component, the larger the change in pH appeared, and the lower the pH. Each bacterium was inoculated and cultured for 3 days in a 30°C incubator. The growth of the bacteria was confirmed as the cultivation time elapsed.

Results and Discussion

1) Extraction of metagenomic DNA from fermentation broth

Metagenomic DNA extraction from traditional fermentation broth contains a large amount of insoluble coloring compounds including indigo and plant residues, so it has a lot of impurities. Therefore, in order to extract only pure bacterial-derived DNA with little damage by the sample or compound used in the extraction process without impurities, high purity DNA was extracted by different extraction methods such as sample fractionation as a preceding process.

2) 16s rRNA amplification and library construction

The 16s rRNA gene is an evolutionarily well-conserved region, and is used for phylogenetic studies of prokaryotes, and is used for microbial identification and community analysis. The 16S rRNA is divided into a conserved sequence and a variable sequence. The conserved sequence is a conserved nucleotide sequence that many organisms have in common due to the low degree of evolution, whereas the variable part is a part with a large diversity according to the differentiation between species and genus of microorganisms. Since it contains a nucleotide sequence that exists only in, it is very useful for understanding the relationship between microorganisms. Therefore, by amplifying the 16S rRNA gene from the genomic DNA of various microorganisms extracted from the indigo fermentation broth, inserting it into a vector, and constructing a library, it is possible to know the types of various microorganisms in the indigo plant.

For the library construction, the entire 16S rRNA gene was amplified using a universal primer that combines genomic DNA extracted from indigo fermentation broth as a template with an indicator gene in general bacteria. At this time, the size of 16S rRNA DNA is about 1500bp, which appears to be a single band, but in fact, genes derived from numerous microorganisms are mixed.

The amplified 16S rRNA gene was inserted into a cloning vector to create a library, transformed into E. coli, and clones into which the 16S rRNA gene was inserted were selected. Clones into which the 16S rRNA gene has been inserted are checked for insertion of the recombinant gene through colony cracking, and each colony is inoculated into a liquid medium and cultured, and then the recombinant plasmid is extracted.

3) Analysis of microbial strains in fermentation broth through 16s rRNA sequencing

When a specific gene is cut using a restriction enzyme, it is cut into pieces of various sizes depending on the presence and frequency of the restriction enzyme recognition site in the nucleotide sequence. This can be confirmed in different patterns depending on the degree of evolution and whether or not mutations occur, even for the same type of microorganism. Using this principle, it is a method mainly used for gene conversion research, phylogenetic analysis, and species identification in the biological field.

An enzyme cocktail solution containing BamH Ⅱ, EcoR Ⅰ, and Hind Ⅲ, a restriction enzyme that recognizes and cleaves six nucleotide sequences using the extracted recombinant plasmid, and a restriction enzyme Sau that recognizes and cleaves four nucleotide sequences. After each reaction with 3AI, electrophoresis was performed. Gene clones showing different patterns were selected by analyzing the cutting pattern. As a result, 19 types of 16S rRNA fragment patterns in stained samples and 5 types of 16S rRNA fragment patterns in stained samples from a total of 76 selected gene clones were identified, and the sequence was analyzed.

The 1500bp 16S rRNA gene sequence obtained through sequence analysis was identified in the fermentation broth through sequence comparison analysis with related strains using the BlastN program provided by NCBI. As a result, in fermentation broth with good dyeing, ① Alkalibacterium species (Alkalibacterium sp.) E-119, ② Alkalibacterium olivoapovliticus WW2-SN4c, ③ Alkalibacterium Cyclotolerance psychrotolerans ), ④ Uncultured bacterium sp. 19 kinds of microorganisms such as SMQ95 were identified, and in fermentation broth with poor staining, ① Alkalibacterium species (Alkalibacterium sp.) E-119, ② Uncultured bacterium clone ambient_alkaline-56, and ③ Bacterium SL3.41. Five major microorganisms were identified. The identified strains confirmed the relationship through the evolutionary tree through the Clustal W program using each sequence characteristic.

As a result of analysis of 16S rRNA gene library, Alkalibacterium species (Alkalibacterium sp.) was confirmed, followed by 14% of uncultured bacterium. In the fermentation broth that could not be dyed, uncultured bacterium was confirmed at a rate of about 50%, followed by Bacillus species ( Bacillus sp.) accounted for about 30%. Based on the above results, the main strains in the fermentation broth are Alkalibacterium species (Alkalibacterium sp.) and uncultured bacterium sp., and it is thought that the difference between the main colonies and the differences between species found in samples that are well stained and samples that are not stained are important causes of providing different reducing power in fermentation broth.

4) Exploration of culture conditions for pure microbial culture and acquisition of strains capable of pure culture

An experiment was conducted to confirm whether the species identified through the above results exist in the fermentation broth and whether they can be cultured. The culture experiment was performed by modifying the composition of the medium by adding the composition to the LB medium. The fermentation broth was spread on a modified LB nutrient medium according to the dilution ratio, and cultured at 30° C. for 4 days, and 5 strains of different phenotypes were separated and cultured from the two fermentation broths. Subsequently, the 16S rRNA gene was amplified through colony PCR and then analyzed. As a result of nucleotide sequence analysis of the 16S rRNA gene, the strains cultured in the fermentation broth were Dietzia natronolimnaea strain W5044, Dietzia natronolimnaea strain TPL19, Nesterenkonia spp. sp.) AC84, Nesterenkonia sp. sp.) Tibet-IBa2, Nesterenkonia sp. sp.) 110-8, Bacillus species (Bacillus sp.) It was confirmed that the strains represented by CNJ826 PL04 were consistent with or closely related to the strain. Some of these are strains that were not identified when analyzing the 16S rRNA gene library, and are thought to be other strains that exist and grow in the fermentation broth.

5) New naming of strains isolated from traditional reduced saline solution

In order to confirm the information on the four strains isolated from the traditional reduced saline solution, which was named through the previous study, the 16s rRNA gene was amplified and sequenced. The 16s rRNA gene sequence obtained through PCR was compared with the GeneBank Database. Dietzia named through prior research natronolimnaea strain TPL19, Nesterenkonia spp. sp.) AC84, Dietzia natronolimnaea strain W5044, Nesterenkonia spp. sp.) 110-8, largely classified into two genera. Through the confirmation experiment, the name was changed according to the similarity of the 16s rRNA gene sequence of each strain. Dietzia The fungus named natronolimnaea strain TPL19 was Dietzia It was confirmed that the similarity was high with the bacteria of the natronolimnaea strain. Nesterenkonia species sp.) AC84, Dietzia natronolimnaea strain W5044, Nesterenkonia spp. sp.) 110-8 is a Nesterenkonia species (Nesterenkonia sp.) Tibet-IBa2, Nesterenkonia sp. sp.) It was confirmed that the similarity to AC84 was high. Dietzia, which was classified into another genus natronolimnaea The 16s rRNA gene sequence of strain W5044 is Nesterenkonia spp. sp.) and high similarity. The similarity between the 16s rRNA genetic sequence obtained through reconfirmation and the registered bacteria was confirmed, and each bacteria was named with a new name and registered in GeneBank.

6) Identification of the morphological characteristics of the strain

By comparing the 16s rRNA gene sequence, it was confirmed that bacteria with high sequence similarity were Gram-positive bacteria. Gram-staining was performed to confirm the exact morphological characteristics of the four strains discovered. All four strains were confirmed to be Gram-positive bacteria through Gram-staining. In the case of Gram-staining, some Gram-positive bacteria are sometimes stained as Gram-negative, so it is difficult to read. Anaerobic bacteria tend to decolorize as negative as gram-positive bacteria are more frequent than other bacteria, so it is also negative depending on the culture period. It is difficult to accurately read because it may be discolored. Therefore, to reinforce these concerns, the KOH test was performed. The discovered four bacteria are mixed with a 3% KOH solution to check the time and degree of viscosity appearing. In the KOH test, the results showing strong viscosity within 15 seconds were read as Gram-negative bacteria, but none of the bacteria used in the experiment showed viscosity. Therefore, it was confirmed that the four strains belong to Gram-positive bacteria as confirmed by 16s rRNA sequence similarity.

<Characterization of strains through Gram staining> Escherichia coli Dietzia sp.
KDB1 Nesterenkonia sp. KDB2 Nesterenkonia sp. KDB3 Nesterenkonia sp. KDB4 Gram dye
(Gram
staining)

Gram voice Gram positive Gram positive Gram positive Gram positive

7) Analysis of physiological activity characteristics of strains

For accurate identification of bacteria, a more accurate lineage can be identified by confirming not only the expressive characteristics but also the characteristics of physiological activity. Based on the characteristics of growing in an alkali of pH 9 or higher, a judgment test was performed to determine whether each bacteria reacts to a specific reagent, and the physiological activity characteristics of the strains were confirmed using the API ZYM kit. All four of the excavated bacteria were Gram-positive bacteria, and the specific colony color shown by each phenotype was confirmed. In [Table 6], each bacteria exhibited different specific phenotypes. It was confirmed that all the bacteria discovered through the Gram staining result and KOH test were Gram-positive bacteria. As a result of biochemical characterization, all four bacteria were positive for indole and catalase, and negative for gelatin and casein resolution. Dietzia species sp.) KDB1 and Nesterenkonia species (Nesterenkonia sp.) KDB2, Nesterenkonia sp. sp.) KDB3 and Nesterenkonia species (Nesterenkonia sp.) KDB4 showed different results. Nestor alkylene zirconia species (Nesterenkonia sp.) KDB2, Nestor alkylene zirconia species (Nesterenkonia sp.) KDB3 and Nestor alkylene zirconia species (Nesterenkonia sp.) All of the phenotypes of KDB4 were different, but the physiological activity characteristics were similar.

<Biochemical properties test of bacteria> Dietzia sp.
KDB1 Nesterenkonia sp.
KDB2 Nesterenkonia sp.
KDB3 Nesterenkonia sp.
KDB4 Colony coloring
(Colony pigmentation) Red White Pale red
(Light red) Yellow Gram reaction
(Gram reaction) + + + + KOH test - - - - Oxidase test - + + + Catalase test
(Catalase test) + + + + Gelatin hydrolysis
(Gelatin hydrolysis) - - - - Casein hydrolysis
(Casein hydrolysis) - - - - Starch hydrolysis
(Starch hydrolysis) - - - - Indole test
(Indole test) + + + +

(+: Positive,-: negative)

<Analysis of biochemical properties using API ZYM kit> Dietzia sp.
KDB1 Nesterenkonia sp.
KDB2 Nesterenkonia sp.
KDB3 Nesterenkonia sp.
KDB4 Alkaline phosphatase
(Alkaline phosphatase) + + + + Esterase [Esterase (C4)] + + + + Esterase Lipase
[Esterase Lipase (C8)] + + + + Lipase [Lipase (C14)] + - - + Leucinearylamidase
(Leucinearylamidase) + + + + Valinearylamidase
(Valinearylamidase) + + + + Cristinarylamidase
(Crystinearylamidase) + - + + Trypsin - + + + α-chymotrypsin - - + + Acid phosphatase
(Acid phospatase) + + + + Naphtol-AS-BI-phosphohydrolase + + + + αgalactosidase - - - - βgalactosidase - - - - β-glucuronidase - - - - αglucosidase + + + + β-glucosidase + - - - N-acetyl-β glucosaminidase - - - - α mannosidase - - - + α fucosidase - - - -

(+: Positive,-: negative)

As a result of the API ZYM kit, Nesterenkonia sp. KDB2, Nesterenkonia sp. KDB3 and Nesterenkonia sp. KDB4 showed similar characteristics, but Dietzia sp. ) KDB1 showed different characteristics from the three strains. All four strains showed no activity against α-galactosidase, β-galactosidase, β-glucuronidase, and α-fucosidase. In this case, information on the carbon source to be used in the medium can be obtained through these results.

8) Optimizing the culture medium of bacteria

In order to establish the culture conditions of the bacteria, the use of the carbon source nitrogen source was confirmed based on the alkaline conditions. A M9 minimal nutrient medium was prepared in 100 mM NaHCO ₃ /Na ₂ CO ₃ solution (pH 10.04), and a carbon source was added to 1%. Glucose, xylose, maltose, sucrose, and starch were added to 1%, respectively, and incubated for 3 days in an incubator at 30°C. A medium containing 1% of glucose, xylose, maltose, sucrose, and starch was not grown in 0% of the control, where no carbon source was added. Even in, all the bacteria did not grow. The composition of the rich medium used in the previous study did not contain a carbon source, and the four species of bacteria discovered because the bacteria did not grow in the minimal nutrient medium containing the carbon source were glucose, xylose, and It is predicted that carbon sources such as maltose, sucrose, and starch are not used as main energy sources. The rich medium that has been used so far is composed mainly of organic nitrogen sources such as yeast extract, beef extract, and tryptone. The degree of growth varies depending on the pH, but growth is generally possible in a medium containing a large amount of organic nitrogen sources. Therefore, a plate medium was prepared based on a commonly used organic nitrogen source 100mM sodium bicarbonate (NaHCO ₃ )/sodium carbonate (Na ₂ CO _{3) solution (pH 10.04).} Depending on the concentration of each nitrogen source (0, 0.1, 0.5, 1, 5%), the pH of the medium is adjusted to 9 to 10, and the higher the concentration of the nitrogen source, the lower the pH. Yeast extract showed that all bacteria were able to grow even at a low concentration containing 0.1%, and clear colonies and specific colors of the bacteria were clearly observed under the 1% condition. Beef extract is Dietzia species in 5% condition. sp.) Nesterenkonia species except KDB1 (Nesterenkonia All three bacteria belonging to sp.) grew with distinct traits. Dietzia species sp.) KDB1 showed that it was possible to grow by beef extract, but the distinctive red colonies were not clearly formed. In the medium to which 1% of peptone was added, it grew with a faint color, and under 5% condition, all the bacteria grew with clear color and colony formation. In the medium containing Tryptone, as opposed to beef extract, Nesterenkonia spp. sp.) was grown with a faint phenotype, and Dietzia spp. sp.) KDB1 was grown with a clear red colony in a medium containing 1% tryptone. In the medium containing 5% Tryptone, Dietzia spp. sp.) KDB1 shows vigorous growth, but Nesterenkonia species (Nesterenkonia The three bacteria belonging to sp.) showed slower growth compared to the medium containing 1%.

<Confirmation of growth of strains by organic nitrogen source> Nutrient Dietzia sp.
KDB1 Nesterenkonia sp.
KDB2 Nesterenkonia sp.
KDB3 Nesterenkonia sp.
KDB4 Yeast extract + + + + Beef extract + + + peptone + + + + Trypton +

(+: Can grow with a clear phenotype)

Experiment for evaluating the reducing power of new strains

1) Culture and quantification of strain

After taking the suspension of the strain cultured under the optimal conditions of the strain culture, using a centrifuge (1580MGR, Gyrogen Co., Ltd., Korea) (12000rpm, 1min, 4°C) to settle the bacteria and then as it was used for indigo reduction. The culture degree of the bacterial suspension was confirmed by the optical density (O.D.) value at 600 nm using a UV absorbance spectrophotometer (Aglient 845, Aglient Technologies, Waldbronm, Germany), and the final O.D. value was 1.8 to 2.0. However, the amount of bacterial strain used was presented as a value obtained by completely drying the strain obtained by centrifugation in an oven and weighing it.

2) Evaluation of reducing power

Reduction was carried out in an incubator at 32° C. using indigo (2.5 g of natural indigo, 0.25 g of synthetic indigo) sterilized at high temperature and high pressure (120° C., 20 min) in 35 mL of filtered 0.2% sodium carbonate aqueous solution (pH 11.32). Natural Indigo is made by immersing the pigment with slaked lime on the side grown in Naju, Jeollanam-do, and then dried in an oven at 50℃ (indigo 10.66% (W/W)) and synthetic indigo (Indigo, Vat Blue 1, Aldrich , Germany) purchased and used a commercially available product.

In order to observe the degree of reduction, a bacterial strain (7-100 mg) was added to the above reducing solution, followed by dyeing of hemp fabric according to the elapsed days. The hemp fabric was immersed in the supernatant of the reducing solution for 20 minutes, oxidized in air, color developed, washed with water, neutralized with 0.1% acetic acid aqueous solution for 10 minutes, washed with water and dried. In addition, the pH of the reducing solution was measured before dyeing.

3) Measurement of dyeing amount and color characteristics

The dyed fabric was evaluated for surface dyeing by using a color difference meter (Color-Eye 3100, Macbeth, USA) with the K/S value at the maximum absorption wavelength, and for color characteristics, Munsell's H V/C value was measured.

Results of evaluating reducing power of new strains

One) Diet Bell( Dietzia sp .) KDB Indigo reduction using 1

① Natural indigo reduction

Bacterial strain Dietzia sp. In the case of reducing natural indigo using KDB 1, changes in the pH and the amount of dyeing of the reducing solution according to the progress of the reduction are shown in [Fig. 2]. In the case of pH, it can be seen that the pH decreases continuously as the reduction day elapses regardless of the amount of strain, and as the amount of strain increases, the degree of pH decrease according to the elapsed day is greater, indicating a lower pH value in the same elapsed day. In the case of the amount of dyeing, the K/S value continuously increased until the maximum dyeing date, regardless of the amount of strain, and thereafter it showed a tendency to gradually decrease. On the same elapsed day, the higher the amount of strain, the higher the K/S value until the elapsed day when the maximum amount of staining was observed, but after that, the higher K/S value was not shown for the larger amount of strain.

[Table 9] shows the dye bath pH, K/S values and H V/C values of the dyed fabrics at the reduction initiation date and maximum dyeing amount at the time of reduction of natural indigo according to the amount of strain. When the strain was reduced by adding 7 mg of strain, the staining was performed from day 2, and when the strains of 20, 40, and 60 mg were added, the staining was performed immediately after 1 day. As described above, the initiation of reduction proceeded faster when the strain was added, but the initial dyeing amount according to the addition of the strain was 0.37 to 0.75, showing no significant difference, and all showed PB series colors. When 7mg of the strain was added, the maximum amount of dyeing was shown on the 5th day, and when the strains of 20, 40, and 60mg were added, the maximum amount of dyeing was shown on the fourth day. Each K/S value is 3.83, 8.02, 9.47. 11.23 and 12.35 showed a higher K/S value as the amount of strain added increased, and the salt bath pH at this time showed a lower value as the amount of strain added increased. All of the dyed fabrics showed PB series, and the more strains were added, the stronger the purple scent was dyed with the PB series. Both the brightness and saturation values continued to decrease as the amount of strain added increased, resulting in a darker and darker color, because the higher the amount of strain added, the higher the K/S value. .

< Natural Indigo Reduction by Dietzia sp. KDB 1> Strain amount
(mg) Start of reduction Maximum amount of dyeing lapse
Number of days pH K/S value H V/C lapse
Number of days pH K/S value H V/C Sample 7 2 10.59 0.61 3.4PB 6.9/3.7 5 10.12 8.02 3.5PB 3.7/5.0

20 One 10.82 0.37 2.6PB 7.6/2.7 4 10.07 9.47 4.0PB 3.4/4.8

40 One 10.82 0.45 2.5PB 7.3/3.0 4 9.91 11.23 4.3PB 3.1/4.4

60 One 10.62 0.75 3.3PB 6.6/3.9 4 9.66 12.35 4.4PB 2.9/4.1

② Synthetic indigo reduction

Bacterial strain Dietzia spp. sp.) When the synthetic indigo was reduced using KDB 1, changes in the pH and the amount of dyeing of the reducing solution according to the reduction progress are shown in [Fig. 3]. In the case of pH, it showed a continuous decrease in pH as the reduction day elapsed regardless of the amount of strain, and as the amount of strain increased, the degree of decrease with elapsed days was greater, indicating a lower pH value in the same elapsed day. In the case of the amount of dyeing, when 7 mg of the strain was added, it was not reduced at all.In the case of adding 20 to 60 mg of the strain, the K/S value was higher as the amount of strain increased until the 6th day, but 40 mg of the strain was added after 7 days. Showed a higher K/S value than when 60 mg was added.

[Table 10] shows the dye bath pH, K/S value and HV/C value of the dyed fabric at the reduction date of the synthetic indigo according to the amount of strain and the reduction date showing the maximum amount of dyeing, except for the case that it was not reduced at all. Presented. When 20mg of the strain was added, it was reduced on the 5th day, and the maximum amount of dyeing (K/S value 1.39) was shown the next day, and the K/S value was 1 or less from the 8th day. When the strains 40 and 60 mg were added, they were all reduced in one day, but when the strain 60 mg was added, the initial staining amount was twice as much as that when the strain 40 mg was added. In addition, it was confirmed that the higher the amount of strain, the higher the maximum amount of dyeing was shown. The salt bath pH at this time showed a lower value as the amount of strain added increased, and all were stained with PB series colors. And as the amount of strain added was higher, the brightness was lower and the saturation was higher, resulting in a dark but vivid color.

< Synthetic Indigo Reduction by Dietzia sp. KDB 1> Strain amount
(mg) Start of reduction Maximum amount of dyeing lapse
Number of days pH K/S value H V/C lapse
Number of days pH K/S value H V/C Sample 20 5 10.60 0.81 2.3PB 6.8/3.5 6 10.48 1.39 2.2PB 6.2/4.1

40 One 10.96 0.85 3.2PB 7.0/3.5 8 9.77 3.12 3.1PB 5.0/4.5

60 One 10.75 1.36 3.2PB 6.3/4.5 6 9.65 4.51 3.0PB 4.7/5.3

2) Nestorenconia Bell( Nesterenkonia sp .) KDB2 Indigo reduction using

① Natural indigo reduction

Bacterial strain Nesterenkonia spp. sp.) When natural indigo is reduced using KDB2, the change in pH and amount of dyeing of the reducing solution according to the reduction progress is shown in [Fig. 4]. In the case of pH, it showed a continuous decrease in pH as the reduction date elapsed regardless of the amount of strain, and the higher the amount of strain, the greater the degree of decrease in pH with the elapsed day, indicating a lower pH value in the same elapsed day. In the case of the amount of dyeing, the higher the amount of strain on the same elapsed day, the higher the amount of dyeing was, but in the case of the amount of strain 7, 20, and 40 mg, the difference in the amount of dyeing according to the amount of strain was not large. In the case of 7 mg of strain, reduction staining was performed only until 5 days, and in the case of strain 20, 40, and 60 mg, reduction staining was performed until 7, 10, and 14 days respectively, and the reducing power was maintained for a longer time as the amount of strain increased, but K/S value Appeared very low.

[Table 11] shows the dye bath pH, K/S values and H V/C values of the dyed fabrics at the reduction initiation date and the maximum dyeing amount at the time of reduction of natural indigo according to the amount of strain. When the strain was reduced by adding 7mg and 20mg strain, the staining was reduced from the 3rd day, and when 40mg and 60mg of the strain were added, the staining was reduced from the 2nd day. The initial salting amount according to the addition of the strain was as low as 0.34 to 0.39 in strain 7, 20, and 40 mg, and the highest in the strain 60 mg as 1.14. All colors were stained with PB series. In the case of the maximum amount of dyeing, the maximum amount of dyeing was shown on the 4th day when 7mg of the strain was added, on the 5th day when 20mg of the strain was added, and on the 3rd day when 40mg and 60mg of the strain were added. Each K/S value was 0.42, 0.82, 1.01, and 2.65, showing a slightly higher K/S value as the amount of strain added increased, and the salt bath pH at this time was 9.50 to 9.69, showing no significant difference depending on the amount of strain added. All of the dyed fabrics showed PB series, and the more strains were added, the more blue-ish was dyed with the PB series. As the amount of strain added increased, the brightness value continued to decrease and the saturation value continued to increase, resulting in a darker but vivid color.

< Nesterenkonia species sp.) Natural indigo reduction by KDB2> Strain amount
(mg) Start of reduction Maximum amount of dyeing lapse
Number of days pH K/S value H V/C lapse
Number of days pH K/S value H V/C Sample 7 3 9.81 0.39 0.4PB 7.5/2.8 4 9.67 0.42 3.5PB 7.4/3.3

20 3 9.75 0.36 2.2PB 7.5/3.0 5 9.69 0.82 3.2PB 6.6/4.0

40 2 9.65 0.34 2.1PB 7.6/2.5 3 9.69 1.01 2.5PB 6.4/4.1

60 2 9.55 1.14 2.6PB 6.4/4.5 3 9.50 2.65 2.3PB 5.2/4.9

② Synthetic indigo reduction

Bacterial strain Nesterenkonia spp. sp.) When the synthetic indigo was reduced using KDB2, changes in the pH and the amount of dyeing of the reducing solution according to the reduction elapsed days until the end of the reduction are shown in [Fig. 5]. The amount of strain was 20, 40, 60, and 100mg. In the case of pH, regardless of the amount of strain, the pH decreased rapidly as the reduction day elapsed, and Na ₂ CO ₃ was added on the 6th day (strain 20 to 60 mg: 0.05 g was added, strain 100 mg: 0.10 g was added) to increase the pH from 10.30 to 10. It was adjusted to 10.65. Up to 60 mg of strain, the larger the amount of strain, the greater the decrease in pH with the elapsed days, indicating a lower pH value in the same elapsed day. Showed a change of. In the case of the amount of dyeing, reduction dyeing began after pH adjustment by addition of alkali, regardless of the amount of strain, and no constant tendency was observed depending on the amount of strain added. Up to the 10th day elapsed, the highest amount of dyeing was shown in the order of 40mg, 60mg, 20mg, and 100mg. After 10 days, the highest at 100mg, followed by 40mg. It was the lowest by value. The reducing power was maintained for a longer time at 100mg with the largest amount of strain added.

[Table 12] shows the dye bath pH, K/S values and H V/C values of the dyed fabrics at the reduction initiation date of synthetic indigo according to the amount of strain and the reduction day showing the maximum dyeing amount. Regardless of the amount of strain, all of the strains began to be reduced on the 7th day, the day after the pH was adjusted by adding alkali, and strains 20, 40, and 60mg showed the maximum amount of dyeing of 2.46, 5.92, and 3.61, respectively, on the 8th day, and then decreasing. A tendency was shown, and 100mg of the strain showed a maximum amount of staining (5.96) similar to that of 40mg of the strain on the 6th or 14th day compared to other strains. All colors were stained with PB series. In the case of samples dyed at 40mg and 100mg with similar maximum dyeing amount, the brightness and saturation of the samples dyed at 100mg were higher.

< Nesterenkonia species sp.) Synthetic indigo reduction by KDB2> Strain amount
(mg) Start of reduction Maximum amount of dyeing lapse
Number of days pH K/S value H V/C lapse
Number of days pH K/S value H V/C Sample 20 7 10.50 1.23 2.5PB 6.1/3.8 8 10.39 2.46 3.0PB 5.5/5.0

40 7 10.22 0.95 2.3PB 6.5/3.2 8 10.05 5.92 3.7PB 3.9/4.3

60 7 9.90 2.28 2.6PB 5.7/4.6 8 9.72 3.61 3.0PB 5.0/5.4

100 7 10.12 1.31 2.6PB 6.0/4.0 14 9.28 5.96 3.1PB 4.3/5.2

3) Nestorenconia Bell( Nesterenkonia sp .) KDB3 Indigo reduction using

① Natural indigo reduction

Bacterial strain Nesterenconia species (Nesterenkoniasp.) When natural indigo is reduced using KDB3, the change in pH and amount of dyeing of the reducing solution according to the reduction progress is shown in [Fig. 6]. In the case of pH, it showed a continuous decrease in pH as the reduction day elapsed regardless of the amount of strain, and the higher the amount of strain, the greater the degree of decrease in pH with elapsed days, indicating a lower pH value in the same elapsed day. In the case of the amount of staining, it showed a tendency to decrease after the maximum staining date regardless of the amount of strain, and the large amount of strain did not necessarily show a high K/S value. The reducing power was maintained similarly for 15 to 16 days without significant difference depending on the amount of strain.

[Table 13] shows the dye bath pH, K/S values and H V/C values of the dyed fabrics at the reduction initiation date and maximum dyeing amount at the time of reduction of natural indigo according to the amount of strain. Regardless of the amount of strain, all were subjected to reduction staining from day 2, and the initial staining amount was 0.32, 2.59, 3.90, and 5.99, respectively, showing higher K/S values as the amount of strain increased. All showed PB series colors. The maximum amount of staining was observed on the 6th day when 7mg of the strain was added, on the 4th day when the strain 20mg was added, and on the 3rd day when 40mg and 60mg of the strain were added, respectively, and the K/S values were 4.81, 6.59, 8.40, and 9.86. As a result, the higher the amount of strain, the higher the maximum amount of staining was shown. All of the dyed fabrics showed PB series, and the more strains were added, the stronger the purple scent was dyed with the PB series. The brightness decreases as the amount of strain added increases, and the saturation increases by 0.1 at 20 mg, and then decreases, resulting in a darker and darker color as the amount of strain increases.

< Nesterenkonia species sp.) Natural indigo reduction by KDB3> Strain amount
(mg) Start of reduction Maximum amount of dyeing lapse
Number of days pH K/S value H V/C lapse
Number of days pH K/S value H V/C Sample 7 2 10.79 0.32 3.1PB 7.4/2.5 6 10.12 4.81 3.2PB 4.4/5.4

20 2 10.78 2.59 3.2PB 5.3/5.0 4 10.37 6.59 3.6PB 4.0/5.5

40 2 10.53 3.90 3.2PB 4.7/5.3 3 10.22 8.40 3.7PB 3.6/5.2

60 2 10.27 5.99 3.3PB 4.3/5.4 3 10.05 9.86 3.9PB 3.4/4.9

② Synthetic indigo restoration

When the synthetic indigo was reduced using the bacterial strain Nesterenkonia sp. KDB3, changes in the pH and the amount of salting of the reducing solution according to the days of reduction until the end of reduction are shown in [Fig. 7]. The amount of strain was added to 7, 20, 60, and 100mg. In the case of pH, as the reduction date elapsed in all strains, the pH decreased continuously, and as the amount of strain increased, the degree of decrease with elapsed days was significantly greater, indicating a lower pH value on the same elapsed day. In the case of the amount of dyeing, when 7 mg of the strain was added, it was not reduced at all, and when strains 20, 60, and 100 mg were added, the K/S value was higher as the amount of strain increased until the 10th day except the 2nd day when reduction was initiated. After 10 days, when 60 mg of the strain was added, the K/S value was higher than that when 100 mg was added. The reducing power was 12 to 13 days, which was similar according to the amount of strain.

[ Table 14]. When 20mg of the strain was added, it was reduced on the 5th day, and after showing the maximum amount of dyeing (K/S value 3.04) on the 7th day, the amount of dyeing continued to decrease. In the case of adding 60mg and 100mg of the strain, both began to be reduced on the 3rd day and showed the maximum amount of dyeing on the 6th day. The K/S value was higher at 100mg at the initiation of reduction, but the maximum amount of dyeing was higher when 60mg of the strain was added. The pH of the salt bath showing the maximum amount of dyeing showed a lower value as the amount of strain added increased. All showed PB series colors, and the higher the maximum dyeing amount, the lower the brightness and the same or lower saturation.

< Nesterenkonia species sp.) Synthetic indigo reduction by KDB3> Strain amount
(mg) Start of reduction Maximum amount of dyeing lapse
Number of days pH K/S value H V/C lapse
Number of days pH K/S value H V/C Sample 20 5 10.65 0.59 3.4PB 7.3/3.1 7 10.40 3.04 3.4PB 5.2/5.1

60 3 10.57 0.71 3.1PB 6.9/3.1 6 9.92 8.93 3.8PB 3.5/4.8

100 3 10.09 2.11 3.1PB 5.6/4.6 6 9.49 7.53 3.2PB 3.9/5.1

4) Nestorenconia Bell( Nesterenkonia sp .) KDB4 Indigo reduction using

① Natural indigo reduction

Bacterial strain Nesterenkonia spp. sp.) When natural indigo was reduced using KDB4, changes in pH and amount of dyeing of the reducing solution according to the reduction progress are shown in [Fig. 8]. In the case of pH, up to 40 mg of the strain amount showed a continuous decrease in pH as the reduction day elapsed, and the greater the amount of strain, the greater the degree of pH decrease with the elapsed days, indicating a lower pH value in the same elapsed day. On the other hand, in the case of the strain amount of 60 mg, although the amount of strain was larger, there was no significant difference from the pH change at the strain amount of 40 mg. In the case of dyeing amount, the K/S value tended to increase until the maximum dyeing date, and decreased thereafter. In the same elapsed day, the larger strain amount did not always show a higher K/S value.

[Table 15] shows the dye bath pH, K/S values and H V/C values of the dyed fabrics at the reduction initiation date and maximum dyeing amount when reducing natural indigo according to the amount of strain. In the case of reduction by adding 7 mg of strain, the dyeing was reduced from day 2, and in the case of adding strains 20, 40, and 60 mg, all of the strains were reduced dyed immediately after 1 day. The initial dyeing amount according to the amount of strain added was 0.30 to 0.59, which did not show a significant difference, and all showed PB series colors. In the case of strain 7, 20, and 60 mg, all showed the maximum amount of dyeing on the 6th day, and in the case of the addition of 40mg of the strain, the maximum amount of dyeing was shown on the 5th day. Each of the K/S values was 6.81, 7.19, 8.35, and 9.93, showing higher K/S values as the amount of strain added increased, and the salt bath pH at this time also showed lower values as the amount of strain added increased. The colors of the dyed fabrics all showed PB series, and both the brightness and saturation values continued to decrease as the amount of strain added increased, resulting in darker and darker colors.

< Nesterenkonia species sp.) Natural indigo reduction by KDB4> Strain amount
(mg) Start of reduction Maximum amount of dyeing lapse
Number of days pH K/S value H V/C lapse
Number of days pH K/S value H V/C Sample 7 2 10.43 0.30 3.2PB 7.8/3.0 6 9.93 6.81 3.4PB 4.0/5.4

20 One 10.59 0.40 3.2PB 7.5/2.4 6 9.50 7.19 3.4PB 3.9/5.2

40 One 10.42 0.42 3.1PB 7.2/2.6 5 9.40 8.35 3.6PB 3.8/5.1

60 One 10.25 0.59 2.9PB 6.9/2.6 6 9.13 9.93 3.6PB 3.4/5.1

② Synthetic indigo restoration

Bacterial strain Nesterenkonia spp. sp.) When the synthetic indigo is reduced using KDB4, changes in pH and amount of dyeing of the reducing solution according to the reduction elapsed days until the end of reduction are shown in [Fig. 9]. Strains 20, 40, 60, and 100mg were used. In the case of pH, with the amount of the strain of 20 mg, the pH continued to decrease as the reduction day elapsed, and in the case of the amount of the strain of 40 mg and 60 mg, there was little decrease in pH from the 7th day. In the strain 100mg, the pH decreased rapidly until the 4th day, whereas after the 5th day, the pH gradually increased. In the case of the amount of staining, in the case of the strains 20mg and 40mg, the amount of staining increased until the maximum staining date and then decreased thereafter, but in the case of the strains 60mg and 100mg, the amount of staining was very low on all elapsed days.

[Table 16] shows the dye bath pH, K/S values and H V/C values of the dyed fabrics at the reduction initiation date of the synthetic indigo according to the amount of strain and the reduction day showing the maximum dyeing amount. When the strain 20mg was added, it was reduced on the 5th day, the strain 40mg was reduced from the 2nd day, and the strains 60mg and 100mg was reduced from the 1st day. Each K/S value was 1.18, 0.41, 0.54, and 0.84, showing the highest amount of staining in the case of 20 mg with the smallest strain. In the case of elapsed days showing the maximum amount of staining, the strain was on day 6 (K/S value 2.75) for strain 20 mg, on day 5 (K/S value 1.77) for strain 40 mg, and on day 4 (K/S value of 1.03) for strain 60 mg. The higher the amount, the faster the maximum dyeing day was displayed, while the maximum amount of dyeing was lower as the amount of strain increased. In particular, in the case of 100mg with the largest amount of strain, it showed the maximum amount of dyeing (K/S value 0.84) with the initiation of reduction, showing the lowest reducing power. The color showed a mode PB series, and as the amount of strain added was small, the maximum dyeing amount was high, and the brightness was low, while the saturation was high, and the color was dark but vivid.

< Nesterenkonia species sp.) Synthetic indigo reduction by KDB4> Strain amount
(mg) Start of reduction Maximum amount of dyeing lapse
Number of days pH K/S value H V/C lapse
Number of days pH K/S value H V/C Sample 20 5 10.17 1.18 3.6PB 6.5/4.4 6 10.11 2.75 3.0PB 5.5/5.1

40 2 10.13 0.41 2.7PB 7.5/2.8 5 9.49 1.77 3.1PB 6.1/4.7

60 One 10.37 0.54 2.0PB 7.2/3.2 4 9.17 1.03 4.0PB 6.4/4.3

100 One 10.04 0.84 2.4PB 6.8/3.6 One 10.04 0.84 2.4PB 6.8/3.6

③ Synthetic indigo Re-reduction

Bacterial strain Nesterenkonia spp. sp.) When the synthetic indigo was reduced using KDB4, after completion of the first reduction, alkali was added to adjust the pH to 10.31 to 10.36, and the degree of re-reduction was checked. Changes in the pH and the amount of salting of the reducing solution according to the elapsed days of re-reduction are shown in [Fig. 10]. On the 9th day, the pH value was 10.12 to 10.23, and there was almost no decrease in pH compared to the first reduction regardless of the amount of strain. In the case of the amount of dyeing, dyeing by re-reduction was not at all at 20 mg, and at the same elapsed day, the higher the amount of strain, the slightly higher dyeing amount was.

[Table 17] shows the dye bath pH, K/S values and H V/C values of the dyed fabrics at the date of re-reduction of synthetic indigo according to the amount of strain and the reduction day showing the maximum amount of dyeing. In the strain amount 40mg, re-reduction staining was performed from the day immediately following pH adjustment, and in the strain 60mg and 100mg, re-reduction staining was performed on the 2nd and 3rd days, respectively. The S value is shown. In the case of maximum staining amount, K/S value of 2.25 on day 3 at 40 mg of strain amount, K/S value of 2.52 on day 4 at 60 mg of strain amount, and K/S value of 2.80 on day 3 at 60 mg. Showed value. In the case of the strain amount of 100 mg, the amount of dyeing continued to decrease after the start of reduction. All colors were stained with PB series.

< Nesterenkonia species sp.) Synthetic indigo re-reduction by KDB3> Strain amount
(mg) Start of reduction Maximum amount of dyeing lapse
Number of days pH K/S value H V/C lapse
Number of days pH K/S value H V/C Sample 40 One 10.34 0.96 0.8PB 6.5/3.2 3 10.26 2.25 3.0PB 5.7/4.8

60 2 10.33 1.72 1.6PB 6.0/4.5 4 10.25 2.52 2.5PB 5.4/5.1

100 3 10.25 2.80 3.1PB 5.3/4.8 3 10.25 2.80 3.1PB 5.3/4.8

Institute of Agricultural Biotechnology KACC91821 20130626 Institute of Agricultural Biotechnology KACC91822 20130626 Institute of Agricultural Biotechnology KACC91823 20130626 Institute of Agricultural Biotechnology KACC91824 20130626

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> The reduction dyeing of indigo using bacteria <130> P13050010601 <160> 4 <170> KopatentIn 2.0 <210> 1 <211> 1463 <212> RNA <213> Artificial Sequence <220> <223> indigo dyeing bacteria <400> 1 agggtttttt aactggctca ggacgaacgc tggcggcgtg cttaacacat gcaagtcgaa 60 cggtaaggcc ctttcggggg tacacgagtg gcgaacgggt gagtaacacg tgggtaatct 120 gccctgcact tcgggataag cctgggaaac cgggtctaat accggatatg agctcctgcc 180 gcatggtggg ggttggaaag tttttcggtg caggatgagt ccgcggccta tcagcttgtt 240 ggtggggtaa tggcctacca aggcgacgac gggtagccgg cctgagaggg tgatcggcca 300 cactgggact gagacacggc ccagactcct acgggaggca gcagtgggga atattgcaca 360 atgggcgaaa gcctgatgca gcgacgccgc gtgggggatg acggtcttcg gattgtaaac 420 tcctttcagt agggacgaag cgaaagtgac ggtacctgca gaagaagcac cggccaacta 480 cgtgccagca gccgcggtaa tacgtagggt gcaagcgttg tccggaatta ctgggcgtaa 540 agagctcgta ggcggtttgt cacgtcgtct gtgaaatcct ccagctcaac tgggggcgtg 600 caggcgatac gggcagactt gagtactaca ggggagactg gaattcctgg tgtagcggtg 660 aaatgcgcag atatcaggag gaacaccggt ggcgaaggcg ggtctctggg tagtaactga 720 cgctgaggag cgaaagcatg gggagcaaac aggattagat accctggtag tccatgccgt 780 aaacggtggg cgctaggtgt ggggtccttc cacggattcc gtgccgtagc taacgcatta 840 agcgccccgc ctggggagta cggccgcaag gctaaaactc aaaggaattg acgggggccc 900 gcacaagcgg cggagcatgt ggattaattc gatgcaacgc gaagaacctt acctaggctt 960 gacatataca ggacgacggc agagatgtcg tttcccttgt ggcttgtata caggtggtgc 1020 atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc 1080 ctgtctcatg ttgccagcac gttatggtgg ggactcgtga gagactgccg gggtcaactc 1140 ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgtctagggc ttcacacatg 1200 ctacaatggc tagtacagag ggctgcgaga ccgcgaggtg gagcgaatcc cttaaagcta 1260 gtctcagttc ggattggggt ctgcaactcg accccatgaa gtcggagtcg ctagtaatcg 1320 cagatcagca ttgctgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacgtc 1380 atgaaagtcg gtaacacccg aagccggtgg cctaaccctt gtgggaggga gccgtcgaaa 1440 gtgggatcgg cattggacac aca 1463 <210> 2 <211> 1518 <212> RNA <213> Artificial Sequence <220> <223> Nesterenkonia sp. KDB 2 <400> 2 agggtttgtt tttcgctcag gatgaacgct ggcggcgtgc ttaacacatg caagtcgaac 60 gatgaagacc gtgcttgcac ggttggatta gtggcgaacg ggtgagtatc acgtgagtaa 120 ccttcccttg actctgggat aagcccggga aactgggtct aataccggat acgaccagtc 180 ctcgcatggg gtgctggtgg aaagatttat cggtcttgga tggactcgcg gcctatcagc 240 ttgttggtga ggtaatggct caccaaggcg atgacgggta gccggcctga gagggtgacc 300 ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt ggggaatatt 360 gcacaatggg cgaaagcctg atgcagcgac gccgcgtgcg ggatgacggc cttcgggttg 420 taaaccgctt tcagcaggga agaagcgaaa gtgacggtac ctgcagaaga agcgccggct 480 aactacgtgc cagcagccgc ggtaatacgt agggcgcgag cgttatccgg aattattggg 540 cgtaaagagc tcgtaggcgg tttgtcacgt ctgctgtgaa agcccgaggc tcaacctcgg 600 gtgtgcagtg ggtacgggca gactagagtg cagtagggga gactggaatt cctggtgtag 660 cggtgaaatg cgcagatatc aggaggaaca ccgatggcga aggcaggtct ctgggctgtt 720 actgacgctg aggagcgaaa gcatggggag cgaacaggat tagataccct ggtagtccat 780 gccgtaaacg ttgggcacta ggtgtggggg acattccacg ttttccgcgc cgtagctaac 840 gcattaagtg ccccgcctgg ggagtacggc cgcaaggcta aaactcaaag gaattgacgg 900 gggcccgcac aagcggcgga gcatgcggat taattcgatg caacgcgaag aaccttacca 960 aggcttgaca tggaccggat cgctgcagag atgcagtttc ccttcggggt cggttcacag 1020 gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc 1080 gcaaccccta tcctatgttg ccagcacgtg atggtgggga ctcatgggag actgccgggg 1140 tcaactcgga ggaaggtggg gacgacgtca aatcatcatg ccccttatgt cttgggcttc 1200 acgcatgcta caatggccgg tacaatgggt tgcgatactg tgaggtggag ctaatcccta 1260 aaagccggtc tcagttcgga tcgaagtctg caactcgact tcgtgaagtt ggagtcgcta 1320 gtaatcgcag atcagcaacg ctgcggtgaa tacgttcccg ggccttgtac acaccgcccg 1380 tcaagtcacg aaagtgggta acacccgaag ccggtggcct aacccttgtg gggggagccg 1440 tcgaaagtgg gactcgcgat tggatatata aaaaggaggg ggatatatat atatataaat 1500 aagagcgccc cgtcttta 1518 <210> 3 <211> 1530 <212> RNA <213> Artificial Sequence <220> <223> Nesterenkonia sp. KDB 3 <400> 3 aaggtttcca ggaaactgct caggatgaac gctggcggcg tgcttaacac atgcaagtcg 60 aacgatgaag accgtgcttg cacggttgga ttagtggcga acgggtgagt atcacgtgag 120 taaccttccc ttgactctgg gataagcccg ggaaactggg tctaataccg gatacgacca 180 gtcctcgcat ggggtgctgg tggaaagatt tatcggtctt ggatggactc gcggcctatc 240 agcttgttgg tgaggtaatg gctcaccaag gcgatgacgg gtagccggcc tgagagggtg 300 accggccaca ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat 360 attgcacaat gggcgaaagc ctgatgcagc gacgccgcgt gcgggatgac ggccttcggg 420 ttgtaaaccg ctttcagcag ggaagaagcg aaagtgacgg tacctgcaga agaagcgccg 480 gctaactacg tgccagcagc cgcggtaata cgtagggcgc gagcgttatc cggaattatt 540 gggcgtaaag agctcgtagg cggtttgtca cgtctgctgt gaaagcccga ggctcaacct 600 cgggtgtgca gtgggtacgg gcagactaga gtgcagtagg ggagactgga attcctggtg 660 tagcggtgaa atgcgcagat atcaggagga acaccgatgg cgaaggcagg tctctgggct 720 gttactgacg ctgaggagcg aaagcatggg gagcgaacag gattagatac cctggtagtc 780 catgccgtaa acgttgggca ctaggtgtgg gggacattcc acgttttccg cgccgtagct 840 aacgcattaa gtgccccgcc tggggagtac ggccgcaagg ctaaaactca aaggaattga 900 cgggggcccg cacaagcggc ggagcatgcg gattaattcg atgcaacgcg aagaacctta 960 ccaaggcttg acatggaccg gatcgctgca gagatgcagt ttcccttcgg ggtcggttca 1020 caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 1080 agcgcaaccc ctatcctatg ttgccagcac gtaatggtgg ggactcatgg gagactgccg 1140 gggtcaactc ggaggaaggt ggggacgacg tcaaatcatc atgcccctta tgtcttgggc 1200 ttcacgcatg ctacaatggc cggtacaatg ggttgcgata ctgtgaggtg gagctaatcc 1260 ctaaaagccg gtctcagttc ggatcgaagt ctgcaactcg acttcgtgaa gttggagtcg 1320 ctagtaatcg cagatcagca acgctgcggt gaatacgttc ccgggccttg tacacaccgc 1380 ccgtcaagtc acgaaagtgg gtaacacccg aagccggtgg cctaaccctt gtggggggag 1440 ccgtcgaaag tgggactcgc gattggatat acaagggggg gggcgcatat atatatataa 1500 aagggggggg cggctctctt cttgtggagg 1530 <210> 4 <211> 1518 <212> RNA <213> Artificial Sequence <220> <223> Nesterenkonia sp. KDB 4 <400> 4 aggtttgatt ctcgctcagg atgaacgctg gcggcgtgct taacacatgc aagtcgaacg 60 atgaagaccg tgcttgcacg gttggattag tggcgaacgg gtgagtatca cgtgagtaac 120 cttcccttga ctctgggata agcccgggaa actgggtcta ataccggata cgaccagtcc 180 tcgcatgggg tgctggtgga aagatttatc ggtcttggat ggactcgcgg cctatcagct 240 tgttggtgag gtaatggctc accaaggcga tgacgggtag ccggcctgag agggtgaccg 300 gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagtg gggaatattg 360 cacaatgggc gaaagcctga tgcagcgacg ccgcgtgcgg gatgacggcc ttcgggttgt 420 aaaccgcttt cagcagggaa gaagcgaaag tgacggtacc tgcagaagaa gcgccggcta 480 actacgtgcc agcagccgcg gtaatacgta gggcgcgagc gttatccgga attattgggc 540 gtaaagagct cgtaggcggt ttgtcacgtc tgctgtgaaa gcccgaggct caacctcggg 600 tgtgcagtgg gtacgggcag actagagtgc agtaggggag actggaattc ctggtgtagc 660 ggtgaaatgc gcagatatca ggaggaacac cgatggcgaa ggcaggtctc tgggctgtta 720 ctgacgctga ggagcgaaag catggggagc gaacaggatt agataccctg gtagtccatg 780 ccgtaaacgt tgggcactag gtgtggggga cattccacgt tttccgcgcc gtagctaacg 840 cattaagtgc cccgcctggg gagtacggcc gcaaggctaa aactcaaagg aattgacggg 900 ggcccgcaca agcggcggag catgcggatt aattcgatgc aacgcgaaga accttaccaa 960 ggcttgacat ggaccggatc gctgcagaga tgcagtttcc cttcggggtc ggttcacagg 1020 tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1080 caacccctat cctatgttgc cagcacgtga tggtggggac tcatgggaga ctgccggggt 1140 caactcggag gaaggtgggg acgacgtcaa atcatcatgc cccttatgtc ttgggcttca 1200 cgcatgctac aatggccggt acaatgggtt gcgatactgt gaggtggagc taatccctaa 1260 aagccggtct cagttcggat cgaagtctgc aactcgactt cgtgaagttg gagtcgctag 1320 taatcgcaga tcagcaacgc tgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1380 caagtcacga aagtgggtaa cacccgaagc cggtggccta acccttgtgg ggggagccgt 1440 cgaaagtggg actcgcgttg gatatacaaa gagggggggg cggagatata tatataaaaa 1500 agggagagag gtgtagta 1518

Claims (8)

Nestenkonia species with indigo reducing ability ( Nesterenkonia sp.) KDB2 (KACC91822P). Nestenkonia species with indigo reducing ability ( Nesterenkonia sp.) KDB3 (KACC91823P). Nestenkonia species with indigo reducing ability ( Nesterenkonia sp.) KDB4 (KACC91824P). A microorganism agent for reducing indigo, which comprises the strain of any one of claims 1 to 3 or a mixed strain thereof as an active ingredient. A method for producing a microorganism agent for reducing indigo, which comprises the strain of any one of claims 1 to 3 or a mixed strain thereof as an active ingredient. A method for reducing and staining indigo using the strain of any one of claims 1 to 3 or a mixed strain thereof. The method according to claim 6,
The method for reducing and dyeing indigo is a method for reducing indigo which is carried out in an aqueous alkali solution. 4. The method according to any one of claims 1 to 3,
The strain is a gram-positive bacterium and has a reducing ability of indigo having activity against a nitrogen source.

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