KR100706175B1 - New bacterium Flavococcus chejuensis producing zeaxanthin and production method of zeaxanthin using thereof - Google Patents

Abstract

The present invention is a novel zeaxanthin-producing bacterium Flavococcus chejuensis isolated from seawater in Jeju. It relates to a strain and a method for producing zeaxanthin using the same.

Flavococcus Jejuensis, Zeaxanthin, Carotenoids, Antioxidants, Geriatric eye disease, Colorants

Description

New bacterium Flavococcus chejuensis producing zeaxanthin and production method of zeaxanthin using lamb}             

1 is a photograph on a solid medium of a novel isolated strain according to the present invention (hereinafter abbreviated as D2). Yellow on the medium indicates zeaxanthin biosynthesis.

2 is a micrograph of a novel isolated strain D2 according to the present invention.

Figure 3 is a graph showing the growth curve according to the temperature of the novel isolated strain D2 according to the present invention.

4 is a taxonomic diagram according to the 16S rRNA nucleotide sequence of the novel isolated strain D2 according to the present invention.

5 is a photograph showing the TLC results of the paracoccus zeaxantinifaciens ATCC 21588 used as a novel isolated strain D2 and a control strain according to the present invention. At this time, lane 1 shows the TLC results of Paracaucus Giacanthinipansias and lane 2 shows the TLC results of the novel isolated strain D2 according to the present invention.

Figure 6 is a photograph showing the HPLC results of the novel isolated strain D2 and Paracoccus zeaxantinifaciens ATCC 21588 according to the present invention.

The present invention relates to a novel zeaxanthin biosynthetic bacterium Flavococcus chejuensis strain isolated from Jeju seawater and a method for producing zeaxanthin using the same.

Zeaxanthin is a C ₄₀ methyl-branched hydrocarbon structure with eight C ₅ isoprene units formed through continuous condensation and has a molecular weight of 568.88. Zeaxanthin is widely distributed in nature and is produced in various plants such as marigold petals, spinach, corn and kale, as well as bacteria and algae such as photosynthetic bacteria, cyanobacteria and non-photosynthetic bacteria.

Zeaxanthin is a yellowish xanthophyll carotenoid family and is one of the most known carotenoid compounds in addition to beta carotene. The carotenoids biosynthesized in plants are orange and red fat soluble pigments inherent in the chloroplast and pigment membranes. Although much is not known about the biosynthesis of zeaxanthin, enzymes involved in the carotenoid biosynthesis pathway in plants have recently been identified to provide information on the biosynthesis of zeaxanthin.

For plants, biosynthesis of zeaxanthin occurs through the following route (see US Pat. No. 6,627,795). The first process of carotenoid biosynthesis in plants is carried out by phytoene synthase. Phytoene synthase converts geranylgeranyl diphosphate to phytoene. The phytoene thus synthesized is converted to phytopoluene by the second enzyme (phytoene desaturase) of the carotenoid biosynthesis involved in the phytoene desaturation process, followed by an additional desaturation process, and then zeta-carotene is produced. do. Thereafter, zeta-carotene is converted into neurosporene by an enzyme that desaturates zeta-carotene (zeta-carotene desaturase), which is further unsaturated to form lycopene. Such lycopene can be converted to alpha-carotene or beta-carotene, of which beta-carotene is biosynthesized to zeaxanthin by beta-carotene dehydroxylase.

Zeaxanthin is known as an antioxidant and a colorant and is known to have some anticancer efficacy with respect to antioxidant function. Zeaxanthin has been found to be significantly more free-radical scavengers, i.e., free-stage oxygen scavenging ability in living tissues under hypoxic conditions with respect to in vivo antioxidant function.

The human body needs a constant supply of oxygen to maintain life phenomena. During normal metabolism, harmful and reactive reactive oxygen compounds (ROS) are produced. These oxygen compounds are largely divided into free radicals and non-radicals, both of which may cause cell damage due to unstable molecular structure. The production of such oxygen compounds may be accelerated due to smoking, ultraviolet rays, ozone, and the like. Therefore, in vivo, an antioxidant system such as antioxidant vitamins, minerals, enzymes, etc., for protecting tissues from oxidative stress caused by oxygen compounds is organized systematically. At this time, zeaxanthin increases antioxidant function through various interactions and suppresses the destruction of antioxidants by oxidation. In addition, it has been reported to reduce the risk of malignant tumors associated with antioxidant function in vivo as a bio-defense against free-radical damage to the body, and mutagenic inhibitory effect on cultured cancer cells, animals Useful results have been reported to be effective in inhibiting cancer in cancer transplantation and cancer-induced trials.

In addition, zeaxanthin is mainly distributed in the retina of the human body and has a prophylactic and delaying effect on degenerative eye diseases caused by aging such as cataracts and macular degeneration, which may cause decreased visual acuity. Moeller et al., J. Am. coll. Natr. 5: 522, (2000).                         

In addition, zeaxanthin is used as a feed additive and has a variety of uses, such as a colorant in the cosmetic and food industries. Zeaxanthin is a bright orange to yellow product that is in the spotlight as a colorant for animal feed for coloring land, skin and egg yolk in dark yellow. In winter, when there is a shortage of zeaxanthin and its isomer xanthophyll in livestock feed, the color of livestock yolks, milk fat and poultry fats is bleached because of the lack of zeaxanthin and xanthophyll pigments in livestock feed. . These pigment deficiencies can be resolved by adding zeaxanthin and xanthophyll pigments to livestock feed.

Zeaxanthin is a natural pigment that has excellent coloring and deposition ability and is less harmful than the toxicity of synthetic pigments. Zeaxanthin is also known to have better coloring and deposition capabilities than other carotenoid pigments. For example, carotenoids or carotenoid-containing compounds such as canthaxanthin, alfalfa, and cayenne pepper may cause abnormal red or purple pigmentation in meat, etc. or streaks in egg yolk when used at high concentrations. On the other hand, lutein is known to have the disadvantage of making the meat and egg yolk green when used in high concentrations.

In contrast, zeaxanthin is known to color yellow-red evenly in meats and the like even at high concentrations, and is a natural pigment useful as a livestock feed, food and beverage, and cosmetic colorant.

Extracting it from plants for industrial use of zeaxanthin has the disadvantages of low yield, high production cost, and difficulty in continuous supply in consideration of seasonal growth of plants. Thus, isolating microorganisms that biosynthesize zeaxanthin would be very useful for continued supply of zeaxanthin in large quantities.

Microorganisms that biosynthesize zeaxanthin were isolated from the ocean by Hoffmann-La Roche in the mid-1960s (US Pat. No. 3,891,504). One microorganism, named R-1512, was deposited with the ATCC organ (accession number ATCC 21588) and classified under Flavobacterium as a microorganism that produces zeaxanthin. Subsequently, further screenings identified several species, including R1534 and R114, that produced higher levels of zeaxanthin (Britton et al., Arch Microbiol. 113: 33 (1977); Goodwin, TW Biochem Soc Symp. 35: 233 (1972); McDermott et al., Biochem J. 144: 231 (1974); Mohanty et al., Helv Chim Acta. 83: 2036 (2000). However, unlike carotenoids such as beta-carotene or asaxanthin, strains that produce zeaxanthin as the main carotenoid are still very rare (Johnson and Schroeder, Adv.). Biochem.Eng. Biotechnol. 53: 119 (1996)).

Therefore, the development of zeaxanthin-producing strains as major cateroids would be very useful in the industry in that it can stably supply large amounts of natural pigments having useful uses as antioxidants, senile eye diseases, coloring agents and feed additives. will be. Thus, the inventors have tried to screen strains that biosynthesize zeaxanthin.

As a result of continuous efforts to isolate strains that biosynthesize zeaxanthin, the present inventors have been able to identify novel strains that produce zeaxanthin from Jeju seawater, thereby completing the present invention.

Accordingly, in one aspect, an object of the present invention is to provide a novel bacterial Flavococcus chejuensis strain that biosynthesizes zeaxanthin.

In another aspect, an object of the present invention is to provide a method for producing zeaxanthin using a novel bacterial Flavococcus Jejuensis strain that biosynthesizes zeaxanthin.

The present invention relates to a novel strain Flavococcus chejuensis that biosynthesizes zeaxanthin as a major carotenoid, and a method for producing zeaxanthin using the same.

Since the bacteria present in seawater live in seawater having a salt concentration of about 3%, there are many basophilic and salt-tolerant bacteria. Examples of seawater bacteria include Vibrio, Pseudomonas, Achromobacter, Aeromonas, and Photobacterium. (Micrococcus), Corynebacterium (Corynebacterium), etc., some are known to be detected. The number of bacteria in seawater germs decreases as they move farther away from the land, and the number of sea germs rapidly decreases in the outer sea. In appearance, the number of bacteria is the highest in the water depth of about 4 to 50m, Vibrio, Pseudomonas, Flavobacterium (Flavobacterium) is known to exist mainly.

Gram-negative, aerobic Bacillus or cocci are Pseudomonas genus, Halobacterium genus, Acetobacter genus, Gluconobacter genus, Alcaligenes genus, Brucella genus, Flavo genus Bacterium genus and the like are known, of which the flavobacterium genus is known to produce yellow to red pigment and to be colored on the meat surface.

In order to identify zeaxanthin biosynthetic bacteria, the present inventors collected and screened soft seawater of Jeju Island and found a strain that forms yellow colonies and named it D2.

Subsequently, the present inventors confirmed the zeaxanthin production ability through TLC and HPLC experiments with the comparison of the known zeaxanthin production ability with respect to the known D2 strain, and the morphological characteristics of the bacterium were Gram-negative bacteria. . In addition, by analyzing the physiological characteristics and carbon source availability of the D2 strain according to the present invention was obtained as shown in Table 1, the 16S rRNA base sequence through the PHYDIT program (molecular sequence editor for phylogeny by Jongsik Chun) Phylogenetic tree) (Saitou N. and Nei M., Mol Biol Evol. 4: 406 (1987)), it was confirmed that it is a renal strain having a similarity of 94% or less with other species (see Fig. 4).

Accordingly, the present inventors named this new strain, Gram-negative, aerobic cocci, as " Flavococcus chejuensis " which means "yellowish cocci isolated from the coast of Jeju." On March 3rd, it was deposited with KCTC 10534BP at KCTC (Korean Collection for Type Cultures), 52 Eun-dong, Yuseong-gu, Daejeon, Korea.

The characteristics of the novel strain Flavococcus chejuensis according to the present invention were verified and characterized by the following method.

end. Isolation and Culture of Strains

1) Isolation of bacteria

Coastal seawater near Daejeong-eup, Jeju Island, Korea was harvested and coated with 100 μl of marine agar plate 2216 (manufactured by Difco Laboratories, MD USA) and incubated at 25 ° C. for 48 hours. After 48 hours a yellow colony was found and this strain was passaged in the same medium to obtain a single strain, which was tentatively named D2 (see FIG. 1).

2) Culture of D2 Strain                     

Marine broth medium 2216 (manufactured by Difco Laboratories, MD USA) was used to culture the isolated strain D2. In the case of liquid medium, 37.4 g of powder medium is added to 1 L of distilled water, then boiled for 2 minutes to completely dissolve all the compounds, followed by sterilization for 15 minutes at 121 ° C. In the case of solid medium, 55.1 g of powder medium is added to 1 L of distilled water. Boil for a while to dissolve all compounds and then used by sterilizing for 15 minutes at 121 ℃. The preparation of the culture was inoculated in a 500ml baffled flask inoculated with a single colony of the strain separated in a liquid medium dispensed by 50ml each and incubated for 2 days at 25 ℃, 150rpm.

I. Identification of Isolated Strain D2

1) Morphological and cultural characteristics of bacteria

Some of the liquid cultures incubated at 25 ° C. for 48 hours using the above-mentioned marine broth medium were found to be morphogenetic under microscopic examination, and Gram-negative bacteria were examined as Gram-negative bacteria (see FIG. 2). In addition, the absorbance was measured at O.D600nm at 12 hours intervals at 20 ℃, 25 ℃, 30 ℃, 37 ℃ using Marine Broth medium to confirm the optimum growth temperature of the strain. As a result, optimal growth was observed at 25 ° C and 30 ° C, and the absorbance was increased faster than 25 ° C due to a short lag phase at 30 ° C. However, the absorbance after 48 hours was confirmed to be the same 30 ℃ and 25 ℃ (see Figure 3).

2) Physiological Characteristics and Carbon Source Availability of Bacteria

API 20NE kit (manufactured by Bio-Merieux Korea Co., Ltd) was used to investigate the physiological characteristics and carbon source availability of the strain according to the present invention. The isolated strain D2 according to the present invention was incubated in marine broth 2261 for 48 hours at 25 ° C., and then inoculated onto the substrate of the kit, and the results are shown in Table 1.

Survey item Characteristics Nitrate reducing power voice Indole generation voice Glucose acidification voice Esculin hydrolysis positivity Gelatin hydrolysis voice Beta-lactate activity positivity Use of arabinos voice Maltose use voice Cytochrome oxidation positivity

3) Sequence analysis of the bacterium 16S rRNA

The nucleotide sequence of 16S rRNA was analyzed for the identification of the isolated strain D2. 16S rRNA can be a good marker for evolving organisms. rRNAs are old molecules, functionally constant, widely distributed, and well conserved over a wide range of phylogenetic distances. In addition, since the number of sequences that can differ from each other in large molecules such as rRNA is very large, the similarity between the two sequences suggests a few systematic relationships.

In order to amplify the 16S rRNA of the isolated strain D2 according to the present invention, a bacterial commonly used 16S rRNA amplification primer (SEQ ID NO: 1 and SEQ ID NO: 2) was prepared, and PCR (polymerase chain reaction) was performed. As a result, a fragment of 1.4 kb was obtained, and the fragment was cloned into the pST-Blue1 vector (Novagen, WI, USA) to analyze the nucleotide sequence (SEQ ID NO: 3).                     

4) Fatty acid analysis of bacteria

Fatty acid composition in the cell was analyzed for identification of the isolated strain D2 according to the present invention, and 16S rRNA sequencing analysis resulted in comparing the fatty acid composition with two species having the highest similarity (similarity). The analytical instrument used a Microbial Identification System MIDI (Microbial ID. Inc., DE, USA), and the analyzed data was retrieved from TSBA40 (Sherlock Version 4.0; manufactured by MIDI, USA). The results are shown in Table 2.

Assay of Zeaxanthin Formation of Isolated Strain D2

1) Zeaxanthin production capacity assay through thin layer chromatography (TLC)

The pigment compound contained in the isolated strain D2 was extracted through acetone. The pigment extract was dried and then dissolved in a methanol: chloroform 1: 1 mixed solvent. The medium of TLC was silica gel (Silica gel 60F ₂₅₄ , Merck, Germany), and the extracted pigment extract was developed in a chloroform: aceton 97: 3 solvent. The results are shown in FIG.

2) Zeaxanthin production ability assay through HPLC (high-pressure liquid chromatography)

The pigment compound contained in the isolated strain D2 was extracted through acetone. The extracted pigment extract was dried and then dissolved in a methanol: chloroform 1: 1 mixed solvent. The column used for HPLC was a C18 reversed phase column (Ultrasphere ODS 4.6 mm × 25 cm, manufactured by Beckman Coulter, Calif., USA) and the solvent was acetonitrile: methanol: isoprophanol 90 It was used as 6: 6. The flow rate was 1 ml / min, and the absorbance was detected at UV 475 nm. The results are shown in FIG.

Thus, as one aspect, the present invention provides a novel Flavococcus chejuensis (Accession No. KCTC 10534BP) having a production capacity of zeaxanthin.

The novel Flavococcus Jejuensis having the production capacity of zeaxanthin of the present invention can be fermented in a variety of media that can promote the production of zeaxanthin, such fermentation methods are batch, fed-batch (fed) -batch) and continuous culture, etc. can be illustrated. Sugars and polymers thereof, polyalcohols, and the like may be used as the assimilation carbon source that may be included in the medium, and inorganic nitrogen compounds, materials of animal, plant and microbial origin may be used as the assimilation nitrogen source, other vitamins, Growth promoters, pigment formation promoters, and the like may be included in the medium.

The novel Flavococcus Jejuensis of the present invention has a pH of 5.5 to 8, preferably 6 to 7.8, more preferably 7 at 20 to 40 ° C, preferably 23 to 35 ° C, more preferably 25 to 30 ° C. In the culture to 7.8, in order to increase the production capacity of zeaxanthin according to the culture thereof, it is possible to adjust the culture conditions, such as oxygen, carbon dioxide.

Zeaxanthin produced from the cultured Flavococcus Jejuensis is present in the strain, and zeaxanthin-containing Flavococcus Jejuensis can be recovered by conventional methods such as centrifugation, filtration and the like. . The recovered ziazine tin-containing Flavococcus Jejuensis can be recovered from the culture and used as an animal feed additive.

Therefore, in another aspect, the present invention is to cultivate Flavococcus chejuensis (Accession No. KCTC 10534BP) and recover zeazanthin-containing Favococcus chejuensis containing zeaxanthin from the culture medium. It provides a method for producing zeaxanthin-containing Flavococcus Jejuensis, including the step of.

Zeaxanthin present in the cultured cells of the Flavococcus Jejuensis can be purified by using a conventional purification method in the art. Zeaxanthin present in the cells can be purified using standard methods in the art, for example using organic solvents.

In still another aspect, the present invention provides a method for culturing Flavococcus chejuensis (Accession No. KCTC 10534BP), recovering Flavococcus Jejuensis containing zeaxanthin from a culture medium, and accumulating from recovered cells. It provides a method for producing zeaxanthin, including extracting zeaxanthin.

Zeaxanthin purified as described above can be used as an antioxidant, colorant, senile eye disease, etc. as is well known in the art.

Example

Example 1 Identification Through 16S rRNA Sequences of Renal Isolate Strain D2

The isolated strain D2 was incubated in Marine Broth 2216 (manufactured by Difco Laboratories, MD USA) for 48 hours at 25 ° C., and the chromosome extracted therefrom was used as a template for PCR reaction, and the following primers were used.

GF1: 5'-TAA CAC ATG CAA GTC GAA CG-3 '(SEQ ID NO: 1)

GR1: 5'-GGT GTG ACG GGC GGT GTG TAC AAG-3 '(SEQ ID NO: 2)

The polymerase chain reaction was performed using a thermal cycler (manufactured by Takara, Japan), reacted at 94 ° C. for 5 minutes, 30 seconds at 94 ° C., 30 seconds at 60 ° C., and 45 seconds at 72 ° C. After the reaction was performed 35 times, a fragment of 1.4 kb was obtained by performing the reaction at 72 ° C. for 15 minutes. The obtained fragment was extracted from 0.8% agarose gel and then cloned into pST-Blue1 (Novagen, WI, USA) and analyzed for sequencing (SEQ ID NO: 3). As a result of confirming the flexible relationship between different species through NCLAST's BLAST-N, it showed 93.99% similarity with Marine algicidal bacterium Cytophaga sp. I-1858. . However, Cytophaga sp. I-1858 is not a type strain and thus cannot be compared, so Gelidibacter algens shows the highest similarity among the type strains. Compared with the ACAM 536T strain showed a similarity of 92.13%. Compared with the type strain, the strain is usually classified as a new genus if it has a similarity of 94% or less. Therefore, the isolated strain D2 according to the present invention was found to be a new trick based on the sequencing results of the 16S rRNA (see FIG. 4), and the inventors of the present invention referred to the strain "2" for the bacterium D2 according to the present invention. It was named "Flavococcus" which means " , and the species name was" c hejuensis " which means the coast of Jeju. This strain was named KCTC (Korean Collection for Type Cultures, Yuseong-gu, Daejeon Metropolitan City, Korea) on November 3, 2003. The deposit was made with accession number KCTC 10534BP to 52, Eun-dong Biotechnology Center, Korea Research Institute of Bioscience and Biotechnology, and 16S rRNA base sequence (SEQ ID NO: 3) was registered in GenBank under the registration number AY455998.

Example 2 Identification through Fatty Acid Analysis of Newly Isolated Strain Flavococcus Jejuensis

For analysis of fatty acid composition in isolated strain Flabococcus jejunsis according to the present invention it was incubated for 48 hours at 25 ℃ in marine agar plate 2216 (Difco Laboratories, MD, USA). 40 mg of the culture was taken in a cap tube with a loop, and then 1 ml of a reagent containing 15% sodium hydroxide was added to a methanol: distilled water (methanol: DW) 1: 1 solution, followed by reaction at 100 ° C. for 25 minutes to extract intracellular fatty acids. . For methylation of the extracted fatty acid, 2 ml of 6N HCl was added and reacted at 80 ° C. for 10 minutes, followed by rapid cooling. In order to convert an aqueous fatty acid into an organic phase, hexane: methyl-tert-butyl ether (Hexane: Methyl -tert Buthyl Ether) After adding 1.25 ml of a 1: 1 mixed solution, the solution was rotated for 10 minutes to obtain only the supernatant. Finally, the sample was washed with 3 ml of saturated sodium hydroxide solution and analyzed through an analyzer. The results of the analysis in the TSBA40 database showed no species with the same fatty acid composition, and 16S rRNA sequencing showed the highest similarity ( Gelidibacter algens and Cyclocerpens ). It was confirmed that the ethanol composition of Psychroserpens burtonensis was different from the fatty acid composition. This is shown in Table 2 below. Therefore, Flavococcus Jejuensis was able to prove a new trick through this fatty acid analysis.

fatty acid Total fatty acid (%) Flavococcus Jeju Nsis Cyclocerpens Bourtonensis Gelidibacter Argens 14: 0 0.5 0.2 0.3 15: 0 2.6 10.1 8.8 16: 0 2.6 0.4 1.4 18: 0 - - 0.2 i13: 0 - <0.1 - i14: 0 - 0.4 0.5 i15: 0 11.6 10.0 7.9 a15: 0 5.3 10.4 13.2 i15: 1 14.5 - - a15: 1 2.7 - - i15: 1w10c - 14.1 11.3 a15: 1w10c - 8.4 8.8 i16: 0 1.0 0.6 8.0 i16: 1 0.5 - - i16: 1w11c - 9.1 - i16: 1w6c - - 7.7 i17: 0 0.5 - - a17: 0 0.5 - 1.5 i17: 1 w7c - 1.5 4.9 i17: 1 w9c 0.6 - - a17: 1 w7c - <0.1 5.2 15: 1 w11c - 6.4 3.5 15: 1 w6c 0.7 3.5 - 15: 1 w4c - 7.6 1.3 16: 1 w9c - 0.7 - 16: 1 w7c - 0.4 5.9 16: 1 w5c - 6.9 1.5 17: 1 w8c 0.4 1.2 - 17: 1 w6c 0.6 1.5 3.7 2-OH 15: 0 1.7 - - 2-OH 17: 0 3.6 - - 3-OH i14: 0 0.4 - - 3-OH i15: 0 8.4 - 0.5 3-OH i16: 0 4.7 - 0.8 3-OH 16: 0 2.2 - 0.3 3-OH i17: 0 13.3 - 0.4 3-OH a17: 0 - - 0.4 2-OH i15: 0/16: 1w7c 12.1 - - 17: 1 ISO I / ANTEI B 2.5 - - unknown 11.543 0.9 - - unknown 13.565 2.1 - - unknown 16.582 1.3 - -

Example 3 Examination of Zeaxanthin Formation Capacity by TLC

According to the present invention, thin layer chromatography (TLC) was performed in order to investigate the zeaxanthin production ability of the novelly named Flavococcus chejuensis . At this time, Paracoccus zeaxantinifaciens known to produce zeaxanthin ATCC 21588 [Alan, Berry et al., Int. J. Syst. Evol. Micro., 53: 231, (2003)] as a control strain. As a result of calculating the Rf values ( Rf = development length of development solvent / development length of strain sample) of the control strain and Flabococcus jejunsis, both strains had the same value as Rf = 1.25 / 8.5 = 0.147 [see FIG. 5]. ]. Therefore, it could be verified that the novel strain Flavococcus Jejuensis according to the present invention produces zeaxanthin.

Example 4 Investigation of Zeaxanthin Production Capacity by HPLC

It was investigated whether Flavococcus Jejuensis produced zeaxanthin through HPLC. At this time, paracocos giazanthinipatians known to produce zeaxanthin ATCC 21588 was used as a control strain. Zeaxanthin extracted from the control strain showed a peak at 7.9 components, and the novel strain Flavococcus Jejuensis isolated according to the present invention also showed the same peak at 7.9 components. As a result, a single 7.9 component peak (see FIG. 6) was also shown in HPLC, which was analyzed by mixing the pigment compounds extracted from the two strains, confirming that Flavococcus Jejuensis according to the present invention produced zeaxanthin like the control strain. Could.

The zeaxanthin biosynthetic bacterium Flavococcus chejuensis strain provided by the present invention can stably supply useful natural pigments zeaxanthin which can be used as antioxidants, colorants, senile eye diseases, animal feed supplements, etc. Very useful.

<110> Korea Research Institute of Bioscience and Biotechnology <120> New bacterium Flavococcus chejuensis producing zeaxanthin and          production method of zeaxanthin using <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer GF1 for amplifying 16S rRNA <400> 1 taacacatgc aagtcgaacg 20 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer GR1 for amplifying 16S rRNA <400> 2 ggtgtgacgg gcggtgtgta caag 24 <210> 3 <211> 1307 <212> DNA <213> Flavococcus chejuensis <220> <221> ZN_FING (222) (1) .. (1307) <223> 16S rRNA <400> 3 gagcttgctc gaccggcgca cgggtgcgta acgcgtatac aatctgcctt gtactggggt 60 atagccttta gaaatgaaga ttaacacccc ataatataca gagcccgcat gggttctata 120 ttaaaggtta cggtacaaga tgagtatgcg tcctattagt tagttggtgc ggtaacggcg 180 caccaagaca gcgataggta ggggccctga gagggggatc ccccacactg gtactgagac 240 acggaccaga ctcctacggg aggcagcagt gaggaatatt ggacaatgga gggaactctg 300 atccagccat gccgcgtgca ggaagactgc cctatgggtt gtaaactgct tttatacggg 360 aagaaaccgt gctacgtgta gcaccttgac ggtaccgtaa gaataaggat cggctaactc 420 cgtgccagca gccgcggtaa tacggaggat ccaagcgtta tccggaatca ttgggtttaa 480 agggtccgta ggtggactgg tcagtcagag gtgaaatcct gcagcttaac tgtagaactg 540 cctttgatac tgctagtctt gaatcattgt gaagtggtta gaatatgtag tgtagcggtg 600 aaatgcatag atattacata gaataccaat tgcgaaggca gatcactaac aatgtattga 660 cactgatgga cgaaagcgta ggtagcgaac gggattagat accccggtag tctacgccgt 720 aaacgatgga tactagctgt ccggcttcgg ctgggtggct aagcgaaagt gataagtatc 780 ccacctgggg agtacgttcg caagaatgaa actcaaagga attgacgggg gcccgcacaa 840 gcggtggagc atgtggttta attcgatgat acgcgaggaa ccttaccagg gcttaaatgt 900 agattgacag gtttagagat agacttttct tcggacaatt tacaaggtgc tgcatggttg 960 tcgtcagctc gtgccgtgag gtgtcaggtt aagtcctata acgagcgcaa cccctgttgt 1020 tagttgccag catgtaaaga tgggaactct agcaagactg ccggtgcaaa ccgtgaggaa 1080 ggtggggatg acgtcaaatc atcacggccc ttacgtcctg ggctacacac gtgctacaat 1140 ggtagggaca gagagcaacc actgggcgac caggagcgaa tctataaacc ctatcacagt 1200 tcggatcgga gtctgcaact cgactccgtg aagctggaat cgctagtaat cggatatcag 1260 ccatgatccg gtgaatacgt tcccgggcct tgtacacacc gcccgtc 1307

Claims (11)

Flavococcus chejuensis (Accession No. KCTC 10534BP) having a production capacity of zeaxanthin. Zeaxanthin-containing, comprising culturing Flavococcus chejuensis (Accession No. KCTC 10534BP) and recovering zeazanthin-containing Flavococcus chejuensis containing zeaxanthin from the culture medium. How to produce Flavococcus Jeju Nsis. The method according to claim 2, wherein the Flavococcus Jejuensis (Accession No. KCTC 10534BP) is incubated under aerobic conditions at 20 to 40 ° C. delete An animal feed additive comprising zeaxanthin-containing flavococcus Jejuensis produced by the method according to claim 2. Culturing Flavococcus chejuensis (Accession No. KCTC 10534BP), recovering Flavococcus Jejuensis containing zeaxanthin from the culture medium and extracting the accumulated zeaxanthin from the recovered cells By, how to produce zeaxanthin. The method according to claim 6, wherein the Flavococcus Jejuensis (Accession No. KCTC 10534BP) is incubated under aerobic conditions at 20 to 40 ° C. delete delete delete delete

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