KR102626229B1 - C50 carotenoid producing optimization method using an extremely halophilic archaeon halorubrum sodomense strain mlba0099 - Google Patents

Abstract

The present invention provides Halorubrum sodomense MBLA0099, a strain that produces C50 carotenoids, provides a method for optimizing carotenoid production by optimizing the culture of the strain, and a method for extracting and confirming the produced carotenoids. By providing, the Halorubrum sodomense MBLA0099 strain according to the present invention was confirmed to have a 2.06-fold increase in carotenoid production compared to the existing one based on 1 ml of culture medium in modified ATCC 1176 medium, and the C50 carotenoid produced through this, bacterioruberin and its precursor It was confirmed that any one of monoanhydrobacterioruberin, bisanhydrobacterioruberin, and 2-isopentenyl-3,4-dihydrorhodopsin could be produced.

Description

Method for producing C50 carotenoids using the extremely halophilic archaebacterium Halorubrum SODOMENSE MBLA0099 strain {C50 CAROTENOID PRODUCING OPTIMIZATION METHOD USING AN EXTREMELY HALOPHILIC ARCHAEON HALORUBRUM SODOMENSE STRAIN MLBA0099}

The present invention relates to a method for optimizing carotenoid production by optimizing the culture of the Halorubrum sodomense MBLA0099 strain, an extremely halophilic archaeon that produces C50 carotenoids.

Carotenoids are a group of pigments of natural isoprenoid compounds synthesized by plants, algae, and microorganisms and are widely distributed in nature. Among the compounds, they are classified into carotenes, which do not contain oxygen in the molecule, and xanthophylls, which contain oxygen. Representative examples of carotenes include lycopene (red), alpha-carotene (orange), There is beta-carotene (orange), and xanthophylls include astaxanthin (orange), zeaxanthin (yellow), and fucoxanthin (brown). Also, depending on the number of carbons constituting the compound, it is divided into C ₃₀ series, C ₄₀ series, and C ₅₀ series. In the natural world, carotenoids play a role in absorbing light energy in the photosynthetic organs of plants, protecting against photo-oxidative damage, and increasing structural stability in the cell walls of microbial cells.

As the various effects of these carotenoids have been revealed, they are being used not only as natural pigments but also in the functional food industry. There are already commercially available products for lutein and zeaxanthin, which are known to be precursors of vitamin A and improve eye health, and lycopene and astaxanthin, which have antioxidant properties and scavenge free radicals in the body. In addition, there are products for anti-cancer, heart disease, immune function improvement, and skin damage. Due to its preventive and whitening effects, it is in the spotlight as a highly functional medicine and cosmetic material.

Carotenoids have mainly been extracted from plants or fruits or produced through chemical synthesis. However, these existing methods have low production yields, generate by-products, and have side effects in the case of synthesis, making them unsuitable for direct use in the food and pharmaceutical industries. Therefore, methods for producing carotenoids using microorganisms such as microalgae, yeast, and bacteria have recently received attention. However, carotenoid production using microorganisms has the disadvantage that the production system has not yet been properly established and the production cost is high. Only a few carotenoids, such as β-carotene and astaxanthin, can be produced industrially. Recently, in order to overcome these limitations, attempts have been made to produce carotenoids using microorganisms by isolating new types of microorganisms that produce carotenoids, establishing culture systems, developing separation and purification systems, and making culture media cheaper.

Haloarchaea are archaea belonging to the phylum euryarchaeota and are widely distributed in high-saline environments such as saline lakes, sea salt, salt pan soils, and saline foods. Most extreme halophilic archaea require 10-30% salt content for proper growth and to protect cells from osmotic stress. In addition, due to the nature of the strain, it biosynthesizes carotenoids such as C ₄₀ series lycopene and C ₅₀ series bacterioruberin, which is known to be used by extreme halophilic archaea to survive by increasing the structural stability of the cell wall in a high-salt environment. In particular, studies have shown that bacterioruberin, a major carotenoid produced by hyperhalophilic archaea such as Halococcus morrhuae and Halobacterium salinarum, prevents DNA damage from ionizing radiation (IR) and UV due to its strong antioxidant ability.

Among nutrients that affect microbial growth, nitrogen source is a basic nutrient required for microbial growth. It is a substance used for biosynthesis and energy production and is an essential growth factor for cell growth. The growth of microorganisms varies depending on the amount and type of nitrogen in the culture medium. You can. In addition, most carotenoids produced by microorganisms are produced through the mevalonic acid pathway, and substances that microorganisms can use as carbon sources, such as precursors (glucose, glycerol, and pyruvate) used in this biosynthetic pathway, can be used as precursors in the carotenoid biosynthetic pathway. Therefore, the type and concentration of carbon source in the culture of extremely halophilic archaea can act as a factor affecting carotenoid production.

Carotenoid production from hyperhalophilic archaea has various advantages over production from other microorganisms. First, it produces a relatively high concentration of carotenoids compared to other common microorganisms known to produce existing carotenoids. Second, the high-salt environment necessary for survival inhibits the growth of other microorganisms, so the possibility of contamination is low. Third, because cells are easily destroyed in a low-salt environment, carotenoids present within the cells can be easily extracted.

However, although carotenoids produced by extreme halophilic archaea are highly novel and applicable, and have various advantages, related research is relatively insufficient compared to other microorganisms. This is due to the high-salt environment, complex media components, and Additionally, there is a problem of producing a low bacterial mass during cultivation. For this reason, although extreme halophilic archaea are excellent microbial resources for research on carotenoid production, they have been recognized as a resource with little utility from an industrial and economic perspective. To overcome these limitations, this research project seeks to isolate extremely halophilic archaea, optimize the production of carotenoids through culture optimization, and extract and confirm the produced carotenoids.

Korean Patent Publication No. 10-2020-0087383

The present invention discloses Halorubrum sodomense MBLA0099, which was isolated from a salt farm in the Sorae region of Korea using a selection medium, and provides a culture optimization method for increasing carotenoid production. In addition, extraction and analysis conditions for bacterioruberin, a produced C50 carotenoid, are provided.

According to one embodiment of the present invention, in order to isolate extremely halophilic archaea that produce carotenoids, seawater, tidal flat samples, halophytes, etc., including domestic salt pan samples, are collected, and then DBCM2, a medium for isolating extremely halophilic archaea, is used from each sample. Enrichment culture was performed using this method. Afterwards, ATCC 1176 (4 g KCl, 20 g MgSO ₄ ·7H ₂ O, 13 g MgCl ₂ ·6H ₂ O, 156 g NaCl, 1 g CaCl ₂ ·6H _{2 O, 0.5 g, which is known to be capable of growing extremely halophilic} archaea) NaBr, 0.2 g NaHCO ₃ , 1 g Gluocse, 5 g Yeast extract, adjusted pH 7.0 using 1 M Tris-base buffer) diluted in solid medium, smeared, and cultured at 37°C to produce microorganisms with red-colored bacterial colonies. Ultrahalophilic archaea were purified by streaking on the same medium at least three times.

To identify the isolated microorganism, identification was performed using PCR primers 0018F (5'-ATTCCGGTTGATCCTGCC-3') and 1518R (5'-AGGAGGTGATCCAGCCGC-3'), which can confirm the 16S rRNA gene base sequence for archaea. As a result, strain MBLA0099 showed the highest similarity (99.05%) to DSM 3755 strain of Halorubrum sodomense, an extremely halophilic archaeon ( Halorubrum sp.).

In order to compare strain growth and carotenoid production according to the nutritional components of the medium, all experiments were conducted based on the absorbance (OD600, OD490) values per 1 ml of culture medium. In addition, all experiments were cultured at 37°C, 80 rpm, for 6 days.

Culture was performed by adding 0.5% (w/v) of each nitrogen source (Yeast extract, Beef extract, Tryptone, Peptone, Fish peptone, Casamino acid, Ammonium sulfate, MSG, Sodium nitrate, Ammonium citrate) to increase strain growth and carotenoid production. As a result of the comparison, the use of yeast extract showed the highest carotenoid production. In addition, 0.1% (w/v) of each carbon source (Glucose, Galactose, Strach, Maltose, Sucrose, Mannitol, Fructose, Mannose, Acetate, Lactose, Glycerol) was added and cultured, and sucrose showed the highest carotenoid production. As a result of experiments conducted by adding carbon sources that affect carotenoid production at various concentrations (0-8 %, w/v), strain growth and carotenoid production were almost the same when the sucrose concentration was 0.2 % (w/v) or more. did. After fixing the sucrose concentration at 0.2% (w/v), the concentration of the nitrogen source was adjusted to check the carotenoid production of the strain according to the C/N ratio, which is the molecular weight ratio of the carbon source and nitrogen source, and the concentration was adjusted to various ratios (10:1, 5:1, As a result of culturing (3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, 1:5, 1:10), the ratio of carbon source and nitrogen source was 1. :1 showed the highest carotenoid production.

Additionally, due to the nature of extremely halophilic archaea, the production of carotenoids may vary depending on the salt concentration, so the production of carotenoids was confirmed by culturing in low-salt to high-salt (5-30%, w/v) environments, and the salt concentration in the medium was 20%. The highest carotenoid production was observed when

Finally, the results of comparing carotenoid production using the modified ATCC 1176 medium to which 0.2 % (w/v) sucrose, 0.77 % (w/v) yeast extract, and 20 % (w/v) NaCl were added to the existing ATCC 1176 medium. It was confirmed that it increased by 2.06 times compared to the existing ATCC 1176 medium.

In order to confirm the carotenoids in the microorganisms, the cells were recovered by culturing them in modified ATCC 1176 liquid medium, and to extract carotenoids from the recovered cells, acetone: methanol (7:3, v/v) was used. After solvent extraction, the production of carotenoids was first confirmed through thin layer chromatography (TLC). To accurately identify the extracted carotenoid, HPLC was used to confirm that the produced carotenoid was bacterioruberin.

According to one embodiment of the present invention, preparing a strain of Halorubrum sodomense MBLA0099 deposited with accession number XXX; Inoculating and culturing the strain in a medium that contains a carbon source and a nitrogen source and whose salinity is controlled by including sodium chloride (NaCl); Recovering the bacterial cells from the medium: and extracting carotenoids from the recovered bacterial cells by solvent extraction.

The present inventors deposited the above strain with the Korea Seed Society on May 25, 2021, under accession number KCCM12990P.

According to one embodiment of the present invention, a method for optimizing carotenoid production by optimizing the type and concentration of carbon and nitrogen sources, which are culture medium components of Halorubrum sodomense MBLA0099, and controlling salt concentration is provided.

According to one embodiment of the present invention, the Halorubrum sodomense MBLA0099 strain was confirmed to mainly produce bacterioruberin (BR), monoanhydrobacterioruberin (MABR), and bis. It was confirmed that bisanhydrobacterioruberin (BABAR) and 2-isopentenyl-3,4-dehydrorhodopsin (IDR) could be produced.

The Halorubrum sodomense MBLA0099 strain according to the present invention is the first case of confirming carotenoid production according to culture medium components and C/N ratio using extremely halophilic archaea of the Halorubrum genus, and is the first to produce C50 carotenoids. This is a case. The representative C50 carotenoids produced are bacterioruberin, monoanhydrobacterioruberin, and bisanhydrobacterioruberin, and the C45 carotenoid 2-isopentenyl-3,4-dihydrorhodopsin.

Figure 1 is a neighbor-joining phylogenetic tree of the 16S rRNA gene sequence of strain MBLA0099 isolated from a sample.
Figure 2 is a graph comparing carotenoid production according to the type and concentration of nitrogen source.
Figure 3 is a graph comparing carotenoid production according to the type and concentration of carbon source.
Figure 4 is a graph comparing carotenoid production according to sucrose concentration.
Figure 5 is a graph comparing carotenoid production according to C/N ratio.
Figure 6 is a graph comparing carotenoid production according to salt concentration.
Figure 7 shows the TLC results of carotenoids derived from Halorubrum sodomense MBLA0099 and the results of spectral analysis using a spectrophotometer.
Figure 8 shows the results of HPLC analysis of four spots confirmed through TLC and analysis of the maximum absorption wavelength of the main peak.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are only intended to aid understanding of the present invention, and the scope of the present invention is not limited to these examples in any way.

Example

1. Isolation and identification of strains

To isolate extreme halophilic archaea, seawater, tidal flat samples, and halophytes, including domestic salt farm samples, were collected, and then enrichment culture was performed from each sample using DBCM2, a medium for isolating extreme halophilic archaea. Afterwards, it was diluted and smeared on ATCC 1176 solid medium, which is known to be capable of growing extremely halophilic archaea, and cultured at 37°C. Microorganisms with red-colored bacterial colonies were streaked on the same medium at least three times to produce extremely halophilic archaea. Purely isolated.

Among the isolated microorganisms, the strain with the reddest colony color was selected, and for identification, 0018F (5'-ATTCCGGTTGATCCTGCC-3') and 1518R (5'-AGGAGGTGATCCAGCCGC-3), PCR primers that can confirm the 16S rRNA gene base sequence for archaea, were used. ') was used for identification.

The fragment was sequenced by Macrogen Co. (Seoul, Korea), and sequence alignment and identification were performed using Bioedit 7.2.6.1 software and Ezbiocloud (https://www.ezbiocloud.net). A phylogenetic tree was constructed using MEGA 7.0 software.

2. Optimization of culture medium components of the strain

Based on ATCC 1176 medium, the One factor at a time (OFAT) method was used to compare strain growth and carotenoid production according to nutritional components to select the type and concentration of nitrogen and carbon sources. All experiments were performed by adding 80 ml of medium to a 200 ml erlenmeyer flask and culturing at 37°C, 80 rpm for 6 days. For bacterial mass and carotenoid production, OD600 and OD600 values were measured using a UV/Vis spectrophotometer based on 1 ml of culture medium. OD490 values were measured and compared.

Yeast extract (0.5 %, w/v), which is used as a nitrogen source in the existing ATCC 1176 medium, and beef extract, tryptone, peptone, fish peptone, casamino acid, ammonium sulfate, MSG, sodium nitrate, ammonium, which are known to be widely used as a nitrogen source by microorganisms. Ten nitrogen sources, including citrate, were added at 0.5% (w/v) each, and strain growth and carotenoid production by nitrogen source were compared.

For the carbon source, the nitrogen source that showed the highest carotenoid production in the nitrogen source experiment was added to the ATCC 1176 medium, and glucose (0.1%, w/v), which is used as an existing carbon source, and galactose, strach, maltose, and sucrose, which are known to be widely used as carbon sources by microorganisms. , 0.1% (w/v) of each of 11 carbon sources including mannitol, fructose, mannose, acetate, lactose, and glycerol were added and the same culture was performed to compare carotenoid production by carbon source. Afterwards, in order to conduct experiments depending on the concentration of the carbon source, the carbon source was added to the medium at various concentrations ranging from 0-8% (w/v), and the carotenoid production by carbon source concentration was compared.

For the C/N ratio experiment to determine the carbon source:nitrogen source ratio by fixing the confirmed carbon source concentration, the experiment was conducted based on molecular weight. Nitrogen sources were added at different ratios of 10:1, 5:1, 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, 1:5, and 1:10. Cultivation was performed in the same manner and carotenoid production was compared.

In addition, to compare strain growth and carotenoid production according to salt concentration, strains were cultured identically at various salt concentrations of 5-30% (w/v) and carotenoid production according to salt concentration was compared.

Through the above experiment, a modified ATCC 1176 medium in which the type and concentration of nitrogen and carbon sources and salt concentration were changed from the existing ATCC 1176 medium was used as a culture medium in later experiments.

3. Carotenoid extraction

To extract carotenoids, the isolated strain was inoculated into modified ATCC 1176 liquid medium and cultured at 37°C while stirring at 180 rpm. After 6 days, the culture was centrifuged at 10,000 rpm for 5 minutes. The culture supernatant was removed, only the settled cells were collected, and extraction was sufficiently performed using acetone:methanol (7:3, v/v) solvent until the red color of the cells disappeared. The solvent was removed from the extracted carotenoid using a reduced pressure concentrator, and the carotenoid in solid state was recovered. Afterwards, it was re-dissolved in 100% methanol and residual cells and salts were removed through a 0.2 μm cellulose acetate syringe filter.

4. Qualitative analysis of carotenoids

The recovered carotenoids were spotted on a silica TLC plate and developed using the developing solvent, heptane:acetone (5:5, v/v). As a result, it was confirmed that four major spots were observed in common. . Samples were prepared by scraping each colored spot on TLC and recovering with 100% methanol, and the spectrum was analyzed in the A300-600 wavelength range using a spectrophotometer to determine bacterioruberin and its Comparative analysis of unique peaks of the derivatives was performed.

Additionally, the production of each carotenoid was analyzed using the YL9100 Plus HPLC System (Younglin, South Korea). Chromatographic separation was performed using an HPLC column (C18 5 um, 4.6*250 mm, ThermoFisher), the flow rate was 1.0 ml/min, the injection volume was 20 μl, and the mobile phase consisted of 100% methanol.

Experiment result

1. Isolation, identification and culture optimization of C50 carotenoid producing bacteria

After collecting seawater, tidal flat samples, and halophytes, including domestic salt farm samples, enrichment culture was performed from each sample using DBCM2, a medium for isolating extremely halophilic archaea. Afterwards, it was diluted and smeared on ATCC 1176 solid medium, which is known to be capable of growing extremely halophilic archaea, and cultured at 37°C. Microorganisms with red-colored bacterial colonies were streaked on the same medium at least three times to produce extremely halophilic archaea. Purely isolated. Among the isolated strains, the strain with the reddest color was selected, and for identification, 0018F (5'-ATTCCGGTTGATCCTGCC-3') and 1518R (5'-AGGAGGTGATCCAGCCGC-3), PCR primers that can confirm the 16S rRNA gene base sequence for archaea, were used. ') was used for identification. As a result of identification based on the 16S rRNA gene sequence, the selected MBLA0099 strain was found to have 99.05% similarity with the Halorubrum sodomense strain of the Halorubrum genus, an extremely halophilic archaea (Figure 1).

To optimize culture, we compared carotenoid production according to the type and concentration of carbon and nitrogen sources among the culture medium components. As a result, yeast extract, a component of the existing medium, had the best carotenoid production at 0.150 (OD490) for the nitrogen source (Figure 2), and ammonium It was confirmed that bacteria did not grow on sulfate, MSG, sodium nitrate, and ammonium citrate. In the case of the carbon source, it was confirmed that when glucose, a component of the existing medium, was replaced with sucrose, carotenoid production increased to 0.176 (OD490) (Figure 3). When the concentration of the carbon source was 0.2-6% (w/v), the carotenoid production was confirmed to be about 0.193 (OD490), and when the concentration of the carbon source was more than 7% (w/v), the carotenoid production was similar, but the bacterial mass decreased. It was confirmed that (Figure 4). Therefore, the concentration of the carbon source was selected at 0.2% (w/v), which is the most efficient compared to the concentration of the added carbon source.

In the case of C/N ratio, based on the molecular weight ratio, the C ratio of sucrose, which is a carbon source, is 0.421 (per g), and the N ratio of yeast extract, which is a nitrogen source, is 0.109 (per g), so based on 2 g/L of sucrose. As a result of cultivating by adding 0.77, 1.55, 2.58, 3.86, 5, 7.73, 11.59, 15.45, 23.18, 38.63, and 77.25 (g/L) of yeast extract, respectively, when 7.73 g/L of yeast extract was added The highest carotenoid production was shown at 0.218 (OD490), and the C/N ratio at this time was 1:1. It was confirmed that when the C/N ratio was more than 1:2, the bacteria could not grow and carotenoids were not produced (Figure 5 ).

When cultured at salt concentrations of 5, 7.5, 10, 12.5, 15, 15.6, 17.5, 20, 22.5, 25, 27.5, and 30 % (w/v), when the salt concentration is 5 % (w/v) Bacteria were unable to grow, and if the concentration was less than 10% (w/v), bacteria hardly grew. At 20% (w/v), carotenoid production was confirmed to be the highest at 0.309 (OD490). It was confirmed that carotenoid production decreased in high salt situations exceeding 25% (w/v) (Figure 6).

Finally, in the existing ATCC 1176 medium, 0.1% (w/v) glucose as a carbon source was changed to 0.2% (w/v) sucrose, 0.5% (w/v) yeast extract as a nitrogen source was changed to 0.773% (w/v), and salt When using modified ATCC 1176 medium whose concentration was changed from 15.6% (w/v) to 20% (w/v), it was confirmed that carotenoid production increased by 2.06 times from 0.150 (OD490) to 0.309 (OD490).

2. Halorubrum sodomense Confirmation of C50 carotenoid production by MBLA0099

Microorganisms in the Halorubrum genus are known to produce bacterioruberin, a C50 carotenoid, and are also known to produce carotenoids such as lycopene and β-carotene, which are precursors of bacterioruberin. Four spots were identified for C50 carotenoid production by the Halorubrum sodomense MBLA0099 strain through TLC analysis, and these were re-extracted in methanol and spectrum analysis was performed (Figure 7). After spectrum analysis, HPLC analysis was performed for each of the four spots (Figure 8). Through comparison of the maximum absorption wavelength of previously reported C50 carotenoids, Halorubrum sodomense MBLA0099 is a C50 carotenoid, bacterioruberin, and its precursors, monoanhydrobacterioruberin, bianhydrobacterioruberin, and 2-isopentenyl-3,4. -Dihydrorhodopsin was identified.

Review results

Halorubrum sodomense MBLA0099 was isolated from Sorae Salt Farm and has red colonies. It was confirmed that carotenoid production significantly increased through culture optimization, and the production of C50 carotenoids was confirmed by solvent extraction from Halorubrum sodomense MBLA0099 and TLC, spectrum, and HPLC analysis.

Korea Microbiological Conservation Center (Overseas) KCCM12990P 20210525

<110> INCHEON NATIONAL UNIVERSITY RESEARCH BUSINESS FOUNDATION <120> C50 CAROTENOID PRODUCING OPTIMIZATION METHOD USING AN EXTREMELY HALOPHILIC ARCHAEON HALORUBRUM SODOMENSE STRAIN MLBA0099 <130>P21-0099 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 1 attccggttg atcctgcc 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 2 gcggctggat cacctcct 18 <210> 3 <211> 1477 <212> DNA <213> Halorubrum sodomense <400> 3 tattcattcc ggttgatcct gccggaggcc attgctattg ggatccgatt tagccatgct 60 agtcgcacga gttcagactc gtggcgaata gctcagtaac acgtggccaa actacccttc 120 ggaacacaat accctcggga aactgaggct aatagtgtat accacatcac cactggaatg 180 agtgatgtgc caaacgctcc ggcgccgaag gatgtggctg cggccgatta ggtagacggt 240 ggggtaacgg cccaccgtgc caataatcgg tacgggtcat gagagtgaga acccggagac 300 ggaatctgag acaagattcc gggccctacg gggcgcagca ggcgcgaaac ctttacactg 360 cacgacagtg cgataggggg atcccaagtg cacaggcata gcgcctgtgc ttttcggtac 420 cgtaaggtgg taccagaata agggctgggc aagaccggtg ccagccgccg cggtaatacc 480 ggcagcccaa gtgatggccg atcttattgg gcctaaagcg tccgtagctg gccgcgcaag 540 tccatcggga aatccacctg ctcaacaggt gggcgcccgg tagaaactgc gtggcttggg 600 accggaaggc gcgacgggta cgtccggggt aggagtgaaa tcccgtaatc ctggacggac 660 cgccgatggc gaaagcacgt cgcgaggacg gatccgacag tgagggacga aagccagggt 720 ctcgaaccgg attagatacc cgggtagtcc tggccgtaaa caatgtctgc taggtgtggc 780 tcccactacg agtgggtgct gtgccgtagg gaagccgcta agcagaccgc ctgggaagta 840 cgtccgcaag gatgaaactt aaaggaattg gcgggggagc actacaaccg gaggagcctg 900 cggtttaatt ggactcaacg ccggacatct caccagcatc gactgtggta atgacgatca 960 ggttgatgac cttatccgag cctcagagag gaggtgcatg gccgccgtca gctcgtaccg 1020 tgaggcgtcc tgttaagtca ggcaacgagc gagacccgca cccttacttg ccagcagtac 1080 cgcgaggtag ctggggacag tagggggacc gccgtggcta acacggagga aggaacgggc 1140 aacggtaggt cagtatgccc cgaatgtgct gggcaacacg cgggctacaa tggtcgagac 1200 aaagggttcc tactccgaaa ggagacggta atctcagaaa ctcgatcgta gttcggattg 1260 tgggctgcaa ctcgcccaca tgaagctgga ttcggtagta atcgcgtgtc acaagcgcgc 1320 ggtgaatacg tccctgctcc ttgcacacac cgcccgtcaa agcacccgag tgaggtccgg 1380 atgaggcgtt ccacgaacgt cgaatctggg cttcgcaagg gggcttaagt cgtaacaagg 1440 tagccgtagg ggaatctgcg gctggatcac ctccacc 1477

Claims (9)

Preparing the Halorubrum sodomense MBLA0099 strain deposited with deposit number KCCM12990P;
The strain is grown in a salt-controlled medium containing sucrose as a carbon source and yeast extract as a nitrogen source, the carbon/nitrogen ratio (C/N ratio) is 1:1, and sodium chloride (NaCl). Inoculating and culturing;
Recovering bacteria from the medium: and
C50 carotenoid production method comprising: extracting carotenoids from the recovered bacterial cells by solvent extraction.
According to paragraph 1,
C50 carotenoid production method, characterized in that the salt concentration of the medium is more than 5% and less than 20%.
According to paragraph 1,
The medium is a modified ATCC 1176 medium containing 0.2% (w/v) sucrose and 0.77% (w/v) yeast extract, and the salt concentration is adjusted to 20% (w/v). C50 carotenoid production method, characterized in that.
According to paragraph 1,
The carotenoid is a C50 carotenoid production method, characterized in that any one of bacterioruberin, monoanhydrobacterioruberin, bisanhydrobacterioruberin, and 2-isopentenyl-3,4-dihydrorhodopsin.
According to paragraph 1,
The step of extracting carotenoids from the recovered bacterial cells by solvent extraction is a C50 carotenoid production method, characterized in that using an acetone:methanol (7:3, v/v) solution.
According to clause 5,
C50 carotenoid production method, characterized in that the extracted carotenoid is analyzed through HPLC.
Halorubrum sodomense MBLA0099 strain deposited with accession number KCCM12990P,
The strain contains sucrose as a carbon source and yeast extract as a nitrogen source, the carbon/nitrogen ratio (C/N ratio) is 1:1, and the salinity is adjusted including sodium chloride (NaCl). A strain characterized by the highest C50 carotenoid production. delete delete