WO2016076454A1

WO2016076454A1 - Method for culturing microorganisms and method for separating and purifying 9-cis β-carotene from cultured microorganisms

Info

Publication number: WO2016076454A1
Application number: PCT/KR2014/010853
Authority: WO
Inventors: 윤태성; 신병철
Original assignee: 한국생명공학연구원; (주)아크에이르
Priority date: 2014-11-10
Filing date: 2014-11-12
Publication date: 2016-05-19
Also published as: KR101907994B1; KR20160055714A

Abstract

The present invention relates to a method for producing 9-cis β-carotene from microorganisms, comprising a method for improving the culture rate of microorganisms using a photochemical gas generated by the micro-spray of gas and the control of a light source and a method for separating and purifying 9-cis β-carotene from the cultured microorganisms. The 9-cis β-carotene according to the present invention is separated and purified from the microorganisms cultured by the micro-spray of gas and the control of the light source, thereby facilitating production, enabling eco-friendly mass production, and exhibiting excellent purity.

Description

Method of culturing microorganisms and separating and purifying 9-cis beta-carotene from the culture microorganism

The present invention includes a method of culturing a microorganism using a photochemical gas generated by gas injection and light source control, and a method for separating and purifying 9-cis beta carotene (9-cis β-carotene) from the culture microorganism. , And a method for producing 9-cis beta-carotene from microorganisms.

Carotenoids are pigments that represent an orange-based (yellow to bright red) color that is primarily present in the peels of green and yellow vegetables such as carrots, old pumpkins, bell peppers, sesame leaves, lettuce, leeks, and spinach. Among them, beta-carotene (β-carotene) is a precursor of almost all carotenoids, which are converted into vitamin A, an essential nutrient in the body. Beta-carotene is widely used as a food additive due to its antioxidant effect, high nutritional value, and unique color to prevent oxidation damage in animal tissues, and is also useful as an anticancer agent, vascular disease agent, and antioxidant related to skin aging. Is being used. In particular, 9-cis beta-carotene (9-cis β-carotene) is known to have an excellent effect as a drug among the isomers of beta-carotene. However, the amount of beta-carotene present in green yellow vegetables and fruits is not so high that its production does not meet the demand, and the purification of beta-carotene from natural products is a mixture of all-trans beta-carotene and 9-cis beta-carotene. Isomers do not separate the situation.

Dunaliella, a natural microalgae, is a eukaryotes belonging to photosynthetic green algae, which can survive in salt concentrations ranging from about one tenth of seawater to 5M near salts. Exhibits high properties. Microalgae of the genus Dunaliella are found in places with low nitrogen content, strong sunlight and high salt concentrations, which can accumulate beta-carotene in the cells and become yellow-green to orange when exposed to such strong environmental stress conditions. Is found. These beta-carotenes have been reported to accumulate with oily granulocytes in the thylakoid interstitial spaces in the chloroplasts (Ben-Amotz et al., J. Phycol. 18: 529-537), and these accumulated beta-carotenes protect the photosynthetic system from strong light. We assume that it is for.

A series of processes in which microorganisms, animals, and plant cells are inoculated in proper breeding and propagated under appropriate conditions such as temperature and oxygen are called cultures, and the type of culture is classified into liquid culture and solid culture. Important external conditions in culture include temperature, humidity, light gas phase composition (partial pressure of carbon dioxide and oxygen), and the most important direct influence on the cultured organisms is the medium. The medium is also known as an incubator and is a direct environment for the organism, and is also a source of various nutrients necessary for survival and proliferation. In general, the liquid culture phase using the liquid medium cultures algae and microorganisms by controlling the temperature, roughness, gas and saturation amount of organic and inorganic matters contained in the medium, that is, nutrition culture method, and in general, the incubation time of algae and microorganisms In order to improve their culture rate, a method of adjusting the nutrient content of the medium is mainly used. However, in the case of such liquid culture, there is a problem in that the aggregation of the culture cells occurs in proportion to the growth of the culture. This agglomeration phenomenon can be found in most algae and microorganisms and is a cause of inhibiting the speed of cell culture. In addition, gas, such as oxygen and carbon dioxide, must be continuously supplied to properly maintain the hydrogen ion index, dissolved oxygen, and dissolved carbon dioxide, which are environmental conditions necessary to promote culture in the incubator. However, simply injecting these gases from the outside does not produce enough dissolved ozone necessary for the growth of certain microorganisms, and it is also difficult to keep constant. Accordingly, Patent Publication No. KR10-2014-0046132 discloses a method of preventing agglomeration of a culture through microspray of air on a liquid medium, and generating a photochemical gas by controlling light sources, thereby accelerating the growth rate of algae and microorganisms. It is adopted.

As described above, by culturing algae and microorganisms using photochemical gas generated by air injection and light source control, 9-cis beta-carotene is isolated and purified from the culture microorganisms. Research on how to produce is urgently needed.

The inventors of the present invention, while searching for the production method of 9-cis beta-carotene (9-cis β-carotene), after culturing the microorganisms using the photochemical gas generated by gas injection and light source control, the culture When separating and purifying 9-cis beta-carotene from the microorganism, it is confirmed that the production of 9-cis beta-carotene is easy, and eco-friendly mass production, 9-cis beta-carotene shows excellent purity, the present invention Was completed.

Accordingly, the present invention provides a method for improving the cultivation rate of microorganisms using photochemical gas generated by microspray of gas, and light source control, and a method for separating and purifying 9-cis beta-carotene from the culture microorganism. It is intended to provide a method for producing 9-cis beta-carotene.

In order to achieve the above object,

The present invention comprises the steps of culturing the microorganisms using the photochemical gas generated by the gas injection and light source control; And separating and purifying 9-cis beta-carotene (9-cis β-carotene) from the cultured microorganisms, which provides a method for producing 9-cis beta-carotene from microorganisms.

Hereinafter, the present invention will be described in detail.

The present invention can facilitate production of 9-cis beta-carotene by varying the composition and culture conditions of the medium, such as salt concentration, concentration of nitrogen source, or pressure, light source control, in order to maximize growth of the strain.

The micro-injection of the gas according to the present invention is controlled by a sensor unit for detecting the index of the aggregation point of the microbial mycelium, using a low pressure mist spray nozzle discharged in the incubator at the time of aggregation is formed as the culture of the microorganism in the medium is grown. By diffusing and spraying gas into the medium, the aggregated culture is pulverized.

The gas injected through the nozzle may be air, oxygen, nitrogen, nitrogen dioxide, carbon dioxide, or the like, and is preferably air. The gas is microsprayed through a nozzle of 0.1 to 0.5 mm under low pressure to pulverize the agglomeration of the culture, resulting in a coagulation resolution index of 0 to 1%.

In addition, in order to cultivate microorganisms well in a liquid medium, a certain amount of dissolved acid tank (DO), dissolved carbon dioxide (DCO ₂ ), and ozone are required. In the present invention, a method of generating a required gas by adjusting the light intensity and illuminance of a light source is adopted. Doing.

The light source adjustment may be performed at an illuminance range of 1000 to 7000 Lux, and a luminance range of 4000 to 12000K, and a required photochemical gas may be ozone or carbon dioxide. That is, by installing a sensor to detect the residual amount of dissolved oxygen, dissolved carbon dioxide and ozone in the incubator, when the ozone content is insufficient, nitrogen dioxide is introduced and in the oxidation process of nitrogen gas, the luminance range is 4000 to 5000K and the illuminance range is 6000 ± 500Lux. Providing a light source produces ozone. In addition, when the dissolved carbon dioxide is insufficient, the infrared light source in the luminous intensity range of 10000 to 12000K and the illuminance range of 3000 ± 500 Lux is irradiated to increase the dissolved carbon dioxide in the medium.

The microorganism is a microorganism of 0.1 mm or less including 9-cis beta-carotene, and is not particularly limited in the present invention, and includes microalgae, protozoa, filamentous fungi, yeasts, viruses, and the like. The microalgae may be Dunaliella, Spirrolina, Chlamydomonas, Simbella, Chlorella, etc., which are cultured in a liquid medium, but Dunaliellane is preferable. Dunaliella contains natural beta-carotene, and beta-carotene beneath the cell wall by synthesizing beta-carotene under the cell wall to protect chlorophyll a, a chlorophyll important for photosynthesis when the culture environment deteriorates, such as strong light irradiation. It is known to form a film.

The culture medium is a supply medium of nutrients necessary for the survival and proliferation of microorganisms, but may be a liquid medium or a solid medium, but is preferably a liquid medium. In addition, in order to improve the culture rate of the microorganism, it may include NaNO ₃ , NaCl, NaH ₂ PO ₄ H ₂ O, Na ₂ SiO ₃ 9H ₂ O, trace metals and vitamins. The trace metals are FeCl ₃ 6H ₂ O, Na ₂ EDTA 4H ₂ O, CuSO ₄ 5H ₂ O, Na ₂ MoO ₄ 2H ₂ O, ZnSO ₄ 7H ₂ O, MnCl ₂ 4H ₂ O and Co (Cl) ₂ · 6H ₂ O and the like, the vitamin is cyanocobalamin (bioano), biotin (biotin), thiamine HCl, vitamin C, vitamin E, vitamin B12, calcium pantothenate (calcium pantothenate) , Folic acid, nicotinamide, and the like.

After the incubation is finished, the cultured microorganisms can recover the cells by spontaneous precipitation of the culture or centrifugation.

Separation and purification of 9-cis beta-carotene produced in the cultured microorganisms, (1) adding hexane to the cultured microbial powder, stirring and evaporating to obtain a semi-solid; (2) obtaining beta carotene extract from the semisolid; (3) purifying the beta carotene extract by chromatography, and then ultrasonically pulverizing the eluate to obtain a powder containing all trans- and 9-cis beta carotene: (4) obtaining a crystal by evaporating the powder. Then, methanol and tetrahydrofuran were added and rotovap to separate all trans- and 9-cis beta carotene; And (5) crystallizing the isolated 9-cis beta-carotene.

Step (1) and (2) is a step of extracting beta carotene from the culture microorganism, according to an embodiment of the present invention, after washing the dry powder of the culture duraellia salina strain with ethanol, the carotenoid component Cyclo-nuxanon is added to separate and the hexane layer is evaporated, followed by addition of hexane and stirring and evaporation at -5 to 5 ° C. for 0.8 to 1.2 hours to obtain a dark brown semisolid. After adding the cyclo-nucleanone to the obtained semi-solid, it is maintained for 11 to 13 hours at -25 ~ -15 ℃ to obtain a beta carotene extract.

Step (3) is a step of purifying beta carotene, and may be performed using silica gel column chromatography, ODS column chromatography, or a combination thereof. According to an embodiment of the present invention, the beta carotene extract obtained above was purified by silica gel column chromatography using cyclohexane-ethyl and acetate (50: 1, v / v) as eluent to remove impurities and reddish brown foam. A solid was obtained, which was ultrasonically pulverized to obtain an orange powder, which was then purified by ODS column chromatography using methanol and tetrahydrofuran (1: 0.2-1: 1, v / v) as eluent to give all trans beta. An orange powder obtained by mixing carotene and 9-cis beta carotene was obtained. The powder thus obtained can be measured for absorbance at 450 nm or 475 nm using HPLC to determine the content of beta-carotene.

Step (4) is a step of separating and crystallizing 9-cis beta-carotene from the purified beta-carotene powder, an orange powder containing all-trans beta-carotene and 9-cis beta-carotene obtained according to an embodiment of the present invention. After evaporation, methanol and tetrahydrofuran can be added at 1: 0.2 to 1: 1 (v / v) and rotovap to separate 9-cis beta-carotene from all trans beta-carotene.

The isolated 9-cis beta-carotene may be crystallized at -25 ~ -15 ℃, preferably -20 ℃.

The present invention also provides 9-cis beta-carotene produced by the above production method. 9-cis beta-carotene produced according to the present invention has an average diameter of 0.1 ~ 0.4㎛, and has the advantage of showing excellent purity.

9-cis beta-carotene according to the present invention is separated and purified from the cultured microorganisms by the micro-injection and light source control of the gas, it is easy to produce, eco-friendly mass production is possible, there is an effect showing excellent purity.

1 is a diagram showing a 9-cis beta carotene according to the present invention.

Figure 2 (a, b, c, d, e, f) shows the UV-B measurements of HPLC for carotenoids and tocopherols of the Dunaliella salina strain according to the present invention It is a graph.

Figure 3a, Figure 3b is a graph of the component analysis at 450 nm absorption band using a sample containing 9-cis beta-carotene purified according to Example 2-2 using HPLC.

Figure 4 is a graph of the component analysis at 470 nm absorption band using a sample containing 9-cis beta carotene purified according to Example 2-3.

5A and 5B are graphs showing ¹ H-NMR and ¹³ C-NMR spectra for 9-cis beta-carotene cultured, isolated and purified from Dunaliella salinaro strain according to the present invention.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

실시예 1. 광 바이오배양기를 이용한 미생물의 배양Example 1 Cultivation of Microorganisms Using an Optical Biocultivator

Microalgae Dunaliella salina (KMMCC-1064) strains were cultured using an optical biocultivator (see KR10-2014-0046132). The Dunaliella salina strain is inoculated in an f / 2 culture medium, pressure control and air injection of the optical bio-incubator is performed using a nozzle having a diameter in the range of 0.1 to 0.5mm under low pressure, the illumination range of the light source is 1000 To 7000Lux, the brightness range was performed by adjusting to 4000 to 12000K. The strain had a size of 13.0 ± 1.6 μm and was cultured for 15 days using the f / 2 culture medium of Tables 1 to 3 below.

Various media for culturing Dunaliella salina are shown in Tables 1-3 (Table 1: culture medium, Table 2: trace metal solution medium, Table 3: vitamin solution medium).

Table 1

Quantity	Compound	Stock solution	Molar concentration in the final medium
1 mL	NaNO ₃	75 g / L dH ₂ O	8.83 × 10 ^-4 M
1 mL	NaH _₂ PO ₄ · H ₂ O	5 g / L dH ₂ O	3.63 × 10 ^-5 M
1 mL	Na _₂ SiO ₃ · 9H ₂ O	30 g / L dH ₂ O	1.07 × 10 ^-4 M
1 mL	f / 2 trace metal solution	See Table 2
0.5 mL	f / 2 vitamin solution	See Table 3

TABLE 2

Quantity	Compound	Stock solution	Molar concentration in the final medium
3.15 g	FeCl ₃ · 6H ₂ O	-	1 × 10 ^-5 M
4.36 g	Na ₂ EDTA2H ₂ O	-	1 × 10 ^-5 M
1 mL	CuSO ₄ · 5H ₂ O	9.8 g / L dH ₂ O	4 × 10 ^-8 M
1 mL	Na ₂ MoO ₄ 2H ₂ O	6.3 g / L dH ₂ O	3 × 10 ^-8 M
1 mL	ZnSO ₄ · 7H ₂ O	22.0 g / L dH ₂ O	8 × 10 ^-8 M
1 mL	CoCl ₂ · 6H ₂ O	10.0 g / L dH ₂ O	5 × 10 ^-8 M
1 mL	MnCl ₂ 4H ₂ O	180.0 g / L dH ₂ O	9 x 10 ^-7 M

TABLE 3

Quantity	Compound	Stock solution	Molar concentration in the final medium
1 mL	Cyanocobalamin	1.0 g / L dH ₂ O	1 × 10 ^-10 M
10 mL	Biotin	0.1 g / L dH ₂ O	2 × 10 ^-9 M
200 mg	ThiamineHCl			3 × 10 ^-7 M

After the incubation was completed, the cultured strain was filtered using a hollow fiber membrane of 0.05 μm size, and the carotenoid content of the strain was analyzed using HPLC (Tosoh, MCPD-3600).

실시예 2. 배양미생물로부터 9-시스 베타카로틴을 분리 및 정제Example 2 Isolation and Purification of 9-cis Beta Carotene from Cultured Microorganisms

Separation and purification of 9-cis beta-carotene (see FIG. 1) from Dunaliella salina strain cultured according to Example 1 was performed.

2-1. 배양미생물의 초기정제2-1. Initial purification of cultured microorganisms

First, the dry powder (1 kg) of Dunaliella salina strain (KMMCC-1064) cultured according to Example 1 was washed twice with ethanol (5L) to remove the vegetable oil component. To separate the carotenoids, cyclo-hexanone (9 L) was added, the hexane layer was evaporated to give a dark brown semisolid and solid (53 g), and then to the obtained sample, hexane (1 L) was added again at 0 ° C. After stirring for 1 hour, the hexane layer was evaporated to give 38 g of a dark brown semisolid containing 14.8 g of all trans beta carotene. Thereafter, cyclo-nuxanone (185 mL) was added to this semisolid, and the mixture was maintained at -20 ° C for about 12 hours. Thereafter, the precipitated solid was removed by filtration, and an extract containing 4 g of all-trans beta-carotene and a large amount of 9-cis beta-carotene isomer was obtained.

2-2. 오픈칼럼 크로마토그래피를 이용한 9-시스 베타카로틴의 분리2-2. Separation of 9-cis Beta-carotene by Open Column Chromatography

The extract according to Example 2-1 was subjected to silica gel column chromatography (silica gel: 900 ml, column: 5.5 cm × × 38 cm, eluent: cyclohexane and ethyl acetate (50: 1, v / v)). Purification removed impurities to obtain 16.6 g of a reddish brown foamy solid. 1660 mL of ethanol was added to the solid, which was then pulverized by ultrasonication. The solid was filtered, washed with ethanol and dried to obtain an orange powder (12 g,> 80%). The ultrasonic mill used (VCX750, Sonic & Materials Inc., USA) was operated for 6 minutes at a speed of 3000 rpm. The obtained powder was subjected to component analysis at 450 nm absorption band using HPLC, and the results are shown in FIGS. 3A and 3B.

As shown in Figure 3a and 3b, the orange powder can be seen that isomers, such as all-trans beta carotene, 9-cis beta carotene are mixed.

2-3. ODS 오픈칼럼 크로마토그래피를 이용한 9-시스 베타카로틴의 분리2-3. Separation of 9-cis Beta-carotene by ODS Open Column Chromatography

11.2 g of the orange powder according to Example 2-2 was mixed with 30 mL of chloroform, followed by ODS open column chromatography (Chromatorex DM1020T: 1.2 L, column: 8 cm ￠ × 24 cm, eluent: methanol-tetrahydrofuran (5: 1). 1: 1, v / v)) to obtain a concentrate eluted with the formation of a pale green band in the column. 1660 mL of ethanol was added to the concentrate, followed by ultrasonication to obtain 10.1 g of an orange powder. The obtained powder was subjected to component analysis at 475 nm absorption band using HPLC, and the results are shown in FIG. 4.

As shown in FIG. 4, the orange powder may be mixed with all-trans beta-carotene and 9-cis beta-carotene.

2-4. 9-시스 베타카로틴의 결정화2-4. Crystallization of 9-cis Beta-carotene

The orange powder containing all trans beta-carotene and 9-cis beta-carotene according to Example 2-3 was placed in a double jacketed reactor maintained at a temperature of 20 ° C. in a bath and 1 ° C. This was evaporated to a decrease of 1/5 to give an orange crystal. Methanol and tetrahydrofuran were added (5: 1-1: 1, v / v) to the crystals, and evaporated to give 75 mg of 9-cis beta-carotene per fraction, which was crystallized by maintaining at about -20 ° C. As a result, the total extraction rate of 7.5g of 9-cis beta-carotene was compared to 1kg of dry Dunalella salina dry powder, and it was confirmed that dark storage was possible at about -20 ° C for 2 weeks.

실험예 1. 배지 및 배양환경에 따른 카로티노이드의 함량변화Experimental Example 1. Changes in the content of carotenoids according to the medium and culture environment

Dunaliella salina strain cultured according to Example 1 was filtered using a 0.05 micron hollow fiber membrane (hollow fiber membrane), using high performance liquid chromatography (HPLC, Tosoh Co. MCPD-3600) The carotenoid content was analyzed. The HPLC comprises Nucleosil 5 μm C18 (250 × 4.6 mm id) (Macherey-Nagel) as the Guard column, Bondaclone 10 μm C18 (3.9 × 150 mm id) as the Analytical column, and Capillary column (15 mm × 0.53 mm id). . In addition, total carotenoid content was analyzed using β-carotene, zeaxanthin, lutein, astaxanthin and cryptoxanthin, tocopherol, ascorbic acid (Vitamin C), catalase, peroxidase, and superocide dismutase as a standard for comparison. The list of standards includes (±) cis-trans abscisic acid, (±) cis-trans ABA, trans-ABA, all-trans-l3-carotene, ABA-Methyl ester, formaldehyde, polychlorinated f1occulant, 2,6-Di-terr -butyl-p-cresol, aqueous scintillant, and N-methyl-N'-nitro-nitrosoguanidine were used.

The change in the content of carotenoids according to the culture medium and the culture conditions is shown in Table 4, and more specific results are shown in Tables 5 to 7.

Table 4

Culture medium and culture conditions	mg g-1	percent(%)	Ratio
1. F / 2 medium without the trace metal solution	9.2	0.92	1.0
2.F / 2 medium + UV (290-320 nm, 2,000 Lux)	14.5	1.45	1.58
3. Nitrogen + 8% NaCl, l2.5 mM Nitrogen + 8% NaCl	31.1	3.11	3.38
4. 8% NaCl without nitrogen supply	45.5	4.55	4.94
5. Add 12% NaCl + 5 mM / L Nitrogen	54.1	5.41	5.88
6. Add 12% NaCl + 2.5 mM / L Nitrogen	105.1	10.5	11.42
7. Add 16% NaCl + 5 mM / L Nitrogen	72.9	7.29	8.05
8. Add 16% NaCl + 2.5 mM / L Nitrogen	115.2	11.5	12.6

[Revision 20.11.2014 under Rule 26]
Table 5

[Revision 20.11.2014 under Rule 26]
Table 6

[Revision 20.11.2014 under Rule 26]
TABLE 7

As shown in Tables 4 to 7, when 16% NaCl was added to the culture medium and 2.5mM / L nitrogen was added, the culture of Dunaliella salina was increased, and the carotenoid production was increased by about 10 to 70%. It can be confirmed that. In addition, UV-B measurements of HPLC of carotenoids and tocopherol of Dunaliella Salina were examined and used as reference values for calculating UV-B values of 9-cis beta-carotene (FIG. 2 (a, b)). , c, d, e, f)).

실험예 2. 9-시스 베타카로틴의 순도 측정Experimental Example 2. Determination of Purity of 9-cis Beta-carotene

The purity of 9-cis beta-carotene obtained according to Example 2 was analyzed using nuclear magnetic resonance (NMR) spectra.

First, a membrane test of the obtained 9-cis beta carotene was performed. The membrane filter of β-carotene content of The United States Pharmacopeial Convention (USP) was 0.45 μm pore size, but the membrane tester was confirmed by passing the 0.4 μm pore size. The membrane filter is made of PTFE and manufactured in the form of hollow fiber. Thereafter, ¹ H-NMR and ¹³ C-NMR spectra were measured for 9-cis beta-carotene filtered through the membrane filter, and the results are shown in FIGS. 5A and 5B.

As shown in Figures 5a and 5b, it can be seen that the obtained 9-cis beta carotene is free from impurities and exhibits excellent purity.

¹ H-NMR: δ ppm (CDCI ₃ ): 1.03 (C1, -CH ₃ ), 1.04 (C1-CH ₃ ), 1.48 (C2-H ₂ and C2′-H ₂ ), 1.62 (C3-H ₂ and C3'-H ₂ ), 1.72 (C5'-CH ₃ ), 1.76 (C5-CH ₃ ), 1.97 (C9-CH ₃ , C9'-CH ₃ , C13-CH ₃ , and C13.-CH ₃ ), 2.03 (C4-H ₂ and C4'-H ₂ ), 6.04 (C10-H), 6.12 (Cs'-H), 6.15 (C10'-H), 6.18 (C7-H and C7'-H), 6.23 (C14-H and C14'-H), 6.28 (C12-H), 6.34 (C12'-H), 6.62 (C15-H and C15'-H), 6.64 (C1F-H), 6.66 (Cs-H ), 6.75 (C11-H).

¹³ C-NMR: (C9'-Me), 12.81 (Cly-Me), 12.87 (C13-Me), 19.28 (C3 and C3 '), 20.77 (C9-Me), 21.76 (Cs'-Me), 21.89 (Cs-Me), 28.98 (CI'-Me × 2), 29.02 (C1-Me × 2), 33.10 (C4 and C4 '), 34.25 (C1), 34.29 (C ￡), 39.59 (C2), 39.68 (C2 '), 123.86 (Cll), 124.99 (CII'), 126.63 (C7 '), 128.43 (C7), 129.38 (C10 and C5'), 129.50 (C5), 129.86 (C15), 130.01 (C8 and C15 '), 130.87 (C) 0'), 132.31 (C14), 132.41 (C14 '), 134.59 (C9), 135.95 (C9'), 136.35 and 136.39 (C13 and C13 '), 136.51 (Cl2), 137.25 ( C12 '), 137.79 (C8'), 137.93 (C6 '), 138.24 (C6).

Claims

Culturing the microorganism using the photochemical gas generated by gas injection and light source control; And separating and purifying 9-cis beta-carotene (9-cis β-carotene) from the cultured microorganisms.
The method of claim 1,

The gas is air, oxygen, nitrogen, nitrogen dioxide, and carbon dioxide, characterized in that at least one member selected from the group consisting of, 9-cis beta carotene production method.
The method of claim 1,

The fine spray is characterized in that through a nozzle of 0.1 to 0.5mm, 9-cis beta carotene production method from the microorganism.
The method of claim 1,

The light source is characterized in that the illuminance range is performed at 1000 to 7000 Lux, and the luminance range is 4000 to 12000K, 9-cis beta carotene production method from microorganisms.
The method of claim 1,

The photochemical gas is ozone or carbon dioxide, characterized in that the production of 9-cis beta carotene from microorganisms.
The method of claim 5,

When the photochemical gas is ozone, it is produced by irradiation in the light intensity range 4000 to 5000K and the illuminance range 6000 ± 500Lux, and when the photochemical gas is carbon dioxide, the amount of dissolved carbon dioxide in the light intensity range 10000 to 12000K and the illuminance range 3000 ± 500Lux Characterized in that it is produced by adjusting, 9-cis beta carotene production method from microorganisms.
The method of claim 1,

The microorganism is at least one selected from the group consisting of microalgae, protozoa, filamentous fungi, yeast, and viruses, 9-cis beta carotene production method from the microorganism.
The method of claim 7, wherein

The microalgae are one or more selected from the group consisting of Dunaliella, spirolina, chlamidomonas, simbella, and chlorella, the method of producing 9-cis beta-carotene from microorganisms.
The method of claim 1,

The culture medium comprises at least one member selected from the group consisting of NaNO 3 , NaCl, NaH 2 PO 4 H 2 O, Na 2 SiO 3 .9H 2 O, trace metals and vitamins. A method of producing cis beta-carotene.
The method of claim 9,

The trace metals are FeCl 3 6H 2 O, Na 2 EDTA 4H 2 O, CuSO 4 5H 2 O, Na 2 MoO 4 2H 2 O, ZnSO 4 7H 2 O, MnCl 2 4H 2 O and Co (Cl) A method for producing 9-cis beta-carotene from microorganisms, characterized in that it comprises one or more selected from the group consisting of 2 · 6H 2 O.
The method of claim 9,

The vitamins are cyanocobalamin, biotin, biotin, thiamine HCl, vitamin C, vitamin E, vitamin B12, calcium pantothenate, folic acid, and nicotinamide. Method for producing 9-cis beta-carotene from microorganisms, characterized in that it comprises one or more selected from the group consisting of.
The method of claim 1,

Separation and purification of the 9-cis beta-carotene,

(1) adding hexane to the cultured microbial powder, stirring and evaporating to obtain a semisolid;

(2) obtaining beta carotene extract from the semisolid;

(3) purifying the beta carotene extract by chromatography, and then ultrasonically grinding the eluate to obtain a powder comprising all trans- and 9-cis beta carotene:

(4) evaporating the powder to obtain crystals, and then adding methanol and tetrahydrofuran and rotating evaporation to separate all trans- and 9-cis beta carotene; And

(5) crystallizing the isolated 9-cis beta-carotene; a method of producing 9-cis beta-carotene from microorganisms.
The method of claim 12,

In the step (1), the stirring is characterized in that it is carried out for 0.8 to 1.2 hours at -5 ~ 5 ℃, 9-cis beta carotene production method.
The method of claim 12,

In the step (2), the extraction of the beta carotene is added to the cyclo-nucleanone, it is carried out by maintaining for 11 to 13 hours at -25 ~ -15 ℃, production of 9-cis beta carotene from microorganisms Way.
The method of claim 12,

In the step (3), the chromatography is silica gel column chromatography, ODS column chromatography, or a combination thereof, 9-cis beta carotene production method from microorganisms.
The method of claim 15,

The silica gel column chromatography is performed using cyclohexane-ethyl and acetate as eluent, 9-cis beta carotene production method from the microorganism.
The method of claim 15,

The ODS column chromatography is performed using methanol and tetrahydrofuran as eluent, 9-cis beta carotene production method from the microorganism.
The method of claim 12,

In the step (4), methanol and tetrahydrofuran are 1: 0.2 ~ 1: 1 (v / v), characterized in that the production of 9-cis beta carotene from microorganisms.
The method of claim 12,

In the step (5), crystallization is characterized in that carried out at -25 ~ -15 ℃, 9-cis beta carotene production method from the microorganism.