KR101907994B1 - A method of cultivating a microorganism and method of separating and purificating 9-cis beta-carotene from the cultivated microorganism - Google Patents
A method of cultivating a microorganism and method of separating and purificating 9-cis beta-carotene from the cultivated microorganism Download PDFInfo
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Abstract
The present invention relates to a method for improving the culture speed of a microorganism using a photochemical gas produced by micro-injection of gas and light source control and a method for separating and purifying 9-cis beta carotene (9-cis? -Carotene) from the cultured microorganism To a method for producing 9-cisbeta carotene from a microorganism. The 9-cisbeta carotene according to the present invention has an effect of facilitating production, mass production in an environmentally friendly manner, and exhibiting excellent purity by being separated and purified from microorganisms cultured by fine injection of gas and light source control.
Description
The present invention relates to a method for culturing a microorganism using a photochemical gas produced by micro-injection of gas and light source control, and a method for separating and purifying 9-cis beta carotene (9-cis? -Carotene) from the cultured microorganism , A method for producing 9-cisbeta carotene from a microorganism.
Carotenoids are greenish-yellow vegetables such as carrots, old pumpkins, bell peppers, sesame leaves, lettuce, leeks and spinach, and orange colorants (yellow to bright red), which are predominant in the bark of fruits. Among them, beta-carotene (beta-carotene) is the precursor of almost all carotenoids and is converted to vitamin A, an essential nutrient in the body. Beta-carotene is widely used as a food additive due to antioxidative effect, high nutritive value and unique color that can prevent oxidation damage in animal tissues, and is also useful as an anticancer agent, a vascular disease agent, and an antioxidant related to skin aging . In particular, 9-cisbeta carotene (9-cis? -Carotene) is known to have excellent effects as a pharmaceutical product among the isomers of beta-carotene. However, the amount of beta-carotene present in greenish-yellow vegetables and fruits is not so high that the 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-cisbeta-carotene It has not been possible to isolate isomers.
Dunaliella, a natural microalgae, is a eukaryote belonging to the photosynthetic green algae. It is a salt adaptation range that can survive even at a salt concentration ranging from 1/10 of the seawater to 5M close to the saturated solution . The microalgae of the Dernalella genus are found in low nitrogen, sunlight, and high salt concentrations, which, when exposed to such strong environmental stress conditions, accumulate beta carotene in the cells and cause yellow- Is found. These beta-carotenes have been reported to accumulate in the cytoplasmic interstitial space with oily granulocytes (Ben-Amotz et al., J. Phycol. 18: 529-537), and the accumulated beta-carotene protects the photosynthetic system from strong light .
A series of processes in which microorganisms, animal and plant cells are inoculated into a proper diet and grown under appropriate conditions such as temperature and oxygen are referred to as culturing, and the culture is classified into liquid culture and solid culture. Important external conditions for culture include temperature, humidity, composition of light gas phase (partial pressure of carbon dioxide and oxygen), and other important factors that directly affect the organism to be cultivated. The medium is also referred to as an incubator and is a supply environment for various nutrients necessary for survival and proliferation as well as a direct environment for the organism. Generally, in a liquid culture medium using a liquid medium, algae and microorganisms are cultured by controlling the saturation amount of organic and inorganic substances contained in temperature, lightness, gas and medium, that is, nutrition culture method. And the nutrient composition of the medium is controlled in order to improve the cultivation speed of the medium. However, in such a liquid culture, there is a problem that aggregation of the cultured cells occurs in proportion to the growth of the cultured product. This coagulation phenomenon can be confirmed in most algae and microorganisms, and causes the rate of cell culture to deteriorate. In addition, gas such as oxygen and carbon dioxide should be continuously supplied in order to properly maintain hydrogen ion index, dissolved oxygen and dissolved carbon dioxide, which are environmental conditions necessary for promoting culture in the incubator. However, there is a problem that it is difficult to sufficiently generate dissolved ozone necessary for the growth of a specific microorganism and to keep it constant by merely injecting these gases from the outside. Korean patent application KR 10-2012-0112175 discloses a method for promoting the growth rate of algae and microorganisms by preventing the agglomeration of cultures through micro-injection of air on a liquid medium and generating a photochemical gas by light source control Method.
As described above, after algae and microorganisms are cultured by using a photochemical gas generated by micro-injection of air and light source control, 9-cisbeta carotene is separated and purified from the cultured microorganism, There is an urgent need for research on a method for producing the polyvinyl alcohol.
The inventors of the present invention have investigated a method for producing 9-cis beta carotene (9-cis beta -carotene), and after culturing a microorganism using a photochemical gas produced by fine injection of gas and light source control, When 9-cisbeta carotene is isolated and purified from microorganisms, 9-cisbeta carotene can be easily produced, can be mass-produced in an environmentally friendly manner, and 9-cisbeta carotene shows excellent purity. .
Accordingly, the present invention relates to a method for improving the culture speed of a microorganism using a photochemical gas produced by micro-injection of gas and light source control, and a method for separating and purifying 9-cisbeta carotene from the cultured microorganism 9-cisbeta carotene. ≪ / RTI >
In order to achieve the above object,
The present invention relates to a method for producing a microorganism, which comprises culturing a microorganism using a photochemical gas produced by micro-injection of a gas and light source control; And separating and purifying 9-cisbeta carotene (9-cis? -Carotene) from the cultured microorganism. The present invention also provides a method for producing 9-cisbeta carotene from a microorganism.
The present invention also provides 9-cisbeta carotene produced by the production method.
The present invention also provides a pharmaceutical product comprising the 9-cisbeta carotene.
The present invention also provides a food composition comprising the 9-cisbeta carotene.
The present invention also provides a cosmetic composition comprising the 9-cisbeta carotene.
Hereinafter, the present invention will be described in detail.
The present invention relates to a method for producing a microorganism, which comprises culturing a microorganism using a photochemical gas produced by micro-injection of a gas and light source control; And separating and purifying 9-cisbeta carotene (9-cis? -Carotene) from the cultured microorganism. The present invention also provides a method for producing 9-cisbeta carotene from a microorganism.
In order to maximize the growth of the strain, the present invention can promote the production of 9-cisbeta carotene by changing the composition of the medium and the culture conditions such as the salt concentration, the concentration of the nitrogen source, or the pressure and the light source control.
The micro-injection of the gas according to the present invention is controlled by a sensor unit which senses the aggregation time index of the microorganism mycelium. When the microorganism grows in accordance with the growth of the microorganism in the medium, a low-pressure mist spray nozzle Thereby spreading the gas on the medium to pulverize the agglutinated culture.
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 finely injected through a nozzle of 0.1 to 0.5 mm under a low pressure to pulverize the agglutination of the culture, resulting in a flocculation resolution index of 0 to 1%.
In order to cultivate microorganisms well in a liquid culture medium, a certain amount of dissolved oxygen (DO), dissolved carbon dioxide (DCO 2 ) and ozone are required. In the present invention, a method of generating necessary gas by adjusting the luminous intensity and illuminance of a light source is adopted .
The light source adjustment may be performed at an illuminance range of 1000 to 7000 Lux, and a luminous intensity range of 4000 to 12000 K, and the required photochemical gas may be ozone or carbon dioxide. That is, a sensor unit for sensing the amount of dissolved oxygen, dissolved carbon dioxide, and ozone in the incubator is installed to introduce nitrogen dioxide when the ozone content is insufficient. In the oxidation process of the nitrogen gas, a light intensity range of 4000 to 5000K, an illuminance range of 6000 ± 500 Lux When a light source is provided, ozone is generated. In addition, when dissolved carbon dioxide is insufficient, an infrared light source having a luminous intensity range of 10000 to 12000 K and an illuminance range of 3000 ± 500 Lux is irradiated to increase dissolved carbon dioxide in the medium.
The microorganism is not particularly limited in the present invention and includes microalgae, protozoa, filamentous fungi, yeast, viruses and the like, which are fine microorganisms of 0.1 mm or less including 9-cisbeta carotene. The microalgae may be Dulnellella, Spirulina, Clemidomonas, Simbella, Chlorella, etc. which are cultured in a liquid medium, but it is preferably Dulnellella. Dnaliola contains natural beta-carotene. When the culture conditions deteriorate as a result of intense light irradiation, beta-carotene is mass-synthesized to protect chlorophyll a, a chlorophyll important for photosynthesis, and beta carotene It is known to form a film.
The culture medium may be a liquid medium or a solid medium as a supply source of nutrients necessary for survival and proliferation of microorganisms, but it is preferably a liquid medium. In addition, NaNO 3 , NaCl, NaH 2 PO 4 .H 2 O, Na 2 SiO 3 .9H 2 O, trace metals and vitamins can be included to improve the culture rate of microorganisms. The trace metals include 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 vitamin may be cyanocobalamin, biotin, thiamine HCl, vitamin C, vitamin E, vitamin B12, calcium pantothenate, Folic acid, nicotinamide, and the like.
After the cultivation is completed, the cultured microorganism can recover the cells by spontaneous precipitation of the culture broth or centrifugation.
The separation and purification of 9-cisbeta carotene produced in the culture microorganism can be carried out by: (1) adding hexane to the cultured microbial powder, stirring and evaporating to obtain a semi-solid; (2) obtaining a beta carotene extract from the semi-solid; (3) purifying the beta carotene extract by chromatography, and ultrasonically pulverizing the eluate to obtain a powder containing allotrans- and 9-cisbeta carotene; (4) subjecting the powder to evaporation to obtain crystals; After that, methanol and tetrahydrofuran are added and rotary evaporation is carried out to separate alltrans- and 9-cisbeta carotene; And (5) crystallizing the separated 9-cisbeta carotene.
The steps (1) and (2) above are for extracting beta carotene from the cultured microorganism. According to one embodiment of the present invention, the dried powder of the cultivated Dynalysella salina strain is washed with ethanol and then the carotenoid component To separate the cyclohexane, the hexane layer is evaporated, hexane is added again, and the mixture is stirred at-5 to 5 ° C for 0.8 to 1.2 hours and evaporated to obtain a dark brown solid. After the cyclohexanone is added to the obtained semi-solid, the β-carotene extract can be obtained by maintaining the mixture at -25 to -15 ° C. for 11 to 13 hours.
The step (3) may be carried out by purifying beta carotene, using silica gel column chromatography, ODS column chromatography or a combination thereof. According to one embodiment of the present invention, the obtained beta carotene extract was purified by silica gel column chromatography using cyclohexane-ethyl and acetate (50: 1, v / v) as an eluent to remove impurities, (1: 0.2 ~ 1: 1, v / v) as an eluent was further purified by ODS column chromatography to obtain an all-trans-β- Orange powder mixed with carotene and 9-cisbeta carotene was obtained. The thus obtained powder can be assayed for its beta-carotene content by measuring the absorbance at 450 nm or 475 nm using HPLC.
The step (4) is a step of separating and crystallizing 9-cisbeta carotene from the purified beta-carotene powder, wherein an orange powder containing all-trans beta-carotene and 9-cisbeta carotene obtained according to an embodiment of the present invention After evaporation, methanol and tetrahydrofuran are added at 1: 0.2 to 1: 1 (v / v) and the mixture is rotary evaporated to separate 9-cisbeta carotene from all-trans-beta carotene.
The separated 9-cisbeta carotene can be crystallized at -25 to -15 占 폚, preferably -20 占 폚.
The present invention also provides 9-cisbeta carotene produced by the production method. The 9-cisbeta carotene produced according to the present invention has an average diameter of 0.1 to 0.4 μm and has an advantage of exhibiting excellent purity.
The present invention also provides a pharmaceutical product comprising the 9-cisbeta carotene.
The present invention also provides a food composition comprising the 9-cisbeta carotene.
The present invention also provides a cosmetic composition comprising the 9-cisbeta carotene.
The present invention also relates to a method for the treatment and / or prevention of cardiovascular diseases which is characterized by converting LDL cholesterol into HDL cholesterol through retinoid X receptor (RXR) binding of Macrophage, including 9-cisbeta carotene. Prophylactic or therapeutic medicines.
The present invention also relates to a method for the treatment of cerebrovascular disease, characterized by the generation of antiplatelet through retinoid acid receptor (RAR) binding of platelets, including 9-cisbeta carotene. Prophylactic or therapeutic medicines.
The present invention also relates to a method for the treatment of acanthamoeba keratitis, which is characterized by an increase in the activity of the macrophage inflammatory chemokine receptor by the Cxcl2 (chemokine ligand 2) gene, including the 9-cisbeta carotene. keratitis). < / RTI >
The present invention also relates to a method for the treatment and / or prophylaxis of cancer or metabolic diseases characterized by the negative regulation of environmental stress of the skin fibroblast through the mRNA inhibition of the dual specificity phosphatase 1 (Dusp1) gene comprising the 9-cisbeta carotene Or a pharmaceutically acceptable salt thereof.
The present invention also relates to a method for the prevention of cancer diseases, which is characterized by the production of mRNA by inhibiting transcripts of microRNA (microRNA, miRNA) by the MIR 8094 gene including 9-cisbeta carotene Or therapeutic medicines.
The present invention also relates to a method for the prophylaxis or treatment of an inflammatory disease, which comprises the step of inhibiting the encoding of protein granulocytes by a colony stimulating factor 3 (Csf3) gene comprising the 9-cisbeta carotene. Provide therapeutic medicines.
The present invention also relates to a pharmaceutical composition comprising a Pfkfb3 (6-phosphofructo-2-kinase / fructose) conjugate, which binds to fructose-2,6-bisphosphate (F2,6BP) -2,6-biphosphatase 3), thereby inhibiting the activity of the cyclin-
The present invention also relates to a method for the treatment and / or prophylaxis of pediatric lymphocytic leukemia, natural lymphoid leukemia, mixed pedigree leukemia, AF4 / X mental retardation syndrome, A cerebellar ataxia, a corneal cell loss-related disease, or a chromosomal predominantly aberrant mutation.
The present invention also relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient for the treatment of neonatal adrenoleukodystrophy (NALD), Zellweger syndrome, or infant refsum disease (IRD), including 9-cisbeta carotene, peroxisomal biogenesis factor 5, PEX5), which is characterized in that it inhibits the neonatal adrenal leukodystrophy, the Zellweger syndrome, or the infant's disease.
The present invention also provides a method for producing a beta-galactosidase-1 or a glycoconjugate encoding substance GLB1 (galactosidase beta 1) gene comprising the 9-cisbeta carotene And a drug for preventing or treating cancer.
In addition, the present invention provides a medicament for the prophylaxis or treatment of cancer diseases, which comprises producing a minichromosome maintenance complex component 5 (MCM5) gene which replicates DNA comprising the 9-cisbeta carotene.
The present invention also relates to a method for producing dUMP and pyrophosphate by hydrolyzing dUTP by subcloning mitochondria and nuclei of DUT (deoxyuridine triphosphatase) mRNA located on chromosome 19 (chromosome 19), including 9-cisbeta carotene, Characterized by the formation of a homotetrameric enzyme in the form of a pharmaceutically acceptable salt thereof.
The present invention also relates to a method of increasing glutathione S-transferase alpha 2 (GSTA2) mRNA comprising the 9-cisbeta carotene to produce cytosolic and membrane binding of glutathione S-transferase Which is characterized by protecting glutathione peroxidase by activating glutathione peroxidase to protect cells and peroxidation of alpha class enzyme active oxygen species.
The present invention also relates to a method for the treatment and / or prophylaxis and / or treatment of cancer diseases, characterized in that the activity of UBE2C (ubiquitin-conjugating enzyme E2C) gene as defined in
The present invention also provides a method for producing a STMN1 (stathmin 1) gene that inhibits phosphoprotein activity involved in stathmin cells, including 9-cisbeta carotene, to degrade microtubules Or a pharmaceutically acceptable salt thereof.
The present invention also provides a medicament for the prophylaxis or treatment of cancer, which comprises activating E2F1, PTGER2, RABL6, TBX2 or MYC protein comprising 9-cisbeta carotene.
The present invention also provides a pharmaceutical composition for preventing or treating cancer diseases, which comprises the activity of inhibiting TP53, CDKN1A, CDKN2A, RABL6, RB1, PTEN1, NUPR1, RBL1 or RBL1 protein including 9-cisbeta carotene .
The 9-cisbeta carotene according to the present invention has an effect of facilitating production, mass production in an environmentally friendly manner, and exhibiting excellent purity by being separated and purified from microorganisms cultured by fine injection of gas and light source control.
BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a diagram showing agglutination dissolution and culture of (a) 9-cisbeta carotene and (b) a culture medium in a liquid medium according to the present invention.
Figure 2 is a diagram showing the UV-B measurement of HPLC on carotenoid and tocopherol of dunaliella salina strain according to the present invention.
FIG. 3 is a graph showing a sample containing 9-cisbeta carotene purified according to Example 2-2, which was analyzed by HPLC at 450 nm absorption band.
FIG. 4 is a graph showing the results of analyzing a sample containing 9-cisbeta carotene purified according to Example 2-3 using a 470 nm absorption band using HPLC. .
FIG. 5 is a graph showing 1 H-NMR and 13 C-NMR spectra of 9-cisbeta carotene cultured, separated and purified from a Dnaliella salinarro strain according to the present invention.
FIG. 6 is a graph showing raw data for transcript sequencing analysis of wild-type mice (control group) in which 9-cisbeta carotene was injected or not (control group), and trimmed read values obtained by preprocessing (a) data amount comparison, (b) Q30 value (phred score,
Figure 7 shows a heatmap for a list of Differentially Expressed Genes (DEG) for transcript sequencing of wild-type mice (control) wild-type mice (Mus musculus) injected with or without 9-cisbeta carotene .
FIG. 8 is a diagram showing a GO term related to a biological process (BP), which is a functional classification of a gene ontology using the DAVID tool for the DEG list of FIG. 7; FIG.
FIG. 9 is a diagram showing a GO term related to a molecular function (MF), which is a functional classification of a gene ontology, using the DAVID tool for the DEG list of FIG.
FIG. 10 is a diagram showing a GO term related to a cellular component (CC), which is a functional classification of a gene ontology, using the DAVID tool for the DEG list of FIG. 7;
11 is a diagram showing a signal transduction mechanism of Polo-like kinase (PLK).
Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.
Example 1. Culture of microorganisms using a photo-bio-incubator
A microalgae, Dunaliella salina (KMMCC-1064), was cultivated using a photo-bio-incubator (KS Application No. 10-2012-0112175). The dunaliella salinas strain was inoculated into a f / 2 culture medium, and the pressure regulation and air injection of the photo-biochemical incubator were carried out using a nozzle of 0.2 to 0.3 mm under a low pressure. The illuminance range of the light source was 1000 to 7000 lux, The range was adjusted from 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 Dulnellella salina are shown in Tables 1 to 3.
After the cultivation was completed, the strain cultured using a hollow fiber membrane having a size of 0.05 μm was filtered and the content of carotenoids of the strain was analyzed using HPLC (Tosoh Corporation, MCPD-3600).
Example 2. Separation and purification of 9-cisbeta carotene from cultured microorganisms
Separation and purification of 9-cisbeta carotene (see Fig. 1) was carried out from the Dulnaliella salina strain cultivated in accordance with Example 1 above.
2-1. Initial purification of cultured microorganisms
First, dry powder (1 kg) of the Dulnaliella salina strain (KMMCC-1064) cultivated according to Example 1 was washed twice with ethanol (5 L) to remove the vegetable oil component. To remove the carotenoid, cyclohexanone (9 L) was added, and the hexane layer was evaporated to obtain a dark brown solid and solid (53 g). To the obtained sample, hexane (1 L) After stirring for 1 hour at < RTI ID = 0.0 > C, < / RTI > the hexane layer was evaporated to give 38 g of a dark brown solid containing 14.8 g of trans-betacarotin. Cyclosuccinone (185 mL) was then added to the semi-solid and the mixture was maintained at -20 <0> C for approximately 12 hours. The precipitated solids were then removed by filtration to give an extract containing 4 g of all trans beta-carotene and a large amount of 9-cisbeta carotene isomer.
2-2. Isolation of 9-cisbeta carotene by open-column chromatography
The extract according to Example 2-1 was purified by silica gel column chromatography (silica gel, 900 ml, column: 5.5 cm 횞 38 cm, eluent: cyclohexane and ethyl acetate (50: 1, v / v) To remove impurities, and 16.6 g of a reddish brown foamy solid was obtained. To this solid was added 1660 mL of ethanol, followed by pulverization with ultrasonic waves, followed by filtration, washing with ethanol, and drying to obtain orange powder (12 g,> 80%). The ultrasonic grinder (VCX750, Sonic & Materials Inc., USA) used at this time was operated at a speed of 3000 rpm for 6 minutes. The obtained powder was subjected to component analysis at 450 nm absorption band using HPLC, and the result is shown in FIG.
As shown in Fig. 3, it can be confirmed that the orange powder contains isomers such as all-trans-beta-carotene and 9-cisbeta-carotene.
2-3. Isolation of 9-cisbeta 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 and subjected to 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 a pale green band formed on the column. To this concentrate was added 1660 mL of ethanol and then pulverized by ultrasonic wave 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 result is shown in FIG.
As shown in FIG. 4, it can be confirmed that the orange powder mixed with all-trans-beta-carotene and 9-cisbeta-carotene.
2-4. Crystallization of 9-cisbeta carotene
The orange powder containing all-trans-beta-carotene and 9-cisbeta carotene according to the above Example 2-3 was placed in a double-jacketed reaction tank maintained at an internal temperature of the bath of 20 ° C and a bath temperature of 1 ° C, Was evaporated until it was reduced to 1/5, and orange crystals were obtained. The crystals were added with methanol and tetrahydrofuran (5: 1 - 1: 1, v / v)), and the mixture was rotary evaporated to obtain 75 mg of 9-cisbeta carotene per fraction, which was crystallized at about- . The total extractive yield was 7.5 g of 9-cisbeta carotene versus 1 kg of dry powder of dunalisella salalina, and it was confirmed that the dark room could be stored at about -20 ℃ for 2 weeks.
Experimental Example 1. Variation of carotenoid content according to medium and culture environment
The Dulnaliella salina strain cultivated in accordance with Example 1 was filtered using a hollow fiber membrane having a size of 0.05 mu m and analyzed by high performance liquid chromatography (HPLC, MCPD-3600 from Tosoh) Carotenoid content was analyzed. The HPLC was carried out using Nucleosil 5 μm C18 (250 × 4.6 mm id) (Macherey-Nagel) as a guard column,
The changes in carotenoid contents according to the culture medium and culture conditions are shown in Table 4, and more specific results are shown in Tables 5 to 7.
As shown in Tables 4 to 7, when 16% NaCl was added to the culture medium and 2.5 mM / L of nitrogen was added and then the strain of Dnalleryella salina was cultured, the carotenoid production was about 10 to 70% , Respectively. In addition, UV-B measurement of HPLC of carotenoid and tocopherol of Dnalariella salina was examined and used as a reference value for calculating UV-B value of 9-cisbeta carotene (see FIG. 2).
Experimental Example 2. Measurement of purity of 9-cisbeta carotene
The purity of 9-cisbeta carotene obtained according to Example 2 was analyzed by nuclear magnetic resonance (NMR) spectroscopy.
First, a membrane test of the obtained 9-cisbeta carotene was carried out. The membrane filter of β-carotene content of The United States Pharmacopoeial Convention (USP) was confirmed to be 0.45 μm pore size, but 0.4 μm pore size in the present membrane test. The membrane filter is made of PTFE and is made in the form of a hollow fiber. Then, 1 H-NMR and 13 C-NMR spectra of 9-cisbeta carotene filtered through the membrane filter were measured, and the results are shown in Fig.
As shown in Fig. 5, it was confirmed that the obtained 9-cisbeta carotene had no impurities and exhibited excellent purity.
1 H-NMR: δ ppm ( CDCI 3): 1.03 (C1, -CH 3), 1.04 (C1-CH 3), 1.48 (C2-
13 C-NMR: (C9'-Me), 12.81 (Cly-Me), 12.87 (C13-Me), 19.28 (C3 and C3 '), 20.77 (C2-Me2), 33.10 (C4 and C4 '), 34.25 (C1), 34.29 (C), 39.59 (C2), 39.68 (C2 '), 123.86 (Cl1), 124.99 (CII'), 126.63 (C7 '), 128.43 (C7), 129.38 (C10 and C5'), 129.50 (C5), 129.86 (C15), 130.01 ), 130.87 (C0 '), 132.31 (C14), 132.41 (C14'), 134.59 (C9), 135.95 (C9 '), 136.35 and 136.39 (C13 and C13'), 136.51 C12 '), 137.79 (C8'), 137.93 (C6 '), 138.24 (C6).
Experimental Example 3. Wild mice ( Mus musculus ) Transcript sequence analysis
After 9-cisbeta carotene isolated and purified according to Example 2 was administered to Raw264.7 cells, which are macrophages of wild-type mice ( Mus musculus ), gene expression values were obtained through transcript sequence analysis to discriminate genes And functional classification and gene annotation were performed based on gene ontology and path information on the significant genes. The results were also compared with a control (control) without 9-cisbeta carotene.
3.1 Experimental Method
First, total RNA was isolated from the cells or tissues of wild-type mice (Mus musculus) in which 9-cisbeta carotene was administered or not (control group), and then DNA contamination was eliminated using DNase. A kit was selected according to the type of RNA to be profiled as a library preparation step. When a mRNA with a Poly-A-tail is studied, an mRNA purification kit is used for not only mRNA but also non- RNA was purified using a ribo-zero rRNA removal kit when studying total RNA containing-coding RNA (lincRNA, etc.).
The purified RNA was randomly fragmented for sequencing into a fragment sequence and cDNA was prepared by reverse transcription on the chopped RNA fragments. A different adapter was attached to both ends of the prepared cDNA fragment and ligation was performed. Thereafter, PCR amplification was performed to the extent that sequencing was possible, and an insert size of 200-400 bp was obtained through a size selection procedure. In the case of a paired-end sequencing, the sequence is sequenced by the length of the read from both ends of the cDNA fragment.
3.2 Analysis Method
We performed quality control analysis of raw read obtained through sequencing. After producing basic statistics such as overall read quality and total bases, total leads, and GC (%), it is recommended to use artifacts such as adapter sequences, contaminated DNA, PCR clones, Was removed. The preprocessed leads were mapped to a standard genome using a TopHat program with a splice, and then an aligned read was generated. Assembly was carried out using the Cufflinks program using paired information of aligned leads based on standard genomes. During this process, we produced information on known transcripts and novel transcripts, as well as alternative splicing transcripts.
For each sample, we can investigate the expression level through the mapped leads for each transcript. At this time, the normalization value considering the transcript length and the depth of coverage is calculated and the expression profiles between samples are compared. In case of paired end sequencing, within normalization is performed with RPKM (Read Per Kilobase of Transcript per Million mapped reads) for single end (ngle end) sequencing with FPKM (Fragments Per Kilobase of Transcript per Million mapped reads) And the expression profile was extracted. The genes or transcripts expressing differentially expressed genes were selected by statistical hypothesis testing. In case of known gene information, function-specific annotation and GSEA (gene set enrichment analysis) based on GO and KEGG databases were conducted on differentially expressed genes. In addition, the deFuse program was used to predict the fusion gene
3.3 Results of analysis
Raw data for the transcript sequencing of wild-type mice (control group) treated with 9-cisbeta carotene and the trimmed read values after preprocessing (a) Data (B) Q30 value (phred score,
In addition, a heatmap for a list of Differentially Expressed Genes (DEG) for transcript analysis of wild-type mice (mice) in which 9-cisbeta carotene was administered or not (control group) , And the DEG list is used for a biological process (BP), a molecular function (MF), a cellular component (CC), and the like, which are functions of the gene ontology, ) GSEA (gene set enrichment analysis), which are shown in FIGS. 8, 9 and 10, respectively. Here, 731 transcripts satisfying the condition | fc | ≥2 were extracted to select genes that differentially expressed between the two groups.
Among the 731 selected genes having a change in the expression level of more than 2-fold, a list of genes with significant differences is shown in Table 9. < tb > < TABLE >
The mechanism of action of 9-cisbeta carotene is due to its metabolite, retinoic acid. Retinoids, such as lipophilic antioxidants, are involved in pathophysiology, including the antiatherosclerotic process, by limiting the oxidation of LDL particles. Retinoids exert their biological effects through high affinity binding to nuclear receptors. The retinoid X receptor (RXR) is activated by the 9-cis retinoid acid, while the retinoic acid receptor (RAR) can be bound by the sms all-trans retinoid acid. Such binding results in the expression of anti-atherosclerotic agents as inhibitors of macrophage cholesterol efflux transporter or macrophage cholesterol influx carrier. In addition, inflammation and platelet activation are reversed by reducing the expression of P-selectin and fibrinogen. In addition, negative and oxidized LDLs induce macrophage foam cell formation leading to the secretion of a variety of inflammatory cytokines and chemokines and promote plaque generation and instability. Oxidative deformation of LDL has been shown to be an essential mechanism to increase inflammation potential. In contrast, inhibition of cholesterol transport protein deficiency (CETP) increases the level of HDL. Thus, CETP inhibitors increase HDL levels, promote plaque stability and reduce atherosclerosis.
The signal transduction mechanism of polo-like kinase (PLK) is shown in Fig. 11 when 9-cis beta carotene is administered to mouse macrophage cell line raw264.7 cell. Polol-like kinase (PLK) has been shown to activate chromosomal mitosis, chromosomal mitosis by estrogen receptors, and cell cycle regulation by cyclin proteins. It was also observed that the expression of the protein involved in the G2 / M phase of chromosomal arrest, the test procedure was reduced, and the expression of the protein involved in the TNFR1 signaling process was reduced.
Claims (41)
Treating the photochemical gas produced by micro-injection of the gas and the light source control to a strain of Dulnalina salina inoculated in the medium and culturing to promote the production of 9-cisbeta carotene (9-cis? -Carotene); And
And separating and purifying 9-cis beta carotene (9-cis? -Carotene) from the cultivated strain, wherein the light source is adjusted to an illuminance range of 1000 to 7000 Lux, and a luminosity range of 4000 to 12000 K ,
The separation and purification
(1) adding hexane to a Dnalidina salina strain powder, stirring and evaporating to obtain a semi-solid;
(2) obtaining a beta carotene extract from the semi-solid;
(3) purifying the beta carotene extract by chromatography, and subjecting the eluate to ultrasonic pulverization to obtain a powder containing allotrans- and 9-cisbeta carotene:
(4) evaporating the powder to obtain crystals, adding methanol and tetrahydrofuran, and evaporating the mixture by rotary evaporation to separate allotrans and 9-cisbeta carotene; And
(5) crystallizing the separated 9-cisbeta carotene. 9. A method for producing 9-cisbeta carotene from a dunaliana salina strain.
Wherein the gas is at least one selected from the group consisting of air, oxygen, nitrogen, nitrogen dioxide, and carbon dioxide.
Characterized in that the micro-injection is carried out through a nozzle of 0.1 to 0.5 mm. ≪ RTI ID = 0.0 > 8. < / RTI >
Wherein the photochemical gas is ozone or carbon dioxide. ≪ RTI ID = 0.0 > 8. < / RTI >
Wherein the culture medium comprises at least one selected from the group consisting of NaNO 3 , NaH 2 PO 4 .H 2 O, Na 2 SiO 3 .9H 2 O, trace metals and vitamins. Lt; RTI ID = 0.0 > 9-cisbeta < / RTI > carotene.
The trace metals include 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 and 6H, characterized in that it comprises at least one member selected from the group consisting of O 2, a method of producing the 9-cis beta-carotene from two analytic elna Salina strain.
The vitamin is selected from the group consisting of cyanocobalamin, biotin, thiamine HCl, vitamin C, vitamin E, vitamin B12, calcium pantothenate, folic acid, and nicotinamide. ≪ RTI ID = 0.0 > 8-cisbeta < / RTI > carotene from a Dulnariella salina strain.
The method for producing 9-cisbeta carotene from a strain of Dulnalina salina, wherein in the step (1), stirring is carried out at -5 to 5 ° C for 0.8 to 1.2 hours.
In step (2), the extraction of beta-carotene is carried out by adding a cyclo-nuclear saponin followed by maintaining at -25 to -15 ° C for 11 to 13 hours. A method for producing beta carotene.
A method for producing 9-cisbeta carotene from a dunaliella salina strain, wherein, in the step (3), the chromatography is silica gel column chromatography, ODS column chromatography or a combination thereof.
Wherein the silica gel column chromatography is carried out using cyclohexane-ethyl and acetate as the eluent. 9. A method for producing 9-cisbeta carotene from a dunaliella salina strain.
Wherein the ODS column chromatography is carried out using methanol and tetrahydrofuran as the eluent. 2. The method according to claim 1, wherein the ODS column chromatography is performed using methanol and tetrahydrofuran as an eluent.
A method for producing 9-cisbeta carotene from a dunaliella salina strain, wherein in step (4), methanol and tetrahydrofuran are added at a ratio of 1: 0.2 to 1: 1 (v / v).
Characterized in that in step (5), the crystallization is carried out at -25 to -15 DEG C. 9. A method for producing 9-cisbeta carotene from a dunaliella salina strain.
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| KR102107989B1 (en) | 2019-12-20 | 2020-05-07 | 한국해양바이오클러스터 주식회사 | Mass production method by medium optimization of deodorized microorganisms for reducing odor in industrial or commercial air conditioners |
| KR20210051646A (en) | 2019-10-31 | 2021-05-10 | (주)아크에이르 | A synthesis method of 9-cis Beta-carotene |
| KR102271364B1 (en) | 2020-12-28 | 2021-06-30 | (주)아크에이르 | Synthesis method of new compounds Potassium all-trans retinoate and Potassium 9-cis retinoate. |
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| KR20210051646A (en) | 2019-10-31 | 2021-05-10 | (주)아크에이르 | A synthesis method of 9-cis Beta-carotene |
| KR102107989B1 (en) | 2019-12-20 | 2020-05-07 | 한국해양바이오클러스터 주식회사 | Mass production method by medium optimization of deodorized microorganisms for reducing odor in industrial or commercial air conditioners |
| KR102271364B1 (en) | 2020-12-28 | 2021-06-30 | (주)아크에이르 | Synthesis method of new compounds Potassium all-trans retinoate and Potassium 9-cis retinoate. |
| KR102311539B1 (en) | 2020-12-28 | 2021-10-12 | (주)아크에이르 | A pharmaceutical composition for cardiovascular treatment comprising the novel compounds Potassium all-trans retinoate and Potassium 9-cis retinoate. |
| KR20230055089A (en) * | 2021-10-18 | 2023-04-25 | 연세대학교 산학협력단 | Method and System for Reducing Nitrogen Oxides from Gas by Using Bacteria |
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