KR101244213B1 - Cultivation method of chlorella and extraction method of beta carotene from chlorella - Google Patents

Abstract

The present invention discloses a method for culturing chlorella with increased beta carotene content, and a method for efficiently extracting beta carotene from chlorella using supercritical fluid extraction.

Description

Method of culturing chlorella and extracting beta carotene from chlorella {CULTIVATION METHOD OF CHLORELLA AND EXTRACTION METHOD OF BETA CAROTENE FROM CHLORELLA}

The present invention relates to a method for culturing chlorella and a method for extracting beta carotene from chlorella, and specifically, to extract beta carotene from chlorella with high efficiency using a method for culturing chlorella with increased beta carotene content and a supercritical fluid extraction method. It is about how to.

Beta-carotene is used industrially as a pharmaceutical, functional food, cosmetics and food additives. Currently, beta-carotene is produced by commercial chemical synthesis process, but recently, the US, Europe and Korea government strictly prohibit the use of synthetic beta-carotene as food and cosmetic additives, and develop biological process that can produce natural beta-carotene cheaply. There is a growing interest in. Natural beta-carotene extracted from green yellow vegetables such as carrots, tomatoes, and pumpkins is very expensive because it takes a long time to grow plants, while the amount of biosynthetic beta carotene is very small. In addition, due to the solid cell wall, a complex extraction process is required to recover it, and since other carotenoid components are also present, an additional separation and purification process is required to obtain pure beta carotene from them.

On the other hand, natural beta-carotene is three to four times higher in price due to the superior antioxidant effect than synthetic products, consumer's preference for natural products, and strengthened regulation of medicines. Imported from. Therefore, research to obtain low-cost natural beta carotene is very urgent situation.

Chlorella vulgaris is a health food that is commercialized as a relatively high value-added microalgae. In foreign countries, studies on the production of fats, proteins, vitamins, and the like from chlorella have been extensively conducted. In particular, studies on culturing chlorella using fungi have been actively conducted until the 80s.

On the other hand, research has recently begun on a good microalgal species with high content of beta carotene per cell. However, most studies have focused on the induction mechanism for beta carotene promotion, and most of the production of natural beta carotene is caused by Dunaliella, a type of microalgae.

However, chlorella (chlorella vulgaris) may vary in productivity of beta carotene (beta-carotene) depending on the culture medium used, the inventors of the present invention in a culture medium for chlorella that can increase the content of beta carotene As a result of intensive research, the present invention has been completed.

On the other hand, extraction from chlorella containing various components by conventional organic solvent extraction method results in low solvent selection, residual organic solvent, difficulty in removing solvent, environmental pollution, and low extraction yield. There are various problems such as long time extraction time of time. Therefore, there is a need for a study on how to extract beta-carotene from chlorella with high efficiency.

On the other hand, Supercritical Fluid Extraction Technology is a technology that uses fluids above the critical temperature and pressure, and can replace the existing processes in the fields of extraction and refining such as medicine, food processing and petrochemical refining. New environmentally friendly clean technologies (CD Bevan and PS Marshall.The use of supercritical fluid in the isolation of natural products, Nat. Prod. Rep. 11, 451-576, 1994). In particular, developed countries have been using gas or liquid in traditional processes for the past three decades due to rising energy resource prices, environmental problems associated with traditional separation processes, and increased demand for special-purpose materials that cannot be manufactured by gas or liquid processes. Breaking away from the concept of using, we have been working hard to develop new process fluid technology that uses supercritical fluid technology as process fluid. As a result, the supercritical fluid process is rapidly spreading to various industries such as fine chemicals, energy, environment, and new materials, and the movement to replace various traditional separation technologies with the supercritical fluid process is accelerating.

Accordingly, the inventors of the present invention have completed the present invention as a result of intensive research on a method of extracting beta carotene from chlorella with high efficiency by applying a supercritical fluid extraction method.

It is an object of the present invention to provide a method for culturing chlorella with an increased content of beta carotene.

Another object of the present invention is to provide a method for extracting beta-carotene from chlorella with high efficiency and high purity by applying a supercritical fluid extraction method.

It is also an object of the present invention to provide conditions under which beta carotene can be optimally extracted from chlorella.

In order to achieve the above object, the chlorella culture method of the present invention comprises the steps of (A) preparing a liquid medium containing sodium nitrate, potassium phosphate dibasic, magnesium sulfate and calcium chloride; (B) adjusting the pH of the liquid medium to 7-8, then sterilizing; And (C) dispensing chlorella in the sterilized liquid medium, followed by culturing; and culturing the chlorella with increased beta carotene content.

In addition, the chlorella culture method of the present invention the liquid medium further comprises citric acid, ferric citrate (III) ammonium, sodium iron EDTA, sodium carbonate, manganese dichloride, zinc sulfate, cupric sulfate, sodium molybdate and cobalt nitrate It is characterized by.

In addition, the method for extracting beta carotene from the chlorella of the present invention (A ') collecting the chlorella cultured by the above method from the liquid medium, lyophilized, and then powdered; (B ') filling the powdered chlorella into a supercritical fluid extraction container; (C ′) extracting chlorella extract by introducing supercritical fluid into the extraction vessel through a heat exchanger; (D ′) separating the supercritical fluid and the chlorella extract under reduced pressure in a reduced pressure separator; And (E ') collecting the separated chlorella extract.

In addition, the method for extracting beta carotene from the chlorella of the present invention is characterized in that the supercritical fluid is carbon dioxide in the step (C ').

In addition, the method for extracting beta carotene from the chlorella of the present invention is characterized in that the pressure of the carbon dioxide is 200bar, the temperature is 30 ℃.

Hereinafter, the method for culturing chlorella of the present invention and the method for extracting beta carotene from chlorella will be described in detail for each step.

1. Method of culturing chlorella

(A) preparing a liquid medium

Chlorella (chlorella vulgaris) may vary in productivity of beta-carotene depending on which medium is cultured. The present invention relates to a liquid medium containing nitrate and phosphate as main components. It can be used to culture chlorella with an excellent increase in the content of beta carotene.

The liquid medium of the present invention contains nitrate and phosphate as main components and various other inorganic salts. It is preferable that the main nitrate and phosphate contain sodium nitrate and potassium phosphate dibasic. In addition to sodium nitrate and potassium phosphate dibasic, magnesium sulfate, calcium chloride, citric acid, ammonium ferric citrate, sodium iron EDTA, sodium carbonate, manganese dichloride, zinc sulfate, cupric sulfate, sodium molybdate and sodium cobalt nitrate It is preferable to further include. When chlorella is cultured in a liquid medium containing all the above components, the beta carotene content of the cultured chlorella is significantly increased.

(B) after adjusting the pH of the liquid medium, sterilizing

The present invention adjusts the pH of the liquid medium prepared in step (A) to 7 to 8, preferably 7.5, it can be obtained a high concentration of chlorella when the pH of the liquid medium satisfies the above range.

Next, the liquid medium of the present invention is sterilized, preferably sterilized at 110 ° C. for 2 hours, more preferably 30 minutes in an autoclave.

(C) dispensing chlorella in sterile liquid medium, and then culturing

The present invention is cooled to room temperature in the liquid medium sterilized in the step (B), and then dispensed Chlorella Vulgaris strain. The chlorella vulgaris strain is preferably dispensed from a clean bench or a sterile box.

Next, the medium to which the Chlorella vulgaris strain is dispensed is transferred to a shaker and incubated for about 7 days under a fluorescent light for 24 hours. In this case, the temperature of the culture chamber is preferably maintained at 25 ° C. If the above range is satisfied, high concentration of chlorella may be cultured. The present invention can repeat these processes to continuously produce chlorella cells.

The chlorella cultured in this way increases the content of beta-carotene compared to conventional commercial chlorella, and thus more effectively extracts beta-carotene from chlorella.

In addition, the present invention is preferably used to collect the cells by centrifuging the chlorella produced in this way. At this time, the centrifugal conditions are preferably 8500 rpm under 4 ° C temperature.

In addition, the present invention is preferably used to make the centrifuged cell in the form of a powder dried through a freeze-drying process.

2. How to Extract Beta Carotene from Chlorella

The present invention can be extracted beta carotene very effectively by applying a supercritical fluid extraction method to chlorella cultured in a liquid medium as described above.

The method for extracting beta carotene from chlorella of the present invention comprises the steps of (A ') collecting chlorella from the liquid medium and lyophilizing, followed by powdering, (B') filling the powdered chlorella into a supercritical fluid extraction vessel. (C ') extracting the chlorella extract by introducing a supercritical fluid through a heat exchanger into the extraction vessel, (D') separating the supercritical fluid and the chlorella extract under reduced pressure in a reduced pressure separator, and (E ' It is preferable to include the step of collecting the separated chlorella extract.

At this time, in the step (A '), chlorella vulgaris is preferably to collect only the cells from the liquid medium through centrifugation, the centrifugation conditions are preferably set to 8500 rpm under 4 ° C temperature. If the centrifugation conditions satisfy the above range, only cells can be effectively collected from the medium without breaking the cells.

In addition, the collected chlorella cells are preferably lyophilized and powdered, and then ground in a mortar to make fine particles. The finer the chlorella cells, the more effective beta carotene can be extracted from chlorella.

In addition, in the step (B '), it is preferable to fill the chlorella powder in the supercritical fluid extraction container as shown in FIG.

In addition, in the step (C ′), it is preferable to use carbon dioxide as the supercritical fluid, and the temperature of the supercritical fluid is preferably 30 ° C. and the pressure is 200 bar.

In addition, the supercritical fluid separated in the step (D ') is circulated and recycled by depressurization by a pump together with the supercritical fluid supplemented from the outside, it is preferable to collect the separated chlorella extract.

By using the supercritical fluid extraction method, the present invention can obtain useful components from chlorella with high extraction efficiency without loss. In particular, the present invention can obtain beta-carotene from chlorella with very high efficiency by using supercritical fluid extraction.

According to the present invention can be cultured chlorella with increased content of beta carotene.

According to the present invention, beta carotene can be extracted from chlorella with high efficiency and high purity by applying a supercritical fluid extraction method.

In addition, according to the present invention, beta carotene can be extracted from chlorella under optimum extraction conditions.

Figure 1 is a chromatogram of HPLC analysis of beta carotene standard.
Figure 2 is a result of preparing a calibration curve by diluting the beta carotene standard sample with acetone (1ppm, 4ppm, 5ppm).
3 is a chromatogram analyzed by HPLC of the acetone extract of Chlorella of Example 1.
Figure 4 is a chromatogram analyzed by HPLC of the methanol extract of Chlorella of Example 2.
5 is a chromatogram analyzed by HPLC of the ethanol extract of Chlorella of Example 3.
6 is a chromatogram analyzed by HPLC of the chloroform extract of Chlorella of Example 4.
7 is a chromatogram analyzed by HPLC of the hexane extract of Chlorella of Example 5.
8 is a chromatogram analyzed by HPLC of the acetone extract of Chlorella of Comparative Example 1.
9 is a chromatogram analyzed by HPLC of the methanol extract of Chlorella of Comparative Example 2.
10 is a chromatogram analyzed by HPLC of the ethanol extract of Chlorella of Comparative Example 3.
11 is a chromatogram analyzed by HPLC of the chloroform extract of Chlorella of Comparative Example 4.
12 is a chromatogram analyzed by HPLC of the hexane extract of Chlorella of Comparative Example 5.
FIG. 13 shows LC / MS / MS analysis results of the hexane extract of Chlorella of Example 5. FIG.
14 is a schematic view of the supercritical fluid extraction apparatus used in Examples 6 to 13;
15 is a chromatogram analyzed by HPLC of the supercritical fluid extract of Chlorella of Example 8. FIG.
FIG. 16 is a chromatogram analyzed by HPLC of the supercritical fluid extract of Chlorella of Example 9. FIG.
17 is a chromatogram analyzed by HPLC of the supercritical fluid extract of Chlorella of Example 10. FIG.
18 is a chromatogram analyzed by HPLC of the supercritical fluid extract of Chlorella of Example 11. FIG.
19 is a result of preparing a calibration curve by diluting a beta carotene standard sample with acetone (1000ppm, 2500ppm, 5000ppm).

Hereinafter, the content of the present invention will be described in more detail in the following examples, but the scope of the present invention is not limited only to the following examples, and includes modifications of equivalent technical ideas.

Manufacturing example  : Culture of Chlorella containing Beta Carotene

In the present Example, after preparing a liquid medium, Chlorella Vulgaris strains were cultured by aliquoting.

As shown in Table 1, the culture medium was prepared by mixing the media components in the Erlenmeyer flask. The pH of the culture was adjusted to 7.50 and the inlet of the Erlenmeyer flask was plugged and sealed with foil. In order to aseptically sterilize the culture solution, the medium was sterilized at 110 ° C. for about 2 hours, preferably for 30 minutes, and then cooled to room temperature. Chlorella vulgaris strains were aliquoted into chilled medium in a clean bench or aseptic box. After dispensing, the medium was transferred to a shaker and incubated for about 7 days under a fluorescent lamp for 24 hours. At this time, the temperature of the culture chamber was maintained at 25 ℃. The produced chlorella was centrifuged to collect only the cells. At this time, the centrifugation conditions were 8500 rpm under 4 ° C temperature. The centrifuged cells were made in the form of dried powder through lyophilization process.

 Medium component mg / L Sodium Nitrate 1500 Potassium phosphate dibasic tetrahydrate 40 Magnesium sulfate heptahydrate 75 Calcium chloride 27.2 Citric acid 6 Ammonium iron (Ⅲ) citrate 6 EDTA iron sodium salt (EDTA) One Sodium carbonate 10 Manganese (II) chloride tetrahydrate 1.81 Zinc sulfate heptahydrate 0.22 Cupric sulfate pentahydrate 0.39 Sodium molybdate 0.05 Cobalt nitrate hexahydrate 0.049

Example  1 to 5: Extraction of Beta Carotene from Chlorella

In this example, a solvent extraction method was performed to extract beta carotene from the chlorella cultured in Preparation Example. Solvents used in the solvent extraction method are acetone, methanol, ethanol, chloroform and hexane. 0.5 g of chlorella vulgaris cultured in the above Preparation Example was added to 100 ml of the solvent and stirred for 4 hours. After the extraction was completed by stirring, the solvent was removed by a rotary box evaporator. Saturated aqueous solution of NaCl: MeOH = 1: 1 was made to dissolve the extract. This was placed in a separatory funnel for liquid-liquid extraction and the layers separated. As time goes by, two clear layers were separated. When the layers were separated, the supernatant was collected and Na ₂ SO ₄ , which is widely used as a dehydrating agent, was added to the extracted sample to remove moisture. The extract was removed by removing the solvent with a rotary box evaporator to remove the moisture.

Comparative example  1 to 5: Extraction of Beta Carotene from Chlorella

Note: Beta carotene was extracted from chlorella in the same manner as in the above example, except that chlorella raw material commercially available from the subject was used.

Types of Chlorella Type of solvent Example 1 Chlorella Powder Cultured in Preparation Acetone Example 2 Chlorella Powder Cultured in Preparation Methanol Example 3 Chlorella Powder Cultured in Preparation ethanol Example 4 Chlorella Powder Cultured in Preparation chloroform Example 5 Chlorella Powder Cultured in Preparation Hexane Comparative Example 1 Chlorella raw material sold by Daesang Co., Ltd. Acetone Comparative Example 2 Chlorella raw material sold by Daesang Co., Ltd. Methanol Comparative Example 3 Chlorella raw material sold by Daesang Co., Ltd. ethanol Comparative Example 4 Chlorella raw material sold by Daesang Co., Ltd. chloroform Comparative Example 5 Chlorella raw material sold by Daesang Co., Ltd. Hexane

Experimental Example  1: Beta-carotene content analysis of chlorella solvent extract

In this experimental example, HPLC analysis was performed to analyze the beta carotene content of the extracts prepared in Examples 1 to 5 and Preparation Examples 1 to 5.

The mobile phase of HPLC was mixed with chloroform: methanol in a ratio of 4:96, and the column was an ODS2 (4.6 × 150 mm) column manufactured by waters. The flow rate of the mobile phase was 1 mL per minute and detected by a UV detector at a wavelength of 450 nm. To confirm that the extracted material was beta-carotene, the retention time was compared by using a beta-carotene standard purchased from TCI.

1 is a chromatogram analyzed by HPLC of the beta carotene standard sample, it can be seen that the retention time of beta carotene is about 18 minutes. In addition, the results of diluting the standard sample with acetone to a concentration of 1 ppm, 4 ppm, and 5 ppm to prepare a calibration curve are shown in FIG. 2.

Extracts (total 10 extracts) extracted from each of the chlorella with five solvents were analyzed by the HPLC conditions. As shown in Figures 3 to 12, the extract of Example 5 contained beta carotene, except for this, it was confirmed that beta carotene extracted at a very low concentration that cannot be quantified by HPLC. In addition, when the amount of beta carotene was calculated from the calibration curve of FIG. 2 with respect to the extract of Example 5, it was found that 2.18 ppm of beta carotene was extracted. From this, chlorella cultured in the liquid sample of the present preparation was confirmed that the content of beta carotene increased compared to the conventional chlorella.

Experimental Example  2: Identification of structural character of active ingredient in chlorella extract

In this Experimental Example, the components of the hexane extract of Chlorella obtained in Example 5 were analyzed. That is, the extraction was confirmed again by confirming the presence or absence of the peak corresponding to the molecular weight 538 of the beta carotene by component analysis of LC / MS / MS. LC / MS / MS was used as TSQ Quantum Ultra of Thermo, and the column was Gemini-NX 5um C18 (100 × 2.0mm). The column flow rate was 200 μl per minute. The injection volume was 20 μl, and the mobile phase was used by mixing methanol and 2-propanol in a 3: 1 ratio. Vaporizer temperature was 100 ° C., Sheath gas pressure was 40 psi, Aux gas pressure was 10 psi, and the filling temperature was 320 ° C.

Results obtained by analyzing by LC / MS / MS are shown in FIG. 10, the molecular weight of the active ingredient of the hexane extract of Chlorella obtained in Example 5 was confirmed that the beta carotene 538.

Example  6-13: Beta-carotene extraction from chlorella by supercritical fluid extraction

In this example, beta-carotene was extracted from Chlorella Vulgaris cultured in the preparation example using a supercritical fluid extraction method. The specific method is as follows, and the schematic diagram of the supercritical fluid apparatus used at this time is shown in FIG.

1) Chlorella vulgaris was collected from the liquid medium by centrifugation, lyophilized, lyophilized, powdered and ground in a mortar to make fine particles.

2) 0.5 g of chlorella in the form of fine powder prepared in step 1) was filled in a supercritical fluid extraction vessel.

3) The supercritical fluid was introduced into the extraction container filled with chlorella powder in step 2) to extract the extract. Here, carbon dioxide was used as the supercritical fluid, and supercritical extraction conditions are shown in Table 3 below.

4) The mixture of the extract extracted in step 3) and the supercritical fluid were separated under reduced pressure through a pressure reducing valve in a pressure reducing separator.

5) The supercritical fluid separated in step 4) was circulated and recycled by depressurization by a pump with the supercritical fluid supplemented from the outside, and the separated chlorella supercritical fluid extract was collected.

CO ₂ CO ₂
(g / min) Modifier (mL / min) Modifier Example 6 150 2 0.1 mL / min Acetone Example 7 150 2 0.1 mL / min - Example 8 200 2 0.1 mL / min Acetone Example 9 200 3 0.1 mL / min Acetone Example 10 200 2 0.1 mL / min - Example 11 200 3 0.1 mL / min - Example 12 250 2 0.1 mL / min Acetone Example 13 250 2 0.1 mL / min -

Experimental Example  3: Chlorella Supercritical  Beta-carotene content analysis of fluid extracts

In this experimental example, HPLC analysis was performed to analyze the beta carotene content of the extracts prepared in Examples 6 to 13. At this time, the extract was concentrated by removing the solvent with a rotary box evaporator and concentrated, followed by HPLC analysis as in the extract of the solvent extraction method.

15 to 18 are chromatograms of the chlorella supercritical fluid extracts of Examples 8 to 11, whereby peaks having retention times of 10 minutes, 11 minutes, 18 minutes, and 19 minutes existed. Compared with the retention time of the beta-carotene standard of 1, beta-carotene is present in the 18 minutes.

On the other hand, when Example 6, was a case where the pressure of the CO ₂ in the 7150, the height of the peak, regardless of the flow rate of CO ₂ lower than in the Examples 6 to 9 and Example 12, the pressure of CO ₂ of 13 In the case of 250, the extraction was not performed smoothly due to the clogging phenomenon of the line in which the extract was recovered when the supercritical fluid was extracted. From this, when the chlorella supercritical fluid extraction, it can be seen that the content of beta carotene is very increased when the pressure of the supercritical fluid is 200bar.

In the following, the amount of extracted beta carotene was compared to the case where the pressure of CO ₂ of Examples 8 to 11 was 200. Since the beta-carotene detected at high concentrations as shown in FIGS. 15 to 18 could not be quantified by the calibration curve of FIG. 2, the calibration curve was rewritten using a standard standard of 1000 ppm, 2500 ppm, and 5000 ppm It is shown in 19. The quantitative results by the calibration curve obtained using the beta carotene standard sample are shown in Table 4.

 Peak area Beta Carotene Concentration (ppm) Example 6 18353109  7971 Example 7 10294376  4862 Example 8 11282532  5244 Example 9 7878894  3932

As shown in Table 4, it was confirmed that beta carotene can be effectively extracted from chlorella when extracted using a supercritical fluid extraction method than a general solvent extraction method. In addition, the solvent extraction method takes about 13 hours in terms of time efficiency, whereas the supercritical fluid extraction method takes about 2 hours and was found to be more effective.

Claims (5)

delete (A) sodium nitrate, potassium phosphate dibasic, magnesium sulfate, calcium chloride, citric acid, ferric citrate (III) ammonium, sodium iron EDTA, sodium carbonate, manganese dichloride, zinc sulfate, cupric sulfate, sodium molybdate and cobalt nitrate Preparing a liquid medium comprising;
(B) adjusting the pH of the liquid medium to 7-8, then sterilizing; And
(C) dispensing chlorella in the sterilized liquid medium, and then culturing;
Method for culturing chlorella, characterized in that the chlorella with increased beta carotene content. (A ') collecting chlorella cultured by the method of claim 2 from the liquid medium, lyophilized and then powdered;
(B ') filling the powdered chlorella into a supercritical fluid extraction container;
(C ′) extracting chlorella extract by introducing supercritical fluid into the extraction vessel through a heat exchanger;
(D ′) separating the supercritical fluid and the chlorella extract under reduced pressure in a reduced pressure separator; And
(E ') a method for extracting beta carotene from chlorella, characterized in that it comprises a; collecting the separated chlorella extract. . The method of claim 3,
In the step (C '),
The supercritical fluid is a method for extracting beta carotene from chlorella, characterized in that the carbon dioxide. 5. The method of claim 4,
Method for extracting beta carotene from chlorella, characterized in that the pressure of the carbon dioxide is 200bar, the temperature is 30 ℃.

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