JP5230244B2 - Extraction method of active ingredients in hard shell - Google Patents

Description

  The present invention relates to a method for extracting an active ingredient using subcritical water, and more particularly to a method suitable for extracting an active ingredient from a plant or algae having a hard shell.

Astaxanthin is a type of carotenoid pigment that exhibits a red color. There are natural products and chemically synthesized products. Chemically synthesized products that can be mass-produced industrially have been added to feed to color fish and shellfish for aquaculture. However, the use of natural products derived from natural products has been limited due to the fact that production methods suitable for commercial use have not been established.
However, in recent years, as it has become clear that its antioxidant effect is very strong, it is expected to be used for general foods, health foods, pharmaceuticals, etc. When added to general foods, natural products Since the use of only a product derived from a natural product is permitted, there is a need for an immediate establishment of a production method suitable for commercial use of a product derived from a natural product.

Among organisms that biosynthesize and accumulate astaxanthin in the body, Haematococcus pluvialis green algae (hereinafter referred to as "Hematococcus algae") have been known to accumulate at a high content, Recently, a pure culture method suitable for commercial use has finally been established.
However, when taking out astaxanthin from a liquid-cultured Haematococcus algae with a high yield, the hard cell wall must be mechanically crushed and then extracted using an organic solvent such as acetone, ethanol, hexane or the like.

The organic solvent to be used is permitted by the Food Sanitation Law, but even if it is permitted, the upper limit of the amount of residual solvent is strictly regulated and it is preferable that it is not included for safety reasons Needless to say.
In addition, it is inevitable that the medium, nutrients, chemicals and proteins in algae, chlorophylls, sterols, phospholipids and other lipids during the cultivation of Haematococcus algae are extracted together. For this reason, it is difficult to obtain a product with high astaxanthin purity.
Furthermore, the above-mentioned solvents are flammable and dangerous under the Fire Service Act, and the safety of the extraction device must also be considered.
In addition, dry crushing is indispensable as a pretreatment for the extraction process, and not only a dedicated device is required and the process becomes long, but also oxides generated after drying are extracted together. Unavoidable.

Japanese Patent Laid-Open No. 9-111139

  The present invention solves the above-mentioned problems, and provides a novel and useful method capable of extracting an active ingredient existing in a hard shell such as astaxanthin in one step and without using an organic solvent. With the goal.

As a result of trial and error, the present inventor has found that if subcritical water is used for extraction of an active ingredient existing in a hard shell such as astaxanthin, the shell can be crushed and the active ingredient can be extracted in one apparatus. It came to propose one extraction method based on a headline and it.
That is, the method for extracting an active ingredient present in a hard shell of the plant or algae of the present invention is characterized by extracting the active ingredient by pulverizing the shell by bringing the shell into contact with subcritical water. This is an active ingredient extraction method.

The extraction method of the present invention is suitable for extracting astaxanthin from green algae, in particular, Haematococcus pluvialis. In addition, it has been found that when extracting astaxanthin from Haematococcus pluvialis, astaxanthin can be extracted with a good yield by raising the temperature of subcritical water to 180 to 230 ° C. and increasing the internal pressure to 3 to 8 MPa. Furthermore, it has been found that the yield of astaxanthin is further improved by raising the temperature of subcritical water to 210 to 220 ° C. and raising the internal pressure to 5 to 6 MPa.
Moreover, when dissolving an extract in oil, it is also possible to add oil to the water used as subcritical water beforehand.

  According to the present invention, a hard shell can be pulverized and extracted with high yield without decomposing or altering the active ingredients present therein, so that the active ingredients present in the hard shell such as astaxanthin can be extracted. Extraction in stages and without the use of organic solvents.

The present invention is a method particularly suitable for extracting active ingredients present in hard shells of plants and algae. As promising treatment objects, for example, chlorella, spirulina, and faffia yeast are assumed as algae, and pollen, mushroom spores and the like are also assumed although they are not algae. Pollen and the like are expected to be promising because they have small details and are difficult to crush mechanically.
In addition, the active substance is dispersed in the shell, not only the active ingredient enclosed in the outer shell, such as the capsule wall of the cell wall or soft capsule, but also the processed object. Something like that.

When the target is an algae such as Haematococcus algae, liquid culture is performed in an appropriate medium according to a known method. A hermetic culture method in which foreign microorganisms are not mixed and propagated and other contaminants are not mixed is preferable.
Then, the strain is collected from the culture solution, and hematococcus algae is separated by a conventional method, for example, filtration or centrifugation, and washed with water to remove the adherent medium or additives.
The pretreatment up to this point is the same as when an organic solvent is used.

After finishing the above pretreatment, an extraction treatment using subcritical water is performed.
Specifically, the hematococcus algae washed with water are kept in a water-containing state and put in an appropriate sealed pressure vessel together with an appropriate amount of water and sealed, and the inside of the vessel is set to a subcritical atmosphere while stirring.
22 MPa, 375 ° C. is a critical point, and a state at a temperature and pressure lower than this critical point but close to it is called a subcritical state. When the inside of the above-described sealed pressure resistant container is heated, the internal pressure increases as the temperature rises, and the water in the container becomes the above-mentioned subcritical water.

  Haematococcus alga is covered with a hard shell, but if it is brought into contact with subcritical water of about 3 to 8 MPa at 180 to 230 ° C., it is preferably in contact with subcritical water of about 5 to 6 MPa at 210 to 220 ° C. Then, the shell is crushed and astaxanthin is rapidly extracted. Since subcritical water was known to hydrolyze, there was a risk of astaxanthin being decomposed, but surprisingly it was hardly decomposed. However, the holding time at 180 ° C. or higher is preferably 5 to 15 minutes. If it is shorter than that, crushing of the shell and extraction of astaxanthin may be insufficient. On the other hand, if it is longer, astaxanthin may be altered by contact with high heat for a long time. It is.

If the final product is a soft capsule, astaxanthin will be dispersed and blended in the oil. After adding the oil to the container in advance, extraction using subcritical water as described above should be performed. You can also.
In addition, if the extraction process is not performed immediately after the above-described pretreatment is completed, it may be dried once.

Subcritical extraction can be performed at the laboratory level using commercially available subcritical extraction equipment.
In this extraction apparatus, an electric heater is attached to a hermetic pressure resistant container, and is configured to increase the pressure while increasing the temperature in the container.
Note that laboratory-level equipment is configured to increase the pressure while raising the temperature in the container, so it is necessary to increase the initial pressure by putting an inert gas such as liquid nitrogen in the container in advance. It is.

The test was conducted as follows.
<Subcritical water extraction (present invention)>
(culture)
The Haematococcus pluvialis N144 strain was cultured for 10 days at 25 ° C. using a KBM medium having the composition shown in Table 1 below under fluorescent light irradiation. On the 4th day, sodium acetate trihydrate (Wako Pure Chemical Industries, Ltd.) was added to a total concentration of 45 mM, and on the 6th day, iron sulfate (2) heptahydrate (Wako Pure) Yakuhin Co., Ltd.) was added to a total concentration of 450 μM.

(Extraction)
After cultivation, Haematococcus pluvialis N144 strain was collected and centrifuged at 6000 rpm to separate and recover Haematococcus algae. The recovered Haematococcus algae was freeze-dried using a commercially available freeze dryer (TAITEC VD-250F).
Thereafter, 1 g of dried Haematococcus algae (on a dry cell basis) was dissolved and dispersed in 100 ml of pure water, and the mixture was placed in the above-mentioned commercially available subcritical extraction apparatus for extraction treatment.

In Tables 2 and 3 above, the extraction temperature is described at one point. However, due to the temperature control accuracy limit of the apparatus used, the above extraction temperature is not always maintained at the temperature itself, but ± 3 ° C. The temperature fluctuates within.
The numerical value on the left side of the pressure column is the initial pressure value.
The holding time is a time during which the inside of the container is held at a constant temperature (within ± 3 ° C.) from when it reaches a predetermined extraction temperature until it is lowered. Immediately after the predetermined holding time, the electric heater is turned off and cooled.
The extraction time is the time from when the container is set and the electric heater is turned on to when the container is cooled to room temperature after being turned off and the container is taken out from the apparatus.

(Quantitative)
When the extraction temperature is 215 ° C., 1.5 ml of the extract, 1 ml of 0.05M Tris buffer (pH 7.0) and 100 μl of cholesterol esterase are placed in a brown test tube and reacted at 37 ° C. for 1 hour. By extracting with ether and distilling off the solvent, a sample in which astaxanthin was made free by deesterification was prepared.
When the extraction temperatures were 181 ° C. and 198 ° C., a free product could not be obtained. Therefore, 90 ml of the extract was extracted with 250 ml of acetone, filtered with suction, and then subjected to the above deesterification treatment to prepare a sample.
Next, the sample was dissolved in a mixed solvent of dichloromethane: methanol 1: 3 and subjected to HPLC analysis, and the amount of astaxanthin per 1 g of dry cell weight was quantified from the peak area near 480 nm which is the absorption maximum wavelength of astaxanthin. .
From this result, it can be considered that a critical extraction effect can be obtained when subcritical water having an extraction temperature of about 215 ° C. and a pressure of 5 to 6 MPa is brought into contact.

<Solvent extraction (conventional product for comparison)>
As a conventional product for comparison, hematococcus algae and glass beads were placed in a disposable tube, incubated, softened, and then removed and physically disrupted by the Bead-beater method. Thereafter, centrifugation was performed, the precipitate was washed with acetone, and the supernatant was collected. The collected supernatant was deesterified with cholesterol esterase, and the free form was quantified.

<Evaluation>
The quantitative values of astaxanthin, the yield, the color of the sample after extraction, and the number of cells after extraction are shown in Tables 4 and 5 below and FIGS. 1 and 2.
The yield was calculated as (quantitative value of the product of the present invention / quantitative value of the conventional product for comparison) × 100 (%).

The above results are also shown in FIG.
From the above results, it can be considered that a critically high extraction effect is obtained when the extraction temperature is around 215 ° C. When the holding time was 1 minute, the holding time at 180 ° C. or higher when the extraction temperature was 215 ° C. was in the range of 5 to 15 minutes. If the extraction time at high temperature becomes longer, it is thought that astaxanthin is destroyed by long-time exposure. If such a yield is obtained, it is suitable for commercial use.
In addition, since the color of the sample after extraction changes in proportion to the quantitative value, it seems that simple determination can be made as to whether or not extraction has been performed successfully.

  From the above results, it is considered that a critically high yield can always be obtained by using subcritical water set at an extraction temperature of 215 ° C. and a pressure of 5 to 6 MPa.

(Number of cells after extraction)
The number of cells per gram of dry cell weight after extraction was counted using a Toma hemocytometer.
FIG. 2 shows the result.
From this result, it is considered that when the extraction temperature is around 215 ° C., many cells, more precisely, the cell walls are crushed and astaxanthin therein is extracted.

  If the extraction method of the present invention is used to extract astaxanthin from Haematococcus algae, it is safe and free from the use of an organic solvent such as acetone, while ensuring a yield suitable for commercial use. High purity can be obtained without impurities.

It is a graph which shows the fixed_quantity | quantitative_assay result of each sample astaxanthin obtained in the Example of this invention. It is the cell number of some samples obtained in the Example of this invention.

Claims (2)

A method for extracting astaxanthin as an active ingredient present in the cell wall is a hard shell of Haematococcus pluvialis extract astaxanthin by contacting the cell wall subcritical water of 180 to 230 ° C. and 3~8MPa An active ingredient extraction method characterized by the above. 2. The method for extracting an active ingredient according to claim 1, wherein the extraction is performed by bringing Haematococcus prubiaris into contact with subcritical water at 210 to 220 [deg . ] C. and 5 to 6 MPa .

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