KR101879506B1 - Extracting method of beta-glucan from Phellinus baumii - Google Patents

Abstract

The present invention relates to a method for extracting and purifying beta-glucan from pine mushroom with high purity and content. Since the beta-glucan extract extracted by the extraction method of the present invention exhibits higher purity and content than those extracted by the conventional extraction method and has excellent anticancer activity, it can be used as a pharmaceutical composition or a functional food composition exhibiting anticancer activity .

Description

{Extracting method of beta-glucan from Phellinus baumii}

The present invention relates to a method for extracting beta-glucan from pine mushroom with high purity and content.

Polysaccharides of mushrooms include glucan, schizophyllan, lentinan, galactomannan, and krestin, among which beta-glucan is present in large numbers. It is known that beta-glucan inhibits the inflammatory reaction and activates immune cell function by nonspecific immune response to inhibit proliferation and recurrence of cancer cells.

Phellinus baumii ) belongs to the genus of pine scales and mud mushrooms and is known to grow mainly in the stem of mulberry and broad - leaved trees. It is said that all except for the surface of the gut, it is yellow, and among the about 220 species in the world, Phellinus baumii and P. linteus are generally cultivated and distributed.

Fruiting bodies of mushroom are thickened to innumerable cell layers. Betaglucan is in protein bound state and is surrounded by a large amount of non - soluble protein.

Conventionally, in extracting useful components such as beta-glucan from mushroom mycelium or fruiting body, extraction with hot water or extraction with alcohol has been attempted. However, this requires a proper pretreatment technique to effectively separate useful components because of low extraction efficiency. Various preprocessing and extraction methods such as ultrahigh pressure (Korean Patent Application No. 102014-0063083) and ultrasound (Korean Patent Application No. 10-2003-0028783) have been studied, but studies by hydrolysis using enzymes have been lacking.

Korean Patent Publication No. 10-2005-0100718 Korean Patent Publication No. 10-2010-0119922

It is an object of the present invention to provide a method for extracting beta-glucan having high purity and high content from a mushroom.

In order to accomplish the above object, the present invention provides a method for producing a mushroom powder, An enzyme pretreatment step of treating the mixture with a viscozyme; Adjusting the pH of the resulting product to 6-7, and then subjecting it to hot water extraction; And purifying the hydrothermal extract with ethanol. The present invention also provides a method for extracting beta-glucan from a mushroom.

In one embodiment of the present invention, the method comprises mixing a mushroom powder with water at a pH of 3.5-5.5 at a solvent ratio of 10-30% by weight; An enzyme pretreatment step of treating the mixture with a viscozyme; Adjusting the pH of the resulting product through the enzyme pretreatment step to 6-7; Adjusting the pH to 6-7, and subjecting the resultant to hot water extraction; And purifying the hot-water extract with ethanol 2-4 times.

In one embodiment of the present invention, the solvent of the enzyme pretreatment step is water having a pH of 4 and is mixed at a weight of 10 to 30 times the weight of the mushroom powder.

In one embodiment of the present invention, the enzyme pretreatment step is performed by adding 0.6 to 0.7% (v / v) of viscose and stirring at a temperature of 40 to 60 ° C for 6-8 hours.

In one embodiment of the present invention, the pretreatment step comprises mixing about 10-30 times of water having a pH of 3.5-5.5 with a mushroom powder pulverized to a size of 25 mesh or more, adding 0.6-0.7% (v / v) of Viscozyme And the mixture is stirred at a temperature of 40-60 ° C for 6-8 hours.

In one embodiment of the present invention, the pH of the solvent is adjusted to 0.01 M citric acid. Thus, it is possible to decompose the cell wall to facilitate the release of constituent substances in the cell. Stirring in the pretreatment step means stirring by mechanical method, and the pretreatment time may be 6-8 hours.

In one embodiment of the present invention, the step of adjusting the pH of the product after the enzyme pretreatment step to 6-7 is characterized by using 0.01 N NaOH.

In an embodiment of the present invention, the hot water extraction step is performed by hot water extraction at a temperature of 80-90 ° C for 24 hours. The hot water extraction time was increased up to 24 hours in the yield and beta glucan contents in proportion to the time increase rate.

In one embodiment of the present invention, the ethanol purification step may be performed after the hot water extraction is completed, The ethanol is repeated 2-3 times, filtered, and then the same amount of ethanol is added 1-2 times to purify.

In one embodiment of the present invention, the ethanol-extracting step comprises: shaking mixing 1-fold volume of ethanol to the extracted hot-water-extracted extract, leaving it at 4 ° C for 4-8 hours, adding 1-fold volume of ethanol, Deg.] C for 24 hours, then filtered, and then one volume of ethanol is added thereto, followed by shaking, followed by allowing to stand for 24 hours. In one embodiment of the present invention, the number of times the ethanol is added is suitably 2-4 times.

In one embodiment of the present invention, the extraction method comprises mixing the mushroom powder with water at a pH of 3.5-5.5 at a solvent ratio of 10-30%, adding Viscozyme of 0.6-0.7% (v / v) ° C. for 6-8 hours, adjusting the pH of the obtained product to pH 6-7, subjecting to hot water extraction at 80-90 ° C. for 24 hours, and then adding ethanol for 2-4 times for purification.

In one embodiment of the present invention, the extraction method may further include a step of concentrating and lyophilizing after completion of the ethanol purification.

The beta-glucan extract extracted by the extraction method of the present invention showed higher purity and content than that extracted by the conventional extraction method, and it was confirmed that it has excellent anticancer activity. Therefore, the extract of the present invention can be utilized as a pharmaceutical composition or a functional food composition exhibiting anticancer activity.

FIG. 1 illustrates a process for extracting beta-glucan from a mushroom according to an embodiment of the present invention.
FIG. 2 shows the results of measurement of cytotoxicity against B16F10 cells of the mushroom beta-glucan extracted by the extraction method of the present invention.
FIG. 3 shows the results of measurement of cytotoxicity of SK-MEL-5 cell of the mushroom beta-glucan extracted by the extraction method of the present invention.
FIG. 4 shows the results of measuring anticancer activity against B16F10 cells of the mushroom beta-glucan extracted by the extraction method of the present invention.
FIG. 5 shows the result of measuring the antitumor activity of SK-MEL-5 cell of the mushroom beta-glucan extracted by the extraction method of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

≪ Example 1 >

Extraction of beta-glucan from mushroom

Phellinus baumii powder crushed at 25 mesh or more was mixed with water at a pH of 3.5-5.5 with 0.01N HCl at a solvent ratio of 10-30 times (weight ratio), and 0.6-0.7% (v / v) Viscozyme The resulting mixture was stirred at a temperature of 40-60 ° C for 6-8 hours and the resulting solution was adjusted to pH 7 with 0.01N NaOH and then extracted with hot water at 80-90 ° C for 24 hours. Ethanol, and the mixture was concentrated and lyophilized to obtain beta-glucan extract.

The reason for limiting the number of times of ethanol purification to 2-4 times as described above is that, when refining 2-4 times, a lot of purified water having yellowish light is produced at the bottom of the extract.

The reason why the extraction time of the hot water extraction step was limited to 24 hours at a temperature of 80-90 DEG C is that the yield and beta-glucan content increased in proportion to the time increase rate until 24 hours as shown in Table 1 below, , The growth rate was decreased, and the optimum extraction time was determined to be 24 hours.

Extraction Yield and Beta Glucan Content of Mushroom According to Extraction Time Extraction time Yield (%) Beta glucan content (g / 100 g) 3 4.08 ± 0.07 1.28 + 0.02 6 4.33 ± 0.06 1.35 + 0.02 12 4.59 ± 0.09 1.38 + 0.03 24 5.84 ± 0.07 2.06 + 0.02 48 6.56 ± 0.24 2.83 ± 0.25

[Comparative Example 1]

The pretreatment process for treating the enzyme and the ethanol refining process were omitted and only the hot water extraction was performed in the same manner as in Example 1 as a whole.

[Comparative Example 2]

The pretreatment process for treating the enzyme was omitted, and the product was extracted with hot water and purified with about 2-4 times ethanol.

[Comparative Example 3]

As a whole, the same procedure as in Example 1 was carried out, except that the ethanol purification process was omitted, followed by pretreatment with an enzyme and hot water extraction.

The purity and content of beta-glucan in the obtained product obtained in Example 1 and Comparative Examples are shown in Table 2 below. The purity of β-glucan was calculated by subtracting the content of α-glucan from the total glucan content measured by Mushroom and Yeast-glucan assay kit (Megazyme, Wicklow, Ireland). The content of beta-glucan is expressed as the amount of beta-glucan per 100 g of the raw material (g).

division Beta Glucan Purity (%) Beta glucan content (g / 100 g) Example 1 59.05 3.3775 Comparative Example 1 18.96 0.9861 Comparative Example 2 26.64 0.2557 Comparative Example 3 11.15 1.8286

As can be seen from the above Table 2, the purity and content of beta-glucan of the extract of the present invention, which has undergone all of the above steps, is much higher than that of the case of omitting the enzyme pretreatment and / or ethanol purification process. Specifically, the beta-glucan extract extracted through the purification method of the present invention has about 3.1 times higher purity and about 3.4 times higher content than the extract obtained by the conventional hot water extraction method (Comparative Example 1) Compared with the extract obtained by the purification method (Comparative Example 2), the purity was about 2.2 times higher and the content was about 13.2 times higher.

< Example  2>

The Beta Glucan  Cytotoxicity measurement of extract

The cytotoxicity of the beta-glucan extract obtained by the method of Example 1 was compared with the cell viability of the control by MTT assay for B16F10 cell and SK-MEL-5 cell (FIGS.

The experimental method of MTT assay is as follows. B16F10 cells and SK-MEL-5 cells were seeded at 5.0 × 10 ⁴ cells / mL in a 96-well plate and cultured in a CO ² incubator at 37 ° C for 24 hours for attachment and stabilization. The concentration of the sample was diluted from 100 μg / mL, and the diluted sample was treated with cultured cells and cultured for another 24 hours. After removing the culture medium, 100 μL of 10% MTT (Thiazolylblue tetrazolium bromide) solution (5 mg / mL in phosphate-buffered saline (PBS)) was added and incubated again for 1 hour. After removing the culture medium, 100 μL of DMSO was added and the resulting formazan was dissolved. The absorbance was measured at 590 nm using a Victor3 1420 multiable counter (PerkinElmer Inc., Boston, Mass., USA). The results were compared with the control.

As a result, when the cell survival rate of the control was 100%, the cell survival rate was more than 80% at a concentration of 1 to 100 μg / mL in all the examples and comparative examples, ) And for SK-MEL-5, cell viability was 75% when treated with the concentration of 100 μg / mL for Comparative Example 3 (FIG. 3).

< Example  3>

The Beta Glucan  Anticancer activity of extract

As a result of the cytotoxicity test on the beta-glucan extract obtained by the method of the present invention (see Example 2 above), the anticancer activity was measured with an extract of 10 μg / mL in the concentration where no toxicity was observed. As a result, .

Anticancer activity was measured by in vitro wound healing assay on B16F10 cells and SK-MEL-5 cells (Fig. 4 and 5). Resveratrol (10 μg / mL) was used as a positive control.

The experimental method of in vitro wound healing assay is as follows. B16F10 cells and SK-MEL-5 cells were seeded at 3 × 10 ⁵ cells / well in a 6-well plate and cultured in a CO ² incubator at 37 ° C for 24 hours. Then, a yellow pipette tip was used to form a wound on the surface of the cell and the culture medium was replaced. The samples were treated at 3, 10, and 30 μg / mL, and incubated at 37 ° C in a CO ² incubator at 0, 6, and 12 hours intervals. The degree of healing was monitored with a microscope (ECLIPSE TE2000-U, Nikon, Japan). Wound healing rate was measured by measuring the distance of wound and the degree of inhibitory activity was compared with the control (Table 3).

As a result, as shown in Figs. 4 and 5, cancer cell proliferation inhibitory activity was higher in all treatments than control. In particular, the anticancer activity of Example 1 and Comparative Example 2 was superior to the positive control, and it was confirmed that the anticancer activity of Example 1 was the best in both of B16F10 cell and SK-MEL-5 cell.

division time Distance of wound (% of 0 hours) B16F10 SK-MEL-5 Control 0 100 ± 1.33 100.00 + - 4.00 6 55.94 + 2.04 59.55 1.54 12 20.87 ± 2.67 27.83 + - 0.77 Positive
control 0 100 ± 4.29 100.00 ± 1.33 6 89.15 + - 4.28 96.34 ± 0.77 12 65.42 + 2.04 70.33 + - 1.33 Example 1 0 100 ± 0.19 100.00 0.77 6 85.79 ± 1.34 84.53 + - 2.77 12 67.32 ± 0.2 77.94 + - 3.36 Comparative Example 1 0 100 ± 3.67 100.00 + 1.34 6 93.16 ± 3.36 96.70 + - 3.36 12 56.6 ± 8.15 58.75 + - 8.15 Comparative Example 2 0 100 ± 0.2 100.00 ± 0.58 6 75.18 + - 2.52 79.91 + 1.73 12 67.1 ± 1.15 72.05 ± 1.00 Comparative Example 3 0 100 ± 2.67 100.00 0.77 6 77.99 + 2.04 75.51 + - 3.36 12 47.48 ± 1.54 33.72 + 1.34

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (5)

Mixing 10 to 30 parts by weight of water having a pH of 3.5 to 5.5 in 1 part by weight of the ground mushroom powder;
An enzyme treatment step of treating the mixture with a viscozyme;
Adjusting the pH of the resulting product to 6-7;
Adjusting the pH to 6-7, and subjecting the resultant to hot water extraction; And
The method of extracting beta-glucan from a mushroom comprising the step of purifying the hot-water extract with ethanol 2-4 times. The method according to claim 1,
Wherein the solvent of the enzyme treatment step is water having a pH of 4. The method according to claim 1,
Wherein the enzyme pretreatment step is performed by adding 0.6 to 0.7% (v / v) viscose and stirring at a temperature of 40 to 60 DEG C for 6-8 hours. The method according to claim 1,
Wherein the hot water extraction step is hydrothermal extraction at a temperature of 80-90 DEG C for 24 hours. The method according to claim 1,
Wherein the ethanol is purified by repeating ethanol twice or more times by weight of the weight of the hot water extract after the hot water extraction, and then filtering the same by adding the same amount of ethanol 1-2 times.

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