KR20100032552A - METHODS OF PRODUCING OF PHELLINUS LINTEUS MYCELLIUM HAVING HIGH β-GLUCAN CONTENT - Google Patents

Abstract

PURPOSE: A method for producing Phellinus linteus mycelium having enhanced beta-glucan is provided to enhance polysaccharide content of mycelium in a short time. CONSTITUTION: A method for producing Phellinus linteus mycelium having enhanced beta glucan content comprises: a step of preparing a medium for culturing Phellinus linteus, which contains 5-15 weight% of medicinal herb extract, 0.5-1.5 weight% of rice bran, and 71.5-92 weight% of distilled water; a step of inoculating Phellinus linteus spawn to the medium and culturing; and a step of culturing at an air injection rate of 0.5-1.1vvm. The medicinal herb extract is isolated from Panax ginseng, Artemisia princeps var., Astragalus membranaceus, and morus bark.

Description

Method for producing Phellinus linteus mycellium having high β-glucan content}

The present invention relates to a method for producing situation mushroom mycelium with increased content of beta glucan and a method for producing situation mushroom mycelium powder.

Mushrooms are microorganisms belonging to basidiomycetes in the classification system of organisms, and have been used for food for a long time and have been introduced as folk traditional medicine for various diseases. There are about 15,000 mushrooms growing naturally on earth, but most of them can be used for food, except those that are toxic. These mushrooms are reported to contain a large amount of polysaccharide glucan and its derivatives, polysaccharides such as glucan is known to have an anticancer action.

Beta-glucan is a type of polysaccharide that activates the immune function of human normal cellular tissues, inhibits the proliferation and recurrence of cancer cells, and promotes the production of interleukin and interferon that activate immune cells. Active beta glucan enters the body where cancer cells are located and produces cytokines (Cytokine) to support the activity of immune cells, T cells and B cells, thereby activating the immune function of cellular tissues.

In addition, mushrooms are mostly used for food, so it is known to be less side effects, safer in terms of toxicity as well as to enhance the body's immune function and excellent anti-cancer effect. In particular, the situation mushroom is known to be excellent as an immune enhancer and anticancer agent compared to other mushrooms, but it is a perennial and does not reproduce well in nature. In addition, there is a problem of resource depletion and ecosystem destruction, so it is very difficult to obtain fruiting bodies. Situation mushrooms are widely distributed throughout the world, but the fruiting bodies are not well formed in nature.

Conventional situation mushroom artificial cultivation method is a variety of wood by-products, such as sawdust, a mixture of grains, there is a method of cultivating fruiting body by using herbal medicine by-products. However, the conventional method for artificially cultivating the situation mushroom fruiting body takes a long period of 3-6 months of cultivation is difficult to mass production and does not meet the demand. On the other hand, the cultivation of the situation mushroom mycelium has the advantage that not only shortens the time required for fruiting body formation, but also obtains a large amount of polysaccharides in a short time. However, the conventional situation mushroom mycelium culture method is difficult to mass production due to the difficult culture conditions. As such, although the situation mushroom is effective as an anticancer agent and an immune enhancer, its supply is limited and mass production and rapid production are not easy, and thus are not widely used.

As a prior art related to artificial mushroom culture, Korean Patent Registration No. 51056 (Registration date 1992.04.24) discloses a method of cultivating mycelial mushroom mycelium with a medium mixed with mulberry and mulberry sawdust rice bran. Mulberry or the old tree as the main raw material of the culture medium is not easily available, so it is difficult to manufacture a large amount of culture medium, and the culture period is 50 to 60 days, which takes a long time.

In addition, Korean Patent Registration No. 204984 (Registration Date 1999.03.31) discloses a method of using cereals as a culture medium component of Felinus linteus mycelium, but this method also requires 60 days of incubation time, which is not economical. have.

Korean Patent Publication No. 10-2007-0121073 (published Dec. 27, 2007) discloses a method of increasing the content of beta glucan in culture as a prior art related to artificial culture of mycelial mushrooms with high content of beta glucan. In addition, the incubation period is 10 to 25 days, the incubation time is long and only the content of beta glucan in the culture medium is increased, but there is no mention of a method for selectively purifying only beta glucan in the culture medium.

Therefore, the present inventors have made a thorough research to overcome the problems of the prior art, as a result of the reaction surface analysis method was able to establish the optimization conditions of the situation mushroom mycelium culture medium with high yield of polysaccharide within a short time using ultrafiltration It was confirmed that the beta glucan purity mushroom extract powder can be obtained.

Therefore, the main object of the present invention is to provide a method for producing a situation mushroom mycelium having a high content of polysaccharides as a main component of the mushroom mushroom mycelium extract.

The present invention also provides a method for producing a situation mushroom mycelium powder having a high content of beta glucan.

In order to achieve the above object, the present invention includes (a) herbal medicine extract, rice bran and distilled water prepared by extracting hot water extract of a herbal mixture consisting of ginseng, Astragalus, larvae (mugwort), Angelica, Sangbaekpi (Mulberry bark) and Ogapi Preparing a situation mushroom culture medium; (b) inoculating the culture medium spawn seed in the medium; And (c) provides a method for producing a situation mushroom mycelium with increased content of beta glucan, characterized in that it comprises the step of culturing with an air injection amount of 0.5-1.1 vvm in the culture. In addition, (a) adding the ionized water to the situation mushroom mycelium culture medium in which the content of the beta glucan produced by the method is increased and extracted by heating; (b) filtering by adding 2-5% by weight of perlite of the mycelium culture medium to the extract; (c) ultrafiltration of the filtrate to remove material of low molecular weight and concentrating in vacuo; (d) precipitating the concentrate with ethanol; (e) centrifuging and recovering the precipitate; And (f) provides a method for producing a situation mushroom mycelium powder with increased content of beta glucan, characterized in that it comprises the step of vacuum drying the recovered product.

The first aspect of the present invention (a) a situation mushroom comprising a medicinal herb extract, rice bran and distilled water prepared by extracting the herbal medicine mixture consisting of ginseng, Astragalus, larvae (mugwort), Angelica, Sangbaekpi (Mulberry bark) and Ogapi Preparing a culture medium; (b) inoculating the culture medium spawn seed in the medium; And (c) provides a method for producing a situation mushroom mycelium with increased content of beta glucan, characterized in that it comprises the step of culturing with an air injection amount of 0.5-1.1 vvm in the culture.

Specifically, the situation mushroom culture medium contains 5-15% by weight of the medicinal herb extract, 0.5-1.5% by weight of rice bran and 71.5-92% by weight of distilled water, and 2-10% by weight of anhydrous crystalline glucose Potato starch is characterized in that it further comprises 0.5-2% by weight.

A second aspect of the present invention comprises the steps of adding the ionized water to the situation mushroom mycelium culture medium in which the content of the beta glucan produced by the method is increased and extracted; (b) filtering by adding 2-5% by weight of perlite of the mycelium culture medium to the extract; (c) ultrafiltration of the filtrate to remove material of low molecular weight and concentrating in vacuo; (d) precipitating the concentrate with ethanol; (e) centrifuging and recovering the precipitate; And (f) provides a method for producing a situation mushroom mycelium powder with increased content of beta glucan, characterized in that it comprises the step of vacuum drying the recovered product. More specifically, the amount of the ionized water is 0.5 to 2 times the mycelium culture medium, the heating temperature is 95 ℃ or more and 100 ℃ or less, the heating time is 1 to 5 hours, low molecular weight material removed during the ultrafiltration Is a low molecular weight substance having a molecular weight of 20,000 or less, and the vacuum concentration is characterized in that it is concentrated to 20 to 25 Brix.

The ultrafiltration is a method of separating and concentrating a high molecular material and a low molecular material using a semipermeable membrane, and is a method of separating, purifying and concentrating colloidal materials that are difficult to separate by general filtration. The molecular weight can be separated up to 500-300,000, and the semi-permeable membrane used here includes a collodion membrane, a cellophane membrane, and a gelatin membrane. Ultrafiltration used in the present invention is characterized by the removal of low molecular weight substances with molecular weights up to 20,000.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are merely to illustrate the present invention, the contents of the present invention

It is not limited to an example.

< Example  1> Optimization by Response Surface Methodology

According to the central composit design ( CCD ), the independent variables of the total sugar content and mycelium content in the culture medium were the herbal extract ratio, rice bran content, and air intake during the culture as shown in Table 1.

The herbal extracts were prepared by adding 200 g of ginseng, 200 g of Astragalus, 200 g of Aegye (mugwort), 200 g, Angelica 200 g, 100 g of Morus bark (mulberry bark) and 100 g of Ogapi in 2 L of purified water and then bathing them at 110 ° C. for 2 hours.

The range of each independent variable was determined through preliminary experiments.

<Table 1> Independent variables according to central composition plan

range encoding -1.68 -One 0 One 1.68 Herbal Medicine Extract (%) 1.6 5 10 15 18.4 Rice bran (%) 0.16 0.5 One 1.5 1.84 Air injection amount (vvm) 0.3 0.5 0.8 1.1 1.3

Seventeen culture conditions based on three independent variables were planned according to the Central Synthesis Planning Method. The cultures were cultured for 7 days according to each condition. The total sugar content and the culture medium were filtered, alcohol was added to the filtrate to precipitate, and the precipitate was recovered and dried. The yield of the polysaccharide is shown. As shown in Table 2, the total sugar content of the culture medium was 38.45-74.16% and the yield of polysaccharide was 0.42-0.81%, as shown in Table 2.

<Table 2> Experimental results according to the culture conditions by each independent variable

Independent variable Dependent variable Herbal Medicine Extract (%) Rice bran (%) Air injection amount (vvm) Total sugar content (mg / mL) Polysaccharide (%) One -One -One -One 49.54 0.43 2 One -One -One 46.27 0.56 3 -One One -One 51.25 0.52 4 One One -One 48.71 0.53 5 -One -One One 55.78 0.66 6 One -One One 53.67 0.61 7 -One One One 60.42 0.64 8 One One One 57.29 0.57 9 -1.68 0 0 53.11 0.42 10 1.68 0 0 49.25 0.56 11 0 -1.68 0 60.82 0.71 12 0 1.68 0 69.47 0.72 13 0 0 -1.68 38.45 0.43 14 0 0 1.68 53.98 0.66 15 0 0 0 74.16 0.79 16 0 0 0 71.54 0.78 17 0 0 0 72.68 0.81

Independent variable (herb medicine extract and rice bran content ratio, incubation of the air injection amount) of the reaction surface regression equation determined in the response surface regression equation as shown in Table 3, the coefficient (R ²⁾ to the total sugar content and the polysaccharide yield are each 0.994 in the culture medium and A response model of 0.970 was appropriate and statistically significant (P-value <0.01).

<Table 3> Reaction equation of response surface of independent variables

Dependent variable Response surface regression formula Coefficient of determination (R ² ) P-value % Per total _{Y 1 = 72.8052 - 1.2845X 1 +} 1.9739X 2 + 4.2109X 3 - 7.6825X 1 2 - 2.7451X 2 2 - 9.4379X 3 2 - 0.0363X 1 X 2 + 0.0712X 1 X 3 + 0.5138X 2 X 3 0.994 0.001 Polysaccharide (%) Y ₂ = 0.7937 + 0.0187X ₁ + 0.0012X ₂ + 0.0605X ₃ - 0.1085X _{1 ²} - 0.0289X ^{2 2} -0.0890X ₃ ² -0.0175X ₁ X _{2 - 0.0325X 1 X 3 - 0.0150X 2} X 3 0.970 0.001

The response surface diagram of each independent variable for the total sugar content and the polysaccharide content in the culture is shown in FIG . 1 . Glucose content in the culture medium tended to increase as the air injection amount neared the center value of 0, and then decreased again after 0. In addition, the glucose content increased as the amount of the medicinal herb extract added up to 10%, but decreased again when added more than 10%. Similarly to the change in glucose content, the polysaccharide yield increased as the closer to the center value of each independent variable, and then decreased again after the center value.

The conditions for separately optimizing the glucose content and the yield of polysaccharide in the culture and the conditions for the optimization of multiple reactions of the two dependent variables are shown in Table 4. Optimization conditions for increasing the glucose content in the culture and polysaccharide yield were similar. It was found that the content of polysaccharide increases as the glucose content in the culture solution increases.

<Table 4> Conditions for Optimizing Multiple Reactions

Dependent variable Independent variable Optimal independent variable Dependent variable encoding Actual value Expected value Goal setting value  % Per total Herbal Medicine Extract (%) -0.07 9.65 73.71 Maximum value Rice bran (%) 0.36 1.18 Air injection amount (vvm) 0.27 0.88  Polysaccharide yield (%) Herbal Medicine Extract (%) -0.03 9.85 0.80 Maximum value Rice bran (%) 0.08 1.04 Air injection amount (vvm) 0.39 0.92  Multiple response optimization Herbal Medicine Extract (%) 0.01 10.05 Rice bran (%) 0.36 1.18 Air injection amount (vvm) 0.25 0.88

Minitab, one of the statistical programs, was used to optimize the multiple reactions to simultaneously satisfy the optimization of total sugar content and mycelium content in the culture medium. Multiple reaction optimization conditions were established for herbal extracts, rice bran content, and air intakes of 10.05%, 1.18%, and 0.88vvm, respectively.

< Example  2> Beta Glucan  Culture of mycelial mycelium with high content

Media and culture conditions for culturing the situation mushroom mycelium having high beta glucan content are shown in Table 5 and Table 6.

The cultures were incubated for 7 days under the conditions of 0.9vvm of air injection using a medium containing 1.18% of rice bran and 10.05% of medicinal herb extracts. Immediately after the end of the culture, the glucose content and the yield of polysaccharide precipitates in the culture medium were 71.84 mg / mL and 0.77%, respectively, slightly lower than expected (Table 7).

<Table 5> Situation mushroom mycelium culture medium composition with high content of beta glucan

Culture composition subject ratio(%) Anhydrous Glucose 5 Potato starch One Rice bran 1.18 Chinese herbal medicine extract 10.05

<Table 6> Culture conditions of mycelial mycelium with high content of beta glucan

Culture condition Culture 15 L Incubation temperature 28 ℃ Stirring speed 150 rpm Incubation time 7 day Spawn volume 1.0 L

<Table 7> Glucose Content and Polysaccharide Sediment Yield of Cultures after Culture

Dependent variable Expected value Actual value % Per total 73.65 71.84 Sediment amount (%) 0.80 0.77

< Example  3> Beta Glucan  Preparation of Extracted Powder Mycelium Mycelium with High Content

Figure 2 shows a process for producing a situation mushroom mycelium extract powder having a high beta glucan content. After the culture was completed, 1 times more ionized water was added to the culture solution, followed by extraction for 1 hour at 95 ° C., followed by 4% of the culture solution and the perlite corresponding to 4% of the culture solution.

After filtration, the culture medium was removed using ultrafiltration to remove low molecular weight substances with molecular weight less than 20,000, concentrated to 25 briquettes using a vacuum concentrator, and repeatedly precipitated polymer materials until alcohol purity reached 92% using alcohol. It was recovered and dried in vacuo.

Table 8 shows the beta glucan content and filtrate of ultra-low molecular weight 20,000 or less after the filtrate filtered with a filter glass and precipitated with alcohol and dried in vacuo, followed by vacuum drying. After removing the low molecular weight of the precipitate by using alcohol and vacuum dried after measuring the content of beta glucan content of the mycelia mushroom mycelia extracts.

It is believed that the purity of beta glucan was increased by removing medium components and low molecular materials other than beta glucan in the culture solution through ultrafiltration.

<Table 8> Glucan Content of Situary Mushroom Mycelia Extract

Total Glucan Alpha Glucan Beta Glucan Culture Alcohol Precipitate 60.53 9.07 51.46 Alcohol precipitate after ultrafiltration 92.38 6.73 85.65

< Experimental Example  1> Measurement method of mycelium extract powder

Beta-glucan, an active ingredient of Example 3, was measured using a megazyme β-glucan assay kit (Megazyme β-glucan assay kit; Megazyme International Ireland Ltd., Ireland), and then the total glucan and sugar content other than glucan were determined. It was obtained by subtracting the values of sugar contents other than alpha-glucan and glucan.

To 100 mg of mycelial powder of 0.5 mm or less, 1.5 mL of hydrochloric acid (37%, v / v) was added, and then the contents were mixed well, followed by mixing at 30 ° C. for 60 minutes at 15 minutes intervals. In order to decompose and remove starch and protein, 10 mL of distilled water was added thereto and then left in boiling water (100 ° C.) for 2 hours.

The contents were then cooled to room temperature, adjusted to pH by addition of 2N KOH, and diluted with 200 mM sodium acetate buffer (pH 5.0). Diluted contents were centrifuged to obtain supernatant, and then, 100 μL of the supernatant was extracted with axole, 3-β-glucanase (exo1,3-β-Glucanase; 100 U / mL) + β-glucosidase (β-Glucosidase; 20). unit / mL) solution was added to each 100uL and left for 1 hour at 40 ℃, 3mL of glucose determination reagent was added and then left at 40 ℃ 20 minutes. Thereafter, absorbance was measured at 510 nm to obtain a total glucan and sugar content values other than glucan. In addition, sugar content values other than alpha-glucan and glucan were added to 2 mg KOH 2mL in 100mg of mycelium powder, and stirred for 20 minutes in an ice-filled water bath using a magnetic bar, followed by 8mL of 1.2M sodium acetate buffer (pH 3.8). Added.

In addition, after adding 200 uL of a solution of amyloglucosidase (1630 unit / mL) + Invertase (500 unit / mL), the mixture was left at 40 ° C for 30 minutes, and 200 nM sodium acetate buffer (sodium acetate buffer) ; pH 5.0). Then, 3 mL of glucose determination reagent was added and left at 40 ° C. for 20 minutes. The absorbance was then measured at 510 nm to obtain sugar content values other than alpha-glucan and glucan.

Beta-glucan content was obtained by the following equation.

Beta Glucan = (Sugar other than Glucan + Glucan)-(Sugar other than Alpha Glucan + Glucan)

 As described above, the inventors of the present invention have established the optimum conditions for cultivating the situation mushroom mycelium having a high content of beta glucan in a short time by using the reaction surface analysis method used in the food manufacturing process or new product development. In addition, the culture medium was subjected to ultrafiltration followed by alcohol precipitation to increase the content of beta glucan of the situation mushroom mycelium extract.

Figure 1 shows the glucose content and the polysaccharide content change in the culture medium according to the change of the independent variable in the culture of mycelium mushroom mycelium.

Figure 2 shows a method for producing a situation mushroom mycelium extract powder with increased beta-glucan content after incubating the situation mushroom mycelium under optimal conditions.

Claims (4)

(a) preparing a situation mushroom culture medium comprising a medicinal herb extract, rice bran, and distilled water prepared by hot water extraction of a herbal mixture consisting of ginseng, Astragalus, Aeroba, ragweed, Sangbaekpi (Mulberry bark) and Ogapi; (b) inoculating the culture medium spawn seed in the medium; And (C) the method of producing a situation mushroom mycelium with increased content of beta glucan, characterized in that it comprises the step of culturing with an air injection amount of 0.5-1.1 vvm in the culture. The method according to claim 1, wherein the mushroom culture medium comprises 5-15% by weight of the medicinal herb extract, 0.5-1.5% by weight of rice bran and 71.5-92% by weight of distilled water compared to the total medium composition, in addition to 2-10 anhydrous glucose Method for producing a situation mushroom mycelium with increased content of beta glucan, characterized in that it further comprises a weight% and 0.5-2% by weight potato starch. (a) adding ionized water to the situation mushroom mycelium culture medium in which the content of beta glucan produced by the method of any one of claims 1 to 2 is increased, and extracting it by heating; (b) filtering by adding 2-5% by weight of perlite of the mycelium culture medium to the extract; (c) ultrafiltration of the filtrate to remove material of low molecular weight and concentrating in vacuo; (d) precipitating the concentrate with ethanol; (e) centrifuging and recovering the precipitate; And (f) a method of producing a situation mushroom mycelium powder in which the content of beta glucan is increased, comprising the step of vacuum drying the recovered product. According to claim 3, wherein the amount of the ionized water is 0.5 to 2 times the mycelium culture medium, the heating temperature is 95 ℃ or more and 100 ℃ or less, the heating time is 1 to 5 hours, the low molecular weight removed during the ultrafiltration The substance is a low molecular weight substance having a molecular weight of 20,000 or less, the vacuum concentration is a method of producing a situation mushroom mycelium powder with an increase in the content of beta glucan, characterized in that concentrated to 20 to 25 Brix.

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