KR20160149696A - Rapid production method of fermented Rhus vernicifula containing high level functional compounds and removing urushiols - Google Patents

Abstract

The present invention relates to a lacquer tree (rhus vernicifula) fermentation process for effective urushiol removal from a lacquer tree and functional element loss minimization. The fermented lacquer preparation process according to the present invention includes inoculum preparation using fomitella fraxinea, spawn preparation based on inoculum culture, and a lacquer tree fermentation process based on inoculation of the prepared spawn. In the above-described process, fermentation can be carried out within a significantly shorter period of time than in existing fermentation processes while functional elements are effectively maintained. Accordingly, the present invention can be useful in related industries.

Description

Technical Field [0001] The present invention relates to a method for preparing a fermented lacquer for removing urushiol and preserving functional ingredients,

The present invention relates to a Rhus The present invention relates to a method for producing a property of fermented lacquer which rapidly removes urushiol and preserves a functional ingredient so as to use vernicifula as a material of a pharmaceutical or the like.

Rhus ( Rhus ) belonging to the family Anacardiaceae vernicifula ) is known to exist in temperate regions, with about 600 species worldwide. Currently, Rhus ( Rhus) verniciflua Stokes) are distributed in Northeast Asia including China and Japan. Domestic plants in the lacquer tree include lacquer tree, lacquer tree, diluted tree, fenugreek, and lacquer tree. Rhus sap is 40 to 60% urushiol, 20 to 25% moisture, 6.5 to 10% vegetable rubber, 1.4 to 2.5% insoluble glycoprotein, and 0.1 to 1.0% laccase Consists of.

The major pharmacological actions of lacquer are anticancer, antioxidant, hangover, etc. (Korean Forest Service, National Institute of Forest Breeding, Chengchi Research Team, Apr.22,297, Food Science, vol 31, 238-245), gastrointestinal diseases, heart disease, arthritis, Diabetes, paralysis, arthritis, and chronic fatigue are known to have efficacy (herbal gangmok, herb medicine). Recently, due to various physiological activity effects of lacquer, it has attracted attention as a health food and pharmaceutical material.

However, in spite of these various physiological activity effects of lacquer, it is known that urushiol contained in Rhus verniciflua induces allergy such as causing rash in skin, inducing itching, and causing blisters. It is becoming a stumbling block. Lacquer and lacquer duck, which have been used for a long time, have been difficult to commercialize due to the risk of allergies, especially in dermatology hospitals.

Urushiol is one of the phenol derivatives, and its chemical structure is a lipid-soluble compound in which the side chain R groups are connected in a linear chain with 15 to 17 carbon atoms. The urushiol is mixed with thirteen mixtures. As a main component of the Rhus verniciflua, . Therefore, in order to be commercialized through the utilization of the pharmacological efficacy of Rhus verniciflua, it is necessary to remove or reduce urushiol from Rhus verniciflua first. However, the conventional technique of lacquer tree is focused on elimination of urushiol, The improvement of the fermentation period and the maintenance of effective functional ingredients have not been considered.

There are several physiologically active substances in Rhus verniciflua, one of which is pentagalloyl glucose (PGG). PGG is a compound in which five molecules of gallic acid and one molecule of glucose are bound in an ester form and is found in some plants such as peony and plant, pomegranate, evening primrose, lacquer plant and green tea. In addition, the results reported in the literature so far suggest that the PGG component is useful as an antioxidant, an antibacterial and antiviral agent, an inhibitor of Helicobacter pylori growth, an improvement in gastric function, an antidiabetic agent, an antimutagenic agent, It has been found out that it has various physiological activities such as anti-inflammatory, anti-allergic, blood cholesterol-lowering, skin whitening and skin aging inhibition effects. Thus, possibility of use as food additives, cosmetic raw materials and specific medicines has been proposed, Is a compound that is currently known to be commercially available.

However, since the existing method of producing or potentiating the lacquer tree has the limitation that the PGG component is inevitably lost, it is impossible to preserve PGG, a useful physiologically active substance of Rhus verniciflua.

Meanwhile, longevity mushroom ( Fomitella fraxinea ) is a fungus belonging to the mushroom family and is also known as Akashi Mori mushroom and has been used for a long time as a private medicine. In North Korea, it has been reported that hepatitis injections are being developed and used to treat hepatitis using mushrooms and longevity mushrooms. Medicinal mushroom contains 3.5% of steroid compound, 1.5% of flavonoid, 0.2% of glycoside, 0.8% of coumarin and 0.5% of alkaloid. In particular, longevity mushrooms have been shown to be effective against various immune diseases including chronic hepatitis B, cancer, nephritis and arthritis, and have been found to have a strong antioxidant effect. Also, the method of fermenting food materials by inoculating the mycelium of long- Research is underway, but there is no known method for producing fermented lacquer using longevity mushroom.

The inventors of the present invention have found that, in the fermentation process for removing urushiol of Rhus verniciflua, the fermentation period is remarkably shorter than that of the conventional process, and the content of PGG, which is a functional substance, is effectively preserved through the process of the present invention using longevity mushrooms Thereby completing the present invention.

It is an object of the present invention to provide a process for producing fermented lacquer wherein urushiol is removed from a lacquer in a short period of time and at the same time functional ingredients are effectively preserved.

In order to achieve the above object,

1) culturing a mushroom strain to produce an inoculum;

2) The inoculation source of step 1) above was divided into Rhus ( Rhus vernicifula ), and culturing the seeds to produce seeds ; And

3) Inoculating the seeds of step 2) into Rhus verniciflua, followed by cultivation or fermentation.

In addition, the present invention provides a composition of a fungicidal composition for producing fermented lacquer comprising a mushroom strain culture liquid and Rhus verniciflua as an active ingredient.

The fermented lacquer with urushiol removed can be produced in a shorter period of time than the conventional process, and the functional ingredient, pentagalloyl glucose (PGG) can be effectively retained by the method of the present invention, INDUSTRIAL APPLICABILITY The present invention can be usefully used for the production of raw lacquer materials for the manufacture of pharmaceuticals.

1 is a diagram showing the molecular weight of pentagalloyl glucose as a single component by performing liquid chromatography / mass spectrometry (LC / MS).
2 is a graph showing the results of ¹ H-NMR measurement of pentagaloylglucose.
Fig. 3 is a chart showing the results of ¹³ C-NMR measurement of pentagaloylglucose. Fig.
Fig. 4 is a diagram showing the chemical structure of pentagaloylglucose. Fig.
FIG. 5 is a graph showing changes in urushiol content with time of fermentation. FIG.
6 is a diagram showing an overall process and sequence of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention

In order to achieve the above object,

1) culturing a mushroom strain to produce an inoculum;

2) The inoculation source of step 1) above was divided into Rhus ( Rhus vernicifula ), and culturing the seeds to produce seeds ; And

3) Inoculating the seeds of step 2) into Rhus verniciflua, followed by cultivation or fermentation.

The mushroom strain of step 1) is preferably, but not limited to, a Fomitella fraxinea strain.

The above mushroom strain is preferably deposited with the deposit number KACC93226P, but is not limited thereto.

In addition, the lacquer of step 2) and step 3) can be used in all areas such as lacquer, outer lumber, roots, stem parts, core materials and shims, and can be used without distinction such as roots, Do not.

The inoculation of step 2) may be performed by inoculating 2 to 10 parts by weight of the inoculum with respect to the total weight of the lacquer of step 2), but the present invention is not limited thereto, and the inoculation of step 3) But it is not limited thereto.

In addition, the culture or fermentation in step 3) is preferably, but not always, cultivated or fermented for 7 to 14 days after inoculation of seedlings into Rhus verniciflua.

In addition, the method preferably removes urushiol from the Rhus verniciflua component but inhibits the decrease of pentagalloyl glucose, but is not limited thereto.

In addition, the present invention provides a composition of a fungi for producing fermented lacquer comprising a mushroom strain culture solution and Rhus verniciflua as an active ingredient.

The composition of the fungicide is preferably prepared through the steps 1) and 2), but is not limited thereto. In addition, the composition of the present invention is preferably, but not limited, to remove urushiol from the Rhus verniciflua component and inhibit reduction of the pentagaloylglucose.

According to a preferred embodiment of the present invention, the present inventors manufactured an inoculum using a long-lived mushroom and cultivated it to produce a seed. The seed was inoculated on a lacquer tree, cultured and fermented to produce fermented lacquer, The process was summarized to complete the method of producing the properties (see FIG. 6).

According to the embodiment of the present invention, the fermented lacquer can be manufactured through the fermentation period of 7 to 14 days through the manufacturing process of the present invention, and as shown in FIG. 5, urushiol, which is an allergen- (Fig. 5). As shown in Fig. 5, it can be effectively removed.

In addition, through remarkably shortening the fermentation period, loss of pentagalloyl glucose (PGG), which is a functional ingredient of fermented lacquer identified and isolated as shown in Figs. 1 to 4, is minimized, (See Figs. 1 to 4 and Table 2).

If the fermentation period is less than 7 days after seedling inoculation, the removal of urushiol may be inadequate and more than 50% of the initial content may remain. If the fermentation period exceeds 14 days, the content of the functional ingredient, pentagaloylglucose It is difficult to preserve the functional ingredient of the fermented lacquer. Therefore, it was confirmed that the optimum fermentation period based on the fermented lacquer production process of the present invention is 7 to 14 days after seedling inoculation.

Hereinafter, embodiments and experimental examples of the present invention will be described in detail.

However, the following Examples and Experimental Examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention by the following Examples and Experimental Examples.

Example 1 Preparation of fermented lacquer according to the present invention

<1-1> Inoculation site preparation process

Longevity mushroom ( Fomitella fraxinea ) were prepared and activated by subculturing two or more times with potato dextrose agar (PDA). The activated mycelium of long-lived mushrooms was cut into 5 × 5 mm size and inoculated on sterilized malt culture and cultured with shaking at 100 rpm at 25 ° C. Then, the microorganisms reaching the logarithmic growth phase were sampled and sterilized aseptically at 1000 rpm for 3 minutes in a homogenizer (Omnimixer, USA) to obtain a liquid seed inoculum.

&Lt; 1-2 >

In the embodiment of the present invention, 2 kg of lacquer of the lacquer trunk is prepared, dried, pulverized to a thickness of about 3 to 4 mm using a sawdust maker, and added with water or dried to obtain 70 % Water content, and then packed in a PP bottle for culture of 600 cc mushroom, sterilized at 121 ° C and 1.2 atm for 1 hour, and then cooled in a clean room. Then, the seedlings were prepared by inoculating 10 mL of Lycopersicum L. inoculum prepared in Example <1-1> and culturing at 25 DEG C for 14 days.

<1-3> Fermentation lacquer manufacturing process

In the example of the present invention, 10 kg of lacquer tree was prepared, and the skin was picked, dried, ground to a size of 3 to 4 mm, adjusted to a moisture content of 70%, and then cultured in a 2 kg mushroom culture The bag was filled with 1.5 kg of the moisture-adjusted silkworm, sterilized at 121 캜 and 1.2 atm for 1 hour, and then cooled in a clean room. Then, 500 g of the seeds cultured in Example <1-2> were pulverized with a seed-mill and then mixed with 1.5 kg of the sterilized lacquer. Followed by incubation and fermentation at 25 DEG C for 7 to 14 days (FIG. 6).

Example 2 Isolation and Identification of Pentagalloyl Glucose in Fermented Lacquer

<2-1> Separation of Pentaerythritol Glucose from Fermented Lacquer

To 500 g of the fermented lacquer obtained in Example <1-3>, 1.5 L of methanol was added, and the mixture was heated in a 70 ° C water bath for 1 hour and then filtered to obtain a supernatant. The residue was further subjected to the above-mentioned procedure two more times, and the obtained supernatant was concentrated under reduced pressure to obtain an extract. 500 mL of distilled water was added to the extract to dissolve it. Then, the extract was adsorbed on a Diaion HP-20 filled column (5 x 40 cm), and a mixed solution of n-hexane and diethyl ether (1: 1 ), Ethyl acetate and methanol, respectively, and the fractions were concentrated under reduced pressure. Among them, ethyl acetate fraction containing free form of flavonoid was subjected to silica gel column chromatography to separate pentagalloylglucose into single components.

<2-2> Structural analysis of isolated pentagaloylglucose

LC / MS (HP1100 series, Agilent, Santa Clara, Calif.) Was performed to analyze the chemical structure of the pentagaloylglucose isolated through Example <2-1> ¹ H-NMR and ¹³ C-NMR were performed using a nuclear magnetic resonance spectrometer (Avance 400 MHz, Bruker, Germany).

As a result, it was found that the molecular ion peak was detected as m / z 939.2 [M-1] ^- and the molecular weight was 940 (Fig. 1). As a result of spectroscopic measurement, Was found to be pentagalloyl glucose (Fig. 2, Fig. 3 and Table 1).

The ¹³ C-NMR chemical shifts (methanol-d4, 600 and 150 MHz) δ _C 隆_H δ _C 隆_H Glucose Galloyl-3 One 93.93 6.24 (1H, d, J = 8.40) One 120.33 6.97 (2H, s) 2 72.30 5.59 (1H, t, J = 9.00) 2 110.52 - 3 74.22 5.91 (1H, d, J = 9.60) 3 146.55 - 4 69.91 5.63 (1H, t, J = 9.60) 4 140.46 - 5 74.53 4.43 (1 H, m) 5 146.55 - 6 63.24 4.51 (1H, m) 6 110.52 - 7 167.13 - Galloyl-1 Galloyl-4 One 119.80 6.91 (2H, s) One 120.46 7.05 (2H, s) 2 110.46 - 2 110.58 - 3 146.40 - 3 146.59 - 4 140.15 - 4 140.51 - 5 146.40 - 5 146.59 - 6 110.46 - 6 110.58 - 7 166.34 - 7 167.40 - Galloyl-2 Galloyl-5 One 120.3 6.95 (2 H, s) One 121.15 7.12 (2 H, s) 2 110.50 - 2 110.74 - 3 146.50 - 3 146.68 - 4 140.26 - 4 140.94 - 5 146.50 - 5 146.68 - 6 110.50 - 6 110.74 - 7 157.04 - 7 168.04 -

Experimental Example 1 Confirmation of urushiol removal of fermented lacquer

In order to confirm the degree of elimination of urushiol present in the lacquer tree, the sample was extracted with methanol from the fermented lacquer of Example <1-3>, and then subjected to high performance liquid chromatography (HPLC) analysis , A 600 controller consisting of Water's system (Milford, MA, USA), 600 pump, 717 plus autosampler and 486 tunable UV detector. Halo column (4.6 mm x 100 mm, 2.7 μm) was used for the column and mobile phase was analyzed in isocratic mode using 85% methanol. Analysis was carried out at room temperature. The flow rate was 0.5 mL / min, the injection volume was 20 μL, and the detector wavelength was 203 nm.

As a result, as shown in Fig. 5, three kinds of common urushiol components present in Rhus verniciflua showed a gradual decrease until 5th day after the start of fermentation of Example <1-3>, and a sharp decrease between 5 and 10 days Respectively.

As shown in Fig. 5, the uriciol content of the control (non-fermented lacquer) was 15 chain-linked carbon bonded side chains to the third carbon of the catechol skeleton (Fig. 7) Urushiol C15: 3 is 678 ug / g, uriciol C15: 1 with one double bond in 15 straight-chain bonded carbons bonded side chains is 560 ug / g, and 15 straight-chain bonded carbons bonded with side chains The uriciol contents of C15: 3, C15: 2, C15: 2 and C15: 2 of the fermented lacquer were higher than those of the control. 1 was degraded by about 91%. From 13 days after fermentation, it was confirmed that more than 90% of urushiol compounds were removed. C15: 1, C15: 2, and C15: 3 can be prevented from allergic reactions. The above results show that the present invention can effectively reduce all three components of urushiol in a short period of time, and can be used as a raw material for food.

< Experimental Example  2> Fermented lacquer Pentagalloyl Glucose ( pentagalloyl  glucose; PGG ) Ingredient preservation confirmation

During the same fermentation period as in <Experiment 1>, the content of PGG as a functional ingredient was analyzed. That is, each sample was extracted with methanol from the fermented lacquer of Example <1-3>, and analyzed by HPLC under the following conditions. The column was analyzed with gradient mode using water (A) containing 0.1% formic acid and water (B) containing 90% acetonitrile, and YMC-Pak C ₁₈ (4.6 mm x 25 cm) UV (280 nm) was used.

As a result, as shown in Table 2, the PGG content of the fermented lacquer of Example <1-3> after 13 days of fermentation with urushiol removed for use as food was 602.90 mg / 100 g wet base and contained in the raw material before fermentation But the content of PGG was 44.86 mg / 100g wet base in 30 days culture of lacquer according to the conventional method, and it was confirmed that the content was 13.4 times higher than that of the conventional method .

Agricultural Biotechnology Research Institute KACC93226 20150406

Claims (10)

1) culturing a mushroom strain to produce an inoculum;
2) The inoculation source of step 1) above was divided into Rhus ( Rhus vernicifula ), and culturing the seeds to produce seeds ; And
3) Inoculating the Rhus verniciflua seeds of step 2) and culturing or fermenting the fermented lacquer property.
The method according to claim 1, wherein the mushroom strain of step 1) is a Fomitella fraxinea strain.
[Claim 3] The method according to claim 2, wherein the long-lived mushroom strain is deposited with the deposit number KACC93226P.
[2] The method according to claim 1, wherein the inoculation of step 2) comprises inoculating the inoculum with 2 to 10 parts by weight of the total weight of the lacquer tree of step 2).
The method according to claim 1, wherein the step 3) is inoculated with 25 to 35 parts by weight of the seedlings based on the total weight of the lacquer tree in step 3).
[7] The method according to claim 1, wherein the culture or fermentation in step 3) is carried out after fermenting the seedlings with Rhus verniciflua for 7 to 14 days or fermenting the fermented lacquer.
The method of claim 1, wherein the method further comprises removing urushiol from the Rhus verniciflua component and inhibiting the reduction of pentagalloyl glucose.
Mushroom strain culture medium and Rhus verniciflua as active ingredients.
[9] The composition according to claim 8, wherein the fungicide has urushiol removed from the Rhus verniciflua component, and the reduction of the pentagaloylglucose is inhibited.
[9] The composition according to claim 8, wherein the fungicide is produced through step 1) and step 2) of claim 1.

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