JP2008212137A - Method for producing new gisenoside from ginseng by liquid culture of phellinus linteus mycelium utilizing biotransformation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide gisenoside Rd, Rg2 and Rh1 produced by liquid culture of mycelium of Phellinus linteus strain (Phellinus linteus, KCTC 0912BP) with excellent gisenoside biotransformation activity, and to provide a method for producing the same comprising mannose which is a specific constituent of the polysaccharides originating from the strain. <P>SOLUTION: The invention relates to the quantity production method for producing gisenoside Rd, Rg2 and Rh1 which are either present or not in ginsengs such as fresh Panax schinseng suisann, dry Panax schinseng suisann, and ginseng radix, etc., in trace amounts, in short time by using biotransformation utilizing liquid culture of mycelium of Phellinus linteus. The method comprises production of a new biotransformed gisenoside available by mass production from a Phellinus linteus strain (KCTC 0912BP) having an excellent biotransformation activity of gisenoside in the components of ginsengs. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a novel method for producing ginsenoside from ginseng by liquid culture of perinus linteus mycelium using a biotransformation method. More specifically, the present invention relates to mulberry among ginseng components containing a large amount of substances having excellent pharmacological activity. The present invention relates to a method for mass-producing a useful saponin component in a short period of time by using a bioconversion method by liquid culture of jaundice mycelium.

  Ginseng, known as 'an oriental mysterious spirit', is recognized as the best quality in the world due to natural conditions suitable for the growth of ginseng and cultivation and processing techniques accumulated for a long time. It is in the limelight as a classic image product. In particular, ginseng contains a large amount of physiologically active substances such as saponins and polyphenols that other plants do not have at all, and has a very excellent pharmacological effect.

  According to recent scientific research results, ginseng contains various physiologically active substances such as ginsenoside (ginseng saponin), polyacetylene, polyphenolic compounds, organic acids, amino acids, peptides, polysaccharides, vitamins, etc. Has the most excellent bioactive function, and its pharmacological actions include central nerve inhibitory action, protein synthesis promoting action, immune function regulating action, insulin-like action, detoxification action, anti-carcinogenesis Anticancer effects have been reported.

  Saponins are composed of a sugar part (glycose) and a non-sugar part (aglycose), and are widely distributed in the plant kingdom, but ginseng saponins are characterized by the fact that the non-sugar part is a dammaran triterpene. It has been classified into protopanaxdiol and protopanaxtriol systems, and 38 kinds of ginsenoside chemical structures have been revealed to date.

  Of these, five major ginsenosides (ginsenoside Rb1, Rb2, Rc, Re, Rg1) are known to occupy over 80% of the total ginsenoside, and glucose, rhamnose, arabinose, and ylose are present in the non-sugar part. It is known that the pharmacological action differs depending on the type and number of saccharides bound and the binding position. Among these ginsenoside derivatives, particularly recently, there have been many studies on the bioactive function of ginsenoside, which is present in a small amount in ginseng. Such ginsenoside, that is, Rd, Rg3, Rh1, and Ck are the five major types. It has been shown to have more excellent physiological activity than ginsenoside (Patent Document 1).

  Therefore, recently, there have been active attempts to convert the main ginsenoside into a trace amount of ginsenoside as described above. The method includes a heat treatment method (Patent Document 2) and an acid and alkali treatment method (Patent Document). 3) An enzyme treatment method (Patent Document 4) is disclosed.

  However, the method of treating with heat or acid and alkali may decompose other ginsenosides and polysaccharides having other excellent activities, resulting in elimination of other physiological activities of ginseng. There is a point. On the other hand, the enzyme treatment method requires a very large amount of enzyme compared to the amount of temperament, and thus has a problem that it is inefficient in reaction time and yield.

  In the case of moss, it is recognized as a material with various physiological activities by various studies. Of these, Perinus linteus, a kind of mulberry yellow moth, has an anticancer activity of 96.7%, and it is reported that the protein polysaccharide of perinus linteus mycelium effectively suppresses Sarcoma 180 multiple cancers and solid cancers Has been. The method of converting ginsenoside by higher fungi can be divided into two methods, solid culture and liquid culture, depending on the culture method of higher fungi. However, when higher fungi are cultured using carrot as a temperament, the production time is long and the production efficiency is inferior. Productivity has been highlighted as a problem.

In particular, in the case of solid culture of higher fungi, it may act as a problem of production space, a labor-consuming process, and a difficulty in the industrialization process. Such solid culture is disclosed in Patent Documents 1, 5, and 6. According to the above patents, a method for producing ginseng products containing higher fungi through fermentation on a solid medium for about 14 to 90 days is known.
Korean Published Patent No. 10-2006-0062083 Korean Patent Publication No. 1996-017670 Korean Patent Registration No. 10-0620107 Korean Patent Publication No. 10-02304002 Korean Patent Registration No. 10-0496666 Korean Published Patent No. 10-2004-0108476

  In the present invention, the mycelium of Mulberry pertuslintheus strain, which has not been reported so far in consideration of the above-mentioned points, can be applied to hydrangea, dry ginseng, red ginseng, etc. using a bioconversion method by liquid culture. It is an object to provide a method capable of mass-producing ginsenoside Rd, Rg2 and Rh1 in a short amount or in a short amount.

  The object of the present invention is to inoculate and inoculate Mulberry yellow perinum Perinus linteus KCTC 0912BP strain, produce red ginseng powder medium and inoculate the inoculum and mass culture, obtain mulberry yellow fermented ginseng dry powder to obtain butanol solution The residue was dried after cooling at reflux, dissolved by adding methanol, filtered, and then HPLC analysis was performed.

  A feature of the present invention is that rare ginsenosides Rd, Rg2, and Rh1, which are present in trace amounts in ginseng / dry ginseng, red ginseng, etc., are biotransformed by liquid mycelia culture of Perinus linteus strain to produce a large amount in a short period of time. It was completed by providing a manufacturing method that can be produced.

  According to the present invention, ginseng / dry ginseng and red ginseng powder are incorporated into the components of ginseng / dry ginseng and red ginseng through liquid culture of mycelium of Perinus linteus KCTC 0912BP by the powerful bioconverting enzyme of this strain. There is an effect of mass-producing saponins containing rare ginsenosides Rd, Rg2 and Rh1 in a short period of time or in a short amount.

  In addition, the present invention is ginseng powder obtained by biotransformation of the Perinus linteus strain (Phellinus linteus KCTC 0912BP) by a liquid culture method includes not only the rare ginsenoside but also mannose which is a specific polysaccharide constituent component derived from the strain. Since it has an excellent effect, it is a very useful invention in the biopharmaceutical industry.

  Hereinafter, the configuration of the present invention will be described in detail.

  The present invention utilizes a perinus linteus mycelium (Phellinus linteus, KCTC 0912BP) that is excellent in biotransformation activity of ginsenoside among ginseng components, and is capable of producing a novel mass-produced rare product that is biotransformed by liquid culture of this strain. A method for producing ginsenoside is provided. More specifically, the present invention selects a perinus linteus (KCTC 0912BP) strain having excellent ginsenoside bioconversion activity, and only ginsenoside Rd, Rg2 and Rh1 obtained by liquid culture of the mycelium of the strain. Provided is a method for producing a polysaccharide in which the constituent component of a specific polysaccharide derived from a mulberry yellow mycelium is composed of mannose.

  The present inventors transferred the mycelium of the Perinus linteus strain to a potato dextrose (PDA) solid medium and purely cultured; the mycelium cultured in the PDA solid medium was again liquefied in a potato dextrose broth liquid medium, respectively. A step of culturing; after washing 300 g of ginseng with running water, it is put into a 5 L closed container and steamed for 5 hours using heated steam, and the steamed ginseng is heated with a hot air dryer 50. A process for producing red ginseng by drying in a drying chamber after drying for 1 hour at a temperature of 15 ° C. and naturally drying the ginseng; including the red ginseng powder obtained in the above process; A step of mass-producing ginseng ginsenoside after putting it in a bioreactor together with the mycelium of the Perinus linteus strain in a liquid medium; and the desired rare ginsenoside Rd, Rg2 and Separates Rh1 and constituent sugars Constructed in accordance with the present invention mulberry yellow fermented ginseng immune effects and toxicity testing step; step and that.

  Another object of the present invention is to provide cocoon saponins characterized in that ginsenoside obtained within a short time from the bioconversion process using the mulberry yellow koji mycelium liquid culture method contains Rd, Rg2 and Rh1.

  Another feature of the present invention is that ginsenoside biotransformed from the above-mentioned Perinus linteus strain (Phellinus linteus KCTC 0912BP) has never been disclosed so far in which Rd, Rg2 and Rh1 coexist. It is in.

  Hereinafter, examples of specific configurations of the present invention will be described in detail. However, the scope of the present invention is not limited only to these examples, and those skilled in the art will be able to make some design changes or numerical values and processes. It can be said that these inventions belong to the scope of the right of the present invention.

[Example 1] Mycelium inoculum culture of Perinus linteus KCTC 0912BP The Perinus linteus KCTC 0912BP strain used in the present invention was collected and selected at Seoraksan, Gangwon-do, Korea. This is a distribution of strains deposited as patented strains at the Research Institute Bioresource Center (KCTC). As a result of the present inventors' research, the strain was confirmed to have the best biotransformation activity. The culture conditions for the mycelium seed culture were as follows: the inoculum was maintained at 25 ° C. in a potato text rose agar (PDA) medium for 7 to 10 days. It was further cultured for 10 days in a 500 ml Erlenmeyer flask containing potato text rose broth complex medium. The mycelium of the strain, which has been sufficiently cultured for 10 days, is sprayed for 1 minute at 4,000 rpm with a pulverizer, and further, 10% After inoculating v / v), liquid culture was carried out for 10 days, and this culture solution was used as a 10% (v / v) inoculum. The liquid cultured mycelium used as the inoculum was placed in a sterilized mixer, homogenized, and inoculated into a container containing 5 L of WIM medium in a 7 L upper fermentor (Marubishi Co. Ltd.). The culture environment of the mycelium of Perinus linteus KCTC 0912BP was supplied while adjusting the air injection rate to 0.1-1 vvm. The air injection rate was adjusted to 1 vvm or less because the mycelium tends to adhere to the incubator wall due to the generation of a large amount of bubbles when the air injection rate was 1 vvm or more. The culture temperature was cultivated for 5 to 10 days by installing a heating / cooling device in the culture chamber and adjusting the room temperature to 20 to 35 ° C. The bioreactor was sterilized at 121 ° C. for 20 minutes using a sterilizer.

[Example 2] Production of red ginseng powder After 4 kg of ginseng from which the brain head had been removed was washed with running water, it was placed in a 20 L closed container and steamed with heated steam for 5 hours. The steamed ginseng was placed in a hot air dryer and dried at 50 ° C. for 1 hour, then transferred to a drying room and naturally dried to a moisture content of 15% or less. In order to use red ginseng as a biotransformation material, red ginseng powder was manufactured to a size of about 20-30 mesh using a sand separator.

[Example 3] Mass production of ginseng ginsenoside using a bioconversion system by liquid culture of mycelium of the perinus linteus strain of the present invention The WIM medium 36L containing red ginseng powder produced in Example 2 was produced to produce a 70L biological reaction. After being transferred to an in situ fermenter, a sterilization process was performed at 121 ° C. for 40 minutes. After the temperature of the bioreactor is lowered to about 28 ° C. using a low-temperature circulation tank, the perinusintheus KCTC 0912BP strain mycelium inoculum 4L cultured in Example 1 is usually used in a 70L bioreactor. Inoculated and cultured using aseptic inoculation method. As a comparative example, in the case of using ginseng powder or dried ginseng powder as a raw material, a WIM medium 36L containing ginseng powder or dried ginseng powder instead of the red ginseng powder was manufactured to produce a 70 L biological reactor (in situ fermenter ), Followed by sterilization at 121 ° C. for 40 minutes. After the temperature of the bioreactor is lowered to about 28 ° C. using a low temperature circulation tank, the perinusintheus KCTC 0912BP strain mycelium inoculum 4L cultured in Example 1 is usually used in a 70L bioreactor. Inoculated and cultured using the existing sterile inoculation method. Fermentation carrot reaction conditions were supplied while adjusting the air injection rate to 0.1-1 vvm. The air injection rate was adjusted to 1 vvm or less because the mycelium tends to adhere to the wall of the incubator due to the generation of a large amount of bubbles when the air injection rate was 1 vvm or more. The culture temperature was cultivated for 1 to 10 days by installing a heating / cooling device in the culture chamber and adjusting the room temperature to 20 to 35 ° C. After completion of the cultivation, the fermented carrot was filtered with a filter paper and dried with a dryer at 60 ° C. for 8 hours, and the weight of the mulberry yellow fermented carrot was measured to obtain about 600 g / L in dry weight. The culture supernatant containing extracellular polysaccharides and ginseng fermentation extract components was concentrated to 60 Brix, mixed with dried mulberry fermented ginseng, and dried in a dryer at 60 ° C. for 5 hours. The final dried product was pulverized to a size of about 100 mesh using a sand separator to obtain 1.6 kg of mulberry yellow fermented carrot powder.

[Example 4] Extraction of ginsenoside of biotransformed ginseng As a result of the above-mentioned Example 3, the analysis of the pattern of ginsenoside of mulberry corn fermented ginseng mass-cultured was conducted based on the example standards and test methods of health functional food corporation ginseng products. It carried out according to. That is, weigh 5 g of the sample that passed through 100 mesh, put it in a 250 ml reflux flask, add 50 ml of water-saturated butanol solution, cool it by refluxing in a 70-80 ° C. water bath for 1 hour, cool it, filter it, It moved to the liquid separation surplus. The above operation was repeated twice or more on the residue. Distilled water (50 ml) was added to the liquid separation, and the mixture was vigorously shaken and allowed to stand until the butanol layer and the butanol layer were completely separated. Remove the aqueous layer (lower layer) and transfer to a concentration flask containing the butanol layer in advance.Concentrate under reduced pressure in a water bath, add 50 ml of ether to the residue, and cool at reflux in a water bath at about 46 ° C for 30 minutes to remove the ether. did. The residue was dried to a content to obtain an analytical dry sample of ginsenoside according to the present invention.

(Experimental Example 1) HPLC analysis of biotransformed mulberry yellow fermented ginseng ginsenoside of the present invention After methanol was completely dissolved in the dried mulberry fermented ginseng ginseng (sample) of Example 4, 0.45 μm Minisart RC- It filtered with 15 (Satorius) filter, inject | poured into HPLC according to the following analysis conditions, and analyzed the carrot ginsenoside and compared the area of the peak. The column uses Platinum C18 (100A, 1.5 μl, 33 mm × 7 mm), the mobile phase starts with a water to acetonitrile ratio of 80:20, 75:25 for the first 10 minutes and 50:15 for the next 15 minutes. The concentration gradient of water and acetonitrile was adjusted at a ratio of 50. The mobile phase flow rate was 1.2 ml / min, and chromatograms were obtained at a wavelength of 203 nm (FIGS. 1 and 2). The raw material ginseng is a dried powder in the present invention, compared with the biotransformed, ginsenoside characteristic change of Mulberry yellow fermented ginseng powder is a significant increase of 2.5 to 10 times in Rd, Rg2 and Rh1 (Table 2).

(Experimental Example 2) Polysaccharide analysis of bioconverted mulberry yellow fermented ginseng of the present invention In order to extract polysaccharides from mulberry yellow fermented ginseng obtained in Example 4, mulberry yellow fermented ginseng dry powder (sample), water 80 g of 20-fold water was added to 4 g of ginseng powder and red ginseng powder, followed by reflux extraction for 3 hours. The extract was filtered, and 95% ethanol was added in an amount 4 times that of the filtrate, and then allowed to stand at 4 ° C. for 12 hours and centrifuged. After centrifugation, the supernatant was removed and the precipitate was dried to obtain a high molecular polysaccharide. Among the polysaccharides, the polysaccharides derived from Mulberry mycelium were measured as the content of methyl-α-D-mannopyranoside contained in the polysaccharides by the following experimental method.

  A sample of 50 mg was precisely measured, placed in a 20 ml glass container, filled with 10 ml of methanolic 0.75N hydrochloric acid test solution and filled with nitrogen, and then reacted in a water bath at 80 ° C. for 24 hours. The semi-reaction solution was cooled at room temperature, transferred to a 100 ml flask for vacuum distillation, and concentrated under reduced pressure at 60 ° C. 20 ml of methanol was completely dissolved in the dried product concentrated under reduced pressure, and after filtering through a 0.45 μm filter, 10 ml of the filtrate was taken and further concentrated at 60 ° C. under reduced pressure. To the residue, 5 ml of an internal standard solution was added, 0.5 ml of trimethylchlorosilane (TMCS) and 1 ml of hexamethyldisilazane (HMDS) were added, and the mixture was shaken vigorously for 3 minutes, and then filtered to prepare a test solution. For the standard solution, weigh accurately 34.5 mg of the special grade reagent methyl-α-D-mannopyranoside, add the internal standard solution to dissolve to make exactly 50 ml, take 5 ml of this solution, add 0.5 ml of TMCS and 1 ml of HMDS, and shake vigorously for 3 minutes. Prepared. The internal standard solution was prepared by precisely measuring 50 mg of rhamnose and adding anhydrous pyridine to dissolve it to make exactly 100 ml.

  The polysaccharide content was calculated from the area of the test solution and the standard solution obtained by the following GC analysis method using the test solution and the standard solution prepared as described above.

  The analytical instrument was GC (Varian 3800), and the analytical conditions were as follows.

Column: RTX-5 Capillary column (0.53mm × 30m)
Detector: Hydrogen flame ionization detector (290 ℃)
Column Temp: Maintain at 180 ° C for 1 minute, then increase at 4 ° C per minute and maintain at 220 ° C.

Injector Temp: 270 ℃
Carrier gas: N ₂ 80psi, Air 60psi
Injection volume: 1.0μl
The calculation method is as follows.

Methyl-α-D-mannopyranoside peak area in specimen
As: Methyl-α-D-mannopyranoside peak area of standard solution
Asi: Internal standard substance peak area of standard solution
AT: Methyl-α-D-mannopyranoside peak area of the test solution
Ati: Internal standard peak area of the test solution
Ws: Standard product collection (mg)
WT: Sample collection amount (mg)
As a result of the analysis, 14 mg of mannose was contained per 1 g of mulberry yellow fermented red ginseng powder, whereas no mannose was analyzed in daffodils, dry ginseng or red ginseng (FIGS. 3 and 4). Therefore, the mulberry yellow fermented ginseng powder according to the method of the present invention contains polysaccharides derived from mulberry yellow mycelium, and the polysaccharides obtained from ginseng, dried ginseng and red ginseng are completely different in the type and content of constituent sugars. I found it different.

(Experimental Example 3) Investigation of immune enhancement effect of biotransformed mulberry yellow fermented red ginseng of the present invention <Culture and administration of test substance>
Using the saponin extracted using the mulberry yellow fermented red ginseng powder produced in Example 3 of the present invention, the immune enhancing effect on macrophages was investigated. In order to measure the activity of macrophages, the amount of nitric oxide (NO) produced was determined. Normally, nitric oxide is a low-molecular radical and is known to play a role such as nerve signal transmission function, blood coagulation and blood pressure regulation function, and immune function against cancer cells. The macrophage line Raw264.7 cell line was purchased from the Korea Cell Line Research Foundation. DMEM containing 10% FBS was cultured in a cell culture medium at 37 ° C in a 5% CO ₂ incubator. Maintained by subculturing twice a week. Raw264.7 cell line was made up to 5 × 10 ⁵ cells / ml with DMEM containing 10% FBS, dispensed into a 96 well plate and adapted for 24 hours, using DMEM medium without FBS The test substance was dissolved and processed. After culturing for 24 hours, the amount of NO released from the culture solution in each well was measured. For quantification of NO, 100 μl of the supernatant was taken, mixed with the same amount of Griess reagent (Sigma: G4410), allowed to react for 15 minutes, and then the absorbance was measured at 540 nm using an ELISA reader. Sodium nitrite was used to create a standard calibration curve. NO concentrations in the test substance-treated group and the negative control group were analyzed using a dominant difference test (Student t-test), and significance was recognized when P <0.01.

<Measurement of immune enhancement effect>
In this experiment, mouse-derived macrophages (Raw 264.7) were treated with 4 types of test substances such as mulberry yellow fermented red ginseng to increase the immune function of macrophages using the amount of nitric oxide (NO) produced as an index. Conducted to evaluate. NO synthesized by activated macrophages is known to act as an important signal substance that activates the immune world and exerts antiviral and anticancer activities. In this experimental result, the NO concentration from all the test substances showed an increasing tendency compared to the negative control group. In particular, in the mulberry yellow fermented red ginseng treatment group, the NO production amount of 14.6 μmol / L higher than the other test substances was shown. Shown (Figure 5). Therefore, in the case of the present mulberry yellow fermented red ginseng, it was judged that the immune function regulating effect of the phagocytic cells was excellent. That is, it was confirmed that all the test substances were statistically significant (P <0.01) when compared with the negative control group.

[Experimental Example 4] Toxicity test of biotransformed fermented red ginseng of the present invention <Dietary of experimental animal and administration of test substance>
Toxicity tests were conducted by single administration to ICR mice using the mulberry yellow fermented red ginseng powder produced in Example 3 of the present invention. After fermented red ginseng product was orally administered to mice once, the toxicity of the product was evaluated by performing mortality for 14 days, observation of clinical symptoms, body weight measurement, feed and water intake measurement, GOT / GPT test, etc. . Experimental animals were used after adapting 40 ICR mice produced by Korea Biolink Co., Ltd. for one week in the animal breeding room (22 ± 2 ° C). For each group, 5 mice were selected, and the litter used during the test period was sterilized at 121 ° C for 15 minutes in a high-pressure steam sterilizer, and water was placed in a clean bottle as a laboratory animal. Supplied freely.

  For the preparation of experimental substances, mulberry yellow fermented red ginseng products were divided into hot water extract and powder that passed through 100 mesh, selected according to dosage and mixed carefully so that bubbles do not occur in physiological saline on the day of administration went. The dose volume was as shown in the table below. The test substance was administered orally using a sonde, and the dose was calculated according to the body weight immediately before administration.

<Analysis sample collection and sample analysis>
All experimental animals were observed once a day at regular intervals for changes in general condition, onset of poisoning, and death. Body weight measurement and feed and water intake were measured once every two days, and body weight was also measured before administration of the test substance and the day before necropsy. For blood biochemical tests, the blood was left in the refrigerator for about 2 hours, and then centrifuged to separate the serum, and the GOT / GPT detection kit (Asayama Pharmaceutical) was used.

  As a result of the experiment, no animals died in all experimental groups and no other clinical symptoms were observed. As a result of measuring body weight and intake of feed and water, no significant change was observed from the female and male administration groups compared to the control group (Table 3). In addition, no abnormal signs were observed in the blood biochemical examination as compared with the control group (Table 4), and no special abnormalities in tissue organs were observed as a result of necropsy.

It is the liquid chromatogram which showed the ginsenoside pattern of the ginseng by this invention. It is the liquid chromatogram which showed the ginsenoside pattern produced by the bioconversion method of this invention. It is the gas chromatogram which showed the pattern of the constituent sugar of the red ginseng polysaccharide by this invention. It is the gas chromatogram which showed the pattern of the saccharide | sugar of the mulberry yellow fermented red ginseng polysaccharide produced by the bioconversion method of this invention. 1 is a diagram showing an immune enhancing effect of mulberry yellow fermented red ginseng of the present invention.

Claims (6)

  A method for producing ginsenoside using a bioconversion method generated from liquid culture of a Perinus strain.   The method for producing ginsenoside according to claim 1, wherein the strain of the genus Perinus is Perinus linteus KCTC 0912BP.   The perinus strain is cultivated in a liquid culture medium containing ginseng powder for biotransformation at an air injection rate of 0.1 to 1 vvm, an agitation rate of 50 to 500 rpm, and a culture temperature of 20 to 35 ° C. for 1 to 10 days. The ginsenoside manufacturing method of Claim 1 or 2.   4. The method for producing ginsenoside according to claim 3, wherein the raw material for biotransformation ginseng includes ginseng, dried ginseng or red ginseng.   Biotransformation ginseng powder comprising ginsenoside Rd, Rg2 or Rh1 produced by the ginsenoside production method according to any one of claims 1 to 4, wherein the polysaccharide component is mannose.   A bioconverted ginseng powder extract comprising ginsenoside Rd, Rg2 or Rh1 produced by the ginsenoside production method according to any one of claims 1 to 4, wherein the polysaccharide component is mannose.

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