TW201346027A - Cultivation method of Taiwanofungus camphorates - Google Patents

Abstract

A cultivation method of Taiwanofungus camphorates comprises: a formulating step, an inoculating step, and a culturing step. The formulating step is by mixing 0.1 to 50% of glucose, 1 to 5% of peptone, 0.1 to 50% of malt extract, 0.1 to 50% of yeast extract, and 0.5 to 10% of solidifying agent with water, and obtaining a column medium, and the depth of column medium is 2 to 20 cm. The inoculating step is by inoculating Taiwanofungus camphorates at several preserved inoculation points inside the column medium, and the several preserved inoculation points is 0.5 to 10 cm away from the surface of the column medium. The culturing step is by culturing the column medium under 23 to 28 DEG C at least 3 months, and obtaining a mass of Taiwanofungus camphorates.

Description

Method for cultivating burdock

The present invention relates to a method for cultivating Antrodia camphorata, and more particularly to a method for cultivating Antrodia camphorata which can produce a triterpenoid compound from Antrodia camphorata.

Antrodia (Taiwanofungus camphorates), also known as mushrooms or Antrodia camphorata, based belonging Basidiomycota (Basidiomycota) in Fungi, Basidiomycotina (Basidiomycotina), with Basidiomycetes (Homobasidiomycetes), no off bacteria mesh (Aphyllophorales ), Polyporaceae ( Polyporaceae ), Antrodia ( Antrodia ), Antrodia camphorata only grows on Taiwan's conservation tree species of burdock, which is a unique mushroom in Taiwan.

From the mycelium or fruit body extract of Antrodia camphorata, a variety of physiologically active substances such as polysaccharides, adenosine or several triterpenoids are found, among which triterpenoids, such as Aminocin A, Aminocin B (Antcin B), Amino C (Antcin C), Amine E (Antcin E), Amine F (Antcin F), Methylantcinate G, Methylamine oxime H ( Methylantcinate H), Antrocin, 4,7-dimethoxy-5-methyl-1,3-benzodioxole and 2 , 2',5,5'-tetramethoxy-3,4,3',4'-bismethyl-endooxy-6,6'-biphenyl (2,2',5,5'- Tetramethoxy-3,4,3',4'-bimethyl-enedioxy-6,6'-dimethylbiphenyl), etc., have been shown to have anti-cancer cell growth ability.

However, because of the fungus growing on the burdock wood, the production is rare and difficult to obtain. Therefore, the industry has artificially cultivated the burdock, or obtained the mycelium or fruiting body of the burdock, and further extracts the physiology of the mycelium or fruit body of the burdock. An active substance (such as a triterpenoid or a polysaccharide).

The conventional method for cultivating Antrodia camphorata includes solid state culture method, liquid fermentation method, eucalyptus cultivation method or space capsule cultivation method. However, the above conventional methods have their own disadvantages.

The conventional solid-state culture method is to dig a piece of the mycelium or fruit body of A. angustifolia, which is about 1 cm square, and attach it to the surface of a solid medium, so that the Astragalus can adhere to the surface growth of the solid medium, although it is conventionally used. The solid culture method can obtain the mycelium or fruit body of Antrodia camphorata, and is less likely to produce bacteria. However, the mycelium or fruit body obtained by the conventional solid culture method contains only a very low amount of triterpenoids. Therefore, the mycelium or fruit body of Antrodia camphorata obtained by the conventional solid culture method cannot be applied to extract physiologically active substances without being used by the industry.

In the liquid fermentation method, the mycelium of Antrodia camphorata is inoculated into a liquid culture solution, which can enhance the physiologically active substance contained in Antrodia camphorata, but a solid substrate such as bagasse or whole grains is added during the cultivation process. As a support for the mycelium of Antrodia camphorata, the liquid fermentation method must be cultured in a large-volume reactor, and the preparation cost thereof is high, and the liquid fermentation method is also prone to contamination by bacteria.

In the cultivation of eucalyptus, the burdock is inoculated on the burdock, and the burdock component obtained by the method is similar to the physiologically active substance contained in the wild burdock, but the burdock is difficult to obtain and the cultivation cost is low. It is high, so it is not possible to produce triterpenoids in large quantities.

The conventional space bag cultivation method, although the overall cost is low, is susceptible to contamination by bacteria in the cultivation process, and the content of physiologically active substances is low, and therefore, triterpenoids cannot be produced in large quantities.

Although the above method can obtain the mycelium or fruit body of the Astragalus membranaceus containing the triterpenoid compound, the cultivation operation is not easy, and the bacteria are easily generated, so that the entire batch of the cultured A. glabra is contaminated and causes a serious loss to the operator, and the used Astragalus sinensis The quality of the mycelium or fruit body of Antrodia camphorata produced by the culture method is not high, and the triterpenoids contained therein are still insufficient.

In view of this, there are still shortcomings in the practice of the cultivation of Antrodia camphorata, which needs further improvement.

The main object of the present invention is to provide a method for cultivating Antrodia camphorata, which is capable of increasing the growth amount of A. angustifolia mycelium or fruit body per unit medium.

A second object of the present invention is to provide a method for cultivating Antrodia camphorata, which is capable of reducing the contamination of bacteria.

In order to achieve the foregoing object, the technical content of the present invention includes:

A method for cultivating Antrodia camphorata comprises: a medium preparation step of 0.1 to 50% by weight of glucose, 1 to 5% of peptone, 0.1 to 50% of barley extract, and 0.1 to 50% of yeast extract And 0.5~10% curing agent is prepared by mixing water into a mixed solution, and the mixed solution is boiled to completely dissolve the above components, and then the mixed solution is solidified into a column culture medium, and the depth of the column medium is 2~ 20 cm; an inoculation step is provided inside the column culture medium with a plurality of predetermined inoculation sites, the predetermined inoculation sites are 0.5 to 10 cm deep from the surface of the column culture medium, and inoculating O. angustifolia in each predetermined inoculation position. And a culture step of culturing the inoculated column culture medium at a temperature of 23 to 28 ° C for at least three months to obtain a burdock cell.

Wherein, after the culturing step, a drying step is further included, wherein the column medium having the burdock bacillus is dried at 30-40 ° C until the water content is less than 2%, and a dry bacterium of the burdock is obtained.

Among them, each predetermined inoculation position is 0.5 to 2 cm apart.

Wherein, the column medium can be selected as a multi-faceted cylinder or a cylinder.

Among them, the curing agent can be selected as agar.

The culture time of the culture step is preferably 4 to 12 months.

The drying time of the drying step is preferably 24 to 72 hours.

The above and other objects, features and advantages of the present invention will become more <RTIgt;

Referring to Fig. 1, a method for culturing an Antrodia camphorata according to the present invention comprises a medium preparation step S1, an inoculation step S2, and a culture step S3.

The medium preparation step S1 is to use 0.1 to 50% by weight of glucose (Glucose), 1 to 5% of peptone, 0.1 to 50% of malt extract, 0.1 to 50% of yeast. The extract (Yeast extract) and 0.5 to 10% of the curing agent are formulated into a mixed solution by using water, and the mixed solution is boiled to completely dissolve the above components, and then the mixed solution is solidified into a column culture medium, and the column is solidified. The depth of the medium is 2 to 20 cm.

More specifically, in the mixed solution of the present invention, glucose provides a carbon source required for the growth of Antrodia camphorata, and peptone, barley extract and yeast extract provide nitrogen sources and other trace elements required for the growth of Antrodia camphorata. The mixed solution can be further added with other ingredients which contribute to the growth of Antrodia camphorata, such as trace elements or vitamins, according to the familiar art of the person skilled in the art, and is not described here; wherein the curing agent is selected as agar (Agar) The mixed solution contains 0.5 to 10% agar to provide the support required for the growth of the mycelium of A. angustifolia, and to prevent the density of the agar from being too high to hinder the growth of the mycelium of Antrodia camphorata.

The column culture medium of the present invention may be selected as a multi-faceted cylinder or a cylinder. The depth of the column medium referred to in the present invention is not limited to the column height and the column width of the column medium, that is, the column height is wider than the column. Between 2 and 20 cm to provide sufficient depth for the growth of Antrodia camphorata in the interior of the column medium without the operation of the inoculation step S2 being too large due to the excessive thickness of the column medium.

Referring to Figures 2a to 2e, the column medium 1 is a multi-faceted column system comprising a bottom surface 11, a top surface 12 and at least one side surface 13, the bottom surface 11 being attached to a culture tray. The top surface 12 and the side surface 13 are provided with a surface for inoculating the burdock, wherein the side surface 13 may be a flat surface or a curved surface. If the side surface 13 is a flat surface, the number of the side surfaces 13 is preferably 3 to 12, such as the four-sided cylinder, the six-sided cylinder, the octahedral cylinder or the twelve-sided cylinder shown in Figures 2a to 2e, or the number of the side surfaces of the cylinder is more than 12, the column The body is close to a cylinder, so the side surface 13 is close to a curved surface.

The inoculation step S2 is provided with a plurality of predetermined inoculation sites in the interior of the column culture medium, and the plurality of predetermined inoculation sites are 0.5 to 10 cm deep from the outer surface of the column medium, and the inoculum is inoculated on each predetermined inoculation site. . More specifically, the Antrodia camphorata is not limited to the existing strains of Burdock , such as Taiwanofungus camphorates KP01 (GenBank accession number: JQ945230) or the strain of Burdock (BCRC 35396, BCRC 35398, BCRC) of the Hsinchu Food Science and Industrial Development Research Institute, Taiwan. 35716, BCRC 36401, BCRC 36711, BCRC 36795, BCRC 37848, BCRC 37849 or BCRC 37850). In the present invention, the mycelium of Antrodia camphorata is inoculated into the inside of the column medium in a three-dimensional space, so that the A. sinensis is comprehensively coated by the column culture medium, and the column culture medium is improved. The nutrient content and the space utilization rate increase the amount of mycelium in the unit medium, thereby increasing the content of physiologically active substances (such as triterpenoids) produced by Antrodia camphorata; the present invention can also be inoculated on the outer surface of the column medium. To make full use of the column culture medium.

For example, as shown in FIG. 2a, the outer surface of the column culture medium 1 of the embodiment includes a bottom surface 11, a top surface 12 and four side surfaces 13, and the top surface 12 is referenced to the bottom surface. 11 extends to a depth of at least 0.5 cm, and the other five faces are deduced by the range of the predetermined inoculation position P, that is, the oblique line portion S as shown in the figure; the tetrahedral column of the embodiment The column height of the body is selected to be 6 cm, and the bottom surface 11 and the top surface 12 are a regular quadrilateral, and the side length of the quadrilateral is selected to be 3 cm (that is, the four side surfaces 13 are 3 cm long and 6 cm long).

Referring to FIG. 2e again, the outer surface of the column culture medium 1 of the embodiment includes a bottom surface 11, a top surface 12 and a side surface 13 extending toward the bottom surface 11 based on the top surface 12 to at least 0.5 cm deep, the top surface 12 and the side surface 13 are deduced by the same extent, and the range of the circumference is the allowable range of the predetermined inoculation position P, that is, the oblique line portion S as shown; the column of the cylinder The height is 6 cm, and the bottom surface 11 and the top surface 12 of the cylinder are circular, and the diameter of the circular shape is 6 cm.

After inoculation of the Astragalus membranaceus mycelium ( Taiwanofungus camphorates KP01 ) with an inoculation loop (or inoculation needle), the inoculating loop with the mycelium of Antrodia camphorata is inserted into the oblique block of the column medium 1, and each predetermined inoculation position A spacing D is provided between the two, and the spacing D is preferably 0.5 to 2 cm to avoid the distance between the inoculated mycelium of the Antrodia camphorata, and inhibit each other from growing.

By the distance D between the predetermined inoculation positions of 0.5 to 2 cm, it is ensured that the mycelium of Antrodia camphorata in each predetermined inoculation position can make full use of the surrounding nutrients, which is beneficial to the growth of the mycelium of Antrodia camphorata, and can avoid the scheduled inoculation. The mycelium of Antrodia camphorata is inhibited from growing due to its inoculation.

In the culturing step S3, the inoculated column culture medium is cultured in an environment of a temperature of 23 to 28 ° C for at least three months to obtain a burdock cell. More specifically, the Astragalus lucidum strain of the present embodiment is cultured for at least 3 months, preferably after 4 to 12 months, to obtain a physiologically active material of the burdock fungus, especially containing a relatively high content. Triterpenoids.

Referring to FIG. 3, the culture method of the Antrodia camphorata of the present invention is preferably carried out after the culturing step S3, and then a drying step S4 is carried out, and the column culture medium containing the burdock bacillus is at 30-40 ° C. Dry down to a moisture content of less than 2% to obtain a dried calf of burdock. Since the burdock is dried to remove most of the moisture, it is easy to store or carry out the extraction operation of the subsequent physiologically active substance.

In order to confirm the burdock cell obtained by the culture method of the burdock of the present invention, it is possible to produce a physiologically active substance such as a triterpenoid compound, and the following are qualitative results of the preferred embodiment of the present invention and the triterpenoid compound thereof.

Please refer to Table 1, which is the composition and weight percentage of the mixed solution in this embodiment. After the mixed solution is boiled, when the mixed solution is cooled and solidified, it is poured into a mold of a four-sided cylinder and at room temperature. After standing to solidify the mixed solution, a tetrahedral cylinder medium as shown in Fig. 2a is obtained, and the column medium is poured out and placed on a culture tray; the operation of the embodiment is preferably performed on an aseptic workstation. It is carried out in order to prevent other microorganisms from contaminating the column culture medium and affecting the cultivation of the subsequent Antrodia camphorata.

Table 1: Formulation of the mixed solution of this example

Please refer to the schematic diagram of the predetermined inoculation position setting shown in Figure 2a. After inoculation in each of the predetermined inoculation positions P, the culture is carried out in an incubator at 25 ° C, and a Bacillus sinensis is obtained in the 4th month. After drying the burdock cell in an oven at 40 ° C for 72 hours, a dried burdock bacterium is obtained, and the burdock dry bacterium is extracted, and then subjected to ultra high pressure liquid chromatography double mass spectrometer (UPLC). -MS/MS) Analysis of a representative substance of the triterpenoids in the extract - Amine A.

The amine hydrazine A extract of the present example was obtained by immersing the dried sirloin (100 g) in 90% ethanol (500 ml), standing for 24 hours, and concentrating into a vacuum concentrator. After extracting the powder, it was further quantified to 95 μl (μL) with 95% ethanol to obtain the amine guanidine A extract.

The detection conditions of UPLC-MS/MS in this example were as follows: mobile phase (A-water, B-acetonitrile), flow rate 0.4 mL/min, injection volume 4 μL, column temperature 35 °C. Dual mass spectrometer LCMS-8030, ion source ESI(+)/ESI(-) (simultaneous and negative ion simultaneous scanning), ion source interface voltage 4.5 kV/-3.5 kV, atomization gas-nitrogen 3.0 L/min, dry gas-nitrogen 15 L/min, collision gas-argon gas 230 kPa, desolvation tube temperature 250 ° C, heating module temperature 400 ° C, scanning range Q3 scan, m/z 200-800.

Please refer to the chromatograms detected by UPLC-MS/MS in the present embodiment, wherein the [m/z] ⁺ of the amine 蕈A is 455.3 (as shown in FIG. 4), [m/z] ^- is 453.3 (as shown in Fig. 5), and the retention time is 13.129 minutes and 13.204 minutes, respectively. From this, it is understood that this example can indeed produce anthraquinone containing triterpenoids. Mycelium or fruiting bodies, in particular, produce mycelium or fruiting bodies of Antrodia camphorata containing amine A.

Therefore, the method for cultivating the Antrodia camphorata of the present invention increases the number of times of inoculating the mycelium of the Antrodia camphorata in the column medium, and inoculating the mycelium of the Antrodia camphorata into the inside of the column medium, so that the Antrodia camphorata can be fully coated with the medium; Compared with the surface of the culture medium under aerobic conditions, the present invention inoculates the mycelium of Antrodia camphorata in a position relative to the internal environment of the anaerobic medium, and promotes the physiological environment of the Antrodia camphorata by providing a relatively anaerobic growth environment of Burdock. Terpenoids, in turn, inhibit the growth of bacteria by the antibiotic substances produced by the burdock.

Thereby, the method for cultivating the Antrodia camphorata of the present invention can increase the growth amount of the mycelium or fruit body of the Antrodia camphorata in the unit medium, and achieve the effect of increasing the content of the physiologically active substance such as the triterpenoid in the unit medium.

The method for culturing the Antrodia camphorata of the present invention is to prevent the growth of the bacteria by increasing the number of times of inoculating the mycelium of the Antrodia camphorata and densely distributing it inside the inoculated medium, thereby preventing the loss due to contamination by the bacteria.

While the invention has been described in connection with the preferred embodiments described above, it is not intended to limit the scope of the invention. The technical scope of the invention is protected, and therefore the scope of the invention is defined by the scope of the appended claims.

S1. . . Medium preparation step

S2. . . Inoculation step

S3. . . Training step

S4. . . Drying step

1. . . Cylinder medium

11. . . Bottom

12. . . Top surface

13. . . Side surface

D. . . spacing

P. . . Scheduled inoculation

S. . . Slash portion

Fig. 1 is a block diagram (1) of the method for cultivating the Antrodia camphorata of the present invention.

Fig. 2a is a perspective view of the cylindrical medium of the present embodiment - a tetrahedral cylinder.

Figure 2b: Stereoscopic view of the column culture medium of the present embodiment - a six-sided cylinder.

Figure 2c: Stereoscopic view of the column culture medium of the present embodiment - an octahedral column.

Fig. 2d is a perspective view of the cylindrical medium of the present embodiment - a twelve-sided cylinder.

Fig. 2e is a perspective view of a cylindrical medium of the present embodiment - a cylinder.

Fig. 3 is a block diagram (2) of the method for cultivating the Antrodia camphorata of the present invention.

Figure 4: UPLC-MS/MS chromatogram ([m/z] ⁺ ) of this example.

Figure 5: UPLC-MS/MS chromatogram ([m/z] ^- ) of this example.

S1. . . Medium preparation step

S2. . . Inoculation step

S3. . . Training step

Claims (7)

A method for cultivating Antrodia camphorata comprises: a medium preparation step of 0.1 to 50% by weight of glucose, 1 to 5% of peptone, 0.1 to 50% of barley extract, and 0.1 to 50% of yeast extract And 0.5~10% curing agent is prepared by mixing water into a mixed solution, and the mixed solution is boiled to completely dissolve the above components, and then the mixed solution is solidified into a column culture medium, and the depth of the column medium is 2~ 20 cm; an inoculation step is to set a plurality of predetermined inoculation sites in the interior of the column culture medium, the predetermined inoculation sites are 0.5 to 10 cm deep from the outer surface of the column medium, and inoculating the Ox And a culture step of incubating the inoculated column culture medium at a temperature of 23 to 28 ° C for at least three months to obtain a burdock cell. The method for cultivating Antrodia camphorata according to Item 1, wherein the culturing step further comprises a drying step of drying the column culture medium with burdock bacteria at 30-40 ° C until The water content is less than 2%, and a dried calf of the calf is obtained. The method for cultivating Antrodia camphorata according to claim 1 or 2, wherein each predetermined inoculation position is 0.5 to 2 cm apart. The method for cultivating Antrodia camphorata according to Item 3 of the patent application, wherein the column medium is a multi-faceted cylinder or a cylinder. The method for cultivating Antrodia camphorata according to claim 1 or 2, wherein the curing agent is agar. The method for cultivating Antrodia camphorata according to claim 1 or 2, wherein the culturing time of the culturing step is 4 to 12 months. The method for cultivating Antrodia camphorata according to Item 2 of the patent application, wherein the drying step has a drying time of 24 to 72 hours.