Patent Publication Number: US-2019169565-A1

Title: Medium and method for culturing antrodia cinnamomea

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     The application claims the benefit of Taiwan application serial No. 106142249, filed Dec. 1, 2017, the entire contents of which are incorporated herein by reference. 
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention generally relates to a medium and, more particularly, to a medium for culturing  Antrodia cinnamomea . The present invention also relates to a method for culturing  Antrodia cinnamomea  using the medium. 
     2. Description of the Related Art 
       Antrodia cinnamomea , a precious traditional Chinese medicine in Taiwan, grows only on inner rotten walls of hollow materials from a conserving species of  Cinnamomum kanehirai . Wild  Antrodia cinnamomea  is rich in physiologically active ingredients such as triterpenoids and polysaccharides. Triterpenoids possess effects such as anti-tumor, liver-protective, anti-dotal, anti-high blood lipid and pressure and immuno-modulating activities. Polysaccharides show effect on stimulation of macrophages, improvement of immune-regulation, clearance of free radicals and anti-hepatitis B virus. As a result, wild  Antrodia cinnamomea  becomes precious and expensive. 
     Due to several problems of wild  Antrodia cinnamomea , such as rare amount and difficulty in obtaining, industries tend to culture  Antrodia cinnamomea  by conventional methods and produce mycelia or fruit bodies of  Antrodia cinnamomea . Furthermore, physiologically active ingredients, such as triterpenes and polysaccharides, can be further extracted from the mycelia and fruit bodies of  Antrodia cinnamomea.    
     As an example, solid state fermentation is one of the conventional method for culturing mycelia of  Antrodia cinnamomea , using a conventional medium including substances such as wood flour, soybean powders, calcium carbonate (CaCO 3 ), glucose, etc. The solid state fermentation is wildly used in the industry due to the following reasons. First, the mycelia of  Antrodia cinnamomea  with similar physiologically active ingredients can be obtained due to the substances of the conventional medium. Second, the solid state fermentation is easy to operate. Third, the substances of the conventional medium are easily obtained. Fourth, the conventional medium used in the solid state should be kept in low water content, and thus is not easy to be used by other microorganisms. However, color of the mycelia of  Antrodia cinnamomea  obtained by the solid state fermentation is not red enough, and thus decreases the consumer&#39;s willingness to purchase the mycelia of  Antrodia cinnamomea  obtained by the solid state fermentation. In light of this, the conventional medium should be improved. 
     SUMMARY OF THE INVENTION 
     It is therefore an objective of the present invention to provide a medium for culturing  Antrodia cinnamomea , which is used to obtain mycelia of  Antrodia cinnamomea  in red color. 
     It is therefore another objective of the present invention to provide a method for culturing  Antrodia cinnamomea  to culture the mycelia of  Antrodia cinnamomea  by the medium. 
     One embodiment of the invention discloses the medium for culturing  Antrodia cinnamomea  includes a carbon source, a nitrogen source, vitamin A, agar and a solvent. The medium includes 8-16 grams/liter of the carbon source, 0.4-1.6 grams/liter of the nitrogen source and 4 grams/liter of vitamin A. Thus, mycelia of  Antrodia cinnamomea  grown on the medium can uptake vitamin A in the medium and the mycelia of  Antrodia cinnamomea  in red color can be obtained. 
     In an example, the carbon source is selected from the group consisting of malt extract, glucose, fructose, galactose, lactose, starch, cellulose, sucrose and combination therof. The nitrogen source is selected from the group consisting of peptone, yeast extract, whey protein, tryptone, amino acids, urea, ammonium acetate, ammonium chloride, ammonium nitrate, potassium nitrate, sodium nitrate and combination therof. 
     In an example, the medium can include 0.8% (w/v) of malt extract, 0.8% (w/v) of glucose and 0.04% (w/v) of peptone. Alternatively, the medium can include 0.8% (w/v) of malt extract, 0.08% (w/v) of peptone and 0.08% (w/v) of yeast extract. 
     Another embodiment of the invention discloses the method for culturing  Antrodia cinnamomea . The method includes inoculating a medium with mycelia of  Antrodia cinnamomea . The mycelia of  Antrodia cinnamomea  inoculated on the medium is then cultured at 20-27° C. for 28-50 days. Thus, mycelia of  Antrodia cinnamomea  grown on the medium can uptake vitamin A in the medium and the mycelia of  Antrodia cinnamomea  in red color can be obtained. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. 
         FIG. 1  depicts a chemical structure of vitamin A. 
         FIG. 2 a    depicts photographs illustrating growth status of mycelia of  Antrodia cinnamomea  grown on medium according to a first embodiment of the present invention. 
         FIG. 2 b    depicts a bar chart illustrating average diameter of the mycelia of  Antrodia cinnamomea  grown on medium according to the first embodiment of the present invention. 
         FIG. 3 a    depicts photographs illustrating growth status of mycelia of  Antrodia cinnamomea  grown on medium according to a second embodiment of the present invention. 
         FIG. 3 b    depicts a bar chart illustrating average diameter of the mycelia of  Antrodia cinnamomea  grown on medium according to the second embodiment of the present invention. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Vitamin A with a chemical structure shown in  FIG. 1  is one of the fat-soluble vitamins. Vitamin A does not easily lose due to metabolism, and can be kept in cells, which is appreciated by a person having ordinary skill in the art. Detail description is not given to avoid redundancy. 
     Specifically, vitamin A can be used as an additive in a medium for culturing  Antrodia cinnamomea . That is, 1 liter of the medium includes 4 grams of vitamin A. Therefore, when the medium is used to culture mycelia of  Antrodia cinnamomea , as an example,  Antrodia cinnamomea  strains with BCRC number of BCRC 35396, BCRC 35716, BCRC 36401 and BCRC 37848, vitamin A in the medium can be uptaken by the mycelia of  Antrodia cinnamomea  and the mycelia of  Antrodia cinnamomea  in red color can thus be obtained. 
     Moreover, the medium can further include a carbon source as well as a nitrogen source for growth of  Antrodia cinnamomea . As an example, the medium can include 0.8-1.6% (w/v) of a carbon source and 0.04-0.16% (w/v) of a nitrogen source. That is, 8-16 grams of the carbon source, 0.4-1.6 grams of the nitrogen source and 4 grams of vitamin A are mixed, and a solvent, such as distilled water, is added until the total volume is 1 liter. The carbon source can be selected from the group consisting of malt extract, glucose, fructose, galactose, lactose, starch, cellulose, sucrose and combination therof. The nitrogen source can be selected from the group consisting of peptone, yeast extract, whey protein, tryptone, amino acids, urea, ammonium acetate (CH 3 COONH 4 ), ammonium chloride (NH 4 Cl), ammonium nitrate (NH 4 NO 3 ), potassium nitrate (KNO 3 ), sodium nitrate (NaNO 3 ) and combination therof. In addition, the medium can also include proper amount of agar, and thus the medium can be formed in a solid state and can be used in the solid state fermentation. 
     The medium according to a first embodiment of the present invention includes 0.8% (w/v) of malt extract, 0.8% (w/v) of glucose, 0.04% (w/v) of peptone, 0.4% (w/v) of vitamin A, 0.8% (w/v) of agar and 97.16% (w/v) of water. Besides, the medium according to a second embodiment of the present invention includes 0.8% (w/v) of malt extract, 0.08% (w/v) of peptone, 0.08% (w/v) of yeast extract, 0.4% (w/v) of vitamin A, 0.8% (w/v) of agar and 97.84% (w/v) of water. 
     Therefore, the medium according to the first and the second embodiments of the present invention can be applied in a method for culturing  Antrodia cinnamomea . The medium is inoculated with the mycelia of  Antrodia cinnamomea , and the mycelia of  Antrodia cinnamomea  inoculated on the medium is cultured at 20-27° C. for 28-50 days. The mycelia of  Antrodia cinnamomea  in red color can thus be obtained. 
     To evaluate the medium with vitamin A as the additive can be used to obtain the mycelia of  Antrodia cinnamomea  in red color, the mycelia of  Antrodia cinnamomea  is inoculated on the medium listed in TABLE 1, and cultured at 20-27° C. for 35 days. Growth status and average diameter of colonies on the medium on days 0, 7, 14, 28 and 35 are shown in  FIGS. 2 a  and 2 b   . 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 1 
               
             
            
               
                   
                   
               
               
                   
                 Medium 
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Malt 
                   
                 Vitamin 
                 Growth status 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 extract 
                 Glucose 
                 Peptone 
                 A 
                 Biomass 
                 Growth rate 
               
               
                 Group 
                 (%) 
                 (%) 
                 (%) 
                 (%) 
                 (mg) 
                 (R 2 ) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 A0 
                 0.8 
                 0.8 
                 0.04 
                 0 
                 3.4 
                 0.2174 
               
               
                   
                   
                   
                   
                   
                   
                 R 2  = 0.9598 
               
               
                 A1 
                 0.8 
                 0.8 
                 0.04 
                 0.4 
                 6.85 
                 0.1767 
               
               
                   
                   
                   
                   
                   
                   
                 R 2  = 0.8968 
               
               
                 A2 
                 0.8 
                 0.8 
                 0.04 
                 0.8 
                 4.49 
                 0.0398 
               
               
                   
                   
                   
                   
                   
                   
                 R 2  = 0.9251 
               
               
                   
               
            
           
         
       
     
     Referring to  FIG. 2 a   , the mycelia of  Antrodia cinnamomea  obtained by using the medium of groups A0-A2 have color with hexadecimal color codes of #FFC78E, #FF5809 and #FFEEDD, respectively. That is, the mycelia of  Antrodia cinnamomea  obtained by using the medium of group A1 (with vitamin A in the concentration of 0.4% (w/v)) is redder than the mycelia of  Antrodia cinnamomea  obtained by using either the medium of group A0 (without vitamin A) or the medium of group A2 (with vitamin A in a concentration of 0.8% (w/v)). Moreover, referring to TABLE 1, on day 35, biomass of the mycelia of  Antrodia cinnamomea  obtained by using the medium of group A1 significantly increases compared to biomass of the mycelia of  Antrodia cinnamomea  obtained by using the medium of group A0 or A2. Referring to  FIG. 2 b   , the mycelia of  Antrodia cinnamomea  obtained by using the medium of group A1 has similar diameter with the mycelia of  Antrodia cinnamomea  obtained by using the medium of group A0. 
     In addition, the mycelia of  Antrodia cinnamomea  is inoculated on the medium listed in TABLE 2, and cultured at 20-27° C. for 35 days. Growth status and average diameter of colonies on the medium on days 0, 7, 14, 28 and 35 are shown in  FIGS. 3 a  and 3 b   . 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 2 
               
             
            
               
                   
                   
               
               
                   
                 Medium 
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Malt 
                   
                 Vitamin 
                 Growth status 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 extract 
                 Glucose 
                 Peptone 
                 A 
                 Biomass 
                 Growth rate 
               
               
                 Group 
                 (%) 
                 (%) 
                 (%) 
                 (%) 
                 (mg) 
                 (R 2 ) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 B0 
                 0.8 
                 0.08 
                 0.08 
                 0 
                 2.60 
                 0.1893 
               
               
                   
                   
                   
                   
                   
                   
                 R 2  = 0.9763 
               
               
                 B1 
                 0.8 
                 0.08 
                 0.08 
                 0.4 
                 4.35 
                 0.2078 
               
               
                   
                   
                   
                   
                   
                   
                 R 2  = 0.9356 
               
               
                 B2 
                 0.8 
                 0.08 
                 0.08 
                 0.8 
                 5.40 
                 0.0908 
               
               
                   
                   
                   
                   
                   
                   
                 R 2  = 0.9693 
               
               
                   
               
            
           
         
       
     
     Referring to  FIG. 3 a   , the mycelia of  Antrodia cinnamomea  obtained by using the medium of groups B0-B2 have color with hexadecimal color codes of #FFD1A4, #FF8040 and #FFEEDD, respectively. That is, the mycelia of  Antrodia cinnamomea  obtained by using the medium of group B1 (with vitamin A in the concentration of 0.4% (w/v)) is redder than the mycelia of  Antrodia cinnamomea  obtained by using either the medium of group B0 (without vitamin A) or the medium of group B2 (with vitamin A in a concentration of 0.8% (w/v)). Moreover, referring to TABLE 2, on day 35, biomass of the mycelia of  Antrodia cinnamomea  obtained by using the medium of group B1 significantly increases compared to biomass of the mycelia of  Antrodia cinnamomea  obtained by using the medium of group B0 or B2. Referring to  FIG. 3 b   , the mycelia of  Antrodia cinnamomea  obtained by using the medium of group B1 has similar diameter with the mycelia of  Antrodia cinnamomea  obtained by using the medium of group B0. 
     Accordingly, the mycelia of  Antrodia cinnamomea  grown on the medium can uptake vitamin A in the medium and the mycelia of  Antrodia cinnamomea  in red color can be obtained. Therefore, the consumer&#39;s willingness to purchase the mycelia of  Antrodia cinnamomea  increases. 
     Although the invention has been described in detail with reference to its presently preferable embodiment, it will be understood by one of ordinary skill in the art that various modifications can be made without departing from the spirit and the scope of the invention, as set forth in the appended claims.