Title:  Method for propagating fungi using solid state fermentation

United States Patent:  6,558,943

Issued:  May 6, 2003

Inventors:  Li; Pei-Jung (Miaoli Hsien, TW); Shen; Chung-Guang (Taipei, TW)

Assignee:  Sun Ten Pharmaceutical Co., Ltd. (Taipei, TW)

Appl. No.:  655435

Filed:  September 5, 2000

A solid state fermentation (SSF) method is provided which is effective for both small- and large-scale fungal cultivation. Also provided is SSF media for fungal cultivation. The media preferably contains a carbon source and nitrogen source to provide a carbon to nitrogen ratio of about 5:1 to about 25:1 by weight. The media may also contain a vitamin and an inorganic substance. A preferred SSF medium contains malt extract, yeast extract, peptone, glucose, water, solid base, and calcium carbonate or gypsum. Before propagating fungal mycelia in the SSF medium, the mycelia may be pre-cultivated in a solid culture medium and then in a liquid medium. Although the SSF method can be used in growing most fungi, preferred fungi include Cordyceps sinensis, Ganoderma lucidum, Antrodia camphorata, Trametes versicolor, and Agaricus blazei. The SSF method not only produces high yield of fungi, but also stimulates the production of fungal metabolites, particularly the kinds with pharmaceutical and medicinal activities. Cordyceps sinensis is preferably grown to produce the active compound H1A which is a derivative of ergosterol.

SUMMARY OF THE INVENTION

The present invention provides a method for propagating fungi, especially the kinds of fungi which can be used for food, medicine, and health purposes. The method applies solid state fermentation (SSF) to propagate fungi. The preferred kinds of fungi that can be used in SSF include, but are not limited to, Cordyceps sinensis, Trametes versicolor, Antrodia camphorata, Agaricus blazei, and Ganoderma lucidum, which are all within the classes of Ascomycotina and Basidomycotina. Among these fungi, Cordyceps sinensis is the most preferred kind.

It is preferred that before SSF is to be applied to the propagation of a fungus, certain pre-cultivation steps are to be followed. First, wild, healthy fungus isolates of proper quality are selected. Alternatively, fungus isolates stored in liquid nitrogen are activated. These fungus isolates are disinfected. Their mycelia are cut into pieces under sterile condition and placed into a solid culture medium in a glass tube or plate. The solid culture medium is made of appropriate culture media such as potato dextrose agar (PDA), yeast extract agar (YEA), malt extract agar (MEA), yeast malt agar (YMA), and peptone yeast glucose agar (PYG).

After the mycelia have multiplied to cover most of the medium, approximately 0.5 cm in diameter of the mycelia are cut and transferred toga flask containing a liquid culture medium. The liquid culture medium is made of appropriate culture media such as potato dextrose broth (PDB), yeast extract broth (YEB), malt extract broth (MEB), yeast malt broth (YMB), and peptone yeast glucose broth (PYGB). The mycelia are grown under rotating shaking conditions for about 8 days, preferably about 5-6 days. Then, the mycelia are transferred to a larger shaker flask containing the same or different liquid culture medium and incubated under reciprocating shaking conditions for no more than 6 days, preferably about 3-4 days. This is followed by aeration/agitation and further incubation for 4-5 days. At this stage, the cultivated mycelia are ready for the SSF.

The SSF medium suitable for fungi propagation comprises a carbon source, a nitrogen source, vitamins, and inorganic substances. Additionally, trace elements and organic substances can be added. The carbon source is derived from at least one of the following: starch, glucose, monosaccharide, polysaccharide, dextrin, maltose, saccharose, methyl cellulose, fructose, turanose, and corn powder. The nitrogen source is derived from at least one of the following: defatted soybean powder, peptone, yeast paste, yeast syrup, peanut cake powder, yeast powder, wheat bran, casein, calcium caseinate, and defatted beancake powder. Vitamins include, but are not limited to, vitamin B₁, vitamin B₆, and nicotinic acid. Inorganic substances include, but are not limited to, calcium sulfate and calcium carbonate. The preferred ratio of the carbon source (C) and the nitrogen source (N) is about 5:1 to 25:1 by weight.

During the SSF, pH, water content in the SSF medium, temperature, relative humidity, and light cycle are properly controlled. The pH is preferred to be controlled at pH 4.5 to 7. The temperature is preferred to be controlled at 22+50^oC. The water content in the SSF medium is preferred to be between 40 and 70%. The relative humidity is preferred to be between 60 and 80%. The light cycle is preferred to be at 30% light and 70% dark.

The incubation period for fungus in the SSF culture is normally between 20 and 60 days, preferably between 30 and 50 days. The longer the incubation period, the greater the production of the mycelium dry weight. However, prolonged incubation of fungus in the SSF culture does not guarantee that the production of active material/metabolite is also proportionally increased. For instance, as shown in FIG. 2a (infra), when Cordyceps sinensis is incubated in the SSF culture, the mycelial dried weight (g) increases during the first 34 days of incubation and plateaued between 34 and 50 days. On the contrary, as shown in FIG. 2b (infra), the amount of H1A, which is a derivative of ergosterol, is peaked at 38 days after incubation in the SSF culture. The amount of H1A decreases significantly during prolonged SSF incubation.

Due to this discrepancy, high performance liquid chromatography (HPLC) analysis of the total nucleoside amount and the amount of H1A/ergosterol is used in addition to the measurements of dried mass to determine when would be the proper time to harvest the fungi. The total nucleoside content not only reflects the active life cycles of the fungus (i.e., the higher the nucleoside content, the greater the replication of the fungus) but also may relate to pharmacological activities (i.e., the amount of adenosine in Cordyceps sinensis has been postulated by Dr. Ming-Shi Shiao of the Veteran Hospital in Taipei, Taiwan, to be related to its pharmacological activities). For purposes of producing higher quantity of active metabolites, such as H1A/ergosterol from Cordyceps sinensis, the amount of H1A/ergosterol is monitored and analyzed by HPLC.

Alternatively, the mycelia of the fungus grown in SSF for about 6 to 14 days can be transferred to another SSF for continuous cultivation of the fungus. In other words, the propagation of fungus in SSF can be continued for many generations as long as fresh SSF is provided.

As a specific example, the present invention also provides a method for propagating Cordyceps sinensis in an SSF culture using an SSF medium. As described above, the SSF culture is preferred to be preceded by pre-cultivation of the mycelia first in a slant culture medium comprising a solid medium such as PDA, YEA, MEA, YMA, and PGY followed by incubation of the mycelia in a liquid culture containing a liquid culture media such as PDB, YEB, MEB, YMB and PYGB.

The SSF medium contains malt extract, yeast extract, peptone, glucose, water, a solid base, and calcium carbonate/gypsum. Examples of a solid base include, but not limited to, rice, coarse rice/unpolished rice, corn, wheat, nude wheat, oat, and oatmeal. It is preferable that the SSF medium contains 0.3-4% by weight of malt extract, 0.3-4% by weight of yeast extract, 0.1-2% by weight of peptone, 1-5% by weight of glucose, 30-70% by weight of water, 40-60% by weight of solid base, and 2% by weight of calcium carbonate or gypsum.

The SSF medium is made by first mixing malt extract, yeast extract, peptone, glucose, water, and a solid base together and heating them to boiling to form a solid base mixture. Then, the solid base mixture is allowed to cool down. Finally, calcium carbonate or gypsum is added to the cooled solid base mixture and the final mixture is granulated. The SSF granules are placed into a SSF bottle and sterilized. After the mycelia have been inoculated, aerated, and thoroughly mixed with the SSF granules, the SSF granules-containing bottle is sat on the shelf in the incubator until harvest time.

The SSF incubation period for Cordyceps sinensis is determined either by the total nucleoside content or the amount of H1A and/or ergosterol in the dried mycelium of Cordyceps sinensis, both monitored and quantified by BPLC. The mycelia of Cordyceps sinensis in SSF can be transferred to a fresh SSF so that the propagation of Cordyceps sinensis can be continued.

The present invention further provides two SSF media. The first SSF comprises a carbon source, a nitrogen source, vitamin, and inorganic substance. The carbon source comprises at least one of the following: starch, glucose, monosaccharide, polysaccharide, dextrin, maltose, saccharose, methyl cellulose, fructose, turanose, and corn powder. The nitrogen source comprises at least one of the following: defatted soybean powder, peptone, yeast paste, yeast syrup, peanut cake powder, yeast powder, wheat bran, casein, calcium caseinate, and defatted beancake powder. The vitamin comprises at least one of the following: vitamin B₁, vitamin B₆, and nicotinic acid. The inorganic substance comprises at least one of the following: calcium sulfate and calcium carbonate. The preferred carbon source (C) and nitrogen source (N) ratio is 5:1 to 25:1 by weight.

The second SSF medium comprises 0.3-4% by weight of malt extract (preferably 0.5-3%, and most favorably 2%), 0.3-4% by weight of yeast extract (preferably 0.5-3%, and most favorably 2%), 0.1-2% by weight of peptone (preferably 0.3-1%, and most favorably 0.5%), 1-5% by weight of glucose (preferably 2-4%, and most favorably 2%), 30-70% by weight of water (preferably 40-60%, and most favorably 50%), 30-70% by weight of solid base (preferably 40-60%, and most favorably 50%), and 0.3-4% by weight of calcium carbonate or gypsum (preferably 0.5-3%, and most favorably 2%).

Claim 1 of 30 Claims

We claim:

1. A method for propagating a fungus comprising:

propagating mycelia of said fungus in a solid state fermentation culture using a solid state fermentation medium;

wherein said fungus is one selected from the group consisting of Trametes versicolor, Agaricus blazei, Ganoderma luciduni, and Antrodia camphorata;

wherein said solid state fermentation medium comprises a carbon source (C), a nitrogen source (N), a vitamin and an inorganic substance; wherein said C: said N is at a ratio of about 5:1 to about 25:1 by weight; and

wherein before propagating said mycelia of said fungus in said solid state fermentation medium, said mycelia are pre-cultivated first in a solid culture medium and then in a liquid culture medium.
 

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