Method for propagating fungi using solid state fermentation

Details Method for propagating fungi using solid state fermentation Method for propagating fungi using solid state fermentation

2000-09-05

2003-05-06

Naff, David M. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Fungi

C424S093500, C435S256800, C514S169000

Reexamination Certificate

active

06558943

ABSTRACT:

I. FIELD OF THE INVENTION
The present invention relates to a method for propagating fungi using solid state fermentation (SSF). SSF is particularly suitable for propagating fungi which are used for food, medicine, or health purposes. The kinds of fungi which can be propagated by SSF include, but not limited to,
Cordyceps sinensis, Trametes versicolor, Antrodia camphorata, Agaricus Blazei
, and
Ganoderma Lucidum
. The present invention also relates to the formulations and preparations of SSF media.
II. DESCRIPTION OF THE RELATED ART
Cordyceps sinensis
is a parasitic fungus that has been used as a traditional Chinese medicine since ancient times. It is particularly famous for treating patients with kidney failure and asthma. It is also known for its anti-tumor effects.
Recently, Lin et al.,
J. Lab. Clin. Med
., 133:55-63 (1999), reported a finding of an active compound named “H1A” in the fruiting body of
Cordyceps sinensis
. H1A is a derivative of ergosterol having the chemical formula of:
Ergosterol is (3&bgr;, 22E)-Ergosta-5,7,22-trien-3-ol. H1A appears to have pharmacological effects on the immune system, renal function, and cardiovascular system. It has also been known to have clinical effects on suppressing the activated human mesangial cells (HMC) and alleviating IgA nephropathy (Berger's Disease), thus, preventing the disease from progressing to the uremia stage. Lin et al.'s
J. Lab. Clin. Med
., 133:55-63 (1999) article is herein incorporated by reference.
The authors of the
J. Lab. Clin. Med
., 133:55-63 (1999) article, Lin et al., have a U.S. patent (U.S. Pat. No. 5,582,828) disclosing a method for identifying and isolating H1A from
Cordyceps sinensis
, which is also herein incorporated by reference.
Wild
Cordyceps sinensis
is extremely rare and difficult to obtain. The conventional cultivation methods for
Cordyceps sinensis
include solid media stationary incubation, liquid media rotating shaking incubation, liquid-state fermentation, and submerged liquid-state fermentation. Solid media stationary incubation uses solid media, such as malt extract agar (MEA), potato dextrose agar (PDA), or YEA (yeast extract agar), as culture media. Liquid media rotating shaking incubation and liquid-state fermentation use liquid media, such as malt extract broth (MEB), potato dextrose broth (PDB), or YEB (yeast extract broth) as culture media. They are known to be expensive and produce low yields of microorganisms. Moreover, it is not known that
Cordyceps sinensis
produced by these methods can generate a sufficient amount of H1A.
Submerged liquid-state fermentation is known for its capability of generating a high yield of microorganisms. However, this method requires high capital investment due to high energy and waste output. Also, because the fermentation cycle of the submerged liquid-state fermentation is generally short, usually five to seven days, it is doubtful that the metabolites generated by this method are sufficient enough to be of significance for medicinal uses.
Solid state fermentation (SSF) has been known for the production of enzymes and organic acids used in the paper pulp bleaching treatment industry and for producing organic pesticides. It is also widely used for producing soy sauce, alcoholic beverages from fruit or unpolished rice, and Chinese sour cabbage. SSF refers to the growth of microorganisms on moist solid substrates in systems with a continuous gas flow without the presence of free liquid between substrate particles. SSF has never been used for fungal propagation, particularly for fungi belonging to the classes of Ascomycotina and Basidomycotina in Eumycota.
There are two common types of solid bases used in an SSF culture. The first type uses “natural solid materials” as a solid base. For example, food stuffs or agricultural byproducts are used as a solid base not only to provide a physical matrix for microbial growth but also to be used as the main source of microbial nutrients. The second type uses an “inert solid support supplemented with nutrients” as a solid base. This type of solid base acts only as a physical support for microbial growth.
An SSF culture contains three phases, which are: (a) a solid phase which comprises the solid base as described above; (b) a liquid phase which is bound to the solid phase where intraparticle mass transfer occurs; and (c) a continuous gas phase. The gas phase controls the temperature and moisture of the SSF culture. It also provides oxygen for fungal growth.
In contrast to other fermentation methods, SSF requires low capital investment due to low energy and waste output. The base materials and media used in SSF are generally cheaper and simpler than other methods. In addition, the SSF medium generally contains low water content which not only reduces the risk of contamination but also offers a favorable condition for fungal growth, because it resembles the natural habitats for fungi. In fact, the SSF culture allows the fungal spores to proliferate because they can lay onto the surface of the solid base.
SSF is also known to have the following disadvantages. First, it is very difficult to monitor and control the parameters (such as moisture, pH, temperature, substrate concentration etc.) of the fermentation process in an SSF culture. Second, thus far the underlying scientific and engineering basis of SSF is still poorly understood. Finally, direct quantitative measurements for biomass in an SSF culture are difficult. In fact, many of the studies done so far on SSF have been done in either qualitative or empirical levels.
In the invention to be presented in the following sections, a method for propagating fungi using SSF will be described. The present invention not only will teach a useful SSF medium to grow fungi with high yields of metabolites but also will teach ways to monitor and control the parameters of the fermentation such as moisture, pH, temperature, and substrate concentration to maximize the production of fungi. The present invention also will provide direct quantitative and qualitative measurements of the biomass and active ingredients. Finally, the present invention will provide a method for effective production of
Cordyceps Sinensis
and its active substance H1A.
III. 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 incub

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