<i>Ganoderma lucidum </i>strain suitable for large-scale liquid fermentation culture and method of using the strain

Abstract

Disclosed is a Ganoderma Lucidum strain suitable for large-scale liquid fermentation, named as Ganoderma Lucidum G2, deposited with China General Microbiological Culture Collection Center under the accession number CGMCC No. 3982 on Jul. 20, 2010, and a method of mutation breeding the same and use of the strain. The Ganoderma Lucidum strain which belongs to Ganodermataceae, genus Ganoderma, species red Ganoderma Lucidum is obtained by artificial mutagenizing and breeding. The production of mycelia using the Ganoderma Lucidum strain G2 is 80120 times higher as compared with the production of mycelia using wild-type nave Ganoderma Lucidum strain. The mycelia produced using the Ganoderma Lucidum strain G2 have higher contents of main pharmaceutical ingredients. The Ganoderma Lucidum strain suitable for large-scale liquid fermentation can be used for manufacturing oral solution or beverage comprising Ganoderma mycelia or extracts of Ganoderma mycelia as main active ingredient.

Claims

1. A method of culturing a Ganoderma lucidum strain named Ganoderma lucidum G2 that was deposited with China General Microbiological Culture Collection Center under the accession number CGMCC No. 3982 on Jul. 20, 2010 in a large-scale liquid fermentation process in manufacturing of an oral solution or a beverage that includes Ganoderma mycelia or extracts of Ganoderma mycelia as a main active ingredient.

2. The method of claim 1, wherein the Ganoderma mycelia is obtained by liquid fermentation with the following culture conditions: the medium for culturing the Ganoderma lucidurn G2 has the following composition: TABLE-US-00010 (NH.sub.4).sub.2HPO.sub.4 0.8 g KH.sub.2PO.sub.4 0.5 g MgSO.sub.4 7H.sub.2O 0.05 g NaCl 1 g FeSO.sub.4 7H.sub.2O 0.03 g sucrose 25 g maltose 5 g peptone 3 g silkworm chrysalis meal 2 g extract of potato 1000 ml; wherein the extract of potato is prepared by boiling 300 g of potato pieces in 1000 ml of purified water for 20 minutes followed by filtration; and the fermentation condition comprises: optimal culture temperature: 321 C.; content of dissolved oxygen: 0.05 mmol/L-0.20 mmol/L; and pH: 4.5-5.5.

3. A method of manufacturing an oral solution or a beverage comprising: selecting the Ganoderma lucidum strain named Ganoderma lucidum G2, deposited with China General Microbiological Culture Collection Center under the accession number CGMCC No. 3982 on Jul. 20, 2010; performing a large-scale fermentation process of the Ganoderma lucidum G2; obtaining one or more of Ganoderma mycelia and extracts of Ganoderma mycelia through the large-scale fermentation process; selecting one or more of the Ganoderma mycelia and extracts of Ganoderma mycelia; and incorporating the selected one or more of the Ganoderma mycelia and extracts of Ganoderma mycelia as a main active ingredient in an oral solution or a beverage.

4. The method according to claim 3, wherein the large-scale fermentation process is a liquid fermentation process.

5. The method according to claim 4, wherein the Ganoderma mycelia are obtained through the liquid fermentation process.

6. The method according to claim 5, wherein the liquid fermentation process includes: using a medium for culturing the Ganoderma lucidum G2, wherein the medium has the following composition: TABLE-US-00011 (NH.sub.4).sub.2HPO.sub.4 0.8 g KH.sub.2PO.sub.4 0.5 g MgSO.sub.4 7H.sub.2O 0.05 g NaCl 1 g FeSO.sub.4 7H.sub.2O 0.03 g sucrose 25 g maltose 5 g peptone 3 g silkworm chrysalis meal 2 g extract of potato 1000 ml.

7. The method according to claim 6, further comprising: preparing the extract of potato by boiling 300 g of potato pieces in 1000 ml of purified water for 20 minutes, followed by filtration thereof.

8. The method according to claim 6, further comprising: performing the fermentation under a fermentation condition that includes: an optimal culture temperature of 321 C.; a content of dissolved oxygen of 0.05 mmol/L-0.20 mmol/L; and a pH of 4.5 to 5.5.

9. The method according to claim 4, wherein the Ganoderma mycelia obtained from the large-scale liquid fermentation is a raw mycelia culture.

10. The method according to claim 9, further comprising: adding one or more adjuvants and excipients to the raw mycelia culture.

11. The method according to claim 10, further comprising: selecting the one or more adjuvants and excipients based on a healthcare activity for which the oral solution or beverage is to be ingested by a person.

12. The method according to claim 11, further comprising selecting one or more of a polysaccharide extract of poria cocos and a polysaccharide extract of polyporus umbellatus as adjuvants.

13. The method according to claim 11, further comprising utilizing one or more of the polysaccharide extract of poria cocos and the polysaccharide extract of polyporus umbellatus for treatment of cancer patients.

14. The method according to claim 11, further comprising selecting one or more of vitamins and trace elements as adjuvants.

15. The method according to claim 11, wherein the healthcare activity for which the one or more adjuvants and excipients are selected include blood viscosity, cleansing blood, promoting cell activation, preventing arteriosclerosis, improving metabolism, enhancing immunity, improving sleep quality, improving male health, calming nerves, scavenging free radicals, treating cancers, preventing cancers, and delaying aging.

16. The method according to claim 3, wherein the extract of Ganoderma obtained from the large-scale fermentation process is one of Ganoderma polysaccharide, Ganoderma organic germanium, and Ganoderma terpenes.

Description

DETAILED DESCRIPTION OF EMBODIMENTS

(1) The embodiments of the present application will be described with reference to the following examples. It should be understood that, the following examples are part, but not all, of the embodiments of the present application. Based on the following examples, a person skilled in the art would conceive of other variations without inventive effort, which are within the scope of the claims.

Example 1

(2) A Ganoderma Lucidum strain suitable for large-scale liquid fermentation, which is named as Ganoderma Lucidum G2, deposited with China General Microbiological Culture Collection Center under the accession number CGMCC No. 3982 on Jul. 20, 2010.

(3) The Ganoderma Lucidum strain suitable for large-scale liquid fermentation of the present application is obtained by artificial mutagenizing and breeding, and is suitable for large-scale liquid fermentation. The Ganoderma Lucidum strain of the present application belongs to Ganodermataceae, genus Ganoderma, species Ganoderma Lucidum (W.curt.:Fr.)Karst.

(4) The condition suitable for growth of mycelia comprises a temperature of 30 C.1 C., pH of 4.55.5, culture duration of 1012 days. The temperature suitable for growth of sporocarp is 25 C.30 C., and its growth cycle is usually 1 to 3 years. Pileus is in semicircle, kidney or circle shape, and in woody texture. Pileus has a width of 515 cm, and a thickness of 0.81 cm. Pileus is reddish-brown and has paint gloss. Pileus has ringlike ridges, radial wrinkles, and a thin edge usually introverted. The flesh is white or light brown. The tube face is initially white, then becomes light brown, and then brown. Tubes have a density of 35/mm. Stalk grow by side and occasionally grow in a deflected direction. Stalk has a length of 315 cm, and a diameter of 13 cm. Stalk is purple-brown and has paint gloss. Spores are brown, and in an egg shape. Spores have sizes of 912 m4.57.5 m. Sporocarp has a middle to large size and may be larger.

(5) The production of mycelia using the Ganoderma Lucidum strain G2 of the present application is 80120 times higher as compared with the production of mycelia using wild-type nave Ganoderma Lucidum strain. The mycelia have contents of main pharmaceutical ingredients higher than those in wild-type nave Ganoderma Lucidum strain, including (in wt/wt %) Ganoderma polysaccharide 16.8%, Ganoderma organic germanium 5.8%, terpenes with 28 or less C atoms 5.6%, small proteins (LZ-8) 16.6%, adenosine 3.6%, and mannitol 4.6%.

(6) The medium used for culturing the Ganoderma Lucidum strain G2 suitable for large-scale liquid fermentation of the present application has the following composition:

(7) TABLE-US-00002 (NH.sub.4).sub.2HPO.sub.4 0.8 g KH.sub.2PO.sub.4 0.5 g MgSO.sub.4 7H.sub.2O 0.05 g NaCl 1 g FeSO.sub.4 7H.sub.2O 0.03 g sucrose 25 g maltose 5 g peptone 3 g silkworm chrysalis meal 2 g extract of potato 1000 ml;

(8) wherein the extract of potato is prepared by cutting 300 g of potato into pieces, boiling the potato pieces in 1000 ml of purified water for 20 min followed by filtration;

(9) and the fermentation condition includes:

(10) optimal culture temperature: 321 C.;

(11) content of dissolved oxygen: 0.05 mmol/L0.20 mmol/L;

(12) pH: 4.55.5.

(13) The large-scale culture leads to the yield of dry mycelia of about 68 g/1000 ml. Main pharmaceutical ingredients in mycelia include (in wt/wt %) Ganoderma polysaccharide 16.8%, Ganoderma organic germanium 5.8%, terpenes with 28 or less C atoms 5.6%, small proteins (LZ-8) 16.6%, adenosine 3.6%, and mannitol 4.6%. The production of mycelia is 80-120 times higher as compared with the production of mycelia using wild-type nave Ganoderma Lucidum strain.

Example 2

(14) The method of obtaining a Ganoderma Lucidum strain suitable for large-scale liquid fermentation by mutagenizing and breeding comprises the following steps.

(15) Step 1: Subjecting pileus and stipe of non-lignified wild Ganoderma fruiting body collected from Prunus mume Apricot to protoplast isolation, inoculating the isolate thus obtained into a basic medium for culturing at a temperature of 28 C.30 C. for 15 days, and subjecting the resultant Ganoderma mycelia to repeated isolation and purification, thereby obtaining wild-type nave Ganoderma mycelia;

(16) wherein the basic medium used in protoplast isolation and purification carried out on pileus and stipe of non-lignified wild Ganoderma fruiting body has the following composition:

(17) TABLE-US-00003 (NH.sub.4).sub.2HPO.sub.4 0.3 g KH.sub.2PO.sub.4 0.2 g MgSO.sub.47H.sub.2O 0.02 g NaCl 0.5 g FeSO.sub.47H.sub.2O 0.03 g sucrose 20 g sterile water 1000 ml.

(18) Step 2: Inoculating the wild-type nave Ganoderma mycelia from Step 1 into a basic medium for culturing, then into a fermentation broth for culturing in a shaker, thereby obtaining a suspension of the wild-type nave Ganoderma mycelia;

(19) wherein (1) the components and their contents of the basic medium was optimized by a single factor orthogonal experiment, in order to obtain a medium suitable for the growth of wild-type nave Ganoderma.

(20) The orthogonal experiment was carried out as follows.

(21) 5000 ml of purified water was divided into 25 flasks with 200 ml for each. The flasks were numbered 125, and divided into five groups, i.e., Nos. 15, Nos. 610, Nos. 1115, Nos. 1620, and Nos. 2125. Basic medium with different composition was added into the flasks respectively, as shown in table 1.

(22) TABLE-US-00004 TABLE 1 (unit: g) basic medium Nos. 1~5 6~10 11~15 16~20 21~25 (NH.sub.4).sub.2HPO.sub.4 0.3 0.6 0.5 0.3 0.8 KH.sub.2PO.sub.4 0.5 0.5 0.2 0.5 0.3 MgSO.sub.47H.sub.2O 0.02 0.05 0.04 0.04 0.05 NaCl 0.5 0.5 0.8 0.5 0.5 FeSO.sub.47H.sub.2O 0.03 0.03 0.03 0.03 0.03 sucrose 20 30 20 30 20 peptone 3 3 2 3 2

(23) The medium was subjected to high pressure sterilization and then used for culturing the nave Ganoderma mycelia for 12 days. Then, the mycelia in flasks No. 125 were filtered, and dried at a temperature of 1051 C. for 24 h. Then, the dried mycelia were weighed, and the average weight of mycelia for each group was calculated and compared with each other. An optimal basic medium with following composition was established.

(24) TABLE-US-00005 (NH.sub.4) .sub.2HPO.sub.4 0.6 g KH.sub.2PO.sub.4 0.5 g MgSO.sub.4 7H.sub.2O 0.05 g NaCl 0.5 g FeSO.sub.4 7H.sub.2O 0.03 g sucrose 30 g peptone 3 g sterile water 1000 ml.

(25) (2) Based on the optimal basic medium established from the above single factor orthogonal experiment, a multiple factor orthogonal experiment was carried out by selecting different nitrogen sources, carbon sources, trace elements, culturing temperature and oxygen requirement as the factors. The optimal fermentation conditions finally established included the following.

(26) The fermentation broth has the following composition:

(27) TABLE-US-00006 (NH.sub.4).sub.2HPO.sub.4 0.8 g KH.sub.2PO.sub.4 0.5 g MgSO.sub.47H.sub.2O 0.05 g NaCl 1 g FeSO.sub.47H.sub.2O 0.03 g sucrose 25 g maltose 5 g peptone 3 g silkworm chrysalis meal 2 g sterile water 1000 ml;
and

(28) the fermentation condition includes:

(29) optimal culture temperature: 321 C.;

(30) content of dissolved oxygen: 0.05 mmol/L0.20 mmol/L;

(31) pH: 4.55.5.

(32) Step 3: subjecting the wild-type nave Ganoderma mycelia from Step 2 to a combinatory mutagenizing and breeding process which combines UV radiation, NaN.sub.3 chemical mutagenesis and transient heating in sterile water at 80 C. 90 C., thereby obtaining a Ganoderma Lucidum strain suitable for efficient production of Ganoderma mycelia in large-scale liquid fermentation.

(33) In Step 3, UV radiation is performed first. In particular, three samples of suspensions containing wild-type nave Ganoderma mycelia (10 wt/wt %) were prepared, and named as Sample 1, Sample 2 and Sample 3. the wild-type nave Ganoderma mycelia were irradiated with a 40 W UV lamp from a distance of 20 cm, and the radiation durations were set as below.

(34) TABLE-US-00007 Sample No. 1 2 3 radiation duration (min) 5 10 20

(35) Next, Sample 2 which is irradiated for 10 min had a lethality rate of 93% was selected in further experiments.

(36) After culturing Sample 2, three samples of suspensions containing mycelia (10 wt/wt %) were prepared, and named Sample 4, Sample 5 and Sample 6. The samples were subjected to chemical mutagenesis with a chemical mutagenesis agent.

(37) NaN.sub.3 is selected as the chemical mutagenesis agent. In particular, 1 g of NaN.sub.3 of 1.2 equivalent was dissolved into a mixture of 5 ml of dimethylformamide (DMF) and 5 ml of dimethyl sulfoxide (DMSO), thereby obtaining the chemical mutagenesis agent. Sample 4, Sample 5 and Sample 6 were added to the chemical mutagenesis agent for chemical mutagenesis treatment. The durations of chemical mutagenesis were set as follows.

(38) TABLE-US-00008 Sample Nos. 4 5 6 duration of chemical mutagenesis (min) 5 10 20

(39) Finally, transient heating in sterile water was carried out. In particular, Sample 4, Sample 5 and Sample 6 were washed with sterile water three times using centrifugation, and then subjected to transient heating in sterile water at 80 C.90 C. The durations of transient heating were set as follows.

(40) TABLE-US-00009 Sample Nos. 4 5 6 duration of transient heating ( sec ) 1 5 10

(41) Sample 6 was subjected to transient heating for 10 sec with a lethality rate of 96%. The survived strain was the Ganoderma Lucidum G2 which is suitable for liquid fermentation and can lead to high production of Ganoderma mycelia. The strain was deposited with China General Microbiological Culture Collection Center under the accession number CGMCC No. 3982.

Example 3

(42) This example relates to a use of a Ganoderma Lucidum strain suitable for large-scale liquid fermentation in manufacturing oral solution or beverage comprising Ganoderma mycelia or extracts of Ganoderma mycelia as main active ingredient.

(43) In Example 3, Ganoderma mycelia are obtained by large-scale liquid fermentation with the medium and conditions described in Example 1.

(44) The Ganoderma Lucidum strain of the present application is subjected to large-scale liquid fermentation to yield Ganoderma mycelia. Then, the Ganoderma mycelia, as main active ingredient, are used to prepare Ganoderma oral solution. The Ganoderma oral solution is enriched in Ganoderma polysaccharide, Ganoderma organic germanium, triterpenes, and other active ingredients, and has a stable composition.

(45) The oral solution or beverage may be a raw mycelia culture obtained from large-scale liquid fermentation of the Ganoderma Lucidum strain of the present application. The oral solution is advantageous in many aspects, e.g., immunity improvement, blood pressure control, lowering of blood viscosity, blood cleaning, cell activation promotion, arteriosclerosis prevention, metabolism improvement, sleep ameliorating, anti-allergy, male health improvement, nerves calming, and delaying aging.

(46) The oral solution or beverage may also be a Ganoderma oral solution or beverage with particular healthcare activity prepared by supplementing a raw mycelia culture obtained from large-scale liquid fermentation of the Ganoderma Lucidum strain of the present application as the main active ingredient with adjuvants or excipients directed to the particular healthcare activity.

(47) For example, polysaccharide extract of poria cocos, or polysaccharide extract of polyporus umbellatus may be added as medical adjuvants to prepare a Ganoderma oral solution especially beneficial to cancer patients. Vitamins or trace elements may be added as energy adjuvants to prepare a functional Ganoderma oral solution for improving physical ability.