WO2014170911A2

WO2014170911A2 - Process for continuous production of mushroom

Info

Publication number: WO2014170911A2
Application number: PCT/IN2014/000217
Authority: WO
Inventors: Karuppan PERUMAL; Velusamy KARUPPURAJ; Subramanian Chandra SEKARENTHIRAN
Original assignee: Shri Amm Murugappa Chettiar Research Centre (Mcrc); Department Of Biotechnology (Dbt)
Priority date: 2013-04-08
Filing date: 2014-04-08
Publication date: 2014-10-23
Also published as: WO2014170911A3

Abstract

The invention is directed to an organic method of cultivation of mushroom and more particularly the fruit bodies of G. lucidum (reishi) on various cellulosic substrates for continuous production throughout the year, by shortening the crop cycle. In this process, multiple context tissue is used for growing mycelia. The spawn is grown using maize grains supplemented with source of calcium, which increases its survival. The grown mycelium in the spawn is transferred by mixing and layer method to a multiple mushroom bed and transferred to a cropping room and then to the harvesting room. The first harvest starts in 22 to 36 days with the total days of crop cycle being 52-54 days, with bio-efficiency up to 90%. In the post-harvesting stage the harvested materials are covered with sheets to get a quality product.

Description

PROCESS FOR CONTINUOUS PRODUCTION OF MUSHROOM

Field of invention

The invention relates to an organic process technology for continuous production of mushroom or fruiting bodies. The invention more particularly provides an economical process technology for growing fruiting bodies G. lucidum (reishi).

Background of the invention

Ganoderma lucidum (G. lucidum) is commonly known as Lingzhi mushroom or reishi mushroom, encompasses several fungal species of the genus Ganoderma, and most commonly refers to the closely related species, Ganoderma lucidum and Ganoderma tsugae. G. lucidum enjoys special veneration in East Asia, where it has been used as a medicinal mushroom in traditional Chinese medicine for more than 2,000 years, making it one of the oldest mushrooms known to have been used medicinally. Lingzhi is listed in the American Herbal Pharmacopoeia and Therapeutic Compendium.

The amount of wild mushroom is not sufficient for commercial exploitation and traditional mushroom cultivation takes several months. Its cultivation on solid substrates, stationary liquid medium or by submerged cultivation has become essential to meet the increasing demands in the international markets (Mizuno et ai, 1995). G. lucidum is being cultivated artificially in over ten countries with an annual production of 4,300 tons.

Henmi et al., 1937 attempted an artificial cultivation of Reishi and Naoi achieved mass production in 1971 by cultivating it in sawdust-containing pots. The cultivation method uses bed logs or sawdust. G. lucidum growing on saw dust produced a bioefficiency of 17.3 % (Tnratana et al., 1991 ; Chen, 1999).

The most popular method of production of known G. lucidum is by wood log method of cultivation. The method of cultivation has limitations such as limited availability of wood logs, saw dust and tree products, requires extensive time for the fruit body harvest (6- 12 months) and it is not very cost-effective. National Research Centre for Mushroom Solan, India has brought out a dossier on the cultivation of reishi. According to the dossier, the mushroom bed is made of saw dust or wheat bran or a combination thereof, for which the crop cycle was four months. The mycelium is grown on a potato dextrose agar medium and for the spawn production, wheat grains and sawdust are to be used.

Korean culture of reishi was cultivated by Veena and Pandey (J Mushroo 15 (2): 125- 128, 2006, Int. J Med Mushrooms 13(4):397-400, 201 1). Sorghum grains were used for the spawn production and the substrate was sawdust or paddy straw or rice bran or a combination thereof. Potato dextrose agar medium was used for the growth of mycelium.

Sumathi (Ph. D. thesis. University of Madras, Chennai, India, 2008) and Karuppuraj 201 1 (Ph. D. thesis. University of Madras, Chennai, India 201 1 ) have grown wild isolates and indigenous isolates of reishi spawns over maize grains for which mycelium was_* first grown on potato dextrose agar medium, and sugarcane bagasse, newspaper, saw dQ^'st or coir pith or a combination thereof were used for the mushroom bed.

In all the above methods, culturing the mycelium over potato dextrose agar for the preparation of mother spawn is an essential step which requires specific scientific skills.

Also, the average harvesting period ranges between 120 days to 240 days or fnore. Therefore, there is a need to develop a process, which is simple and efficient for yearlong continuous commercial production of G. lucidum of consistent quality using locally available low cost bioresources.

Ob jects of invention

Objective of the present invention is to provide an improved process for the organic and cost effective cultivation of fruit bodies of Ganoderma lucidum var. lucidum and Ganoderma lucidum var. capense basidiomata (fruit body) with shorter time crop cycle.

Another objective of the present invention is to provide an improved process for the cultivation of fruit bodies of Ganoderma lucidum var. lucidum and Ganoderma lucidum var. capense by elimination of surface sterilization by using context tissue directly. One more objective of the present invention is to provide an improved process for the cultivation of fruit bodies of Ganoderma lucidum var. lucidum and Ganoderma lucidum var. capense mycelium production by the elimination of Potato Dextrose Agar (PDA) medium or any such specified medium required conventionally for the growth of mycelium.

One another objective of the present invention is to provide an improved process for the cultivation of fruit bodies of Ganoderma lucidum var. lucidum and Ganoderma lucidum var. capense by modifications in the spawn bed preparation for increased survival and efficiency of the spawn.

Yet another objective of the present invention is to provide an improved process for the cultivation of fruit bodies of Ganoderma lucidum var. lucidum and Ganoderma lucidum var. capense using locally available agriwastes/ bioresources/ agri industry based wastes/ forest litter such as paddy straw, pine tree leaves, sorghum husk, sugarcane leaves/ tops, sugarcane bagasse, reeds, banana stem etc.

Further one of the objectives of the present invention is to provide an improved process for the cultivation of fruit bodies of Ganoderma lucidum var. lucidum and Ganoderma lucidum var. capense with high bio-efficiency.

Still another objective of the present invention is to provide an improved process for the cost effective cultivation of fruit bodies of Ganoderma lucidum var. lucidum and Ganoderma lucidum var. capense which is simple, economical, organically, environmentally friendly, safe and of good quality.

Brief description of the accompanying drawing

Figure 1 represents a flow chart showing the different steps of the cultivation of present invention.

Room 1 - inoculation room

Room 2 - cropping room Room 3 - harvesting shed

Room 4 - post harvest area

Definitions:

Sterilisation is not particularly limited, as long as it is a step for killing all microorganisms. Usually, in the case of using steam, sterilization at 101 - 125°C preferably at 1 18°C for 30-120 minutes under high pressure or UV treatment or chemical means or boiling and includes pasteurization.

Surface sterilisation is the process of sterilization of the wild mushrooms and its inoculation to a prescribed medium like potato dextrose agar to obtain mycelium. Context tissue is the flesh tissue of the mushroom cap or stalk.

Mycelium propagated vegetatively is known as spawn.

Mother spawn is the mycelium, or primary filamentous growth of the mushroom used for propagation of the mushroom.

Bed spawn is the production of large scale spawns from mother spawn for inoculation in mushroom beds.

Cropping room consists of conditions required for growth of the mycelium to pin head, such as relative humidity, temperature, carbon dioxide, oxygen.

Harvesting room consists of conditions required for growth of the pin head to mature fruit bodies, such as relative humidity, temperature, carbon dioxide, oxygen. Cropping cycle/life cycle starts from the day of preparation of mushroom bed to harvest of mature fruit bodies until no more fruit bodies can be formed in a mushroom bed.

Bio-efficiency (BE) was calculated by using the following formula:

BE = (Fresh weight of the produced mushrooms / Weight of the dry substrate) x 100 Survival bio-efficiency here defined as the number of bags survived without any contamination in the sterilized spawn bags inoculated with context tissue of the mushroom.

Inoculation room consists of inoculation chamber where the inoculation of context tissue into the sterilized spawn bags is done.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the present invention provides a method for production of mushrooms by growing context tissue under controlled condition in a sterilized spawn having one or more substrate in presence of a mixture of calcium source to produce mycelia and transferring the mycelia to multiple mushroom beds; allowing said mycelia to form complete colonization of mycelium in cropping room, and transferring the mushroom-beds to harvest room to allow pin heads to form fruit body, and harvesting the fruit body for one or more crops.

One more aspect of the invention provides a three stage production of G. lucidum and its variants, involving: first stage which allows growing context tissue obtained from G. lucidum or its variants under controlled condition in a sterilized spawn having one or more substrate in presence of a mixture of calcium source to produce mycelia and transferring the mycelia to multiple mushroom bed. In second stage the mycelia is allowed to fully colonize the mushroom beds in cropping room for 7- 13 days.

Finally, in the third stage the fully colonized mushroom-beds is transferred to harvest room in order to develop pin heads and to form fruit body; and multiple harvesting the fruit body wherein during each harvest no residual bud is left un- harvested. In another aspect of the invention provides a unique process to produce G. lucidum and its variants, by eliminating culturing the mycelia in potato dextrose agar medium. As per all conventional and reported process, this step is an essential step. One embodiment of the present invention, the inoculation room is kept under sterilized conditions, with temperature in the range of 20- 35°C; relative humidity in the range of 70 to 85% and lux in the range of 0 to 2800.

Another embodiment of the present invention, the substrate is selected from a group consisting of grains, saw dust, paddy straw, sorghum husk, sugarcane leaves, reeds, banana stem, sugarcane bagasse milled, sugarcane bagasse crushed, sugarcane bagasse pelleted, wheat bran, rubberwood, stumps, compost, paper, paper products, cardboard, used coffee grounds, used tea leaves, gardening debris, seed shells, corncobs and banana fronds or a combination thereof. Yet another embodiment of the present invention, the substrate is grain which is selected from sorghum, maize, barley, rice, paddy, paddy husk or wheat or a combination thereof.

One of the embodiments of the present invention is the calcium source which is either natural or synthetic or a combination thereof. One another embodiment of the present invention, the natural source of calcium is selected from group consisting of hemi-hydrate, plaster-of-paris, gypsum, anhydrite, oyster shells, skeleton of coral, shells of aquatic creatures, snails shells, seashells, coal balls, pearls, eggshells, rocks, marble, limestone, calcite, aragonite, caterite, chalk, hydroxy apatite, slaked lime, quicklime and travertine or a combination thereof. One more embodiment of the present invention, the synthetic source of calcium is selected from group consisting of calcium carbonate, calcium bicarbonate, calcium sulfate, calcium sulfide, calcium nitrate, calcium chloride, calcium hypochlorite, calcium fluoride, calcium carbide, calcium hydroxide, calcium oxide, calcium phosphate and calcium phosphide or a combination thereof. Still another embodiment of the present invention the calcium source is present in the range of 0.5 to 3% by weight. Further, one more embodiment of the present invention, the substrate for the mushroom bed is selected from locally available substrates such as agriwastes, bioresources, agri-industry based wastes and forest litter or a combination thereof.

Still one more embodiment of the present invention the agriwaste is selected from a group consisting of paddy straw, sorghum husk, sugarcane leaves, sugarcane tops, reeds and banana stem or a combination thereof.

One more embodiment of the present invention, the bioresource is selected from group consisting of paddy straw, sorghum husk, sugarcane leaves, sugarcane tops, reeds, and banana stem or a combination thereof. Still one another embodiment of the present invention, the agri-industry based waste is selected from group consisting of sugarcane bagasse, reeds, coir pith and mango waste, or a combination thereof.

Another embodiment of the present invention is the forest litter is pine tree leaves.

Further, one of the embodiment of the present invention, the mushroom bed is prepared by conventional method such as mixing and layering method.

In yet another embodiment of the present invention the mushroom is selected from group consisting of to GL 01 , GL 02, GL 03 and GL 04 and the strains had been deposited with TCC, Chandigarh.

In one more embodiment of the present invention the mushroom is selected from group consisting of Ganoderma lucidum, Ganoderma lucidum (Var) lucidum, and Ganoderma lucidum (Var) capense or their variants.

The invention is directed to an organic method of cultivation of mushroom products and more particularly G. lucidum (reishi), for continuous production through out the year, by shortening the crop cycle (first and final harvest 22-36 and 52 - 54 days respectively). In this process surface sterilization is replaced by the multiple context tissue method. The spawn is grown using maize grains supplemented with source of calcium, which increases its survival. The grown mycelium in the spawn is transferred by mixing and layer method to a multiple mushroom bed and transferred to a cropping room and then to the harvesting room. The multiple mushroom bed consists of any locally available agriwastes/ bioresources such as paddy straw, sorghum husk, sugarcane leaves/ tops, reeds, banana stem etc; agri industry based wastes in any form such as sugarcane bagasse, coir pith, etc.; forest litter, such as pine tree leaves, etc.; where the temperature, light, carbon dioxide levels and the relative humidity are maintained as required for the growth. The carbon dioxide level in the cropping room is maintained at 1300- 1800 ppm and in the harvest room at 100-800 ppm.

The first harvest starts in 22 to 36 days with the total days of crop cycle being 52- 54 days, with bio-efficiency up to 90% and in the post-harvesting stage the harvested materials are covered with sheets to get a quality product.

Examples

Example 1 : Preparation spawn bed or spawn bag

A known quantity of good quality of maize grains were washed thoroughly in running water until the grains, were free from dust and impurities. The washed grains were half boiled until they became soft. The excessive water was drained and the grains were cooled in clean trays and in sterile environment.

The cooled grains were mixed optionally with different sources of calcium natural or synthetic or a combination thereof which is about 0.5 to 3% by weight.

The pH of the said mixture was maintained between 5 and 9.

The grains were packed in suitable spawn containers and were subjected to sterilisation.

Example 2: Method of growing mycelia in spawn bag using context tissue under laboratory conditions

Context tissues from the indigenous basidiocarp of Ganoderma lucidum (GL 01, 02, 04) and G. lucidum (GL 03) procured from Microbial Type Culture Collection, Chandigarh were removed for inoculating in the spawn containers. The inoculated multiple spawn containers were labeled and placed in the inoculation room for the development of mycelium (Refer to Room-1 in figure 1 ). Mycelial growth was initiated on 3^rd day in all the isolates of G. lucidum spawn. The days for mycelial run and days of mycelial life are given in the table 1 below.

Table 1: Mycelial growth of G. lucidum on spawn bags and their life cycle

Key: MG: maize grains, DFMR: days for mycelial run, DML: days of mycelial life GL 01 - Ganoderma lucidum (Var) lucidum

GL 02-Ganoderma lucidum (Var) lucidum

GL 03 -Ganoderma lucidum (MTCC 1039)

GL 04-Ganoderma lucidum (Var) capense

Example 3: Method of growing mycelia in spawn bag using context tissue

The multiple inoculated spawn containers were maintained in the inoculation room till the mycelia grew and the optimum spawn growth temperature is 20- 30°C. The relative humidity should be 80 -85% for spawn bag maintenance.

The days for spawn run and the days of spawn life is presented in the table2.

Example 4: Survival efficiency of mycelia grown in spawn bag

The survival efficiency of the spawn with the addition of different amounts and source of calcium is given in table 2 below. Table2: Survival efficiency of the Ganoderma spawns with and without Gypsum

ote: a - Cacum carbonate

Example 5: Preparation of mushroom bed - Refer to Room-2 in fig: 1

The mushroom bed was prepared as follows:

Any locally available low cost agriwastes/ bioresources/ agri industry based wastes/ forest litter such as paddy straw, pine tree leaves, sorghum husk, sugarcane leaves/ tops, reeds, banana stem etc., can be used for the preparation of the mushroom bed.

The collected substrates were first shade dried for 48 hours.

The dried substrates were cut into pieces by using agri shedder cutting machines. The cut substrates were washed to remove any dust materials. The chopped substrates were soaked in fresh water, excess water drained and the substrates were sterilized. The sterilized substrates were shade dried to maintain the required moisture for utilization in the mushroom bed.

Sources of calcium can be optionally added to the mushroom bed.

Example 6: Inoculation of mycelia in mushroom bed

The spawns were then transferred by mixing and layer method to multiple mushroom beds and kept in the inoculation room. The inoculated beds were maintained in a clean culture room and maintained temperature at 20-30 °C with relative humidity of 70-85 % and observed for the complete colonization of the mushroom bed. The inoculated bags were perforated (12 nos) with sterilized teasing needles. Matured spawn (1 1- 14 days old) were used for mushroom bed preparation. Mycelial growth in the mushroom bed was initiated on the 2^nd day of inoculation. The bags were kept in the semi dark condition for full colonization of the mycelium and complete colonization of the bag was recorded from 9 - 10 days for sugarcane bagasse milled substrate, 7- 1 1 days for pine tree leaves, 8- 13 days for paddy straw, of inoculation.

Example 7: Growing of mycelia in mushroom bed kept in harvesting room - Room 3 in Fig: 1

The multiple mushroom beds were moved to the harvesting room and were maintained at 20-35°C, with relative humidity of 80 - 90 % by sprinkling water through fogger and in 13-19 days multiple pinheads develop in the mushroom bed. On an average it takes 13- 18 days for pine tree leaves, 16-19 days for paddy straw 15- 19 for sugarcane bagasse milled substrate.

Example 8: Growing of mycelia in mushroom bed kept in Harvest room

Once the multiple pinheads grow up enough to form the cap, indicated by the flattening of whitish top of the pinhead, the mushroom beds in the harvest room, the relative humidity is reduced to less than or equal to 80% and more fresh air is introduced to achieve around 100 ppm of carbon dioxide.

The browning of the outer most ring of the fruit body indicates the maturation of fruit body and hence the fruit bodies are harvested at this time period after which sporulation of the mushroom begins. The size of the matured fruit body varies from 3 to 14 cm broad and the individual fruit body weight ranges from 15 gm to 105 gm

The fruit body was matured and harvested from pine tree leaves, paddy straw, sugarcane bagasse in 32-35 days, 35-46 and 43-54 days respectively. In the post-harvesting stage the harvested materials were dried by conventional methods (refer to room 4 in figure 1). During post-harvesting care was taken by covering the harvested material with sheets to get a quality product.

Example 9: Use of various substrates and their crop cycle

Growing stages of G. lucidum on various substrates and their crop cycle, where the cultivation was carried out during the month of June for paddy straw, July - August for Pine tree leaves and July - August for sugarcane bagasse is given in table 3 below.

Table 3: Growing stages of G. lucidum on various substrates and their crop cycle

Substrates GL02(days)

DFSR Pinhead FH SH TH TD

PTL 8-9 15-16 24-36 34-35 - 34-35

PDS 10- 1 1 18- 19 30-32 38-39 - 38-39

SBM 8 - 9 16- 17 23-24 32-34 48-50 48-50

Key: PTL: pine tree leaves, PDS: paddy straw, SBM: sugarcane bagasse milled, DFSR: days for spawn run. FH: first harvest, SH: second harvest, TH: third harvest, TD: total days

GL 01 - Ganoderma lucidum (Var) lucidum

GL 02-Ganoderma lucidum (Var) lucidum

GL 03 -Ganoderma lucidum (MTCC 1039)

GL 04-Ganoderma lucidum (Var) capense

Example 10: Bio-efficiency of fruit bodies of G. lucidum

Bioefficeincy of fruit bodies of G. lucidum on various substrates, where the cultivation was carried out during the month of June for paddy straw, July - August for Pine tree leaves and July - August for sugarcane bagasse is given in table 4 below. Table 4: Bio-efficiency of fruit bodies of G. lucidum

Key: PTL: pine tree leaves, PDS: paddy straw, SBM: sugarcane bagasse milled FH: first harvest, SH: second harvest, TH: third harvest, TW: total weight, BE: bioefficiency,

GL 01 - Ganoderma lucidum (Var) lucidum

GL 02-Ganoderma lucidum (Var) lucidum

GL 03-Ganoderma lucidum (MTCC 1039)

GL 04-Ganoderina lucidum (Var) capense Example 11

Yield performance of fruit bodies of G lucidum during different periods of year using sugar cane bagasse milled as substrate cultivated by conventional methods and present method is given in table 5 and 6 below Table 5: Yield performance of fruit bodies of G. lucidum during different periods of year using sugar cane bagasse crushed. Cultivation carried out by conventional method (i.e., with PDA medium)

Key: GL 01 - Ganoderma lucidum (Var) lucidum

GL 02-Ganoderma lucidum (Var) lucidum

GL 03-Ganoderma lucidum (MTCC 1039)

GL 04-Ganoderma lucidum (Var) capense Table 6: Yield performance of fruit bodies of G. lucidum during different periods of year using sugar cane bagasse milled. Cultivation carried out by present method (i.e without PDA medium)

Key: GL 01 - Ganoderma lucidum (Var) lucidum

GL 02-Ganoderma lucidum (Var) lucidum

GL 03-Ganoderma lucidum (MTCC 1039)

GL 04-Ganoderma lucidum (Var) capense

Claims

claim:

A process for production of mushroom, said process comprising:

growing context tissue in an inoculation room under controlled condition in a sterilized spawn made of one or more substrates in presence of a mixture of calcium source to produce mycelia and transferring the mycelia to multiple mushroom beds;

allowing said mycelia to form complete colonization of mycelium in cropping room, wherein the cropping room is maintained at a temperature in the range 20-35°C, relative humidity of 70-90% and carbon dioxide level at 1300- 1800 ppm and lux in the range of 250-1000;

transferring the mushroom-beds to harvest room to allow pin heads to form, and the pin heads to develop into fruit body, wherein the harvest room is maintained at a temperature in the range 20-35°C, relative humidity 80-90% and carbon dioxide at 100-800 ppm and lux 100-2800; and

harvesting the fruit body for one or more crops,

wherein during each harvest no residual bud is left un-harvested.

A process of producing fruit bodies of Ganoderma lucidum and its variants, said process comprising:

i. growing context tissue obtained from Ganoderma lucidum or its variants under controlled condition in a sterilized spawn made of one or more substrate in presence of a mixture of calcium source to produce mycelia and transferring the mycelia to multiple mushroom bed;

ii. allowing said mycelia to fully colonize the mushroom beds in cropping room;

iii. transferring the fully colonized mushroom-beds to harvest room to develop pin heads and said pin heads develops into fruit body; and iv. harvesting the fruit body one or more crops wherein during each harvest no residual bud is left un-harvested;

wherein the said process is devoid of culturing the mycelia in PDA medium.

3. The process as claimed in claims 1 or 2, wherein is the inoculation room is under sterilized conditions, with temperature in the range of 20- 35°C; relative humidity in the range of 70 to 85% and lux in the range of 0 to 2800. Note:

4. The process as in claims 1 or 2 wherein the substrate is selected from group consisting of grains, saw dust, paddy straw, Sorghum husk, sugarcane leaves, reeds, banana stem, sugarcane bagasse milled, sugarcane bagasse crushed, sugarcane bagasse pelleted, wheat bran, rubber wood, stumps, compost, paper, paper products, cardboard, used coffee grounds, used tea leaves, gardening debris, seed shells, corncobs and banana fronds or a combination thereof.

5. The process as in claim 4 wherein the grain is selected from sorghum, maize, barley, rice, paddy, paddy husk or wheat or a combination thereof.

6. The process as in claims 1 or 2 wherein the calcium source is either natural or synthetic or a combination thereof.

7. The process as claimed in claim 6, the natural source of calcium is selected from group consisting of hemihydrate, plaster of paris, gypsum, anhydrite, oyster shells, skeleton of coral, shells of aquatic creatures, snails shells, seashells, coal balls, pearls, eggshells, rocks, marble, limestone, calcite, aragonite, caterite, chalk, hydroxy apatite, slaked lime, quicklime and travertine or a combination thereof.

8. The process as claimed in claim 6, the synthetic source of calcium is selected from group consisting of calcium carbonate, calcium bicarbonate, calcium sulfate, calcium sulfide, calcium nitrate, calcium chloride, calcium hypochlorite, calcium fluoride, calcium carbide, calcium hydroxide, calcium oxide, calcium phosphate and calcium phosphide or a combination thereof.

9. The process as claimed in claims 1 or 2, the calcium source present is in the range of 0.5 to 3% by weight.

10. The process as in claims 1 or 2, wherein the mushroom bed is selected from locally available group consisting of agriwaste, bioresources, agri industry based wastes and forest litter or a combination thereof, wherein the mushroom bed is prepared by mixing and layering method.

1 1. The process as claimed in claim 10, the agriwaste is selected from group consisting of paddy straw, sorghum husk, sugarcane leaves, sugarcane tops, reeds and banana stem or a combination thereof.

12. The process as claimed in claim 10 wherein the bioresource is selected from group consisting of paddy straw, sorghum husk, sugarcane leaves, sugarcane tops, reeds, and banana stem or a combination thereof.

13. The process as claimed in claim 10 wherein the agri industry based waste is selected from group consisting of sugarcane bagasse, reeds, coir pith and mango waste, or a combination thereof.

14. The process as claimed in claim 10 wherein the forest litter is pine tree leaves.

15. The process as claimed in claims 1 or 2, wherein the context tissue of the mushroom is selected from group consisting of GL 01, GL 02, GL 03 and GL 04. 16. The process as claimed in claims 1 or 2, wherein the context tissue of the mushroom or fruiting body is selected from group consisting of Ganoderma lucidum, Ganoderma lucidum (Var) lucidum, and Ganoderma lucidum (Var) capense or their variants.