WO2020017949A1

WO2020017949A1 - Ophiocordyceps sinensis cultivation in low land and tropical region with local ingredients and automated system

Info

Publication number: WO2020017949A1
Application number: PCT/MY2019/000024
Authority: WO
Inventors: Julian Wei Lung TIONG
Original assignee: Tiong Julian Wei Lung
Priority date: 2018-07-19
Filing date: 2019-07-16
Publication date: 2020-01-23
Also published as: WO2020017949A9

Abstract

The present invention relates to a method of cultivating Ophiocordyceps sinensis in low land and tropical region comprising steps of producing Ophiocordyceps sinensis inoculum; producing a host unit, wherein the host unit is fed with organic food via an automated feeding system under sterile condition; inoculating the host unit with the Ophiocordyceps sinensis inoculum; incubating the inoculated host unit until the inoculated host unit dies; incubating Ophiocordyceps sinensis in the dead inoculated host unit for 9 hours at 17ºC under bright condition; incubating Ophiocordyceps sinensis in the dead inoculated host unit for 15 hours at 3ºC under dark condition; irradiating the dead inoculated host unit; with ultraviolet ray with a wavelength of 275-420 nm for 10 minutes upon emergence of a primordium of Ophiocordyceps sinensis from the dead inoculated host unit; and harvesting the Ophiocordyceps sinensis once stroma of the Ophiocordyceps sinensis reached a pre-determined height or length.

Description

OPHIOCORDYCEPS SINENSIS CULTIVATION IN LOW LAND AND TROPICAL REGION WITH LOCAL INGREDIENTS AND AUTOMATED SYSTEM

Field of the Invention

The present invention relates to Ophiocordyceps sinensis cultivation. More particularly the present invention relates to a method of cultivating Ophiocordyceps sinensis in low land and tropical region with local ingredients and automated system.

Background of the Invention

Ophiocordyceps sinensis, or formerly known as Cordyceps sinensis, is a fungi and a type of cordyceps fungi that is widely accepted to be originated from wild sources of the Tibetan regions. There are another kind of cordyceps which is known as Cordyceps militaris which has been widely cultivated and commercialized. Ophiocordyceps sinensis is also known as “winter worm” and “summer grass” in Chinese. The difference between the two types of cordyceps is that Cordyceps militaris can be grown on artificial man made substrate as well as insects while Ophiocordyceps sinensis grows solely on its original host. The larva which act as hosts and will eventually die as the fungi’s mycelium grows inside the body of the hosts.

Ophiocordyceps sinensis are mainly found in Himalayas and the Tibetan region. Regulation of certain government requires harvesting of Ophiocordyceps sinensis to be done in a sustainable manner and harvesting of the Ophiocordyceps sinensis from the wild are regulated particularly in areas such as in Tibet. This regulation will ultimately reduce Ophiocordyceps sinensis supply to the market. In order to ease the demand pressure, domestication and cultivation of Ophiocordyceps sinensis is necessary. Cultivation of Ophiocordyceps sinensis has to be done in such way that the cultivated Ophiocordyceps sinensis is 100% similar to the Ophiocordyceps sinensis harvested in the wild. There have been methods carried out to cultivate and harvest Ophiocordyceps sinensis in the market. US 8,008,060 B2 is a patent publication that discloses a method of growing Cordyceps sinensis, comprising the steps of growing said fungus in a substrate at approximately 17 to 25° C at an atmospheric pressure which is in the range of approximately 10% below to approximately 10% above normal sea level atmospheric pressure for approximately 25 to 33 days in diffuse light, and thereafter growing said fungus in an atmosphere containing approximately 45 to 55% of oxygen of normal sea level atmosphere, at approximately 2 to 4° C. with exclusion of light, for approximately 13 to 22 weeks. CN 101659578 B is a patent publication that discloses an artificial culture method of Kyushu Cordyceps sinensis and a culture medium thereof. The artificial culture method comprises the following steps: preserving a Kyushu Cordyceps sinensis strain on the inclined surface of a PDA culture medium of a test tube; sterilizing and cooling a bottled activating culture medium, and inoculating the Kyushu Cordyceps sinensis strain saved on the inclined surface of the PDA culture medium to culture for 7-20 days at a temperature of between 20°C and 30°C; sterilizing and cooling an induced culture medium to the room temperature and inoculating the cultured Kyushu Cordyceps sinensis strain under aseptic condition; putting inoculated Kyushu cordyceps into a thermotank for culturing by scattered illumination; and after picking up sub-sinensis, ventilating and drying at a temperature of between 40°C and 60°C to obtain the Kyushu Cordyceps sinensis.

Ophiocordyceps sinensis requires to grow on a host that is well fed and free from diseases. Prior art described above does not suggest a safe and effective way of feeding a host for Ophiocordyceps sinensis. There appears a need for the present invention to develop a method of cultivating Ophiocordyceps sinensis that allows for an effective feeding of host in the cultivation of Ophiocordyceps sinensis.

Summary of the Invention

The present invention relates to Ophiocordyceps sinensis cultivation. More particularly the present invention relates to a method of cultivating Ophiocordyceps sinensis in low land and tropical region with local ingredients and automated system. Accordingly, in an aspect of the present invention, there is provided a method of cultivating Ophiocordyceps sinensis in low land and tropical region, characterized in that, the method comprising steps of producing Ophiocordyceps sinensis inoculum; producing a host unit, wherein the host unit is fed with organic food via an automated feeding system under sterile condition; inoculating the host unit with the Ophiocordyceps sinensis inoculum; incubating the inoculated host unit until the inoculated host unit dies; incubating Ophiocordyceps sinensis in the dead inoculated host unit for 9 hours at 17°C under bright condition; incubating Ophiocordyceps sinensis in the dead inoculated host unit for 15 hours at 3°C under dark condition; irradiating the dead inoculated host unit with ultraviolet ray with a wavelength of 275-420 nm for 10 minutes upon emergence of a primordium of Ophiocordyceps sinensis from the dead inoculated host unit; and harvesting the Ophiocordyceps sinensis once stroma of the Ophiocordyceps sinensis reached a pre-determined height or length.

The organic food is a pesticide-free and pathogen-free food and preferably a pesticide-free and pathogen-free carrot. The pesticide-free and pathogen-free carrot is obtainable by steps of washing a carrot with 75% aqueous alcohol; and rinsing the carrot with sterile water. The automated feeding system comprising a chamber for receiving a container of the host unit; a disinfection unit for disinfecting the container of the host unit within the chamber; a lid operator for opening and closing a lid of the container of the host unit; and a food insertion unit for inserting organic food into the container of the host unit. The disinfection unit of the automated feeding system carries out disinfection using 75% ethanol. The transporting unit of the automated feeding system further comprising a transporting unit that transports the container of the host unit to and from the chamber. The host unit of the method is a larva of a moth selected from a group comprising bat moth.

A second aspect of the present invention provided an automated feeding system for feeding a host unit in a method of cultivating Ophiocordyceps sinensis in low land and tropical region, characterized in that, the system comprising a chamber for receiving a container of the host unit; a disinfection unit for disinfecting the container of the host unit within the chamber; a lid operator for opening and closing a lid of the container of the host unit; and a food insertion unit for inserting organic food into the container of the host unit. In a third and final aspect of the present invention, there is provided a product obtainable by a method of cultivating Ophiocordyceps sinensis in low land and tropical region comprising steps of producing Ophiocordyceps sinensis inoculum; producing a host unit, wherein the larval is fed with organic food via an automated feeding system under sterile condition; inoculating the host unit with the Ophiocordyceps sinensis inoculum; incubating the inoculated host unit until the inoculated host unit dies; incubating Ophiocordyceps sinensis in the dead inoculated host unit for 9 hours at 17°C under bright condition; incubating Ophiocordyceps sinensis in the dead inoculated host unit for 15 hours at 3°C under dark condition; inadiating the dead inoculated host unit with ultraviolet ray with a wavelength of 275-420 nm for 10 minutes upon emergence of a primordium of Ophiocordyceps sinensis from the dead inoculated host unit; and harvesting the Ophiocordyceps sinensis once stroma of the Ophiocordyceps sinensis reached a predetermined height or length.

It is an object of the present invention to provide a method of cultivating Ophiocordyceps sinensis that minimize labour reliance in particular during feeding of hosts. It is also an object of the present invention to provide a method of cultivating

Ophiocordyceps sinensis that reduces chances of cross-contamination during feeding of hosts.

It is an object of the present invention to provide a method of cultivating Ophiocordyceps sinensis that is sustainable, reliable and cost effective.

The present invention consists of certain novel features and a combination of parts hereinafter fully described and illustrated in the accompanying drawings and particularly pointed out in the appended claims; it being understood that various changes in the details may be without departing from the scope of the invention or sacrificing any of the advantages of tie present invention. Brief Description of the Drawings

For the purpose of facilitating an understanding of the invention, there is illustrated in the accompanying drawings the preferred embodiments from an inspection of which when considered in connection with the following description, the invention, its construction and operation and many of its advantages would be readily understood and appreciated.

Figure 1 is a flowchart illustrating an overall process of the method of cultivating Ophiocordyceps sinensis in low land and tropical region with local ingredients and automated/mechanized system according to the present invention.

Figure 2 is a flowchart illustrating an automated/mechanized feeding and rearing system used in the method of cultivating Ophiocordyceps sinensis in low land and tropical region with local ingredients and automated/mechanized system according to the present invention.

Detailed Description of the Invention The present invention relates to Ophiocordyceps sinensis cultivation. More particularly the present invention relates to a method of cultivating Ophiocordyceps sinensis in low land and tropical region with local ingredients and automated system. Hereinafter, the method, system and the product shall be described according to the preferred embodiments of the present invention and by referring to the accompanying description and drawing(s). However, it is to be understood that limiting the description to the preferred embodiments of the invention and to the drawings is merely to facilitate discussion of the present invention and it is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claim.

The term “Cordyceps sinensis" used herein refers to Ophiocordyceps sinensis.

The present invention will now be described in further detail by way of preferred embodiments and non-limiting exa ple(s). Accordingly, in an aspect of the present invention, there is provided a method of cultivating Ophiocordyceps sinensis in low land and tropical region, characterized in that, the method comprising steps of producing Ophiocordyceps sinensis inoculum; producing a host unit, wherein the host unit is fed with organic food via an automated feeding system under sterile condition; inoculating the host unit with the Ophiocordyceps sinensis inoculum; incubating the inoculated host unit until the inoculated host unit dies; incubating Ophiocordyceps sinensis in the dead inoculated host unit for 9 hours at 17°C under bright condition; incubating Ophiocordyceps sinensis in the dead inoculated host unit for 15 hours at 3°C under dark condition; irradiating the dead inoculated host unit with ultraviolet ray with a wavelength of 275-420 nm for 10 minutes upon emergence of a primordium of Ophiocordyceps sinensis from the dead inoculated host unit; and harvesting the Ophiocordyceps sinensis once stroma of the Ophiocordyceps sinensis reached a pre-determined height or length.

It is preferred that the organic food is a pesticide-free and pathogen-free food and preferably a pesticide-free and pathogen-free carrot. The pesticide-free and pathogen-free carrot is obtainable by steps of washing a carrot with 75% aqueous alcohol; and rinsing the carrot with sterile water.

It is also preferred that the automated feeding system comprises a chamber for receiving a container of the host unit; a disinfection unit for disinfecting the container of the host unit within the chamber; a lid operator for opening and closing a lid of the container of the host unit; and a food insertion unit for inserting organic food into the container of the host unit. Preferably, the disinfection unit of the automated feeding system carries out disinfection using 75% ethanol. The transporting unit of the automated feeding system further comprising a transporting unit that transports the container of the host unit to and from the chamber.

It is preferred that the host unit of the method is a larva of a moth, produced from selected bat moth egg.

A second aspect of the present invention provided an automated feeding system for feeding a host unit in a method of cultivating Ophiocordyceps sinensis in low land and tropical region, characterized in that, the system comprising a chamber for receiving a container of the host unit; a disinfection unit for disinfecting the container of the host unit within the chamber; a lid operator for opening and closing a lid of the container of the host unit; and a food insertion unit for inserting organic food into the container of the host unit.

In a third and final aspect of the present invention, there is provided a product obtainable by a method of cultivating Ophiocordyceps sinensis in low land and tropical region comprising steps of producing Ophiocordyceps sinensis inoculum; producing a host unit, wherein the host unit is fed with organic food via an automated feeding system under sterile condition; inoculating the host unit with the Ophiocordyceps sinensis inoculum; incubating the inoculated host unit until the inoculated host unit dies; incubating Ophiocordyceps sinensis in the dead inoculated host unit for 9 hours at 17°C under bright condition; incubating Ophiocordyceps sinensis in the dead inoculated host unit for 15 hours at 3°C under dark condition; irradiating the dead inoculated host unit with ultraviolet ray with a wavelength of 275-420 nm for 10 minutes upon emergence of a primordium of Ophiocordyceps sinensis from the dead inoculated host unit; and harvesting the Ophiocordyceps sinensis once stroma of the Ophiocordyceps sinensis reached a pre-determined height or length.

It is an advantage of the present invention to provide a method of cultivating Ophiocordyceps sinensis that minimize labour reliance in particular during feeding of hosts. The method as described herein reduces chances of cross-contamination during feeding of hosts. The method of cultivating Ophiocordyceps sinensis is sustainable, reliable and cost effective.

The present invention will now be described in further detail by way of an example. EXAMPLE 1

In this example, materials and methods of cultivating Ophiocordyceps sinensis or Cordyceps sinensis are described. Project premises

It is required to have a suitable under roof premises for cultivating Ophiocordyceps sinensis. The entire premises is indoor and with air filtration system. The premises are described below:- a) Breeding Section - This section is for adult moth mating that has climate and light control to enable the species to mate and lay egg. Egg collection tray is needed to simplify egg collection. b) Laboratory Unit - This section includes all the laboratory equipment, working area, egg hatching incubator which is the egg hatchery unit, egg storing system, and Ophiocordyceps sinensis seedling production and preparation. c) Larva Management Center - This section which manage the hatched larva, growing, incubating the larva from young larva until mature; which produce larva for infestation process, as well as pupal development to produce adult moth for egg laying and breeding purpose. d) Ophiocordyceps sinensis Cultivation Room - This section is where the mature larva that has been infested are placed and observed to produce dead larva. The dead larva will be collected, disinfected and placed in separate area to encourage primordial and growth until Ophiocordyceps sinensis is produced. Breeding Section

Equipment

List of equipment needed for the breeding section is as follows: a) Adult Moth Cage - Baby mosquito nets were obtained from a market with a size of 104cm x 50cm x 50cm. The nets are washed and dried before being used as the adult moth cage. Sterilized cotton cloth is placed at the bottom of the adult moth cage to simplify egg collection. b) Plastic Flower - The plastic flower is essential inside the adult moth cage which is prepared for egg laying purpose. The plastic flower used shall be treated with UV ray for a whole night and disinfected. c) Tray - The tray is essential for the cage which has sterilized cotton cloth underneath. This is used as bottom support, and as collector of the egg which failed to be collected inside the cage. d) Petri Dish - The petri dish is clean and sterilized before use. Sterile paper is then added to the petri dish in preparation for egg storage.

Mating Process

A cage which was prepared by using baby mosquito nets was obtained. Sterilized plastic flower was placed inside the cage to mimic a “grass land-like” environment. The purpose of the plastic flower is to encourage mating between male and female moths. In accordance to a ratio of male to female of 1 :1 , 100 to 200 adult moth are placed inside each cage.

The moths were incubated under temperatures of 12°C to 15°C, 80% humidity and 15W scattering light ( light intensity 50 - 100 lux ) in cage. The light helps the adult moth in flying, stretching their wings and mate.

The adult moth will not eat anything. Mating has to be done very fast as they can only live 3 to 7 days. The female moth will only mate once in her life. About 5 to 40 minutes after mating, the female began to lay eggs. Eggs were originally milky white in colour and turns black in 3 to 5 hours. The eggs were to be collected as soon as possible. The best time to collect is 8 to 10 hours postpartum. Upon collection, the eggs were placed in a cultivation dish (petri dish) which was laid with sterile paper. The petri dish was sealed and placed in storage for later use. The collected eggs will be transferred to the laboratory unit, stored and prepared for next round hatching when required.

Different type of female bat moths yields varying amounts of eggs. However, it is typical that moths yield between 200 - 800 eggs per female moth. Laboratory Unit

Petri Dish Preparation PDA was added to the petri dish. The petri dish was then placed in an autoclave at 121 degree Celsius for 60 minutes. The petri dish was then kept refrigerated at 9 to 16 degree Celsius before use.

Jar Preparation

Specially designed 250ml glass jar was prepared as the accommodation for the larva. Firstly, the jar was washed and bleached with detergent. The jar with compost inside was put in an autoclave at a temperature of 121 degree Celsius for 60 minutes. The jar was then kept refrigerated at 9 to 16 degree Celsius before use.

Hatching Container Preparation

Plastic container which can sustain autoclave pressure and heat is used for this purpose. Initially, a sterile paper was placed underneath the container. The container with compost inside was placed in an autoclave at a temperature of 121 degree Celsius for 60 minutes. The container was then kept refrigerated at 9 to 16 degree Celsius before use.

Larval Food Preparation

The larvae are to be fed with carrot. Larval deaths are mostly caused by poisonous effects of chemical pesticides or some biological diseases. In view of this, it is necessary to use carrot which is free from pesticides and biological contamination. In order to guarantee the feed quality, the carrot was bleached with sterile water and then disinfected with 75% ethanol. In order to ensure the baby carrot is safe for larval consumption, the carrot was sliced into tiny thin slices which consist of 10 grams each piece. The carrot pieces were then washed with 75% aqueous ethanol for 3 times and further rinsed with sterile water for 5 times. Each batch of carrots that were brought in to be made into the feed for the larva is different from one another. Careless work can cause deathly effects which bring damages to the production line. Thus, safe precautionary steps are to be taken. In order to ensure the carrots are safe, each batch of carrot underwent a sampling process. Disinfection and cleaning process are to be carried out on a sample of carrot from the batch before being fed to 1 or 2 live larvae. The larvae were then observed for 2 days. The remains of the carrot shall be stored in the storage at 4°C. If the testing larva shows no fatal effects, the batch of the carrot “imported” is considered safe for larval consumption. This steps is proven effective in testing the quality of the carrot, which helps to bring down the risk associates to the larva which cause deathly damages due to contamination of pesticides and microbiological contaminants, yet increases the survival rate of the larva.

Egg Disinfection Solution

The solution was prepared by adding 15ml 4M Sodium Hydroxide and 5ml sodium hypochlorite (which contains activated chlorine 7.5%) into 10ml sterile water.

Producing Inoculum

A completely developed and healthy Ophiocordyceps sinensis was selected and bleached with sterile water before being left overnight. On a clean and sterile work site, stroma of the cordyceps was sliced open and a strain of the cordyceps was isolated.

Culture of the cordyceps was inoculated on a slant medium (PDA) in a petri dish at 9°C to 16°C for 90 to 120 days until the PDA has been fully covered by mycelium. The mycelium covered PDA was obtained and triturated with a shredder.

The triturated mycelium was then diluted with a sterile 5% glucose solution until a ratio of 1 :600. The mixture was then filtered using a 2 layered gauze filter and the filtered fluid was collected. The filtered fluid was then centrifuged for 30 minutes at a temperature of 4°C and at a speed of 10,000 revolutions / minute. As soon as the centrifugation ended, it was observed that the centrifuge tube contains a precipitate layer of mycelial cake, a supernatant layer of liquid above the mycelial cake layer and another supernatant layer above the first supernatant layer. A syringe was used to extract the first supernatant layer liquid above the mycelial cake. This liquid will be used as needling invasion (infestation) dye. The liquid contains tiny spores which is almost invisible to the naked eyes. This process can be repeated to produce sustainable inoculum continuously.

Egg Collection and Storage

Eggs that were collected during mating and during egg laying by the adult moth were transferred into a petri dish that was laid with sterile paper. The petri dish containing the eggs was stored in an incubator under 4 °C. This would enable the eggs to be kept as a stock for up to 1 year so that the cultivation cycle can run anytime of the year.

Egg Hatching

Under low temperature condition (between 9°C to 16°C), eggs obtained from the storage were placed in a sterile mesh gauze. The mesh was then rinse with sterile water for 3 times. The sterile mesh gauze containing the eggs was then placed in a disinfection solution for 3 to 6 minutes before rinsing it with sterile water for 3 times.

The cleaned eggs were transferred into a hatching container which was underlaid with sterile paper. The eggs were then incubated and hatched in temperatures ranging from 9°C to 16°C for 35 to 40 days. Sterile water was added to ensure the sterile paper is wet in order to provide moisture to the eggs during incubation and hatching period. Successfully hatched eggs will exhibit activity inside the container and were prepared to be transferred into the cultivation jar.

Larval Placement

Successfully hatched larva was relocated from the hatching container into a cultivation jar with one larva per jar. Robotic System

Mechanization and automation of fixed and routine work is essential to minimize labour reliance and to increase precision. The following is done via robotic system:

Cultivation Jar Preparation

This process is carried out to add cultivation substrate into the jar in accordance to a pre-determined weight set. The process starts by placing the jar on a feeding system. The system then opens up the cover of the jar, followed by insertion of compost. The jar was then covered by a lid and tighten. The sealed jar was then prepared to be autoclaved as per the requirement. Carrot Slice Feeding

The larva which remains in the jar needs to be fed with carrot every 30 days. Thus, the jar with the larva needs to be placed on the feeder system. The entire group of jar entered a“storage chamber” of the system and disinfection process took place. The jar was sprayed and cleaned with ethanol 75% to kill all the bacteria and other microbiological contaminants which might be on the jar and to ensure a clean jar entering the sterile environment.

The jar’s cover was then opened and a prepared carrot slice was inserted into the jar. After the carrot was inserted, the system will re-seal the jar and then allow the jar to leave the system.

Larva Management Center The purpose of this center is to control larval growth, feeding, and to provide an adequate environment for the larva to grow. Larvae Rearina/lncubation Container

Specially designed 400ml glass jar was prepared as the accommodation for the larva. 40g compost soil which has 35% to 40% humidity was added into the jar. The jar containing the compost was put in an autoclave at a temperature of 121 degree Celsius for 60 minutes. The jar was then kept refrigerated at 9 to 16 degree Celsius before use.

Larva Management Unit

Once the larva has entered into the cultivation container, the container was then transferred to this unit. Here, the larva was fed with baby carrot, which was prepared as mentioned earlier. The larva will be fed with carrot throughout its growing life journey. The larva was fed at 30 days interval. Routine checks were carried out to ensure that food are available throughout the larva’s life. The amount of food given to the larva are based on their age, which is roughly between 10g to 30 gram per container for each time.

Yunnan larva ( Thitarodes jianchuanensis) was used in this example, which matures between 136 to 263 days. Maturity of the larva can also be defined as the larva reached age stages between 7 and 10. A mature larva is ready for Ophiocordyceps sinensis production use.

An optimal environmental condition of larva growth is between 9-13°C with a humidity of 50% and under normal day or night condition.

At the end of this stage, all the mature larvae will enter breeding stock selection stage. Those un-selected larvae will be sent for Ophiocordyceps sinensis section for infestation process. Breeding Stock Selection

In order to select a breeding stock, it is normal to choose those which grow and mature faster, healthy and have good weight.

At this stage, male pupae generally are smaller in size compare to female pupae. Distinguishing feature includes ventral cracks and protrusions in the last somite ventral. The tail somite is observed by observing both sides of the center line that features two distal dark ventral arc. For the male pupae, there are two distinct black spot between the two distal dark ventral arcs. For the female pupae, there is a vertical fracture line between the two distal dark ventral arcs.

In order to manage this, the selected stock was relocated to a bigger jar, which is specially designed for larva, prepared as per prescribe above. The species will show the following characteristics:

i) having a slim oval shape body;

ii) having a length between 23mm to 31 mm;

iii) having a“horn shaped” tumor head;

iv) having a colour of reddish brown or reddish light brown;

v) having a size of head shell of 4mm to 6mm;

vi) having an abdomen a 3 short segments that are obvious in the abdomen area; vii) having an“underside thorn” segment;

viii) having a“tumor” shape structure protruding on the seventh segment; and ix) having an enlarged breeding organ at 8th and 9th segment.

The above characteristics indicate that the larva is entering the pupa stage. During this time, male and female pupa has to be incubated separately and under different condition in order to synchronize emergence of the male and female pupa later on and to ensure a high success rate of male and female adult moth mating process and simultaneous eclosion. Feeding was carried out as usual.

The male pupa was placed under 10°C while the female pupa was placed at 13°C. Both are placed at 50% humidity in normal day and night light intensities. Under 9-13 ⁰ C, the Yunnan bat moth pupa period is generally 34-42 days. Ophiocordvceps sinensis Cultivation

Forcing Primordial Trav Preparation A tray was placed on a cultivation rack. Dead larvae from Ophiocordyceps sinensis inoculum infestation were collected and placed on the tray. A plastic tray which can sustain autoclave pressure and heat was used. In order to prepare a cultivation medium, compost of up to 2 inches thick was filled in the tray. The compost comprised of 50% humidity

Infestation

For this part, there are 2 ways of conducting infestation as per describe in the following.

In the first option, the inoculum liquid for infestation purpose was obtained. The liquid was mixed with the compost medium at 1 :1 ratio. 20g of the infested compost was added into each jar. The inoculum liquid contains spores and tiny particles of Ophiocordyceps sinensis. This infestation process relies on the food intake for the spores/tiny particles to enter its body; or alternatively, infestation were done when there is“skin changing” whereas the outer layer of the larva is weak and easy to infest, and the spores will enter the body via the pore holes on the skin, which achieves infestation objective. In the second option, artificial infestation were carried out using specially designed syringe to conduct injection of the inoculum liquid into the body of the moth. The outer wall of syringe have needle accommodating chamber. Using manual approach, grasp the depth during needle, the head of a silica gel pad to touch the subject.

Dead Larva Collection

3 to 4 months after infestation, dead larva will start to appear. As dead larva starts to appear the compost medium was open up and dead larva was collected. Surface of the dead larva was sterilized with 75% alcohol. After disinfection process, the dead larva was placed in the forcing primordial tray which was prepared earlier. The dead larva was placed with the head up and the head was covered up with compost for up to 2mm below the surface. Forcing Primordial on Artificial Ophiocordyceps sinensis Cultivation

All the forcing primordial tray which has dead larva was placed on the steel rack that was equipped with normal lighting and UV lighting. The infested dead larva“buried” in the sterilized compost was incubated with 50% humidity and with bright light for 9 hours a day at 17°C. The infested dead larva“buried” in the sterilized compost was then further incubated in the dark for 15 hours a day at 3°C.

As a successful primordial starts to emerge (small stroma) during “noon time”, ultraviolet ray with a wave length of 275-420nm were radiated on the tray for 10 minutes. Once the stroma grows to a suitable height, Ophiocordyceps sinensis is ready to be cultivated

EXAMPLE 2

In this example, an overall process of the method of cultivating Ophiocordyceps sinensis in low land and tropical region with local ingredients and automated/mechanized system is simplified with reference to Figure 1.

EXAMPLE 3

In this example, an automated/mechanized feeding and rearing system used in the method of cultivating Ophiocordyceps sinensis in low land and tropical region with local ingredients and automated/mechanized system is simplified with reference to Figure 2.

Industrial Application

The present invention is industrially applicable. The method of cultivating Ophiocordyceps sinensis helps to minimize labour reliance in particular during feeding of hosts. The method as described herein reduces chances of cross- contamination during feeding of hosts. The method of cultivating Ophiocordyceps sinensis of the present invention is sustainable, reliable and cost effective.

While in the foregoing specification this invention has been described in relation to certain preferred exemplary embodiments thereof and many details have been set forth for purpose of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention.

Claims

1. A method of cultivating Ophiocordyceps sinensis in low land and tropical region, characterized in that, the method comprising steps of:

i) producing Ophiocordyceps sinensis inoculum (10);

ii) producing a host unit, wherein the host unit is fed with organic food via an automated feeding system under sterile condition (20);

iii) inoculating the host unit with the Ophiocordyceps sinensis inoculum (30); iv) incubating the inoculated host unit until the inoculated host unit dies; v) incubating Ophiocordyceps sinensis in the dead inoculated host unit for 9 hours at 17°C under bright condition;

vi) incubating Ophiocordyceps sinensis in the dead inoculated host unit for 15 hours at 3°C under dark condition;

vii) irradiating the dead inoculated host unit with ultraviolet ray with a wavelength of 275-420 nm for 10 minutes upon emergence of a primordium of Ophiocordyceps sinensis from the dead inoculated host unit (40); and viii) harvesting the Ophiocordyceps sinensis once stroma of the

Ophiocordyceps sinensis reached a pre-determined height or length (50).

2. The method according to claim 1 , wherein the organic food in the step of producing the host unit (10) is a pesticide-free and pathogen-free food.

3. The method according to claim 2, wherein the pesticide-free and pathogen- free food is selected from a group comprising a pesticide-free and pathogen-free carrot.

4. The method according to claim 3, wherein the pesticide-free and pathogen- free carrot is obtainable by steps of:

i) washing a carrot with 75% aqueous alcohol; and

ii) rinsing the carrot with sterile water.

5. The method according to claim 1 , wherein the automated feeding system comprising:

a chamber for receiving a container of the host unit; a disinfection unit for disinfecting the container of the host unit within the chamber;

a lid operator for opening and closing a lid of the container of the host unit; and

a food insertion unit for inserting organic food into the container of the host unit.

6. The method according to claim 5, wherein the disinfection unit carries out disinfection using 75% ethanol.

7. The method according to claim 5, wherein the automated feeding system further comprising a transporting unit that transports the container of the host unit to and from the chamber.

8. The method according to claims 1 to 7, wherein the host unit is a larva of a moth selected from a group comprising bat moth.

9. An automated feeding system for feeding a host unit in a method of cultivating Ophiocordyceps sinensis in low land and tropical region, characterized in that, the system comprising:

a chamber for receiving a container of the host unit;

a disinfection unit for disinfecting the container of the host unit within the chamber;

10. A product obtainable by a method of cultivating Ophiocordyceps sinensis in low land and tropical region comprising steps of:

i) producing Ophiocordyceps sinensis inoculum (10);

iii) inoculating the host unit with the Ophiocordyceps sinensis inoculum (30); iv) incubating the inoculated host unit until the inoculated host unit dies; v) incubating Ophiocordyceps sinensis in the dead inoculated host unit for 9 hours at 17^eC under bright condition;

vii) irradiating the dead inoculated host unit with ultraviolet ray with a wavelength of 275-420 nm for 10 minutes upon emergence of a primordium of Ophiocordyceps sinensis from the dead inoculated host unit (40); and viii) harvesting the Ophiocordyceps sinensis once stroma of the Ophiocordyceps sinensis readied a pre-determined height or length (50).