Cordyceps sinensis, also known as Cordyceps sinensis, is referred to as Cordyceps sinensis. At present, it has been reported that more than 400 species of Cordyceps sinensis parasitize insects, spiders and other organisms and produce fruiting bodies. 68 species have been recorded in China. In the concept of some people and scholars at home and abroad, all fungal combinations that are parasitic by Cordyceps sinensis and can produce fruiting bodies are collectively called Cordyceps sinensis. In Chinese medicine, the vast majority of scholars and people in our country and the Cordyceps referred to in this paper refer to the combination of insects and bacteria formed by Cordyceps sinensis as (Berkeley) saccardo (1878), which is only distributed in alpine meadows in the Qinghai-Tibet Plateau and marginal areas of China and parasitic on the larvae of Insecta, Lepidoptera and Hepialidae. ; Other groups are only called Cordyceps sinensis, not authentic Cordyceps sinensis.
First, the medicinal history and development status of Cordyceps sinensis and its host insects
Medicinal Cordyceps sinensis, the earliest text can be found in Wang An's Compendium of Materia Medica (1694) in Qing Dynasty: "Cordyceps sinensis is sweet, which can protect lung and kidney, stop bleeding and eliminate phlegm, stop fatigue and relieve cough. Jiading, Sichuan produces the best. Winter in the soil, like an old silkworm, hairy, unearthed in summer, turned into grass If you don't eat it, it will become a bug in winter. Later, Zhao Xuemin's Compendium of Materia Medica (1756) recorded that "Cordyceps sinensis and ginseng can work together to cure various debilitating diseases", and the book also described the origin, eating method and dosage of Cordyceps sinensis in detail. In 1757, Wu Xu's book Materia Medica also pointed out: "Cordyceps sinensis protects the lungs and kidneys, stops bleeding and eliminates phlegm. "Later, hundreds of ancient Chinese medicine books such as Qiannang, Textual Research, Sichuan Tongzhi and Materia Medica recorded Cordyceps sinensis.
The history of exporting Cordyceps sinensis as a medicinal material to foreign countries in China is longer than recorded in writing. From the middle of the Ming Dynasty, it spread from Zhejiang to Japan during 1400- 1465, and was widely eaten by nobles. 1723, Jean-Gartley Tzku, a European missionary, brought Cordyceps collected from northwest China to France. Reaumur introduced this kind of Cordyceps at the bachelor's conference of French Academy of Sciences and included it in the minutes of the conference. 1943, Berkley identified China Cordyceps sinensis and officially named it China Cordyceps sinensis; 1878 was classified as Cordyceps by Sacado. The study of Cordyceps sinensis has attracted attention abroad, and the Cordyceps sinensis in China has become famous all over the world.
Cordyceps sinensis enjoys a high reputation because it is only distributed in China. For hundreds of years, it has been regarded as a precious nourishing Chinese herbal medicine comparable to natural ginseng and velvet antler by people in China and Asia. It has mild medicinal effects and is more widely used in medicine and food than other kinds of tonics. In China, it has long been a high-grade tonic with the same origin of medicine and food, which can be eaten all year round. It is suitable for the sick, the disabled and the weak, and has no side effects. The effects of more than 70 ancient Chinese medicine documents on Cordyceps sinensis are summarized as follows: "It can replenish yin and yang, cure fatigue and cough, and all kinds of deficiency; Its advantages are the same as ginseng and pilose antler, but its nature is mild, and it is suitable for people of all ages who are weak and sick ... "Since the founding of the People's Republic of China, there are more than 200 kinds of medicine books in China that record the efficacy and use of Cordyceps sinensis in detail. People's Republic of China (PRC) Pharmacopoeia records that its function is "nourishing lung and kidney, stopping bleeding and resolving phlegm". It can be used for chronic cough and asthma, cough and hemoptysis, impotence and nocturnal emission, and soreness of waist and knees. " With the development of medicine at home and abroad and the gradual popularization of traditional Chinese medicine in the world, people have found that the functions and functions of Cordyceps sinensis are more and more extensive in recent years. Studies have proved that it has the functions of sedation, hemostasis, anticonvulsant, lowering blood pressure, improving myocardial ischemia, anti-platelet aggregation, anti-aging, regulating human immunity, and resisting lung cancer, lymphoma and liver cancer. At present, it has attracted more and more attention from researchers in the fields of pharmacy and biology at home and abroad, and the related research reports have increased greatly.
In China, the earliest scientific description and publication of the distribution of the genus Hepialus on the Qinghai-Tibet Plateau is Poujade 1886, which is a kind of Hepialus. It was found on the snowy mountain in Baoxing County, Sichuan Province. Later, scholars such as Alpharaky and Staudinger discovered two species in Tibet at 1889 and 1895 respectively, namely, the nebula H.nebulosus (homonym: H.luteus) and H.varians. 1909, Oberthur collected medicinal materials and plant specimens from China, Tibet and Sichuan on the French Armorica Peninsula, and found an adult bat moth named H.armoricanus after the peninsula. However, when the new species was published, it was explained that the origin of this bat moth may be the Sichuan-Tibet Plateau in China, because it has not been found on the peninsula. Later, until 1959, researchers from the Institute of Zoology, Chinese Academy of Sciences collected a large number of adults and larvae of hepialus in Kangding, Sichuan, which were identified by Zhu Hongfu as the same species as the genus hepialus found in Ka Peninsula, ammari, France, so it was named Cordyceps hepialus and was identified as one of the host insects of Cordyceps sinensis, a special product in China. Up to now, 37 host insects of Cordyceps sinensis endemic to China have been found in the Qinghai-Tibet Plateau and its marginal areas.
Second, the current situation of artificial cultivation and industrialization of Cordyceps sinensis and hepialus
(A) the status of artificial cultivation of sex Cordyceps sinensis
Over the past 20 years, more than 60 scientific research units and thousands of people have carried out a lot of research, experiments and explorations on artificial cultivation of Cordyceps sinensis. Yunnan, Sichuan and other places have achieved successful experience in cultivating sexual types in small areas at high altitude, but due to many factors such as long cycle, few promotion objects, large investment and lower income than natural collection at high altitude, it has not been popularized in large areas. In the middle and low altitude areas of Yunnan, Sichuan, Zhejiang, Gansu, Qinghai, Beijing, Fujian, Guangdong, Guizhou, Guangxi and other provinces (cities, districts), the research on artificial cultivation of sexual Cordyceps sinensis has also been carried out for many years, although many of them have achieved very gratifying results in some aspects, such as the successful breeding of moth insects in Kunming, Chengdu and Chongqing in Yunnan and Sichuan provinces. In Zhejiang and other places, bats and moths have successfully propagated indoors; However, it can not be called successful culture because it does not solve the difficulties of inoculation and batch and repeated growth of progeny. It is still far from the goal of artificially cultivating sex Cordyceps sinensis in small area, small batch and many times at low altitude. Although several companies claim that they have successfully cultivated sex Cordyceps sinensis in batches at low and medium altitudes, the scientific credibility is extremely low in fact. Because:
(1) At present, no one has been able to isolate and cultivate authentic Cordyceps fungi stably for a long time (that is, a large number of daughter bases have been repeatedly grown on the culture medium). Substrates have been cultivated in Qinghai, Yunnan, Sichuan, Beijing and other places, but their ascospores are few or none, and their growth is unstable, which does not mean that they have mastered the sexual artificial cultivation technology at middle and low altitudes.
(2) The artificial asexual culture technology of Cordyceps sinensis is relatively easy to master. Inoculation of hepialus larvae indoors can lead to infection and enter into a stiff stage, but it is rare to complete the sexual form and grow the stroma and ascospores.
(3) Apart from environmental factors such as altitude, climate, soil and air pressure, are there multi-bacterial infections among bacteria? If it is a compound infection, which bacteria are playing a key role? When did it work with Cordyceps sinensis? Wait, the question is not clear.
Therefore, it needs a lot of research and experimental work to batch and repeatedly cultivate Cordyceps sinensis in middle and low altitude areas. Even if large-scale artificial cultivation of Cordyceps sinensis is carried out in high altitude areas, many technical experiments are needed. Therefore, it is still very arduous and far away to cultivate sex Cordyceps sinensis artificially and realize large-scale or industrial production.
(2) Current situation of artificial culture of asexual Cordyceps sinensis.
Because it is too difficult to cultivate sexual Cordyceps sinensis artificially, in recent years, most units and researchers who have cultivated Cordyceps sinensis artificially have turned to asexual research and application. Because the asexual culture technology of Cordyceps sinensis is easy to master, and most of the fungi isolated from Cordyceps sinensis collected in nature have similar physical and chemical properties and pharmacological effects to the sexual components of Cordyceps sinensis, all localities have identified the self-isolated fungi as the asexual stage of Cordyceps sinensis, and set up factories to produce bacterial powder for development and application. At present, there are 9 genera and 3 1 species of Cordyceps sinensis isolated from the body of Cordyceps sinensis in nature, including 16 species found in more than 40 provinces (cities) such as Beijing, Jilin, Shanxi, Shanghai, Qinghai, Zhejiang, Fujian, Jiangxi, Guangdong, Yunnan and Guizhou. Submerged large-scale fermentation culture and solid culture of mycelium and conidia are substitutes for sex Cordyceps sinensis, and some manufacturers have entered large-scale industrial production, such as more than 20 kinds of products such as Lexus capsule, Jinshuibao capsule, Ningxinbao capsule, Xinganbao, Cordyceps sinensis chicken essence, Cordyceps sinensis capsule, Cordyceps sinensis wine, Cordyceps sinensis ginseng tonic wine, etc., with an annual output value of10 million yuan to hundreds of millions. Moreover, due to the short artificial propagation cycle and high yield, mycelium and meristem robe have great development and application potential as sexual substitutes of Cordyceps sinensis in the future.
Three, Cordyceps sinensis and hepialus insect artificial cultivation techniques
(1) Host insect breeding technology of Cordyceps sinensis In order to cultivate Cordyceps sinensis artificially, a large number of hosts-hepialus insects must be artificially raised first. Batmoths spend 98% of their life in the soil, and only 2.5- 13d adults live on the ground. Therefore, the artificial feeding management of such insects is mainly carried out in the soil.
1. Feeding tools for bat moths
(1) Feeding tools for hepialus eggs. There are two ways to raise bat moth eggs. One is to use a glass Petri dish with a diameter of 15-20cm, pad it with 2-3 layers of filter paper, and put a piece of absorbent cotton in the dish to keep the humidity. Each plate can raise 200-500 eggs. Liu Fa can't observe this, but it is difficult to grasp the humidity. Secondly, raise the jar with l0cm xX5-8cm or 25cm×30cm glass, put it in fine humus soil with a thickness of 3-5cm, plant 1-2 bulbs, and put the eggs in the soil layer from the surface of the soil to the depth of 1cm. Each jar can feed 300- 1000 eggs. When feeding, as long as the plants are kept from wilting, no humidity can be added. This method has high hatching rate, but it is difficult to observe and transplant larvae.
(2) There are four feeding tools for larvae.
A. Glass insect tank: use a glass tank of 18-25cmX30-35cm, or use a specimen to soak the tank, place the soil of 15-25cm, seed the larvae for feeding, and move the newly hatched larvae into the tank, each tank can support 10-30 larvae.
B. Underground insect rearing box: The box is generally 30-50 cm high, 20-25 cm wide and 3-5 cm thick. The frame of the feeding box is welded with steel bars, and the steel bars on it are made into handles for easy lifting; The wide sides are 3-5mm glass, which is convenient for observing insect activities; The bottom and both sides are equipped with 16 mesh copper mesh, which is convenient for moisture penetration. Then filling soil, planting feed plants, and stocking 20-50 larvae in each box; Dig a groove slightly larger and deeper than the feeding box on the solid ground, put the feeding box into the groove, lift the feeding box off the ground during observation, and wipe off the soil on the outer glass so as to clearly see the activities of the larvae.
C. Large insect cage: under semi-natural conditions, choose a site with good drainage and loose soil, dig a deep ditch with a length of 12- 15m, a width of 2-3m and a depth of 4cm, pad nylon gauze on the bottom and around it (it is better to use double layers, so that insects are not easy to get out), then shovel off loose humus to fill it, and plant larval food. This method is beneficial to large-scale feeding, but it is difficult to observe.
D. Various pots for bare cultivation: the plant feed is washed and put into the pot, and the larvae are put into the pot for breeding, and the cover of the pot is covered with a layer of black cloth or black paper for shading. Observe Liu Fa's life history, but pay attention to changing feed frequently and removing feces.
(3) Feeding tools for pupae and adults. Make an insect box, 50cm long, 50cm wide and 55-60cm high. The bottom is sealed with thin iron plate or double-layer copper net; The top and periphery are wrapped with iron wire or nylon wire, and a movable gauze door can be made in front. Put 10-20cm soil at the bottom of the box, plant, and put pupae and adults in the soil. This method can be used for close planting and feeding.
2. Key points of feeding technology
(1) Select soil. Soil is the most important activity place for hepialus in her life. Whether the soil conditions are suitable for the life of insects is one of the keys to the survival rate of artificial feeding. Alpine meadow soil is most suitable for the growth of hepialus; Followed by mixed soil of flowing stone beach and meadow; The third is brown and dark brown forest soil in the mountains, and other soils are not good for this insect.
(2) the choice of food. Larvae like to eat tender roots and buds of Polygonum viviparum, Polygonum viviparum, Astragalus membranaceus and rhubarb. Because hepialus is an omnivorous insect, Gramineae (such as highland barley, wheat, rice bud, etc. ) can be used in places where there is no such plant. Tender root breeding of Cruciferae and Cyperaceae. In the middle and low altitude areas (the temperature suitable for living), rhubarb, carrots, radishes, sweet potatoes (sweet potatoes), potatoes and apples can also grow normally. And it is best to use rhubarb, carrots and sweet potatoes.
(3) Selection of temperature and humidity. Batmoth is an insect that is resistant to low temperature and afraid of high temperature. Most suitable for living and growing in 15- 19C; This insect is most suitable for air humidity of 75%-85% and soil water content of 40%-45%.
3. Management technology
(1) Adult stage. Adults don't eat any food, as long as they keep moisture and proper temperature, they can survive and lay eggs normally. But the moth's habitat is related to the amount of eggs laid. Adults prefer to lay eggs in the roots of Polygonaceae and other plants, feeding on rhododendrons and hepialus larvae, so it is best to plant several related plants before moths mate and lay eggs. In addition, there can be no strong light.
(2) Egg stage. In order to hatch eggs normally, we must master the appropriate temperature and humidity.
(3) larval stage. Due to the long growth period and thin wall of larvae, besides necessary observation, soil change and food change, they must be carefully raised to minimize turnover. The key to feeding larvae is the initial incubation period, and the newly hatched larvae should be given enough delicate and low-humidity food; It is best to screen the soil to avoid rough and hard objects scratching the main body. In addition, in order to prolong food and soil from mildewing, ultraviolet lamp should be used for sterilization for l5min before replacement, and soil can also be exposed to the sun 1-2h before use.
(4) pupal stage. When raising pupae, first compact the soil in the pupa-raising vessel, and then artificially build a soil chamber with a wooden stick the thickness of pupa. The earth room is slightly inclined, about 2-3 cm deep, and a pupa is placed in each room. When the pupa is released, the pupa head faces the soil surface and the top is covered with fine soil. The temperature and humidity of pupa can be controlled between 80%-85% of atmospheric humidity and 42%-45% of soil humidity, 18-22℃.
[2] Artificial culture technology of Cordyceps sinensis
In order to obtain pure strains of grass in winter and early summer, it is necessary to separate, purify, rejuvenate and preserve them artificially.
1. separation technology and conditions of cordyceps sinensis
(1) Material requirements. Obtaining pure strains requires strict materials; Generally speaking, if the tissue block separation method of Cordyceps sinensis is adopted, the best separation material is the material collected from the end of 65438+1October every year to165438+1October, when the soil in the alpine meadow on the Qinghai-Tibet Plateau has just begun to freeze. During this period, Cordyceps sinensis just infected moth larvae, and entered the stage of zombie for a short time. The tender bud just grew a worm head of 0.2-0.5cm, and there were few other miscellaneous bacteria in the worm. If the samples are taken in May and June of the following year, there are many kinds of miscellaneous bacteria living or saprophytic in the stiff worm and its sub-seat, so it is difficult to obtain pure strains. If the ascospores are used for isolation and culture, it is best to collect the isolation materials in the middle and late July of each year when the ascospores are just partially mature.
(2) Operation method. There are three steps.
A. separation of stiff worms (i.e. sclerotia): before separation, the surface of the main body is washed with water, and then washed with sterile water for 2-3 times; Disinfect the surface of the separated material with 0.65min438+0%-0.2% mercuric chloride solution for about 3-5min; Then wash with sterile water. Select the front part bounded by the chest and feet. Cut off the epidermis with a surgical knife, avoid the digestive tract, cut the hyphae in the blood cavity into sesame seeds, and press them into the plate culture medium, each dish contains 1-2 capsules. Cultured at 15- 19℃. When the colony grows to 0.2-0.5cm, a small amount of mycelium is selected and purified repeatedly on the plate medium for 2-4 times. After confirming that there are no other miscellaneous bacteria, transfer to a test tube for preservation and culture.
B, separating the bud: cutting the washed bud from the top of the stiff worm, putting it into 0. 1% mercuric chloride solution for disinfection for 2-3min, washing it with sterile water, cutting off the middle tissue block and pressing it into the culture medium; The culture conditions are the same as a.
C. Ascomycete spore separation: covering the mature substrate of Ascomycete spores with a transparent paper bag, and allowing the Ascomycete spores to bounce and stick to the paper bag. Then, the paper bag containing ascomycete spores was immersed in 25% glucose solution, washed off the spores, and cultured at 15- 19℃, and examined microscopically every day. When the robe germinates, a single spore is sucked onto the plate with a micropipette for culture. The culture conditions are the same as a.
Or, you can bring the whole mature Cordyceps sinensis indoors, wrap the larvae of stiff worms or even worms with cotton paper, leaving only the pregnant part, and place glass slides horizontally in the sterile room to keep the humidity of the main body at any time; Microscopic examination every day, when ascospores bounce on the glass slide, they are sucked into the culture medium of Petri dish with a micropipette for culture; The culture conditions are the same as a.
(3) Selection of culture medium.
A. Potato glucose agar medium (PDA) is a universal medium for all Cordyceps fungi, which can be used in the initial stage of isolation and culture, but the strain growth is not very vigorous and it is easy to age and degenerate.
B enrichment medium (1): multifunctional protein umbilical cord 10g, glucose 50g, dipotassium hydrogen phosphate 1g, magnesium sulfate 0.5g, live silkworm chrysalis 30g, auxin 0.5μg, agar 20g, water 1000mL, pH: 5.0.
Enrichment medium (II): 40g of preserved protein, 40g of glucose, peeled fresh potato 100g, dipotassium hydrogen phosphate 1g, 0.5g of magnesium sulfate, beef paste 10g, 0.5μg of auxin, 30g of bat moth live larvae (powder), 20g of agar and alpine meadow soil leachate/kl.
On the intensive medium, the colonies grew more vigorously and rapidly than on PDA medium. Jiafu Ⅱ is superior to Ⅰ.
2. Growth characteristics In the separation and culture of Cordyceps sinensis, it is necessary to master the utilization of temperature, pH value, illumination, carbon and nitrogen sources and inorganic salts.
(1) Effects of different temperatures on mycelium growth. Cordyceps likes low temperature. It can grow slowly at 0-4℃; The growth rate is accelerated at 5-8℃; 10- 19℃ is the suitable growth and development range of this strain; The optimum growth temperature is 15- 19℃. When the temperature exceeds 20℃, the mycelium grows rapidly, the colony changes from white to grayish black or brownish yellow, and begins to mutate, so it is not suitable for high temperature culture.
(2) Requirements for pH value. Cordyceps sinensis is an acid fungus, and its optimum pH value is 5.0-6.0. When the pH value is below 4.5 and above 6, it will increase slowly or not with the increase or decrease.
(3) the influence of light. The strain adapts to weak light and short light in the early stage of ascospore germination and mycelium growth, and adapts to strong light in the later stage. In artificial culture, hyphae, conidia and substrate have obvious phototaxis, which grow densely in the sunny side and sparsely in the backlight side. All kinds of bacteria cultured in the all-black environment are delicate, slender and sparse.
(4) Utilization of carbon source. Cordyceps sinensis can use a variety of carbon sources, but when glucose and maltose are used together, it grows fastest, and when glucose is used alone, it can grow well, followed by starch such as potato and sucrose.
Cordyceps sinensis is a fungus that grows from a parasitic host to a saprophytic stage, so it can make good use of various organic nitrogen sources. And live worms are the best; The mixed use of peptone and yeast extract is also very ideal for growth, followed by their own use, followed by beef sauce and so on. However, the utilization of inorganic nitrogen such as ammonium sulfate and potassium nitrate is poor.
There is a certain demand for ash nutrients, which grow vigorously in the medium containing trace amounts of magnesium sulfate and dipotassium hydrogen phosphate, followed by potassium dihydrogen phosphate, and other inorganic salts such as sodium, calcium, iron and copper can also be used.
3. After separation and purification, Cordyceps sinensis can be used as various experiments and expanded production applications.
(1) There are usually three methods to increase its yield: solid static culture, shake flask culture and large tank aeration fermentation culture.
A. Static culture: mainly used for solid culture, such as tube slant culture, triangular flask culture, rice culture, etc. In static culture, bacteria can grow normally as long as the temperature and light are well controlled. The conidia on the inclined plane can be stored in the refrigerator at 1-2℃ for 8- 14 months after maturity, and can also be directly used as seeds for production.
B. shaking culture: both liquid culture and small-scale propagation culture can be used. Shake the medium and subtract agar from the solid medium. It is best to choose a constant temperature oscillating culture machine, put the liquid culture medium into a triangular bottle and inoculate the test-tube solid strain for culture. After continuous oscillation, the components in the liquid medium are mixed evenly without precipitation, and at the same time, gas-liquid contact and exchange are promoted, so that oxygen enters the liquid medium, which is beneficial to mycelium growth and conidia formation.
C. aeration fermentation culture in large tanks: When producing mycelia, conidia and other bacterial powders on a large scale, aeration fermentation culture in large tanks must be used. In this method, aeration is carried out through suction or vacuum pump decompression, and bacteria are removed through a filter, and then they are sent to a liquid culture medium in a tank for the growth and development of Cordyceps sinensis.
(2) Fermentation process. Separation and purification of natural Cordyceps sinensis → slant seeds in test tubes → primary seeds of oscillating liquid → secondary seeds → small-scale seed fermentor → production fermentor → concentration → powder spraying and drying → finished bacterial powder.
The fermentation temperature of Cordyceps sinensis in the tank is 20-25℃, the tank pressure is 392.3-686.5kPa(0.4-0.7kg/cmz), and the ventilation rate is 0.5- 1.ovvm *. The liquid culture medium injected into the fermentor should be 65%-75% of the tank capacity. The inoculation amount is 65438 00%, the stirring speed is 65438 080 rpm, the culture time is 72-96 hours, and then it is put into a tank for concentration and powder spraying.
(3) Multi-purpose fermentation medium. Fresh potato (peeled) 8%, sucrose 2%, corn starch 0.5%, silkworm chrysalis powder 1%, peptone 0.4%, ammonium sulfate 0.2%, and pH value 5.5-6.0.
(4) the standard of fermentation culture products. Conidia are almost completely separated female spore stalks. When the number of spore stalks did not increase obviously, the number of conidia per ml was 654.38 0.8 billion ~ 2.5 billion. Residual sugar is less than1%; When the amino nitrogen is lower than 0.2mg/mL, it can become the finished bacterial liquid.
4. Inoculation points of Cordyceps sinensis: Inoculating host insects with sexual or asexual Cordyceps sinensis spores is a key measure to artificially cultivate sexual Cordyceps sinensis and rejuvenate asexual strains.
(1) tieback period. The key to the reconnection of Cordyceps sinensis is to choose the weak antibacterial period of the host insect, one is the molting period; The second is the period when the larval feeding activity is intense and the friction damage rate is high.
(2) tieback method. There are two kinds.
A. Spraying method: When the hepialus moth larvae molt at the age of 4-6 and above 65,438+0/3, the larvae are concentrated and sprayed with 5%-65,438+00% glucose solution on the insect body or food with ascospores or conidia. After inoculation, the bacterial liquid is slightly dried on the insect body for about 30 minutes, and the larvae are put back into the soil to live naturally. This method has a high infection rate, but the disadvantage is that the larvae are not easy to be taken out of the soil and concentrated, and the larvae have the habit of killing each other when concentrated. After being bitten, bugs are easily infected by miscellaneous bacteria and die. When the mycelium of Cordyceps sinensis does not fill the body cavity, the injured worm will rot and cannot form a rigid worm (sclerotium).
B. Natural contact method: feeding hepialus moth larvae in a large area of semi-natural soil, and regularly taking small samples to observe the growth of the larvae. When most of the larvae are suitable for infection, fine soil is planted and inoculated with feed plant soaking solution, which is evenly spread on the surface of the raised insects, and then water is sprayed to make bacteria penetrate into the soil, so that the larvae are exposed to infection during feeding activities. The infection rate of this method is not too high, but once infected, Cordyceps sinensis grows well, and uninfected hepialus larvae can also grow and reproduce normally.
(Excerpted from Yang's Utilization and Industrialization of Insect Resources in China)
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