(1) At present, no one has been able to isolate and culture authentic Cordyceps sinensis fungi for a long time and stably (that is, repeatedly grow stroma in batches on the culture medium). In Qinghai, Yunnan, Sichuan, Beijing and other places, the stroma has been cultivated, but its ascospores are few or no, and the growth is unstable, which does not mean that the cultivation technology of sexual artificial in middle and low altitude has been mastered.
(2) The artificial asexual culture technology of Cordyceps sinensis is relatively easy to master, and it can be infected into a stiff stage when inoculated into hepialus larvae indoors, but it is rare to complete the sexual form and grow a daughter and an ascospore.
(3) At present, apart from many environmental factors such as altitude, climate, soil, air pressure, etc., is there a multi-bacterial infection among the bacteria? If it is a compound infection, which bacteria are playing a key role? In what period did it work with Cordyceps sinensis? Wait, the question is not clear.
Therefore, a lot of research and experimental work are needed to cultivate sex Cordyceps sinensis artificially in batches and repeatedly 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 need to be carried out. Therefore, the artificial cultivation of sexual Cordyceps sinensis is still very arduous and far away to achieve large-scale or industrial production.
(II) Current situation of artificial cultivation of asexual Cordyceps sinensis
Due to the great difficulty in artificial cultivation of sexual Cordyceps sinensis, in recent 1 years, most units and researchers who have carried out artificial cultivation of Cordyceps sinensis have turned to the research and application of asexual cordyceps sinensis. 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 functions to the sexual components of Cordyceps sinensis, so all localities have identified the self-isolated fungi as the asexual stage of Cordyceps sinensis, and have set up factories to produce bacterial powder and develop and apply them. At present, 31 species belonging to 9 genera have been isolated from Cordyceps sinensis in nature, among which 16 species have been used in more than 4 provinces (cities) such as Beijing, Jilin, Shanxi, Shanghai, Qinghai, Zhejiang, Fujian, Jiangxi, Guangdong, Yunnan, Guizhou, etc. for large-scale fermentation culture and solid culture of mycelium and conidia as sexual Cordyceps sinensis. Such as Zhiling Capsule, Jinshuibao Capsule, Ningxinbao Capsule, Xinganbao, Cordyceps Chicken Essence, Cordyceps Capsule, Cordyceps Wine, Cordyceps Ginseng tonic wine, etc., the annual output value of more than 2 products has reached 1 million yuan to several hundred million yuan. Moreover, because of the short artificial propagation cycle and high yield, mycelium and meristem robe have great potential for development and application as sexual substitutes of Cordyceps sinensis in the future.
In order to obtain pure strains of winter, early summer and grass, manual separation, purification, rejuvenation and preservation must be carried out.
1. Technology and conditions for isolating Cordyceps sinensis
(1) Material requirements. Obtaining pure strains requires strict materials; Generally speaking, if the method of tissue block separation of Cordyceps sinensis is used, the best separation material is the material collected from the end of October to November every year when the soil in alpine meadow in Qinghai-Tibet Plateau has just begun to freeze. In this period, Cordyceps sinensis has just infected hepialus larvae and entered the stage of stiff insects for a short time, and the tender daughter buds just grow .2-.5cm from the head of the insect, and there are few other miscellaneous bacteria in the insect body, so it is the easiest to separate. If the material is collected in May and June of the following year, there are many kinds of miscellaneous bacteria living or saprophytic in the body and sub-seat of the stiff worm, so it is difficult to obtain pure strains. If the ascospores are used for isolation and culture, it is best to collect and isolate the materials in the middle and late July of each year when the ascospores have just partially matured.
(2) operation method. There are three steps.
a. separation of stiff worms (i.e. sclerotia): before separation, wash the surface of the main body with water, and then wash it with sterile water for 2-3 times; Disinfect the surface of the separated materials with .1%-.2% mercuric chloride solution for about 3-5min minutes; Then clean with sterile water. Select the front part bounded by chest and foot. Cut off the epidermis with a scalpel, avoid the digestive tract, take hyphae from the blood cavity and cut them into sesame seeds, and press them into a flat culture medium, with 1-2 grains per dish. Culture at 15-19℃, when the colony grows .2-.5cm, select a small amount of hyphae and repeatedly purify them on the plate medium for 2-4 times. After confirming that there are no other miscellaneous bacteria, transfer them into test tubes for preservation and culture.
B. daughter bud separation: cut off the washed daughter bud from the top of the stiff worm, put it 3minto .1% mercuric chloride solution for disinfection for 2-3 minutes, wash it with sterile water, cut off the tissue block in the middle part and press it into the culture medium; The culture conditions are the same as a.
C. Ascomycete spore separation: cover the mature stroma of Ascomycete spores with a transparent paper bag, and let the Ascomycete spores bounce and stick on the paper bag. Then, immerse the paper bag with Ascomycete spores in 25% glucose solution, wash the spores, put them in 15-19℃ for culture, and make microscopic examination every day. When the robe germinates, use a micropipette to suck a single spore into a flat plate for culture; The culture conditions are the same as a. Alternatively, the whole mature Cordyceps sinensis can be brought indoors, and the sub-seats of stiff worms and even worms are wrapped with cotton paper, leaving only the pregnant part, and a glass slide is placed horizontally in the sterile room to keep the main body humidity at any time; Microscopic examination every day, when ascospores are seen bouncing on the glass slide, they are sucked into the culture medium of the 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), which is a universal medium for all Cordyceps fungi, can be used in the initial stage of isolation and culture, but the strain is not very vigorous and easy to age and degenerate.
B. Enriched culture medium (1): 1g of protein umbilical cord, 5g of glucose, 1g of dipotassium hydrogen phosphate, .5g of magnesium sulfate, 3g of live silkworm chrysalis, .5μg of auxin, 2g of agar, 1mL of water, and pH： 5..
enrichment medium (ⅱ): 4g preserved protein, 4g glucose, 1g peeled fresh potato, 1g dipotassium hydrogen phosphate, .5g magnesium sulfate, 1g beef paste, .5μg auxin, 3g live hepialus moth larva (ground), 2g agar, 1OOmL alpine meadow soil leaching solution, pH: 5..
On enriched medium, 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, light, carbon and nitrogen sources and inorganic salts.
(1) Effects of different temperatures on mycelium growth. Cordyceps sinensis likes low temperature. It can grow slowly at -4℃; The growth rate is accelerated at 5-8℃; 1-19℃ is the suitable growth and development range of this strain; The optimum growth temperature is 15-19℃. When the temperature exceeds 2℃, the hyphae grow rapidly, and the colonies change from white to grayish black or brownish yellow, and begin to mutate, so it is not suitable for high temperature culture.
(2) requirements for pH value. Cordyceps sinensis is a kind of acid fungus, and its optimum pH value is 5.-6.. When the pH value is below 4.5 and above 6, it will grow slowly or not with the increase or decrease.
(3) the influence of light. In the early stage of ascospore germination and mycelium growth, this strain was adapted to weak light and short light, and in the later stage it was adapted to strong light. In artificial culture, hyphae, conidia and stroma have obvious phototaxis, and they grow densely on the sunny side, sparsely on the backlight side, and all kinds of bacteria cultured in the all-black environment are delicate, slender and sparse.
(4) Utilization of carbon sources. 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 develops from a parasitic living host to a saprophytic stage, so it can make good use of various organic nitrogen sources. And live worms are the best; The combination of peptone and yeast extract is also very ideal for growth, followed by their separate use, followed by beef paste 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 with 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. Culture method Cordyceps sinensis can be used as various experiments and expanded production applications after separation and purification.
(1) There are three methods to expand its production: solid static culture, shaking culture and aeration fermentation culture in large tanks.
a. static culture: mainly used in solid culture, such as tube slant culture, triangular flask culture, rice culture and so on. In static culture, as long as the temperature and light are well mastered, the bacteria can grow normally. When the conidia on the inclined plane are mature, they can be stored in a refrigerator at 1-2℃ for 8-14 months, and can also be directly used as seeds for production.
B. oscillation culture: both liquid culture and small-scale propagation culture can be used. Shake the culture medium and subtract agar from the solid culture medium. It is best to choose a constant temperature oscillation culture machine, put liquid culture medium in a triangular bottle, and inoculate the test tube solid strain to culture. After continuous oscillation, all components in the liquid culture medium are mixed evenly without precipitation, and at the same time, the contact and exchange between gas and liquid are promoted, so that oxygen enters the liquid culture 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 by suction or vacuum pump decompression, and bacteria are removed through a filter, which is 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 seed in test tube → primary seed of oscillator liquid → secondary seed → small seed fermentation tank → production fermentor → concentration → powder spraying and drying → finished bacterial powder.
The fermentation temperature of Cordyceps sinensis in the tank is 2-25℃, the tank pressure is 392.3-686.5kPa(.4-.7kg/cmz), and the ventilation rate is .5-1.ovvm *. The liquid culture medium injected into the fermentor should be 65%-75% of the tank capacity. When the inoculation amount is 1%, the stirring speed is 18r/min, and the culture time is 72-96h, it can be concentrated and sprayed with powder.
(3) The fermentation medium is versatile. Fresh potato (peeled) 8%, sucrose 2%, corn starch .5%, silkworm chrysalis powder 1%, peptone .4%, ammonium sulfate .2%, pH value 5.5-6..
(4) the standard of fermentation culture products. Conidia, i.e., almost completely detached female spore stalks, when the number of spore stalks does not increase obviously, the number of conidia per milliliter is 1.8 billion to 12.5 billion. Residual sugar is less than 1%; When the amino nitrogen is lower than .2mg/mL, it can become the finished bacterial liquid.
4. Key points of Cordyceps sinensis inoculation: Inoculating sexual or asexual Cordyceps sinensis spores to host insects is a key measure for artificially cultivating sexual Cordyceps sinensis and rejuvenating asexual strains.
(1) tieback period. The key to the reconnection of Cordyceps sinensis is to choose the weak antibacterial period of host insects, one is 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. spray method: when the larvae of hepialus hepialus are 4-6 years old and more than 1/3 of the larvae molt, concentrate the larvae, spray 5%-1% glucose solution with ascospores or conidia on the worms or food, and after about 3min minutes of inoculation, the bacterial solution will be slightly dried on the worms, and the larvae will be returned to the soil for natural life. 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 when concentrated, the larvae have the habit of killing each other. After being bitten, the worms are easily infected by miscellaneous bacteria and die. When the hyphae of Cordyceps sinensis are not full of body cavities, the injured worms will rot and cannot form rigid worms (sclerotia).
B. natural contact method: raise hepialus moth larvae in a large area of semi-natural soil, regularly take small samples to observe the growth status of the larvae, when most larvae are suitable to be infected, plant them with feed plant fungus soaking solution and inoculate them with fine soil, evenly spread them on the surface where the insects are raised, and then spray water to make the 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 the uninfected hepialus larvae can grow normally and reproduce.
(selected from Utilization and Industrialization of Insect Resources in China by Yang Darong) Extracted from Utilization and Industrialization of Insect Resources in China
Cultivation Techniques of Early Summer Grass in Winter
(1) Overview of alias Cordyceps sinensis, Cordyceps sinensis and Cordyceps sinensis. It is the daughter of Cordyceps sinensis (Berk.) Sacc., a plant belonging to the genus Cordyceps of ergot family, and the dried insect body of its strange owner. Gan, Wen. Nourishing lung and tonifying kidney, it is mainly used for treating pulmonary tuberculosis, hemoptysis, asthenic asthma, night sweats, nocturnal emission, impotence and soreness of waist and knees. Mostly born in alpine mountains, grasslands, river valleys and grass. Distributed in Gansu, Qinghai, Sichuan, Yunnan, Tibet and other provinces and autonomous regions.
(2) Plant characteristics Cordyceps sinensis is a combination of fungi belonging to Ascomycetes, Caryopteris, Ergodiaceae and Cordyceps that parasitize the larvae of Lepidoptera moth. The whole shape is made up of insect body and fruiting body, with yellow-brown or yellowish brown appearance. The insect body is very similar to silkworm, slender and cylindrical, 3 ~ 5 cm long and .3 ~ 1 cm thick, with 2 ~ 3 obvious links, and there are 8 pairs in the abdomen, especially 4 pairs in the middle. The fruiting body refers to the extension outside the host insect. A reproductive organ that can be recognized by the naked eye after repeated knots and differentiation of hyphae. The texture is crisp, the section is yellow-white, the head is reddish brown, and there are daughter seats. The daughter seats are mostly solitary, slender like sticks, 4 ~ 1 cm long, the top is swollen, the surface is brown, the texture is slightly tough, and the section is yellow-white and fibrous. The head of the daughter seat is scattered or densely packed with ascomycetes, and numerous ascomycetes are formed in the ascomycetes, which are ascospores that can reproduce sexually.
(3) Growth characteristics of Cordyceps sinensis