WO2015003547A1

WO2015003547A1 - Culture method for natural species rejuvenation of artificially bred cordyceps strains

Info

Publication number: WO2015003547A1
Application number: PCT/CN2014/080068
Authority: WO
Inventors: 刘飞
Original assignee: 重庆市中药研究院
Priority date: 2013-07-12
Filing date: 2014-06-17
Publication date: 2015-01-15
Also published as: CN103404368A; CN103404368B

Abstract

Disclosed is a method for natural species rejuvenation of artificially bred strains, comprising the following steps: 1) infecting cordyceps host larvae with cordyceps strains artificially bred for more than 2 generations, screening the contaminated larvae which are firstly rejuvenated, and placing the larvae in a rejuvenation base after continuously culturing for 1 to 2 stages; 2) managing the transition periods so that the contaminated larvae adapt to the wild environment; 3) managing the specie nature exercise period of the bombyx insects; 4) rejuvenation of natural species of sexual spores and mycodermas; 5) collecting cordyceps with mature sexual spores, and transplanting to a sterilized culture medium to continuously culture after washing the insect-strain complexes; 6) after-ripening of the cordyceps; and 7) harvesting cordyceps strain materials. The method provides a strain guarantee for large-scale sustainable production of cordyceps.

Description

Breeding and breeding method for artificially breeding Cordyceps species

Technical field

The invention relates to a strain breeding technique, and more particularly to a method for rejuvenating and cultivating artificially breeding Cordyceps species. technical background

Cordyceps mainly refers to Cordyceps sinensis. It also refers to other combinations of fungi (C. oxysporum) with host larvae or mites under special conditions, including Asian worms, Cordyceps militaris and Cordyceps militaris; Cordyceps sinensis, It is a rare and precious Chinese herbal medicine in China. It has the functions of nourishing the lungs and kidneys, relieving cough and phlegm, treating cough, hemoptysis, impotence and nocturnal emission, physical weakness after illness, and chronic cough in the elderly. Cordyceps sinensis is mainly produced in the meadows of the alpine mountainous areas of 3500-5200m above sea level in Tibet, Qinghai, Yunnan, Sichuan and Gansu provinces. Natural resources are very limited. In recent years, excessive excavation has caused the deterioration of the ecological environment, and natural resources have become increasingly exhausted. Therefore, artificial cultivation of Cordyceps sinensis is imperative. The key to artificial culture is whether Cordyceps sinensis can infect the host insects, enter the host's body cavity, and grow and grow in the body cavity, and finally produce a sub-seat to form a winter insect summer herb. The infection path of the wild worms of the larvae of the larvae is that the larvae of the genus Batae fly through the body surface or the esophagus to infect the genus Corydalis in the wild, but the above method has a low infection rate, a long period, and cannot be scaled up. produce.

At present, the host artificial infection method is to apply the Chinese spores of the spores to the host body, inject into the host, infuse or pass the word material from the host esophagus into the host. Tu Yongqin et al., “Experimental study on the infection of Cordyceps sinensis by the larvae of the genus Bismuth” published in the journal Edible Fungus 2010 (3), discloses a variety of strains of the genus Corydalis sinensis as the host of Cordyceps sinensis. However, the above method has the problems of separation and purification of the species of Trichosporon sinensis, and the larvae infection rate and the survival rate after infection are low. In the process of artificially cultured Cordyceps, the applicant found that the ascospores of the Cordyceps sinensis were incompletely developed from the collection of ascospores, conidia and fungus in the Cordyceps sinensis after several generations of artificial culture. The rate of infection of the collected reproductive material against the host larvae of Cordyceps sinensis was lower than that of the wild material. Cordyceps species are bred under artificial conditions. After 3-5 generations, the offspring of the offspring are underdeveloped or not developed, and the invasiveness of the strains is reduced. For the problem of various degeneration of Cordyceps in production, the traditional solution is to continuously collect strains from the native environment every year. Due to the limited resources of wild strains, uncontrolled indiscriminate excavation not only makes the species resources endangered, but also seriously destroys the natural ecological environment. Therefore, in order to achieve sustainable cultivation of the strains and sustainable development of the Cordyceps industry, it must be adopted. The breeding and breeding techniques of artificial breeding strains.

Summary of the invention

In order to solve the problem of species degradation of artificially propagated Cordyceps species, the present invention provides a method for breeding a species of artificially propagated Cordyceps species, comprising the following steps:

1) Artificially propagated second-generation (or third-generation, fourth-generation, fifth-generation, etc.) Cordyceps species to Cordyceps host larvae (Artificial or wild larvae can be used) to carry out bacterial infection, screening the first rejuvenated susceptible larvae, continue to culture 1-2 years old, and then put them into the rejuvenation base;

2) The larvae adapt to the transitional management of the wild environment;

3) Management of the exercise period of the zombie species;

4) Rejuvenation of sexual spores and fungus species;

5) Collecting the sexually active spores and the mature fungus Cordyceps sinensis, and after washing the worm complex, put it into the sterilized medium and continue to culture;

6) Post-ripening of Cordyceps;

7) Harvesting Cordyceps species.

Screen the first viable larvae that rejuvenate. The standard for rejuvenation is to start eating and start attacking other companions, starting with the first rejuvenated larvae to 40% of the entire larvae to be rejuvenated as the reinforced Cordyceps host larvae.

The transition period described in the above step 3) is the first 1-2 months after the rejuvenated susceptible larvae are placed in the native environment; during this period, the temperature should be controlled at 5-25 °C, and the soil moisture is controlled in quality. The water content is 50-80%, avoiding the rodents and birds from killing the larvae of the Cordyceps host, and avoiding the animal husbandry stepping on the larvae of the Cordyceps host.

The above-mentioned step 4) management of the zombie species is as follows: In the larvae of the larvae, the rodents and the birds should be prevented from catching and killing the larvae of the Cordyceps sinensis, but the susceptibility should be squeezed in the grazing larvae. The soil matrix in which the larva lives, thereby insulting the companion or natural enemies with a tight soil matrix; keeping the base of the vegetation bare, increasing the oxygen supply and light of the soil.

The rejuvenation of the spores and the fungus species mentioned in the above step 5) is to control the soil moisture to be 60-80% of the mass water content; avoid rodents and birds to kill the larvae of the Cordyceps host, avoid animal treading, and develop into sexuality. Spores and fungus mature Cordyceps.

Step 6) The post-ripening of the Cordyceps is to wash the Cordyceps mature grass and then transplant it into the indoor 60-10CTC high temperature disinfection substrate, the indoor temperature is controlled at 8-25 ° C, using 60-100 ° C After the high temperature disinfection treatment, the cold water is moisturized, and the natural photoperiod and natural light intensity are used for 7-15 days to collect the strain material.

The above-mentioned sign of sexual spore maturation is that the sub-seat begins to appear as a white filament.

The beneficial technical effects of the present invention are as follows: The seed rejuvenation method of the artificial breeding strain provided by the invention not only effectively solves the problem of species degradation of the artificial breeding strain, but more importantly solves the artificial breeding in the actual production process of Cordyceps. The problem of sustainable production of strains provides a kind of strain protection for large-scale and sustainable production of Cordyceps, which can effectively avoid wild species of Cordyceps Resource dependence. detailed description

Example 1 Screening of host larvae infected with bacteria

1500 larvae of bat moth artificial vocabulary were collected for 1-2 months, respectively, named experimental group I, experimental group II, experimental group III, experimental group IV and experimental group. The method of cultivating Cordyceps sinensis by the method described in the patent application "A low-altitude artificial culture method of Cordyceps sinensis" (Application No.: CN201210292550. 6) is collected, and ascospores, conidia and fungus of Cordyceps sinensis cultivated by the method are used as bacteria. According to the method, the Cordyceps sinensis is repeatedly cultured according to the method; the above operations are repeated three times or more, and the obtained ascospores, conidia and fungus are used as propagation materials, and these propagation materials are mixed as artificial propagation materials. Transfer artificial propagation materials to environmental conditions with air cleanliness requirements of not less than 100,000; workers (in accordance with cleanliness requirements) are required to wear overalls, masks and hats.

The experimental group I, the experimental group II, the experimental group III, the experimental group IV and the experimental group V were respectively subjected to artificial expansion and propagation material infection treatment. The method of infection is described in the method described in "A method for the infection of host plants of the Cordyceps sinensis and host infection method" (Application No.: CN201210338583.X). The infected larvae are continuously collected after infection; the criteria for screening are: 1) the host larvae have reduced activity, and 2) do not eat. Each group was selected 500; as each experimental group.

The above-mentioned larvae infected with the genus Trichosporon sinensis are concentrated, and in addition to providing a conventional vocabulary amount, it is necessary to provide sufficient water. Inoculation by spraying and inoculation by irrigation requires an increase in the amount of subsequent water supply; it is necessary to increase the humidity of the word or substrate by inoculating the word or mixing into the substrate. Intensive wording is done at a temperature of 8-18 °C. During the concentration of words, the larvae with active recovery are continuously selected until the specified number is selected. The criteria for judging are: 1) starting to resume eating; 2) starting to attack other host larvae. The amount of screening in the experimental group I was 100, the amount in the experimental group II was 150, the amount in the experimental group III was 200, the amount in the experimental group IV was 250, and the amount in the experimental group V was 300.

Experimental group I, experimental group II, experimental group III, experimental group IV, and experimental group V were respectively placed in the area of the Cordyceps sinensis producing area to prevent the large animals such as cattle and sheep from trampling at any time, and the surrounding area of the culture area was surrounded by barbed wire. The slope of the rejuvenation base is preferably between 10 and 30 degrees, and the drainage is good. There are other shrubs other than turf plants to avoid high-intensity sunshine and bird damage to adults. The survival rate of the host larvae of each group was counted after 50 days of culture. The details are shown in Table 1. Table 1 Effect of different screening ratios on survival rate of host larvae

Group Experimental group I Experimental group II Experimental group III Experimental group IV Experimental group V

Number of survivors (bars) 73 111 141 161 186

Survival rate (%) 73. 0 74. 0 70. 5 64. 4 62. 2

The contents in Table 1 demonstrate that the survival rate of host larvae is greatly reduced after the selection of host larvae is higher than 40%, so the proportion of screening strong larvae is 40%.

The host larvae to be tested in the experimental group I, the experimental group II, the experimental group III, the experimental group IV, and the experimental group V gradually adapted to the living environment in the field and completed the transition period.

After the larva crawls from the putting area to the surrounding native humus soil, the exercise period begins. The larvae at this stage will receive natural exercise from the temperature, humidity and light of the natural world and will develop resistance to various diseases, insects and rodents until the larva develops into a zombie. When the base grass begins to grow and gradually covers the surface, it is possible to allow the herbivores such as cattle and sheep to enter the base to feed the grass, so that the base of the turf is exposed to the outside, which will provide sufficient oxygen for the larvae of the underground growth and development. Conditions, while light will also have a positive impact on larval growth and development.

After the exercise period, the insects enter the rejuvenation period and begin to germinate the fruiting bodies. During this period, large animals such as cattle and sheep are strictly prohibited from entering the base; physical or chemical measures should be taken to prevent the harm of rodent and bird natural enemies against susceptible larvae. The formation of the zombies into the sub-seat begins to germination. The matricity of the substrate to prevent its growth is insufficient. Watering should be done in the morning and evening on a sunny day. The standard is to wet the soil layer where the zombie is located. Adequate moisture conditions not only facilitate the growth of the sub-seat, but also help to produce a stronger skin. The development of the sub-seat at this stage will be subjected to natural cycles of temperature, humidity and light in nature, together with the fungus to produce resistance to various diseases. During the rejuvenation period, large animals such as cattle and sheep should be strictly prohibited from entering the base; physical or chemical measures should be taken to prevent the harm of rodent and bird natural enemies to susceptible larvae until they develop into sexual spores (including ascospores and meristems). Spore) Mature Cordyceps sinensis.

When the sexual spores and fungus of Cordyceps sinensis mature in the seed rejuvenation base, it is necessary to go through a post-maturing period to adapt to the process of returning from the field to the interior. During the operation, the Cordyceps sinensis is transplanted into the collection tray of the strain material by means of a spade or the like for post-cultivation. Remove the substrate or other impurities carried during transplanting. During the post-ripening period, it is moisturized with cold water sterilized at 60-100 °C, and placed at 5-25 °C, and cultured with natural photoperiod and natural light intensity. After about 1 week of rejuvenation, the sexual spores (including ascospores and conidia) and fungus of Cordyceps sinensis gradually adapted to the indoor environment. The spores and the fungus which have undergone the post-ripening cycle are collected and used as a propagation material for the rejuvenating bacteria.

Example 2 Activity of Reproductive Materials of Rejuvenated Strains

Refer to the patent application "A Low-Elevation Artificial Culture Method of Cordyceps sinensis" (Application No.: CN201210292550. 6) to cultivate Cordyceps sinensis; collect Asipialus xiao jinensisTu etMa larvae, 4000 insects and aliquots were placed in 8 lm ² clean sterilized enamel dishes, each group of 500 worms, covered with some sterilized dry leaves, to facilitate insect activity and concealment, spray some steamed water Keep humidity. First, use sterile bead buds, carrots, red peony, and leaf characters to feed for 7 days to restore the growth of the worms. Then remove all remaining food. After stopping feeding for 2 to 3 days, take any of the above three groups of words to feed the artificially propagated (more than 2 generations) material suspension sprayed bead buds, carrots, red peony, and vegetable leaves as a control group.

Take any 4 groups of words from the suspension of the rejuvenating strain propagation material obtained in Example 1 and spray the bead buds, carrots, red peony, and vegetable leaves as the experimental group; remove the residue every day, and use the same for the second day (continued) The method word is fed, even for 30 days, to observe the growth and activity of the larvae, then the growth is robust. Agile, responsive larvae, then covered with 10cm of alpine meadow soil and turf recovered from the larval growth area, controlled at 1~5 °C, continue to feed, at 1 month, 3 months, 6 After one month and one year, the experimental group and the control group were each excavated from the worm body, and the number of infected and dead worms was calculated. The data of the mixed vocabulary feeding method group was obtained.

Twenty larvae were randomly selected from the larval population of each experimental group for investigation at the time of 1 month, 3 months, 6 months and 1 year. After disinfecting the surface of the larvae, a small amount of hemolymph was taken under sterile conditions using a special tool, and the presence or absence and morphology of the cells in the hemolymph were recorded by microscopic examination. Follow up to observe the larval rigidity. While observing the cells, a small amount of hemolymph was taken and applied to a sterilized PDA medium plate, and cultured in an 18 ° C incubator. Take out the culture dish once a day to observe the growth and development of the cells, or pick the cells and their culture to observe. Colonies were formed at the cells to be coated, and the hyphae of the colony surface was taken for microscopic examination. The specific results are shown in Table 2.

Infection of larvae from different strains

Strain material Detection time Survival larvae / head Average infection rate /%

1 month later 385 33

Control group 3 months later 340 46

6 months later 297 80

1 year later 263 90

1 month later 384 81

Experimental group 3 months later 321 95

6 months later 295 100

1 year later 261 100

The results showed that the host larvae infected with Cordyceps sinensis was a complicated process that took a certain amount of time to complete the entire infection process. As can be seen from Table 2, using the regenerative strain propagation material described in Example 1, the strain has the strongest infection ability to the host larvae, and the host larvae can be infected with the Cordyceps sinensis after one month of inoculation. The infection reached a peak after 6 months of inoculation, and the infection rate was 100%. After a year of word cultivation, the larvae can become rigid, and then sprout out of the young shoots and grow into fruit bodies. The experimental group obtained by infecting the larvae of the rejuvenating bacterial propagation material provided by the present invention is compared with the control group obtained by infecting the larva without the rejuvenated propagation material.

It was found that the morphological characteristics of ascospores, ascospores and fruit bodies of Cordyceps sinensis and wild Cordyceps sinensis obtained by the bacterium of the cultivar obtained by the present invention are almost identical; compared with Cordyceps sinensis which has not been regenerated by artificial expansion and propagation materials, There are large differences in morphology between Cordyceps sinensis spores, ascospores and fruiting bodies cultivated by artificially expanded and propagated materials. Wherein, the ascospore fruit fruit rate is the fullness rate of the ascospores on the fruit body; the yield of each group of Cordyceps sinensis is shown in Table 3.

Table 3 Effect of Different Strain Materials on the Yield and Morphology of Cordyceps Sinensis

Group Ascospore fruit rate Ascospore sp. per 100 bars > 5000 bars / kg

(%) (%) (Article) Control group 76. 5 68 52

Experimental group 92. 4 94 71

Wild group 92. 1 99 77

Studies have shown that after 3-5 generations of artificially propagated Cordyceps species, there will be species-like degradation; the appearance is the obvious degeneration of the offspring with incomplete or undeveloped ass of the offspring and the reduced invasiveness of the species. Using the method provided by the present invention to reproduce the Cordyceps sinensis produced by the strain, the Cordyceps grade and the ascospore filling rate are close to those of the wild Cordyceps.

Claims

1. A method for breeding and breeding a Cordyceps species, characterized in that: the following steps are included:

1) Inoculation of Cordyceps host larvae by artificially bred 2 or more generations of Cordyceps sinensis, screening the first larvae that have revived vitality, continuing to culture 1-2 years old, and placing them in the rejuvenation base;

2) The transition period management of susceptible larvae adapted to the wild environment;

3) Management of the exercise period of the snakes;

4) Rejuvenation of sexual spores and fungus species;

5) Collecting the mature spores of the spores, cleaning the worm complex, transplanting them into the sterilized culture medium and continuing the cultivation;

6) Post-ripening of Cordyceps;

7) Harvesting Cordyceps species.

2. The method for rejuvenating a species of artificially propagated Cordyceps sinensis according to claim 1, wherein: the criterion for rejuvenation is to start eating and start attacking other companions; the larvae that recover from the first article start to collect It is a susceptible larvae that rejuvenates and cultures until it accounts for 40% of the larvae of the entire susceptible larvae.

The method according to claim 1, wherein the transition period management in the step 2) is the first one after the rejuvenating susceptible larvae are placed in the native environment. 2 months; During this period, the temperature should be controlled at 8-25 °C, and the soil moisture should be controlled at 50-80% of the water content. Avoid rodents and birds to kill the larvae of Cordyceps host, and avoid animal husbandry to step on the larvae of Cordyceps host.

The method for rejuvenating the artificial breeding Cordyceps species according to claim 1, wherein: step 3) the training period of the zombie species is: avoiding rodents in the larvae of the larvae, The birds are killed by the host larvae of the Cordyceps sinensis, but the soil matrix of the larvae of the larvae should be squeezed in the grazing area of the larvae to bury the companion or natural enemies with a tight soil matrix; keep the base of the vegetation exposed and increase the soil. Oxygen supply and light.

The method for rejuvenating the artificial breeding Cordyceps sinensis according to claim 1, wherein: step 4) the sexual rejuvenation of the spores and the fungus is controlled to control the soil moisture as a mass water content of 60- 80%; Avoid trampling and feeding of rodents and birds to Cordyceps until it develops into Cordyceps with mature spores and mature skin.

6. The method for rejuvenating a species of artificially propagated Cordyceps species according to claim 1, wherein: Step 5) The post-ripening of the Cordyceps is to wash the cordyceps and the matured Cordyceps, and then transplant it into a matrix of 60-100 ° C high temperature disinfection treatment, and the indoor temperature is controlled at 8-25 ° C. 60_10 (TC cold water moisturizing after high temperature sterilization, cultured for 7-15 days using natural photoperiod and natural light intensity, collect strains.

7. The method for rejuvenating a species of artificially propagated Cordyceps sinensis according to claim 6, wherein: the signs of sexual spores and bacterial ripening begin to appear as white filaments in the stalk.