CN111527987B - Method for improving yield of cordyceps militaris sporocarp by two-step inoculation - Google Patents
Method for improving yield of cordyceps militaris sporocarp by two-step inoculation Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/40—Cultivation of spawn
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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Abstract
The invention discloses a method for improving the yield of cordyceps militaris sporocarp by two-step inoculation, which comprises the steps of inoculating a cordyceps militaris mononuclear body strain to a cordyceps militaris cultivation medium, and culturing for a period of time; inoculating another Cordyceps militaris monokaryon, heterokaryon or homokaryon strain containing opposite mating type gene with the inoculated monokaryon strain to the first inoculated Cordyceps militaris culture, and continuously culturing until Cordyceps militaris fruiting body is obtained. The two-step inoculation method adopted by the invention can reduce the strain degeneration influence caused by karyotype change in large-scale production and greatly improve the biotransformation rate of the cordyceps militaris sporocarp, so that the biological yield of the cordyceps militaris sporocarp can be effectively improved.
Description
Technical Field
The invention relates to the technical field of edible fungus cultivation, in particular to a method for improving the biological yield of cordyceps militaris sporocarp by manually regulating and controlling the inoculation time and mode of cordyceps militaris strains.
Background
Cordyceps militaris (Cordyceps militaris), also known as Cordyceps militaris, belongs to Ascomycotina, Pyrenomycetes, Clavicipitales, Clavicipitaceae, Cordyceps, and is a model species of Cordyceps fungi. Cordyceps fungi are a generic term for a class of entomogenous fungi complexes formed by parasitizing the underground fruiting bodies of insects, arachnids or macropolybia. Cordyceps militaris hosts in the wild on larvae and pupae of some insects of the orders Lepidoptera, Coleoptera and Diptera. The Cordyceps militaris contains abundant nutritional ingredients such as protein and vitamins, Cordyceps polysaccharide, adenosine, cordycepin, cordycepic acid, and N6A large number of bioactive substances such as- (2-hydroxyethyl) adenosine, pentostatin and the like, and is a medical and edible fungus with great development prospect.
The improvement of the biotransformation rate, namely the mushroom yield of the cultivation material per unit weight, always solves the core technical problem in the field of edible mushroom cultivation, and is directly related to the market competitiveness of cultivation enterprises. Improving the yield of the cordyceps militaris fruiting body, namely the biotransformation rate, is also a core technical problem in the large-scale cultivation of the cordyceps militaris. The technology relates to different aspects of strain quality, nutrition regulation, cultivation management and the like. At present, in the large-scale cultivation of cordyceps militaris, due to the fact that competition is very violent, all cultivation enterprises have endeavored to perfect relevant cultivation technologies in the aspects of strain quality, nutrition regulation, cultivation management and the like so as to survive in the environments with high quality and poor quality. However, the cordyceps militaris strains are easier to mutate and degenerate than most edible fungi, so the breeding of high-quality strains and the control of the quality of the strains are always the most difficult technical field in the field of cordyceps militaris cultivation.
In general, the degeneration of cordyceps militaris can be classified into the following types:
1. the strain degeneration caused by karyotype changes, and the manifestation fails to initiate the sexual reproduction process, and thus fruiting bodies cannot be formed.
The mating type system of fungi is known to be the genetic basis for controlling their mating affinity and sexual reproduction. The mating type system of cordyceps militaris is a two-polarity mating type system of different mating types. The mating type system is characterized in that sexual reproduction cannot be independently completed by primary hyphae (called monokaryons for short) formed by germination of sexual spores of the same kind, and sexual reproduction can be started and completed only when the primary hyphae formed by germination of sexual spores of two different mating types are mated (namely the two monokaryons of different types are mated) to form heterokaryons, so that normal sporocarps are produced. Meanwhile, the heterokaryon strains with the capability of producing sporocarps are easy to mutate in the process of culturing and subculturing, the heterokaryon is decomposed into two homokaryons without mating capability, although hyphae of the two homokaryons contain mating type genes of different types, the mating capability is lost, and the heterokaryon strains cannot produce the sporocarps even if the heterokaryon strains grow together. The strain degeneration phenomenon that the karyotype change causes the abnormal production of the fruiting body is very high in occurrence frequency in the large-scale cultivation of the cordyceps militaris, and the quality of the produced strain can be influenced to a great extent. According to the degree of heterokaryon decomposition and homokaryon accumulation, the yield of sporocarps can be reduced to different degrees, even the sporocarps are not produced at all, and great loss is brought to scale production. In terms of the characteristics of the fungal mating type system, a remedy is provided, namely, if a monokaryon with sexual affinity exists, the heterokaryon or homokaryon with self asexual affinity and containing different mating type genes can be mated to form a new heterokaryon with the capability of generating sporocarps, which is called double monokaryon for short. There are two existing solutions to heterokaryon decomposition and homokaryon accumulation degradation. One method is to continuously purify the strains, and maintain the quality of the strains by continuously removing homokaryons and reserving heterokaryons, but the method can only reduce the risks of heterokaryon decomposition and homokaryon accumulation to a certain extent and can not radically avoid degeneration. The other method utilizes the characteristic that the karyotype of the monokaryon strain is more stable, two monokaryon strains are respectively prepared during seed production, and the two strains are mixed and inoculated during inoculation. If mixed inoculation is carried out by double mating, the result is poorer than that of single mating mixed inoculation.
2. The spontaneous high-frequency variation of the strains related to high-level active oxygen is expressed as serious degeneration phenomena such as damaged mitochondrial function, overgrowth of hyphae, no color change, no fruiting body generation or great reduction of fruiting body generation capacity and the like. The plant physiology and ecology research institute of Chinese academy of sciences, the King Tree team has made intensive studies on the Degeneration mechanism of this type of strain (see Li, L, Hu, X, Xia, Y, et al. Linkage of Oxidative Stress and microbial dyefunctions to porous Culture degradation in Aspergillus nidulans [ J ]. Molecular & Cellular proteins Mcp,13(2): 449-461).
3. The strain degeneration caused by malnutrition and other factors is represented by the reduction of the capability of the strain to decompose the culture material and the reduction of the biological yield.
The general solution is to culture the strain on host (such as silkworm pupa) or to add components similar to the host nutrition into the culture medium to simulate the field culture condition as much as possible to culture the cordyceps militaris, and then to separate the strain from the cordyceps militaris, in order to achieve the purpose of rejuvenating the strain character.
The above three bacterial degeneration, the first one, is the least easily monitored, because the karyotype change is originally in a different way that fungi naturally exist, the hyphae are still normal, and the color conversion ability is not changed. From the karyotype monitoring, although it is possible to determine whether a single mating type is present in a single bacterial strain culture (slant or liquid bacterial suspension) from the single hyphal level, it is difficult to determine whether two mating types are heterokaryons or the mixed expression of two homokaryons that have been decomposed from the population level of the bacterial strain. If the influence caused by the degradation can be reduced or avoided in the large-scale production, the cultivation efficiency of the cordyceps militaris can be effectively improved.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a method for improving the yield of cordyceps militaris sporocarp by two-step inoculation. The method is a method for improving the biological yield of cordyceps militaris sporocarp by manually regulating and controlling the inoculation time and mode of cordyceps militaris strains. The two-step inoculation method adopted by the invention can reduce the strain degeneration influence caused by karyotype change in large-scale production, and can greatly improve the biotransformation rate of the cordyceps militaris sporocarp, so that the biological yield of the cordyceps militaris sporocarp can be effectively improved. The invention is an important innovation in the field of edible fungus strain inoculation.
The technical scheme is as follows: in order to achieve the above purpose, the method for improving the yield of the cordyceps militaris sporocarp by two-step inoculation comprises the following steps:
(1) inoculating the cordyceps militaris mononuclear body strain to a cordyceps militaris cultivation medium, and culturing;
(2) inoculating another cordyceps militaris monokaryon, heterokaryon or homokaryon strain containing opposite mating type genes with the monokaryon strain inoculated in the step (1) to the cordyceps militaris culture inoculated in the step (1), and continuously culturing until cordyceps militaris sporocarp is obtained, wherein the culture is a culture medium containing the inoculated cordyceps militaris monokaryon strain.
Wherein, the cordyceps militaris mononuclear body strain is inoculated to a cordyceps militaris cultivation medium in the step (1) and cultivated for a period of time. The method for obtaining the cordyceps militaris single karyon strain comprises the following steps: separating and obtaining a certain amount of ascospore monospore strains from mature cordyceps militaris sporocarp by methods such as ascospore ejection and the like, and then pairwise culturing the obtained ascospore monospore strains to generate paired strains of sporocarp, which show that the strains have sexual affinity with each other, wherein at least one strain is a monokaryon strain; from the results of the multiple pairings, it can be concluded whether each of the monospore strains is a monokaryon species; two matched strains capable of producing fruiting bodies, one of which must be a monokaryon species and the other a monokaryon, heterokaryon or homokaryon species containing the opposite mating type genes (employed in step 2). The specific operation method and principle can refer to the published literature [ gaoxin 2008, mating type research of Cordyceps militaris (Cordyceps militaris), edible fungi article 15 (1): 1-5 ] the related content; or other related methods available in the art may be used.
And (3) inoculating another cordyceps militaris monokaryon, heterokaryon or homokaryon strain containing a mating type gene opposite to the monokaryon strain inoculated in the step (1) to the cordyceps militaris culture inoculated in the step (1), and continuously culturing until cordyceps militaris sporocarp is harvested. Obtaining of monocaryon, heterokaryon or homokaryon strains of Cordyceps militaris with opposite mating type genes, as described in the above references, or by other related methods available in the art.
Wherein, the cordyceps militaris mononuclear body strain is inoculated to a cordyceps militaris cultivation medium in the step (1) and cultivated for a period of time, and the time is specifically 3-20 days. This incubation time is one of the core steps of the present invention, and as will be understood from the inventive principles described hereinafter, is critical to the utilization of the high purity of monokaryon species to enhance medium utilization and ultimately yield of fruiting body organisms.
Preferably, the culture time in step (1) is 8 to 15 days. Due to the diversity of cordyceps militaris strains, the growth period of different strains can be different, so that the optimal selection time of different strains is slightly different when the method is implemented. According to the growth characteristics of different strains, the optimal time interval between two steps of inoculation is found, and the optimal scale production benefit can be obtained.
Wherein, another cordyceps militaris monokaryon, heterokaryon or homokaryon strain containing a mating type gene opposite to that of the monokaryon strain inoculated in the step (1) is inoculated to the cordyceps militaris culture inoculated in the step (1), and the inoculated strain has the capability of producing a sexual mating reaction of single mating or double mating with the strain inoculated in the step (1). This is also seen from the method of obtaining monokaryon, heterokaryon or homokaryon species described in step (1), and the sexual mating ability is judged by the principle of single mating and double mating. And continuing culturing after inoculation until cordyceps militaris sporocarp is harvested.
The formula of the culture medium is that different grain raw materials such as wheat kernels, rice, millet, corn and the like are used singly or in combination according to the mass ratio of 1: 1.2-1.8, and the culture medium can also comprise more nutrients familiar to the technical personnel in the field, and the culture medium needs to be sterilized for use.
In the invention, other culture conditions and the like in the cordyceps militaris culture process are all adopted by technologies and conditions which are common in the cordyceps militaris culture field, and can be implemented by technicians in the field, which will be described in more detail in the embodiment.
The design principle is as follows: the two-step method is compared with the two best known inoculation methods which are used in the prior cultivation: a conventional heterokaryon inoculation method (hereinafter, referred to as a conventional method) and a one-step inoculation method (hereinafter, referred to as a one-step method) in which two kinds of monokaryons are mixed. The comparison of the merits of the three methods is shown in Table 1. Among them, the degree of influence by karyotype change is most influenced by the conventional method because the monokaryon is most stable and the heterokaryon is most easily decomposed. In terms of effective bioconversion, homokaryon hyphae also consume nutrients in the culture, but this nutrient consumption does not translate into fruiting body yield and is therefore negative, with a relatively lower effective bioconversion. The traditional method has the possibility of mixing a certain proportion of homokaryons, so the effective biotransformation rate cannot reach the highest. In the process of one-step mating, repeated mating (double-single mating) may cause a certain proportion of homokaryon byproducts to appear, and the effective biotransformation rate cannot reach the highest. The two-step method adopts a method of delaying mating, and the core principle is that the monokaryon inoculated in the first step is cultured for a period of time and has absolute advantages in the culture material, and as a pure species in a stricter sense, the energy utilization and conversion are completely unified, the loss caused by incompatible hypha nutrition does not exist, and the strain inoculated in the second step only plays a role in providing heterokaryon on the surface of a culture medium to start sexual reproduction and basically does not influence the effective biotransformation rate, so that the effective biotransformation rate of the whole culture can be maximized, and the biological yield of sporocarp can also be maximized.
TABLE 1 comparison of the quality of three inoculation methods, the conventional method, the one-step method and the two-step method
A large number of cultivation tests prove that the two-step method can greatly improve the biological yield of the sporocarp compared with other two methods. The two-step method has the disadvantage of relatively prolonging the growth period, but experimental observation shows that the growth speed of the sporocarp in the sporocarp development stage of the two-step method culture is obviously faster than that in the traditional method and the one-step method, so the growth period of the sporocarp is relatively less prolonged, and the benefit is obviously more than the disadvantage. Table 2 provides experimental data for a dry weight of compost of 20 grams and a number of culture replicates per procedure of 500 flasks. Wherein the two-step method improves the biotransformation rate by 77.8 percent compared with the traditional method and improves the biotransformation rate by 66.7 percent compared with the one-step method. While the prolongation effect of the growth period is less than 15% (as in example 1 of the present invention).
Therefore, the invention improves the yield of the cordyceps militaris sporocarp by using the two-step inoculation method, can reduce the strain degradation influence in large-scale production, and can improve the biotransformation rate of the cordyceps militaris sporocarp, thereby greatly improving the biological yield of the cordyceps militaris sporocarp.
The two-step inoculation method adopted by the invention is not seen to be applied in literature and production practice. In the field of microbial fermentation, a two-step fermentation method for converting solid culture into liquid culture, a two-step biological fermentation method for vitamin C and the like exist, but the two-step biological fermentation method is obviously different from the two-step biological fermentation method of the invention. Therefore, the invention is a brand new method for improving the yield of the cordyceps militaris sporocarp by two-step inoculation. The invention can also be used for reference to other edible fungi with similar strain degeneration characteristics and cultivation characteristics with cordyceps militaris, so that the invention is an important innovation in the field of edible fungi cultivation.
TABLE 2 comparison of fruiting body yield for three inoculation methods, conventional method, one-step method and two-step method
Has the advantages that: compared with the prior art, the invention has the following advantages:
the invention provides a brand new method for improving the yield of cordyceps militaris sporocarp by two-step inoculation, the yield of cordyceps militaris sporocarp is improved by the two-step inoculation method, not only can the influence of strain degradation in scale production be reduced, but also the biotransformation rate of cordyceps militaris sporocarp can be improved, so that the biological yield of cordyceps militaris sporocarp is greatly improved.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.
Example 1
(1) Inoculating Cordyceps militaris monokaryon strain to Cordyceps militaris culture medium by conventional method
Preparing single-core liquid strain of Cordyceps militaris by conventional method, inoculating into culture medium for culturing Cordyceps militaris, and culturing for 3 days by conventional culturing processes such as dark culture and visible light culture. The conventional culture method and the method for obtaining the strains of the monokaryon are well established in the field, and can be implemented by technical personnel in the field and improved to different degrees, and the specific operation method in the embodiment refers to the related contents described in (Gaoxinhua 2008, mating type research of Cordyceps militaris (Cordyceps militaris), edible fungi academic reports 15 (1): 1-5, and specifically, 1.3). The formula of the culture medium used in the present example is: each bottle contained 20 g of wheat kernel and 30 ml of water, and was used after autoclaving, 50 bottles were repeated for each treatment. The liquid culture adopts PDB culture medium, after the liquid strain is shaken for 3 days under the conditions of 150rpm and 22 ℃, the liquid strain is diluted by 100 times of volume by sterile water and then inoculated to the culture medium, and 4 ml of diluted bacterial liquid is inoculated to each bottle. Other examples were carried out under the same conditions as the method.
(2) Inoculating another cordyceps militaris monokaryon strain containing a mating type gene opposite to that of the monokaryon strain inoculated in the first step to the cordyceps militaris cultivation medium inoculated in the first step according to a conventional method. In the step, the obtaining, preparation and inoculation methods of the strains are the same as those in the step (1), and the related contents can be referred to in the literature (Gaoshenhua 2008, mating type research of Cordyceps militaris (Cordyceps militaris), edible fungi academic newspaper 15 (1): 1-5.). Other examples the culture conditions were the same as the method.
After two-step inoculation, culturing according to a conventional cordyceps militaris culture method, managing fruiting bodies until the fruiting bodies are mature, harvesting and measuring the fresh weight and the dry weight of the fruiting bodies. The conventional culture method described herein is well established in the art, and can be carried out by those skilled in the art, and specifically, the related contents can be found in the literature (Gaoshenhua 2008, mating type research of Cordyceps militaris (Cordyceps militaris), reports on edible fungi 15 (1): 1-5.). The cultivation temperature in the whole cultivation process of this example was 22 ℃ and the light intensity in the light cultivation stage was 500 lux. Other examples the culture conditions were the same as in the present method.
(3) Two-step culture results
The cultivation results from 9 to 11 months in 2019 show that the growth period of the cultivation is 50 days (until harvest), the average fresh weight of the fruiting body is 28.4 g/bottle, the average dry weight is 3.9 g/bottle, and the average biotransformation rate is 142%. The growth period of the culture by using the same materials in the same period in a contrast traditional method is 50 days, the average fresh weight of the sporocarp is 19.4 g/bottle, the average dry weight is 3.3 g/bottle, and the average biological conversion rate is 97 percent. The growth period of the one-step method adopting the same materials in the same period is 50 days, the average fresh weight of the sporocarp is 20.7 g/bottle, the average dry weight is 3.5 g/bottle, and the average biological conversion rate is 103.5%. The conventional method used in the control culture is the most commonly used conventional inoculation method in the field (Gaoshenhua 2008, mating type research of Cordyceps militaris (Cordyceps militaris), section 1.3 of the edible fungi bulletin 15 (1): 1-5); the one-step method is a method for pairing and inoculating two strains with different mating types and having sexual affinity at one time; specifically, all the related contents can be referred to in the literature (Gaoshenhua 2008, mating type research of Cordyceps militaris (Cordyceps militaris), school report of edible fungi 15 (1): section 1.5 of 1-5). The culture medium formula, strain preparation conditions and cultivation conditions adopted by the traditional method and the one-step method in the embodiment are the same as those of the two-step method in the embodiment.
Example 2
(1) Inoculating Cordyceps militaris monokaryon strain to Cordyceps militaris culture medium by conventional method
Preparing single-core liquid strain of Cordyceps militaris by conventional method, inoculating into culture medium for culturing Cordyceps militaris, and culturing for 8 days by conventional culturing processes such as dark culture and visible light culture.
(2) Inoculating another Cordyceps militaris monokaryon strain containing a mating type gene opposite to that of the monokaryon strain inoculated in the first step to the Cordyceps militaris culture inoculated in the first step according to a conventional method.
After two-step inoculation, culturing according to a conventional cordyceps militaris culture method, managing fruiting bodies until the fruiting bodies are mature, harvesting and measuring the fresh weight and the dry weight of the fruiting bodies. (the other steps are the same as in example 1)
(3) Two-step culture results
The culture result from 9 to 11 months in 2019 shows that the growth period of the culture is 53 days, the average fresh weight of the sporocarp is 32.7 g/bottle, the average dry weight is 4.6 g/bottle, and the average biological conversion rate is 163.5%. The growth period of the culture of the same period by the traditional method is 50 days, the average fresh weight of the sporocarp is 19.4 g/bottle, the average dry weight is 3.3 g/bottle, and the average biological conversion rate is 97 percent. The growth period of the one-step method is 50 days, the average fresh weight of the sporocarp is 20.7 g/bottle, the average dry weight is 3.5 g/bottle, and the average biological conversion rate is 103.5%.
Example 3
(1) Inoculating Cordyceps militaris monokaryon strain to Cordyceps militaris culture medium by conventional method
Preparing single-core liquid strain of Cordyceps militaris by conventional method, inoculating into culture medium for culturing Cordyceps militaris, and culturing in dark and light conditions until day 15.
(2) Inoculating another Cordyceps militaris monokaryon strain containing a mating type gene opposite to that of the monokaryon strain inoculated in the first step to the Cordyceps militaris culture inoculated in the first step according to a conventional method.
After two-step inoculation, culturing according to a conventional cordyceps militaris culture method, managing fruiting bodies until the fruiting bodies are mature, harvesting and measuring the fresh weight and the dry weight of the fruiting bodies. (the other steps are the same as in example 1)
(3) Two-step culture results
The culture result from 9 to 11 months in 2019 shows that the growth period of the culture is 57 days, the average fresh weight of the sporocarp is 34.5 g/bottle, the average dry weight is 4.9 g/bottle, and the average biological conversion rate is 172.5%. The growth period of the culture of the same period by the traditional method is 50 days, the average fresh weight of the sporocarp is 19.4 g/bottle, the average dry weight is 3.3 g/bottle, and the average biological conversion rate is 97 percent. The growth period of the one-step method is 50 days, the average fresh weight of the sporocarp is 20.7 g/bottle, the average dry weight is 3.5 g/bottle, and the average biological conversion rate is 103.5%.
Example 4
(1) Inoculating Cordyceps militaris monokaryon strain to Cordyceps militaris culture medium by conventional method
Preparing single-core liquid strain of Cordyceps militaris by conventional method, inoculating into culture medium for culturing Cordyceps militaris rice, and culturing in dark place and under visible light for 20 days.
(2) Inoculating another Cordyceps militaris monokaryon strain containing a mating type gene opposite to that of the monokaryon strain inoculated in the first step to the Cordyceps militaris culture inoculated in the first step according to a conventional method.
After two-step inoculation, culturing according to a conventional cordyceps militaris culture method, managing fruiting bodies until the fruiting bodies are mature, harvesting and measuring the fresh weight and the dry weight of the fruiting bodies. (the other steps are the same as in example 1)
(3) Two-step culture results
The culture result from 9 to 11 months in 2019 shows that the growth period of the culture is 60 days, the average fresh weight of the sporocarp is 32.1 g/bottle, the average dry weight is 4.5 g/bottle, and the average biological conversion rate is 160.5%. The growth period of the culture of the same period by the traditional method is 50 days, the average fresh weight of the sporocarp is 19.4 g/bottle, the average dry weight is 3.3 g/bottle, and the average biological conversion rate is 97 percent. The growth period of the one-step method is 50 days, the average fresh weight of the sporocarp is 20.7 g/bottle, the average dry weight is 3.5 g/bottle, and the average biological conversion rate is 103.5%.
Example 5
(1) Inoculating Cordyceps militaris monokaryon strain to Cordyceps militaris culture medium by conventional method
Preparing single-core liquid strain of Cordyceps militaris by conventional method, inoculating into culture medium for culturing Cordyceps militaris, and culturing in dark and light conditions until day 15.
(2) Inoculating another Cordyceps militaris heterokaryon strain containing a mating type gene opposite to that of the monokaryon strain inoculated in the first step to the Cordyceps militaris culture inoculated in the first step according to a conventional method. The heterokaryon strains used here are the most commonly used strains in cordyceps militaris cultivation and are obtained without additional methods. Meanwhile, the heterokaryon strain itself means that it contains two different mating types, and thus necessarily contains a mating type different from that of the monokaryon strain inoculated in the first step.
After two-step inoculation, culturing according to a conventional cordyceps militaris culture method, managing fruiting bodies until the fruiting bodies are mature, harvesting and measuring the fresh weight and the dry weight of the fruiting bodies. (the other steps are the same as in example 1)
(3) Two-step culture results
The culture result from 9 to 11 months in 2019 shows that the growth period of the culture is 57 days, the average fresh weight of the sporocarp is 32.9 g/bottle, the average dry weight is 4.7 g/bottle, and the average biotransformation rate is 164.5%. The growth period of the culture of the same period by the traditional method is 50 days, the average fresh weight of the sporocarp is 19.4 g/bottle, the average dry weight is 3.3 g/bottle, and the average biological conversion rate is 97 percent. The growth period of the one-step method is 50 days, the average fresh weight of the sporocarp is 20.7 g/bottle, the average dry weight is 3.5 g/bottle, and the average biological conversion rate is 103.5%.
Example 6
(1) Inoculating Cordyceps militaris monokaryon strain to Cordyceps militaris culture medium by conventional method
Preparing single-core liquid strain of Cordyceps militaris by conventional method, inoculating into culture medium for culturing Cordyceps militaris, and culturing in dark and light conditions until day 15.
(2) Inoculating another Cordyceps militaris homokaryon strain containing a mating type gene opposite to that of the monokaryon strain inoculated in the first step to the culture of the Cordyceps militaris after the first step inoculation according to a conventional method. In this step, the homokaryon strain is obtained by the same method as that for the monokaryon strain described in example 1, and the homokaryon is a single cross-type strain which can supply nuclei but cannot accept nuclei in the type affinity reaction, and the related contents can be specifically described in the literature (gaoxin 2008, research on the mating type of Cordyceps militaris (Cordyceps militaris), report on edible fungi 15 (1): 1 to 5.).
After two-step inoculation, culturing according to a conventional cordyceps militaris culture method, managing fruiting bodies until the fruiting bodies are mature, harvesting and measuring the fresh weight and the dry weight of the fruiting bodies. (the other steps are the same as in example 1)
(3) Two-step culture results
The culture result from 9 to 11 months in 2019 shows that the growth period of the culture is 57 days, the average fresh weight of the sporocarp is 33.2 g/bottle, the average dry weight is 4.8 g/bottle, and the average biological conversion rate is 166%. The growth period of the culture of the same period by the traditional method is 50 days, the average fresh weight of the sporocarp is 19.4 g/bottle, the average dry weight is 3.3 g/bottle, and the average biological conversion rate is 97 percent. The growth period of the one-step method is 50 days, the average fresh weight of the sporocarp is 20.7 g/bottle, the average dry weight is 3.5 g/bottle, and the average biological conversion rate is 103.5%.
From the results of the 6 examples, the invention can greatly improve the biological yield of the cordyceps militaris sporocarp, the optimal time interval is 8-15 days, and the effect of the monokaryon strain, the heterokaryon strain and the homokaryon strain which contain the mating types different from those of the first-step inoculated strain is the same in the second-step inoculation (see table 2).
Example 7
Example 7 the same culture method as in example 1 was used except that: the formula of the culture medium used in the present example is: each bottle contained 20 g of rice and 24 ml of water, and was used after autoclaving, 50 bottles were repeated for each treatment.
Example 8
Example 8 the same culture method as in example 1 was used except that: the formula of the culture medium used in the present example is: each bottle contained 20 g of rice and 36 ml of water, and was used after autoclaving, 50 bottles were repeated for each treatment. The results for examples 7 and 8 are similar to example 1.
Claims (4)
1. A method for improving the yield of cordyceps militaris sporocarp by two-step inoculation is characterized by comprising the following steps:
(1) inoculating the cordyceps militaris mononuclear body strain to a cordyceps militaris cultivation medium, and culturing;
(2) inoculating another cordyceps militaris monokaryon, heterokaryon or homokaryon strain containing opposite mating type genes with the monokaryon strain inoculated in the step (1) to the cordyceps militaris culture inoculated in the step (1), and continuously culturing until cordyceps militaris sporocarp is obtained;
inoculating the cordyceps militaris mononuclear body strain to the cordyceps militaris cultivation medium, namely inoculating a cordyceps militaris single cross type strain with sexual affinity to the cordyceps militaris cultivation medium, and culturing for 3-20 days.
2. The method for improving the yield of the fruiting bodies of cordyceps militaris through two-step inoculation according to claim 1, wherein the culture time in the step (1) is 8-15 days.
3. The method for improving the yield of fruiting bodies of Cordyceps militaris through two-step inoculation as claimed in claim 1, wherein the step (2) comprises inoculating another species of Cordyceps militaris (L.) Link, heterokaryon or homokaryon containing a mating type gene opposite to that of the single karyon inoculated in step (1) onto the inoculated culture medium of Cordyceps militaris in step (1), wherein the inoculated species has a mating reaction ability to produce single mating or double mating with the inoculated species in step (1), and culturing is continued after inoculation until fruiting bodies of Cordyceps militaris are harvested.
4. The method for improving the yield of the fruiting bodies of cordyceps militaris through two-step inoculation according to claim 1, wherein the formula of the culture medium is any one or more of wheat kernels, rice, millet and corn according to a mass ratio of 1: 1.2-1.8, and adding water to prepare the composition for use after sterilization.
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