CN107629967B - Liquid culture medium of germination bacteria for gastrodia elata planting and gastrodia elata planting method - Google Patents
Liquid culture medium of germination bacteria for gastrodia elata planting and gastrodia elata planting method Download PDFInfo
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Abstract
The invention relates to a liquid culture medium of germination bacteria for planting gastrodia elata and a gastrodia elata planting method. Specifically, the liquid culture medium for the germination bacteria for planting the gastrodia elata comprises, by weight, every 250 mL: 8-12 g of potatoes, 8-12 g of bran, 8-12 g of bean pulp, 4-6 g of white sugar, 0.5-1 g of peptone, 0.4-0.6 g of monopotassium phosphate, 0.2-0.3 g of magnesium sulfate, 0.8-1.2 mg of thiamine and a proper amount of water are added to 250 mL. In addition, the invention also provides a gastrodia elata planting method which comprises the steps of using the germination bacterium liquid culture medium to culture germination bacteria and using the armillaria mellea liquid culture medium to culture armillaria mellea. The liquid culture medium of the germinating bacteria has excellent performance such as high hypha growth speed.
Description
Technical Field
The invention relates to the technical field of gastrodia elata planting, relates to a culture medium of germination bacteria and armillaria mellea required for gastrodia elata seed germination and gastrodia elata growth, and particularly relates to a liquid culture medium used by the germination bacteria and the armillaria mellea. In particular to a liquid culture medium of germination bacteria for planting gastrodia elata and a gastrodia elata planting method by using the liquid culture medium.
Background
Gastrodia elata Bl, also known as Thymus chinensis, Pleurospermum, Lygodium japonicum, Angelica furcijuga, Eupatorium fortunei, Gastrodia elata, Angelica sinensis, Pop, Gastrodia elata, Clinopodium chinense, white dragon skin, and the like, is a perennial herb of Gastrodia genus of Orchidaceae. The rhizome is thick and has no green leaf, and the capsule is oval and is usually propagated as tuber or seed. The rhizome of the Chinese medicinal composition is used for treating dizziness, numbness of limbs, infantile convulsion and other symptoms, is a rare Chinese medicament, and is used together with Qiongzhen lucid ganoderma for treating headache and insomnia.
The gastrodia elata seeds germinate and the gastrodia elata grows, and the germination and the growth can be realized only by providing nutrition for germination bacteria and armillaria mellea. The gastrodia elata has no root and leaves with assimilation function, so that the gastrodia elata cannot directly absorb nutrients in the environment. The basic nutrient source for the growth of the gastrodia elata is armillaria mellea, and the gastrodia elata cannot grow without the armillaria mellea. However, the relationship between Gastrodia elata and Armillaria mellea is very special and complicated. There is a nutritional relationship between digestion and digestion. Gastrodia elata and Armillaria mellea live together and show interdependence and mutual benefits of symbiosis. This relationship varies with the growth phase and environmental conditions.
"Trees" are the nutritional basis for the growth of Armillaria mellea and Gastrodia elata, which is a parasitic fungus that can survive on over 600 trees or herbaceous plants in nature. It is parasitic not only on the roots of living trees and grasses, but also on the roots and stems of dead trees. Gastrodia elata does not have the ability to produce nutrients by itself, and must grow on Armillaria mellea as a nutrient source. A food chain is formed between gastrodia elata, armillaria mellea and trees (green plants). Armillaria mellea is a 'nutritional bridge' among them, and trees are the material basis among them. The theoretical basis of Gastrodia elata GAP cultivation is the understanding of the relationship among the three.
The condition that the armillaria mellea invades the tuber of the gastrodia elata, when the growing armillaria mellea rhizomes touch the gastrodia elata, growing points at tips of mycorrhizal branches invade protocorms or tubers of the gastrodia elata, and symbiotic combination of the gastrodia elata and the armillaria mellea starts from the growing points. When the growth point of the armillaria mellea mycelium penetrates through the epidermis of the hemp body, the fasciculate mycelium enters the cortex and enters the inside of the cell through the intercellular space or the cell wall pores in a way of dispersing the mycelium, so that the cell nucleus is changed, the cell nucleus is enlarged and then decomposed, organelles, polysaccharide particles and the like disappear, and nutrient substances are absorbed by the mycelium. This process is commonly referred to as the nutrient intake period of Armillaria mellea. The condition that the armillaria mellea invades the tuber of the gastrodia elata is that the tuber of the gastrodia elata is in a dormant or germinating stage, the armillaria mellea rhizomorph is yellow white or brownish red, and the armillaria mellea is in a tender stage. The dark brown senescent rhizomorph cannot invade the tuber of the gastrodia elata, and the tuber of the gastrodia elata or the new tuber in the growth stage cannot be invaded by the armillaria mellea.
Rhizoma Gastrodiae has the ability to assimilate and digest mycelium of Armillaria mellea. In the tissue of underground tuber close to the central column, there are several rows of cells with large size and strong vitality, which have the function of dissolving bacteria and assimilating and digesting armillaria mellea mycelium, called digestion layer. When Armillaria mellea mycelium gradually enters into cells deep in cortex, it is surrounded by protoplasm, twisted into balls, and gradually expanded to be decomposed by rhizoma Gastrodiae cells. Then, cells of the degerming layer become large obviously, protoplasm is thick, the invaded mycelia are digested and assimilated to become nutrient substances for the growth of the gastrodia elata, and the white hemp which is not combined with the armillaria mellea is in a hungry state.
In the aspect of the feedback of the gastrodia elata on the nutrition of the armillaria mellea, along with the growth and development of the gastrodia elata, after the gastrodia elata blooms in a stem or seeds of the gastrodia elata grow, the original hemp body (seeds of the gastrodia elata) gradually ages and loses the capacity of dissolving the armillaria mellea, at the moment, the armillaria mellea is dominant, the armillaria mellea is propagated in large quantity in the gastrodia elata body, the nutrition of the armillaria mellea is absorbed, the original hemp body becomes the nutrition source of. This stage is manifested by the parasitism of Armillaria mellea on Gastrodia elata.
In conclusion, the gastrodia elata grows normally by digesting the armillaria mellea hyphae invading the cortex and the pericycle cells of the gastrodia elata as a nutrient source, which is the aspect that the gastrodia elata depends on armillaria mellea. Once the environment is changed by drought, high temperature, low temperature and other factors, the physiological function and growth trend of the gastrodia elata are weakened. The relationship between Gastrodia elata and Armillaria mellea is also unfavorable for transformation of Gastrodia elata, for example, when the temperature is lowered to below 15 deg.C in the later stage of growth of Gastrodia elata, Gastrodia elata will enter into dormancy stage, but Armillaria mellea can still grow at the temperature. In GAP cultivation, too much watering or rainfall causes excessive growth of Armillaria mellea, which in turn damages Gastrodia elata and also causes rottenness of newly-grown Gastrodia elata. Therefore, the symbiotic relationship between the gastrodia elata and the armillaria mellea changes along with the changes of different growth periods and environmental conditions. The expected cultivation target can be achieved only by creating or utilizing proper environmental conditions and reasonable cultivation techniques.
The gastrodia elata is propagated through sexual propagation, namely seed propagation. This is one of the main ways to prevent the degeneration of gastrodia elata. The gastrodia seeds are tiny, have no endosperm and only consist of proembryogenic cells. The germination of the gastrodia elata seeds is provided with nutrition by infecting the seed embryos with hypha of a fungus of a lentinus (Mycena), and the fungus promoting the germination of the gastrodia elata seeds is called gastrodia elata seed germinating fungus.
The literature reports that the gastrodia elata seed germination bacteria belong to fungi of the genus lentinus (Mycena), and the germination bacteria are partially applied to indoor and outdoor gastrodia elata seed germination experiments and gastrodia elata planting seed germination popularization production. The Japanese herbal handsome helps in 1911 to issue a statement of 'Gastrodia elata and Armillaria mellea symbiosis', and it is generally thought later that Gastrodia elata can not leave Armillaria mellea in the whole growth cycle. According to researches such as Xujintang, the Armillaria mellea can establish a good symbiotic relationship with the rhizoma gastrodiae tuber to provide nutrition for the rhizoma gastrodiae, but the Armillaria mellea has no promotion effect on the germination of the rhizoma gastrodiae seeds but has an inhibition effect. The fungi such as osmunda japonica and the like are separated from the protocorm of the germinated gastrodia elata seeds, and the fungi have a promoting effect on the germination of the gastrodia elata seeds. Tests show that the gastrodia elata seeds are infected by hypha of symbiotic germination fungi to provide nutrition for gastrodia elata embryo. The gastrodia elata seed germinating bacteria are deeply researched into shiitake mushrooms and dendrobium mushrooms. The germination fungi are mainly nutritional type and are saprophytic life, but also have the characteristic of facultative parasitism.
Gastrodia elata seed symbiotic germination bacteria are aerobic fungi which are mainly distributed in dry branches and fallen leaves layers and surface soil in forests, and the growth of the bacteria can be influenced if too much culture material is filled or a bottle cover is too tight in the culture process, the growth speed is delayed, and the culture time is long. The germinant bacteria are weak parasitic bacteria which only infect the basal cells of the gastrodia elata embryo. Protocorm and vegetative propagation stem, rice hemp and white hemp which infect the germination of the gastrodia elata seeds are not observed. When Armillaria mellea invades the vegetative propagation stem differentiated from the protocorm, the germination bacteria and the Armillaria mellea can exist in the same vegetative propagation stem at the same time, and the nutritional effect on the rhizoma Gastrodiae is gradually replaced by the Armillaria mellea. The germinal bacteria can grow in the temperature range of 15-30 ℃. The hyphae grow fastest at 25 ℃, and the growth speed of the hyphae is obviously reduced at the temperature lower than 20 ℃ or higher than 25 ℃.
With the rapid development of the gastrodia elata planting, the demand for germination bacteria and armillaria mellea is larger and larger, and the traditional solid production process has the disadvantages of long production period, high production cost, season control in production, high strain transfer generation, difficulty in controlling strain quality, serious resource waste and the like, so that the development of the strain industry is seriously restricted, and the development of the gastrodia elata planting industry is influenced. In recent years, liquid strain culture technology is rapidly developed in strain industrial application, and the important reason is to overcome the unfavorable factors of the conventional solid strain production, so that the research on the application of the liquid strain culture technology in the production of armillaria mellea and germinant is very important, and a suitable liquid culture medium in the production of liquid strains is a key element in the production process.
Therefore, the liquid medium formula for armillaria mellea and germinant bacteria in gastrodia elata planting is still expected in the field, and the formula is expected to have excellent culture performance.
Reference documents:
【1】 Xu jintang, chinese gastrodia elata cultivation [ M ]. beijing: united press of china cooperative medical university, beijing medical university, 1993: 1-99.
【2】 And 2, Caodebin: gastrodia elata, lucid ganoderma, cordyceps militaris and stropharia rugoso-annulata production technology.
【3】 Caodebin, Rong Guangrong, applies: the basic edible fungus and the seed production technology.
【4】 Gazania, zhangguanmin, majiuxin original plant survey and identification of zhaotong gastrodia elata [ J ] modern traditional Chinese medicine research and practice, 2005, 19 (1): 29-31.
【5】 Research progress of poplar, lanzhong, xu jin tang, gastrodia elata [ J ] Chinese herbal medicine, 2000, 3 (1): 66-69.
【6】 Study of symbiotic germination of gastrodia elata seeds and lentinus fungi [ J ] fungus systems, 2001, 20 (1): 137-141.
【7】 Excellent strain characteristics and effects of Pleurotus dendrobii for promoting germination of Gastrodia elata seeds [ J ] fungus system, 2001, 20 (3): 408-412.
【8】 Forest, Zhao Nu, He Li, etc. design of liquid spawn incubator for edible fungi [ J ] agro-computerized research, 2007 (9): 105-108
【9】 Lu doing boat, Lu Xiao Jue, Li are people: and 300 d, edible fungi.
【10】 Zhang Shengyou: a new technique for producing Chinese liquid spawn.
Disclosure of Invention
The invention aims to provide a liquid culture medium formula for armillaria mellea and germinant bacteria in gastrodia elata planting, and the formula is expected to have excellent culture performance. It has been surprisingly found that a liquid medium for armillaria mellea and germinant bacteria in gastrodia elata planting with a formulation according to the characteristics of the invention, presents excellent technical effects as described in the present invention.
To this end, in a first aspect of the present invention, there is provided a liquid culture medium of Armillaria mellea for Gastrodia elata planting, comprising per 250 mL: 8-12 g of corn flour, 8-12 g of bran, 5.5-7 g of soybean meal, 4-6 g of white sugar, 0.5-1 g of peptone, 0.4-0.6 g of monopotassium phosphate, 0.2-0.3 g of magnesium sulfate, 0.8-1.2 mg of thiamine and a proper amount of water are added to 250 mL.
The liquid culture medium of the armillaria mellea for planting the gastrodia elata comprises the following components in each 250 mL: 9-11 g of corn flour, 9-11 g of bran, 6-6.5 g of soybean meal, 4.5-5.5 g of white sugar, 0.65-0.85 g of peptone, 0.45-0.55 g of monopotassium phosphate, 0.225-0.275 g of magnesium sulfate, 0.9-1.1 mg of thiamine and a proper amount of water are added to 250 mL.
The liquid culture medium of the armillaria mellea for planting the gastrodia elata comprises the following components in each 250 mL: 10 g of corn flour, 10 g of bran, 6.25 g of soybean meal, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine and a proper amount of water are added to 250 mL.
The liquid culture medium of the armillaria mellea for planting the gastrodia elata comprises the following components in each 250 mL: 38 g of potato, 2g of bran, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine and a proper amount of water are added to 250 mL.
The liquid culture medium of the armillaria mellea for planting the gastrodia elata comprises the following components in each 250 mL: 25 g of potato, 12 g of corn flour, 2.5 g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine and a proper amount of water are added to 250 mL.
The liquid culture medium of the armillaria mellea for planting the gastrodia elata comprises the following components in each 250 mL: 25 g of potato, 6g of bean pulp, 2.5 g of white sugar, 2.5 g of brown sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine and a proper amount of water are added to 250 mL.
The liquid culture medium of the armillaria mellea for planting the gastrodia elata comprises the following components in each 250 mL: 25 g of potato, 11 g of bran, 3.7 g of white sugar, 3.7 g of brown sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine and a proper amount of water are added to 250 mL.
The liquid culture medium of the armillaria mellea for planting the gastrodia elata comprises the following components in each 250 mL: 7.5 g of soybean meal, 3.7 g of sawdust, 2.5 g of white sugar, 2.5 g of brown sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine and a proper amount of water are added to 250 mL.
The liquid culture medium of the armillaria mellea for planting the gastrodia elata comprises the following components in each 250 mL: 30 g of potato, 6g of bran, 2.5 g of white sugar, 5g of wood chip, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine and a proper amount of water are added to 250 mL.
The liquid culture medium of the armillaria mellea for planting the gastrodia elata comprises the following components in each 250 mL: 25 g of potato, 12 g of bran, 10 g of corn flour, 5g of white sugar, 5g of wood chips, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine and a proper amount of water are added to 250 mL.
The liquid culture medium of the armillaria mellea for planting the gastrodia elata comprises the following components in each 250 mL: 25 g of potato, 6g of bean pulp, 2.5 g of white sugar, 2.5 g of brown sugar, 10 g of wood chip, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine and a proper amount of water are added to 250 mL.
The liquid culture medium of the armillaria mellea for planting the gastrodia elata comprises the following components in each 250 mL: 12 g of bran, 10 g of bean pulp, 5g of sawdust, 2.5 g of white sugar, 2.5 g of brown sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine and a proper amount of water are added to 250 mL.
The liquid culture medium of Armillaria mellea for planting Gastrodia elata according to the present invention further comprises 2.5 g of maltose and 0.05 g of cupric chloride per 250 mL. It has been surprisingly found that when maltose and cupric chloride are added simultaneously in the above amounts to the liquid medium of Armillaria mellea, the dry quality of the mycelia can be significantly improved.
The liquid culture medium of the armillaria mellea for planting the gastrodia elata is prepared by the following method: weighing each solid material, adding a proper amount of water (generally about 70-80% of the volume of the preparation solution), uniformly stirring, boiling for 20-30 minutes, adding water to a specified volume, continuously boiling for 10 minutes, subpackaging in conical bottles, each bottle being 125mL, adding 5 glass balls into each bottle, sealing with cotton, and then autoclaving for 30 minutes at 121 ℃ in a sterilization pot to obtain the product.
Further, the second aspect of the present invention provides a liquid culture medium of germination bacteria for gastrodia elata planting, wherein the liquid culture medium comprises per 250 mL: 8-12 g of potatoes, 8-12 g of bran, 8-12 g of bean pulp, 4-6 g of white sugar, 0.5-1 g of peptone, 0.4-0.6 g of monopotassium phosphate, 0.2-0.3 g of magnesium sulfate, 0.8-1.2 mg of thiamine and a proper amount of water are added to 250 mL.
The liquid culture medium of the germination bacteria for gastrodia elata planting comprises the following components in each 250 mL: 9-11 g of potatoes, 9-11 g of bran, 9-11 g of soybean meal, 4.5-5.5 g of white sugar, 0.65-0.85 g of peptone, 0.45-0.55 g of monopotassium phosphate, 0.225-0.275 g of magnesium sulfate, 0.9-1.1 mg of thiamine and a proper amount of water are added to 250 mL.
The liquid culture medium of the germination bacteria for gastrodia elata planting comprises the following components in each 250 mL: 10 g of potato, 10 g of bran, 10 g of bean pulp, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine and a proper amount of water are added to 250 mL.
The liquid culture medium of the germination bacteria for gastrodia elata planting comprises the following components in each 250 mL: 25 g of potato, 6g of bean pulp, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine and a proper amount of water are added to 250 mL.
The liquid culture medium of the germination bacteria for gastrodia elata planting comprises the following components in each 250 mL: 25 g of potato, 6g of bean pulp, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine, 5g of wood chips and a proper amount of water are added to 250 mL.
The liquid culture medium of the germination bacteria for gastrodia elata planting comprises the following components in each 250 mL: 10 g of corn flour, 10 g of bran, 6.25 g of soybean meal, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine and a proper amount of water are added to 250 mL.
The liquid culture medium of the germination bacteria for gastrodia elata planting comprises the following components in each 250 mL: 10 g of corn flour, 10 g of bran, 6.25 g of soybean meal, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine, 5g of wood chips and a proper amount of water are added to 250 mL.
The liquid culture medium of the germination bacteria for gastrodia elata planting comprises the following components in each 250 mL: 10 g of potato, 6g of bran, 16 g of corn flour, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine, 5g of wood chips and a proper amount of water are added to 250 mL.
The liquid culture medium of the germination bacteria for gastrodia elata planting comprises the following components in each 250 mL: 20 g of potato, 6g of bran, 10 g of corn flour, 5g of white sugar, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine and a proper amount of water are added to 250 mL.
The liquid culture medium of the germination bacteria for gastrodia elata planting comprises the following components in each 250 mL: 10 g of potato, 15 g of corn flour, 6g of bean pulp, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine and a proper amount of water are added to 250 mL.
The liquid culture medium of the germination bacteria for gastrodia elata planting comprises the following components in each 250 mL: 10 g of potato, 15 g of corn flour, 6g of bean pulp, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine, 5g of wood chips and a proper amount of water are added to 250 mL.
The liquid culture medium of the germination bacteria for gastrodia elata planting comprises the following components in each 250 mL: 16 g of corn flour, 6g of bean pulp, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine, 5g of wood chips and a proper amount of water are added to 250 mL.
The liquid culture medium of the germination bacteria for gastrodia elata planting further comprises 2g of fructose and 0.02 g of copper chloride per 250 mL. It has been surprisingly found that the growth rate of the mycelium can be significantly increased when the above amounts of fructose and copper chloride are added simultaneously to the liquid medium of the germinating bacteria.
The liquid culture medium for the germination bacteria for the gastrodia elata planting is prepared by the following method: weighing each solid material, adding a proper amount of water (generally about 70-80% of the volume of the preparation solution), uniformly stirring, boiling for 20-30 minutes, adding water to a specified volume, continuously boiling for 10 minutes, subpackaging in conical bottles, each bottle being 125mL, adding 5 glass balls into each bottle, sealing with cotton, and then autoclaving for 30 minutes at 121 ℃ in a sterilization pot to obtain the product.
Further, the third aspect of the invention relates to a gastrodia elata planting method, which comprises the steps of using a germination bacterium liquid culture medium to culture germination bacteria and using an armillaria mellea liquid culture medium to culture armillaria mellea.
The gastrodia elata planting method according to the third aspect of the invention, wherein the liquid culture medium of the germination bacteria comprises, per 250 mL: 8-12 g of potatoes, 8-12 g of bran, 8-12 g of bean pulp, 4-6 g of white sugar, 0.5-1 g of peptone, 0.4-0.6 g of monopotassium phosphate, 0.2-0.3 g of magnesium sulfate, 0.8-1.2 mg of thiamine and a proper amount of water are added to 250 mL.
The gastrodia elata planting method according to the third aspect of the invention, wherein the liquid culture medium of the germination bacteria comprises, per 250 mL: 9-11 g of potatoes, 9-11 g of bran, 9-11 g of soybean meal, 4.5-5.5 g of white sugar, 0.65-0.85 g of peptone, 0.45-0.55 g of monopotassium phosphate, 0.225-0.275 g of magnesium sulfate, 0.9-1.1 mg of thiamine and a proper amount of water are added to 250 mL.
The gastrodia elata planting method according to the third aspect of the invention, wherein the liquid culture medium of the germination bacteria comprises, per 250 mL: 10 g of potato, 10 g of bran, 10 g of bean pulp, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine and a proper amount of water are added to 250 mL.
According to the third aspect of the invention, the gastrodia elata planting method, wherein the liquid culture medium of the germination bacteria further comprises 2g of fructose and 0.02 g of copper chloride per 250 mL.
According to the third aspect of the invention, the gastrodia elata planting method is characterized in that the germination bacterium liquid culture medium is prepared by the following method: weighing each solid material, adding a proper amount of water (generally about 70-80% of the volume of the preparation solution), uniformly stirring, boiling for 20-30 minutes, adding water to a specified volume, continuously boiling for 10 minutes, subpackaging in conical bottles, each bottle being 125mL, adding 5 glass balls into each bottle, sealing with cotton, and then autoclaving for 30 minutes at 121 ℃ in a sterilization pot to obtain the product.
The gastrodia elata planting method according to the third aspect of the invention, wherein the armillaria mellea liquid culture medium comprises, per 250 mL: 8-12 g of corn flour, 8-12 g of bran, 5.5-7 g of soybean meal, 4-6 g of white sugar, 0.5-1 g of peptone, 0.4-0.6 g of monopotassium phosphate, 0.2-0.3 g of magnesium sulfate, 0.8-1.2 mg of thiamine and a proper amount of water are added to 250 mL.
The gastrodia elata planting method according to the third aspect of the invention, wherein the armillaria mellea liquid culture medium comprises, per 250 mL: 9-11 g of corn flour, 9-11 g of bran, 6-6.5 g of soybean meal, 4.5-5.5 g of white sugar, 0.65-0.85 g of peptone, 0.45-0.55 g of monopotassium phosphate, 0.225-0.275 g of magnesium sulfate, 0.9-1.1 mg of thiamine and a proper amount of water are added to 250 mL.
The gastrodia elata planting method according to the third aspect of the invention, wherein the armillaria mellea liquid culture medium comprises, per 250 mL: 10 g of corn flour, 10 g of bran, 6.25 g of soybean meal, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine and a proper amount of water are added to 250 mL.
The gastrodia elata planting method according to the third aspect of the invention, wherein the armillaria mellea liquid culture medium further comprises 2.5 g of maltose and 0.05 g of copper chloride per 250mL of the armillaria mellea liquid culture medium.
According to the third aspect of the invention, the gastrodia elata planting method is characterized in that the armillaria mellea liquid culture medium is prepared by the following method: weighing each solid material, adding a proper amount of water (generally about 70-80% of the volume of the preparation solution), uniformly stirring, boiling for 20-30 minutes, adding water to a specified volume, continuously boiling for 10 minutes, subpackaging in conical bottles, each bottle being 125mL, adding 5 glass balls into each bottle, sealing with cotton, and then autoclaving for 30 minutes at 121 ℃ in a sterilization pot to obtain the product.
Gastrodia elata is a saprophytic herb of the genus Gastrodia of the family Orchidaceae. The plant can reach 2 m; tuber-shaped, oval to near-dumbbell-shaped, fleshy, upright stem, orange yellow, grayish brown or blue-green, no green leaf, raceme, long round bud sheet, coniform, flower torsion, orange yellow, light yellow, blue-green or yellowish white, combined generation of sepals and petals, sepal-generated oval triangle, capsula-shaped oval, and flowering and fruiting in 5-7 months. Is distributed in Jilin, Liaoning, inner Mongolia, Hebei, Shanxi, Gansu, Jiangsu, Anhui, Zhejiang, Jiangxi, Taiwan, Henan, Hubei, Hunan, Sichuan, Guizhou, Yunnan and Tibet in China. Living in the open forest, hollow ground, forest edge, shrub edge, nipaler, Plumbum preparatium, India, Japan, Korean peninsula to Siberian also have distribution. Gastrodia elata is one of rare Chinese medicinal materials. Gastrodia elata has been used as a medicine for more than one thousand years, and health wine brewed by Gastrodia elata is regarded as a treasure in palace. Both ancient medical books "Shen nong Ben Cao Jing" and "Ben Cao gang mu" have records of Tian Ma to eliminate diseases and prolong life.
Rhizoma Gastrodiae is thick, tuber-shaped, elliptical to near dumbbell-shaped, fleshy, 8-12 cm long, 3-5(7) cm in diameter, sometimes larger, with denser nodes, and surrounded by many triangular wide oval sheaths. Upright stem, orange yellow, gray brown or blue green, without green leaves, and several membranous sheaths at the lower part. The total inflorescence of rhizoma Gastrodiae is 5-30(50) cm long, and usually has 30-50 flowers; the long round shape of the bud is coated with needle shape, the length is 1-1.5 cm, and the film quality is good; the flower stalks and the ovary are 7-12 mm long and are slightly shorter than the flower buds; twisted flowers, orange, pale yellow, bluish green or yellowish white, near-upright; the length of the perianth cylinder generated by the combination of sepals and petals is about 1cm, the diameter is 5-7 mm, the perianth cylinder is nearly in an inclined egg shape, the top end of the perianth cylinder is provided with 5 splinters, but the front part, namely the cleft at the joint of two side sepals, is as deep as 5 mm, and the base part of the cylinder protrudes forwards; the outer lobe (sepal dissociation part) is in an egg-shaped triangle shape, and the front end is blunt; the inner wheel lobe (petal-growing part) is approximately long and round and small; the labial lobe is oval, 6-7 mm long, 3-4 mm wide, 3 cracks, the base part is attached to the end of the stamen poda and the inner wall of the comforter barrel and is provided with a pair of fleshy callus bodies, the upper part is separated from the head, the upper part is provided with mastoid, and the edge is provided with irregular short tassels; the length of the pistil is 5-7 mm, and the pistil foot is short. The capsule is oval, 1.4-1.8 cm long and 8-9 mm wide. The flower and fruit period is 5-7 months.
Gastrodia elata has no root and leaves, cannot perform photosynthesis, and depends on the nutrition supplied by Armillaria mellea for growth and multiplication. Therefore, the first step of planting the gastrodia elata is to culture a certain amount of high-quality Armillaria mellea strains, and the second step is to buy gastrodia elata seeds. And is timely cultivated with the armillaria mellea. Environment: the proper temperature for the growth of the gastrodia elata is 10-30 ℃, the optimal temperature is 20-25 ℃, the relative air humidity is about 80%, the water content of the soil is 50% -55%, and the pH value is 5-6, namely the subacid ecological environment.
The gastrodia tuber propagation method is well known, and the gastrodia tuber is propagated mainly by using tuber or seed, ⑴ tuber propagation, i.e. gastrodia tuber cultivated in winter or spring, gastrodia tuber cultivated in winter has high inoculation rate and fast growth speed, and the gastrodia tuber cultivated in spring for 3-4 months before cultivation, fungus bed is cultured, Armillariella mellea tree suitable for growth is cultivated, broad-leaved tree with thick bark, hard wood, large rough wood pores, no aromatic substance and strong corrosion resistance is good, ⑵ seed propagation, i.e. gastrodia tuber weighing over 100 g is selected, planted along with picking according to the method, sun light irradiation is prevented when bolting, artificial pollination is performed when flowering, the pollination time can be selected to be about 10 days in sunny days, the pollination is performed when the edge of a medicine cap slightly appears, fruit ears are sleeved, when a small amount of seeds are scattered from the lower fruit, gastrodia tuber is harvested along with maturity from the lower part, and the harvested seeds are sown in time because the service life of the harvested gastrodia tuber is short.
The cultivation of Gastrodia elata is generally from 10 months later to 4 months next. Sowing before the soil is frozen in the last ten days of 10 months to the last 12 months is winter cultivation, sowing after the soil is thawed in 3-4 months in spring is spring cultivation, and sowing time in mountainous areas at high altitude can be prolonged to 5 months.
Gastrodia elata is a rare traditional Chinese medicine and is used for treating symptoms such as dizziness, numbness of limbs, infantile convulsion and the like. Rhizoma gastrodiae tuber: sweet and neutral. Pacify liver and stop wind, stop spasm. Can be used for treating headache, vertigo, numbness of limbs, infantile convulsion, epilepsy, convulsion, and tetanus. [ Jingpo medicine ]: it is used to treat hypertension, vertigo, insomnia, headache, and infantile convulsion (Dehong's medical record). [ Achang drug ] Maoze: the action is similar to the Dehong Biao nationality. [ DEANG MEDICINE ] rhizoma Gastrodiae: the action is similar to the Dehong Biao nationality. [ Lisu drug ] hanging and supplementing door: the tuber is used for treating vertigo, dark eyes, headache, numbness of limbs, hemiplegia, slurred speech, infantile convulsion, and epilepsy. [ BAIYAO ] rhizoma Gastrodiae, and rhizoma Gastrodiae: tuber tuber is used for hypertension, vertigo and headache, which is recorded in Dali Zhi (records of materia Medica). [ HANYAO ] rhizoma Gastrodiae: tuber tuber is used for treating gastropathy (Hani medicine). [ Mongolian medicine ] Wulan-Suomo, Dongbuqiang, Suoshler-Ga: the tuber is used for treating dizziness, numbness of limbs, infantile convulsion, epilepsy, hypertension, and aural vertigo. The sound of the rhododendron molle-hari ga: it can be used for treating hypertension, headache, vertigo, apoplexy, facial distortion, hemiplegia, infantile convulsion, convulsive epilepsy, convulsion, tetanus, rheumatalgia, and numbness of limbs. [ Miao nationality medicine ] tuber is used for treating dizziness, numbness of limbs, infantile convulsion, epilepsy, hypertension, and aural vertigo (Xiang lan examination). Potatoes have, high rising days: root and stem of the same species are indicated for dizziness, stomach ache, dizziness, headache due to general deviation. Therefore, the important application value of the gastrodia elata in clinic can be seen, and the method has great practical significance for developing the planting technology of the gastrodia elata. The invention obtains the liquid culture medium capable of improving the production efficiency of the gastrodia elata by optimizing the liquid culture medium of the armillaria mellea and the germinant bacteria for planting the gastrodia elata.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. The following examples further illustrate the invention without limiting it.
Example 1: screening of armillaria mellea and germination bacterium liquid culture medium
1. Experimental Material
1.1, devices and materials
Air conditioner, reciprocating shaking table, sterilizing pot, conical flask, cotton, electric stove, pot, electronic balance, Xin Bao Xiang wild Armillaria mellea, germinating bacteria (Shanxi province small dendrobium mushroom), super clean bench, inoculating spoon, alcohol lamp, glass ball, filter sieve, Armillaria mellea bacteria culture medium (self-made), germinating bacteria leaf culture medium (self-made).
1.2, 1-10 formulas of armillaria mellea culture medium, wherein the culture medium is prepared into 250mL of water:
formula of a armillaria mellea culture medium 1, 38 g of potatoes, 2g of bran, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate and 1 mg of thiamine;
formula 2 of a shoestring fungus culture medium, 25 g of potato, 12 g of corn flour, 2.5 g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate and 1 mg of thiamine;
formula 3 of a shoestring fungus culture medium, 25 g of potatoes, 6g of bean pulp, 2.5 g of white sugar, 2.5 g of brown sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate and 1 mg of thiamine;
formula 4 of a shoestring fungus culture medium, 25 g of potatoes, 11 g of bran, 3.7 g of white sugar, 3.7 g of brown sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate and 1 mg of thiamine;
5. the formula of the culture medium of the armillaria mellea comprises 7.5 g of soybean meal, 3.7 g of wood chips, 2.5 g of white sugar, 2.5 g of brown sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate and 1 mg of thiamine;
formula 6 of a culture medium of the armillaria mellea, 10 g of bran, 10 g of corn flour, 6.25 g of soybean meal, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate and 1 mg of thiamine;
the formula of the armillaria mellea culture medium is 7, 30 g of potatoes, 6g of bran, 2.5 g of white sugar, 5g of sawdust, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate and 1 mg of thiamine;
the formula of the armillaria mellea culture medium is 8, 25 g of potatoes, 12 g of bran, 10 g of corn flour, 5g of white sugar, 5g of wood chips, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate and 1 mg of thiamine;
the formula of the armillaria mellea culture medium is 9, 25 g of potatoes, 6g of soybean meal, 2.5 g of white sugar, 2.5 g of brown sugar, 10 g of wood chips, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate and 1 mg of thiamine;
10 g of culture medium of the armillaria mellea, 12 g of bran, 10 g of soybean meal, 5g of sawdust, 2.5 g of white sugar, 2.5 g of brown sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate and 1 mg of thiamine.
1.3, 1-10 of a germination bacterium culture medium formula, and preparing 250mL of water:
a germination bacterium culture medium formula 1, potato 25 g, soybean meal 6g, white sugar 5g, peptone 0.75 g, potassium dihydrogen phosphate 0.5g, magnesium sulfate 0.25 g and thiamine 1 mg;
a germination bacterium culture medium formula 2, potato 25 g, soybean meal 6g, white sugar 5g, peptone 0.75 g, potassium dihydrogen phosphate 0.5g, magnesium sulfate 0.25 g, thiamine 1 mg and wood dust 5 g;
a germination bacterium culture medium formula 3, 10 g of corn flour, 10 g of bran, 6.25 g of soybean meal, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate and 1 mg of thiamine;
a germination bacterium culture medium formula 4, 10 g of corn flour, 10 g of bran, 6.25 g of soybean meal, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine and 5g of wood chips;
5g of germination bacteria culture medium, 10 g of potato, 6g of bran, 16 g of corn flour, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine and 5g of wood chips;
a germination bacterium culture medium formula 6. 10 g of potatoes, 10 g of bran, 10 g of soybean meal, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate and 1 mg of thiamine;
the formula of the germination bacteria culture medium is 7, 20 g of potatoes, 6g of bran, 10 g of corn flour, 5g of white sugar, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate and 1 mg of thiamine;
the formula of the germination bacteria culture medium is 8, 10 g of potatoes, 15 g of corn flour, 6g of soybean meal, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate and 1 mg of thiamine;
9 g of germination bacteria culture medium, 10 g of potato, 15 g of corn flour, 6g of soybean meal, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine and 5g of wood chips;
the formula of the germination bacteria culture medium is 10, 16 g of corn flour, 6g of soybean meal, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine and 5g of wood chips.
2. Method of producing a composite material
2.1, strain pretreatment:
if the strain is stored at low temperature, the strain needs to be placed at room temperature and placed for 2-3 days until the strain activity is recovered, and then inoculation can be carried out; the strain cultured in the culture chamber can be directly inoculated into the liquid culture medium without activation.
2.2, preparing a culture medium:
weighing each solid material according to a culture medium formula, adding a proper amount of water (generally about 70-80% of the volume of a prepared solution), uniformly stirring, boiling for 20-30 minutes, adding water to a specified volume, continuously boiling for 10 minutes, subpackaging in conical flasks with each flask being 125mL, adding 5 glass balls into each flask, sealing with cotton, and carrying out high-pressure sterilization at 121 ℃ for 30 minutes in a sterilization pot to obtain the culture medium.
2.3, inoculation and culture:
inoculating Armillaria mellea and germination bacteria into Armillaria mellea liquid culture medium and germination bacteria liquid culture medium respectively, wherein each bottle is inoculated with 1 cubic centimeter and the strain block is as small as possible. And after inoculation, putting the seeds into a shaking table for dark culture, wherein the rotation speed of the Armillaria mellea shaking table is 143 revolutions per minute, the rotation speed of the germination bacteria shaking table is 155 revolutions per minute, and the culture temperature is controlled at 16-24 ℃ by an air conditioner.
After the culture medium becomes clear, inoculating the armillaria mellea and the germination bacterium liquid strain into an armillaria mellea strain culture medium and a germination bacterium leaf culture medium respectively, then putting the armillaria mellea strain culture medium and the germination bacterium leaf culture medium into a light-proof culture room for culture, controlling the temperature to be 16-25 ℃, and observing the growth recovery conditions of the armillaria mellea and the germination bacterium.
It is well known that for seed culture, the main requirements of the cultured liquid seed culture are: the bacteria balls are uniform and small as much as possible, the density of the bacteria balls is moderate, the density of the bacteria balls is preferably just 80% -90% when the culture medium is clarified, and if the density of the bacteria balls is too high, the subsequent operation is adversely affected; the culture time is as short as possible; the liquid strain has strong growth recovery adaptability when being transferred into a solid culture medium.
3. As a result:
3.1, culturing and growing the armillaria mellea in different culture media for different time, and inspecting/evaluating the diameter (mm) of a bacterial ball, the volume density of a strain, the suspension force of the bacterial ball, the roundness of the bacterial ball and the clarification of the culture media, wherein the results are respectively shown in tables 1, 2 and 3.
Table 1: effect of different media on the growth of Armillaria mellea (3 days of culture)
In table 1 and in the present invention, the two parameters of the suspension force of the mushroom ball and the roundness of the mushroom ball are evaluated in a subjective scoring manner, and the score is 0-10, wherein the score is the worst and the best, i.e. the score is the higher and the better.
Table 2: effect of different media on the growth of Armillaria mellea (5 days of culture)
| Culture medium | Diameter of fungus ball (mm) | Volume density of strain | Suspension force of bacterial ball | Roundness of mushroom ball | Clarity of the culture Medium |
| 1 | 1-3 | 12％ | 1 | 4 | Turbidity |
| 2 | 1-5 | 34％ | 4 | 3 | Turbidity |
| 3 | 1-3 | 42％ | 2 | 3 | Turbidity |
| 4 | 1-4 | 43％ | 4 | 4 | Turbidity |
| 5 | 1-3 | 45％ | 4 | 5 | Turbidity |
| 6 | 1-3 | 87％ | 10 | 10 | Clarification |
| 7 | 1-3 | 19％ | 2 | 3 | Turbidity |
| 8 | 1-5 | 47％ | 5 | 5 | Turbidity |
| 9 | 1-3 | 68％ | 7 | 5 | Turbidity |
| 10 | 1-3 | 65％ | 6 | 6 | Turbidity |
Table 3: effect of different media on the growth of Armillaria mellea (7 days of culture)
3.2, the growth conditions of the Armillaria mellea cultured in different culture media are examined/evaluated from the two aspects of hypha recovery growth condition and funicular growth condition after different times of the growth of the fungus culture media, and the results are respectively shown in tables 4, 5 and 6.
Table 4: growth conditions of Armillaria mellea cultured in different culture media in the culture medium (culturing for 12 hr)
| Strains cultured in different culture media | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Restoration of hypha growth | 0 | 2 | 1 | 3 | 0 | 7 | 1 | 3 | 2 | 0 |
| Growth of the fungal cord | 0 | 0 | 0 | 0 | 0 | 4 | 0 | 0 | 0 | 0 |
In table 4 and in the present invention, the two parameters of the growth recovery of hyphae and the growth of hyphae were evaluated in a subjective scoring manner, with a score of 0-10 being the worst score of 0 and the best score of 10, i.e., the higher the score, the better the score.
Table 5: growth conditions of Armillaria mellea cultured in different culture media in the culture medium (3 days for culture)
| Strains cultured in different culture media | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Restoration of hypha growth | 3 | 4 | 2 | 5 | 2 | 10 | 3 | 5 | 4 | 3 |
| Growth of the fungal cord | 0 | 0 | 0 | 3 | 0 | 8 | 1 | 4 | 2 | 3 |
Table 6: growth conditions of Armillaria mellea cultured in different culture media in the culture medium (6 days for culture)
| Strains cultured in different culture media | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Restoration of hypha growth | 5 | 6 | 4 | 6 | 4 | 10 | 5 | 6 | 6 | 4 |
| Growth of the fungal cord | 1 | 2 | 1 | 5 | 1 | 10 | 2 | 5 | 2 | 4 |
According to the results in tables 1 to 6 above, it can be seen that the culture time of the Armillaria mellea No. 6 liquid culture medium is shortest, the size and density of the mycelium pellets are moderate, and the hypha with strongest adaptability recovers and the hypha grows out quickly when the Armillaria mellea No. 6 liquid culture medium is transferred into the planting culture medium, so that the Armillaria mellea No. 6 liquid culture medium is more suitable for being applied to the planting culture of the Armillaria mellea liquid strains of the Gastro.
3.3, the germinating bacteria are cultured in different culture media for different times, and the results are respectively shown in tables 7, 8 and 9 according to the investigation/evaluation of the diameter (mm) of a bacteria ball, the volume density of a strain, the suspending power of the bacteria ball, the roundness of the bacteria ball and the clarification of the culture media.
Table 7: effect of different media on growth of germinating bacteria (3 days of cultivation)
| Culture medium | Diameter of fungus ball (mm) | Volume density of strain | Suspension force of bacterial ball | Roundness of mushroom ball | Clarity of the culture Medium |
| 1 | 1-2 | 3％ | 2 | 3 | Turbidity |
| 2 | 1-2 | 2％ | 3 | 3 | Turbidity |
| 3 | 1-3 | 5％ | 3 | 1 | Turbidity |
| 4 | 1-3 | 4％ | 2 | 2 | Turbidity |
| 5 | 1-4 | 10％ | 1 | 3 | Turbidity |
| 6 | 1-2 | 7％ | 7 | 8 | Turbidity |
| 7 | 1-4 | 6％ | 2 | 4 | Turbidity |
| 8 | 1-4 | 3％ | 1 | 0 | Turbidity |
| 9 | 1-4 | 2％ | 2 | 0 | Turbidity |
| 10 | 1-4 | 3％ | 2 | 1 | Turbidity |
Table 8: effect of different media on growth of germinating bacteria (7 days of cultivation)
| Culture medium | Diameter of fungus ball (mm) | Volume density of strain | Suspension force of bacterial ball | Roundness of mushroom ball | Clarity of the culture Medium |
| 1 | 2-3 | 75％ | 3 | 3 | Clarification |
| 2 | 2-3 | 73％ | 4 | 5 | Clarification |
| 3 | 2-3 | 82％ | 4 | 3 | Clarification |
| 4 | 4-8 | 78％ | 3 | 4 | Clarification |
| 5 | 4-8 | 87％ | 2 | 4 | Clarification |
| 6 | 3-4 | 86％ | 10 | 10 | Clarification |
| 7 | 4-10 | 46％ | 3 | 5 | Turbidity |
| 8 | 5-12 | 48％ | 2 | 1 | Turbidity |
| 9 | 5-12 | 44％ | 3 | 2 | Turbidity |
| 10 | 5-12 | 47％ | 3 | 1 | Turbidity |
Table 9: effect of different media on growth of germinating bacteria (10 days of cultivation)
| Culture medium | Diameter of fungus ball (mm) | Volume density of strain | Suspension force of bacterial ball | Roundness of mushroom ball | Clarity of the culture Medium |
| 1 | 2-4 | 77％ | 5 | 4 | Clarification |
| 2 | 2-4 | 75％ | 5 | 6 | Clarification |
| 3 | 2-4 | 84％ | 5 | 5 | Clarification |
| 4 | 4-9 | 79％ | 5 | 6 | Clarification |
| 5 | 4-8 | 91％ | 6 | 6 | Clarification |
| 6 | 3-4 | 88％ | 10 | 10 | Clarification |
| 7 | 5-16 | 62％ | 6 | 7 | Clarification |
| 8 | 5-14 | 59％ | 4 | 3 | Clarification |
| 9 | 5-17 | 65％ | 5 | 3 | Clarification |
| 10 | 5-16 | 67％ | 4 | 3 | Clarification |
3.4, the growth conditions of the germinating fungi cultured by different culture media are inspected/evaluated from the aspect of restoring the growth condition of hyphae after different times in the growth of the leaf culture medium, and the results are respectively shown in tables 10 and 11.
Table 10: growth of germinating bacteria cultured in different media in leaf media (12 hr culture)
| Strains cultured in different culture media | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Restoration of hypha growth | 1 | 0 | 1 | 3 | 2 | 7 | 1 | 2 | 0 | 1 |
Table 11: the growth conditions of germinal bacteria cultured in different culture media in leaf culture medium (3 days for culture)
| Strains cultured in different culture media | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Restoration of hypha growth | 2 | 1 | 3 | 5 | 4 | 9 | 3 | 4 | 1 | 3 |
From the above tables 7 to 11, it can be seen that the liquid culture medium of germination bacteria No. 6 has short culture time, the size and density of the bacterial balls are the best, and strong adaptability is good for the recovery of hypha to grow well when the germination bacteria No. 6 is transferred into the germination bacteria planting culture medium, so that the liquid culture medium of germination bacteria No. 6 is most suitable for being used for planting and culturing the liquid strains of germination bacteria in gastrodia elata.
Example 2: armillaria mellea and GerminationScreening of liquid Medium for bacteria
Reference example 1 was repeated except that the test was conducted using only the culture medium formulation 6 for armillaria mellea and the culture medium formulation 6 for germinating bacteria, but
The formula 6 of the armillaria mellea culture medium is changed into the following formula: every 250mL of the corn meal comprises 12 g of corn flour, 8g of bran, 5.5 g of soybean meal, 6g of white sugar, 0.5g of peptone, 0.6 g of monopotassium phosphate, 0.2 g of magnesium sulfate, 1.2 mg of thiamine and a proper amount of water which are added to 250 mL; and the formula 6 of the germination bacteria culture medium is changed into the following formula: every 250mL of the potato/soybean milk comprises 8g of potatoes, 12 g of bran, 8g of soybean meal, 6g of white sugar, 0.5g of peptone, 0.4 g of monopotassium phosphate, 0.3 g of magnesium sulfate, 0.8 mg of thiamine and a proper amount of water which are added to 250 mL.
The results shown in tables 1 to 11 obtained in this example are substantially the same as those in the corresponding tables of example 1, and are not significantly different from each other.
Example 3: screening of armillaria mellea and germination bacterium liquid culture medium
Reference example 1 was repeated except that the test was conducted using only the culture medium formulation 6 for armillaria mellea and the culture medium formulation 6 for germinating bacteria, but
The formula 6 of the armillaria mellea culture medium is changed into the following formula: every 250mL of the corn powder comprises 8g of corn flour, 12 g of bran, 7 g of soybean meal, 4g of white sugar, 1 g of peptone, 0.4 g of monopotassium phosphate, 0.3 g of magnesium sulfate, 0.8 mg of thiamine and a proper amount of water, and the balance is added to 250 mL; and the formula 6 of the germination bacteria culture medium is changed into the following formula: every 250mL of the potato/soybean milk contains 12 g of potato, 8g of bran, 12 g of soybean meal, 4g of white sugar, 1 g of peptone, 0.6 g of monopotassium phosphate, 0.2 g of magnesium sulfate, 1.2 mg of thiamine and a proper amount of water which are added to 250 mL.
The results shown in tables 1 to 11 obtained in this example are substantially the same as those in the corresponding tables of example 1, and are not significantly different from each other.
Example 4: screening of armillaria mellea and germination bacterium liquid culture medium
Reference example 1 was repeated except that the test was conducted using only the culture medium formulation 6 for armillaria mellea and the culture medium formulation 6 for germinating bacteria, but
The formula 6 of the armillaria mellea culture medium is changed into the following formula: every 250mL of the corn meal comprises 9 g of corn flour, 11 g of bran, 6g of soybean meal, 4.5 g of white sugar, 0.85 g of peptone, 0.45g of monopotassium phosphate, 0.225 g of magnesium sulfate, 0.9 mg of thiamine and a proper amount of water, and the balance is added to 250 mL; and the formula 6 of the germination bacteria culture medium is changed into the following formula: every 250mL contains 11 g of potato, 11 g of bran, 9 g of soybean meal, 4.5 g of white sugar, 0.85 g of peptone, 0.45g of monopotassium phosphate, 0.275 g of magnesium sulfate, 0.9 mg of thiamine and a proper amount of water which are added to 250 mL.
The results shown in tables 1 to 11 obtained in this example are substantially the same as those in the corresponding tables of example 1, and are not significantly different from each other.
Example 5: screening of armillaria mellea and germination bacterium liquid culture medium
Reference example 1 was repeated except that the test was conducted using only the culture medium formulation 6 for armillaria mellea and the culture medium formulation 6 for germinating bacteria, but
The formula 6 of the armillaria mellea culture medium is changed into the following formula: the corn meal comprises 11 g of corn flour, 9 g of bran, 6.5 g of soybean meal, 5.5 g of white sugar, 0.65 g of peptone, 0.55 g of monopotassium phosphate, 0.275 g of magnesium sulfate, 1.1 mg of thiamine and a proper amount of water which are added to 250 mL; and the formula 6 of the germination bacteria culture medium is changed into the following formula: every 250mL contains 9 g of potato, 9 g of bran, 11 g of soybean meal, 5.5 g of white sugar, 0.65 g of peptone, 0.55 g of monopotassium phosphate, 0.225 g of magnesium sulfate, 1.1 mg of thiamine and a proper amount of water which are added to 250 mL.
The results shown in tables 1 to 11 obtained in this example are substantially the same as those in the corresponding tables of example 1, and are not significantly different from each other.
Example 6: hyphal biomass assay for Armillaria mellea
Measurement of hyphal biomass: after fermentation (i.e., culturing armillaria mellea for 7 days), mycelium is collected by vacuum filtration, the culture medium attached to the strongylocentrotus [ Liubing, Niuyu, Zhang, etc.. optimization of the stock culture and liquid culture medium of symbiotic armillaria mellea of rhizoma gastrodiae [ J ]. edible fungus of China, 2012, 31(3):28-31], dried at 65 ℃ to constant mass, and the dry mass (g/100mL) of the mycelium is measured. Through determination, the dry mass of the mycelium of the Armillaria mellea culture medium No. 6 in the example 1 is 0.44g/100mL, the dry mass of the mycelium of the rest 9 Armillaria mellea culture media is within the range of 0.24-0.42 g/100mL, and no obvious difference is found among different culture medium groups; in addition, the dry mass of the mycelium in the culture medium of Armillaria mellea similar to No. 6 in examples 2 to 5 is in the range of 0.42 to 0.45g/100mL and is not different from each other. Generally speaking, the culture medium is considered to have excellent performance if the dry mass of the mycelium can exceed 0.5g/100mL, and the culture medium can meet the requirement of general use if the dry mass of the mycelium is in the range of 0.4-0.5 g/100 mL. In the supplementary experiments conducted in accordance with the procedures of examples 1 to 5, 2.5 g of maltose and 0.05 g of cupric chloride (per 250 mL) were supplemented to the culture medium for Armillaria mellea No. 6, and as a result, it was found that the dry mass of mycelia was not significantly affected by other indexes except that the dry mass of mycelia was significantly improved (the dry mass of mycelia was within the range of 0.68 to 0.74g/100 mL); and when the above two substances were added to 9 kinds of culture media other than the culture medium of Armillaria mellea No. 6 in the supplementary test of reference example 1, no significant improvement in the dry mass of the mycelia was seen, neither exceeding 0.48g/100 ml. However, in the supplementary experiments conducted with reference to examples 1 to 5, when only maltose or only one of copper chloride was added to the culture medium, the dry mass of the mycelia was not improved, for example, in the case where only maltose or only one of copper chloride was added to the culture medium of Armillaria mellea No. 6 in example 1 and the culture medium of examples 2 to 5, the dry mass of the mycelia was in the range of 0.42 to 0.46g/100 mL. Therefore, the quality of the dried mycelium can be obviously improved after the two substances are supplemented and added into the armillaria mellea liquid culture medium.
Example 7: examination of hyphal growth Rate
The germination bacteria leaf culture medium used in the above examples 1 to 5 is a self-prepared product, which is a classical germination bacteria leaf culture medium, and the ratio is as follows: the content of leaf, wood dust and wheat bran of Fagaceae plant is 60%, 30% and 10%. As in the second stage of "2.3, inoculation and cultivation" of example 1, a fixed amount of leaf medium of germinating fungi was placed in 17cm X33 cm polypropylene bags, sterilized and cooled, and then inoculated with 20mL of liquid strains of germinating fungi per bag, and after cultivation for 7 days at 25 ℃ a line of growth of the hyphae was drawn on the polypropylene bags with a marker pen, and using this as a baseline, the hyphae growth rate (cm/day) was recorded after one month, and 5 replicates were placed for each treatment. It has been found that the hypha growth rate of the bacterial liquid obtained from the culture medium of 10 germination bacteria of example 1 after the inoculation is in the range of 0.33-0.41 cm/day, and the hypha growth rate of the bacterial liquid obtained from example 2-example 5 after the inoculation is in the range of 0.34-0.40 cm/day, which shows a generally acceptable growth rate. However, in the supplementary experiments, when 2g of fructose and 0.02 g of copper chloride were added to the liquid culture medium of the germination bacteria in examples 1 to 5, the growth rate of the mycelium was significantly increased, and particularly, the growth rate of the mycelium in the range of 0.68 to 0.72 cm/day after the liquid culture medium of the germination bacteria in example 1 and the liquid culture medium of the germination bacteria in examples 2 to 5 was inoculated to the leaf culture medium of the germination bacteria was surprisingly increased. However, in the supplementary experiments, if fructose or copper chloride alone is added to the culture medium of the liquid culture medium of the germinating fungi in the supplementary experiments conducted with reference to examples 1 to 5, the growth rate of the hyphae cannot be increased, and for example, the growth rate of the hyphae is in the range of 0.36 to 0.41 cm/day when fructose or copper chloride alone is added to the culture medium of Armillaria mellea No. 6 in example 1 and the culture medium of examples 2 to 5. It can be seen that the growth rate of hyphae can be remarkably improved by simultaneously supplementing the two substances into the liquid culture medium of the germinating fungi. It should be noted that the hypha growth rate reflects the growth rate of the germinal fungi in the leaf culture medium of the germinal fungi, and the two substances are added in the liquid culture medium, and the effects of the two substances on the germinal fungi in the liquid culture medium extend to the leaf culture medium and influence the hypha growth rate, which cannot be explained by the inventor so far. In addition, although fructose used in example 7 was a saccharide as well as maltose used in example 6, the above-mentioned effects of increasing the growth rate of mycelia and improving the dry quality of mycelia were not obtained at all when these two saccharides were used in the same manner as in example 6 and example 7.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (9)
1. The liquid culture medium of the germination bacteria for planting the gastrodia elata comprises 8 ~ 12 g of potatoes, 8 ~ 12 g of bran, 8 ~ 12 g of bean pulp, 4 ~ 6g of white sugar, 0.5 ~ 1 g of peptone, 0.4 ~ 0.6 g of monopotassium phosphate, 0.2 ~ 0.3.3 g of magnesium sulfate, 0.8 ~ 1.2.2 mg of thiamine, 2g of fructose, 0.02 g of copper chloride and a proper amount of water which are added to 250 mL.
2. The liquid culture medium according to claim 1, wherein the medium comprises, per 250mL, 9 ~ 11 g of potato, 9 ~ 11 g of bran, 9 ~ 11 g of soybean meal, 4.5 ~ 5.5.5 g of white sugar, 0.65 ~ 0.85.85 g of peptone, 0.45 ~ 0.55 g of monopotassium phosphate, 0.225 ~ 0.275.275 g of magnesium sulfate, 0.9 ~ 1.1.1 mg of thiamine, 2g of fructose, 0.02 g of copper chloride, and a proper amount of water added to 250 mL.
3. The liquid medium according to claim 1, which comprises per 250 mL: 10 g of potato, 10 g of bran, 10 g of bean pulp, 5g of white sugar, 0.75 g of peptone, 0.5g of monopotassium phosphate, 0.25 g of magnesium sulfate, 1 mg of thiamine, 2g of fructose, 0.02 g of copper chloride and a proper amount of water are added to 250 mL.
4. The liquid medium according to claim 1, which comprises per 250 mL: 8g of potato, 12 g of bran, 8g of bean pulp, 6g of white sugar, 0.5g of peptone, 0.4 g of monopotassium phosphate, 0.3 g of magnesium sulfate, 0.8 mg of thiamine, 2g of fructose, 0.02 g of copper chloride and a proper amount of water are added to 250 mL.
5. The liquid medium according to claim 1, which comprises per 250 mL: potato 12 g, bran 8g, bean pulp 12 g, white sugar 4g, peptone 1 g, potassium dihydrogen phosphate 0.6 g, magnesium sulfate 0.2 g, thiamine 1.2 mg, fructose 2g, copper chloride 0.02 g and water in a proper amount to 250 mL.
6. The liquid medium according to claim 1, which comprises per 250 mL: 11 g of potato, 11 g of bran, 9 g of bean pulp, 4.5 g of white sugar, 0.85 g of peptone, 0.45g of monopotassium phosphate, 0.275 g of magnesium sulfate, 0.9 mg of thiamine, 2g of fructose, 0.02 g of copper chloride and a proper amount of water are added to 250 mL.
7. The liquid medium according to claim 1, which comprises per 250 mL: 9 g of potato, 9 g of bran, 11 g of bean pulp, 5.5 g of white sugar, 0.65 g of peptone, 0.55 g of monopotassium phosphate, 0.225 g of magnesium sulfate, 1.1 mg of thiamine, 2g of fructose, 0.02 g of copper chloride and a proper amount of water are added to 250 mL.
8. The liquid culture medium according to claim 1, which is prepared by weighing each solid material, adding an appropriate amount of water, stirring well, boiling for 20 ~ 30 minutes, adding water to a prescribed volume, continuing to boil for 10 minutes, subpackaging in conical flasks, 125mL per flask, adding 5 glass spheres per flask, sealing with cotton, and autoclaving at 121 ℃ for 30 minutes in a sterilizer.
9. A method for planting Gastrodia elata, comprising the steps of culturing Germination bacteria with the Germination bacteria liquid culture medium of any one of claims 1 ~ 8 and culturing Armillaria mellea with the Armillaria mellea liquid culture medium.
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