CN110663454B - Artificial cultivation method of black ginseng - Google Patents

Abstract

The invention discloses an artificial cultivation method of black ginseng, which comprises the steps of black ginseng strain preparation, black ginseng liquid strain preparation and black ginseng cultivation, and specifically comprises the following steps: preparing a Wulingshen strain: sterilizing fresh radix Linderae, cutting off internal tissues, inoculating into a strain separation solid culture medium, culturing tissue germination, and purifying for 1-3 times to obtain target radix Linderae strain; preparing a Wulingshen liquid strain: inoculating the Wuling ginseng strain into a liquid culture medium, and performing shake culture to obtain a target Wuling ginseng liquid strain; the cultivation of the Wulingshen comprises the following steps: liquid inoculation is carried out on the Wuling ginseng liquid strain, the Wuling ginseng is cultivated until the Wuling ginseng is mature, and the Wuling ginseng is picked; the invention successfully domesticates and cultivates the Wuling ginseng, has the advantages of simple method, sufficient raw materials and the like, and is used for further research and exerts greater value. Multiple groups of experiments prove that the cultivation method can successfully domesticate the Wuling ginseng.

Description

Artificial cultivation method of black ginseng

Technical Field

The invention belongs to the technical field of medicinal fungus cultivation, and particularly relates to an artificial cultivation method of lindera aggregata.

Background

Xylaria nigripes (A. nigripes (B.))Xylaria nigripes) Belonging to the kingdom fungi, the phylum Ascomycota, the subdivision Ascomycota, class, subclass, order Calophyllales, family Calophyllaceae, genus Calophyllum. Xylaria nigripes grows in the waste nests of termites, sclerotia is formed in the gaps between soil and the termite nests, commonly called Wuling ginseng,is a rare traditional Chinese medicine. Wuling ginseng, also called Wuling fungus, Leizzi, chicken tuckahoe, egg of Collybia albuminosa, charcoal stick, Wuli ginseng, etc., spherical, ellipsoidal or egg-shaped to irregular shape, occasionally flat block-shaped, with brown to black outer skin, hard, diameter of 1-8 cm, one end connected with upper fruiting body to form false root, hung in the white ant nest, the other end slightly concave, the interior white, fleshy, soft, with flavor similar to Collybia albuminosa; is widely distributed in Yunnan province, Sichuan province, Jiangxi province, Hunan province, Fujian province and the like. Wild plants are mostly mined in summer or winter, sporocarp of xylaria nigripes on the ground appears in summer, the mining position is easy to find, and the quality is poor when the summer is the primary season; in winter or spring, the ground stroma withers and disappears, but the sclerotium is firmer and has better quality. In summer, the method is mostly applied to sunny slopes such as Chinese red pines, China fir forests and green thick-stick forests, deep soil layers or areas with termite activity such as old ridges, dams and river banks, seedling searching and marking can be carried out in summer, and digging is carried out again in winter and spring. Generally, the more the sub-seats on the ground, the more the sclerotia (more than ten, up to 60 more) there are underground, and the more the sclerotia are dug from the center of the distribution, which can be dug about 1 m.

Wulingshen as a medicinal fungus has the effects of strengthening spleen, removing dampness, tranquilizing and allaying excitement, tonifying qi, reinforcing kidney, nourishing blood and the like, can treat diseases such as spleen deficiency, poor appetite, excessive blood loss after delivery and operation, less milk after delivery, gastroptosis, hernia, heart-kidney imbalance type palpitation, insomnia, neurasthenia, depression, infantile convulsion, traumatic injury and the like, and improves the body immunity and the like, and has a health-preserving function superior to other fungus products. The processed product, WULING Capsule, has been widely used in clinic. The disease types reported and treated by related documents are mainly psychologic diseases, and are secondarily diseases of nerves and digestive systems, and the ginseng flower-like qi tonifying effect is obtained. The Qing Dynasty 'Zu county Zhi' calls that the seedling of Wu Ling Shen is easy to grow out of the earth, the root of the Wu Ling Shen is long, the seedling is firm and is hung in the cave, so the seedling is called thunder Shake when thunder shakes. Round and black with white color, can tonify qi. "the morphology, growth habit and efficacy.

In recent years, many researches on Xylaria nigripes and sclerotium thereof, namely, Xylaria nigripes, and important medicinal fungi are known to have higher medicinal value by many people, so that wild resources can be seriously damaged and the price is high and the like because the Xylaria nigripes cannot be artificially cultured at present. At present, no artificial cultivation report of the Wuling ginseng exists, and the invention completes the artificial cultivation of the Wuling ginseng for the first time. The value of the artificial cultivation can be better utilized only after the artificial cultivation is achieved, and the artificial cultivation method is applied to more people and exerts the highest value.

Disclosure of Invention

The invention aims to provide an artificial cultivation method of lindera aggregata.

The invention aims to realize the artificial cultivation method of the black ginseng, which comprises the steps of black ginseng strain preparation, black ginseng liquid strain preparation and black ginseng cultivation, and specifically comprises the following steps:

A. preparing a Wulingshen strain: sterilizing fresh radix Linderae, cutting off internal tissues, inoculating into a strain separation solid culture medium, culturing tissue germination, and purifying for 1-3 times to obtain target radix Linderae strain;

B. preparing a Wulingshen liquid strain: inoculating the Wuling ginseng strain into a liquid culture medium, and performing shake culture to obtain a target Wuling ginseng liquid strain;

C. the cultivation of the Wulingshen comprises the following steps: liquid inoculation is carried out on the Wuling ginseng liquid strain, the Wuling ginseng is cultivated until the Wuling ginseng is mature, and the Wuling ginseng is picked;

the raw materials of the strain separation solid culture medium comprise peeled potatoes, polygonum multiflorum thunb, glucose, agar powder, peptone, monopotassium phosphate, magnesium sulfate and vitamin B;

the raw materials of the liquid culture medium comprise peeled potatoes, glucose, peptone, potassium dihydrogen phosphate, magnesium sulfate and vitamin B.

The invention aims to achieve artificial domestication and cultivation of the Wuling ginseng, and in recent years, with continuous research on the Wuling ginseng, the Wuling ginseng is found to have higher medicinal value, but wild resources far cannot meet the requirements of people, so that the artificial domestication and cultivation of the Wuling ginseng is inevitable. The invention successfully domesticates and cultivates the Wuling ginseng, has the advantages of simple method, sufficient raw materials and the like, and is used for further research and exerts greater value. Multiple groups of experiments prove that the cultivation method can successfully domesticate the Wuling ginseng.

The invention has the following advantages:

(1) can successfully separate the high-quality Wulingshen strains.

(2) The sporophore of the Wuling ginseng can be obtained by artificial cultivation.

(3) The domestication cultivation method is simple and easy to master.

(4) Less investment, low cost and simple and easy operation.

(5) High yield and short period.

Detailed Description

The present invention is further illustrated by the following examples, which are not intended to be limiting in any way, and any modifications or alterations based on the teachings of the present invention are intended to fall within the scope of the present invention.

The artificial cultivation method of the black ginseng comprises the steps of black ginseng strain preparation, black ginseng liquid strain preparation and black ginseng cultivation, and specifically comprises the following steps:

A. preparing a Wulingshen strain: sterilizing fresh radix Linderae, cutting off internal tissues, inoculating into a strain separation solid culture medium, culturing tissue germination, and purifying for 1-3 times to obtain target radix Linderae strain;

B. preparing a Wulingshen liquid strain: inoculating the Wuling ginseng strain into a liquid culture medium, and performing shake culture to obtain a target Wuling ginseng liquid strain;

C. the cultivation of the Wulingshen comprises the following steps: liquid inoculation is carried out on the Wuling ginseng liquid strain, the Wuling ginseng is cultivated until the Wuling ginseng is mature, and the Wuling ginseng is picked;

the raw materials of the strain separation solid culture medium comprise peeled potatoes, polygonum multiflorum thunb, glucose, agar powder, peptone, monopotassium phosphate, magnesium sulfate and vitamin B;

the raw materials of the liquid culture medium comprise peeled potatoes, glucose, peptone, potassium dihydrogen phosphate, magnesium sulfate and vitamin B.

The raw materials of the strain isolation medium comprise, by weight, 100-300 parts of peeled potatoes, 10-30 parts of fresh lindenbergia root, 10-30 parts of glucose, 10-30 parts of agar powder, 4-6 parts of peptone, 1-3 parts of monopotassium phosphate, 1-3 parts of magnesium sulfate and 1-3 parts of vitamin B.

The preparation method of the strain isolation culture medium comprises the following steps:

1) respectively cutting peeled potatoes and fresh radix linderae into small pieces and uniformly mixing to obtain a material a, adding water into the material a, boiling with water, and filtering to obtain a filtrate b;

2) and adding glucose, agar powder, peptone, potassium dihydrogen sulfate, magnesium sulfate and vitamin B into the filtrate B according to the formula ratio, adding water to a constant volume, stirring for dissolving, subpackaging, sterilizing and pouring into a flat plate to obtain the target strain separation culture medium.

The specific operation is as follows:

step 1: cutting peeled potato 200 g into small pieces, cutting fresh radix Linderae 20g into small pieces, adding 500ml water, and cooking;

step 2: filtering, taking a filtrate, and adding 20g of glucose, 20g of agar powder, 5g of peptone, 2g of monopotassium phosphate, 2g of magnesium sulfate and 1-3 vitamin B tablets into the filtrate;

and step 3: adding water to a constant volume of 1000 mL;

and 4, step 4: stirring for dissolving, subpackaging into triangular flasks, and packaging with culture dish and newspaper;

and 5: sterilizing in a sterilizing pot at 121 deg.C for 20 min;

step 6: taking out the sterilized materials, putting the sterilized materials into a super-sterilization workbench, and pouring a flat plate;

and 7: and (5) solidifying and cooling the flat plate.

The liquid culture medium comprises, by weight, 200-400 parts of peeled potatoes, 20-40 parts of glucose, 4-6 parts of peptone, 1-3 parts of monopotassium phosphate, 1-3 parts of magnesium sulfate and 1-3 parts of vitamin B.

The preparation method of the liquid culture medium comprises the following steps:

1) cutting peeled potatoes in a formula ratio into small pieces, adding crystal water, boiling, and filtering to obtain filtrate a;

2) adding glucose, peptone, potassium dihydrogen phosphate, magnesium sulfate and vitamin B into the filtrate a according to the formula ratio, adding water to a constant volume, stirring for dissolving, subpackaging and sterilizing to obtain the target liquid culture medium.

The specific operation is as follows:

step 1: cutting 300 g peeled potatoes into small pieces, adding 500ml of water, and cooking;

step 2: filtering the potatoes, taking filtrate, and adding 30g of glucose, 5g of peptone, 2g of monopotassium phosphate, 2g of magnesium sulfate and 1-3 vitamin B tablets into the filtrate;

and step 3: adding water to a constant volume of 1000 mL;

and 4, step 4: stirring for dissolving, and packaging into triangular flask;

and 5: sterilizing the liquid triangular flask at 121 deg.C for 20 min;

step 6: taking out the sterilized materials and putting the sterilized materials into a super-sterilization workbench for cooling.

The cultivation material comprises main materials and auxiliary materials.

The main materials comprise sawdust, wheat bran and cottonseed hulls.

The ingredients are gypsum, monopotassium phosphate and magnesium sulfate.

The raw materials of the cultivation material for cultivation comprise, by weight, 60 parts of sawdust, 24 parts of wheat bran, 14 parts of wheat, 1 part of gypsum, 0.5 part of monopotassium phosphate and 0.5 part of magnesium sulfate.

The raw materials of the cultivation material comprise 40 parts of cottonseed hulls, 20 parts of sawdust, 14 parts of wheat, 1 part of gypsum, 0.5 part of monopotassium phosphate and 0.5 part of magnesium sulfate.

The cultivation method comprises the following specific operations:

step 1: the cultivation material is prepared by mixing sawdust, wheat bran, cottonseed hulls and the like as main materials, and gypsum, potassium dihydrogen phosphate, magnesium sulfate and the like as auxiliary materials (formula 1: 60% of sawdust, 24% of wheat bran, 14% of wheat, 1% of gypsum, 0.5% of potassium dihydrogen phosphate, 0.5% of magnesium sulfate and formula 2: 40% of cottonseed hulls, 20% of sawdust, 14% of wheat, 1% of gypsum, 0.5% of potassium dihydrogen phosphate and 0.5% of magnesium sulfate);

step 2: the ingredients are stirred uniformly according to the formula, and the water content is 60 percent;

and step 3: taking a 240 mL-500 mL tissue culture bottle as a cultivation container;

and 4, step 4: compacting each bottle charge in the bottle to one third;

and 5: sterilizing by adopting high-pressure steam at the temperature of 121 ℃ for 2 to 3 hours;

step 6: taking out and cooling after the sterilization is finished;

and 7: cooling and inoculating, namely cooling the culture medium to about 25 ℃ and then carrying out liquid inoculation, wherein liquid strains of the lindera aggregata are used, and the inoculation amount is 5-8% of the strains in the mass of the culture material;

and 8: culturing hyphae, culturing the hyphae in the dark at 24 ℃ and 30-50% of air humidity for 15-20 days;

and step 9: the thalocycline is cultured in the dark for 2 to 5 days under the conditions of 15 to 25 ℃ and 50 to 60 percent of air humidity by utilizing the temperature difference;

step 10: culturing the black ginseng in dark for 40-60 days at 18-25 ℃ and 50-60% of air humidity;

step 11: mature Wuling ginseng and pick it.

The invention is further illustrated by the following specific examples:

example 1

The formula is as follows: 60% of sawdust, 24% of wheat bran, 14% of wheat, 1% of gypsum, 0.5% of monopotassium phosphate and 0.5% of magnesium sulfate are used as cultivation materials, and the domestication and cultivation of the wulingshen comprises the following steps:

(1) the ingredients are stirred uniformly according to the formula, and the water content is 60 percent;

(2) taking a 650mL tissue culture bottle as a cultivation container;

(3) compacting each bottle charge in the bottle to one third;

(4) sterilizing by adopting high-pressure steam at the temperature of 121 ℃ for 2 to 3 hours;

(5) taking out and cooling after the sterilization is finished;

(6) cooling and inoculating, namely cooling the culture medium to about 25 ℃ and then carrying out liquid inoculation, wherein liquid strains of the prepared lindera aggregata are used, and the inoculation amount is 5-8% of the strains in the mass of the culture material;

(7) culturing hyphae, wherein the temperature is controlled at 24 ℃, and the hyphae are cultured in dark for 10-15 days under the condition of 30-50% of air humidity;

(8) the method comprises the following steps of (1) catalyzing a strongpoint, culturing for 10 hours at 15 ℃ and 14 hours at 24 ℃ by utilizing temperature difference, culturing for 2-5 days in a dark environment under the condition that the air humidity is 50% -60%, and gradually twisting a large amount of hyphae to form the strongpoint at the upper half part of a tissue culture bottle;

(9) culturing the black ginseng, wherein the thalli cable gradually grows to form small and slender black ginseng under dark culture for 40-60 days at the temperature of 18-25 ℃ and the air humidity of 50-60 percent, and the post culture gradually expands, wherein the average number of the black ginseng in each bottle is about 3;

(10) culturing until the black ginseng is mature, and picking.

Example 2

The formula is as follows: 40% of cotton seed hulls, 20% of sawdust, 14% of wheat, 1% of gypsum, 0.5% of monopotassium phosphate and 0.5% of magnesium sulfate are used as cultivation materials, and the domestication and cultivation of the wuling ginseng comprise the following steps:

(1) the ingredients are stirred uniformly according to the formula, and the water content is 60 percent;

(2) taking a 650mL tissue culture bottle as a cultivation container;

(3) compacting each bottle charge in the bottle to one third;

(4) sterilizing by adopting high-pressure steam at the temperature of 121 ℃ for 2 to 3 hours;

(5) taking out and cooling after the sterilization is finished;

(6) cooling and inoculating, namely cooling the culture medium to about 25 ℃ and then carrying out liquid inoculation, wherein liquid strains of the prepared lindera aggregata are used, and the inoculation amount is 5-8% of the strains in the mass of the culture material;

(7) culturing hyphae, wherein the temperature is controlled at 24 ℃, and the hyphae are cultured in dark for 10 to 15 days under the condition of 30 to 50 percent of air humidity;

(8) the thalli-accelerating agent is cultured in the dark for 2-5 days under the conditions of 10h at 15 ℃, 14h at 24 ℃ and 50% -60% of air humidity by utilizing the temperature difference, and a large amount of hyphae are gradually twisted at the upper half part of the tissue culture bottle to form the thalli;

(9) culturing the black ginseng, controlling the temperature to be 18-25 ℃, controlling the air humidity to be 50-60%, carrying out dark culture for 40-60 days, gradually growing rhizomorph to form small and slender black ginseng, and gradually expanding after post-culture, wherein the average number of the small and slender black ginseng in each bottle is about 4;

(10) culturing until the black ginseng is mature, and picking.

The effect of domesticating and cultivating wulingshen in different raw material formulas in the embodiment 1 and the embodiment 2 of the invention is shown in the following table:

	radix Linderae	Average diameter
			Formulation 1 (i.e., example 1)	Average about 3 per bottle	Average 3 cm
Formulation 2 (i.e., example 2)	Average 4 per bottle	Average 1.5 cm

Claims (3)

1. An artificial cultivation method of black ginseng is characterized by comprising the steps of black ginseng strain preparation, black ginseng liquid strain preparation and black ginseng cultivation, and specifically comprises the following steps:

A. preparing a Wulingshen strain: sterilizing fresh radix Linderae, cutting off internal tissues, inoculating into a strain separation solid culture medium, culturing tissue germination, and purifying for 1-3 times to obtain radix Linderae strain; the strain separation culture medium is prepared from 100-300 parts by weight of peeled potatoes, 10-30 parts by weight of fresh lindeny ginseng, 10-30 parts by weight of glucose, 10-30 parts by weight of agar powder, 4-6 parts by weight of peptone, 1-3 parts by weight of monopotassium phosphate, 1-3 parts by weight of magnesium sulfate and 1-3 parts by weight of vitamin B;

B. preparing a Wulingshen liquid strain: inoculating the black ginseng strain into a liquid culture medium, and performing shake culture to obtain a black ginseng liquid strain; the liquid culture medium is prepared from 200-400 parts by weight of peeled potatoes, 20-40 parts by weight of glucose, 4-6 parts by weight of peptone, 1-3 parts by weight of monopotassium phosphate, 1-3 parts by weight of magnesium sulfate and 1-3 parts by weight of vitamin B;

C. the cultivation of the Wulingshen comprises the following steps: the cultivation material is prepared from 60 parts by weight of sawdust, 24 parts by weight of wheat bran, 14 parts by weight of wheat, 1 part by weight of gypsum, 0.5 part by weight of monopotassium phosphate and 0.5 part by weight of magnesium sulfate, or is prepared from 40 parts by weight of cottonseed hull, 20 parts by weight of sawdust, 14 parts by weight of wheat, 1 part by weight of gypsum, 0.5 part by weight of monopotassium phosphate and 0.5 part by weight of magnesium sulfate;

c1, cooling the cultivation material to 25 ℃ for liquid strain inoculation, wherein the inoculation amount is 5-8% of the mass of the strain in the cultivation material;

c2, hypha culture: controlling the temperature at 24 ℃ and culturing hyphae in dark for 10-15 days under the condition of 30-50% air humidity;

c3, oxytocin: dark culture is carried out for 2-5 days under the conditions of 10h at 15 ℃, 14h at 24 ℃ and 50-60% of air humidity by utilizing temperature difference, and a large amount of hyphae are gradually twisted to form a hypha rope at the upper half part of the tissue culture bottle;

c4, culturing black ginseng: controlling the temperature to be 18-25 ℃, controlling the air humidity to be 50-60%, carrying out dark culture for 40-60 days, gradually growing the rhizomorph to form small and slender wulingshen, and gradually expanding after-culture;

c5, culturing until the black ginseng is mature, and picking.

2. The artificial cultivation method of wuling ginseng as claimed in claim 1, wherein the preparation method of the strain isolation medium comprises the steps of:

1) respectively cutting peeled potatoes and fresh radix linderae into small pieces and uniformly mixing to obtain a material a, adding water into the material a, boiling with water, and filtering to obtain a filtrate b;

2) and adding glucose, agar powder, peptone, potassium dihydrogen sulfate, magnesium sulfate and vitamin B into the filtrate B according to the formula ratio, adding water to a constant volume, stirring for dissolving, subpackaging, sterilizing and pouring into a flat plate to obtain the target strain separation culture medium.

3. The artificial cultivation method of wuling ginseng as claimed in claim 1, wherein the preparation method of the liquid culture medium comprises the steps of:

1) cutting peeled potatoes in a formula ratio into small pieces, adding crystal water, boiling, and filtering to obtain filtrate a;

2) adding glucose, peptone, potassium dihydrogen phosphate, magnesium sulfate and vitamin B into the filtrate a according to the formula ratio, adding water to a constant volume, stirring for dissolving, subpackaging and sterilizing to obtain the target liquid culture medium.