Armillaria mellea (Oué l.), also known as Armillaria mellea and Corylus heterophylla. It belongs to Basidiomycetes, Kelp, Ascomycetes, Mushrooms, Tricholomataceae and Armillaria. The fruiting body is an annual fungus, which is used for food and medicine, and is also the pathogen of root rot disease that harms forests. It is distributed all over the world. China is mainly distributed in Heilongjiang, Jilin, Liaoning, Hebei, Henan, Shanxi, Shandong, Gansu, Shaanxi, Qinghai, Xinjiang, Sichuan, Anhui, Zhejiang, Hunan, Hubei, Yunnan, Guizhou, Inner Mongolia, Tibet and Taiwan Province provinces. Fruiting body, funicle and mycelium can all be used as medicine. Warm and sweet. The fruiting body contains many kinds of amino acids, such as mannitol, lecithin, ergosterol, vitamin A (with high content), B 1, B2, C, PP, etc., among which aspartic acid, glutamic acid and leucine are the highest (strains, fermented mycelium and gastrodia elata are also the highest, as shown in Table 2 1-9). Has the effects of expelling pathogenic wind, activating collaterals, and strengthening tendons and bones. Can be used for treating lumbago and leg pain, epilepsy, etc. It can also prevent vision loss, night blindness and dry skin. At present, it has been successfully cultivated in the Botanical Garden of Guizhou Academy of Sciences and Qixia County of Shandong Province. In view of the special relationship between Armillaria mellea and Gastrodia elata, the clinical application of Armillaria mellea instead of Gastrodia elata in 1976 was also successful, and the results were identified. Now there are several pharmaceutical factories in China. At this dose, Armillaria fermentans has the same biological activity as Gastrodia elata, such as sedation and anticonvulsant effects on the central nervous system, improving blood circulation and increasing blood flow in brain and coronary artery. Clinical observation results show that the curative effect on vertigo symptoms caused by different reasons (hypertension, vertebrobasilar insufficiency, Meniere's disease, autonomic nerve dysfunction and patients with yin deficiency and yang hyperactivity) is similar to that of Gastrodia elata, and it also has certain curative effect on neurasthenia, insomnia, tinnitus, limb numbness, epilepsy, cerebral arteriosclerosis and other diseases. Mannitol, Armillaria mellea A, Armillaria mellea B, uracil, uridine, erythritol, arabitol, adenine, adenosine, sucrose, hydroxymethylfurfural, methyl octadecanoate, etc. The same components as gastrodia elata were extracted and separated from the fermentation products of gastrodia elata.

Table 2 1-9 Determination of Amino Acids in Armillaria mellea and Gastrodia elata

* Analysis results: Glutamate content is the highest, followed by aspartic acid and leucine, and methionine content is the lowest.

Analytical instrument: 835-50 automatic amino acid analyzer.

I. Morphological characteristics

(A) fruiting body

Summer and autumn are mostly clustered at the base of old stumps or dead trees, and can also be parasitic on living trees. The thallus is about 10- 15 cm high, the lid is fleshy, hemispherical and flat, sometimes slightly umbilical, with a diameter of 5- 12 cm, honey yellow or light khaki, slightly dark at the center, with small scales or tufted scales on the surface or completely smooth, wavy edges and radial stripes, and the handle is 65438 long. The cylinder is slightly curved, sometimes the base is slightly enlarged (the base of bottle culture is underdeveloped), fibrous, soft inside, hollow in the later stage, and light brown. The upper part of the white stalk of the pearl stalk is not obvious or soft. The base of the pearl stalk is connected with that of the rhizoid, and the pearl stalk is attached and extended, nearly white, with white spores. The spores are colorless and smooth, oval, and 8-9.5 × 5.5-6.5 μ m.

(2) Mycelium

It exists in the form of mycelium, fungus ball, furcation fungus and fungus rope.

1. mycelium

Microscopically, the mycelium is colorless and transparent, with filaments of diaphragm, and does not form a lock-like union. In pure culture, it is white carpet-like at first, then it gradually becomes rusty yellow and brownish red, and becomes dark brown when aging. At this time, the bacteria are crisp and thin, with wrinkles on the surface, and sometimes camel-colored curly and fluffy hyphae grow on the bacteria. Fluorescence can be seen in the dark from the growth to aging of hyphae, and the fluorescence of white hyphae is the strongest. Mycelium often invades the tissues of roots or old trunks, causing trees to rot.

2. Spherical and Bifurcated Bacteria

In shake flask culture, mycelia are mostly spiny spheres or irregularly branched bacteria, and the size of bacterial spheres is about 3-7 mm, which is hollow and light brown. Both the fungus ball and the Bifidobacterium have fluorescence in the dark, and the brightness is the strongest after 4-5 days, and gradually decreases after 7 days.

Step 3: Bacterial rope

It is a kind of organized mycelium, and it is a root-like structure twisted by countless white or white-yellow parallel slender hyphae. The outer bread has a sheath, which is white when tender, then turns brown gradually, and the growing point at the tip is white or white-yellow, and turns dark brown when mature, which is several meters long and bifurcated. In pure culture, the suspensor extending into the substrate does not change color or changes color slowly, while the suspensor outside the substrate changes color quickly. White and brittle when young, and fluorescent in the dark. After aging, it is tough and dark brown. Microscopic observation shows that there are short hyphae and septa on the surface of the suspensor, and sometimes colorless rod-shaped crystals can be seen around the hyphae (Figure 2 1- 15).

Fig. 21-15 morphological diagram of Armillaria mellea

1. fruiting body 2. Longitudinal section of fruiting body 3. spore

Second, biological characteristics.

Armillaria mellea is more common in summer and autumn, and it is mostly saprophytic on old stumps and dead trees, or parasitic on living trees. It can infect more than 200 kinds of trees, such as pine, spruce, fir and larch. The parasitism or saprophy of Armillaria mellea can also be found in impatiens, Paeonia lactiflora, American ginseng, edible rhubarb, sugarcane, strawberry and potato.

(1) temperature

According to Roitsma( 1932), Armillaria mellea mycelium keeps astigmatism in a certain culture medium and can form fruiting bodies after 3-4 months at 18-22℃. Li Liangsheng et al. (1987) reported that fruiting bodies can be formed under the general conditions of 12-20℃ in China, and the optimum temperature is 15- 18℃.

Mycelia can grow at 5-30℃, but 24-27℃ is the most suitable. The strain can be formed at 15-28℃, and 25℃ is suitable. The mycelium of Armillaria mellea can emit light at 10-30℃, with the strongest luminescence at 24-28℃. If it exceeds 37℃, it will lose its luminous ability.

(2) Air

Armillaria mellea is an aerobic bacterium, which can grow well only if it is well ventilated. In the process of submerged fermentation culture, ventilation and proper air stirring are also needed.

(3) Humidity

The growth and development of Armillaria mellea need sufficient water and high air humidity. When the water content of the substrate is 50-60% and the relative humidity of the air is 80%, the mycelium grows best. It is necessary to keep the relative humidity of air between 85-95% to form fruiting bodies.

(4) pH value

Armillaria mellea can grow at pH 4-7, but pH6 is more suitable.

(5) Lighting

Light inhibits the growth of Armillaria mellea. Mycelium and suspensor do not need light when growing, but scattered light is needed to form fruiting bodies. Li Liangsheng and others reported that direct light is not needed in the artificial cultivation process, and generally 100-500 LX can grow well and form fruiting bodies.

(6) Nutrition

Armillaria mellea is a facultative parasitic fungus, which can rot on fallen trees or stumps and parasitize on living trees. Cottonseed hull, corncob, sawdust, wheat bran and rice bran can all be used to cultivate fruiting bodies according to local conditions. Glucose is the best carbon source and silkworm chrysalis powder and peptone are the best nitrogen sources for mycelium liquid culture.

Third, cultivation techniques.

There are few literature reports on the cultivation techniques of Armillaria mellea fruiting body. At present, only Shandong Li Liangsheng and others (1988) reported the success of artificial cultivation of Armillaria mellea, but it was not popularized. In 1970s, Armillaria mellea mycelium was successfully produced by fermentation, and it has been applied in clinic, which has achieved great social and economic benefits.

(1) Cultivation of Armillaria mellea fruiting bodies with mushroom bricks

Preparation of 1. culture medium

Expose fresh cottonseed hull for 2-3 days, take 100kg cottonseed hull, and add 20kg corn meal residue, 1kg gypsum, 1kg phosphate fertilizer, and 0. 15kg carbendazim. After mixing, hold it tightly with your hands and drop 65,438+0-2 drops between your fingers. The PH value is 6.5.

2. Preparation before sowing

The moving die is 58×45× 12cm, and the plastic sheet 100cm2. Indoor and outdoor areas of culture should be scrubbed with quicklime water with pH value above 12, and all utensils and plastic sheets should be soaked in 0. 1% potassium permanganate solution for 3-5 minutes.

sow seeds

Put the plastic lining into a movable mold, pile the mixed cottonseed shells on a plastic cloth, about 6cm high, sprinkle a layer of strains, add another layer of material, sprinkle a layer of strains, sprinkle three layers of strains, bury the big strains in the four corners and center of the culture material, sprinkle a layer of broken strains on the surface, properly compact, level off, cover the newspaper, wrap it with plastic cloth, and then compact it with a small wooden board.

4. Management of mycelium growth period

The mycelium can grow at 22-28℃, but 25℃ is suitable, so the stage temperature should be controlled between 24-26℃. It germinated 24 hours after sowing, and it seemed to eat a piece of material 48 hours after sowing. After 7 days, white hyphae can cover the surface of the material. At this time, the film should be uncovered and ventilated every morning and evening. After 25 days, it can penetrate, that is, turn to the differentiation stage.

5. Management of fruiting body growth period

It was found that Armillaria mellea could produce fruiting bodies at 2-20℃, but it grew fastest at 15- 18℃. The relative humidity of the air should be kept between 85-95%, and direct sunlight is strictly prohibited, and it grows well under the illumination of 100-500 LX. It can be harvested in 3-5 days.

After harvesting a batch of fruiting bodies, the mushroom bricks are air-dried for 2-3 days, the surface is dry and the inside is moist, and the water content is about 50%. Then spray nutrient solution until the water content of the mushroom brick reaches about 70%, and cover it with plastic sheets. Mushroom buds can be formed after 3-5 days, and 5 batches can be harvested.

Preparation of nutrient solution: dissolve 250g glucose, 300 yeast tablets and vitamin tablets 1 100 in appropriate amount of water, then add water to 50kg, and the water temperature is 25-30℃.

According to the above production method, 15 truffle brick * * * harvested 37.25kg of Armillaria mellea fruiting body.

The fruiting body of Armillaria mellea can be produced in spring and autumn. It only takes 45-50 days from planting to collecting 5 batches.

6. Harvest and processing

When the cap reaches 80%, it's time to harvest. First, turn it over in the sun, bake it at 60-65℃ after the fruiting body is slightly dry, and turn it over frequently until the stem is dry and covered, then put it into plastic bags, then put it into cartons (or wooden cases) and store it in a cool and dry place.

In addition, there is a method to cultivate fruiting bodies by using waste fungus materials cultivated for 2-4 years.

(2) producing Armillaria mellea mycelium by fermentation

In 1970s, the Institute of Pharmacology of China Academy of Medical Sciences had successfully fermented Armillaria mellea preparation. The methods of producing Armillaria mellea by fermentation can be divided into liquid fermentation and solid fermentation.

1. Liquid fermentation

(1) liquid fermentation production process

(2) Preparation of culture medium

① Inclined culture medium: PDA culture medium can be used. (2) Dissolve (or not add) 50g boiled juice of S wheat bran, 20g glucose, 0.5 g potassium dihydrogen phosphate/kloc-0, 0.75g magnesium sulfate and 0/00 mg vitamin B/kloc-0, then add tap water to 1000ml, and the pH is natural (6-0. 500ml triangular bottle, each bottle is added with 0.5g silkworm chrysalis powder, and the above culture medium 100ml is primary seed culture medium or 5000ml triangular bottle 1000ml culture medium, and 5g silkworm chrysalis powder is secondary seed culture medium. ③ Fermentation medium: glucose 1%, sucrose 2%, bean cake powder 1%, silkworm chrysalis powder 1%, potassium dihydrogen phosphate 0. 15%, magnesium sulfate 0.075%, tap water, natural pH, and all the above media were subjected to/kloc-0.

(3) culture ① inclined plane culture: the inclined plane of the new rotating tube can grow well after 25-26℃ culture 15-20 days. At this time, the mycelium is reddish brown with white edges, fluorescent in the dark, wrinkled on the surface, and sometimes a slightly curly camel-colored aerial mycelium group grows. White and yellow funiculus growing in the culture medium can be seen on the back, and funiculus growing outside the culture medium is dark brown after aging. (2) seed culture: first-class seed culture, in which the slant strain is connected with a bottle of seed culture medium, and the culture is carried out in shake flask at 25-26 DEG C for 5-6 days. Secondary seed culture: inoculate a bottle of primary seeds into secondary seed culture medium, and shake culture for 3-4 days. Third-level seeds are expanded seed cultures in seed tanks. Generally, 25-30L is put into a 40l tank, sterilized at 12 1℃ for half an hour, cooled, inoculated with 5- 10%, stirred and ventilated at 26-28℃, and the stirring speed is 150- 180 rpm. ③ Fermentation culture: aeration and stirring culture in a fermentor. The inoculation amount was 10%, and the fermentation conditions were the same as those of seed culture in seed tank. After 7-9 days of culture, the fermentation broth turns brown and the mycelium is dense, and then it can be taken out of the tank. (4) harvesting: after fermentation, filtering with a centrifuge or a plate frame, concentrating the filtrate under reduced pressure to paste, mixing with strains, drying at 65-75 DEG C, crushing, quality inspection and tabletting.

(4) strain preservation

Armillaria mellea can be stored at 4-5℃ for 8 months to 1 year. But when you use it, you need to turn it again.

2. Solid fermentation culture

The methods and conditions of slant culture and seed culture are the same as those of liquid fermentation.

Preparation of (1) solid fermentation medium

Put 20g of corn flour, 20g of wheat bran and 90ml of tap water into 500ml Kjeldahl nitrogen fixing bottle (or canned bottle), shake well, sterilize at 1 kg/cm2 for 30min, and then spread out while it is hot.

(2) solid fermentation culture

Inoculate secondary seeds into solid culture medium, and the inoculation amount of each bottle is 10-20ml, depending on the surface area. After shaking, Armillaria mellea began to grow at 25-27℃ for about 3 days, hyphae grew on the surface of the culture medium in 8- 10 days, and expanded to the whole culture medium in 15-20 days to form bacteria.

(3) Harvest

When it is ripe, take it out of the bottle and dry it before baking. Drying temperature and other procedures are the same as those of liquid fermentation.

At present, there are 10 pharmaceutical factories in China, with the minimum fermentor of 3t and the maximum of 20t, and two pharmaceutical factories are engaged in solid fermentation. Besides tablets, it also produces granules and oral liquid. In addition to meeting domestic demand, there are also some exports.