CN1279005A - Plastic gyro or its mycelium artificial cultivation method and application - Google Patents

Abstract

The present invention relates to a method for artificially cultivating gyro or its mycelium and its application. Firstly, the gyro mycelium is separated from the fruiting bodies of the gyro fungus gyro, and then the enriched medium is used to carry out the first-level culture of the strain; and then The secondary and tertiary cultures were carried out on the culture medium, and the culture temperature was 15-35°C. The medium can be potato medium, corn grain medium, sawdust medium and corncob medium. Its cultivation process is reasonable, its growth is fast and its yield is high, and it solves the difficult problem of artificial separation and domestication of Glycerus for the first time. The gum gyro or its mycelium active ingredients can be used to make oral medicines for treating psoriasis, dermatitis, vitiligo or eczema and other skin diseases.

Description

Jiaotuoluo or its mycelium artificial culturing method and application

The present invention relates to the mushroom-cultivating technical field.

Natural world has wild Jiaotuoluo, but there is no both at home and abroad relevant for the report of using it for clinical treatment, does not also see the report of useful manual method cultivation cultivation Jiaotuoluo, also it is not carried out remarkable work point and does from the instruction chemical industry.Have very high clinical treatment to be worth owing to find Jiaotuoluo, and at present natural wild and Jiaotuoluo seldom, has important use value so solve its artificial instructionization culture technique problem of separating.

Task of the present invention provides a kind of Jiaotuoluo or its mycelium artificial culturing method and application, it has solved the problem that Jiaotuoluo or its mycelium manually separate the instructionization cultivation well, can artificially improve weight, and available Jiaotuoluo is used for the treatment of skin diseases such as psoriasis, leucoderma, dermatitis or eczema as the dermopathic specific medicament of treatment.

One of the object of the invention is achieved in that

A, carry out strain separating, from the whole fruit body of Jiaotuoluo section fungi Jiaotuoluo, isolate the Jiaotuoluo mycelium, as the female original seed of planting of Jiaotuoluo;

B, carry out female kind of first class inoculum and cultivate, adopt enriched medium to carry out mother and plant and separate the back switching, bacterial classification is grown along media surface after sprouting, form obvious bacterium colony, bacterium colony is grown more than 10 days at least, and as the Jiaotuoluo bacterial classification, the parts by weight proportioning of above-mentioned enriched medium is:

Potato class 100-300 glucose or sucrose 10-30

Vitamin B1 5-15 agar 10-30 water 1000

Method is that the potato class is cut into little or thin slice, is put in that to add water boil in the pot thoroughly well cooked but not mushy to the work bar, filters, and vitaminize B1 is in filtrate, filtrate is gone in the pot, and sugaring and agar with the slow fire heating, stir, agar is dissolved, add hot water and supply moisture, be sub-packed in while hot in the incubator;

Medium is made in c, Jiaotuoluo secondary and three grades of cultivations earlier, the inoculation of sterilization back, and cultivation temperature is 15-35 ℃, waits to grow fine, and gets final product.

Above secondary and three grades of cultural methods are same, and three grades of cultivation results are to grow fruit body.

Above-mentioned medium can be used maize culture medium, and its parts by weight proportioning is: cook corn 60-95, and rice bran 10-30, gypsum 0.5-1.5, material-water ratio are 1: 1.5-2.0, sterilization.

The also available sawdust medium of medium, its parts by weight proportioning is hardwood crumbs 60-90, rice bran or wheat skin 10-30, sucrose 0.5-1.5, calcium carbonate or land plaster 0.1-1.5, material-water ratio are 1: 1.5-2.0 takes by weighing raw material by proportioning, sugar is added in the entry, and other raw material blendings add syrup.

The also available corncob medium of medium, its parts by weight proportioning is corncob 60-95, the sugared 10-30 of rice, gypsum 0.5-1.5, material-water ratio are 1: 1.5-2.0, sterilization.

Two of the object of the invention is: a kind of Jiaotuoluo or its mycelial application, Jiaotuoluo or its mycelium medicinal material are extracted its active ingredient with conventional Chinese medicine alcohol immersion method, reclaim ethanol, this active ingredient is used for making treatment psoriasis, dermatitis, leucoderma or the dermopathic oral drugs of eczema.

One of the present invention's advantage is that the artificial separation domestication culture technique of design is reasonable, can reach predetermined purpose, growth is fast, and the output height is fit to a large amount of cultivations, adopt manual method to solve the difficult problem that Jiaotuoluo or its mycelium manually separate domestication first, have higher actual application value.Utilize Jiaotuoluo or its mycelial active ingredient, make dermopathic medicines such as treatment psoriasis, dermatitis, leucoderma or eczema, have good result of treatment.

Elaborate by the following examples, but not as a limitation of the invention.

Embodiment 1:

One, Jiaotuoluo source and biological property.

(1) source: Jiaotuoluo is the whole fruit body of Jiaotuoluo section fungi Jiaotuoluo.

(2) place of production: Jilin Province's Changbaishan area.

(3) morphological feature: prematurity apothecium intermediate recess, to go up slightly, end is thin, is the drabon look in the conical ascus back side, and the top look shallow, and bottom color depth, card are pitchy.Fruit body is single gives birth to or grows thickly, grow thickly many persons 6-9, also has 3-4, ripe apothecium is open and flat, and the apothecium back side becomes pitchy, and color is darker, it is basidiospore that the black powder is arranged, many Dan Sheng, and it is less to grow thickly, the ascus card out-of-flatness that has, the edge undulate launches out a large amount of black ascospores, powdery after the parachute-opening, it is short conical that the black handle is, and apothecium is as the open and flat cap shape of agaric.The high 1.93cm of prematurity fruit body, standby face width 1.34cm, ripe parachute-opening fruit body, high 2.13cm, the wide 3.95cm of card, the long 1.12cm of handle.

Two, strain separating

Adopt bark (length has mycelium), Ru Mu, meat bacteria organization and spore launch four kinds of modes,, wherein adopt meat bacteria organization's method and adopt strict sterile working from the Jiaotuoluo mycelium in the fruit body intermediate section, cover with the female original seed of planting of Jiaotuoluo of pipe in 20 days.

Three, the female kind cultivated (first class inoculum)

Adopt enriched medium to carry out mother's kind and separate the back switching, the place growth of the globule is arranged along media surface after bacterial classification is sprouted, this bacterium sprouts fast, and it is the obvious bacterium colony of basic point that bacterial classification connects 3-5 days interior the formation with globule point in back.Bacterium colony is not of uniform size, the place is the most obvious with the inoculation point, and bacterium colony is bigger, bacterium colony point sheet takes place, there is lime look mycelium on the surface, sees that from the bacterium colony back side bacterium colony bottom becomes brown class piece, and class's block size is close with the bacterium colony size, and old bacterium colony color depth, the petite look shallow, and old bacterium colony has minority than top class in a kindergarten's piece associating in the generation later stage, and petite independently becomes point-like, left and right sides bacterium colony covered with pipe in about 20 days, but be not at the whole medium of the open and flat covering of media surface, still have the not long bacterium colony of more blank space, colony growth progenesis speed is fast, late growing stage speed is slack-off, may reduce relevant gradually with media surface moisture content.Separate female the kind and differentiate that mutually be defined as the Jiaotuoluo bacterial classification, bacterial classification is insensitive to pH value by eating wood and fruit body,, now can carry out preservation and breed by the domestication that mother is planted.

Four, the Jiaotuoluo second class inoculum is cultivated

Design 3 kinds of prescriptions: prescription 1 is a maize culture medium, and prescription 2 is the weed tree sawdust medium, and prescription 3 is corncob medium (seeing appendix), and pH value is designed to 5,6,7 three numerical value normal-pressure sterilization cooling back inoculations, and cultivation temperature is 22-25 ℃ or 15 ℃-35 ℃.Can find out prescription 1 fast growth, mycelia is strong, the general growth of 500ml Cans about 45 days, prescription 2 prescriptions 3 mycelia a little less than, but fast growth can cover with full bottle in general 35-40 days, find that the Jiaotuoluo mycelia is insensitive to pH value, 5,6,7 three numerical value do not have obvious grown cultures.

Above-mentioned 4 kinds of formulation Example are as follows, and its compound method can be by aforesaid operations.

The enriched medium component

Annotate: potato can be used replacements such as Ipomoea batatas, sweet potato, Chinese yam.

The corn culture medium prescription

The sawdust medium prescription

The corncob culture medium prescription

Five, three utmost points are cultivated.It is the cultivation of Jiaotuoluo

Screen 3 prescriptions carry out respectively bottle plant and the bag plant.The same secondary of method, it is good to grow.

Embodiment 2:

Extraction process:

Get Jiaotuoluo or its fungal mycelium and pulverize (3-5 order), successively with 5 times of amount alcohol refluxs extract 3 times (2h, 1h, 1h), filter, merging filtrate, ethanol, 1 part of taking liquid are added in decompression, 1.5 parts of mixing of auxiliary material, stir evenly, dry (about 60 ℃) pulverized 80 mesh sieves, and were encapsulated or make tablet, pill, electuary, oral liquid etc.

The little hardship of this product, lung liver the heart channel of Hang-Shaoyin is returned in little acid, can be clearing heat and detoxicating, promoting blood circulation is used for the treatment of skin diseases such as psoriasis, dermatitis, leucoderma, eczema.

Below be that the present invention is used for the treatment of dermopathic zoology test data:

Certain medical university's conclusion (of pressure testing): capsule is inhibited to epidermal cell model of hyperplasia (mitosis of mouse oestrogenic hormone phase vaginal epithelial cell); (formation of mouse tail epidermis granular cell) has the promotion cutinization to epidermal cell parakeratosis model; And mix inhibited to the synthetic lymphocyte 3H-TdR of a sending and receiving display list chrotoplast DNA; Explanation can alleviate maybe can reverse the psoriasis pathology change.This capsule can obviously improve the thin amine itch-threshold of cavy phosphoric acid; To rat Irish moss pedal swelling acute inflammation model and granuloma induced by implantation of cotton pellets chronic inflammation model, all has obvious antagonism; To the Mice Auricle capilary arteriole, thin vein bore are significantly increased, capillary is opened the quantity showed increased, and can obviously alleviate hemorheological abnormal change the blood stasis model rat; Show and have obviously antipruritic, anti-inflammatory and function of promoting blood circulation to disperse blood clots.Immunological testing shows that the skin delayed allergy to system's phagocytic function in the net of reflection nonspecific immunity function and reflection T cell-specific immunologic function does not have obvious influence, but the serum immunoglobulin IgG and the IgA of reflection humoral immunity are obviously increased, and IgM does not have obvious influence.

1, to the influence of psoriasis animal model

Purpose: psoriatic basic pathology characteristics are that skin decreases position epidermal cell hyper-proliferative and parakeratosis.Certain medicine if can suppress the too fast hyperplasia of epidermal cell or induce the epidermis granular cell to generate, and promotes Keratoderma, then may have the psoriatic pharmacological action of treatment.The vaginal epithelial cell propagation that is in oestrogenic hormone phase mouse is active, and the epithelial cell conversion is accelerated.The angling of mouse tail epidermis forms similar to people parapsoriasis person, lacks granular cell.Therefore, Chinese scholars as the psoriasis animal model, is estimated the effect of treatment psoriasis medicine with it.In addition, the synthetic skin epithelial cells DNA that reflects to a certain extent of lymphocyte DNA synthesizes and cell proliferation, shows the influence to epidermis and dermal cell propagation indirectly.For this reason, choose above-mentioned three kinds of these capsules of experimental observation to psoriatic drug effect.

(1) to the mitotic influence of mouse oestrogenic hormone phase vaginal epithelial cell

Method: 60 of Kunming mouses, 20-30g.Lumbar injection diethylstilbestrol 0.2mg/ only every day 1 time, continuous 3 days, makes it be in the oestrogenic hormone phase.Be divided into 5 groups at random on 4th, 12 every group.Every day, gastric infusion was 1 time, irritated stomach volume 0.25ml/10g, continuous 3 days.2 hours lumbar injection colchicins are put to death mouse and are got vagina behind the last medicine after 2mg/kg4 hour, 10% formalin fixed, the conventional film-making of paraffin embedding, HE dyeing.Light microscopic is the mitosis number of 300 vagina epithelium basal cells of counting down, and its percentage is a mitotic index.

Result: capsule 0.28 of the present invention, 0.56g/kg (7,14 times of quite clinical consumption per days, down together).Mitosis has obvious suppression effect (P＜0.05,0.01) to mouse oestrogenic hormone vaginal epithelial cell.

(2) influence that mouse tail epidermis granular cell is generated

Method: 60 of Kunming mouses, 20-30g.Be divided into 6 groups at random by table 2,10 every group, male and female half and half.Every day, gastric infusion was 1 time, and irritating the stomach volume is 0.25ml/10g, successive administration 20 days.Put to death whole mouse in 2 hours after the last administration, get apart from mouse root of the tail portion 1.8cm place dorsal part skin one rectangularly, use 10% formalin fixed, the conventional film-making of paraffin embedding, HE dyeing.(400X) observes and counts eyepiece micrometer scope (132 μ m) endoparticle cell number in the scale (10) under light microscopic.

The result: this capsule 0.28,0.56g/kg have obvious facilitation (P＜0.05,0.01) to the generation of mouse tail granular cell.Increase 0.56g/kg add illumination group granular cell, and the Keratoderma layer also obviously thickens.

(3) influence that mouse T lymphocyte DNA is synthesized

Method: 70 of Kunming mouses, 20-22g, male and female half and half.Be divided into 7 groups at random by sex, body weight.Blank group (co-content distilled water is irritated stomach), (co-content distilled water is irritated stomach to positive drug control group (cycli phosphate amine 15mg/kg lumbar injection) illumination control group *, and accepts solar radiation 20 minutes after 2 hours, 8 joules/cm of illumination dose ²), this capsule is little, heavy dose adds illumination group * (capsule dosage and illumination condition are the same), 10 every group.Every day, gastric infusion was 1 time, and irritating the stomach volume is 0.25ml/10g), successive administration 7 days.Last administration and light are put to death, and get spleen.Mix method mensuration by ConA pouring commentaries on classics test 3H-TdR and mix radioactive intensity (cpm), reflect the DNA synthesis rate with this.

When this capsule was used for clinical treatment, patient's medication beyond the region of objective existence of taking off mourning clothes was also accepted solar radiation 20 minutes in back 12 hours in taking medicine, so set up the illumination group and capsule adds the illumination group in this test.

This capsule 0.56g/kg and 0.28,0.56g/kg add illumination person and all can obviously suppress lymphocyte DNA synthetic (P＜0.01 or 0.001) due to the ConA as a result.Adding its inhibitory action of illumination group strengthens to some extent.

Conclusion: (mitosis of mouse oestrogenic hormone phase vaginal epithelial cell) has the inhibition proliferative effect to this capsule to the epidermal cell model of hyperplasia; To epidermal cell parakeratosis model (generation of mouse tail epidermis granular cell), has the promotion cutinization; And the synthetic lymphocyte 3H-TdR of indirect demonstration epidermal dna being mixed inhibited, illumination can strengthen its inhibitory action behind the medicine.To sum up, this capsule can alleviate and maybe can reverse psoriasis pathology and change.

Claims (5)

1, a kind of plastic top or its mycelium artificial cultivation method, it is characterized in that: A, carry out bacterial classification separation, isolate the gyro mycelium from the whole fruiting body of the gyroaceae fungus gyro, as the original species of gyro; b. Carry out the cultivation of the mother species of the first-level strains, use the enriched medium to separate the mother species and then transfer them. After the strains germinate, they grow along the surface of the medium and form obvious colonies. The colonies grow for at least 10 days, and they are used as the strains of the gyro. , the proportioning of parts by weight of the above-mentioned rich culture medium is: Potatoes 100-300 Glucose or sucrose 10-30 Vitamin B1 5-15 Agar 10-30 Water 1000 The method is to cut the potatoes into small strips or thin slices, put them in a pot, add water and boil until the strips are cooked but not rotten, filter, add vitamin B1 to the filtrate, put the filtrate into the pot, add sugar and agar, heat with a slow fire, Stir to melt the agar, heat the water to supplement the water, and distribute it in the incubator while it is hot; c. For the secondary and tertiary cultivation of plastic gyro, first prepare the medium, sterilize it and then inoculate it. The cultivation temperature is 15-35°C, and wait until the growth is good. 2. The method according to claim 1, characterized in that the culture medium is corn grain culture medium, and the ratio of parts by weight is: steamed corn 60-95, rice bran 10-30, gypsum 0.5-1.5 , the ratio of material to water is 1:1.5-2.0, and it is sterilized. 3. The method according to claim 1, characterized in that the medium uses wood chip culture medium, and its weight ratio is 60-90 of hard sawdust, 10-30 of rice bran or wheat husk, and 0.5-1 of sucrose. 5. Calcium carbonate or gypsum powder 0.1-1.5, the ratio of material to water is 1:1.5-2.0, weigh raw materials according to the ratio, add sugar to water, mix other raw materials, add sugar water. 4. The method according to claim 1, characterized in that the culture medium is corncob medium, and its weight ratio is corncob 60-95, rice sugar 10-30, gypsum 0.5-1.5, The ratio of material to water is 1:1.5-2.0, and it is sterilized. 5. The application of a colloidal top or its mycelium, which is characterized in that the colloidal top or its mycelium is soaked in ethanol to extract its active ingredients, reclaiming ethanol, and using the active ingredients to make a treatment for psoriasis, Oral medication for dermatitis, vitiligo, or eczema skin conditions.