CN104164369A - Method for cultivating cordyceps militaris liquid spawn - Google Patents

Abstract

The invention relates to a method for cultivating cordyceps militaris liquid spawn. The method comprises the following steps: preparing fluid nutrient medium, namely subpackaging the prepared fluid nutrient medium into conical flasks, adding 4-5 drops of rapeseed oil to each bottle, and wrapping up the opening of each bottle by brown paper or sealing the opening by non-woven fabrics after filling the opening with a tampon; and sterilizing, namely taking out the sterilized conical flasks, cooling the conical flasks to below 30 DEG C, inoculating 3-4 cant strains of 0.4-0.6cm<3> under an aseptic condition, standing and cultivating the cant strains for 48-72 hours at 20-22 DEG C, germinating hypha on strain blocks, and putting the conical flasks on a reciprocating table to carry out shaking culture, wherein the fluctuating frequency is 80-100 per minute, and the amplitude is 6-10cm; or putting the conical flasks on a rotary table, wherein the fluctuating frequency is 120-200r/minute, and the amplitude is 4cm; and cultivating in a culture chamber at 20-21 DEG C for 3-5d. The cordyceps militaris liquid spawn is cultivated by adopting a shake flask, and the method is small in investment, simple in equipment technology, and suitable for production of a general strain plant.

Description

A kind of cultural method of Cordyceps militaris bacterial classification

Technical field

The present invention relates to Cordyceps production technical field, be specifically related to a kind of cultural method of Cordyceps militaris bacterial classification.

Background technology

Cordyceps militaris (L.) Link. is Ascomycotina, ergot Zoopagales, and the type species of Clavicipitaceae, Cordyceps, have another name called Cordyceps militaris (L.) Link., Cordyceps militaris etc.Cordyceps militaris (L.) Link. morphological specificity stroma list is raw or severally from head or the joint minister of parasitic pupal cell, go out together, and color is orange or orange, 2～8 centimetres of total lengths, and pupal cell color is purple, is about 1.5～2 centimetres.

Cordyceps sinensis and ginseng, pilose antler are equally celebrated for their achievements for for three traditional large tonics of China have won fame both at home and abroad, and domestic and international many experts are to Cordyceps militaris (L.) Link. nutrient chemistry composition, the research of pharmacology and clinical effectiveness, all substantially identical with Cordyceps sinensis, mainly contain and protect lung kidney-nourishing, hemostasis and phlegm, regulate physical function, cure mainly pulmonary tuberculosis hemoptysis, impotence and seminal emission, soreness of waist and knee joint, beauty and skin care, psychasthenia, diabetes, rise white cell, anti-cancer, anticancer is also very large to Chemotherapy.The effect of tame Cordyceps militaris (L.) Link. medicinal efficacy and wild Chinese caterpillar fungus is very nearly the same, and the medical performance of some aspect is even better than wild Chinese caterpillar fungus.Medicine, pharmacology and clinical trial certificate Cordyceps militaris (L.) Link. can be used as the surrogate of Cordyceps sinensis completely.Prove after deliberation, the Cordyceps militaris (L.) Link. medicine clinical effectiveness of artificial culture, identical with natural Cordyceps sinensis, can regulate equally whole body function, improve immunological competence, strengthen the phagocytic function of scavenger cell, the formation of enhancing antibody, mainly contain and protect the nourishing the liver of lung kidney-nourishing, relieving cough and asthmaly eliminate the phlegm, moisturizing wrinkle resistant anti-ageing, antimicrobial antiphlogistic, calmness, vasodilation, reduce blood pressure, reduce the effects such as blood sugar, antifatigue, hypoxia tolerance.Artificial Chinese caterpillar fungus cures mainly: consumptive disease hemoptysis, soreness of waist and knee joint, impotence and seminal emission, neurasthenia, palpitation and insomnia, night sweat, heart disorder, poor appetite, nasopharyngeal carcinoma, pulmonary emphysema, pulmonary tuberculosis, trachitis, asthma, hepatitis, ephritis, suffer from a deficiency of the kidney, the disease such as oligoleukocythemia after diabetes, frequent micturition and Radiotherapy chemotherapy.

Cordyceps militaris spawn culture common are and adopts the shaking table shaking culture of producing and the submerged-culture method that adopts fermentor tank to produce, if a small amount of production can adopt shaking culture, deep layer is cultivated needs a whole set of industrial fermentation equipment, as boiler, air compressor, air-purification system, fermentor tank etc., therefore investment is large, is only applicable to the scale operation of batch production.

Summary of the invention

The cultural method that the object of this invention is to provide a kind of Cordyceps militaris bacterial classification, produces to be applicable to general strain plant.

The present invention is by the following technical solutions:

A cultural method for Cordyceps militaris bacterial classification, comprises the steps:

Step 1, liquid nutrient medium preparation: 200g potato is put into 1000ml water, boil 25min, cross leaching cleaner liquid, add afterwards 10g glucose, 3g peptone, 2g potassium primary phosphate, 1g magnesium sulfate, 0.5g agar, 20mgVB1, low baking temperature fusing, regulates pH to 6.8; Or 15g peptone, 10g glucose, 2g potassium primary phosphate, 1g magnesium sulfate, 0.5 agar, 20mgVB1 are put into 1000ml water, mix, regulate pH to 6.8;

Step 2, the liquid nutrient medium preparing is distributed in the Erlenmeyer flask of 500ml capacity, every bottled amount is 300～350ml, and every bottle drips 4～5 rape oils, and bottleneck is wrapped up kraft paper or non-woven fabrics sealing after adding tampon again, at 1.4kg/m ²sterilizing 30～35min under pressure;

Step 3, Erlenmeyer flask after sterilizing is taken out, be cooled to below 30 ℃, under aseptic condition, access 3～4 0.4～0.6cm ³slant strains, standing cultivation 48～72h at 20～22 ℃, on bacterial classification piece, mycelium germination is placed in concussion cultivation on reciprocating shaking table by Erlenmeyer flask, concussion frequency is 80～100 times/min, amplitude 6～10cm or Erlenmeyer flask is placed in to rotary shaking table, concussion frequency is 120～200 turn/min, amplitude 4cm, culturing room's temperature, at 20～21 ℃, is cultivated 3～5d.

Beneficial effect of the present invention: the present invention adopts shake-flask culture Cordyceps militaris bacterial classification, less investment, equipment and technology is simple, is applicable to general strain plant and produces.

Embodiment

Below in conjunction with embodiment, the present invention is done further and explained.Following embodiment does not limit the present invention in any form, and all employings are equal to replaces or technical scheme that the mode of equivalent transformation obtains, all among protection scope of the present invention.

A cultural method for Cordyceps militaris bacterial classification, comprises the steps:

Step 1, liquid nutrient medium preparation: 200g potato is put into 1000ml water, boil 25min, cross leaching cleaner liquid, add afterwards 10g glucose, 3g peptone, 2g potassium primary phosphate, 1g magnesium sulfate, 0.5g agar, 20mgVB1, low baking temperature fusing, regulates pH to 6.8; Or 15g peptone, 10g glucose, 2g potassium primary phosphate, 1g magnesium sulfate, 0.5 agar, 20mgVB1 are put into 1000ml water, mix, regulate pH to 6.8;

Step 2, the liquid nutrient medium preparing is distributed in the Erlenmeyer flask of 500ml capacity, every bottled amount is 300～350ml, and every bottle drips 4～5 rape oils and plays froth breaking effect, and bottleneck is wrapped up kraft paper or non-woven fabrics sealing after adding tampon again, at 1.4kg/m ²sterilizing 30～35min under pressure;

Step 3, Erlenmeyer flask after sterilizing is taken out, be cooled to below 30 ℃, under aseptic condition, access 3～4 0.4～0.6cm ³slant strains, standing cultivation 48～72h at 20～22 ℃, on bacterial classification piece, mycelium germination is placed in concussion cultivation on reciprocating shaking table by Erlenmeyer flask, concussion frequency is 80～100 times/min, amplitude 6～10cm or Erlenmeyer flask is placed in to rotary shaking table, concussion frequency is 120～200 turn/min, amplitude 4cm, culturing room's temperature, at 20～21 ℃, is cultivated 3～5d.

If there is no shaking table equipment, can solve with hand, shake every day 2～5 times, shake 1～3min at every turn, shake to such an extent that many mycelia also grow soon.Tens bottles can be placed on cradle or hammock and shake together.

Embodiment 1

1, liquid nutrient medium preparation: 200g potato is put into 1000ml water, boil 25min, cross leaching cleaner liquid, add afterwards 10g glucose, 3g peptone, 2g potassium primary phosphate, 1g magnesium sulfate, 0.5g agar, 20mgVB1, low baking temperature fusing, regulates pH to 6.8;

2, the liquid nutrient medium preparing is distributed in the Erlenmeyer flask of 500ml capacity, every bottled amount is 300ml, and every bottle drips 4 rape oils, and bottleneck is wrapped up kraft paper sealing after adding tampon again, at 1.4kg/m ²sterilizing 30min under pressure;

3, the Erlenmeyer flask after sterilizing is taken out, be cooled to below 30 ℃, under aseptic condition, access 3 0.4～0.6cm ³slant strains, standing cultivation 48h at 20 ℃, on bacterial classification piece, mycelium germination is placed on reciprocating shaking table concussion by Erlenmeyer flask and cultivates, concussion frequency is 80 times/min, amplitude 6cm, culturing room's temperature, at 20 ℃, is cultivated 3d.

Embodiment 2

1, liquid nutrient medium preparation: 15g peptone, 10g glucose, 2g potassium primary phosphate, 1g magnesium sulfate, 0.5 agar, 20mgVB1 are put into 1000ml water, mix, regulate pH to 6.8;

2, the liquid nutrient medium preparing is distributed in the Erlenmeyer flask of 500ml capacity, every bottled amount is 350ml, and every bottle drips 5 rape oils, and bottleneck is wrapped up kraft paper or non-woven fabrics sealing after adding tampon again, at 1.4kg/m ²sterilizing 35min under pressure;

3, the Erlenmeyer flask after sterilizing is taken out, be cooled to below 30 ℃, under aseptic condition, access 4 0.4～0.6cm ³slant strains, standing cultivation 72h at 22 ℃, on bacterial classification piece, mycelium germination is placed on reciprocating shaking table concussion by Erlenmeyer flask and cultivates, concussion frequency is 100 times/min, amplitude 10cm, culturing room's temperature, at 21 ℃, is cultivated 5d.

Embodiment 3

1, liquid nutrient medium preparation: 200g potato is put into 1000ml water, boil 25min, cross leaching cleaner liquid, add afterwards 10g glucose, 3g peptone, 2g potassium primary phosphate, 1g magnesium sulfate, 0.5g agar, 20mgVB1, low baking temperature fusing, regulates pH to 6.8;

2, the liquid nutrient medium preparing is distributed in the Erlenmeyer flask of 500ml capacity, every bottled amount is 320ml, and every bottle drips 5 rape oils, and bottleneck is wrapped up non-woven fabrics sealing after adding tampon again, at 1.4kg/m ²sterilizing 35min under pressure;

3, the Erlenmeyer flask after sterilizing is taken out, be cooled to below 30 ℃, under aseptic condition, access 4 0.4～0.6cm ³slant strains, standing cultivation 56h at 21 ℃, on bacterial classification piece, mycelium germination is placed in rotary shaking table by Erlenmeyer flask, concussion frequency is 200 turn/min, amplitude 4cm, culturing room's temperature is at 21 ℃, cultivates 4d.

Claims (1)

1. a cultural method for Cordyceps militaris bacterial classification, is characterized in that, comprises the steps:

Step 1, liquid nutrient medium preparation: 200g potato is put into 1000ml water, boil 25min, cross leaching cleaner liquid, add afterwards 10g glucose, 3g peptone, 2g potassium primary phosphate, 1g magnesium sulfate, 0.5g agar, 20mgVB1, low baking temperature fusing, regulates pH to 6.8; Or 15g peptone, 10g glucose, 2g potassium primary phosphate, 1g magnesium sulfate, 0.5 agar, 20mgVB1 are put into 1000ml water, mix, regulate pH to 6.8;

Step 2, the liquid nutrient medium preparing is distributed in the Erlenmeyer flask of 500ml capacity, every bottled amount is 300～350ml, and every bottle drips 4～5 rape oils, and bottleneck is wrapped up kraft paper or non-woven fabrics sealing after adding tampon again, at 1.4kg/m ²sterilizing 30～35min under pressure;

Step 3, Erlenmeyer flask after sterilizing is taken out, be cooled to below 30 ℃, under aseptic condition, access 3～4 0.4～0.6cm ³slant strains, standing cultivation 48～72h at 20～22 ℃, on bacterial classification piece, mycelium germination is placed in concussion cultivation on reciprocating shaking table by Erlenmeyer flask, concussion frequency is 80～100 times/min, amplitude 6～10cm or Erlenmeyer flask is placed in to rotary shaking table, concussion frequency is 120～200 turn/min, amplitude 4cm, culturing room's temperature, at 20～21 ℃, is cultivated 3～5d.