CN104663252A - Breeding method and production process for cordyceps militaris strain with high cordycepin content - Google Patents

Abstract

The invention relates to a breeding method and a production method for a cordyceps militaris strain with high cordycepin content. The cordyceps militaris strain with large ascostroma expansion and large high cordycepin content is obtained by strain breeding, is shaped as a tadpole and has a good market prospect. Owing to the specificity of the strain, the strain is inoculated according to the industrial production requirements, is then germinated for 8-15 days in a dark place at 16-20 DEG C, is sterilized and is subjected to illumination coloring for 1-3 days, and 6-18 holes are drilled in a plastic film with a tool after coloring is normal when the temperature is increased to 20-23 DEG C, so that air exchange is facilitated, the humidity is kept at 65-85 percent, and the total culture time is controlled at 50-70 days. According to the breeding method and the production method, micromo-nospora purpurea pairing hybridization can be carried out on ascospores, so that the cordyceps militaris yield and the high strain purity can be ensured; a stable strain source can be provided for production through sexual propagation from the view of ascospore pairing hybridization.

Description

A kind of Cordceps militaris method for strain breeding thereof of high cordycepin content and production technology

Technical field

The present invention relates to Cordceps militaris method for strain breeding thereof field, particularly relate to a kind of Cordceps militaris method for strain breeding thereof and production technology of high cordycepin content.

Background technology

Cordceps militaris is also called Cordyceps militaris, northern Chinese caterpillar Fungus, is under the jurisdiction of the Ascomycota of mycota, Hypocreales, Clavicipitaceae, and be the type sepecies of Cordyceps, of the same race with Cordyceps, the whole world extensively distributes.The ascostome of Cordceps militaris on lepidopterous insects is similar to Cordyceps sinensis to the medical value of polypide, is the medicinal nourishing fungi that China has Development volue, can as the substitute of Cordyceps sinensis.In recent years along with researcher is to the understanding of Cordceps militaris tonic effect and multiple medical value, it develops research and receives much attention, and makes great progress in each side such as pharmacology active ingredient and artificial cultivations.It is a lot of that present main cultivated edible fungi breeding for quality work is carried out, but the seed selection work of Cordceps militaris new strains report is less.Now commercially Cordceps militaris commodity mostly are tip and bar-shaped kind, and market is expanded the shape commodity grass that simultaneously cordycepin is high to ascostome and had demand.

The mating type of fungi controls its Mating type and amphigenetic hereditary basis.The mating-type system of fungi known is divided into P.drechsleri and the large class of heterothallism 2.P.drechsleri be a kind of autologous can be pregnant sexual reproduction mode, namely this fungi does not need the mycelia mating of two separate sources, and only sprouting by same sexual spore the primary hypha generated just can complete independently sexual intercourse history.P.drechsleri is divided into elementary P.drechsleri (primary homothallism) and secondary P.drechsleri (secondary homothallism).Heterothallism is a kind of autologous infertile sexual reproduction mode, namely sprouting by same sexual spore the primary hypha that formed can not complete independently sexual intercourse history, only have and sprout mating between the primary hyphae that generates by the sexual spore of two different mating types, just can complete sexual intercourse history.Heterothallism is according to the incompatibility factor of hand breeding type a pair or two right, also can be divided into two classes: have two polarity heterothallism (bipolarheterothallism) of a pair incompatibility factor and have the quadripolarity heterothallism (tetrapolar heterothallism) of two pairs of incompatibility factors.With regard to the mating between the sexual spore that same bacterial strain produces, two polarity heterothallism bacterium can affinity rate be 50%, and quadripolarity heterothallism bacterium can affinity rate be 25%, Cordceps militaris belongs to two polarity heterothallic fungus, Genetic Recombination deletion mutation etc. can be brought at sexual reproduction, make sexual progeny that various variation occur, by thecasporous separation and pairing hybridization, make us likely therefrom screen ascostome and expand the high Cordceps militaris of cordycepin.

Often there is the variation on Chromosome level and gene level on genetic variation Chromosome level in Cordceps militaris, mainly because Chinese caterpillar fungus occurs caused by plasmogamy caryogamy and reduction division in sexual reproduction in sexual reproduction; And the variation on gene level is mainly because Cordyceps militaris is subject to caused by the mutagenesis of nutritional condition and environmental condition in process of growth, even so between different ascospore bacterial strains from same fruit body, in the proterties such as colony colour form growth rate, also there is some difference, and the ability producing fruit body separately is also not quite similar.Test shows colony colour to be the orange single spore of apricot be generally can be pregnant and carpogenic ability is stronger, stronger than carpogenic ability when cultivating separately after pairing, colony colour is that the single spore of light lemon yellow generally can not be pregnant, pairing rear portion be can be pregnant some carpogenic ability also very strong, to find to have the Pairing culture of fan-shaped hybridization region to produce the ability of fruit body by force after single spore opposite culture between single spore, and not have the ability of the single spore Pairing culture of fan-shaped hybridization region generation fruit body weak.

Prior art is separated (breeding method of Cordceps militaris bacterial classification, CN102550290A by tissue; Cordceps militaris the best organizes separation period screening technique, CN103250566A; The screening of cordyceps militaris excellent species and stable breeding method, CN1710063), conidium hybridization (utilizes mating type to cordyceps militaris link bacterial strain rejuvenation and the method improving fruiting body yield, CN103609331A; ), many spores hybridization (a kind of cultural method of Cordyceps militaris spawn, CN102017866A) can realize the production of common Cordceps militaris and conidial powder substantially.But tissue is separated due to vegetative propagation repeatedly, containing a large amount of decline variation bacterium in bacterial strain, spawn activity is obviously reduced, shows as that growth is slack-off, annesl is slack-off, production declining etc.; Utilize conidium and the hybridization of many spores to there is bacterial strain purity not enough, go out the problems such as careless yields is low.

In view of above-mentioned defect, creator of the present invention obtains this creation finally through long research and practice.

Summary of the invention

The object of the present invention is to provide a kind of Cordceps militaris method for strain breeding thereof and production technology of high cordycepin content, in order to overcome above-mentioned technological deficiency.

For achieving the above object, the invention provides a kind of Cordceps militaris method for strain breeding thereof of high cordycepin content, this detailed process is:

Step a, excellent single spore strain breeding thereof;

Hung by the feet above PDA medium by mature sporophore, naturally launch, 18-22 DEG C of lucifuge cultivates 2-3 days, and visible asterism shape germination point, chooses single germination point, picking more than 100 strains on PDA slant medium; After 20-23 DEG C of lucifuge cultivates 15 days, illumination 3 days, selects that 30 strains are sprouted soon on PDA slant medium, growing way is prosperous, and mycelia is sturdy, and see that light annesl is fast, the bacterial strain that look dense, is forwarded to culture dish, and 20-23 DEG C of lucifuge is cultivated, and selects 20 strains and prepares fruit bodys and cultivate;

Step b, preparation liquid spawn;

Get 500ml triangular flask, every bottle adds 200ml liquid nutrient medium, inoculates about 0.5cm ²slant strains, puts shaking table 16-23 DEG C of lucifuge and cultivates, 100rmin ^-1concussion cultivation 4 days; Whether muddyly observe liquid, if muddiness, reject;

Step c, goes out grass experiment;

The liquid spawn of gained is transferred on bottled wheat broth by certain proportion pairing, 7-15 days is cultivated in 18-20 DEG C of lucifuge, after mycelia covers with medium, cultivation temperature modulation 20-23 DEG C, see that light is cultivated, intensity of illumination 500lx-2000lx, continuous light 1-3 days, after annesl, on film, punching is ventilated, illumination changes 12 hours every days into, humid control, at 65-85%, is cultivated after 40 days, selects ascostome to expand and significantly gathers and measure cordycepin content;

Steps d, selects ascus to expand shape bacterial strain;

Observe mature sporophore ascostome and expand degree, choose ascostome and expand significantly and the high bacterial strain of cordycepin content, organize separation to obtain purifying bacterial strain from ascostome, be transferred to PDA slant medium, adopt wheat broth again to carry out grass test; If the bacterial strain that ascostome expands does not appear in above-mentioned 20 strain bacterial strains, then continue remaining bacterium, proceed out grass test, screening ascostome expands cordycepin content height bacterial strain;

Observe mature sporophore ascostome and expand degree, choose ascostome and expand significant bacterial strain, organize separation to obtain purifying bacterial strain from ascostome, be transferred to PDA slant medium, adopt wheat broth again to carry out grass test; If the bacterial strain that ascostome expands does not appear in above-mentioned 20 strain bacterial strains, then continue remaining bacterium, proceed out grass test, screening ascostome expands cordycepin content height bacterial strain.

Further, the liquid nutrient medium in above-mentioned steps b is: corn flour 1-3%, potato 1-3%, glucose 1-3%, peptone 0.1-3%, potassium dihydrogen phosphate 0.05-0.5%, magnesium sulfate 0.01-0.05%, water 89-96.9%, 121 DEG C of sterilizing 30min.

Further, the solid culture medium of the wheat in above-mentioned steps c: wheat 1000g, dregs of beans 10-100g, glucose 10-50g, peptone 10-50g, potassium dihydrogen phosphate 1-5g, magnesium sulfate 0.1-0.5g, potassium dihydrogen phosphate 1-5g, magnesium sulfate 0.1-0.5g, VB ₂a slice, VB ₆a slice, water content 55-65%, 121 DEG C of sterilizing 30min.

The present invention also provides a kind of production technology of Cordceps militaris bacterial strain of high cordycepin content, and this detailed process is:

Step a1, make medium, its formula is: wheat 1 kilogram, dregs of beans 0.05 kilogram, the special growth-promoting agent of Chinese caterpillar fungus 35 grams, 1.5 kilograms, water;

Step a2, compound method: special for Chinese caterpillar fungus growth-promoting agent is put into water, stir to completely dissolve nutrient solution; Every box wheat by 450 grams, 0.7 liter, water is distributed into plastic casing; Point to install rear high-temperature resistance plastice film each plastic casing is covered tightly and pricks one with high temperature resistant bungee;

Step a3, sterilizing: add suitable quantity of water in autoclave, puts into autoclave cultivation magazine, is warming up to 100 DEG C-121 DEG C, keep 1-8 hour; Take out, after cooling, move into transfer room; With high-quality aerial fog disinfectant or the airtight sterilization of conventional method 30 minutes, namely wearable sterilization clothes enters transfer room inoculation;

Step a4, inoculation: entering transfer room should shut up the door carefully rapidly; Inoculating tool, bacterial classification outer wall, operating personnel's both hands, with 75% alcohol wipe or dipping sterilization; Adopt liquid spawn; Aseptically, open joint filling material, liquid strain is sprayed even with inoculating gun; Inoculum concentration depending on container size, or adds in liquid spawn that a certain proportion of sterile water or sterile nutrient solution inoculate;

Step a5, management: sprout indoor and temperature kept 16-20 DEG C of shading to sprout 8-15 days at first; When mycelial growth is to medium 1/2-2/3, temperature is risen to 20 DEG C and carry out drawing bacterium; By box from sprouting room proceed to culturing room, every day 24 h light annesl 1-3 days, when medium transfer to Exocarpium Citri Rubrum and there is mycelia projection time, give the thermal stimulation of 10-15 DEG C, temperature brings up to 20-23 DEG C; Sterilize 30 minutes to culturing room, then use tool point 6-18 hole on the plastic film, humidity keeps 65-85%, and indoor appropriate ventilation provides enough oxygen for stroma grows;

Step a6, gathers: when stroma is the spherical not regrowth of Chinese red or crocus, total incubation time controls at 50-70 days; Open basin mouth pocket knife or it adopts from base portion by scissors, put into clean utensil and dry in time or dry, be not exposed to the sun in order to avoid fade, deposit after drying.

Compared with prior art beneficial effect of the present invention is, the technology of the present invention can realize the variation of Cordceps militaris kind, optimizes ascostome and expands and the high Cordceps militaris bacterium of cordycepin content; Outer likeness in form tadpole, has good market prospects.Due to the particularity of bacterial classification, after industrialization production requirements inoculation, at 16-20 DEG C, lucifuge sprouts 8-15 days, carry out drawing bacterium, then illumination annesl 1-3 days, temperature brings up to 20-23 DEG C, tool point 6-18 hole is used on the plastic film after annesl is normal, be beneficial to air exchange, humidity keeps 65-85%, and total incubation time controls at 50-70 days.

Carry out sporangium pairing hybridization by ascospore, can ensure out that carelessness can make again bacterial classification purity high, the present invention starts with from ascospore pairing hybridization can for producing the bacterial classification source providing stable by sexual propagation.

Embodiment

Below, to above-mentioned being described in more detail with other technical characteristic and advantage of the present invention.

Ascostome of the present invention expands the detailed process of the high Cordceps militaris method for strain breeding thereof of cordycepin content and is:

Step a, excellent single spore strain breeding thereof;

Hung by the feet above PDA medium by mature sporophore, naturally launch, 18-22 DEG C of lucifuge cultivates 2-3 days, and visible asterism shape germination point, chooses single germination point, picking more than 100 strains on PDA slant medium; After 20-23 DEG C of lucifuge cultivates 15 days, illumination 3 days, selects that 30 strains are sprouted soon on PDA slant medium, growing way is prosperous, and mycelia is sturdy, and see that light annesl is fast, the bacterial strain that look dense, is forwarded to culture dish, and 20-23 DEG C of lucifuge is cultivated.Select 20 strains and prepare fruit body cultivation.

Step b, preparation liquid spawn;

Get 500ml triangular flask, every bottle adds 200ml liquid nutrient medium, inoculates about 0.5cm ²slant strains, puts shaking table 16-23 DEG C of lucifuge and cultivates, 100rmin ^-1concussion cultivation 4 days.Whether muddyly observe liquid, if muddiness, reject.

Liquid nutrient medium: corn flour 1-3%, potato 1-3%, glucose 1-3%, peptone 0.1-3%, potassium dihydrogen phosphate 0.05-0.5%, magnesium sulfate 0.01-0.05%, water 89-96.9%.121 DEG C of sterilizing 30min.

Step c, goes out grass experiment;

The liquid spawn of gained is transferred on bottled wheat broth by certain proportion pairing, 7-15 days is cultivated in 18-20 DEG C of lucifuge, after mycelia covers with medium, cultivation temperature modulation 20-23 DEG C, see that light is cultivated, intensity of illumination 500lx-2000lx, continuous light 1-3 days, after annesl, on film, punching is ventilated, illumination changes 12 hours every days into, humid control, at 65-85%, is cultivated after 40 days, selects ascostome to expand and significantly gathers and measure cordycepin content.

Solid culture medium: wheat 1000g, dregs of beans 10-100g, glucose 10-50g, peptone 10-50g, potassium dihydrogen phosphate 1-5g, magnesium sulfate 0.1-0.5g, VB ₂a slice, VB ₆a slice, water content 55-65%.121 DEG C of sterilizing 30min.

Steps d, selects ascus to expand shape bacterial strain;

Observe mature sporophore ascostome and expand degree, choose ascostome and expand significantly and the high bacterial strain of cordycepin content, organize separation to obtain purifying bacterial strain from ascostome, be transferred to PDA slant medium, adopt wheat broth again to carry out grass test.If the bacterial strain that ascostome expands does not appear in above-mentioned 20 strain bacterial strains, then continue remaining bacterium, proceed out grass test, screening ascostome expands cordycepin content height bacterial strain.

Observe mature sporophore ascostome and expand degree, choose ascostome and expand significant bacterial strain, organize separation to obtain purifying bacterial strain from ascostome, be transferred to PDA slant medium, adopt wheat broth again to carry out grass test.If the bacterial strain that ascostome expands does not appear in above-mentioned 20 strain bacterial strains, then continue remaining bacterium, proceed out grass test, screening ascostome expands cordycepin content height bacterial strain.

Following table 1 is parent control bacterial strain in the present embodiment and the ascostome diameter of seed selection bacterial strain fruit body when adopting above-mentioned seed selection step and the contrast table of cordycepin content.

Experimentally result is known, and the ascostome diameter of the breeding method in the present embodiment and cordycepin content are apparently higher than parent control bacterial strain.

The difference of table 1 parent control bacterial strain and seed selection bacterial strain fruit body and cordycepin

Bacterial strain number	Single bottle of dry weight/g	Length/mm	Ascostome diameter/mm	Cordycepin content/%
					Parent	35.3	92.4	3.5	0.68
No. 2	34.8	52.1	8.5	1.18
					No. 23	34.7	50.8	8.2	1.23
No. 39	35.2	52.8	7.6	1.21
					No. 7	33.9	51.3	8.9	1.15
No. 17	34.5	52.2	8.1	1.26
					No. 79	35.1	53.1	7.3	1.19

Industrialized producing technology flow process of the present invention is as follows:

Step a1, make medium, its formula is: wheat 1 kilogram, dregs of beans 0.05 kilogram, the special growth-promoting agent of Chinese caterpillar fungus 35 grams, 1.5 kilograms, water;

Step a2, compound method: special for Chinese caterpillar fungus growth-promoting agent is put into water, stir to completely dissolve nutrient solution; Every box wheat by 450 grams, 0.7 liter, water is distributed into plastic casing; Point to install rear high-temperature resistance plastice film each plastic casing is covered tightly and pricks one with high temperature resistant bungee;

Step a3, sterilizing: add suitable quantity of water in autoclave, puts into autoclave cultivation magazine, is warming up to 100 DEG C-121 DEG C, keep 1-8 hour; Take out, after cooling, move into transfer room; With high-quality aerial fog disinfectant or the airtight sterilization of conventional method 30 minutes, namely wearable sterilization clothes enters transfer room inoculation.

Step a4, inoculation: enter transfer room and should shut up the door carefully rapidly (preventing the air containing miscellaneous bacteria from entering); Inoculating tool, bacterial classification outer wall, operating personnel's both hands etc., with 75% alcohol wipe or dipping sterilization; Adopt liquid spawn (preparing same strain improvement); Aseptically, open joint filling material, liquid strain is sprayed even with inoculating gun; Inoculum concentration, depending on container size, also can add in liquid spawn that a certain proportion of sterile water or sterile nutrient solution inoculate;

Step a5, management: sprouting indoor are generally initial keeps 16-20 DEG C of shading to sprout 8-15 days by temperature; When mycelial growth is to medium 1/2-2/3, temperature can be risen to 20 DEG C and carry out drawing bacterium; By box from sprouting room proceed to culturing room, every day 24 h light annesl 1-3 days, when medium transfer to Exocarpium Citri Rubrum and there is mycelia projection time, give the thermal stimulation of 10-15 DEG C, temperature brings up to 20-23 DEG C; Sterilize 30 minutes to culturing room, then use tool point 6-18 hole on the plastic film, be beneficial to air exchange, humidity keeps 65-85%, and indoor appropriate ventilation provides enough oxygen for stroma grows;

Step a6, gathers: when stroma is the spherical not regrowth of Chinese red or crocus, total incubation time controls at 50-70 days; Open basin mouth pocket knife or it adopts from base portion by scissors, put into clean utensil and dry in time or dry, be not exposed to the sun in order to avoid fade.Leave in sealed plastic bag after drying or sell.

The foregoing is only preferred embodiment of the present invention, is only illustrative for invention, and nonrestrictive.Those skilled in the art is understood, and can carry out many changes in the spirit and scope that invention claim limits to it, amendment, even equivalence, but all will fall within the scope of protection of the present invention.

Claims (4)

1. a Cordceps militaris method for strain breeding thereof for high cordycepin content, is characterized in that, this detailed process is:

Step a, excellent single spore strain breeding thereof;

Hung by the feet above PDA medium by mature sporophore, naturally launch, 18-22 DEG C of lucifuge cultivates 2-3 days, and visible asterism shape germination point, chooses single germination point, picking more than 100 strains on PDA slant medium; After 20-23 DEG C of lucifuge cultivates 15 days, illumination 3 days, selects that 30 strains are sprouted soon on PDA slant medium, growing way is prosperous, and mycelia is sturdy, and see that light annesl is fast, the bacterial strain that look dense, is forwarded to culture dish, and 20-23 DEG C of lucifuge is cultivated, and selects 20 strains and prepares fruit bodys and cultivate;

Step b, preparation liquid spawn;

Get 500ml triangular flask, every bottle adds 200ml liquid nutrient medium, inoculates about 0.5cm ²slant strains, puts shaking table 16-23 DEG C of lucifuge and cultivates, 100rmin ^-1concussion cultivation 4 days; Whether muddyly observe liquid, if muddiness, reject;

Step c, goes out grass experiment;

The liquid spawn of gained is transferred on bottled wheat broth by certain proportion pairing, cultivate 7-15 days in 18-20 DEG C of lucifuge, after mycelia covers with medium, cultivation temperature is adjusted to 20-23 DEG C, see that light is cultivated, intensity of illumination 500lx-2000lx, continuous light 1-3 days, after annesl, on film, punching is ventilated, illumination changes 12 hours every days into, humid control, at 65-85%, is cultivated after 40 days, selects ascostome to expand and significantly gathers and measure cordycepin content;

Steps d, selects ascus to expand shape bacterial strain;

Observe mature sporophore ascostome and expand degree, choose ascostome and expand significantly and the high bacterial strain of cordycepin content, organize separation to obtain purifying bacterial strain from ascostome, be transferred to PDA slant medium, adopt wheat broth again to carry out grass test; If the bacterial strain that ascostome expands does not appear in above-mentioned 20 strain bacterial strains, then continue remaining bacterium, proceed out grass test, screening ascostome expands cordycepin content height bacterial strain;

Observe mature sporophore ascostome and expand degree, choose ascostome and expand significant bacterial strain, organize separation to obtain purifying bacterial strain from ascostome, be transferred to PDA slant medium, adopt wheat broth again to carry out grass test; If the bacterial strain that ascostome expands does not appear in above-mentioned 20 strain bacterial strains, then continue remaining bacterium, proceed out grass test, screening ascostome expands cordycepin content height bacterial strain.

2. the Cordceps militaris method for strain breeding thereof of high cordycepin content according to claim 1, it is characterized in that, liquid nutrient medium in above-mentioned steps b is: corn flour 1-3%, potato 1-3%, glucose 1-3%, peptone 0.1-3%, potassium dihydrogen phosphate 0.05-0.5%, magnesium sulfate 0.01-0.05%, water 89-96.9%, 121 DEG C of sterilizing 30min.

3. the Cordceps militaris method for strain breeding thereof of high cordycepin content according to claim 2, it is characterized in that, the solid culture medium of the wheat in above-mentioned steps c: wheat 1000g, dregs of beans 10-100g, glucose 10-50g, peptone 10-50g, potassium dihydrogen phosphate 1-5g, magnesium sulfate 0.1-0.5g, VB2 a slice, VB6 a slice, water content 55-65%, 121 DEG C of sterilizing 30min.

4. a production technology for the Cordceps militaris bacterial strain of high cordycepin content, is characterized in that, this detailed process is:

Step a1, make medium, its formula is: wheat 1 kilogram, dregs of beans 0.05 kilogram, the special growth-promoting agent of Chinese caterpillar fungus 35 grams, 1.5 kilograms, water;

Step a2, compound method: special for Chinese caterpillar fungus growth-promoting agent is put into water, stir to completely dissolve nutrient solution; Every box wheat by 450 grams, 0.7 liter, water is distributed into plastic casing; Point to install rear high-temperature resistance plastice film each plastic casing is covered tightly and pricks one with high temperature resistant bungee;

Step a3, sterilizing: add suitable quantity of water in autoclave, puts into autoclave cultivation magazine, is warming up to 100 DEG C-121 DEG C, keep 1-8 hour; Take out, after cooling, move into transfer room; With high-quality aerial fog disinfectant or the airtight sterilization of conventional method 30 minutes, namely wearable sterilization clothes enters transfer room inoculation;

Step a4, inoculation: entering transfer room should shut up the door carefully rapidly; Inoculating tool, bacterial classification outer wall, operating personnel's both hands, with 75% alcohol wipe or dipping sterilization; Adopt liquid spawn; Aseptically, open joint filling material, liquid strain is sprayed even with inoculating gun; Inoculum concentration depending on container size, or adds in liquid spawn that a certain proportion of sterile water or sterile nutrient solution inoculate;

Step a5, management: sprout indoor and temperature kept 16-20 DEG C of shading to sprout 8-15 days at first; When mycelial growth is to medium 1/2-2/3, temperature is risen to 20 DEG C and carry out drawing bacterium; By box from sprouting room proceed to culturing room, every day 24 h light annesl 1-3 days, when medium transfer to Exocarpium Citri Rubrum and there is mycelia projection time, give the thermal stimulation of 10-15 DEG C, temperature brings up to 20-23 DEG C; Sterilize 30 minutes to culturing room, then use tool point 6-18 hole on the plastic film, humidity keeps 65-85%, and indoor appropriate ventilation provides enough oxygen for stroma grows;

Step a6, gathers: when stroma is the spherical not regrowth of Chinese red or crocus, total incubation time controls at 50-70 days; Open basin mouth pocket knife or it adopts from base portion by scissors, put into clean utensil and dry in time or dry, be not exposed to the sun in order to avoid fade, deposit after drying.