CN108410854A - A method of preparing cordyceps and hickory chick fusant bacterial strain - Google Patents
A method of preparing cordyceps and hickory chick fusant bacterial strain Download PDFInfo
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Abstract
The invention discloses a kind of methods preparing cordyceps and hickory chick fusant bacterial strain.The method disclosed by the invention for preparing cordyceps and hickory chick fusant bacterial strain includes：Cordyceps merge screening with hickory chick and obtain fusant bacterial strain；Wherein, screening includes fusant bacterial strain of the screening with following characteristics：Compared with cordyceps and hickory chick, the biomass of fusant bacterial strain increases, and the product species and Product yields of fusant bacterial strain also increase；Product species are embodied on monosaccharide type, adenosine and the cordycepin of composition polysaccharide.Fusant bacterial strain is prepared using the method for the present invention, cordyceps militaris link bacterial strain and hickory chick bacterial strain can be carried out merges election effects, enrich the active constituent of rare integration of drinking and medicinal herbs fungi, realize that a bacterium multiple-effect and Cordyceps militaris resource and hickory chick biological resources active material are effectively supplemented, it develops simultaneously to which how excellent physiologically active ingredient is, the purpose of physiological active functions multiple-effect superposition, has high realistic meaning and application value.
Description
Technical field
The present invention relates in biotechnology, a method of preparing cordyceps and hickory chick fusant bacterial strain.
Background technology
The ascomycetes fungi that desinsection can be infected in nature is certified up to more than 1500 kinds, is referred to as broad sense cordyceps sinensis
Bacterium, these fungies play an important role in the natural regulation and control of insect population.And the wherein most representational winter worm summer
Grass, Cordyceps militaris and cicada fungus etc. have long edible medicinal history for the Cordyceps Militaris of representative.Cordyceps militaris (Cordyceps
Militaris) also known as northern Chinese caterpillar Fungus, belong to mycota, Eumycota, Ascomycotina, gang pyrenomycetes, ball are belonged on taxology
Shell Zoopagales, Clavicipitaceae, Cordyceps.Cordyceps militaris distribution is wide, growth is fast, easily cultivates, contains a variety of active ingredients, has to human body
There are many physiological functions.In recent years, scientific workers are in the biosynthesis pathway of Cordyceps militaris active material, the life of phorozoon
Object feature, artificial cultivation, liquid deep layer fermenting culture, culture medium composition, training systern, mycelium and metabolite
Chemical composition detection, separation, extraction, purifying and its function, strain excellent selection and breeding, functional food development, health medicine open
Hair etc. has carried out a large amount of research, achieves many gratifying achievements.Numerous studies have detected or have been separated to Cordyceps militaris and contained
Some multiclass active constituents, such as cordycepin, adenosine, cordycepic acid (PEARLITOL 25C), Cordyceps sinensis polysaccharide, superoxide dismutase
(superoxide dismutase, SOD), ergosterol etc. improve body's immunity to promoting body metabolism
Deng with positive effect.In addition, Cordyceps militaris also contains abundant protein, amino acid, unsaturated fatty acid, vitamin and micro-
The ingredients such as secondary element.It is new resource food that the 2009 Nian Yuan Ministry of Public Health, which ratify Cordyceps militaris, assert that it, as new health food, is the winter
The favorable substitutes of worm summer grass.National health State Family Planning Commission in 2014 ratifies Cordyceps militaris as new raw-food material, eliminates to pupa worm
The limitations such as the amount of grass and use scope, further embody safety and feasibility of the Cordyceps militaris as healthy food.
With the development of modern technologies, cordyceps mycelium both can quickly be produced using liquid deep layer fermenting engineering technology, it can also
Zymotic fluid is obtained, while can be enriched with purpose physiological activator with modified technique parameter, optimum culture condition, improving biology
The content of active material provides facility for large-scale development novel health food.
Hickory chick equally be used as world-renowned wild food (medicine) use bacterium, because its surface have it is cellular can pregnant head, outside
Pole is seen to gain the name like sheep tripe.Hickory chick meat it is tender and crisp it is delicious, nutritive and medicinal value is high, therefore food, medicine, health care,
The fields such as cosmetics show great potentiality to be exploited.Hickory chick (Mor chella esculenta), is commonly called as sheep tripe mushroom, sheep
Tripe dish, positive sparrow bacterium etc., belong to Ascomycotina (Ascomyco tina), Pezizale (Pezizales), Morchellaceae
(Morchellaceae), morchella (Mor chel la).Hickory chick is full of nutrition, and amino acid content is each edible mushroom
First of.Morchella esculenta (L.) Pers polysaccharide is one of its main physiologically active ingredient, has enhancing body immunity, promotes adrenal gland skin hormone
Secretion, reduce cholesterol, prevention of arterial hardening, it is antifatigue, antiviral, inhibit all multiactions such as flesh cancer -180.
Invention content
The technical problem to be solved by the present invention is to how prepare the bacterial strain of high yield and multi-products, the product is more
Sugar, adenosine and cordycepin, the polysaccharide are made of a variety of monosaccharide, including glucose, mannose, galactolipin, xylose and Arab
The monosaccharide such as sugar.
In order to solve the above technical problems, present invention firstly provides a kind of preparation method of fusant bacterial strain, the method packet
It includes：Cordyceps are merged with hickory chick to obtain fusant bacterial strain；Compared with the cordyceps and the hickory chick, institute
The biomass for stating fusant bacterial strain increases.
The biomass can be mycelial biomass.
In the above method, the screening may include fusant bacterial strain of the screening with following characteristics：With the cordyceps and
The hickory chick is compared, and the product species and/or Product yields of the fusant bacterial strain increase.
The increase of the fusant bacterial strain product species may be embodied in following A 1) and/or A2) on：
A1) compared with the cordyceps and the hickory chick, the component of the polysaccharide of the fusant bacterial strain synthesis increases；
A2) compared with the hickory chick, the fusant bacterial strain energy synthesizing adenosine and/or cordycepin.
The component of the polysaccharide of the fusant bacterial strain synthesis increases specifically to can express to be contained in the polysaccharide of the fusant bacterial strain
The xylose and arabinose not contained in the polysaccharide of the cordyceps are also free of in the polysaccharide containing the hickory chick
Some galactolipins.
The fusant bacterial strain Product yields increase may be embodied in following B1) and/or B2) on：
B1) compared with the cordyceps, the polysaccharide yield and/or cordycepin output of the fusant bacterial strain increase；
B2) compared with the hickory chick, the polysaccharide yield of the fusant bacterial strain increases.
In the above method, the cordyceps can be that the deposit number of China typical culture collection center is CCTCC
NO:The bacterial strain of M2016220.
The hickory chick can be hickory chick CICC 14033.
In the above method, it is described cordyceps with hickory chick merge may include the plasm of the cordyceps
Body is merged with the protoplast of the hickory chick.
The protoplast of the cordyceps can be by digesting the cordyceps mycelium in cordyceps mycelium
Wall breaking enzyme solution is carried out in liquid to obtain.The cordyceps mycelium enzymolysis liquid is to be added into the potassium chloride solution of 45g/L
The concentration of lywallzyme, glusulase and cellulase, obtained lywallzyme, glusulase and cellulase is respectively 0.5%-2.5%
(mass percent concentration), 0.5%-2.5% (mass percent concentration) and 0.2%-1.5%'s (mass percent concentration)
Solution, pH 5.0-7.0.
The concentration of lywallzyme, glusulase and cellulase further may respectively be in the cordyceps mycelium enzymolysis liquid
0.5%-1.0% (such as 0.8%), 0.5%-1.0% (such as 0.8%), 0.5%-1.0% (such as 0.8%).
The pH of the cordyceps mycelium enzymolysis liquid can be further 6.2-6.8, such as 6.5.
When carrying out broken wall enzymolysis, the protoplast of the cordyceps and the cordyceps mycelium enzymolysis liquid are matched
Than the protoplast that can be cordyceps described in 2.5g：Cordyceps mycelium enzymolysis liquid described in 100mL.
The mycelial enzymolysis of cordyceps can be incubated 2-8h completions, further, the Cordyceps militaris at 24-35 DEG C
The mycelial enzymolysis of bacterium can be incubated 3-5h completions at 26-30 DEG C, and further, the mycelial enzymolysis of cordyceps can
6h is incubated at 28 DEG C to complete.
The protoplast of the hickory chick by by the hickory chick bacterium mycelium in hickory chick bacterium mycelium enzymolysis liquid
Wall breaking enzyme solution is carried out to obtain.The hickory chick bacterium mycelium enzymolysis liquid is that molten wall is added into the potassium chloride solution of 45g/L
Enzyme and cellulase, the concentration of obtained lywallzyme and cellulase be respectively 1%-2.5% (mass percent concentration) and
The solution of 0.5%-2% (mass percent concentration), pH 5.0-7.0.
The concentration of lywallzyme and cellulase further may respectively be 1.5%- in the hickory chick bacterium mycelium enzymolysis liquid
2% (such as 1.8%), 1.5%-2% (such as 1.8%).
The pH of the hickory chick bacterium mycelium enzymolysis liquid can be further 6.5-6.8, such as 6.5.
When carrying out broken wall enzymolysis, the proportioning of the protoplast of the hickory chick and the hickory chick bacterium mycelium enzymolysis liquid
It can be the protoplast of hickory chick described in 1.8g：Hickory chick bacterium mycelium enzymolysis liquid described in 100mL.
The mycelial enzymolysis of hickory chick bacterium can be incubated 2-8h completions, further, the hickory chick at 24-35 DEG C
The mycelial enzymolysis of bacterium can be incubated 4-5h completions at 26-30 DEG C, and further, the mycelial enzymolysis of hickory chick bacterium can
4h is incubated at 28 DEG C to complete.
In the above method, the protoplast of the protoplast of the cordyceps and the hickory chick is the primary of inactivation
Plastid.
The protoplast of the cordyceps of inactivation can be prepared by a method comprising the following steps：By the pupa
The protoplast of Cordyceps Militaris vertical irradiation 10-15min, the cordyceps inactivated at 5-10cm under 30W ultraviolet lamps
Protoplast.Specifically, the protoplast of the cordyceps of inactivation can be prepared into according to the method included the following steps
It arrives：By the protoplast of the cordyceps under 30W ultraviolet lamps vertical irradiation 10min, the pupa inactivated at 10cm
The protoplast of Cordyceps Militaris.
The protoplast of the hickory chick of inactivation can be prepared by a method comprising the following steps：By the sheep tripe
The protoplast of bacterium is incubated 15-20min at 50-60 DEG C, the protoplast of the hickory chick inactivated.Specifically, going out
The protoplast of the hickory chick living can be prepared by a method comprising the following steps：By the primary of the hickory chick
Plastid is incubated 20min at 50 DEG C, the protoplast of the hickory chick inactivated.
In the above method, the temperature of the fusion can be 35 DEG C.The time of the fusion can be 30 minutes.
In the above method, the fusion can carry out in fusogen, and the fusogen is made of solute and solvent, described
Solute is PEG-6000, and the solvent is 0.05mol/L calcium chloride waters.
The fusant bacterial strain that the preparation method or the method for the fusant bacterial strain are prepared prepare polysaccharide, adenosine and/
Or the application in cordycepin, also belong to protection scope of the present invention.
In above application, the polysaccharide can be the polysaccharide being made of at least two in following monosaccharide：Glucose, sweet dew
Sugar, galactolipin, xylose and arabinose.
That carries out cordyceps militaris link bacterial strain and hickory chick bacterial strain merges election effects, enrich rare integration of drinking and medicinal herbs fungi it is active at
Point, it realizes a bacterium multiple-effect, is the completely new class realized Cordyceps militaris resource and hickory chick biological resources active material and effectively supplemented
Topic develops simultaneously so that how excellent physiologically active ingredient is, the purpose of physiological active functions multiple-effect superposition, have high realistic meaning with
Application value.The fusant bacterial strain FJR1181 that profit is obtained by the present invention is compared with parents' bacterial strain, biomass, polysaccharide production
Amount, adenosine yield and cordycepin output increase, which has greatly production meaning.
That carries out cordyceps militaris link bacterial strain and hickory chick bacterial strain merges election effects, enrich rare integration of drinking and medicinal herbs fungi it is active at
Point, it realizes a bacterium multiple-effect, is the completely new class realized Cordyceps militaris resource and hickory chick biological resources active material and effectively supplemented
Topic develops simultaneously so that how excellent physiologically active ingredient is, the purpose of physiological active functions multiple-effect superposition, have high realistic meaning with
Application value.Using the present invention fusant bacterial strain the obtained fusant bacterial strain FJR1181 of preparation method compared with parents' bacterial strain,
Biomass and product species increase；Compared with one of parent cordyceps, biomass, polysaccharide yield and cordycepin output are equal
Increase, and the monosaccharide type for forming polysaccharide increases, and can synthesize the xylose not contained in parent's cordyceps polysaccharide component and Ah
Draw uncle's sugar；Compared with one of parent hickory chick, biomass and polysaccharide yield increase, and the monosaccharide type for forming polysaccharide increases, can
To synthesize the galactolipin not contained in Morchella esculenta (L.) Pers polysaccharide component, the adenosine and worm that hickory chick cannot synthesize can also be synthesized
Careless element.Show that fusant bacterial strain of the invention has abundant product species, also there is higher Product formation ability, there is pole
Big production meaning.
Biomaterial preservation explanation
The Classification And Nomenclature of biomaterial：Cordyceps militaris FJR1181 (Cordyceps militaris FJR1181)
The strain number of biomaterial：FJR1181
Depositary institution's title of biomaterial：China typical culture collection center (cctcc)
The depositary institution of biomaterial is referred to as：CCTCC
The depositary institution address of biomaterial：Wuhan City, Hubei Province Wuchang District Bayi Road 299, in Wuhan University's preservation
Heart postcode：430072
The preservation date of biomaterial：On December 22nd, 2017
The collection of biomaterial is registered on the books number：CCTCC No：M2017824
Biomaterial preservation explanation
The Classification And Nomenclature of biomaterial：Cordyceps FY1421 (Cordyceps militaris FY1421)
The strain number of biomaterial：FY1421
Depositary institution's title of biomaterial：China typical culture collection center (cctcc)
The depositary institution of biomaterial is referred to as：CCTCC
The depositary institution address of biomaterial：Wuhan City, Hubei Province Wuchang District Bayi Road 299, in Wuhan University's preservation
Heart postcode：430072
The preservation date of biomaterial：On April 21st, 2016
The collection of biomaterial is registered on the books number：CCTCC No：M2016220
Specific implementation mode
The present invention is further described in detail With reference to embodiment, the embodiment provided only for
The present invention is illustrated, the range being not intended to be limiting of the invention.Experimental method in following embodiments, unless otherwise specified,
For conventional method.Material as used in the following examples, reagent, instrument etc., unless otherwise specified, commercially
It arrives.Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.
Cordyceps FY1421 (Cordyceps militaris FY1421) in following embodiments is in 2016 4
The moon is preserved in China typical culture collection center (abbreviation CCTCC on the 21st；Address：Wuhan, China, Wuhan University；Postcode：
430072), deposit number is CCTCC NO:M2016220.
Hickory chick CICC 14033 (Morchella sp CICC 14033) in following embodiments is that Chinese industrial is micro-
Biological inoculum preservation administrative center (abbreviation CICC) product.
The selection and breeding of embodiment 1, fusant bacterial strain FJR1181
The present embodiment is by cordyceps FY1421 (Cordyceps militaris FY1421) and hickory chick CICC
14033 protoplast is merged, and the blending decision of one plant of new biomass, polysaccharide, adenosine and the equal high yield of cordycepin is obtained
Strain.Concrete operation step is as follows：
1, it sets out the mycelial collection of parents' bacterial strain
By one of parent to set out, bacterial strain cordyceps FY1421 (Cordyceps militaris FY1421) inoculations
Slant pore is eluted with sterile saline, is eluted after 24-28 DEG C is cultivated 5-7 days to PDA solid slope culture mediums
Then liquid filters the eluent using aseptic filter paper and obtains spore suspension, spore suspension is transferred by 10-20% inoculum concentrations
In PDA liquid medium, (PDA liquid medium component includes：Potato leaches powder 20g/L, glucose 20g/L, peptone
15g/L, potassium dihydrogen phosphate 0.8g/L, magnesium sulfate 0.3g/L, vitamin B120mg/L, surplus are water, adjust pH to 6.5-7.0,
121 DEG C of high pressure steam sterilization 20-30min) it cultivates, it is 50mL to turn liquid measure in the triangular flask of 250mL, includes 10 beades, in
24-28 DEG C, 100-150rpm of constant temperature oscillator, cultivate 24-36h, after culture, obtain liquid culture, 8000rpm from
Heart 10min collects mycelium, is used in combination sterile saline washing secondary, and centrifugation, aseptic filter paper suck dry moisture are finally collected
To cordyceps FY1421 mycelium.
Another parent strain that will be set out, hickory chick CICC 14033 (Morchella sp CICC 14033) are seeded to
(wherein, comprehensive PDA solid slope composition includes for the test tube slants 18 × 180mm of comprehensive PDA solid medium：Potato is leached
Powder 20g/L, glucose 20g/L, KH2PO43.0g/L, MgSO4·7H2O 1.5g/L, vitamin B1100 μ g/L, agar 15-
20g/L, surplus are water, natural pH, 121 DEG C of high pressure steam sterilization 20-30min), 24-26 DEG C is cultivated 5-7 days, then with sterile
Physiological saline prepares spore eluent under washing the spore in solid slope, and filters the eluent using aseptic filter paper and obtain
To spore suspension, the spore suspension is inoculated in PDA fluid nutrient mediums by the inoculum concentration of 10-20%, the triangular flask of 250mL
It is 50mL inside to turn liquid measure, includes 10 beades, with rotating speed constant temperature oscillator culture 24-36 hours of 100-150r/min, is obtained
Liquid culture is obtained, 8000rpm centrifuges 10min, collects mycelium and obtains 14033 mycelium of hickory chick CICC.
2, the preparation of this starting strain of parents protoplast suspension
Mycelium is prepared as raw material using step 1, using the potassium chloride solution of a concentration of 45g/L as homeo-osmosis agent, is adopted
With lywallzyme (Guangdong Province's Culture Collection), glusulase (Sangon Biotech (Shanghai) Co., Ltd.) and
The enzymolysis liquid of cellulase (Sangon Biotech (Shanghai) Co., Ltd.) mixing.
Wherein, the mycelial enzymolysis wall-breaking methods of cordyceps FY1421 are as follows：Into the potassium chloride solution of 45g/L
It is respectively 0.8% (matter to add lywallzyme, glusulase and cellulase, the concentration for obtaining lywallzyme, glusulase and cellulase
Measure percent concentration), the cordyceps mycelium enzyme of 0.8% (mass percent concentration) and 0.8% (mass percent concentration)
Solve liquid；The cordyceps FY1421 mycelium that step 1 obtains are added to (cordyceps in cordyceps mycelium enzymolysis liquid
The proportioning of FY1421 mycelium and cordyceps mycelium enzymolysis liquid is the cordyceps FY1421 mycelia that 2.5g steps 1 obtain
Body：100mL cordyceps mycelium enzymolysis liquid) after, 6h is digested at 28 DEG C, enzymolysis pH is 6.5, and it is primary to obtain cordyceps
Plastid, protoplast concentration reach 1 × 107A/mL, and the efficient (microexamination of the protoplast obtained under this method
Show mycelium substantially all formation protoplast), the regeneration rate of protoplast also preferable (regeneration rate=regeneration clump count/original
Raw plastid sum × 100%), reach 30%.After obtained protoplast is centrifuged, supernatant is removed, the chlorination of 45g/L is utilized
Protoplast is resuspended in aqueous solutions of potassium, obtains cordyceps protoplast suspension.
14033 bacterial strains of hickory chick CICC digest wall-breaking method：Lywallzyme is added into the potassium chloride solution of 45g/L
And cellulase, the concentration for obtaining lywallzyme and cellulase is respectively 1.8% (mass percent concentration) and 1.8% (quality
Percent concentration) Morchella esculenta (L.) Pers mycelium enzymolysis liquid；14033 mycelium of hickory chick CICC that step 1 obtains are added to sheep
(14033 mycelium of hickory chick CICC and the proportioning of Morchella esculenta (L.) Pers mycelium enzymolysis liquid walk for 1.8g in tripe bacterium mycelium enzymolysis liquid
Rapid 1 obtained 14033 mycelium of hickory chick CICC：100mL Morchella esculenta (L.) Pers myceliums enzymolysis liquid) after, 4h, enzyme are digested at 28 DEG C
It is 6.5 to solve pH, obtains hickory chick protoplast, protoplast concentration reaches 1 × 107A/mL, and the original obtained under this method
Efficient (microexamination shows the substantially all formation protoplast of mycelium) of raw plastid, the regeneration rate of protoplast also compared with
Good (regeneration rate=regeneration clump count/protoplast sum × 100%), reaches 30%.After obtained protoplast is centrifuged,
Supernatant is removed, protoplast is resuspended using the potassium chloride solution of 45g/L, obtains hickory chick protoplast suspension.
3, the inactivation treatment of parents' protoplast
The inactivation condition of cordyceps militaris link bacterial strain FY1421 protoplasts is ultraviolet irradiation.The pupa worm for taking 300 μ L steps 2 to obtain
Careless bacterium protoplast suspension, is spread evenly across on regeneration culture medium that (regeneration culture medium each component is as follows：7.5 g/L of glucose, sugarcane
Sugared 7.5g/L, maltose 5.0g/L, peptone 0.5g/L, yeast powder 1.0g/L, KH2PO4 0.5g/L、 MgSO4·7H2O
0.25g/L、CaCl20.2g/L, VB1 0.2g/L, agar 10.00g/L, 109.30 g/L of mannitol, surplus are water, 121 DEG C
High pressure sterilization 20min), it uncaps at 5-10cm under distance 30W ultraviolet lamps, avoids vertical irradiation 10- under white light disturbed condition
15min carries out ultraviolet inactivation, i.e., irradiates 10min at ultraviolet lamp 10cm, irradiates 5min, distance purple at ultraviolet lamp 5cm
8min is irradiated at outer lamp 8cm.Best inactivation condition is：At ultraviolet lamp 10cm, 10min is irradiated, inactivation ratio reaches
100%, inactivation cordyceps protoplast will be obtained and suspended using the potassium chloride solution of 45g/L, inactivation cordyceps are obtained
Protoplast suspension.
The hot inactivation condition of 14033 Strain Protoplasts of hickory chick CICC is：Take the hickory chick that 1mL steps 2 obtain former
Raw plastid suspension is put into 5mL centrifuge tubes, and 50-60 DEG C of hot inactivation treatment is carried out in water bath with thermostatic control, and inactivation time is respectively
15-20min, i.e., inactivation treatment 20min at 50 DEG C, hot inactivation treatment 15min at 55 DEG C, inactivation treatment 10min at 60 DEG C, often
3 repetitions of gradient, and untreated control group is set.Centrifuge tube is gently vibrated every 2min 1 time.Best hot inactivation condition is：It goes out
Temperature 50 C living, time 20min, inactivation ratio reach 100%, obtain inactivation hickory chick protoplast suspension.
4, protoplast fusion
Then, the fusion and regeneration of inactivation parents' protoplast are carried out：With 35% (mass percent concentration) polyethylene glycol
6000 (PEG-6000) solution (solvent of the solution is 0.05mol/L calcium chloride waters, solute PEG-6000) are to promote to melt
Agent takes inactivation cordyceps protoplast suspension and inactivation each 1mL of hickory chick protoplast suspension to mix and centrifuge, abandons respectively
The resuspension of 1mL fusogen is added into precipitation for supernatant, and obtained mixing protoplast suspension is pre- in 30 DEG C of water-baths
It after hot 5min, then is immediately placed in 35 DEG C of shaking baths, 60 revs/min, oscillation fusion 30min.After cell fusion terminates,
Supernatant is abandoned, then oozes steady agent with 0.6mol/L KCl and washs precipitation three times in 2000r/min, 4 DEG C of centrifugation 10min, removal
Obtained protoplast pellet is diluted to 10 by PEG with 0.6mol/L KCl homeo-osmosis agents5A cell/mL, is then coated with
In on regeneration culture medium tablet, being placed in 25 DEG C of constant incubators, it is protected from light culture, the namely regenerated fusant bacterial strain grown.
5, the screening of fusant bacterial strain
35 plants of fusant bacterial strains of the fast growing on the tablet of step 4 are turned to be inoculated into comprehensive PDA culture medium slant
(test tube slants 18 × 180mm), cultivation temperature are 26 DEG C, cultivate 4 days, each fusant bacterial strain is further activated.
After culture, 5mL sterile salines are added to each test tube slant, elution obtains seed suspension, will plant
Sub- suspension inoculation fermentation shaking flask carries out shake flask fermentation screening, 250ml shaking flask liquid amount 50ml, and seed suspension inoculum concentration is 1mL,
Fermentation fermentation medium used carries out, and condition of culture is 26 DEG C, 180rpm, is cultivated 4 days, and tunning is obtained, and collects mycelia
Body, it is 7% (mass percent) that the mycelium of obtained each bacterial strain is dried to moisture at 60 DEG C, after being dried
Mycelium, it is mycelium dry weight to weigh mycelial weight at this time, then detect the polyoses content of each bacterial strain, adenosine and
Cordycepin content.
In 35 plants of fusant bacterial strains, 34 plants of biomass is in 25g/L tunnings hereinafter, polyoses content (accounts for mycelia 15%
Soma weight percentage) hereinafter, adenosine content in 3.0g/kg mycelium dry weights hereinafter, cordycepin content is in 0.5g/kg mycelia
Soma weight is hereinafter, the biomass of other 1 plant of bacterium (i.e. the fusant bacterial strain of number FJR1181) reaches 35g/L tunnings, polysaccharide
Content reaches 27.5% (percentage for accounting for mycelium dry weight), and adenosine content reaches 4.5g/kg mycelium dry weights, and cordycepin contains
Amount reaches 1.5g/kg mycelium dry weights.
Fermentation medium used in above-mentioned shaking flask is calculated as according to volume is weighed：Glucose 8g/100mL, 0.5 g/ of peptone
100mL, yeast powder 1.0g/100mL, analysis for soybean powder (powder made of being ground after soya bean peeling) 2.0g/100mL, corn flour are (beautiful
Powder made of being ground after rice peeling) 5.5g/100mL, wheat flour (powder made of being ground after wheat peeling) 0.5g/
100mL, amine sulfate 0.5g/100mL, potassium dihydrogen phosphate 0.75g/100mL, dipotassium hydrogen phosphate 0.25g/100mL, magnesium sulfate
0.3g/100mL, amino acid nutrient mother liquor 0.5ml/100ml, micro- mother liquor 10ml/100ml, vitamin B1 2mg/
100ml, vitamin B21mg/100ml, vitamin B61mg/100ml, surplus are water, and tune pH is 6.8,121 DEG C of high pressure sterilizations
25min。
The component and concentration of above-mentioned amino acid nutrient mother liquor are as follows：Glycine 0.6g/L, threonine 0.5g/L, figured silk fabrics ammonia
Sour 0.3g/L, leucine 0.1g/L, isoleucine 0.2g/L, phenylalanine 0.5g/L, serine 0.2g/L, proline 0.1g/
L, hydroxyproline 0.06g/L, cysteine 0.04/L, tryptophan 0.05g/L, methionine 0.1g/L, lysine 0.2g/L,
Glutamic acid 0.1g/L, surplus are water, and 4 DEG C of storages are spare.
The component and concentration of above-mentioned micro- mother liquor are as follows：Ferrous sulfate 16.5mg/L, calcium chloride 16.5mg/L,
Copper sulphate 0.3mg/L, zinc sulfate 3.5mg/L, surplus are water, and 4 DEG C of storages are spare.
The detection of above-mentioned fermentation mycelium polyoses content：According to People's Republic of China (PRC) agricultural industry criteria NYT 1676-
The measurement of Thick many candies content carries out in 2008 edible mushrooms.
The detection of above-mentioned fermentation mycelium cordycepin and adenosine：According to People's Republic of China (PRC) agricultural industry criteria NYT
The measurement high performance liquid chromatography of cordycepin and adenosine carries out in 2116-2012 cordyceps products.
6, the identification and preservation of fusant bacterial strain FJR1181
The identification for the fusant bacterial strain that number is FJR1181 in step 5：
Morphological Identification：Colonial morphology --- it is produced in PDA solid medium tablets, initial stage subiculum white, fine and closely woven suede
Hairy, center can form protrusion, edge clear, and mycelia is after culture basal growth, later stage illumination cultivation, hyphal surface and flat
The formation and accumulation of the equal visible yellow color pigment of back.
Microscopic morphology is shown：Mycelia is transparent and hyperbranched, and conidiophore, sporophore top can be formed on mycelium top
End is not expanded, no branch, and conidium is in spherical or ellipticity, is born on sporophore in a cluster, also sees the spore of some evacuations
Son.In conjunction with《Fungal identification handbook》And colonial morphology and microscopic appearance it is identical as Cordyceps sinensis fungus, mould pole is especially drawn up with pupa
It is similar.
Molecular Identification：Using fungi 18S ribosomal universal primer ITS1 and ITS4 (raw work bioengineering (Shanghai) stock
Part Co., Ltd) PCR amplification is carried out to the ITS sequence of the rDNA genes of fusant bacterial strain FJR1181, obtaining amplification length is about
The genetic fragment of 580bp measures the genetic fragment of acquisition, and measurement result shows that its size is 585bp, and particular sequence is sequence
Sequence 1 in list.The sequence is logged in into NCBI (https://www.ncbi.nlm.nih.gov/) carry out sequence alignment
(Blast), as a result display and the nearest bacterial strain of its affiliation are Cordyceps militaris strain C-5 (Cordyceps militaris
Bacterial strain C-5), but its similitude is 93%.
The fusant bacterial strain (fusant bacterial strain FJR1181) that number is FJR1181 is named as Cordyceps militaris FJR1181
(Cordyceps militaris FJR1181), and carry out inclined-plane preservation and glycerine conservation.Cordyceps militaris FJR1181 is in 2017
On December 22, in is preserved in China typical culture collection center (abbreviation CCTCC；Address：Wuhan, China, Wuhan University；Postal
It compiles：430072), deposit number is CCTCC NO:M2017824.
7, the genetic stability of Cordyceps militaris FJR1181
Cordyceps militaris FJR1181 is subjected to secondary culture to investigate its genetic stability, passage in every 3 days is primary, passes on for 15 generations,
Shake flask fermentation is carried out every a generation and measures mycelium content, polyoses content and adenosine and cordycepin content, as a result shows Cordyceps militaris
Mycelial each index content has no significant change in FJR1181 succeeding generations, shows good genetic stability.
The fermentation application of embodiment 2, Cordyceps militaris FJR1181
One, the seed activation culture of Cordyceps militaris FJR1181
1, Cordyceps militaris FJR1181 is seeded to comprehensive PDA solid slope culture medium, 26 DEG C are cultivated 6 days.
2, the spore in the cultured comprehensive PDA solid slope culture medium of step 1 is eluted with sterile physiological saline,
It transfers in comprehensive PDA solid slope culture medium, culture 4 days is protected from light for 26 DEG C in incubator, it is primary to continue activation passage.
Two, the shake-flask seed culture of Cordyceps militaris FJR1181
1, with 5ml sterile salines by the conidium in step 12 cultured PDA solid slope culture mediums
Elution, prepares bacteria suspension.
2, the bacteria suspension prepared is transferred in shaking flask containing seed culture medium by 2% inoculum concentration, in constant-temperature shaking culture
Lower 26 DEG C of 200r/min is cultivated 4 days in device, is obtained seed culture fluid 1, is further increased mycelia number, while strengthening mycelia vigor,
Achieve the purpose that bacterial strain seed culture.
The wherein component of seed culture medium and a concentration of：White sugar (white granulated sugar, the white granulated sugar concretely Suzhou Ou Yang
Chemical Industry Science Co., Ltd's product) 40.0g/L, peptone 10.0g/L, yeast powder 5.0g/L, cornstarch 20.0g/L, sulfuric acid
Magnesium 0.5g/L, potassium dihydrogen phosphate 1.0g/L, dipotassium hydrogen phosphate 0.75/L, VB1 0.05g/L、VB2The ammonia of 0.02g/L, embodiment 1
Base acid nutritious mother liquid 5ml/L, micro- mother liquor 10ml/L, surplus is water, and tune pH value is 6.3,121 DEG C of high pressure sterilizations
25min。
Three, the seed amplification culture of Cordyceps militaris FJR1181
The seed culture fluid 1 that step 2 obtains is seeded to the seeding tank containing seed culture medium, inoculum concentration 2%.
Initial period is cultivated, control fermentation tank ventilatory capacity is 250ml/min, and mixing control 180rpm, temperature control is at 26 DEG C, just
The concentration control of beginning glucose is in 50-100g/L, and Whole Process Control dissolved oxygen (DO) is 40%, by controlling stream plus 70% (matter
Measure percentage) glucose solution, maintain fermentation tank in glucose content be 1.0-1.5g/100mL.Fermentation period control
System was at 6 days, and overall process does not need illumination, and culture terminates, and the mycelia bulk concentration in seeding tank reaches 20%, this is that seed is put
Big culture solution.
Four, Cordyceps militaris FJR1181 liquid deep layer fermentings culture
By culture transferring amount 20%, the seed amplification culture solution of step 3 is inoculated in the fermentation medium containing embodiment 1
In fermentation tank.
Overall process of fermenting (fermentation time is 6 days)：Front 2 days is dark culturing, starts within the 3rd day to fermentation ends to be light
According to culture (intensity of illumination：300lux)；Reduced sugar (glucose) concentration in monitoring cultivating system in real time, by being added 70%
Concentration of reduced sugar in the glucose solution control cultivating system of (mass percent) is in 0.5-1.0g/100mL (whenever training
When concentration of reduced sugar in the system of supporting is less than 0.5g/100mL, 70% glucose solution is added and makes in cultivating system
Concentration of reduced sugar reaches 0.5-1.0g/100mL)；4 hours before fermentation ends, 70% glucose solution is not added.Hair
At the end of ferment, the concentration of reduced sugar 0.1g/100ml in cultivating system.Cultivating global parameters is：Fermentation tank ventilatory capacity is 20L/
Min, mixing control 200rpm, tank pressure are 0.8Mpa, and temperature is controlled at 25 DEG C, and control dissolved oxygen (DO) is 35%.
Five, the mycelial collection of Cordyceps militaris FJR1181 fermentation culture mediums
After completing step 4, rounding cultivating system, using flat centrifugal collection mycelium, (3000rpm is centrifuged
30min), it is 7% that moisture is then dried in 60 DEG C, and sample is Cordyceps militaris FJR1181 products after drying.
Six, the mycelial preparation of control strain
By cordyceps militaris link bacterial strain FY1421 replace Cordyceps militaris FJR1181 using test group condition according to Step 1: two, three,
Four, it five is operated, collects cordyceps militaris link bacterial strain FY1421 mycelium, and carry out 60 DEG C to be dried to moisture being 7%, obtain control 1
Bacterial strain mycelium.
By hickory chick CICC 14033 replace Cordyceps militaris FJR1181 using test group condition according to Step 1: two, three,
Four, it five is operated, collects 14033 mycelium of hickory chick CICC, and carry out 60 DEG C to be dried to moisture being 7%, obtain control 2
Bacterial strain mycelium.
Seven, the measurement of mycelial biomass, polyoses content, adenosine and cordycepin content
1, the measurement of mycelial biomass, polyoses content, adenosine and cordycepin content
By 2 bacterium of Cordyceps militaris FJR1181 products and the obtained 1 bacterial strain mycelium of control of step 6 and control in step 5
The mycelial progress biomass of strain is weighed, and polysaccharide, adenosine and cordycepin content are then measured.Experiment is in triplicate.
Testing result is：The average biomass of Cordyceps militaris FJR1181 products reaches 3.5g dry weights/100ml zymotic fluids, more
Sugared average content reaches 275g/kg mycelium dry weights, and adenosine average content reaches 4.5g/kg mycelium dry weights, and cordycepin is average
Content reaches 1.5g/kg mycelium dry weights.1421 average biomass of cordyceps militaris link bacterial strain FY is 3.2g dry weights/100ml zymotic fluids,
Polysaccharide average content is 112g/kg, and adenosine average content is 6.0g/kg mycelium dry weights, and cordycepin average content reaches
1.3g/kg mycelium dry weight.The average biomass of hickory chick CICC 14033 is 2.5g dry weights/100ml zymotic fluids, and polysaccharide is flat
Equal content is 206g/kg mycelium dry weights, and adenosine content 0, cordycepin content is also 0.
The result shows that：Cordyceps militaris FJR1181 has concentrated the advantage of parents' sheet, under the conditions of liquid deep layer fermenting, Neng Gouyou
On the basis of effect synthesis and a certain amount of adenosine of accumulation and cordycepin, the specific synthesis for having excellent complex polysaccharide and product
Tired ability has good potentiality of the exploitation rich in Rare edible fungus polysaccharide.
2, the measurement of the monosaccharide type and ratio of composition polysaccharide
The polysaccharide for extracting each bacterial strain, using high effective liquid chromatography for measuring Cordyceps militaris FJR1181, cordyceps militaris link bacterial strain FY
Each monosaccharide of 1421 and hickory chick CICC, 14033 polysaccharide forms.The condition of sample pretreatment is：Weigh 6mg polysaccharide samples
In ampoule bottle, 1.0mL distilled water is added, adds isometric 2mol/L trifluoroacetic acids, after tube sealing in 110 DEG C of baking ovens
Hydrolyze 90min；Lid is opened after cooling, 1.0mL methanol is added, use N2Drying.Determination condition is：Chromatographic condition is as follows:Detection
Device:RI (differential refraction detector)；Chromatographic column:AminexHPX-87H HPX-87H analytical columns (300 × 7.8mm)；Column temperature:50
℃；Mobile phase:The dilute sulfuric acid of 0.005mol/L, flow velocity：1mL/min.Experiment is in triplicate.The testing result of polysaccharide is averaged
Value such as the following table 1：
The monosaccharide composition of table 1, each bacterial strain polysaccharide
As a result it shows：Cordyceps militaris FJR1181 has more compared with cordyceps militaris link bacterial strain FY1421 and hickory chick bacterial strain CICC 14033
The monosaccharide of horn of plenty forms, it is shown that the synthesis capability of specific polysaccharide.
<110>Fujian Normal University, Xiamen Yuan Zun bioengineering Co., Ltd
<120>A method of preparing cordyceps and hickory chick fusant bacterial strain
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 585
<212> DNA
<213>Fusant bacterial strain FJR1181
<400> 1
ctccgtaggt gaacctgcgg agggatcatt agcaagttaa ccaactccca accctttgtg 60
aacataccta tcgttgcttc ggcggactcg cccagcgcct ggacgcgggc ctggcgcggc 120
ggccgatcgg ggccccaaac actgtatcta ccagtttttc tgaatccgcc gcaaggcatt 180
acaaatgaat caaaactttc aacaacggat ctcttggctc tggcatcgat gaagaacgca 240
gggaaatgcg ataacatatg tgaattgcag aattcagtga atcatcgaat ctttgaacgc 300
acattgcgcc cgccagcatt ctcccgggca tgcctgttcg aagcgtcatt tcaaccctcg 360
acgtcccctc ctggatgtcg gcgttggggt tcgcgcagca caccgccgcc cccgaaatga 420
agttggcggc ccgtccgcgg cgacctcatc gtagtactcc aactcgcacc gggaagtagg 480
cacgtggcca cgccgttgat gaacgcttaa ctctgaacgt tgacctcgga tcaggtagga 540
atagtcgctg aactgcagca tatcagcata tcaataagcg gagga 585
Claims (10)
1. a kind of preparation method of fusant bacterial strain, including：Cordyceps merge screening with hickory chick and obtain fusant bacterial strain；
Compared with the cordyceps and the hickory chick, the biomass of the fusant bacterial strain increases.
2. according to the method described in claim 1, it is characterized in that：The screening includes blending decision of the screening with following characteristics
Strain：Compared with the cordyceps and the hickory chick, the product species and/or Product yields of the fusant bacterial strain increase.
3. according to the method described in claim 2, it is characterized in that：The increase of the fusant bacterial strain product species is embodied in following
A1 on) and/or A2)：
A1) compared with the cordyceps and the hickory chick, the component of the polysaccharide of the fusant bacterial strain synthesis increases；
A2) compared with the hickory chick, the fusant bacterial strain energy synthesizing adenosine and/or cordycepin；
And/or the fusant bacterial strain Product yields increase is embodied in following B1) and/or B2) on：
B1) compared with the cordyceps, the polysaccharide yield and/or cordycepin output of the fusant bacterial strain increase；
B2) compared with the hickory chick, the polysaccharide yield of the fusant bacterial strain increases.
4. according to any method in claim 1-3, it is characterised in that：The cordyceps are Chinese Typical Representative culture
The deposit number of collection is CCTCC NO:The bacterial strain of M2016220；
And/or the hickory chick is hickory chick CICC 14033.
5. method according to any one of claims 1-4, it is characterised in that：It is described to melt cordyceps and hickory chick
Conjunction includes merging the protoplast of the cordyceps with the protoplast of the hickory chick.
6. according to the method described in claim 5, it is characterized in that：The protoplast of the cordyceps and the hickory chick
Protoplast is the protoplast of inactivation.
7. according to any method in claim 1-6, it is characterised in that：The temperature of the fusion is 35 DEG C, and/or,
The time of the fusion is 30 minutes.
8. according to any method in claim 1-7, it is characterised in that：The fusion carries out in fusogen, described
Fusogen is made of solute and solvent, and the solute is PEG-6000, and the solvent is 0.05mol/L calcium chloride waters.
9. any the method or its fusant bacterial strain being prepared are preparing polysaccharide, adenosine and/or cordyceps sinensis in claim 1-8
Application in element.
10. according to the method described in claim 9, it is characterized in that：The polysaccharide is by least two groups in following monosaccharide
At polysaccharide：Glucose, mannose, galactolipin, xylose and arabinose.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110591924A (en) * | 2019-09-03 | 2019-12-20 | 西北农林科技大学 | Morchella strain and application thereof |
| CN115093972A (en) * | 2022-04-26 | 2022-09-23 | 上海市农业科学院 | Cordyceps militaris strain and application thereof |
| TWI819429B (en) * | 2021-12-10 | 2023-10-21 | 賴孟煜 | Fungal fusant strain, method of manufacturing the same and composition including the same |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103266101A (en) * | 2013-04-18 | 2013-08-28 | 天津师范大学 | Fusion method for protoplast of Cordyceps sinensis and Cordyceps militaris |
| US20150257406A1 (en) * | 2014-03-15 | 2015-09-17 | Mycotechnology, Inc. | Myceliated cacao products and methods for making myceliated products from green cacao and other agricultural substrates |
| CN105647820A (en) * | 2016-03-22 | 2016-06-08 | 广西生众生物科技开发有限公司 | Efficient preparation method of morchella protoplast |
| CN106479899A (en) * | 2016-06-08 | 2017-03-08 | 厦门元尊生物工程有限公司 | A kind of cordyceps militaris link bacterial strain and its application in cordycepin is prepared |
| CN107502624A (en) * | 2017-09-08 | 2017-12-22 | 上海市农业科学院 | A kind of method that hickory chick genetic transformation is carried out using electric shocking method |
| CN107624513A (en) * | 2017-10-25 | 2018-01-26 | 山东惠民齐发果蔬有限责任公司 | It is a kind of rich in the edible and medical fungi cultural hypha method of polysaccharide and application |
-
2018
- 2018-03-21 CN CN201810234445.4A patent/CN108410854B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103266101A (en) * | 2013-04-18 | 2013-08-28 | 天津师范大学 | Fusion method for protoplast of Cordyceps sinensis and Cordyceps militaris |
| US20150257406A1 (en) * | 2014-03-15 | 2015-09-17 | Mycotechnology, Inc. | Myceliated cacao products and methods for making myceliated products from green cacao and other agricultural substrates |
| CN105647820A (en) * | 2016-03-22 | 2016-06-08 | 广西生众生物科技开发有限公司 | Efficient preparation method of morchella protoplast |
| CN106479899A (en) * | 2016-06-08 | 2017-03-08 | 厦门元尊生物工程有限公司 | A kind of cordyceps militaris link bacterial strain and its application in cordycepin is prepared |
| CN107502624A (en) * | 2017-09-08 | 2017-12-22 | 上海市农业科学院 | A kind of method that hickory chick genetic transformation is carried out using electric shocking method |
| CN107624513A (en) * | 2017-10-25 | 2018-01-26 | 山东惠民齐发果蔬有限责任公司 | It is a kind of rich in the edible and medical fungi cultural hypha method of polysaccharide and application |
Non-Patent Citations (5)
| Title |
|---|
| DU,XH等: "Mixed-reproductive strategies, competitive mating-type distribution and life cycle of fourteen black morel species", 《SCIENTIFIC REPORTS》 * |
| 付晓燕等: "粗柄羊肚菌原生质体制备及再生", 《现代农业科学》 * |
| 余知和: "蕈菌与生物工程技术", 《食用菌学报》 * |
| 郭成金等: "冬虫夏草与蛹虫草原生质体融合初探", 《食品科学》 * |
| 陈芳草等: "羊肚菌原生质体育种技术及研究进展", 《庆祝中国土壤学会成立60年专刊》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110591924A (en) * | 2019-09-03 | 2019-12-20 | 西北农林科技大学 | Morchella strain and application thereof |
| CN110591924B (en) * | 2019-09-03 | 2021-10-29 | 西北农林科技大学 | Morchella strain and application thereof |
| TWI819429B (en) * | 2021-12-10 | 2023-10-21 | 賴孟煜 | Fungal fusant strain, method of manufacturing the same and composition including the same |
| CN115093972A (en) * | 2022-04-26 | 2022-09-23 | 上海市农业科学院 | Cordyceps militaris strain and application thereof |
| CN115093972B (en) * | 2022-04-26 | 2023-06-30 | 上海市农业科学院 | Cordyceps militaris strain and application thereof |
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