CN106479899B - A kind of cordyceps militaris link bacterial strain and its preparing the application in cordycepin - Google Patents
A kind of cordyceps militaris link bacterial strain and its preparing the application in cordycepin Download PDFInfo
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Abstract
The invention discloses a kind of cordyceps militaris link bacterial strain and its preparing the application in cordycepin.The present invention provides a kind of cordyceps FY1421, is CCTCC NO:M2016220 in the deposit number of China typical culture collection center.The present invention also protects application of the cordyceps FY1421 in production cordycepin.The present invention also protects a kind of method and method for producing cordycepin of culture cordyceps FY1421.Cordyceps FY1421 provided by the present invention has excellent cordycepin synthesis and build-up properties, is one plant of great production cordycepin and the production bacterial strain rich in cordycepin mycelium application value.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of cordyceps militaris link bacterial strain and its is preparing answering in cordycepin
With.
Background technique
Cordyceps militaris (Cordyceps militaris) also known as northern Chinese caterpillar Fungus, belong to mycota, belong on taxology true
Bacterium door, Ascomycotina, gang pyrenomycetes, Spheeriales, Clavicipitaceae, Cordyceps belong to be equal with wild cordyceps, are us
Traditional one of the important edible and medical fungi of state.
Currently, Cordyceps militaris has realized extensive artificial cultivation, achieve the research and development of its resource in recent decades
Remarkable progress has been increasingly becoming the substitute that wild resource constantly reduced, needed the wild cordyceps of protection；Again because it has
High nutritive value and health-care efficacy, Related product are received by more and more consumers.Data is shown according to the study,
Cordyceps militaris contains the various bioactivators such as cordycepin, cordycepic acid, Cordyceps sinensis polysaccharide, adenosine, sterol, unsaturated fatty acid, tool
There is strengthen immunity, adjust endocrine, antifatigue, anti-oxidant, anti-aging, antibacterial, antitumor, anti anoxia, sedation, drop blood
Sugar, the protection different physiological roles such as liver kidney and respiratory system.Its chemical component and function and famous medicinal fungi cordyceps sinensis
Extremely similar, the substances such as certain active constituents such as cordycepin, adenosine class are also significantly higher than cordyceps sinensis.2005, state food
Drug Administration should be given with the mycelium of artificial propagation clearly stipulate that the raw material of health food has used cordyceps sinensis
With replacement.2009, Cordyceps militaris was approved as new resource food through the national former Ministry of Public Health, is can be referred to as now as food new raw material, is permitted
Perhaps directly edible as food.
Modern research shows that cordycepin is most important one kind, in clinical medicine and health care in Active Components in Various Cordyceps militaris Strains
In play an important role.In recent years, the effective component as Cordyceps militaris class product marking, the physiological function of cordycepin
And the research of pharmacological action, cause the highest attention of biological educational circles and medicine sector scientific worker, scientific worker is to worm
The developmental research of careless element product is also paid attention to and specific further.As the active function to cordycepin gradually recognizes and mechanism of action
It discloses, antibacterial, anti-inflammatory, the significant function that is antitumor, adjusting human endocrine and enhancing immune function of human body etc. showed,
Will further it be paid attention to.Therefore the Cordyceps militaris product of high cordycepin content, either medical rehabilitation or diet are developed
Health will all possess huge economic value and social effect.
Summary of the invention
The object of the present invention is to provide a kind of cordyceps militaris link bacterial strain and its preparing the application in cordycepin.
Cordyceps FY1421 (Cordyceps militaris FY1421) provided by the invention, in April, 2016
It is preserved within 21st China typical culture collection center (abbreviation CCTCC；Address: Wuhan, China, Wuhan University；Postcode:
430072), deposit number is CCTCC NO:M2016220.Cordyceps FY1421 (Cordyceps militaris
FY1421) CCTCC NO:M2016220 is referred to as cordyceps FY1421.
Application of the cordyceps FY1421 in production cordycepin also belongs to protection scope of the present invention.
The method that the present invention also protects culture cordyceps FY1421 includes the following steps: using liquid fermentation medium
Cultivate cordyceps FY1421.
Using the condition of liquid fermentation medium culture cordyceps FY1421 are as follows: ventilatory capacity 15-20L/min, stirring
Revolving speed is 150-200rpm, and temperature is 24-28 DEG C, dissolved oxygen (DO) is 25-45%, and the time is 96-120 hours.
Use the method for liquid fermentation medium culture cordyceps FY1421 concretely: the time for 96 hours, wherein
Be within 0-24 hours dark culturing, be within 25-96 hours illumination cultivation (intensity of illumination concretely: 300lux)；0-92 is small
When, it is (every in 0.8-1.5g/100mL by the way that the concentration of reduced sugar that 70g/100ml glucose solution controls in cultivating system is added
When the concentration of reduced sugar in cultivating system is lower than 0.8g/100mL, 70g/100ml glucose solution is added and to cultivate body
Concentration of reduced sugar in system reaches 1.5g/100mL)；Cultivate global parameters: ventilatory capacity 20L/min, speed of agitator are
200rpm, temperature are 25 DEG C, dissolved oxygen 35%.
Use the method for liquid fermentation medium culture cordyceps FY1421 concretely: the time for 120 hours, wherein
Be within 0-24 hours dark culturing, be within 25-120 hours illumination cultivation (intensity of illumination concretely: 200lux)；0-115
Hour, the concentration of reduced sugar in cultivating system is controlled in 0.8-1.5g/100mL by addition 70g/100ml glucose solution
(when the concentration of reduced sugar in cultivating system is lower than 0.8g/100mL, 70g/100ml glucose solution is added and to cultivate
Concentration of reduced sugar in system reaches 1.5g/100mL)；Cultivate global parameters: ventilatory capacity 15L/min, speed of agitator are
150rpm, temperature are 24 DEG C, dissolved oxygen 25%.
Use the method for liquid fermentation medium culture cordyceps FY1421 concretely: the time for 96 hours, wherein
Be within 0-24 hours dark culturing, be within 25-96 hours illumination cultivation (intensity of illumination concretely: 500lux)；0-92 is small
When, it is (every in 0.8-1.5g/100mL by the way that the concentration of reduced sugar that 70g/100ml glucose solution controls in cultivating system is added
When the concentration of reduced sugar in cultivating system is lower than 0.8g/100mL, 70g/100ml glucose solution is added and to cultivate body
Concentration of reduced sugar in system reaches 1.5g/100mL)；Cultivate global parameters: ventilatory capacity 17.5L/min, speed of agitator are
175rpm, temperature are 28 DEG C, dissolved oxygen 45%.
The method that the present invention also protects culture cordyceps FY1421, in turn include the following steps (a) and step (b):
Step (a): seed enriched medium culture cordyceps FY1421 is used；
Step (b): the product of step (a) is seeded to liquid fermentation medium and is cultivated.
In step (b), the condition of the culture are as follows: ventilatory capacity 15-20L/min, speed of agitator 150-200rpm, temperature
Degree is 24-28 DEG C, dissolved oxygen (DO) is 25-45%, and the time is 96-120 hours.
In step (b), the condition of the culture concretely: the time is 96 hours, wherein 0-24 hour train to be dark
Support, be within 25-96 hour illumination cultivation (intensity of illumination concretely: 300lux)；0-92 hours, by the way that 70g/ is added
100ml glucose solution controls the concentration of reduced sugar in cultivating system in 0.8-1.5g/100mL (whenever in cultivating system
When concentration of reduced sugar is lower than 0.8g/100mL, 70g/100ml glucose solution is added and makes the reduced sugar in cultivating system dense
Degree reaches 1.5g/100mL)；Global parameters: ventilatory capacity 20L/min, speed of agitator 200rpm are cultivated, temperature is 25 DEG C, molten
Solving oxygen is 35%.
In step (b), the condition of the culture concretely: the time is 120 hours, wherein 0-24 hour train to be dark
Support, be within 25-120 hour illumination cultivation (intensity of illumination concretely: 200lux)；0-115 hours, by the way that 70g/ is added
100ml glucose solution controls the concentration of reduced sugar in cultivating system in 0.8-1.5g/100mL (whenever in cultivating system
When concentration of reduced sugar is lower than 0.8g/100mL, 70g/100ml glucose solution is added and makes the reduced sugar in cultivating system dense
Degree reaches 1.5g/100mL)；Global parameters: ventilatory capacity 15L/min, speed of agitator 150rpm are cultivated, temperature is 24 DEG C, molten
Solving oxygen is 25%.
In step (b), the condition of the culture concretely: the time is 96 hours, wherein 0-24 hour train to be dark
Support, be within 25-96 hour illumination cultivation (intensity of illumination concretely: 500lux)；0-92 hours, by the way that 70g/ is added
100ml glucose solution controls the concentration of reduced sugar in cultivating system in 0.8-1.5g/100mL (whenever in cultivating system
When concentration of reduced sugar is lower than 0.8g/100mL, 70g/100ml glucose solution is added and makes the reduced sugar in cultivating system dense
Degree reaches 1.5g/100mL)；Global parameters: ventilatory capacity 17.5L/min, speed of agitator 175rpm are cultivated, temperature is 28 DEG C,
Dissolved oxygen is 45%.
Step (a) is concretely: cordyceps FY1421 is seeded to seed enriched medium, and (cordyceps FY1421 exists
Initial concentration in cultivating system is 104A/ml), 26 DEG C are cultivated 4 days, the inoculum that strengthened (cordyceps FY1421
Spore concentration in cultivating system is 1012A/gram culture).
Step (b) is concretely: the product of 100g step (a) being seeded to 35L liquid fermentation medium and is cultivated.
The present invention also protects a kind of method for producing cordycepin, includes the following steps: to cultivate cordyceps FY1421.
The method of the production cordycepin includes the following steps: using liquid fermentation medium culture cordyceps
FY1421。
Using the condition of liquid fermentation medium culture cordyceps FY1421 are as follows: ventilatory capacity 15-20L/min, stirring
Revolving speed is 150-200rpm, and temperature is 24-28 DEG C, dissolved oxygen (DO) is 25-45%, and the time is 96-120 hours.
Use the method for liquid fermentation medium culture cordyceps FY1421 concretely: the time for 96 hours, wherein
Be within 0-24 hours dark culturing, be within 25-96 hours illumination cultivation (intensity of illumination concretely: 300lux)；0-92 is small
When, it is (every in 0.8-1.5g/100mL by the way that the concentration of reduced sugar that 70g/100ml glucose solution controls in cultivating system is added
When the concentration of reduced sugar in cultivating system is lower than 0.8g/100mL, 70g/100ml glucose solution is added and to cultivate body
Concentration of reduced sugar in system reaches 1.5g/100mL)；Cultivate global parameters: ventilatory capacity 20L/min, speed of agitator are
200rpm, temperature are 25 DEG C, dissolved oxygen 35%.
Use the method for liquid fermentation medium culture cordyceps FY1421 concretely: the time for 120 hours, wherein
Be within 0-24 hours dark culturing, be within 25-120 hours illumination cultivation (intensity of illumination concretely: 200lux)；0-115
Hour, the concentration of reduced sugar in cultivating system is controlled in 0.8-1.5g/100mL by addition 70g/100ml glucose solution
(when the concentration of reduced sugar in cultivating system is lower than 0.8g/100mL, 70g/100ml glucose solution is added and to cultivate
Concentration of reduced sugar in system reaches 1.5g/100mL)；Cultivate global parameters: ventilatory capacity 15L/min, speed of agitator are
150rpm, temperature are 24 DEG C, dissolved oxygen 25%.
Use the method for liquid fermentation medium culture cordyceps FY1421 concretely: the time for 96 hours, wherein
Be within 0-24 hours dark culturing, be within 25-96 hours illumination cultivation (intensity of illumination concretely: 500lux)；0-92 is small
When, it is (every in 0.8-1.5g/100mL by the way that the concentration of reduced sugar that 70g/100ml glucose solution controls in cultivating system is added
When the concentration of reduced sugar in cultivating system is lower than 0.8g/100mL, 70g/100ml glucose solution is added and to cultivate body
Concentration of reduced sugar in system reaches 1.5g/100mL)；Cultivate global parameters: ventilatory capacity 17.5L/min, speed of agitator are
175rpm, temperature are 28 DEG C, dissolved oxygen 45%.
The method of the production cordycepin includes the following steps (a) and step (b):
Step (a): seed enriched medium culture cordyceps FY1421 is used；
Step (b): the product of step (a) is seeded to liquid fermentation medium and is cultivated.
In step (b), the condition of the culture are as follows: ventilatory capacity 15-20L/min, speed of agitator 150-200rpm, temperature
Degree is 24-28 DEG C, dissolved oxygen (D0) is 25-45%, and the time is 96-120 hours.
In step (b), the condition of the culture concretely: the time is 96 hours, wherein 0-24 hour train to be dark
Support, be within 25-96 hour illumination cultivation (intensity of illumination concretely: 300lux)；0-92 hours, by the way that 70g/ is added
100ml glucose solution controls the concentration of reduced sugar in cultivating system in 0.8-1.5g/100mL (whenever in cultivating system
When concentration of reduced sugar is lower than 0.8g/100mL, 70g/100ml glucose solution is added and makes the reduced sugar in cultivating system dense
Degree reaches 1.5g/100mL)；Global parameters: ventilatory capacity 20L/min, speed of agitator 200rpm are cultivated, temperature is 25 DEG C, molten
Solving oxygen is 35%.
In step (b), the condition of the culture concretely: the time is 120 hours, wherein 0-24 hour train to be dark
Support, be within 25-120 hour illumination cultivation (intensity of illumination concretely: 200lux)；0-115 hours, by the way that 70g/ is added
100ml glucose solution controls the concentration of reduced sugar in cultivating system in 0.8-1.5g/100mL (whenever in cultivating system
When concentration of reduced sugar is lower than 0.8g/100mL, 70g/100ml glucose solution is added and makes the reduced sugar in cultivating system dense
Degree reaches 1.5g/100mL)；Global parameters: ventilatory capacity 15L/min, speed of agitator 150rpm are cultivated, temperature is 24 DEG C, molten
Solving oxygen is 25%.
In step (b), the condition of the culture concretely: the time is 96 hours, wherein 0-24 hour train to be dark
Support, be within 25-96 hour illumination cultivation (intensity of illumination concretely: 500lux)；0-92 hours, by the way that 70g/ is added
100ml glucose solution controls the concentration of reduced sugar in cultivating system in 0.8-1.5g/100mL (whenever in cultivating system
When concentration of reduced sugar is lower than 0.8g/100mL, 70g/100ml glucose solution is added and makes the reduced sugar in cultivating system dense
Degree reaches 1.5g/100mL)；Global parameters: ventilatory capacity 17.5L/min, speed of agitator 175rpm are cultivated, temperature is 28 DEG C,
Dissolved oxygen is 45%.
Step (a) is concretely: cordyceps FY1421 is seeded to seed enriched medium, and (cordyceps FY1421 exists
Initial concentration in cultivating system is 104A/ml), 26 DEG C are cultivated 4 days, the inoculum that strengthened (cordyceps FY1421
Spore concentration in cultivating system is 1012A/gram culture).
Step (b) is concretely: the product of 100g step (a) being seeded to 35L liquid fermentation medium and is cultivated.
Any description above liquid fermentation medium is made of solute and solvent；The solute and its in the liquid fermentation
Concentration in culture medium is as follows: glucose 2-4g/100ml, peptone 1-2g/100ml, yeast powder 1-2g/100ml, corn flour
5-6g/100ml, beancake powder 1-3g/100ml, wheat flour 0.4-0.6g/100ml, potassium dihydrogen phosphate 0.4-0.6g/100ml, sulphur
Sour magnesium 0.2-0.4g/100ml, vitamin B11-3mg/100ml, vitamin B20.5-1.5mg/100ml, glycine 0.2-
0.4mg/100ml, threonine 0.2-0.3mg/100ml, valine 0.1-0.2mg/100ml, leucine 0.04-0.06mg/
100ml, isoleucine 0.05-0.15mg/100ml, phenylalanine 0.2-0.3mg/100ml, serine 0.05-0.15mg/
100ml, proline 0.04-0.06mg/100ml, hydroxyproline 0.02-0.04mg/100ml, cysteine 0.01-0.03mg/
100ml, tryptophan 0.02-0.03mg/100ml, methionine 0.04-0.06mg/100ml, lysine 0.05-0.15mg/
100ml, glutamic acid 0.04-0.06mg/100ml；The solvent is water.
The solute and its concentration in the liquid fermentation medium is concretely: glucose 3g/100ml, albumen
Peptone 1.5g/100ml, yeast powder 1.5g/100ml, corn flour 5.5g/100ml, beancake powder 2.0g/100ml, wheat flour 0.5g/
100ml, potassium dihydrogen phosphate 0.5g/100ml, magnesium sulfate 0.3g/100ml, vitamin B12mg/100ml, vitamin B2 1mg/
100ml, glycine 0.3mg/100ml, threonine 0.25mg/100ml, valine 0.15mg/100ml, leucine 0.05mg/
100ml, isoleucine 0.1mg/100ml, phenylalanine 0.25mg/100ml, serine 0.1mg/100ml, proline
0.05mg/100ml, hydroxyproline 0.03mg/100ml, cysteine 0.02mg/100ml, tryptophan 0.025mg/100ml, first
Methyllanthionine 0.05mg/100ml, lysine 0.1mg/100ml, glutamic acid 0.05mg/100ml.
The solute and its concentration in the liquid fermentation medium is concretely: glucose 2g/100ml, albumen
Peptone 1.0g/100ml, yeast powder 1.0g/100ml, corn flour 5.0g/100ml, beancake powder 1.0g/100ml, wheat flour 0.4g/
100ml, potassium dihydrogen phosphate 0.4g/100ml, magnesium sulfate 0.2g/100ml, vitamin B11mg/100ml, vitamin B2 0.5mg/
100ml, glycine 0.2mg/100ml, threonine 0.2mg/100ml, valine 0.1mg/100ml, leucine 0.04mg/
100ml, isoleucine 0.05mg/100ml, phenylalanine 0.2mg/100ml, serine 0.05mg/100ml, proline
0.04mg/100ml, hydroxyproline 0.02mg/100ml, cysteine 0.01mg/100ml, tryptophan 0.02mg/100ml, first
Methyllanthionine 0.04mg/100ml, lysine 0.05mg/100ml, glutamic acid 0.04mg/100ml.
The solute and its concentration in the liquid fermentation medium is concretely: glucose 4g/100ml, albumen
Peptone 2.0g/100ml, yeast powder 2.0g/100ml, corn flour 6.0g/100ml, beancake powder 3.0g/100ml, wheat flour 0.6g/
100ml, potassium dihydrogen phosphate 0.6g/100ml, magnesium sulfate 0.4g/100ml, vitamin B13mg/100ml, vitamin B2 1.5mg/
100ml, glycine 0.4mg/100ml, threonine 0.3mg/100ml, valine 0.2mg/100ml, leucine 0.06mg/
100ml, isoleucine 0.15mg/100ml, phenylalanine 0.3mg/100ml, serine 0.15mg/100ml, proline
0.06mg/100ml, hydroxyproline 0.04mg/100ml, cysteine 0.03mg/100ml, tryptophan 0.03mg/100ml, first
Methyllanthionine 0.06mg/100ml, lysine 0.15mg/100ml, glutamic acid 0.06mg/100ml.
The pH=6.5-7.0 of any description above liquid fermentation medium.
The raw material proportioning of any description above seed enriched medium are as follows: 1L nutrient solution: 0.5-1.5kg millet；The battalion
Nutrient solution is made of solute and solvent；The solute and its concentration in the nutrient solution are as follows: potato leaches powder 1-3g/
100ml, glucose 0.5-1.5g/100ml, peptone 0.4-0.8g/100ml, potassium dihydrogen phosphate 0.05-0.15g/100ml, sulphur
Sour magnesium 0.04-0.06g/100ml, vitamin B11-3mg/100ml, vitamin B20.5-1.5mg/100ml, glycine 0.2-
0.4mg/100ml, threonine 0.2-0.3mg/100ml, valine 0.1-0.2mg/100ml, leucine 0.04-0.06mg/
100ml, isoleucine 0.05-0.15mg/100ml, phenylalanine 0.2-0.3mg/100ml, serine 0.05-0.15mg/
100ml, proline 0.04-0.06mg/100ml, hydroxyproline 0.02-0.04mg/100ml, cysteine 0.01-0.03mg/
100ml, tryptophan 0.02-0.03mg/100ml, methionine 0.04-0.06mg/100ml, lysine 0.05-0.15mg/
100ml, glutamic acid 0.04-0.06mg/100ml；The solvent is water.
The solute and its concentration in the nutrient solution is concretely: potato leaches powder 2g/100ml, glucose
1g/100ml, peptone 0.6g/100ml, potassium dihydrogen phosphate 0.1g/100ml, magnesium sulfate 0.05g/100ml, vitamin B12 mg/
100ml, vitamin B2 1mg/100ml, glycine 0.3mg/100ml, threonine 0.25mg/100ml, valine 0.15mg/
100ml, leucine 0.05mg/100ml, isoleucine 0.1mg/100ml, phenylalanine 0.25mg/100ml, serine
0.1mg/100ml, proline 0.05mg/100ml, hydroxyproline 0.03mg/100ml, cysteine 0.02mg/100ml, color ammonia
Sour 0.025mg/100ml, methionine 0.05mg/100ml, lysine 0.1mg/100ml, glutamic acid 0.05mg/100ml.
The raw material proportioning of the seed enriched medium is concretely: 1L nutrient solution: 1.0kg millet.
The preparation method of the seed enriched medium is concretely: nutrient solution is uniformly mixed with millet, Steam by water bath (when
Between concretely 30min).
The present invention is also protected for cultivating the cordyceps FY1421 and/or preparing the kit of cordycepin, the examination
Agent box includes the liquid fermentation medium.
The kit further includes the seed enriched medium.
The present invention provides cordyceps FY1421 and cultivates the method for cordyceps FY1421 and pass through culture pupa worm
The method that careless bacterium FY1421 prepares cordycepin.Cordycepin content in the mycelium of cordyceps FY1421 is than existing bacterium germination out
Strain improves nearly 170 times, is one plant of great production cordycepin and the production bacterial strain rich in cordycepin mycelium application value.This hair
The method for preparing cordycepin of bright offer has the advantages that fermentation period is short, cordycepin output is high.The present invention has great answer
With value and industrial prospect.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
Cordyceps militaris link bacterial strain 50383: Chinese agriculture Microbiological Culture Collection administrative center (www.accc.org.cn, referred to as
ACCC), ACCC is numbered: 50383.
Lywallzyme: Guangdong Culture Collection.
Glusulase: Sangon Biotech (Shanghai) Co., Ltd., article No.: A600870.
Cellulase: Sangon Biotech (Shanghai) Co., Ltd., article No.: A002598.
Beancake powder: Zibo Kun Xing grain and oil Co., Ltd.
PDA solid medium: potato leaches powder 20g, glucose 20g, agar 15-20g, and distilled water is settled to
1000mL, natural pH；121 DEG C of high pressure steam sterilization 20-30min.
PDA liquid medium: potato leaches powder 20g, glucose 20g, peptone 15g, potassium dihydrogen phosphate 0.8g, sulfuric acid
Magnesium 0.3g, vitamin B120mg, distilled water are settled to 1000ml, adjust pH to 6.5-7.0；121 DEG C of high pressure steam sterilization 20-
30min。
Amino acid nutrient mother liquor the preparation method comprises the following steps: taking glycine, threonine, valine, leucine, isoleucine, benzene
Alanine, serine, proline, hydroxyproline, cysteine, tryptophan, methionine, lysine and glutamic acid add distillation
Water is settled to 1000ml；4 DEG C of storages, it is spare.
Seed enriched medium: potato leaches powder 20g, glucose 10g, peptone 6.0g, potassium dihydrogen phosphate 1.0g, sulphur
Sour magnesium 0.5g, amino acid nutrient mother liquor 5ml, vitamin B1 20mg, vitamin B2 10mg, distilled water are settled to 1000ml, system
At nutrient solution；1000ml nutrient solution is uniformly mixed with 1000g millet, and Steam by water bath 30min obtains seed enriched medium；121℃
High pressure sterilization 20-30min.Concentration of each solute in nutrient solution is as follows: potato leaches powder 2g/100ml, glucose 1g/
100ml, peptone 0.6g/100ml, potassium dihydrogen phosphate 0.1g/100ml, magnesium sulfate 0.05g/100ml, vitamin B1 2mg/
100ml, vitamin B21mg/100ml, glycine 0.3mg/100ml, threonine 0.25mg/100ml, valine 0.15mg/
100ml, leucine 0.05mg/100ml, isoleucine 0.1mg/100ml, phenylalanine 0.25mg/100ml, serine
0.1mg/100ml, proline 0.05mg/100ml, hydroxyproline 0.03mg/100ml, cysteine 0.02mg/100ml, color ammonia
Sour 0.025mg/100ml, methionine 0.05mg/100ml, lysine 0.1mg/100ml, glutamic acid 0.05mg/100ml.
The preparation method (pH=6.5-7.0) of liquid fermentation medium: taking glucose, peptone, yeast powder, corn flour,
Beancake powder, wheat flour, potassium dihydrogen phosphate, magnesium sulfate, amino acid nutrient mother liquor, vitamin B1, vitamin B2 add distilled water constant volume
It is 6.5-7.0 to 100ml, tune pH；121 DEG C of high pressure sterilization 25-30min, are cooled to 24-28 DEG C, spare.Divide according to the method described above
Liquid fermentation medium first, liquid fermentation medium second and liquid fermentation medium third are not prepared.
In liquid fermentation medium first, the concentration of each solute is as follows: glucose 3g/100ml, peptone 1.5g/100ml,
Yeast powder 1.5g/100ml, corn flour 5.5g/100ml, beancake powder 2.0g/100ml, wheat flour 0.5g/100ml, biphosphate
Potassium 0.5g/100ml, magnesium sulfate 0.3g/100ml, vitamin B12mg/100ml, vitamin B21mg/100ml, glycine
0.3mg/100ml, threonine 0.25mg/100ml, valine 0.15mg/100ml, leucine 0.05mg/100ml, isoleucine
0.1mg/100ml, phenylalanine 0.25mg/100ml, serine 0.1mg/100ml, proline 0.05mg/100ml, hydroxyl dried meat ammonia
Sour 0.03mg/100ml, cysteine 0.02mg/100ml, tryptophan 0.025mg/100ml, methionine 0.05mg/100ml,
Lysine 0.1mg/100ml, glutamic acid 0.05mg/100ml.
In liquid fermentation medium second, the concentration of each solute is as follows: glucose 2g/100ml, peptone 1.0g/100ml,
Yeast powder 1.0g/100ml, corn flour 5.0g/100ml, beancake powder 1.0g/100ml, wheat flour 0.4g/100ml, biphosphate
Potassium 0.4g/100ml, magnesium sulfate 0.2g/100ml, vitamin B11mg/100ml, vitamin B20.5mg/100ml, glycine
0.2mg/100ml, threonine 0.2mg/100ml, valine 0.1mg/100ml, leucine 0.04mg/100ml, isoleucine
0.05mg/100ml, phenylalanine 0.2mg/100ml, serine 0.05mg/100ml, proline 0.04mg/100ml, hydroxyl dried meat ammonia
Sour 0.02mg/100ml, cysteine 0.01mg/100ml, tryptophan 0.02mg/100ml, methionine 0.04mg/100ml,
Lysine 0.05mg/100ml, glutamic acid 0.04mg/100ml.
In liquid fermentation medium third, the concentration of each solute is as follows: glucose 4g/100ml, peptone 2.0g/100ml,
Yeast powder 2.0g/100ml, corn flour 6.0g/100ml, beancake powder 3.0g/100ml, wheat flour 0.6g/100ml, biphosphate
Potassium 0.6g/100ml, magnesium sulfate 0.4g/100ml, vitamin B13mg/100ml, vitamin B21.5mg/100ml, glycine
0.4mg/100ml, threonine 0.3mg/100ml, valine 0.2mg/100ml, leucine 0.06mg/100ml, isoleucine
0.15mg/100ml, phenylalanine 0.3mg/100ml, serine 0.15mg/100ml, proline 0.06mg/100ml, hydroxyl dried meat ammonia
Sour 0.04mg/100ml, cysteine 0.03mg/100ml, tryptophan 0.03mg/100ml, methionine 0.06mg/100ml,
Lysine 0.15mg/100ml, glutamic acid 0.06mg/100ml.
The method for detecting cordycepin content: the efficient liquid of measurement of cordycepin and adenosine in NY/T 2116-2012 cordyceps product
Phase chromatography.
One, the mycelial collection of cordyceps militaris link bacterial strain 50383
1, cordyceps militaris link bacterial strain 50383 is seeded to PDA solid slope culture medium, 24-28 DEG C culture 5-7 days.
2, the spore in the cultured PDA solid slope culture medium of step 1 is eluted with sterile physiological saline, and passed through
Spore suspension is obtained by filtration in aseptic filter paper, and it is 1.8 × 10 that spore concentration is contained in spore suspension7A/mL.
3, the spore suspension for preparing step 2 by 5-20% inoculum concentration transfer in PDA liquid medium culture (24-28 DEG C,
100-150rpm, 24-36h), obtain liquid culture.
4, the liquid culture 8000rpm centrifugation 10min obtained step 3 collects mycelium, is washed with sterile saline
It washs secondary, with aseptic filter paper suck dry moisture, obtains mycelium.
Two, the preparation of 50383 protoplast suspension of cordyceps militaris link bacterial strain
1, the mycelium for taking step 1 to obtain, using the enzymolysis liquid being mixed to get by lywallzyme, glusulase and cellulase
Enzyme digestion reaction is carried out to mycelium, the enzymolysis liquid after being reacted.In enzyme digestion reaction: the use concentration of lywallzyme is 0.5%-
2.5%, optium concentration 0.5%-1.0%；Use the concentration 0.5%-2.5%, optium concentration 0.5%- of glusulase
1.0%；The use concentration of cellulase is 0.2%-1.5%, optium concentration 0.5-1.0%%；Hydrolysis temperature range is 24-
35 DEG C, best hydrolysis temperature is 26-30 DEG C；Enzymolysis time is 2-8h, and best enzymolysis time is 3-5h；Adjusting enzymatic hydrolysis pH is 5.0-
7.0, the best pH that digests is 6.2-6.8.
2, the protoplasm somatocyte that step 2 obtains is resuspended using the potassium chloride solution of 45g/L to precipitate, obtains protoplast
Suspension.
Three, atmospheric pressure at room plasma (ARTP) mutagenic treatment of cordyceps militaris link bacterial strain protoplast
1, it carries out preliminary experiment and selects the mutagenic treatment time, the method is as follows:
Protoplast suspension prepared by the step of taking 10-20 μ L two is uniformly coated on the upper surface of metal slide glass, after dry
Slide glass is transferred to objective table with tweezers.Fungus slide glass, setting are handled as the working gas of plasma using high-purity helium
Different processing groups is arranged in power 80W, irradiation distance 4mm, 26-30 DEG C of the temperature of plasma, throughput 10L/min,
The processing time of each group is respectively 0 (control), 5,10,15,20,25,30,35,40,45,50,55s, and every group of setting weighs three times
It is multiple.By treated, slide glass is transferred in the EP pipe equipped with 1mL regeneration culture medium, on the oscillator regeneration culture 45min, attached
Microorganism on slide glass be eluted in regeneration culture medium, form bacteria suspension.Bacteria suspension is applied to regenerated plate to be placed on
Culture counted flat-plate bacterial colony after 2-3 days in 24-28 DEG C of incubator, calculated lethality, the following institute of lethality calculation method
Show:
Lethality %=(without mutagenic treatment clump count-through mutagenic treatment clump count)/without mutagenesis clump count × 100%
By counting the lethality of each processing group, lethality is selected to carry out for the treatment with irradiation time of 75-85% formal real
It tests, the processing time is 20s.
2, according to step 1 as a result, formally being tested, the method is as follows:
Protoplast suspension prepared by the step of taking 15 μ L two is uniformly coated on the upper surface of metal slide glass, uses after dry
Slide glass is transferred to objective table by tweezers.Fungus slide glass, setting electricity are handled as the working gas of plasma using high-purity helium
Source power 80W, irradiation distance 4mm, 26-30 DEG C of the temperature of plasma, throughput 10L/min, processing time are 20s.Sample
After being disposed, slide glass is put into the EP pipe equipped with 1mL regeneration culture medium with aseptic nipper, regeneration culture on the oscillator
45min is eluted to the microorganism being attached on slide glass in regeneration culture medium, forms new bacteria suspension.
Four, the screening of excellent efficient accumulation cordycepin cordyceps militaris link bacterial strain
1, the new bacteria suspension for obtaining step 3 dilutes 1000 times, and 100-300 μ L dilution is taken to be applied to regeneration culture
Base plate is placed in culture in incubator.
2, the mutant strain of the fast growing on the plate of step 1 is inoculated into 96 equipped with 1mL PDA liquid medium
Hole microwell plate shaken cultivation, cultivation temperature are 24-28 DEG C, revolving speed 150-200rpm, cultivate 2 days, mycelium is collected by centrifugation, examine
Survey mycelium cordycepin content.
Statistics obtains 4562 plant mutant bacterial strains, and the direct mutation bacterial strain that wherein cordycepin content improves is 311 plants, and cordycepin contains
The negative mutant strain that amount reduces is 4251 plants.Compared with starting strain, 300 plants of cordycepin content in direct mutation bacterial strain is improved
Amplitude is 150%-500%, and 11 plants of cordycepin content increase rate is 500% or more.
3, the direct mutation bacterial strain (11 plants) that cordycepin content significantly improves in collection step 2, continues shaking flask secondary screening,
250ml shaking flask, liquid amount 50ml, condition of culture are 24-28 DEG C, 150-200rpm, cultivate 2 days, collect mycelium, detect its life
Object amount (mycelium weight in wet base), cordycepin content.
In 11 plants of direct mutation bacterial strains, 10 plants of cordycepin content is " 2.5g/kg mycelium dry weight " hereinafter, other 1 plant of bacterium
The cordycepin content of (i.e. the bacterial strain of number FY1424) reaches " 42.3g/kg mycelium dry weight ".The bacterial strain of number FY1424 is ordered
Entitled cordyceps FY1421 carries out inclined-plane preservation and glycerol conservation.
Five, the preservation of cordyceps FY1421
Cordyceps FY1421 (Cordyceps militaris FY1421) is preserved in Chinese allusion quotation on April 21st, 2016
Type culture collection (abbreviation CCTCC；Address: Wuhan, China, Wuhan University；Postcode: 430072), deposit number is
CCTCC NO:M2016220.Cordyceps FY1421 (Cordyceps mi1itaria FY1421) CCTCCNO:M2016220
Referred to as cordyceps FY1421.
Six, the genetic stability of cordyceps FY1421
Cordyceps FY1421 is subjected to secondary culture to investigate its genetic stability, passage in every 3 days is primary, passage 15
In generation, carries out the cordycepin content of shake flask fermentation measurement bacterial strain every a generation, as the result is shown in cordyceps FY1421 succeeding generations
Ferment cordyceps sinensis cellulose content has good genetic stability without significant change.
The fermentation application of embodiment 2, cordyceps FY1421
One, the seed hot housing of cordyceps FY1421
1, cordyceps FY1421 is seeded to PDA solid slope culture medium, 24-28 DEG C culture 5-7 days.
2, the conidium in the cultured PDA solid slope culture medium of step 1 is eluted with sterile physiological saline, is turned
It is connected to PDA solid slope culture medium, is protected from light culture 3-5 days for 24-28 DEG C in incubator.
3, the conidium in the cultured PDA solid slope culture medium of step 2 is washed with 10ml sterile physiological saline
It is de-, obtain bacteria suspension.
4, by bacterial suspension inoculation that 10ml step 3 obtains into the eggplant bottle that 100g seed enriched medium is housed (pupa worm
Initial concentration of the careless bacterium FY1421 in cultivating system is 104A/ml), it is cultivated 4 days for 26 DEG C in incubator, strengthened kind
Sub- culture (spore concentration 1012A/gram culture).
Two, cordyceps FY1421 liquid deep layer fermenting culture
The reinforcing inoculum for taking 100g step 1 to prepare is inoculated in the fermentation of the 50L containing 35L liquid fermentation medium
It is cultivated in tank.
The liquid fermentation medium that test group first uses is liquid fermentation medium first.
The liquid fermentation medium that test group second uses is liquid fermentation medium second.
The liquid fermentation medium that test group third uses is liquid fermentation medium third.
The incubation (fermentation time be 96 hours) of test group first: 0-24 hours being dark culturing, 25-96 hours
For illumination cultivation (intensity of illumination: 300lux)；Concentration of reduced sugar in real-time monitoring cultivating system, 0-92 hours, by adding
Enter the concentration of reduced sugar in 70% glucose solution control cultivating system in 0.8-1.5g/100mL (whenever in cultivating system
Concentration of reduced sugar when being lower than 0.8g/100mL, the glucose solution for being added 70% makes concentration of reduced sugar in cultivating system
Reach 1.5g/100mL)；To fermentation ends, 70% glucose solution is not added within 93rd hour.When fermentation ends, culture
Concentration of reduced sugar in system is 0.1g/100ml.Cultivate global parameters are as follows: fermentor ventilatory capacity is 20L/min, mixing control
For 200rpm, temperature is controlled at 25 DEG C, and control dissolved oxygen (DO) is 35%.
The incubation (fermentation time is 120 hours) of test group second: 0-24 hours are dark culturing, and 25-120 is small
When be illumination cultivation (intensity of illumination: 200lux)；Concentration of reduced sugar in real-time monitoring cultivating system 0-115 hours, passes through
The concentration of reduced sugar in 70% glucose solution control cultivating system is added in 0.8-1.5g/100mL (whenever cultivating system
In concentration of reduced sugar be lower than 0.8g/100mL when, the glucose solution for being added 70% makes the reduced sugar in cultivating system dense
Degree reaches 1.5g/100mL)；To fermentation ends, 70% glucose solution is not added within 116th hour.When fermentation ends,
Concentration of reduced sugar in cultivating system is 0.01g/100ml.Cultivate global parameters are as follows: fermentor ventilatory capacity is 15L/min, stirring
Control is 150rpm, and temperature is controlled at 24 DEG C, and control dissolved oxygen (DO) is 25%.
The incubation (fermentation time be 96 hours) of test group third: 0-24 hours being dark culturing, 25-96 hours
For illumination cultivation (intensity of illumination: 500lux)；Concentration of reduced sugar in real-time monitoring cultivating system, 0-92 hours, by adding
Enter the concentration of reduced sugar in 70% glucose solution control cultivating system in 0.8-1.5g/100mL (whenever in cultivating system
Concentration of reduced sugar when being lower than 0.8g/100mL, the glucose solution for being added 70% makes concentration of reduced sugar in cultivating system
Reach 1.5g/100mL)；To fermentation ends, 70% glucose solution is not added within 93rd hour.When fermentation ends, culture
Concentration of reduced sugar in system is 0.045g/100ml.Cultivate global parameters are as follows: fermentor ventilatory capacity is 17.5L/min, stirring
Control is 175rpm, and temperature is controlled at 28 DEG C, and control dissolved oxygen (DO) is 45%.
Three, the mycelial separation of cordyceps FY1421 fermentation culture medium
After completing step 2, it is rounded cultivating system, mycelium is collected and is washed with water and (30min is centrifuged by 3000rpm
Mode collect mycelium).
The mycelium weight in wet base yield about 80% (i.e. 80g/100ml) of test group first, dry weight yield are about 8.0% (i.e. 8g/
100ml)。
The mycelium weight in wet base yield about 50% (i.e. 50g/100ml) of test group second, dry weight yield are about 4.0% (i.e. 4g/
100ml)。
The mycelium weight in wet base yield about 65% (i.e. 65g/100ml) of test group third, dry weight yield are about 6.0% (i.e. 6g/
100ml)。
The mycelium that test group first obtains is most.
Four, the mycelial preparation of control strain
Replace cordyceps FY1421 using the condition of test group first according to Step 1: two, three cordyceps militaris link bacterial strain 50383
It is operated, is collected 50383 mycelium of cordyceps militaris link bacterial strain (comparison liquid bacterial strain mycelium).
Five, the measurement of cordycepin content
The cordyceps militaris link bacterial strain that the cordyceps FY1421 mycelium of test group first and step 4 will be used to obtain in step 3
50383 mycelial cordycepin contents are measured.
Testing result are as follows: the cordycepin content in cordyceps FY1421 mycelium reaches 42.3g/kg mycelium dry weight.
Cordycepin content in 50383 mycelium of cordyceps militaris link bacterial strain reaches 0.25g/kg mycelium dry weight.
The result shows that: on the basis of seed hot housing, fermentation period significantly shortens cordyceps FY1421, synthesis
It is significantly increased with the ability of accumulation cordycepin, has excellent production cordycepin and the mycelial ability of worm summer herb with high-content of cordycepin.
Claims (7)
1. cordyceps FY1421(Cordyceps militaris FY1421), deposit number is CCTCC NO:
M2016220。
2. application of the cordyceps FY1421 described in claim 1 in production cordycepin.
3. the method for cordyceps FY1421 described in culture claim 1 a kind of, includes the following steps: using liquid fermentation and culture
Cordyceps FY1421 described in base culture；
The liquid fermentation medium is made of solute and solvent；The solute and its dense in the liquid fermentation medium
It spends as follows: glucose 2-4g/100ml, peptone 1-2g/100ml, yeast powder 1-2g/100ml, corn flour 5-6g/100ml, beans
Cake powder 1-3g/100ml, wheat flour 0.4-0.6g/100ml, potassium dihydrogen phosphate 0.4-0.6g/100ml, magnesium sulfate 0.2-0.4g/
100ml, vitamin B11-3mg/100ml, vitamin B20.5-1.5mg/100ml, glycine 0.2-0.4mg/100ml, Soviet Union's ammonia
Sour 0.2-0.3mg/100ml, valine 0.1-0.2mg/100ml, leucine 0.04-0.06mg/100ml, isoleucine 0.05-
0.15mg/100ml, phenylalanine 0.2-0.3mg/100ml, serine 0.05-0.15mg/100ml, proline 0.04-
0.06mg/100ml, hydroxyproline 0.02-0.04mg/100ml, cysteine 0.01-0.03mg/100ml, tryptophan 0.02-
0.03mg/100ml, methionine 0.04-0.06mg/100ml, lysine 0.05-0.15mg/100ml, glutamic acid 0.04-
0.06mg/100ml；The solvent is water.
4. the method for cordyceps FY1421 described in culture claim 1 a kind of, in turn include the following steps (a) and step (b):
Step (a): seed enriched medium culture cordyceps FY1421 is used；
Step (b): the product of step (a) is seeded to liquid fermentation medium and is cultivated；
The raw material proportioning of the seed enriched medium are as follows: 1L nutrient solution: 0.5-1.5kg millet；The nutrient solution by solute and
Solvent composition；The solute and its concentration in the nutrient solution are as follows: potato leaches powder 1-3g/100ml, glucose
0.5-1.5g/100ml, peptone 0.4-0.8g/100ml, potassium dihydrogen phosphate 0.05-0.15g/100ml, magnesium sulfate 0.04-
0.06g/100ml, vitamin B11-3mg/100ml, vitamin B20.5-1.5mg/100ml, glycine 0.2-0.4mg/
It is 100ml, threonine 0.2-0.3mg/100ml, valine 0.1-0.2mg/100ml, leucine 0.04-0.06mg/100ml, different
Leucine 0.05-0.15mg/100ml, phenylalanine 0.2-0.3mg/100ml, serine 0.05-0.15mg/100ml, dried meat ammonia
Sour 0.04-0.06mg/100ml, hydroxyproline 0.02-0.04mg/100ml, cysteine 0.01-0.03mg/100ml, color ammonia
Sour 0.02-0.03mg/100ml, methionine 0.04-0.06mg/100ml, lysine 0.05-0.15mg/100ml, glutamic acid
0.04-0.06mg/100ml；The solvent is water；
The liquid fermentation medium is made of solute and solvent；The solute and its dense in the liquid fermentation medium
It spends as follows: glucose 2-4g/100ml, peptone 1-2g/100ml, yeast powder 1-2g/100ml, corn flour 5-6g/100ml, beans
Cake powder 1-3g/100ml, wheat flour 0.4-0.6g/100ml, potassium dihydrogen phosphate 0.4-0.6g/100ml, magnesium sulfate 0.2-0.4g/
100ml, vitamin B11-3mg/100ml, vitamin B20.5-1.5mg/100ml, glycine 0.2-0.4mg/100ml, Soviet Union's ammonia
Sour 0.2-0.3mg/100ml, valine 0.1-0.2mg/100ml, leucine 0.04-0.06mg/100ml, isoleucine 0.05-
0.15mg/100ml, phenylalanine 0.2-0.3mg/100ml, serine 0.05-0.15mg/100ml, proline 0.04-
0.06mg/100ml, hydroxyproline 0.02-0.04mg/100ml, cysteine 0.01-0.03mg/100ml, tryptophan 0.02-
0.03mg/100ml, methionine 0.04-0.06mg/100ml, lysine 0.05-0.15mg/100ml, glutamic acid 0.04-
0.06mg/100ml；The solvent is water.
5. a kind of method for producing cordycepin includes the following steps: to cultivate cordyceps FY1421 described in claim 1.
6. method as claimed in claim 5, it is characterised in that: using the hair of liquid described in claim 3 in the method
Cordyceps FY1421 described in ferment culture medium culture.
7. method as claimed in claim 5, it is characterised in that: the method in turn includes the following steps (a) and step (b):
Step (a): using the seed enriched medium culture cordyceps FY1421 described in claim 4；
Step (b): the product of step (a) is seeded to liquid fermentation medium described in claim 4 and is cultivated.
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