CN107151632B - Hirsutella hepiali Chen et Shen filament and its fermentation technique - Google Patents
Hirsutella hepiali Chen et Shen filament and its fermentation technique Download PDFInfo
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Abstract
The invention discloses a kind of Hirsutella hepiali Chen et Shen filament and its fermentation techniques, the invention Hirsutella hepiali Chen et Shen filament and its fermentation technique, it is the unique cordyceps mycelia fermentation technique in current Tibet, in the case of 3600 meters of height above sea level, growth bad border with wild Chinese caterpillar fungus is closest, to make various effects of product be more nearly wild Chinese caterpillar fungus.The ferment cordyceps sinensis D-mannitol advantage fairly obvious in everyways such as active ingredient, content of beary metal, produces huge Social benefit and economic benefit compared with wild Chinese caterpillar fungus.The fermentation technique includes the following steps：(1) it is inoculated with；(2) first class seed pot culture；(3) secondary seed tank culture；(4) fermentation tank culture；(5) it centrifuges；(6) it dries, crushes.
Description
Technical field
The present invention relates to technical field of microbial fermentation, specifically, be related to a kind of Hirsutella hepiali Chen et Shen filament and its
Fermentation technique.
Background technology
Cordyceps sinensis (Cordyceps (Berk) Sacco.) is stroma and its host cordyceps sinensis bat of aweto
The mushroom seat of the complex of the larva corpses shells such as moth (HepialusarmoricanusOberthur), cordyceps sinensis is ergot
Section (Clavicipiraceae) Cordyceps (Cordyceps).Cordyceps sinensis mainly originates in Qinghai in China, Tibet, Yunnan, expensive
It is the rare traditional Chinese medicine of special product of China in meadow soil in the mountains height above sea level 3500-5000m such as state, Sichuan.Cordyceps sinensis passes to west by China
It is square that oneself has 270 years history.Cordyceps sinensis scientific name is set to Cordyceps sinensis by Italy scholar Saccardo within 1878
(Berkeley) Sacc means Chinese cordyceps sinensis.Its is sweet in flavor mild-natured, with all herbal medicine.Primary efficacy can protect lung kidney, mend marrow, change
Phlegm stops phthisical cough.For deficiency of both the lung and kidney disease, treatment pulmonary tuberculosis, old man's chilly, cough, weak, puerpera and old man's anaemia, chronic nephritis
Deng.The often feeding cordyceps summer may additionally facilitate digestion, adjust immune function, resistance of the enhancing human body to a variety of diseases.
There are some to think that cordyceps sinensis is exactly parasitic by Cordyceps sinensis fungus and can generate the fungus of stroma both at home and abroad
Combination common name.However the cordyceps sinensis specified by the traditional traditional Chinese medicine and pharmacy in China and most of scholar is distribution
Alpine meadow in China Yunnan-Guizhou Plateau, Qinghai-Tibet Platean and border district, by Hirsutella sinensis fungi autoeciousness in cordyceps sinensis bat
Moth insect larvae is formed by complex later, and other kinds of entomogenous fungi complex can only be named " cordyceps sinensis ".
Modern medicine believes that, active material of the cordyceps sinensis because of physiology and pharmacology containing there are many, in clinical application research
Have effects that many-sided.The Chinese caterpillar fungus hypha that fortunately artificial eye-liquid fermented and cultured obtains is studied by pharmacology, toxicity, plant,
Prove that its pharmacological action and chemical composition and natural cs are essentially identical, the substitute of clinical application proof cordyceps sinensis for many years
Can be aweto mycelium, the Pharmacopoeia of People's Republic of China of version in 2000 has this specific regulation.
Invention content
The technical issues of solution：
The object of the present invention is to provide a kind of Hirsutella hepiali Chen et Shen filament and its fermentation technique, 3600 meters of height above sea level in Tibet
In the case of, the growth bad border with wild Chinese caterpillar fungus is closest, to make various effects of product be more nearly wild Chinese caterpillar fungus, with open country
The infested grass advantage fairly obvious in everyways such as active ingredient, content of beary metal compared to ferment cordyceps sinensis D-mannitol.
Technical solution：
A kind of fermentation technique of Hirsutella hepiali Chen et Shen filament, includes the following steps：
(1) it is inoculated with；
(2) first class seed pot culture；
(4) secondary seed tank culture；
(4) fermentation tank culture；
(5) it centrifuges；
(6) it dries, crushes.
A kind of fermentation technique of Hirsutella hepiali Chen et Shen filament, includes the following steps：
(1) it is inoculated with：It is packed into seed culture medium in triangular flask, sterilizes, is cooled to room temperature, adjusts pH value to 5.5-6.5, then
Hirsutella hepiali Chen et Shen kind is seeded in the triangular flask equipped with seed culture medium, the constant temperature that temperature is 12-20 DEG C is then placed in
It is 20-30wt% to be cultivated to mycelial concentration with 130-170 revs/min on shaking table, obtains triangular flask zymotic fluid；
(2) first class seed pot culture：First class seed pot culture medium is packed into stirred-tank fermenter, sterilizing is cooled to room
Temperature adjusts pH value and is added in stirred-tank fermenter to 5.5-6.5, then by triangular flask zymotic fluid, is 12-20 DEG C in temperature, stirring
Speed is 130-170 revs/min, ventilatory capacity 30-50L/min, and it is 0.03-0.1MPa, incubation time 10-15 to keep tank pressure
It, obtains first class seed pot zymotic fluid；
(5) secondary seed tank culture：Secondary seed tank culture medium is packed into stirred-tank fermenter, sterilizing is cooled to room
Temperature adjusts pH value and is added in stirred-tank fermenter to 5.5-6.5, then by first class seed pot zymotic fluid, is 12-20 DEG C in temperature,
Mixing speed is 130-170 revs/min, ventilatory capacity 30-50L/min, and it is 0.03-0.1MPa, incubation time 10- to keep tank pressure
15 days, obtain secondary seed tank zymotic fluid；
(4) fermentation tank culture：Fluid nutrient medium is fitted into fermentation tank, sterilizes, is cooled to room temperature, adjusts pH value to 5.5-
6.5, then secondary seed tank zymotic fluid is added in fermentation tank, it is 12-20 DEG C in temperature, mixing speed is 130-170 revs/min,
Ventilatory capacity is 30-50L/min, and it is 0.03-0.1MPa to keep tank pressure, and incubation time is 10-15 days, obtains ferment tank liquid；
(5) it centrifuges：Ferment tank liquid is centrifuged, supernatant is abandoned, obtains sediment；
(6) sediment is dried, crushed to obtain the final product.
A kind of fermentation technique of Hirsutella hepiali Chen et Shen filament, includes the following steps：
(1) it is inoculated with：400-600mL seed culture mediums are packed into capacity is 1000mL triangular flasks, are 120-125 in temperature
Sterilize 25-35min at DEG C, is cooled to room temperature, and adjusts pH value to 5.5-6.5, then Hirsutella hepiali Chen et Shen kind is seeded in and is equipped with
The capacity of 400-600mL seed culture mediums be in 1000mL triangular flasks be then placed in temperature be on 12-20 DEG C of constant-temperature table with
130-170 revs/min of culture to mycelial concentration is 20-30wt%, obtains triangular flask zymotic fluid；
(2) first class seed pot culture：8-12L first class seed pot cultures are packed into the stirred-tank fermenter that capacity is 20L
Base, sterilize 25-35min at being 120-125 DEG C in temperature, is cooled to room temperature, adjusts pH value and sent out to 5.5-6.5, then by triangular flask
Zymotic fluid is added in the stirred-tank fermenter that capacity is 20L, is 12-20 DEG C in temperature, mixing speed is 130-170 revs/min, ventilation
Amount is 30-50L/min, and it is 0.03-0.1MPa to keep tank pressure, and incubation time is 10-15 days, obtains first class seed pot zymotic fluid；
(6) secondary seed tank culture：The training of 80-120L secondary seed tanks is packed into the stirred-tank fermenter that capacity is 200L
Base is supported, sterilizes 25-35min, is cooled to room temperature at being 120-125 DEG C in temperature, adjust pH value to 5.5-6.5, then by level-one kind
Sub- tank zymotic fluid is added in the stirred-tank fermenter that capacity is 200L, is 12-20 DEG C in temperature, mixing speed be 130-170 turn/
Point, ventilatory capacity 30-50L/min, it is 0.03-0.1MPa to keep tank pressure, and incubation time is 10-15 days, obtains secondary seed tank
Zymotic fluid；
(4) fermentation tank culture：1000-1500L fluid nutrient mediums are fitted into 2000L fermentation tanks, are 120-125 in temperature
Sterilize 25-35min at DEG C, is cooled to room temperature, and adjusts pH value to 5.5-6.5, then is by secondary seed tank zymotic fluid addition capacity
It it is 12-20 DEG C in temperature, mixing speed is 130-170 revs/min, ventilatory capacity 30-50L/min, is kept in 2000L fermentation tanks
Tank pressure is 0.03-0.1MPa, and incubation time is 10-15 days, obtains ferment tank liquid；
(5) it centrifuges：Ferment tank liquid is centrifuged, supernatant is abandoned, obtains sediment；
(6) sediment is dried, crushed to obtain the final product.
The drying is to be dried 24-72 hours in the case where temperature is 70-90 DEG C, vacuum degree is 110-150Pa.
The seed culture medium, first class seed pot culture medium, secondary seed tank culture medium, fluid nutrient medium are by following heavy
Amount part raw material is prepared：Glucose, peptone, corn flour, potassium dihydrogen phosphate, magnesium sulfate, yeast extract, antifoaming agent, soya-bean oil,
Water.
The seed culture medium, first class seed pot culture medium, secondary seed tank culture medium, fluid nutrient medium are by following heavy
Amount part raw material is prepared：1-4 parts of glucose, 1-4 parts of peptone, 1-4 parts of corn flour, 0.1-1.0 parts of potassium dihydrogen phosphate, sulfuric acid
0.01-0.1 parts of magnesium, 0.5-2.5 parts of yeast extract, 0.1-0.5 parts of antifoaming agent, 0.5-2.5 parts of soya-bean oil, 950-1050 parts of water.
Further, the seed culture medium, first class seed pot culture medium, secondary seed tank culture medium, fluid nutrient medium
It is prepared by following weight parts raw material：1-4 parts of glucose, 0.2-2 parts of mulberry-leaf extract, 1-4 parts of peptone, corn flour 1-
4 parts, 0.1-1.0 parts of potassium dihydrogen phosphate, 0.01-0.1 parts of magnesium sulfate, 0.5-2.5 parts of yeast extract, 0.1-0.5 parts of antifoaming agent, soya-bean oil
0.5-2.5 parts, 0.2-0.6 parts of polysaccharide, 950-1050 parts of water.
The preparation method of the mulberry-leaf extract is：Mulberry leaf are crushed, 10-20 mesh sieve is crossed, obtains Mulberry Leaf；By mulberry leaf
Powder and mass fraction are the ethanol water of 40-60% with solid-liquid mass ratio 1：(18-22) is added to three necks after mixing
In flask, in 55-65 DEG C of water-bath reflux extraction 1.5-2.5 hour, reflux extraction while with rotating speed for 50-70 revs/min into
Row stirring, centrifugation, take supernatant vacuum degree 0.05-0.1MPa, temperature be 65-75 DEG C at be concentrated under reduced pressure, be concentrated into
Density is 1.02-1.12g/mL (25 DEG C), obtains mulberry-leaf extract.
Further, the preparation method of the mulberry-leaf extract is：Mulberry leaf are crushed, 10-20 mesh sieve is crossed, obtains mulberry leaf powder
End；By Mulberry Leaf and water with solid-liquid mass ratio 1：(18-22) is uniformly mixed；In ultrasonic power 100-200W, supersonic frequency 35-
In 55-65 DEG C of ultrasonic extraction 1.5-2.5 hours under conditions of 45kHz, centrifugation obtains supernatant A and lower sediment；By lower layer
The ethanol water that the mass fraction of 18-22 times of precipitation and Mulberry Leaf weight is 40-60% is added to three necks after mixing
In flask, in 55-65 DEG C of water-bath reflux extraction 1.5-2.5 hour, reflux extraction while with rotating speed for 50-70 revs/min into
Row stirring, centrifugation obtain supernatant B；By supernatant A and supernatant B be uniformly mixed vacuum degree 0.05-0.1MPa,
Temperature is to be concentrated under reduced pressure at 65-75 DEG C, and it is 1.02-1.12g/mL (25 DEG C) to be concentrated into density, obtains mulberry-leaf extract.
The polysaccharide is or mixtures thereof one kind in Blackfungus polyhexose, lentinan.In an embodiment of the invention,
The polysaccharide is made of 65-75wt% Blackfungus polyhexoses and 25-35wt% lentinans.
The present invention also provides a kind of Hirsutella hepiali Chen et Shen filaments, are made using the above method.
Technique effect：
Hirsutella hepiali Chen et Shen filament and its fermentation technique of the present invention are the unique cordyceps mycelia fermentation skills in current Tibet
Art, in the case of 3600 meters of height above sea level, the growth bad border with wild Chinese caterpillar fungus is closest, to make various effects more adjunction of product
Nearly wild Chinese caterpillar fungus.Ferment cordyceps sinensis D-mannitol is clearly demarcated in the everyways such as active ingredient, content of beary metal ten compared with wild Chinese caterpillar fungus
Aobvious advantage produces huge Social benefit and economic benefit.
Specific implementation mode
Cordycepin and Determination of Adenosine：With reference to Guo Tao, Zhang Long, Wang Bing, Shen Hong《Different culture media is cultivated in Cordyceps militaris
Cordycepin and Determination of Adenosine》It is tested.
Cordyceps sinensis polysaccharide assay：With reference to Xu Feng, Wu Lingfang, the kind, Wang Hongyan of woods etc.《Worm in fermented cordyceps sinensis mycelia
The detection of grass polysaccharide content and Structural Identification》It is tested.
Glucose is the powdered glucose that the sources Shandong Zibo Yu Feng sugaring Co., Ltd provides in embodiment.
Peptone provides pharmaceutical grade peptone for Fujian Xianyou three and bio tech ltd in embodiment.
Magnesium sulfate is the epsom salt that Lianyungang of Jiangsu peristoma Mei Ye Co., Ltds provide in embodiment.
Potassium dihydrogen phosphate is provided by Chengdu Lan Jian Chemical Co., Ltd.s in embodiment.
Antifoaming agent provides SXP-106 organic silicon defoamers for Suzhou City China Resources chemical industry Co., Ltd in embodiment.
Soya-bean oil is the golden dragonfish soybean oil that Zhejiang benefit Hai Jiali food industry Co., Ltd provides in embodiment.
Yeast extract is the yeast powder that Zhejiang east is provided at pharmaceutcal corporation, Ltd in embodiment (fermentation medium is special).
Corn flour is the cornstarch that Binzhou Jin Hui corns development corporation, Ltd. provides in embodiment.
Mulberry leaf are the frosted mulberry leaf that Bozhou Bai Chuan pharmaceutcal corporation, Ltds provide in embodiment.
It is 30wt%'s that Blackfungus polyhexose, which is the content that Hangzhou Zhong Zhikang mushrooms Bioisystech Co., Ltd provides, in embodiment
Blackfungus polyhexose.
Lentinan is the perfume (or spice) that the content that Shaanxi Teng Mai biotechnologies Co., Ltd provides is 30wt% in embodiment
Mushroom polysaccharide.
Hirsutella hepiali Chen et Shen, Hirsutella hepiali Chen et Shen.The strain system state food drug surveilance
The Mycophyta health food that management board 2005.5.20 is promulgated is declared and is evaluated in regulation (tentative), and the true of health food is can be used for
One of bacterium strain list.And the whole nation uniquely obtains the Chinese fungus academia expert academicians generally acknowledged real winter worm summer at present
Careless fungi strain.Hirsutella hepiali Chen et Shen is drawn from Microbiological Culture Collection Administrative Center of the Chinese Academy of Sciences in the embodiment of the present invention
Kind.
Embodiment 1
The fermentation technique of Hirsutella hepiali Chen et Shen filament, includes the following steps：
(1) it is inoculated with：500mL seed culture mediums are packed into capacity is 1000mL triangular flasks, are sterilized at being 121 DEG C in temperature
30min is cooled to room temperature, and is adjusted pH value and is seeded in equipped with 500mL seed culture mediums to 6.0, then by Hirsutella hepiali Chen et Shen kind
Capacity be that be then placed in temperature be on 18 DEG C of constant-temperature table with 150 revs/min of cultures to mycelial concentration in 1000mL triangular flasks
For 25wt%, triangular flask zymotic fluid is obtained；
(2) first class seed pot culture：10L first class seed pot culture mediums are packed into the stirred-tank fermenter that capacity is 20L,
Sterilize 30min at being 121 DEG C in temperature, is cooled to room temperature, adjusts pH value to 6.0, then is by triangular flask zymotic fluid addition capacity
It it is 18 DEG C in temperature in the stirred-tank fermenter of 20L, mixing speed is 150 revs/min, and the ventilatory capacity of filtrated air is 40L/
Min, it is 0.08MPa to keep tank pressure, and incubation time is 11 days, obtains first class seed pot zymotic fluid；
(7) secondary seed tank culture：100L secondary seed tank cultures are packed into the stirred-tank fermenter that capacity is 200L
Base, sterilize 30min at being 121 DEG C in temperature, is cooled to room temperature, adjusts pH value and be added to 6.0, then by first class seed pot zymotic fluid
Capacity is in the stirred-tank fermenter of 200L, is 18 DEG C in temperature, mixing speed is 150 revs/min, and the ventilatory capacity of filtrated air is
40L/min, it is 0.06MPa to keep tank pressure, and incubation time is 13 days, obtains secondary seed tank zymotic fluid；
(4) fermentation tank culture：1200L fluid nutrient mediums are fitted into 2000L fermentation tanks, are sterilized at being 121 DEG C in temperature
30min is cooled to room temperature, adjust pH value to 6.0, then by secondary seed tank zymotic fluid be added capacity be 2000L fermentation tanks in,
Temperature is 18 DEG C, and mixing speed is 150 revs/min, and the ventilatory capacity of filtrated air is 40L/min, and it is 0.08MPa, training to keep tank pressure
It is 12 days to support the time, obtains ferment tank liquid；
(5) it centrifuges：Ferment tank liquid is 5000 revs/min with rotating speed and carries out centrifugation 20min, supernatant is abandoned, is sunk
Starch；
(6) sediment is dried 48 hours in the case where temperature is 80 DEG C, vacuum degree is 120Pa, is crushed to 16 mesh up to bat
Moth hair spore mycelium.
The seed culture medium, first class seed pot culture medium, secondary seed tank culture medium, fluid nutrient medium are by following heavy
Amount part raw material is prepared：3 parts of glucose, 2.5 parts of peptone, 3 parts of corn flour, 0.6 part of potassium dihydrogen phosphate, magnesium sulfate 0.04
Part, 1.5 parts of yeast extract, 0.2 part of antifoaming agent, 1.5 parts of soya-bean oil, 1000 parts of distilled water.Each raw material is added to the water and is uniformly mixed i.e.
.
Embodiment 2
The fermentation technique of Hirsutella hepiali Chen et Shen filament, includes the following steps：
(1) it is inoculated with：500mL seed culture mediums are packed into capacity is 1000mL triangular flasks, are sterilized at being 121 DEG C in temperature
30min is cooled to room temperature, and is adjusted pH value and is seeded in equipped with 500mL seed culture mediums to 6.0, then by Hirsutella hepiali Chen et Shen kind
Capacity be that be then placed in temperature be on 18 DEG C of constant-temperature table with 150 revs/min of cultures to mycelial concentration in 1000mL triangular flasks
For 25wt%, triangular flask zymotic fluid is obtained；
(2) first class seed pot culture：10L first class seed pot culture mediums are packed into the stirred-tank fermenter that capacity is 20L,
Sterilize 30min at being 121 DEG C in temperature, is cooled to room temperature, adjusts pH value to 6.0, then is by triangular flask zymotic fluid addition capacity
It it is 18 DEG C in temperature in the stirred-tank fermenter of 20L, mixing speed is 150 revs/min, and the ventilatory capacity of filtrated air is 40L/
Min, it is 0.08MPa to keep tank pressure, and incubation time is 11 days, obtains first class seed pot zymotic fluid；
(8) secondary seed tank culture：100L secondary seed tank cultures are packed into the stirred-tank fermenter that capacity is 200L
Base, sterilize 30min at being 121 DEG C in temperature, is cooled to room temperature, adjusts pH value and be added to 6.0, then by first class seed pot zymotic fluid
Capacity is in the stirred-tank fermenter of 200L, is 18 DEG C in temperature, mixing speed is 150 revs/min, and the ventilatory capacity of filtrated air is
40L/min, it is 0.06MPa to keep tank pressure, and incubation time is 13 days, obtains secondary seed tank zymotic fluid；
(4) fermentation tank culture：1200L fluid nutrient mediums are fitted into 2000L fermentation tanks, are sterilized at being 121 DEG C in temperature
30min is cooled to room temperature, adjust pH value to 6.0, then by secondary seed tank zymotic fluid be added capacity be 2000L fermentation tanks in,
Temperature is 18 DEG C, and mixing speed is 150 revs/min, and the ventilatory capacity of filtrated air is 40L/min, and it is 0.08MPa, training to keep tank pressure
It is 12 days to support the time, obtains ferment tank liquid；
(5) it centrifuges：Ferment tank liquid is 5000 revs/min with rotating speed and carries out centrifugation 20min, supernatant is abandoned, is sunk
Starch；
(6) sediment is dried 48 hours in the case where temperature is 80 DEG C, vacuum degree is 120Pa, is crushed to 16 mesh up to bat
Moth hair spore mycelium.
The seed culture medium, first class seed pot culture medium, secondary seed tank culture medium, fluid nutrient medium are by following heavy
Amount part raw material is prepared：3 parts of glucose, 1 part of mulberry-leaf extract, 2.5 parts of peptone, 3 parts of corn flour, potassium dihydrogen phosphate 0.6
Part, 0.04 part of magnesium sulfate, 1.5 parts of yeast extract, 0.2 part of antifoaming agent, 1.5 parts of soya-bean oil, 0.4 part of Blackfungus polyhexose, distilled water 1000
Part.Each raw material is added to the water and is uniformly mixed to obtain the final product.
The preparation method of the mulberry-leaf extract is：Mulberry leaf are crushed, 16 mesh sieve is crossed, obtains Mulberry Leaf；By mulberry leaf powder
The ethanol water that end is 50% with mass fraction is with solid-liquid mass ratio 1：20 are added in three-neck flask after mixing, 60
Reflux extraction 2 hours in DEG C water-bath, reflux extraction while, is 60 revs/min with rotating speed and is stirred, with rotating speed for 5000 revs/min
Carry out centrifugation 20min, take supernatant vacuum degree 0.06MPa, temperature be 70 DEG C at be concentrated under reduced pressure, being concentrated into density is
1.08g/mL (25 DEG C), obtains mulberry-leaf extract.
Embodiment 3
The fermentation technique of Hirsutella hepiali Chen et Shen filament, includes the following steps：
(1) it is inoculated with：500mL seed culture mediums are packed into capacity is 1000mL triangular flasks, are sterilized at being 121 DEG C in temperature
30min is cooled to room temperature, and is adjusted pH value and is seeded in equipped with 500mL seed culture mediums to 6.0, then by Hirsutella hepiali Chen et Shen kind
Capacity be that be then placed in temperature be on 18 DEG C of constant-temperature table with 150 revs/min of cultures to mycelial concentration in 1000mL triangular flasks
For 25wt%, triangular flask zymotic fluid is obtained；
(2) first class seed pot culture：10L first class seed pot culture mediums are packed into the stirred-tank fermenter that capacity is 20L,
Sterilize 30min at being 121 DEG C in temperature, is cooled to room temperature, adjusts pH value to 6.0, then is by triangular flask zymotic fluid addition capacity
It it is 18 DEG C in temperature in the stirred-tank fermenter of 20L, mixing speed is 150 revs/min, and the ventilatory capacity of filtrated air is 40L/
Min, it is 0.08MPa to keep tank pressure, and incubation time is 11 days, obtains first class seed pot zymotic fluid；
(9) secondary seed tank culture：100L secondary seed tank cultures are packed into the stirred-tank fermenter that capacity is 200L
Base, sterilize 30min at being 121 DEG C in temperature, is cooled to room temperature, adjusts pH value and be added to 6.0, then by first class seed pot zymotic fluid
Capacity is in the stirred-tank fermenter of 200L, is 18 DEG C in temperature, mixing speed is 150 revs/min, and the ventilatory capacity of filtrated air is
40L/min, it is 0.06MPa to keep tank pressure, and incubation time is 13 days, obtains secondary seed tank zymotic fluid；
(4) fermentation tank culture：1200L fluid nutrient mediums are fitted into 2000L fermentation tanks, are sterilized at being 121 DEG C in temperature
30min is cooled to room temperature, adjust pH value to 6.0, then by secondary seed tank zymotic fluid be added capacity be 2000L fermentation tanks in,
Temperature is 18 DEG C, and mixing speed is 150 revs/min, and the ventilatory capacity of filtrated air is 40L/min, and it is 0.08MPa, training to keep tank pressure
It is 12 days to support the time, obtains ferment tank liquid；
(5) it centrifuges：Ferment tank liquid is 5000 revs/min with rotating speed and carries out centrifugation 20min, supernatant is abandoned, is sunk
Starch；
(6) sediment is dried 48 hours in the case where temperature is 80 DEG C, vacuum degree is 120Pa, is crushed to 16 mesh up to bat
Moth hair spore mycelium.
The seed culture medium, first class seed pot culture medium, secondary seed tank culture medium, fluid nutrient medium are by following heavy
Amount part raw material is prepared：3 parts of glucose, 1 part of mulberry-leaf extract, 2.5 parts of peptone, 3 parts of corn flour, potassium dihydrogen phosphate 0.6
Part, 0.04 part of magnesium sulfate, 1.5 parts of yeast extract, 0.2 part of antifoaming agent, 1.5 parts of soya-bean oil, 0.4 part of Blackfungus polyhexose, distilled water 1000
Part.Each raw material is added to the water and is uniformly mixed to obtain the final product.
The preparation method of the mulberry-leaf extract is：Mulberry leaf are crushed, 16 mesh sieve is crossed, obtains Mulberry Leaf；By mulberry leaf powder
End is with water with solid-liquid mass ratio 1：20 are uniformly mixed；In 60 DEG C of ultrasounds under conditions of ultrasonic power 150W, supersonic frequency 40kHz
Extraction 2 hours, with rotating speed be 5000 revs/min carry out centrifugation 20min, take supernatant vacuum degree 0.06MPa, temperature be 70 DEG C
Under be concentrated under reduced pressure, be concentrated into density be 1.08g/mL (25 DEG C), obtain mulberry-leaf extract.
Embodiment 4
The fermentation technique of Hirsutella hepiali Chen et Shen filament, includes the following steps：
(1) it is inoculated with：500mL seed culture mediums are packed into capacity is 1000mL triangular flasks, are sterilized at being 121 DEG C in temperature
30min is cooled to room temperature, and is adjusted pH value and is seeded in equipped with 500mL seed culture mediums to 6.0, then by Hirsutella hepiali Chen et Shen kind
Capacity be that be then placed in temperature be on 18 DEG C of constant-temperature table with 150 revs/min of cultures to mycelial concentration in 1000mL triangular flasks
For 25wt%, triangular flask zymotic fluid is obtained；
(2) first class seed pot culture：10L first class seed pot culture mediums are packed into the stirred-tank fermenter that capacity is 20L,
Sterilize 30min at being 121 DEG C in temperature, is cooled to room temperature, adjusts pH value to 6.0, then is by triangular flask zymotic fluid addition capacity
It it is 18 DEG C in temperature in the stirred-tank fermenter of 20L, mixing speed is 150 revs/min, and the ventilatory capacity of filtrated air is 40L/
Min, it is 0.08MPa to keep tank pressure, and incubation time is 11 days, obtains first class seed pot zymotic fluid；
(10) secondary seed tank culture：100L secondary seed tank cultures are packed into the stirred-tank fermenter that capacity is 200L
Base, sterilize 30min at being 121 DEG C in temperature, is cooled to room temperature, adjusts pH value and be added to 6.0, then by first class seed pot zymotic fluid
Capacity is in the stirred-tank fermenter of 200L, is 18 DEG C in temperature, mixing speed is 150 revs/min, and the ventilatory capacity of filtrated air is
40L/min, it is 0.06MPa to keep tank pressure, and incubation time is 13 days, obtains secondary seed tank zymotic fluid；
(4) fermentation tank culture：1200L fluid nutrient mediums are fitted into 2000L fermentation tanks, are sterilized at being 121 DEG C in temperature
30min is cooled to room temperature, adjust pH value to 6.0, then by secondary seed tank zymotic fluid be added capacity be 2000L fermentation tanks in,
Temperature is 18 DEG C, and mixing speed is 150 revs/min, and the ventilatory capacity of filtrated air is 40L/min, and it is 0.08MPa, training to keep tank pressure
It is 12 days to support the time, obtains ferment tank liquid；
(5) it centrifuges：Ferment tank liquid is 5000 revs/min with rotating speed and carries out centrifugation 20min, supernatant is abandoned, is sunk
Starch；
(6) sediment is dried 48 hours in the case where temperature is 80 DEG C, vacuum degree is 120Pa, is crushed to 16 mesh up to bat
Moth hair spore mycelium.
The seed culture medium, first class seed pot culture medium, secondary seed tank culture medium, fluid nutrient medium are by following heavy
Amount part raw material is prepared：3 parts of glucose, 1 part of mulberry-leaf extract, 2.5 parts of peptone, 3 parts of corn flour, potassium dihydrogen phosphate 0.6
Part, 0.04 part of magnesium sulfate, 1.5 parts of yeast extract, 0.2 part of antifoaming agent, 1.5 parts of soya-bean oil, 0.4 part of Blackfungus polyhexose, distilled water 1000
Part.Each raw material is added to the water and is uniformly mixed to obtain the final product.
The preparation method of the mulberry-leaf extract is：Mulberry leaf are crushed, 16 mesh sieve is crossed, obtains Mulberry Leaf；By mulberry leaf powder
End is with water with solid-liquid mass ratio 1：20 are uniformly mixed；In 60 DEG C of ultrasounds under conditions of ultrasonic power 150W, supersonic frequency 40kHz
Extraction 2 hours is 5000 revs/min with rotating speed and carries out centrifugation 20min, obtains supernatant A and lower sediment；By lower sediment with
The ethanol water that the mass fraction that 20 times of Mulberry Leaf weight is 50% is added in three-neck flask after mixing, at 60 DEG C
In water-bath reflux extraction 2 hours, reflux extraction while with rotating speed be 60 revs/min be stirred, with rotating speed be 5000 revs/min into
Row centrifugation 20min, obtains supernatant B；Supernatant A and supernatant B are uniformly mixed in vacuum degree 0.06MPa, temperature
It is to be concentrated under reduced pressure at 70 DEG C, it is 1.08g/mL (25 DEG C) to be concentrated into density, obtains mulberry-leaf extract.
Embodiment 5
The fermentation technique of Hirsutella hepiali Chen et Shen filament, includes the following steps：
(1) it is inoculated with：500mL seed culture mediums are packed into capacity is 1000mL triangular flasks, are sterilized at being 121 DEG C in temperature
30min is cooled to room temperature, and is adjusted pH value and is seeded in equipped with 500mL seed culture mediums to 6.0, then by Hirsutella hepiali Chen et Shen kind
Capacity be that be then placed in temperature be on 18 DEG C of constant-temperature table with 150 revs/min of cultures to mycelial concentration in 1000mL triangular flasks
For 25wt%, triangular flask zymotic fluid is obtained；
(2) first class seed pot culture：10L first class seed pot culture mediums are packed into the stirred-tank fermenter that capacity is 20L,
Sterilize 30min at being 121 DEG C in temperature, is cooled to room temperature, adjusts pH value to 6.0, then is by triangular flask zymotic fluid addition capacity
It it is 18 DEG C in temperature in the stirred-tank fermenter of 20L, mixing speed is 150 revs/min, and the ventilatory capacity of filtrated air is 40L/
Min, it is 0.08MPa to keep tank pressure, and incubation time is 11 days, obtains first class seed pot zymotic fluid；
(11) secondary seed tank culture：100L secondary seed tank cultures are packed into the stirred-tank fermenter that capacity is 200L
Base, sterilize 30min at being 121 DEG C in temperature, is cooled to room temperature, adjusts pH value and be added to 6.0, then by first class seed pot zymotic fluid
Capacity is in the stirred-tank fermenter of 200L, is 18 DEG C in temperature, mixing speed is 150 revs/min, and the ventilatory capacity of filtrated air is
40L/min, it is 0.06MPa to keep tank pressure, and incubation time is 13 days, obtains secondary seed tank zymotic fluid；
(4) fermentation tank culture：1200L fluid nutrient mediums are fitted into 2000L fermentation tanks, are sterilized at being 121 DEG C in temperature
30min is cooled to room temperature, adjust pH value to 6.0, then by secondary seed tank zymotic fluid be added capacity be 2000L fermentation tanks in,
Temperature is 18 DEG C, and mixing speed is 150 revs/min, and the ventilatory capacity of filtrated air is 40L/min, and it is 0.08MPa, training to keep tank pressure
It is 12 days to support the time, obtains ferment tank liquid；
(5) it centrifuges：Ferment tank liquid is 5000 revs/min with rotating speed and carries out centrifugation 20min, supernatant is abandoned, is sunk
Starch；
(6) sediment is dried 48 hours in the case where temperature is 80 DEG C, vacuum degree is 120Pa, is crushed to 16 mesh up to bat
Moth hair spore mycelium.
The seed culture medium, first class seed pot culture medium, secondary seed tank culture medium, fluid nutrient medium are by following heavy
Amount part raw material is prepared：3 parts of glucose, 1 part of mulberry-leaf extract, 2.5 parts of peptone, 3 parts of corn flour, potassium dihydrogen phosphate 0.6
Part, 0.04 part of magnesium sulfate, 1.5 parts of yeast extract, 0.2 part of antifoaming agent, 1.5 parts of soya-bean oil, 0.4 part of lentinan, 1000 parts of distilled water.
Each raw material is added to the water and is uniformly mixed to obtain the final product.
The preparation method of the mulberry-leaf extract is：Mulberry leaf are crushed, 16 mesh sieve is crossed, obtains Mulberry Leaf；By mulberry leaf powder
End is with water with solid-liquid mass ratio 1：20 are uniformly mixed；In 60 DEG C of ultrasounds under conditions of ultrasonic power 150W, supersonic frequency 40kHz
Extraction 2 hours is 5000 revs/min with rotating speed and carries out centrifugation 20min, obtains supernatant A and lower sediment；By lower sediment with
The ethanol water that the mass fraction that 20 times of Mulberry Leaf weight is 50% is added in three-neck flask after mixing, at 60 DEG C
In water-bath reflux extraction 2 hours, reflux extraction while with rotating speed be 60 revs/min be stirred, with rotating speed be 5000 revs/min into
Row centrifugation 20min, obtains supernatant B；Supernatant A and supernatant B are uniformly mixed in vacuum degree 0.06MPa, temperature
It is to be concentrated under reduced pressure at 70 DEG C, it is 1.08g/mL (25 DEG C) to be concentrated into density, obtains mulberry-leaf extract.
Embodiment 6
The fermentation technique of Hirsutella hepiali Chen et Shen filament, includes the following steps：
(1) it is inoculated with：500mL seed culture mediums are packed into capacity is 1000mL triangular flasks, are sterilized at being 121 DEG C in temperature
30min is cooled to room temperature, and is adjusted pH value and is seeded in equipped with 500mL seed culture mediums to 6.0, then by Hirsutella hepiali Chen et Shen kind
Capacity be that be then placed in temperature be on 18 DEG C of constant-temperature table with 150 revs/min of cultures to mycelial concentration in 1000mL triangular flasks
For 25wt%, triangular flask zymotic fluid is obtained；
(2) first class seed pot culture：10L first class seed pot culture mediums are packed into the stirred-tank fermenter that capacity is 20L,
Sterilize 30min at being 121 DEG C in temperature, is cooled to room temperature, adjusts pH value to 6.0, then is by triangular flask zymotic fluid addition capacity
It it is 18 DEG C in temperature in the stirred-tank fermenter of 20L, mixing speed is 150 revs/min, and the ventilatory capacity of filtrated air is 40L/
Min, it is 0.08MPa to keep tank pressure, and incubation time is 11 days, obtains first class seed pot zymotic fluid；
(12) secondary seed tank culture：100L secondary seed tank cultures are packed into the stirred-tank fermenter that capacity is 200L
Base, sterilize 30min at being 121 DEG C in temperature, is cooled to room temperature, adjusts pH value and be added to 6.0, then by first class seed pot zymotic fluid
Capacity is in the stirred-tank fermenter of 200L, is 18 DEG C in temperature, mixing speed is 150 revs/min, and the ventilatory capacity of filtrated air is
40L/min, it is 0.06MPa to keep tank pressure, and incubation time is 13 days, obtains secondary seed tank zymotic fluid；
(4) fermentation tank culture：1200L fluid nutrient mediums are fitted into 2000L fermentation tanks, are sterilized at being 121 DEG C in temperature
30min is cooled to room temperature, adjust pH value to 6.0, then by secondary seed tank zymotic fluid be added capacity be 2000L fermentation tanks in,
Temperature is 18 DEG C, and mixing speed is 150 revs/min, and the ventilatory capacity of filtrated air is 40L/min, and it is 0.08MPa, training to keep tank pressure
It is 12 days to support the time, obtains ferment tank liquid；
(5) it centrifuges：Ferment tank liquid is 5000 revs/min with rotating speed and carries out centrifugation 20min, supernatant is abandoned, is sunk
Starch；
(6) sediment is dried 48 hours in the case where temperature is 80 DEG C, vacuum degree is 120Pa, is crushed to 16 mesh up to bat
Moth hair spore mycelium.Hirsutella hepiali Chen et Shen filament test result：Cordycepin content is 0.75%, and Cordyceps sinensis polysaccharide content is
27.9%, cordycepic acid content 16.3%, amino acid content 181mg/g, adenosine content 0.78%.
The seed culture medium, first class seed pot culture medium, secondary seed tank culture medium, fluid nutrient medium are by following heavy
Amount part raw material is prepared：3 parts of glucose, 1 part of mulberry-leaf extract, 2.5 parts of peptone, 3 parts of corn flour, potassium dihydrogen phosphate 0.6
Part, 0.04 part of magnesium sulfate, 1.5 parts of yeast extract, 0.2 part of antifoaming agent, 1.5 parts of soya-bean oil, 0.28 part of Blackfungus polyhexose, lentinan
0.12 part, 1000 parts of distilled water.Each raw material is added to the water and is uniformly mixed to obtain the final product.
The preparation method of the mulberry-leaf extract is：Mulberry leaf are crushed, 16 mesh sieve is crossed, obtains Mulberry Leaf；By mulberry leaf powder
End is with water with solid-liquid mass ratio 1：20 are uniformly mixed；In 60 DEG C of ultrasounds under conditions of ultrasonic power 150W, supersonic frequency 40kHz
Extraction 2 hours is 5000 revs/min with rotating speed and carries out centrifugation 20min, obtains supernatant A and lower sediment；By lower sediment with
The ethanol water that the mass fraction that 20 times of Mulberry Leaf weight is 50% is added in three-neck flask after mixing, at 60 DEG C
In water-bath reflux extraction 2 hours, reflux extraction while with rotating speed be 60 revs/min be stirred, with rotating speed be 5000 revs/min into
Row centrifugation 20min, obtains supernatant B；Supernatant A and supernatant B are uniformly mixed in vacuum degree 0.06MPa, temperature
It is to be concentrated under reduced pressure at 70 DEG C, it is 1.08g/mL (25 DEG C) to be concentrated into density, obtains mulberry-leaf extract.
Test case 1
The embodiment 1-5 Hirsutella hepiali Chen et Shen filaments prepared are tested, specific test result is shown in Table 1.
Table 1：Test result table
The present invention is the unique cordyceps mycelia fermentation technique in current Tibet, in the case of 3600 meters of height above sea level, with wild
The growth bad border of cordyceps sinensis is closest, to make various effects of product be more nearly wild Chinese caterpillar fungus.It ferments compared with wild Chinese caterpillar fungus
The cordyceps mycelia advantage fairly obvious in everyways such as active ingredient, content of beary metal, produces huge social benefit
And economic benefit.By a large number of experiments, the optimum growing condition for filtering out Hirsutella hepiali Chen et Shen is investigated, is determining optimum growh item
After part, creative selection Extracts from Plant Recourses investigates type, additive amount and the thalline harvest time of Extracts from Plant Recourses to bat
The influence of bat moth hair spore growth, and orthogonal test is carried out using the optimal level in these three factors, to realize bat moth quilt
The culture of hair spore.
Claims (2)
1. a kind of fermentation process of Hirsutella hepiali Chen et Shen filament, which is characterized in that include the following steps：
(1) it is inoculated with：400-600mL seed culture mediums are packed into capacity is 1000mL triangular flasks, in the case where temperature is 120-125 DEG C
Sterilize 25-35min, is cooled to room temperature, and adjusts pH value and is seeded in equipped with 400- to 5.5-6.5, then by Hirsutella hepiali Chen et Shen kind
The capacity of 600mL seed culture mediums is to be then placed on the constant-temperature table that temperature is 12-20 DEG C with 130- in 1000mL triangular flasks
170 revs/min of cultures to mycelial concentration is 20-30wt%, obtains triangular flask zymotic fluid；
(2) first class seed pot culture：8-12L first class seed pot culture mediums are packed into the stirred-tank fermenter that capacity is 20L,
Temperature is the 25-35min that sterilizes at 120-125 DEG C, is cooled to room temperature, and adjusts pH value and adds to 5.5-6.5, then by triangular flask zymotic fluid
Enter in the stirred-tank fermenter that capacity is 20L, is 12-20 DEG C in temperature, mixing speed is 130-170 revs/min, and ventilatory capacity is
30-50L/min, it is 0.03-0.1MPa to keep tank pressure, and incubation time is 10-15 days, obtains first class seed pot zymotic fluid；
(3) secondary seed tank culture：80-120L secondary seed tank cultures are packed into the stirred-tank fermenter that capacity is 200L
Base sterilizes 25-35min at being 120-125 DEG C in temperature, is cooled to room temperature, adjusts pH value to 5.5-6.5, then by first order seed
Tank zymotic fluid be added capacity be 200L stirred-tank fermenter in, temperature be 12-20 DEG C, mixing speed be 130-170 turn/
Point, ventilatory capacity 30-50L/min, it is 0.03-0.1MPa to keep tank pressure, and incubation time is 10-15 days, obtains secondary seed tank
Zymotic fluid；
(4) fermentation tank culture：1000-1500L fluid nutrient mediums are fitted into 2000L fermentation tanks, in the case where temperature is 120-125 DEG C
Sterilize 25-35min, is cooled to room temperature, and adjusts pH value to 5.5-6.5, then it is 2000L that capacity, which is added, in secondary seed tank zymotic fluid
It it is 12-20 DEG C in temperature in fermentation tank, mixing speed is 130-170 revs/min, ventilatory capacity 30-50L/min, and holding tank pressure is
0.03-0.1MPa, incubation time are 10-15 days, obtain ferment tank liquid；
(5) it centrifuges：Ferment tank liquid is centrifuged, supernatant is abandoned, obtains sediment；
(6) sediment is dried, crushed to obtain the final product；
The seed culture medium, first class seed pot culture medium, secondary seed tank culture medium, fluid nutrient medium are by following weight parts
Raw material is prepared：1-4 parts of glucose, 0.2-2 parts of mulberry-leaf extract, 1-4 parts of peptone, 1-4 parts of corn flour, potassium dihydrogen phosphate
0.1-1.0 parts, 0.01-0.1 parts of magnesium sulfate, 0.5-2.5 parts of yeast extract, 0.1-0.5 parts of antifoaming agent, 0.5-2.5 parts of soya-bean oil, 0.2-
950-1050 parts of 0.6 part of polysaccharide, water；
The preparation method of the mulberry-leaf extract is：Mulberry leaf are crushed, 10-20 mesh sieve is crossed, obtains Mulberry Leaf；By Mulberry Leaf
With water with solid-liquid mass ratio 1：(18-22) is uniformly mixed；Under conditions of ultrasonic power 100-200W, supersonic frequency 35-45kHz
In 55-65 DEG C of ultrasonic extraction 1.5-2.5 hours, centrifugation obtained supernatant A and lower sediment；By lower sediment and mulberry leaf powder
The mass fraction of 18-22 times of last weight is that the ethanol water of 40-60% is added in three-neck flask after mixing, in 55-
Reflux extraction 1.5-2.5 hours in 65 DEG C of water-baths, are 50-70 revs/min with rotating speed while reflux extracts and are stirred, centrifuge,
Obtain supernatant B；It is 65-75 DEG C that supernatant A and supernatant B, which are uniformly mixed in vacuum degree 0.05-0.1MPa, temperature,
Under be concentrated under reduced pressure, it is 1.02-1.12g/mL to be concentrated into density at 25 DEG C, obtains mulberry-leaf extract；
The polysaccharide is or mixtures thereof one kind in Blackfungus polyhexose, lentinan.
2. the fermentation process of Hirsutella hepiali Chen et Shen filament as described in claim 1, it is characterised in that：The drying is in temperature
It is to be dried 24-72 hours under 110-150Pa for 70-90 DEG C, vacuum degree.
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| CN109370914B (en) * | 2018-11-28 | 2021-07-27 | 马兰 | Cordyceps sinensis mycelium powder and production method thereof |
| CN109355204A (en) * | 2018-11-28 | 2019-02-19 | 马兰 | A kind of method of fermenting and producing cordyceps sinensis mycelium powder |
| CN110885759A (en) * | 2019-11-07 | 2020-03-17 | 西藏藏草宜生生物科技有限公司 | Hirsutella hepiali Chen et Shen liquid fermentation method |
| CN112262709A (en) * | 2020-10-16 | 2021-01-26 | 浙江理工大学 | Cordyceps militaris liquid culture medium and preparation method and application thereof |
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