CN107418902A - A kind of mushroom ferment supplemented medium and the continuous cultural method of mushroom deep liquid - Google Patents
A kind of mushroom ferment supplemented medium and the continuous cultural method of mushroom deep liquid Download PDFInfo
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Abstract
The invention belongs to edible fungus liquid deep layer fermenting technical field, and in particular to a kind of supplemented medium of fermenting and producing mushroom and the method for culture of continuously fermenting using supplemented medium progress mushroom deep liquid.The supplemented medium by weight percentage, including 4 6% glucose, 0.5 1% polyprotein peptone, 0.5 1% yeast extract, 0.1 0.3% potassium dihydrogen phosphate, 0.05 0.15% magnesium sulfate, 0.05 0.15% calcium chloride, 0.3 0.5% soya-bean oil, 0.01 0.03% Tween 80,0.05 0.15% acetic acid and 91 93% water.The supplemented medium nutrition general equilibrium, is continuously cultivated for mushroom deep liquid, can effectively extend mushroom exponential phase of growth, improves mushroom Submerged liquid culturation efficiency and biomass, improves lentinan yield.
Description
Technical field
The invention belongs to edible fungus liquid deep layer fermenting technical field, and in particular to a kind of feed supplement of fermenting and producing mushroom
The method that culture medium and application supplemented medium progress mushroom deep liquid are continuously cultivated.
Background technology
Lentinan (Lentinan) (molecular formula：(C6H10O5)n) it is the effective active extracted from quality xianggu fructification
Composition, there is the effects such as immunological regulation, antitumor, anti-infective, antiviral, anti-oxidant, anti-aging, protection liver, mainly should
For medicine and field of health care products.Lentinan can strengthen humoral immunity and cellular immune function, can be one to resisiting influenza virus
The untapped anti influenza health food of kind.
The production of lentinan includes extraction and the bacterium from fermented and cultured from mushroom fruiting body that is natural or manually cultivating
Two ways is extracted in filament and zymotic fluid.Have that the cycle is short, cost is low, yield using liquid fermentation technology production mycelium
Greatly, and there are the advantages such as industrial production prospect, be the main production process of lentinan.
Current existing mushroom liquid fermentation is all the form using batch fermentation.Batch fermentation is also known as batch culture,
Refer to after putting into the nutriment of limited quantity in a closed system, access a small amount of microorganism fungus kind and cultivated, made
Microbial growth, the microbial culture method of a growth cycle is only completed under given conditions.Batch fermentation it is excellent
Point is：Wide in range is required to fermentation condition, without intended distinction types of spawn, similar side can be used to different strain type
Formula is fermented.
Although batch fermentation can effectively obtain shiitake mushroom hypha, and then extract lentinan.But mushroom is as edible true
Bacterium, it compared with simple unicellular bacteria there is period of delay in incubation to grow, and exponential phase of growth increasess slowly what is increased in arithmetic stage
Feature.Therefore, mushroom Liquid batch fermentation big, the adherent growth that easily shows viscosity, does not tolerate the technical problems such as shearing, so as to
Cause mushroom Liquid batch fermentation production efficiency low.
The content of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of mushroom ferment supplemented medium and mushroom
The continuous cultural method of deep liquid.
In order to solve the above-mentioned technical problem, technical scheme is as follows：
The invention provides a kind of mushroom ferment supplemented medium, it is characterised in that by weight percentage, including 4-
6% glucose, 0.5-1% polyprotein peptone, 0.5-1% yeast extract, 0.1-0.3% potassium dihydrogen phosphate, 0.05-
0.15% magnesium sulfate, 0.05-0.15% calcium chloride, 0.3-0.5% soya-bean oil, 0.01-0.03% Tween 80,0.05-
0.15% acetic acid and 91-93% water.
Preferably, described supplemented medium by weight percentage, includes 0.5-1.5% lignin.
Present invention also offers a kind of continuous cultural method of mushroom deep liquid, comprise the following steps：
Mushroom seed liquor, the ferment at constant temperature culture under the conditions of 22-28 DEG C, the fermentation are inoculated with liquid fermentation medium
During control zymotic fluid dissolved oxygen be not less than 25%, pH value be 3.5~4.0；
After fermentation 8-12 days, added using dilution rate as 0.01-0.04/h feed rate into zymotic fluid claim 1 or
Supplemented medium described in 2, continue culture of continuously fermenting.
Preferably, the preparation method of the mushroom seed liquor comprises the following steps：
(1) mushroom spore is inoculated with liquid fermentation medium with spore liquid inocalation method, activation culture obtains work after 8-12 days
Change strain；
(2) activated spawn is inoculated with liquid fermentation medium in the method for flame inoculation, expands numerous culture 4-6 days
Afterwards, mushroom seed liquor is obtained.
Preferably, the liquid fermentation medium by weight percentage, independently includes 4-6% glucose, 0.2-
0.3% polyprotein peptone, 0.2-0.3% yeast extract, 0.1-0.3% potassium dihydrogen phosphate, 0.05-0.15% sulfuric acid
Magnesium, 0.05-0.15% calcium chloride and 93-95% water.
Preferably, filtrated air is passed through in fermentation process into zymotic fluid, the ventilation ratio of the filtrated air is 0.4-
0.6vvm。
Preferably, zymotic fluid is stirred in fermentation process, the initial velocity of stirring is 120-180rpm.
Preferably, the sodium hydroxide solution that the pH mass-volume concentrations of zymotic fluid are 15~25% in the fermentation process
Regulation.
Preferably, the liquid fermentation medium and/or supplemented medium pass through sterilization treatment；The condition of the sterilizing is only
Stand and be：120-122 DEG C of temperature, pressure 103-115kpa；The time of the sterilizing stands alone as 40-60min.
Preferably, the inoculum concentration of the mushroom seed liquor is 0.5-2%.
Beneficial effect：
The invention provides a kind of mushroom ferment supplemented medium, and the supplemented medium nutrition general equilibrium can be effective
Extend mushroom exponential phase of growth, improve mushroom Submerged liquid culturation efficiency and biomass, improve lentinan yield.By the benefit
The deep liquid that material culture medium carries out mushroom is continuously fermented, and can effectively shorten the period of delay of mushroom ferment, extends the life of mushroom index
For a long time to 20 days, mushroom Submerged liquid culturation biomass 72g/L is improved, improves lentinan output to 10.8g/L.
Continuously ferment cultural method present invention also offers a kind of mushroom deep liquid, solve existing mushroom liquid batch
The viscosity easily occurred of fermenting is big, adherent growth, the technical problem for not tolerating shearing.Significantly improve the production effect of shiitake mushroom hypha
Rate, and then improve the yield of lentinan.
Further, the present invention also adds lignin during continuous culture, to improve the resistant to shearing of mushroom mycelium
Power, mushroom growth is stimulated, improve biomass.
The continuously ferment effect of culture of mushroom deep liquid of the present invention refers to table 1：
Table 1：Mushroom deep liquid of the present invention is continuously fermented culture effect table
Embodiment
The invention provides a kind of mushroom ferment supplemented medium, by weight percentage, includes 4-6% grape
Sugar, 0.5-1% polyprotein peptone, 0.5-1% yeast extract, 0.1-0.3% potassium dihydrogen phosphate, 0.05-0.15%
Magnesium sulfate, 0.05-0.15% calcium chloride, 0.3-0.5% soya-bean oil, 0.01-0.03% Tween 80,0.05-0.15%
Acetic acid and 91-93% water.
Preferably, the supplemented medium by weight percentage, including 5% glucose, 0.75% polyprotein
Peptone, 0.75% yeast extract, 0.2% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 0.1% calcium chloride, 0.4% beans
Oil, 0.02% Tween 80,0.1% acetic acid and 92.58% water.
In the present invention, the glucose of supplemented medium 5%, 0.75% polyprotein peptone, 0.75% yeast extract,
0.2% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 0.1% calcium chloride are consistent with fermentation medium to be used to ensure mushroom just
It is frequently grown, soya-bean oil can be used as carbon source or play defoaming effect, and Tween 80 plays the effect of soya-bean oil solubilising, and acetic acid both may be used
So that certain bacteriostasis can also be played as carbon source, ensure supplemented medium in long-time culture of continuous cultivation not by miscellaneous bacteria
Pollution.
The present invention is not particularly limited to the source of above-mentioned each component, conventional commercial.
The supplemented medium nutrition general equilibrium formed is combined by above-mentioned each amount ratio component, can effectively extend the finger of mushroom
Number growth period, and then the continuous harvest of shiitake mushroom hypha is realized, mushroom Submerged liquid culturation efficiency and biomass are improved, is improved fragrant
Mushroom polysaccharide yield.
Present invention also offers a kind of more preferable supplemented medium of effect, by weight percentage, includes 4-6% grape
Sugar, 0.5-1% polyprotein peptone, 0.5-1% yeast extract, 0.1-0.3% potassium dihydrogen phosphate, 0.05-0.15%
Magnesium sulfate, 0.05-0.15% calcium chloride, 0.3-0.5% soya-bean oil, 0.01-0.03% Tween 80,0.05-0.15%
Acetic acid, 0.5-1.5% lignin and 91-93% water.
Preferably, the supplemented medium by weight percentage, including 5% glucose, 0.75% polyprotein
Peptone, 0.75% yeast extract, 0.2% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 0.1% calcium chloride, 0.4% beans
Oil, 0.02% Tween 80,0.1% acetic acid, 1% lignin and 91.58% water.
In the present invention, the effect of the lignin is to stimulate mushroom growth, improves the fingerprinting stress of mushroom mycelium, side
Just the harvesting of shiitake mushroom hypha, mycelium deformation and yield caused by shearing force influences is avoided to decline, so as to further carry
The specific yield of high mushroom mycelium and lentinan.
The present invention is not particularly limited to the source of lignin, conventional commercial.
The present invention is not particularly limited to the preparation method of above-mentioned supplemented medium, using the routine operation of this area, only
Above-mentioned each component can be configured to required mixed aqueous solution to scale, both obtained feed-batch culture of the present invention
Base.
Present invention also offers a kind of continuous cultural method of mushroom deep liquid, comprise the following steps：Trained in liquid fermentation
Support and mushroom seed liquor is inoculated with base, the ferment at constant temperature culture under the conditions of 22-28 DEG C, control the dissolved oxygen of zymotic fluid not in fermentation process
Less than 25%, pH value is 3.5~4.0；It is 0.01-0.04/h feed rate to zymotic fluid using dilution rate after fermentation 8-12 days
Mushroom ferment supplemented medium described in middle addition above-mentioned technical proposal, continue culture of continuously fermenting, after 8-12 days, proceed by
Continuous mycelia harvesting.
The present invention is inoculated with mushroom seed liquor in liquid fermentation medium, carries out the ferment at constant temperature culture of early stage.
In the present invention, the liquid fermentation medium is from the conventional mushroom liquid fermentation medium in this area.
As preferable technical scheme, described liquid fermentation medium by weight percentage, including 4-6% glucose, 0.2-
0.3% polyprotein peptone, 0.2-0.3% yeast extract, 0.1-0.3% potassium dihydrogen phosphate, 0.05-0.15% sulfuric acid
Magnesium, 0.05-0.15% calcium chloride and 93-95% water.It is furthermore preferred that the liquid fermentation medium is by weight percentage,
Including 5% glucose, 0.25% polyprotein peptone, 0.25% yeast extract, 0.2% potassium dihydrogen phosphate, 0.1%
Magnesium sulfate, 0.1% calcium chloride and 94.1% water.Combining the fluid nutrient medium formed by the component of above-mentioned each amount ratio can expire
The nutritional need at sufficient mushroom ferment initial stage, be advantageous to shorten mushroom ferment period of delay.
In the present invention more specifically embodiment, the liquid fermentation medium inoculates mushroom after sterilization treatment
Seed liquor.The temperature of the liquid fermentation medium sterilizing is 120-122 DEG C, preferably 121 DEG C；Pressure is 103-115kpa,
Preferably 110kpa；Sterilization time is 40-60min, preferably 50min.Sterilized under these conditions, will not be to culture medium
Component damage, while can effectively ensure that mushroom inoculation after be not bacterial contamination.
The present invention is not particularly limited to the specific preparation method of the mushroom seed liquor or source, as long as mushroom can be used as
The strain source of fermentation.As preferable technical scheme, the preparation method of the mushroom seed liquor comprises the following steps：
(1) mushroom spore is inoculated with liquid fermentation medium with spore liquid inocalation method, activation culture obtains work after 8-12 days
Change strain；
(2) activated spawn is inoculated with liquid fermentation medium in the method for flame inoculation, after expanding numerous culture 4-6 days, obtained
Mushroom seed liquor.
The present invention is not particularly limited to the source of the mushroom strain, conventional commercial.
In the present invention, the temperature of the activation culture is 22-28 DEG C, preferably 25 DEG C；Controlled during activation culture
The dissolved oxygen amount of zymotic fluid is not less than 25%, preferably not lower than 30%；The pH value of zymotic fluid is 3.5~4.5；Preferably 4.Upper
Activation culture is carried out under the conditions of stating, mushroom can reach exponential phase of growth within the most short time.
Liquid fermentation medium during activation culture is from the conventional mushroom liquid fermentation medium in this area.
As preferable technical scheme, described liquid fermentation medium by weight percentage, including 4-6% glucose, 0.2-
0.3% polyprotein peptone, 0.2-0.3% yeast extract, 0.1-0.3% potassium dihydrogen phosphate, 0.05-0.15% sulfuric acid
Magnesium, 0.05-0.15% calcium chloride and 93-95% water.It is furthermore preferred that the liquid fermentation medium is by weight percentage,
Including 5% glucose, 0.25% polyprotein peptone, 0.25% yeast extract, 0.2% potassium dihydrogen phosphate, 0.1%
Magnesium sulfate, 0.1% calcium chloride and 94.1% water.
In the present invention, the inoculum concentration of the spore liquid inocalation method is 5~15%, preferably 10%.The spore liquid
Spore density is 105Individual/ml.
In the present invention, the activation culture time of mushroom strain is 8~12 days, preferably 10 days.
In the present invention, the temperature for expanding numerous culture is 22-28 DEG C, preferably 25 DEG C of the optimum growth temperature of mushroom；
The dissolved oxygen amount of zymotic fluid is controlled to be not less than 25% during activation culture, preferably not lower than 30% (throughput 0.5vvm, adjusts
Turn over speed 100-500rpm)；The pH value of zymotic fluid is 3.5~4.5；The preferably the most suitable growth pH=4 of mushroom.In above-mentioned condition
Under carry out expanding numerous culture, to ensure that mushroom reaches exponential phase within the shortest time.
The liquid fermentation medium expanded in numerous incubation selects the conventional mushroom liquid fermentation medium in this area i.e.
Can.As preferable technical scheme, described liquid fermentation medium by weight percentage, including 4-6% glucose,
0.2-0.3% polyprotein peptone, 0.2-0.3% yeast extract, 0.1-0.3% potassium dihydrogen phosphate, 0.05-0.15%
Magnesium sulfate, 0.05-0.15% calcium chloride and 93-95% water.It is furthermore preferred that the liquid fermentation medium is by weight percentage
Meter, including 5% glucose, 0.25% polyprotein peptone, 0.25% yeast extract, 0.2% potassium dihydrogen phosphate,
0.1% magnesium sulfate, 0.1% calcium chloride and 94.1% water.
In the present invention, the inoculum concentration of the flame inoculation method is 5~15%, preferably 10%.
In the present invention, the numerous incubation time of the expansion of activated spawn is 4~6 days, preferably 5 days, can make mushroom thalline weight in wet base
Reach 30g/L.
In the present invention, the inoculum concentration for mushroom seed liquor being inoculated with liquid fermentation medium is preferably 0.5-2%, is preferably
1%, the inoculum concentration can effectively shorten the time that mycelia breeding peaks in fermentation tank, make the formation of product advance to, and
The growth machine meeting of miscellaneous bacteria can be reduced, and it is more economical.
After the present invention is inoculated with mushroom seed liquor in liquid fermentation medium, ferment at constant temperature culture is carried out.The constant temperature hair
The condition of ferment culture is as follows：Fermentation temperature is 22-28 DEG C, preferably 25 DEG C；Dissolved oxygen amount is controlled to be not less than 25% in fermentation process,
Preferably not lower than 30%；The pH value of zymotic fluid is 3.5~4.5；Preferably 4.Fermented and cultured under these conditions, can effectively it contract
The fermentation period of delay of short mushroom.
In the present invention, it is preferred to mass-volume concentration is used to control the zymotic fluid for 15~25% sodium hydroxide solution
PH value, more preferably 20% sodium hydroxide solution, to ensure that zymotic fluid pH environment maintains mushroom the most suitable growth pH.
In embodiments of the invention 2, as culture starts, the pH and dissolved oxygen amount in zymotic fluid are as shown in table 2：
Table 2：PH and dissolved oxygen the change table of zymotic fluid
The present invention, into exponential phase of growth, now adds above-mentioned mushroom hair after ferment at constant temperature 8-12 days into zymotic fluid
Ferment supplemented medium, coordinate emissions operation, keep fermentating liquid volume constant, carry out continuous fermented and cultured.
The condition of the culture of continuously fermenting is as follows：Fermentation temperature is 22-28 DEG C, preferably 25 DEG C；Controlled in fermentation process
Dissolved oxygen amount processed is not less than 25%, preferably not lower than 30%；The pH value of zymotic fluid is 3.5~4.5；Preferably 4.In above-mentioned condition
Lower continuous culture, can effectively extend mushroom exponential phase of growth, improve the efficiency and biomass of mushroom deep drainpipe, while improve perfume (or spice)
The yield of mushroom polysaccharide.
In the present invention, the dilution rate of the addition supplemented medium is 0.01-0.04/h, preferably 0.02-0.03/h.
Supplemented medium is persistently added under the speed, can effectively extend the exponential phase of growth of mushroom to 20 days, it is deep to improve mushroom liquid
Layer culture biomass, improves lentinan output.
In the present invention more specifically embodiment, the supplemented medium is then added to zymotic fluid after sterilization treatment
In.The temperature of the supplemented medium sterilizing is 120-122 DEG C, preferably 121 DEG C；Pressure is 103-115kpa, is preferably
110kpa；Sterilization time is 40-60min, preferably 50min.Sterilized under these conditions, will not be to the component of culture medium
Damage, at the same can effectively ensure that supplemented medium add zymotic fluid after, miscellaneous bacteria will not be caused to zymotic fluid.
The present invention proceeds by the harvesting work of continuous mycelia after culture 8-12 days of continuously fermenting.
Mushroom ferment provided by the invention is continuously trained with supplemented medium and mushroom deep liquid with reference to embodiment
Foster method is described in detail, but they can not be interpreted as limiting the scope of the present invention.
Embodiment 1
Liquid fermentation medium weight percentages of components is as follows：Glucose：5%；Polyprotein peptone：0.25%, yeast leaching
Powder：0.25%, potassium dihydrogen phosphate：0.2%, magnesium sulfate：0.1%, calcium chloride：0.1%, remaining is running water.
It is prepared by seed bottle：1000ml liquid fermentation mediums are made by recipe requirements, are dispensed to 500ml conical flask
In, liquid amount 100ml, dispense 10 bottles.Mushroom spore liquid is inoculated into triangular flask with spore liquid inocalation method respectively, control temperature
Spend for 25 DEG C, rotating speed 150rpm, shaken cultivation can be used for 10 days.
The preparation of seeding tank：Using 30L tanks as seeding tank, 20L culture mediums, at 121 DEG C, 110kpa bar are prepared on request
Under part, keep 50min to be sterilized, 25 DEG C are cooled to after sterilizing.By above-mentioned 1000ml seed liquors, connect in the method for flame inoculation
Enter seeding tank, filtrated air, ventilation ratio 0.5vvm are passed through into seeding tank.The rotating speed of seeding tank stirring is adjusted, makes zymotic fluid
Dissolved oxygen be not less than 30%, culture 5 days i.e. switchable fermentation tank.
The preparation of fermentation tank：Using 100L tanks as fermentation tank, 60L culture mediums, at 121 DEG C, 110kpa's are prepared on request
Under the conditions of, keep 50min to be sterilized, 25 DEG C are cooled to after sterilizing.By seed tank culture liquid with 1% inoculum concentration, access hair
Fermentation tank.Filtrated air, ventilation ratio 0.5vvm are passed through into fermentation tank.The rotating speed stirred in adjustment fermentation tank, makes zymotic fluid
Dissolved oxygen is not less than 30%, in incubation with 20% sodium hydroxide control zymotic fluid pH 4.0, cultivating 10 days to enter
Continuously ferment the stage.
Supplemented medium weight percentages of components is as follows：Glucose：5%；Polyprotein peptone：0.75%, yeast extract：
0.75%, potassium dihydrogen phosphate：0.2%, magnesium sulfate：0.1%, calcium chloride：0.1%, soya-bean oil：0.4%, Tween 80：0.02%, vinegar
Acid：0.1%, remaining is running water.
The preparation of feed supplement tank：Using 30L tanks as feed supplement tank, 20L supplemented mediums are prepared by recipe requirements, at 121 DEG C,
Under conditions of 110kpa, keep 50min to be sterilized, 25 DEG C are cooled to after sterilizing.When fermentation tank culture was to 10 days, feed supplement tank
With dilution rate D=0.03h-1Speed start feed supplement, continuous culture starts to harvest mycelia after 10 days.
Embodiment 2
Liquid fermentation medium weight percentages of components is as follows：Glucose：5%；Polyprotein peptone：0.25%, yeast leaching
Powder：0.25%, potassium dihydrogen phosphate：0.2%, magnesium sulfate：0.1%, calcium chloride：0.1%, remaining is running water.
It is prepared by seed bottle：1000ml liquid fermentation mediums are made by recipe requirements, are dispensed to 500ml conical flask
In, liquid amount 100ml, dispense 10 bottles.Mushroom spore liquid is inoculated into triangular flask with spore liquid inocalation method respectively, control temperature
Spend for 25 DEG C, rotating speed 150rpm, shaken cultivation can be used for 8 days.
The preparation of seeding tank：Using 30L tanks as seeding tank, 20L culture mediums, at 121 DEG C, 110kpa bar are prepared on request
Under part, keep 50min to be sterilized, 25 DEG C are cooled to after sterilizing.By above-mentioned 1000ml seed liquors, connect in the method for flame inoculation
Enter seeding tank, filtrated air, ventilation ratio 0.5vvm are passed through into seeding tank.The rotating speed of seeding tank stirring is adjusted, makes zymotic fluid
Dissolved oxygen be not less than 30%, culture 4 days i.e. switchable fermentation tank.
The preparation of fermentation tank：Using 100L tanks as fermentation tank, 60L culture mediums, at 121 DEG C, 110kpa's are prepared on request
Under the conditions of, keep 40min to be sterilized, 22 DEG C are cooled to after sterilizing.By seed tank culture liquid with 0.5% inoculum concentration, access
Fermentation tank.Filtrated air, ventilation ratio 0.4vvm are passed through into fermentation tank.The rotating speed stirred in adjustment fermentation tank, makes zymotic fluid
Dissolved oxygen be not less than 25%, in incubation with 15% sodium hydroxide control zymotic fluid pH 3.5, cultivating 10 days to enter
Enter to continuously ferment the stage.
Supplemented medium weight percentages of components is as follows：Glucose：4%；Polyprotein peptone：0.5%, yeast extract：
0.5%, potassium dihydrogen phosphate：0.1%, magnesium sulfate：0.05%, calcium chloride：0.05%, soya-bean oil：0.3%, Tween 80：0.01%,
Acetic acid：0.05%, remaining is running water.
The preparation of feed supplement tank：Using 30L tanks as feed supplement tank, 20L supplemented mediums are prepared by recipe requirements, at 121 DEG C,
Under conditions of 110kpa, keep 40min to be sterilized, 22 DEG C are cooled to after sterilizing.When fermentation tank culture was to 10 days, feed supplement tank
With dilution rate D=0.01h-1Speed start feed supplement, continuous culture starts to harvest mycelia after 10 days.
Embodiment 3
Liquid fermentation medium weight percentages of components is as follows：Glucose：5%；Polyprotein peptone：0.25%, yeast leaching
Powder：0.25%, potassium dihydrogen phosphate：0.2%, magnesium sulfate：0.1%, calcium chloride：0.1%, remaining is running water.
It is prepared by seed bottle：1000ml liquid fermentation mediums are made by recipe requirements, are dispensed to 500ml conical flask
In, liquid amount 100ml, dispense 10 bottles.Mushroom spore liquid is inoculated into triangular flask with spore liquid inocalation method respectively, control temperature
Spend for 25 DEG C, rotating speed 150rpm, shaken cultivation can be used for 12 days.
The preparation of seeding tank：Using 30L tanks as seeding tank, 20L culture mediums, at 121 DEG C, 110kpa bar are prepared on request
Under part, keep 50min to be sterilized, 25 DEG C are cooled to after sterilizing.By above-mentioned 1000ml seed liquors, connect in the method for flame inoculation
Enter seeding tank, filtrated air, ventilation ratio 0.5vvm are passed through into seeding tank.The rotating speed of seeding tank stirring is adjusted, makes zymotic fluid
Dissolved oxygen be not less than 30%, culture 6 days i.e. switchable fermentation tank.
The preparation of fermentation tank：Using 100L tanks as fermentation tank, 60L culture mediums, at 121 DEG C, 110kpa's are prepared on request
Under the conditions of, keep 50min to be sterilized, 28 DEG C are cooled to after sterilizing.By seed tank culture liquid with 2% inoculum concentration, access hair
Fermentation tank.Filtrated air, ventilation ratio 0.6vvm are passed through into fermentation tank.The rotating speed stirred in adjustment fermentation tank, makes zymotic fluid
Dissolved oxygen is not less than 25%, in incubation with 25% sodium hydroxide control zymotic fluid pH 4.5, cultivating 10 days to enter
Continuously ferment the stage.
Supplemented medium weight percentages of components is as follows：Glucose：6%；Polyprotein peptone：1%, yeast extract：1%,
Potassium dihydrogen phosphate：0.3%, magnesium sulfate：0.15%, calcium chloride：0.15%, soya-bean oil：0.5%, Tween 80：0.03%, acetic acid：
0.15%, remaining is running water.
The preparation of feed supplement tank：Using 30L tanks as feed supplement tank, 20L supplemented mediums are prepared by recipe requirements, at 121 DEG C,
Under conditions of 110kpa, keep 60min to be sterilized, 28 DEG C are cooled to after sterilizing.When fermentation tank culture was to 10 days, feed supplement tank
With dilution rate D=0.04h-1Speed start feed supplement, continuous culture starts to harvest mycelia after 10 days.
Embodiment 4
Liquid fermentation medium weight percentages of components is as follows：Glucose：5%；Polyprotein peptone：0.25%, yeast leaching
Powder：0.25%, potassium dihydrogen phosphate：0.2%, magnesium sulfate：0.1%, calcium chloride：0.1%, remaining is running water.
It is prepared by seed bottle：1000ml liquid fermentation mediums are made by recipe requirements, are dispensed to 500ml conical flask
In, liquid amount 100ml, dispense 10 bottles.Mushroom spore liquid is inoculated into triangular flask with spore liquid inocalation method respectively, control temperature
Spend for 25 DEG C, rotating speed 150rpm, shaken cultivation can be used for 10 days.
The preparation of seeding tank：Using 30L tanks as seeding tank, 20L culture mediums, at 121 DEG C, 110kpa bar are prepared on request
Under part, keep 50min to be sterilized, 25 DEG C are cooled to after sterilizing.By above-mentioned 1000ml seed liquors, connect in the method for flame inoculation
Enter seeding tank, filtrated air, ventilation ratio 0.5vvm are passed through into seeding tank.The rotating speed of seeding tank stirring is adjusted, makes zymotic fluid
Dissolved oxygen be not less than 30%, culture 5 days i.e. switchable fermentation tank.
The preparation of fermentation tank：Using 100L tanks as fermentation tank, 60L culture mediums, at 121 DEG C, 110kpa's are prepared on request
Under the conditions of, keep 50min to be sterilized, 25 DEG C are cooled to after sterilizing.By seed tank culture liquid with 1% inoculum concentration, access hair
Fermentation tank.Filtrated air, ventilation ratio 0.5vvm are passed through into fermentation tank.The rotating speed stirred in adjustment fermentation tank, makes zymotic fluid
Dissolved oxygen is not less than 30%, in incubation with 20% sodium hydroxide control zymotic fluid pH 4.0, cultivating 10 days to enter
Continuously ferment the stage.
Supplemented medium weight percentages of components is as follows：Glucose：5%；Polyprotein peptone：0.75%, yeast extract：
0.75%, potassium dihydrogen phosphate：0.2%, magnesium sulfate：0.1%, calcium chloride：0.1%, soya-bean oil：0.4%, Tween 80：0.02%, vinegar
Acid：0.1%, lignin：1%, remaining is running water.
The preparation of feed supplement tank：Using 30L tanks as feed supplement tank, 20L supplemented mediums are prepared by recipe requirements, at 121 DEG C,
Under conditions of 110kpa, keep 50min to be sterilized, 25 DEG C are cooled to after sterilizing.When fermentation tank culture was to 10 days, feed supplement tank
With dilution rate D=0.03h-1Speed start feed supplement, continuous culture starts to harvest mycelia after 10 days.
Embodiment 5
After harvesting mycelia in the way of embodiment 1-4, the extraction of lentinan is carried out as follows：Weigh perfume (or spice)
Mushroom silk 20g, adding water 400ml, decoct 60 minutes, filtering, filter residue adds water 300ml, decocts 60 minutes, filtering, merging filtrate,
40ml or so (room temperature) is concentrated into, addition ethanol makes alcohol content, and side edged stirs, and stands overnight up to 60%.Filtering, takes precipitation, first
Add the ethanol of 50ml 60% to wash, then add appropriate 95% ethanol washing, 80 DEG C of vacuum drying, obtain 3g lentinans.
Comparative example 1
In addition to the component of supplemented medium is different, other are the same as embodiment 1.
Supplemented medium weight percentages of components is as follows：Glucose：3%；Polyprotein peptone：0.6%, yeast extract：
0.6%, potassium dihydrogen phosphate：0.1%, magnesium sulfate：0.07%, calcium chloride：0.06%, soya-bean oil：0.2%, Tween 80：0.01%,
Acetic acid：0.05%, remaining is running water.
Comparative example 2
In addition to the component of supplemented medium is different, other are the same as embodiment 1.
Supplemented medium weight percentages of components is as follows：Glucose：6%；Polyprotein peptone：0.9%, yeast extract：
0.9%, potassium dihydrogen phosphate：0.4%, magnesium sulfate：0.2%, calcium chloride：0.2%, soya-bean oil：0.6%, Tween 80：0.04%, vinegar
Acid：0.2%, remaining is running water.
Above-described embodiment 1~4 and the methods described of comparative example 1~2 carry out mushroom deep liquid and continuously fermented the process of culture
With result such as table 3：
The culture effect table that continuously ferments of the embodiment 1~4 of table 3 and the methods described of comparative example 1~2
As a result show：Using the supplemented medium described in the claims in the present invention 1, it can effectively extend mushroom exponential growth
Phase, the efficiency and biomass of mushroom deep drainpipe are improved, while improve the yield of lentinan.Mushroom mycelium does not add lignin
Rotating speed upper limit 500rpm, the rotating speed upper limit is improved to 600rpm after adding lignin.Illustrate lignin addition improve it is mycelial
Degeneration-resistant intensity.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
- A kind of 1. mushroom ferment supplemented medium, it is characterised in that by weight percentage, including 4-6% glucose, 0.5-1% polyprotein peptone, 0.5-1% yeast extract, 0.1-0.3% potassium dihydrogen phosphate, 0.05-0.15% sulfuric acid Magnesium, 0.05-0.15% calcium chloride, 0.3-0.5% soya-bean oil, 0.01-0.03% Tween 80,0.05-0.15% acetic acid With 91-93% water.
- 2. supplemented medium according to claim 1, it is characterised in that by weight percentage, in addition to 0.5-1.5% Lignin.
- 3. a kind of continuous cultural method of mushroom deep liquid, it is characterised in that comprise the following steps：Mushroom seed liquor, the ferment at constant temperature culture under the conditions of 22-28 DEG C, the mistake of the fermentation are inoculated with liquid fermentation medium The dissolved oxygen of zymotic fluid is controlled to be not less than 25% in journey, pH value is 3.5~4.0；After fermentation 8-12 days, the institute of claim 1 or 2 is added as 0.01-0.04/h feed rate into zymotic fluid using dilution rate The supplemented medium stated, continue culture of continuously fermenting.
- 4. the continuous cultural method of mushroom deep liquid according to claim 3, it is characterised in that the mushroom seed liquor Preparation method comprises the following steps：(1) mushroom spore is inoculated with liquid fermentation medium with spore liquid inocalation method, activation culture must activate bacterium after 8-12 days Kind；(2) activated spawn is inoculated with liquid fermentation medium in the method for flame inoculation, after expanding numerous culture 4-6 days, obtained Mushroom seed liquor.
- 5. the continuous cultural method of mushroom deep liquid according to claim 3 or 4, it is characterised in that the liquid fermentation Culture medium is by weight percentage, independent to include 4-6% glucose, 0.2-0.3% polyprotein peptone, 0.2-0.3% Yeast extract, 0.1-0.3% potassium dihydrogen phosphate, 0.05-0.15% magnesium sulfate, 0.05-0.15% calcium chloride and 93- 95% water.
- 6. the continuous cultural method of mushroom deep liquid according to claim 3, it is characterised in that to fermentation in fermentation process Filtrated air is passed through in liquid, the ventilation ratio of the filtrated air is 0.4-0.6vvm.
- 7. the continuous cultural method of mushroom deep liquid according to claim 3, it is characterised in that to fermentation in fermentation process Liquid is stirred, and the initial velocity of stirring is 120-180rpm.
- 8. the continuous cultural method of mushroom deep liquid according to claim 3, it is characterised in that sent out in the fermentation process The sodium hydroxide solution that the pH mass-volume concentrations of zymotic fluid are 15~25% is adjusted.
- 9. the continuous cultural method of mushroom deep liquid according to claim 3, it is characterised in that the liquid fermentation and culture Base and/or supplemented medium pass through sterilization treatment；The conditional sampling of the sterilizing is：120-122 DEG C of temperature, pressure 103- 115kpa；The time of the sterilizing stands alone as 40-60min.
- 10. the continuous cultural method of mushroom deep liquid according to claim 3, it is characterised in that the mushroom seed liquor Inoculum concentration be 0.5-2%.
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| CN108624636A (en) * | 2018-05-21 | 2018-10-09 | 湖北创力药业有限公司 | A kind of preparation method of lentinan |
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| CN110283731A (en) * | 2019-07-30 | 2019-09-27 | 河北省微生物研究所 | Mushroom strain and the method that shiitake mushroom hypha is simply prepared using it |
| CN112219640A (en) * | 2020-10-16 | 2021-01-15 | 武汉迪奥药业有限公司 | Continuous preparation culture medium for shiitake mushroom fermentation liquid seeds and preparation method |
| CN114262241A (en) * | 2022-01-18 | 2022-04-01 | 平泉市瀑河源食品有限公司 | Nutrient solution for planting shiitake mushrooms and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108624636A (en) * | 2018-05-21 | 2018-10-09 | 湖北创力药业有限公司 | A kind of preparation method of lentinan |
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| CN112219640A (en) * | 2020-10-16 | 2021-01-15 | 武汉迪奥药业有限公司 | Continuous preparation culture medium for shiitake mushroom fermentation liquid seeds and preparation method |
| CN114262241A (en) * | 2022-01-18 | 2022-04-01 | 平泉市瀑河源食品有限公司 | Nutrient solution for planting shiitake mushrooms and preparation method thereof |
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