CN104404102B - A kind of preparation method of lentinan - Google Patents
A kind of preparation method of lentinan Download PDFInfo
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- CN104404102B CN104404102B CN201410698305.4A CN201410698305A CN104404102B CN 104404102 B CN104404102 B CN 104404102B CN 201410698305 A CN201410698305 A CN 201410698305A CN 104404102 B CN104404102 B CN 104404102B
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- 229920001491 Lentinan Polymers 0.000 title claims abstract description 61
- 229940115286 lentinan Drugs 0.000 title claims abstract description 61
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 240000000599 Lentinula edodes Species 0.000 claims abstract description 67
- 238000000605 extraction Methods 0.000 claims abstract description 64
- 238000000855 fermentation Methods 0.000 claims abstract description 59
- 230000004151 fermentation Effects 0.000 claims abstract description 59
- 239000012530 fluid Substances 0.000 claims abstract description 37
- 150000004676 glycans Chemical class 0.000 claims abstract description 35
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 35
- 239000005017 polysaccharide Substances 0.000 claims abstract description 35
- 241000894006 Bacteria Species 0.000 claims abstract description 17
- 238000003809 water extraction Methods 0.000 claims abstract description 13
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 5
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 5
- 238000004090 dissolution Methods 0.000 claims abstract description 3
- 239000007788 liquid Substances 0.000 claims description 61
- 235000015097 nutrients Nutrition 0.000 claims description 28
- 235000001674 Agaricus brunnescens Nutrition 0.000 claims description 26
- 239000002054 inoculum Substances 0.000 claims description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- 238000001035 drying Methods 0.000 claims description 20
- 239000012141 concentrate Substances 0.000 claims description 19
- 238000001556 precipitation Methods 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 10
- 235000019733 Fish meal Nutrition 0.000 claims description 10
- 244000017020 Ipomoea batatas Species 0.000 claims description 10
- 235000002678 Ipomoea batatas Nutrition 0.000 claims description 10
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 10
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 10
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 10
- 239000012043 crude product Substances 0.000 claims description 10
- 239000008367 deionised water Substances 0.000 claims description 10
- 229910021641 deionized water Inorganic materials 0.000 claims description 10
- 239000004467 fishmeal Substances 0.000 claims description 10
- 238000009630 liquid culture Methods 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 7
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims description 2
- 238000004176 ammonification Methods 0.000 claims 1
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 238000011084 recovery Methods 0.000 abstract description 2
- 230000001105 regulatory effect Effects 0.000 abstract description 2
- 235000001715 Lentinula edodes Nutrition 0.000 description 18
- 239000000306 component Substances 0.000 description 9
- 239000000843 powder Substances 0.000 description 4
- 241000233866 Fungi Species 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000009834 vaporization Methods 0.000 description 3
- 230000008016 vaporization Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000008512 biological response Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000009567 fermentative growth Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The present invention relates to a kind of preparation method of lentinan, comprise the following steps：Fermented and cultured, pH value is adjusted in fermentation process；Supercritical extract, the shiitake mushroom hypha bacterium mud that fermented and cultured is obtained, does not dry and is placed directly within supercritical CO2Extracted in extraction kettle, remove the fat-soluble memebrane protein of shiitake mushroom hypha and fat；Polysaccharide is extracted, dissolution fluid after extraction is transferred in hot water extraction's kettle, carries out the extraction of polysaccharide.Beneficial effects of the present invention are shown：PH value is regulated and controled in fermented and cultured, maximize shiitake mushroom hypha amount of fermentation, and the shiitake mushroom hypha bacterium mud for obtaining fermentation, which is not dried, directly carries out supercritical extract, removes the fat-soluble memebrane protein of shiitake mushroom hypha and fat, improves the recovery rate of lentinan.
Description
Technical field
The present invention relates to the extracting method field of polysaccharide, more particularly to a kind of preparation method of lentinan.
Background technology
Lentinan is a kind of physiologically active polysaccharide material isolated from mushroom, as with " biological response effect
Thing " fungi polysaccharide, there are the multiple functions such as antiviral, antitumor, enhancing body immunity, have in exploitation and suppress tumour, control
Had broad application prospects in terms for the treatment of angiocardiopathy, antiviral drug and relevant healthcare food.
Mushroom medicinal fungus fructification has cultivation period length, the big feature of occupied ground, bioactive polysaccharide thing in fructification
Matter is accumulative to be influenceed by environment, cultivation matrix etc., while the high mushroom Polyose extraction of the hard pigment content of fructification quality cell is difficult,
The problems such as active component difficulty is big, the production cycle is long, cost is high is thus extracted from entity.
, can be in the confined space, not by natural environment influence as one of modern biotechnology scale submerged fermentation technology
The various culture parameters orientation high efficiency of manual control obtain target product, have the cycle using the technology culture mushroom filamentous
It is short, cost is low, yield greatly can industrialized production feature.It is external to start shiitake mushroom hypha fermentation training early in the forties in last century
Support, for example Humfeld (1947) starts fermented and cultured mushroom mycelium.The domestic seventies in last century is also begun to shiitake mushroom hypha
Studied for the macro fungi Mycelium culture of representative.Lentinan bioactivity and fructification mushroom biology are living in shiitake mushroom hypha
Property it is consistent, optimizing lentinan content in Lentinus edodes fermented liquid by fermentation parameter, identical or even content is more with mushroom fruiting body
It is high.
PH value often changes with course of fermentation in shiitake mushroom hypha fermentation process, and it is fast to be unfavorable for shiitake mushroom hypha
Fast-growing is grown, and the most suitable Fermentative growth pH of invention shiitake mushroom hypha is simultaneously controlled by undoubtedly to improving the mushroom mycelium in unit zymotic fluid
Body supply, the yield for improving lentinan have positive effect.
The content of the invention
In view of this, it is an object of the invention to exist for prior art the problem of, there is provided a kind lentinan
Preparation method, improve the yield of shiitake mushroom hypha weight in wet base yield and lentinan.
The invention provides a kind of preparation method of lentinan, comprise the following steps：Fermented and cultured, adjust in fermentation process
Save pH value；Supercritical extract, the shiitake mushroom hypha bacterium mud that fermented and cultured is obtained, does not dry and is placed directly within supercritical CO2Extraction
Extracted in kettle, remove the fat-soluble memebrane protein of shiitake mushroom hypha and fat；Polysaccharide is extracted, dissolution fluid after extraction is transferred to hot water extraction
In kettle, the extraction of polysaccharide is carried out.
Further, the fermented and cultured includes：Choose the mushroom strain activated to be fitted into fluid nutrient medium, 25-28
Under the conditions of DEG C, 150r/min shaking table cultures are prepared into first order seed in 4-6 days, are connect the first order seed with 6-8% inoculum concentration
In fluid nutrient medium, shaking table culture, fermentation seed liquid is prepared into；, will with 6-8% inoculum concentration under certain fermentation culture conditions
The fermentation seed liquid is inoculated in equipped with the solution culture fermentation tank to have sterilized, adjusts pH value, fermented and cultured, using tubular type
Centrifuge collects shiitake mushroom hypha mud to the Lentinus edodes fermented liquid centrifugal treating.
Further, the sterilized liquid medium component is：Sweet potato leachate 200g/L, fish meal 80g/L, brewer's yeast
Leachate 50g/L, pH6.5.
Further, certain fermentation culture conditions are：5%-10% seed liquor inoculum concentration, 25 DEG C of fermentation temperature-
28 DEG C, tank pressure is 0.04-0.06MPa, throughput 0.3-0.9vvm, maintains DO to exist by adjusting fermentation tank rotating speed and throughput
10%-70%.
Further, the regulation pH value includes：Fermentation originates pH value in 12 hours and is down to 4.2-4.5 by originating pH6.5,
Into fermentation tank, Feeding ammonia water makes zymotic fluid pH in fermentation tank be returned to 5.2-5.5；Carried out with fermentation, pulsed is into fermentation tank
Feeding ammonia water keeps the zymotic fluid pH to be maintained at 5.2-5.5, until stopping fermentation, collects the shiitake mushroom hypha in zymotic fluid.
Further, the tube centrifuge rotating speed 12000r/min, sample introduction frequency are 3L/min.
Further, the supercritical extract condition is：Extracting pressure is controlled in 10-12MPa, 40 DEG C -42 of extraction temperature
℃。
Further, the extraction polysaccharide includes：The shiitake mushroom hypha bacterium mud handled through supercritical extract is weighed, adds 5
Times volumes of deionized water, soda acid adjust pH6-7,86 DEG C of hot water extraction 55min of temperature, filter off except extraction bacterium residue collected filtering
Liquid.
Further, the preparation method also includes：The clear liquid is transferred to and is evaporated under reduced pressure in kettle, 40 DEG C of -60 DEG C of temperature
Lower vacuum concentration, which is filtered to 1/3rd of original volume, obtains lentinan concentrate；Add the 95%-98% ethanol of 3 times of volumes
The Precipitation polysaccharide in the lentinan concentrate, filtering obtain lentinan precipitation, and drying obtains lentinan crude product.
Further, the vacuum concentration pressure is not more than 0.001MPa.
Beneficial effects of the present invention are shown：
PH value is regulated and controled in fermented and cultured, maximizes shiitake mushroom hypha amount of fermentation, and will ferment what is obtained
Shiitake mushroom hypha bacterium mud, which is not dried, directly carries out supercritical extract, removes the fat-soluble memebrane protein of shiitake mushroom hypha and fat, improves
The recovery rate of lentinan.
Embodiment
In the following detailed description, a large amount of specific details are proposed, in order to provide thorough understanding of the present invention.But
It will be understood by those within the art that it can also implement the present invention even if without these specific details.
Following preferred embodiment, is described in detail to the present invention.
Embodiment 1
Liquid Culture based component：Sweet potato leachate 200g/L, fish meal 80g/L, brewer's yeast leachate 50g/L, adjust
pH6.5。
From the mushroom strain access 150mL fluid nutrient mediums activated (500mL triangular flasks), 25 DEG C, 150r/min shakes
Bed culture is prepared into primary seed solution in 4 days, first order seed is connected in fluid nutrient medium with 6% inoculum concentration, shaking table culture 6
My god, it is prepared into fermentation seed liquid；It is inoculated in 7% inoculum concentration in fluid nutrient medium, fermentation temperature control is at 26 DEG C, tank pressure
0.05MPa, throughput 0.6vvm, maintain DO to cultivate 5 days 30% by adjusting fermentation tank rotating speed, obtain Lentinus edodes fermented liquid, adopt
Lentinus edodes fermented liquid centrifugal treating (tube centrifuge centrifugal rotational speed 13000r/min, sample introduction with tube centrifuge to above-mentioned acquisition
Frequency 5L/min, collect shiitake mushroom hypha mud.Shiitake mushroom hypha is put in drying box and dried 12 hours, is transferred to pulverizer crushing
Into mushroom powder.A certain amount of mushroom powder is weighed in supercritical CO2In extraction kettle, extraction 60min obtains supercritical CO2At extraction
Manage shiitake mushroom hypha (critical CO2 extracting pressures control is in 10MPa, 42 DEG C of extraction temperature).SCF-CO 2 is taken to handle mycelia
Body, 5 times of volumes of deionized water are added, adjust PH6.4,85 DEG C of hot water extraction 60min, filtered off except extraction bacterium residue must cross filtering
Liquid.The lentinan of above-mentioned acquisition is crossed into cleaner liquid to be transferred in reduction vaporization kettle, be concentrated in vacuo at a temperature of 45 DEG C (concentration pressure≤
0.001MPa) cross cleaner liquid and obtain lentinan concentrate to 1/3rd of original volume.Add 3 times of volumes 95% ethanol in
Precipitation polysaccharide in lentinan concentrate, filtering obtain lentinan and are deposited in 40 DEG C of drying boxes drying to obtain mushroom more
Sugared crude product, polysaccharide yield 10.3%.
Embodiment 2
Liquid Culture based component：Sweet potato leachate 200g/L, fish meal 80g/L, brewer's yeast leachate 50g/L, adjust
pH6.5。
From the mushroom strain access 150mL fluid nutrient mediums activated (500mL triangular flasks), 25 DEG C, 150r/min shakes
Bed culture is prepared into primary seed solution in 4 days, first order seed is connected in fluid nutrient medium with 6% inoculum concentration, shaking table culture 6
My god, it is prepared into fermentation seed liquid；It is inoculated in 7% inoculum concentration in fluid nutrient medium, fermentation culture conditions are：Fermentation temperature control
System is at 26 DEG C, tank pressure 0.05MPa, throughput 0.6vvm, by adjust fermentation tank rotating speed maintain DO 30%, 0-12 hours pH from
So be down to 4.3,12-120 hours controls pH5.2-5.5 by pulse Feeding ammonia water mode, obtains Lentinus edodes fermented liquid；Using tubular type
Lentinus edodes fermented liquid centrifugal treating (tube centrifuge centrifugal rotational speed 13000r/min, sample introduction frequency 5L/ of the centrifuge to above-mentioned acquisition
Min, collect shiitake mushroom hypha mud.A certain amount of shiitake mushroom hypha mud is weighed in supercritical CO2In extraction kettle, extraction 60min is obtained
Supercritical CO2Extraction processing shiitake mushroom hypha mud (critical CO2Extracting pressure is controlled in 10MPa, 42 DEG C of extraction temperature).Take super face
Boundary CO2Extraction processing mycelium mud, 5 times of volumes of deionized water are added, adjust PH6.4,85 DEG C of hot water extraction 60min, filter off and remove
Extraction bacterium residue must cross cleaner liquid.The lentinan of above-mentioned acquisition is crossed into cleaner liquid to be transferred in reduction vaporization kettle, at a temperature of 45 DEG C
It is concentrated in vacuo (concentration pressure≤0.001MPa) and crosses cleaner liquid, lentinan concentrate is obtained to 1/3rd of original volume.Add
Enter 95% ethanol of the 3 times of volumes Precipitation polysaccharide in lentinan concentrate, filtering obtains lentinan and is deposited in 40 DEG C
Drying obtains lentinan crude product, polysaccharide yield 13.8% in drying box.
Embodiment 3
Liquid Culture based component：Sweet potato leachate 200g/L, fish meal 80g/L, brewer's yeast leachate 50g/L, adjust
pH6.5。
From the mushroom strain access 150mL fluid nutrient mediums activated (500mL triangular flasks), 26 DEG C, 150r/min shakes
Bed culture is prepared into primary seed solution in 4 days, first order seed is connected in fluid nutrient medium with 6% inoculum concentration, shaking table culture 7
My god, it is prepared into fermentation seed liquid；It is inoculated in 6% inoculum concentration in fluid nutrient medium, fermented and cultured 3 days, condition is：Fermentation temperature
Degree control maintains DO in 30%, 0-12 hours at 26 DEG C, tank pressure 0.05MPa, throughput 0.6vvm, by adjusting fermentation tank rotating speed
PH is down to 4.2,12-72 hours and controls pH5.2-5.5 by pulse Feeding ammonia water mode naturally, obtains Lentinus edodes fermented liquid；Using
Lentinus edodes fermented liquid centrifugal treating (tube centrifuge centrifugal rotational speed 12000r/min, sample introduction frequency of the tube centrifuge to above-mentioned acquisition
Rate 4L/min, collect shiitake mushroom hypha mud (weight in wet base yield 110.2g/L).A certain amount of shiitake mushroom hypha mud is weighed in overcritical
CO2In extraction kettle, extraction 60min obtains supercritical CO2Extraction processing shiitake mushroom hypha mud (critical CO2Extracting pressure control exists
10MPa, 42 DEG C of extraction temperature).Take supercritical CO2Extraction processing mycelium mud, 5 times of volumes of deionized water are added, adjust PH6.4,
85 DEG C of hot water extraction 60min, filter off except extraction bacterium residue must cross cleaner liquid.The lentinan of above-mentioned acquisition is crossed into cleaner liquid to turn
Enter to be evaporated under reduced pressure in kettle, (concentration pressure≤0.001MPa) is concentrated in vacuo at a temperature of 45 DEG C and crosses cleaner liquid, to three points of original volume
One of obtain lentinan concentrate.95% ethanol of 3 times of volumes of addition Precipitation polysaccharide, mistake in lentinan concentrate
Filter acquisition lentinan is deposited in drying in 38 DEG C of drying boxes and obtains lentinan crude product, polysaccharide yield 14.3%.
Embodiment 4
Liquid Culture based component：Sweet potato leachate 200g/L, fish meal 80g/L, brewer's yeast leachate 50g/L, adjust
pH6.5。
From the mushroom strain access 150mL fluid nutrient mediums activated (500mL triangular flasks), 26 DEG C, 150r/min shakes
Bed culture is prepared into primary seed solution in 4 days, first order seed is connected in fluid nutrient medium with 6% inoculum concentration, shaking table culture 7
My god, it is prepared into fermentation seed liquid；It is inoculated in 6% inoculum concentration in fluid nutrient medium, fermented and cultured 3 days, condition is：Fermentation temperature
Degree control maintains DO in 30%, 0-12 hours at 26 DEG C, tank pressure 0.05MPa, throughput 0.6vvm, by adjusting fermentation tank rotating speed
PH is down to 4.2,12-72 hours and controls pH5.2-5.5 by pulse Feeding ammonia water mode naturally, obtains Lentinus edodes fermented liquid；Using
Lentinus edodes fermented liquid centrifugal treating (tube centrifuge centrifugal rotational speed 12000r/min, sample introduction frequency of the tube centrifuge to above-mentioned acquisition
Rate 4L/min, collect shiitake mushroom hypha mud (weight in wet base yield 110.2g/L).A certain amount of shiitake mushroom hypha mud is weighed in overcritical
CO2In extraction kettle, extraction 60min obtains supercritical CO2Extraction processing shiitake mushroom hypha mud (critical CO2Extracting pressure control exists
10MPa, 42 DEG C of extraction temperature).Take supercritical CO2Extraction processing mycelium mud, 5 times of volumes of deionized water are added, adjust PH6.4,
85 DEG C of hot water extraction 60min, filter off except extraction bacterium residue must cross cleaner liquid.The lentinan of above-mentioned acquisition is crossed into cleaner liquid to turn
Enter to be evaporated under reduced pressure in kettle, (concentration pressure≤0.001MPa) is concentrated in vacuo at a temperature of 45 DEG C and crosses cleaner liquid, to three points of original volume
One of obtain lentinan concentrate.95% ethanol of 3 times of volumes of addition Precipitation polysaccharide, mistake in lentinan concentrate
Filter acquisition lentinan is deposited in drying in 38 DEG C of drying boxes and obtains lentinan crude product, polysaccharide yield 14.0%.
Embodiment 5
Liquid Culture based component：Sweet potato leachate 200g/L, fish meal 80g/L, brewer's yeast leachate 50g/L, adjust
pH6.5。
From the mushroom strain access 150mL fluid nutrient mediums activated (500mL triangular flasks), 26 DEG C, 150r/min shakes
Bed culture is prepared into primary seed solution in 4 days, first order seed is connected in fluid nutrient medium with 6% inoculum concentration, shaking table culture 7
My god, it is prepared into fermentation seed liquid；It is inoculated in 6% inoculum concentration in fluid nutrient medium, fermented and cultured 3 days, condition is：Fermentation temperature
Degree control maintains DO in 26%, 0-12 hours at 25 DEG C, tank pressure 0.04MPa, throughput 0.3vvm, by adjusting fermentation tank rotating speed
PH is down to 4.2,12-72 hours and controls pH5.2-5.5 by pulse Feeding ammonia water mode naturally, obtains Lentinus edodes fermented liquid；Using
Lentinus edodes fermented liquid centrifugal treating (tube centrifuge centrifugal rotational speed 12000r/min, sample introduction frequency of the tube centrifuge to above-mentioned acquisition
Rate 4L/min, collect shiitake mushroom hypha mud (weight in wet base yield 110.2g/L).A certain amount of shiitake mushroom hypha mud is weighed in overcritical
CO2In extraction kettle, extraction 60min obtains supercritical CO2Extraction processing shiitake mushroom hypha mud (critical CO2Extracting pressure control exists
10MPa, 42 DEG C of extraction temperature).Take supercritical CO2Extraction processing mycelium mud, 5 times of volumes of deionized water are added, adjust PH6.4,
85 DEG C of hot water extraction 60min, filter off except extraction bacterium residue must cross cleaner liquid.The lentinan of above-mentioned acquisition is crossed into cleaner liquid to turn
Enter to be evaporated under reduced pressure in kettle, (concentration pressure≤0.001MPa) is concentrated in vacuo at a temperature of 45 DEG C and crosses cleaner liquid, to three points of original volume
One of obtain lentinan concentrate.95% ethanol of 3 times of volumes of addition Precipitation polysaccharide, mistake in lentinan concentrate
Filter acquisition lentinan is deposited in drying in 38 DEG C of drying boxes and obtains lentinan crude product, polysaccharide yield 14.2%.
Embodiment 6
Liquid Culture based component：Sweet potato leachate 200g/L, fish meal 80g/L, brewer's yeast leachate 50g/L, adjust
pH6.5。
From the mushroom strain access 150mL fluid nutrient mediums activated (500mL triangular flasks), 28 DEG C, 150r/min shakes
Bed culture is prepared into primary seed solution in 4 days, first order seed is connected in fluid nutrient medium with 5% inoculum concentration, shaking table culture 6
My god, it is prepared into fermentation seed liquid；It is inoculated in 5% inoculum concentration in fluid nutrient medium, fermented and cultured 4 days, condition is：Fermentation temperature
Degree control maintains DO in 34%, 0-12 hours at 28 DEG C, tank pressure 0.06MPa, throughput 0.9vvm, by adjusting fermentation tank rotating speed
PH is down to 4.4,12-96 hours and controls pH5.2-5.5 by pulse Feeding ammonia water mode naturally, obtains Lentinus edodes fermented liquid；Using
Lentinus edodes fermented liquid centrifugal treating (tube centrifuge centrifugal rotational speed 11000r/min, sample introduction frequency of the tube centrifuge to above-mentioned acquisition
Rate 3L/min, collect shiitake mushroom hypha mud (weight in wet base yield 112.7g/L).A certain amount of shiitake mushroom hypha mud is weighed in overcritical
CO2In extraction kettle, extraction 60min obtains supercritical CO2Extraction processing shiitake mushroom hypha mud (critical CO2Extracting pressure control exists
10MPa, 42 DEG C of extraction temperature).Take supercritical CO2Extraction processing mycelium mud, 5 times of volumes of deionized water are added, adjust PH6.6,
85 DEG C of hot water extraction 55min, filter off except extraction bacterium residue must cross cleaner liquid.The lentinan of above-mentioned acquisition is crossed into cleaner liquid to turn
Enter to be evaporated under reduced pressure in kettle, (concentration pressure≤0.001MPa) is concentrated in vacuo at a temperature of 45 DEG C and crosses cleaner liquid, to three points of original volume
One of obtain lentinan concentrate.95% ethanol of 3 times of volumes of addition Precipitation polysaccharide, mistake in lentinan concentrate
Filter acquisition lentinan is deposited in drying in 38 DEG C of drying boxes and obtains lentinan crude product, polysaccharide yield 14.1%.
Embodiment 7
Liquid Culture based component：Sweet potato leachate 200g/L, fish meal 80g/L, brewer's yeast leachate 50g/L, adjust
pH6.5。
From the mushroom strain access 150mL fluid nutrient mediums activated (500mL triangular flasks), 28 DEG C, 150r/min shakes
Bed culture is prepared into primary seed solution in 4 days, first order seed is connected in fluid nutrient medium with 5% inoculum concentration, shaking table culture 6
My god, it is prepared into fermentation seed liquid；It is inoculated in 5% inoculum concentration in fluid nutrient medium, fermented and cultured 4 days, condition is：Fermentation temperature
Degree control maintains DO in 29%, 0-12 hours at 28 DEG C, tank pressure 0.04MPa, throughput 0.7vvm, by adjusting fermentation tank rotating speed
PH is down to 4.4,12-96 hours and controls pH5.2-5.5 by pulse Feeding ammonia water mode naturally, obtains Lentinus edodes fermented liquid；Using
Lentinus edodes fermented liquid centrifugal treating (tube centrifuge centrifugal rotational speed 11000r/min, sample introduction frequency of the tube centrifuge to above-mentioned acquisition
Rate 3L/min, collect shiitake mushroom hypha mud (weight in wet base yield 112.7g/L).A certain amount of shiitake mushroom hypha mud is weighed in overcritical
CO2In extraction kettle, extraction 60min obtains supercritical CO2Extraction processing shiitake mushroom hypha mud (critical CO2Extracting pressure control exists
10MPa, 42 DEG C of extraction temperature).Take supercritical CO2Extraction processing mycelium mud, 5 times of volumes of deionized water are added, adjust PH6.6,
85 DEG C of hot water extraction 55min, filter off except extraction bacterium residue must cross cleaner liquid.The lentinan of above-mentioned acquisition is crossed into cleaner liquid to turn
Enter to be evaporated under reduced pressure in kettle, (concentration pressure≤0.001MPa) is concentrated in vacuo at a temperature of 45 DEG C and crosses cleaner liquid, to three points of original volume
One of obtain lentinan concentrate.95% ethanol of 3 times of volumes of addition Precipitation polysaccharide, mistake in lentinan concentrate
Filter acquisition lentinan is deposited in drying in 38 DEG C of drying boxes and obtains lentinan crude product, polysaccharide yield 13.9%.
Embodiment 8
Liquid Culture based component：Sweet potato leachate 200g/L, fish meal 80g/L, brewer's yeast leachate 50g/L, adjust
pH6.5。
From the mushroom strain access 150mL fluid nutrient mediums activated (500mL triangular flasks), 28 DEG C, 150r/min shakes
Bed culture is prepared into primary seed solution in 4 days, first order seed is connected in fluid nutrient medium with 5% inoculum concentration, shaking table culture 6
My god, it is prepared into fermentation seed liquid；It is inoculated in 5% inoculum concentration in fluid nutrient medium, fermented and cultured 4 days, condition is：Fermentation temperature
Degree control maintains DO in 30%, 0-12 hours at 27 DEG C, tank pressure 0.04MPa, throughput 0.6vvm, by adjusting fermentation tank rotating speed
PH is down to 4.4,12-96 hours and controls pH5.2-5.5 by pulse Feeding ammonia water mode naturally, obtains Lentinus edodes fermented liquid；Using
Lentinus edodes fermented liquid centrifugal treating (tube centrifuge centrifugal rotational speed 11000r/min, sample introduction frequency of the tube centrifuge to above-mentioned acquisition
Rate 3L/min, collect shiitake mushroom hypha mud.Shiitake mushroom hypha is put in drying box and dried 12 hours, pulverizer is transferred to and is ground into
Mushroom powder.A certain amount of mushroom powder is weighed in supercritical CO2In extraction kettle, extraction 60min obtains supercritical CO2Extraction processing
Shiitake mushroom hypha (critical CO2Extracting pressure is controlled in 10MPa, 42 DEG C of extraction temperature).Take supercritical CO2Extraction processing mycelium,
5 times of volumes of deionized water are added, adjust pH6.4,85 DEG C of hot water extraction 60min, are filtered off except extraction bacterium residue must cross cleaner liquid.Will
The lentinan of above-mentioned acquisition is crossed cleaner liquid and is transferred in reduction vaporization kettle, be concentrated in vacuo at a temperature of 45 DEG C (concentration pressure≤
0.001MPa) cross cleaner liquid and obtain lentinan concentrate to 1/3rd of original volume.Add 3 times of volumes 95% ethanol in
Precipitation polysaccharide in lentinan concentrate, filtering obtain lentinan and are deposited in 40 DEG C of drying boxes drying to obtain mushroom more
Sugared crude product, polysaccharide yield 11.6%.
As shown in embodiment 1-8, embodiment 1 is used lentinan preparation method in the prior art, embodiment 2-7
For lentinan preparation method used in the present invention, the present invention uses pH value during control fermentation, improves the yield of polysaccharide,
Embodiment 3 is most preferred embodiment, polysaccharide yield 14.3%；PH value when embodiment 8 is fermented for control, to obtained Lenlinus edodes
The preparation method that filament mud is dried, polysaccharide yield 11.6%, compared with embodiment 2-7 results, the present invention is used and not dried
Shiitake mushroom hypha mud, the yield of polysaccharide is higher.
Those skilled in the art can be directed to each application-specific, and described function is realized in a manner of flexible, still,
It is this to realize that decision-making should not be construed as the protection domain away from the disclosure.
Claims (7)
1. a kind of preparation method of lentinan, it is characterised in that comprise the following steps：
Fermented and cultured, choose the mushroom strain activated and be fitted into fluid nutrient medium, under the conditions of 25-28 DEG C, 150r/min shaking tables
Culture is prepared into first order seed in 4-6 days, and the first order seed is connected in fluid nutrient medium with 6-8% inoculum concentration, shaking table training
Support, be prepared into fermentation seed liquid；The fermentation seed liquid is inoculated in equipped with the Liquid Culture to have sterilized with 5-7% inoculum concentration
In base fermentation tank, 25 DEG C -28 DEG C of fermentation temperature, tank pressure is 0.04-0.06MPa, throughput 0.3-0.9vvm, is fermented by adjusting
Tank rotating speed and throughput maintain DO in 26%-34%, and fermentation originates pH value in 12 hours and is down to 4.2-4.5 by originating pH6.5, to
Feeding ammonia water makes zymotic fluid pH in fermentation tank be returned to 5.2-5.5 in fermentation tank；Carried out with fermentation, pulsed flows into fermentation tank
Ammonification water keeps the zymotic fluid pH to be maintained at 5.2-5.5, until stopping fermentation, using tube centrifuge to the mushroom ferment
Liquid centrifugal treating, collect shiitake mushroom hypha mud；
Supercritical extract, the shiitake mushroom hypha bacterium mud that fermented and cultured is obtained, does not dry and is placed directly within supercritical CO2Extraction
Extracted in kettle, remove the fat-soluble memebrane protein of shiitake mushroom hypha and fat；
Polysaccharide is extracted, dissolution fluid after extraction is transferred in hot water extraction's kettle, carries out the extraction of polysaccharide.
2. preparation method according to claim 1, it is characterised in that the Liquid Culture based component is：Sweet potato leachate
200g/L, fish meal 80g/L, brewer's yeast leachate 50g/L, pH6.5.
3. preparation method according to claim 1, it is characterised in that the tube centrifuge rotating speed 12000r/min, enter
Sample frequency is 3L/min.
4. preparation method according to claim 1, it is characterised in that the supercritical extract condition is：Extracting pressure control
System is in 10-12MPa, 40 DEG C -42 DEG C of extraction temperature.
5. preparation method according to claim 1, it is characterised in that the extraction polysaccharide includes：Weigh through overcritical extraction
The shiitake mushroom hypha bacterium mud of processing is taken, adds 5 times of volumes of deionized water, soda acid adjusts pH6-7,86 DEG C of hot water extraction 55min of temperature,
Filter off except extraction bacterium residue collected cleaner liquid.
6. preparation method according to claim 5, it is characterised in that the preparation method also includes：The clear liquid is turned
Enter to be evaporated under reduced pressure in kettle, vacuum concentration filters to 1/3rd of original volume and obtains lentinan concentration at a temperature of 40 DEG C -60 DEG C
Liquid；The 95%-98% ethanol of 3 times of volumes of addition Precipitation polysaccharide in the lentinan concentrate, filtering obtain mushroom
Polysaccharide precipitation, drying obtain lentinan crude product.
7. preparation method according to claim 6, it is characterised in that the vacuum concentration pressure is not more than 0.001MPa.
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