CN104073531B - A kind of deep layer liquid fermentation produces the method for Marasmius androsaceus (L.ex Fr.) Fr. extracellular polysaccharide - Google Patents
A kind of deep layer liquid fermentation produces the method for Marasmius androsaceus (L.ex Fr.) Fr. extracellular polysaccharide Download PDFInfo
- Publication number
- CN104073531B CN104073531B CN201410335964.1A CN201410335964A CN104073531B CN 104073531 B CN104073531 B CN 104073531B CN 201410335964 A CN201410335964 A CN 201410335964A CN 104073531 B CN104073531 B CN 104073531B
- Authority
- CN
- China
- Prior art keywords
- marasmius androsaceus
- fermentation
- marasmius
- androsaceus
- liquid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000233062 Gymnopus androsaceus Species 0.000 title claims abstract description 63
- 238000000855 fermentation Methods 0.000 title claims abstract description 56
- 230000004151 fermentation Effects 0.000 title claims abstract description 56
- 239000007788 liquid Substances 0.000 title claims abstract description 34
- 150000004676 glycans Chemical class 0.000 title claims abstract description 24
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 24
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 24
- 238000000034 method Methods 0.000 title claims abstract description 21
- 239000001963 growth medium Substances 0.000 claims abstract description 16
- 239000002609 medium Substances 0.000 claims abstract description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- 238000011218 seed culture Methods 0.000 claims description 20
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 16
- 239000008103 glucose Substances 0.000 claims description 16
- 229920002472 Starch Polymers 0.000 claims description 12
- 235000019698 starch Nutrition 0.000 claims description 12
- 239000008107 starch Substances 0.000 claims description 12
- 239000013530 defoamer Substances 0.000 claims description 11
- 238000001035 drying Methods 0.000 claims description 11
- 239000012531 culture fluid Substances 0.000 claims description 10
- 239000000706 filtrate Substances 0.000 claims description 10
- 239000002054 inoculum Substances 0.000 claims description 10
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 10
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 10
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 10
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 10
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 10
- 239000002244 precipitate Substances 0.000 claims description 8
- 241000255789 Bombyx mori Species 0.000 claims description 6
- 239000012141 concentrate Substances 0.000 claims description 6
- 235000012054 meals Nutrition 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 244000068988 Glycine max Species 0.000 claims description 5
- 235000010469 Glycine max Nutrition 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 5
- 230000000694 effects Effects 0.000 claims description 5
- 210000000582 semen Anatomy 0.000 claims description 5
- 102000011759 adducin Human genes 0.000 claims description 4
- 108010076723 adducin Proteins 0.000 claims description 4
- 230000001476 alcoholic effect Effects 0.000 claims description 4
- 102000004139 alpha-Amylases Human genes 0.000 claims description 4
- 108090000637 alpha-Amylases Proteins 0.000 claims description 4
- 229940024171 alpha-amylase Drugs 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- 238000001291 vacuum drying Methods 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 2
- 239000006228 supernatant Substances 0.000 claims description 2
- 241001480581 Marasmius Species 0.000 abstract description 9
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 6
- 229910052799 carbon Inorganic materials 0.000 abstract description 6
- 238000005516 engineering process Methods 0.000 abstract description 5
- 239000002028 Biomass Substances 0.000 abstract description 4
- 238000000605 extraction Methods 0.000 abstract description 3
- 230000007812 deficiency Effects 0.000 abstract description 2
- 239000000284 extract Substances 0.000 abstract description 2
- 230000000813 microbial effect Effects 0.000 abstract description 2
- 238000005457 optimization Methods 0.000 abstract description 2
- 238000011020 pilot scale process Methods 0.000 abstract description 2
- 238000009630 liquid culture Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 14
- 230000008901 benefit Effects 0.000 description 7
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000008719 thickening Effects 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 239000000356 contaminant Substances 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 150000002333 glycines Chemical class 0.000 description 3
- 238000000386 microscopy Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 208000004296 neuralgia Diseases 0.000 description 3
- 241000222485 Agaricales Species 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 239000006200 vaporizer Substances 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 241000221198 Basidiomycota Species 0.000 description 1
- 208000006373 Bell palsy Diseases 0.000 description 1
- 229920002444 Exopolysaccharide Polymers 0.000 description 1
- 206010063006 Facial spasm Diseases 0.000 description 1
- 206010072132 Fracture pain Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010024229 Leprosy Diseases 0.000 description 1
- 206010054949 Metaplasia Diseases 0.000 description 1
- 208000019695 Migraine disease Diseases 0.000 description 1
- 206010050031 Muscle strain Diseases 0.000 description 1
- OHVGNSMTLSKTGN-BTVCFUMJSA-N [C].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O Chemical compound [C].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O OHVGNSMTLSKTGN-BTVCFUMJSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 230000036592 analgesia Effects 0.000 description 1
- 230000003356 anti-rheumatic effect Effects 0.000 description 1
- 239000003435 antirheumatic agent Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002061 histotrophic effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 210000002239 ischium bone Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- HYYBABOKPJLUIN-UHFFFAOYSA-N mefenamic acid Chemical compound CC1=CC=CC(NC=2C(=CC=CC=2)C(O)=O)=C1C HYYBABOKPJLUIN-UHFFFAOYSA-N 0.000 description 1
- 230000015689 metaplastic ossification Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 206010027599 migraine Diseases 0.000 description 1
- 230000000116 mitigating effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 201000003068 rheumatic fever Diseases 0.000 description 1
- 230000001932 seasonal effect Effects 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000012879 subculture medium Substances 0.000 description 1
- 229940098465 tincture Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000008736 traumatic injury Effects 0.000 description 1
- 206010044652 trigeminal neuralgia Diseases 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to microbial technology field, be specifically related to a kind of method that deep layer liquid fermentation produces Marasmius androsaceus (L.ex Fr.) Fr. extracellular polysaccharide.The specifically production method of the Marasmius androsaceus (L.ex Fr.) Fr. extracellular polysaccharide of a kind of industrially scalable, the method is that the Marasmius androsaceus (L.ex Fr.) Fr. strain activated is carried out expansion joined by being linked into seed tank, again the liquid seeds that expansion prepares is linked in the special liquid culture medium producing polysaccharide and ferments, then fermentation liquid is separated, extract extracellular polysaccharide.The present invention is directed to above-mentioned the deficiencies in the prior art study, on the basis of lab scale and pilot scale continue to optimize fermentation medium (especially carbon source), promote Biomass and extracellular polysaccharide, solve be amplified to industrialized production runs into by lab scale Fermentation Process of Parameter control, extraction equipment type selecting, the difficult problem such as optimization, finally being successfully grafted onto 10 20t fermentation tanks carries out industrialization production, and produce the Marasmius polysaccharide of high-quality smoothly, be truly realized continuously, automatization, the production of industrialization.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to a kind of deep layer liquid fermentation and produce Marasmius androsaceus (L.ex Fr.) Fr. born of the same parents
The method of exo polysaccharides.
Background technology
Marasmius androsaceus (L.ex Fr.) Fr., formal name used at school is Marasmius androsaceus (L.ex Fr.) Fr. another name Marasmius equicrinis Muell., dark brown
Marasmiuses etc., belong to Basidiomycetes, Agaricales (Agaricales), Bai Mo section (ThichoLomataceae), cuticle
Umbrella belongs to (Marasmius).It is born on the deadwood above remote, thickly forested mountains, cool, damp, acid ground, is mainly distributed
Save in China Jilin, Hunan, Hubei, Guangdong etc..China's morning among the people useful wild Marasmius androsaceus (L.ex Fr.) Fr. root shape bacterium
Rope treats fracture pain, traumatic injury etc..According to Guangdong institute of microbiology (1975), this institute is with wide
State the first pharmaceutical factory etc. uses artificial fermentation culture method, it is thus achieved that Marasmius androsaceus (L.ex Fr.) Fr. fermentation culture medium, then is concentrated
" the marasmius androsaceus capsule " made and medicated wine, 600 example according to clinical verification, it was demonstrated that to trigeminal neuralgia, ischium
Neuralgia, migraine, supraorbital neuralgia, facial nerve paralysis, facial spasm and rheumatic arthritis etc. all have
Certain curative effect.Centering, light leprosy neuralgia, lumbar muscle strain etc. also have certain mitigation, are to pacify human body
Entirely, the analgesia having no side effect and antirheumatic.The medicine made with it at present, except " marasmius androsaceus capsule "
Outward, also have " marasmius androsaceus tincture ", " marasmius androsaceus injection " and " CNNS's gardan " etc..Clinic and market effect
Very well.Recent studies indicate that the healthy nutritive value of Marasmius androsaceus (L.ex Fr.) Fr. is the highest, can promote nervous tissue and
The inflammation of fibrous connective tissue disappears, and improves blood circulation and the histotrophic nutrition situation of local, recovers function of nervous system.
Human body can be exempted from by the compositions such as aminoacid that bacterium is contained within, saccharide as food and the adding ingredient of health product
Epidemic disease power improves and defying age has certain effect.The function of other component of Marasmius androsaceus (L.ex Fr.) Fr. is also in studying
Stage.
Increasing scholar begins one's study the active ingredient of Marasmius androsaceus (L.ex Fr.) Fr. and pharmacological action in recent years, but mesh
Before domestic wild Marasmius androsaceus (L.ex Fr.) Fr. resource scarcity, the artificial culture success rate of sporophore is low and poor repeatability, Gu
Body cultivation period length, pollution rate are high, are easily limited by surrounding, it is impossible to meet the growing market demand.
And deep layer liquid fermentation technology has the advantages such as fermentation period is short, high yield, pollution rate are low, not by surrounding
Limit, resource apparatus utilization rate advantages of higher, thus favored by increasing researchers.Both at home and abroad
Also the researcher having a small amount of universities and colleges carried out research to the submerged fermentation of Marasmius androsaceus (L.ex Fr.) Fr., but often it is main
Emphasis is how to obtain higher Biomass, and only small part researcher pays close attention to the functional component of Marasmius androsaceus (L.ex Fr.) Fr.
The research of polysaccharide, but mainly extracted by mycelium and obtain polysaccharide, the byproduct fermentation filtrate as fermentation is big
All directly drain as waste liquid, waste substantial amounts of precious resources, and add environmental protection pressure, the row of improving
Dirty cost.
Summary of the invention
The invention aims to solve the technical bottleneck of industrial-scale production Marasmius polysaccharide, use
Liquid fermentation produce Marasmius polysaccharide overcome solid fermentation cycle length, investment is big, be subject to seasonal restrictions, difficult
With drawbacks such as industrialization, use this invention can continuously, automatization, the CNNS producing high-quality of industrialization
Marasmius polysaccharide.
To achieve these goals, the present invention is carried out essentially according to following step:
The industrialization deep layer liquid fermentation of the present invention produces the method for Marasmius androsaceus (L.ex Fr.) Fr. extracellular polysaccharide and includes following step
Rapid:
A kind of method preparing Marasmius androsaceus (L.ex Fr.) Fr. fermentation liquid, after accessing sterilizing by Marasmius androsaceus (L.ex Fr.) Fr. seed culture fluid
Fermentation medium, inoculum concentration is 10～20%, and ventilating ratio is 1:0.5 1.5vvm, tank pressure 0.02～0.05Mpa,
Cultivate 46 days at 22～30 DEG C of bottom fermentations, obtain Marasmius androsaceus (L.ex Fr.) Fr. fermentation liquid；Wherein said fermentation medium
Formula is: glucose 1-3%, starch 1-3%, yeast powder 0.2-1%, Semen Maydis pulp 0.5-2%, potassium dihydrogen phosphate
0.1-0.5%, Magnesium sulfate heptahydrate 0.1-0.5%, defoamer 0.01-0.05%, 20000u/ml high temperature resistant α-
Amylase 0.001-0.007% (v/v), regulates pH value 6.5-7.5.Except amylase in above-mentioned culture medium composition
" % " in addition, all represents g/100ml.
Described defoamer can be vegetable oil (Oleum Glycines, the Oleum Brassicae campestris) material such as class, organic ethers.
Wherein, described Marasmius androsaceus (L.ex Fr.) Fr. seed culture fluid is preferably prepared by the following method and obtains:
1) first order seed is cultivated: activated shaking flask strain accesses the liquid seed culture medium after sterilizing, inoculation
Amount is 0.1～2%, and ventilating ratio is 1:0.5 1.5vvm, tank pressure 0.02～0.05Mpa, cultivates at 22～30 DEG C
5～6 days, obtain Marasmius androsaceus (L.ex Fr.) Fr. first order seed culture fluid；
2) secondary seed is cultivated: by step 1) the first order seed culture fluid of gained accesses the liquid strain after sterilizing
Sub-culture medium, inoculum concentration is 10～20%, and ventilating ratio is 1:0.5 1.5vvm, tank pressure 0.02～0.05Mpa,
Cultivate 3～4 days at 22～30 DEG C, obtain described Marasmius androsaceus (L.ex Fr.) Fr. seed culture fluid.
Described liquid seed culture medium formula is preferably: glucose 2-4%, dried silkworm chrysalis meal 0.1-0.5%, Semen Glycines
Cake powder 1-3%, potassium dihydrogen phosphate 0.1-0.5%, Magnesium sulfate heptahydrate 0.1-0.5%, defoamer 0.01-0.05%,
Regulation pH value 6.5-7.5." % " in above-mentioned culture medium composition all represents g/100ml.
Described fermentation culture terminal is: fungus ball becomes broken, filtrate is muddy；Reducing sugar is down to minimum and slightly fluctuates,
Reducing sugar controls 0.3% (g/100g) below；Microscopy mycelia is in small, broken bits, without living contaminants.
A kind of method that Marasmius androsaceus (L.ex Fr.) Fr. extracellular polysaccharide extracts, the method comprises the steps:
1) separate: by separated for the Marasmius androsaceus (L.ex Fr.) Fr. fermentation culture of claim 1 gained, obtain CNNS's cuticle
Umbrella supernatant；
2) concentrating: by step 1) filtrate of gained carries out using two grade low-temps to concentrate, and obtain concentrating proportion and be
The concentrated pulp of 1.25～1.40；
3) precipitate with ethanol, drying: add 95% ethanol of concentrated pulp weight 3-5 times amount, with alcohol meter detection mixing
Liquid alcoholic strength about 65-80% (v/v), stands 8-16h, precipitate carries out vacuum drying, temperature 50-75 DEG C,
Vacuum is not less than 0.085MPa；Drying to dried object moisture is 3～8% (g/100g), obtains outside born of the same parents
Sugar.
Wherein, described step 1) described in separation use horizontal screw centrifuge to carry out, rotating speed controls
2500‐3500r/min。
Described step 2) employed in secondary concentration be: one-level use 1 3t double effect evaporator concentrate, will send out
It is 1.15 1.20 (50 DEG C of heat are surveyed) that ferment liquid is concentrated to proportion, and two grades use 1 3t haplo-effect concentrator to concentrate, will
Proportion is that the concentrated solution of 1.1 1.20 (50 DEG C of heat are surveyed) continues to be concentrated to proportion to be 1.25～1.40 (50 DEG C of heat are surveyed)
Concentrated pulp；Concentration process is required to control temperature and is not higher than 75 DEG C, and vacuum is not less than 0.085MPa.
In the present invention, precipitate with ethanol is to use 1 3t Alcohol-settling tank, and precipitate with ethanol number of times is the most, and the content of the outer sugar of born of the same parents is the highest.Very
The empty vacuum drying oven used of drying, born of the same parents' outer sugar content of present invention extraction is 20 50%.
The present invention has a following prominent advantage:
The present invention is directed to above-mentioned the deficiencies in the prior art study, continue to optimize fermentation culture at lab scale and pilot scale
On the basis of base (especially carbon source), lifting Biomass and extracellular polysaccharide, solve and be amplified to industry by lab scale
Fermentation Process of Parameter controls that metaplasia runs in producing, extraction equipment type selecting, the difficult problem such as optimization, the most successful final grafting
Carry out industrialization production to 10 20t fermentation tanks, and produce the Marasmius polysaccharide of high-quality smoothly, very
Just achieving continuously, automatization, the production of industrialization.
In order to improve the yield of purpose product extracellular polysaccharide, the present invention has carried out excellent in the carbon source of fermentation medium
Change and combination: use the compound prescription of fugitive glucose and long-acting starch.Earlier fermentation, to grow mycelium, carries
High biological content is main, therefore with the addition of the glucose carbon source that thalline is preferentially selected in culture medium, breeds rapidly；
The content of fermentation later stage purpose to be improved product, therefore carbon source provides thalline relatively to utilize slower starch, and
Adding appropriate amount of starch enzyme in culture medium, control thalline quantity is at proper size, and the raw metabolite born of the same parents of pregnancy ceased are outer sugared.
In order to provide born of the same parents the yield of outer sugar, the present invention optimizes the formula of fermentation medium, uses for carbon source part
Compound prescription, both ensure that the Biomass of early stage thalline, had ensured again the yield of later stage metabolite.
The employing present invention according to the market demand and product standard, can prepare the polysaccharide of the high-quality of different content, many
Sugar content can be 20 50%.
Detailed description of the invention
The present invention is further illustrated below by way of specific embodiment.But the detail of embodiment is only used for explaining
The present invention, should not be construed as limited overall technical solution.
The Marasmius androsaceus (L.ex Fr.) Fr. that following example use is bought in Institute of Microorganism, Academia Sinica's common micro-organisms
Center, strain coding CGMCC NO5.0152.As a example by this strain bacterium, describe the concrete side of the present invention in detail
Case.But the inventive method is common to all of Marasmius androsaceus (L.ex Fr.) Fr., it is not limited in this bacterial strain.
Embodiment 1
Industrialization deep layer liquid fermentation of the present invention produces the method for Marasmius androsaceus (L.ex Fr.) Fr. extracellular polysaccharide and enters in the steps below
OK:
1, the preparation of Marasmius androsaceus (L.ex Fr.) Fr. fermentation liquid: by the Marasmius androsaceus (L.ex Fr.) Fr. shaking flask bacterial strain that activated by inoculum concentration 1%
Being inoculated in seed culture medium (300L), wherein seed culture medium consists of glucose 3%, dried silkworm chrysalis meal 0.3%, Huang
Soybean cake powder 1.8%, potassium dihydrogen phosphate 0.3%, Magnesium sulfate heptahydrate 0.15%, defoamer 0.02%, regulate pH value
7.0, ventilating ratio is 1:1.0vvm, tank pressure 0.03Mpa, 26～28 DEG C of shaken cultivation 5 Tian Ji get CNNS cuticles
Umbrella primary seed solution；Primary seed solution is accessed secondary seed medium (3000L) by inoculum concentration 15%, wherein two
Level seed culture medium consist of glucose 3%, dried silkworm chrysalis meal 0.3%, soybean cake powder 1.8%, potassium dihydrogen phosphate 0.3%,
Magnesium sulfate heptahydrate 0.15%, defoamer 0.02%, regulate pH value 7.0, and ventilating ratio is 1:1.0vvm, tank pressure 0.03Mpa,
26～28 DEG C of cultivations i.e. obtain Marasmius androsaceus (L.ex Fr.) Fr. secondary seed solution in 3 days；Secondary seed solution is accessed by inoculum concentration 18%
Fermentation medium (7500L), wherein fermentation medium consist of glucose 2%, starch 2%, yeast powder 0.5%,
Semen Maydis pulp 1.5%, potassium dihydrogen phosphate 0.2%, Magnesium sulfate heptahydrate 0.2%, defoamer Oleum Glycines 0.03%, high temperature resistant
Alpha amylase (Zaozhuang outstanding person promise enzyme biology company limited, 20000u/ml) 0.002% (v/v), regulates pH value
7.0；Ventilating ratio is 1:0.5vvm, tank pressure 0.03Mpa, 26～28 DEG C cultivate 4 days, ferment to fungus ball become broken,
Filtrate is muddy；Reducing sugar is down to minimum 0.2% and slightly fluctuates；Microscopy mycelia is in small, broken bits, without living contaminants.I.e.
Obtain Marasmius androsaceus (L.ex Fr.) Fr. fermentation liquid 8850Kg.
2, Marasmius androsaceus (L.ex Fr.) Fr. separation of fermentative broth: above-mentioned Marasmius androsaceus (L.ex Fr.) Fr. fermentation liquid 8850Kg is centrifuged by sleeping spiral shell
Machine separates, and rotating speed controls at 3000r/min, and disengaging time, at 2h, obtains Marasmius androsaceus (L.ex Fr.) Fr. filtrate 7524Kg
With the wet mycelium (water content is 80%) of 1105Kg, wet mycelium directly cold drying can pulverize prepared fermentation
Marasmius androsaceus (L.ex Fr.) Fr. mycopowder.
4, cryoconcentration: the Marasmius androsaceus (L.ex Fr.) Fr. filtrate 7524Kg vacuum of step 2 gained is transferred to 3t economic benefits and social benefits
Vaporizer, is 0.085MPa in vacuum, and thickening temperature is concentrated into proportion under the conditions of being 65 DEG C be 1.20 (50
DEG C heat is surveyed) concentrated solution, then concentrated solution vacuum is transferred to 1.5t single-action ball-type concentrator, in vacuum is
0.09MPa, thickening temperature continues under the conditions of being 60 DEG C to be concentrated into the concentration that proportion is 1.30 (50 DEG C of heat are surveyed)
Slurry 280kg.
5, precipitate with ethanol, drying: add 95% wine of weight 1100kg in the Alcohol-settling tank filling 280kg concentrated pulp
Essence, with alcohol meter detection mixed liquor alcoholic strength 72% (v/v), stands 12h, separates bottom sediment with true
Empty drying baker is dried, temperature 60 C, vacuum 0.09MPa；Dry 12h and obtain born of the same parents outer sugar 132.8kg.
Detection moisture is 4%, and polyoses content is 30%.
Embodiment 2
1, the preparation of Marasmius androsaceus (L.ex Fr.) Fr. fermentation liquid: by the Marasmius androsaceus (L.ex Fr.) Fr. shaking flask bacterial strain that activated by inoculum concentration
1.5% is inoculated in seed culture medium (300L), wherein seed culture medium consist of glucose 3%, dried silkworm chrysalis meal 0.3%,
Soybean cake powder 1.8%, potassium dihydrogen phosphate 0.3%, Magnesium sulfate heptahydrate 0.15%, defoamer 0.02%, regulate pH
Value 7.0, ventilating ratio is 1:1.0vvm, tank pressure 0.03Mpa, and 25～27 DEG C of shaken cultivation 6 Tian Ji get CNNSs are little
Skin umbrella primary seed solution；Primary seed solution is accessed secondary seed medium (3000L) by inoculum concentration 15%, wherein
Secondary seed medium consist of glucose 3%, dried silkworm chrysalis meal 0.3%, soybean cake powder 1.8%, potassium dihydrogen phosphate 0.3%,
Magnesium sulfate heptahydrate 0.15%, defoamer 0.02%, regulate pH value 7.0, and ventilating ratio is 1:1.0vvm, tank pressure
0.03Mpa, 25～27 DEG C of cultivations i.e. obtain Marasmius androsaceus (L.ex Fr.) Fr. secondary seed solution in 3 days；By secondary seed solution by inoculation
Amount 18% access fermentation medium (15000L), wherein fermentation medium consists of glucose 2%, starch 2%, ferment
Female powder 0.7%, Semen Maydis pulp 1.5%, potassium dihydrogen phosphate 0.2%, Magnesium sulfate heptahydrate 0.2%, defoamer Oleum Brassicae campestris
0.03%, thermostable α-amylase (Zaozhuang outstanding person promise enzyme biology company limited, 20000u/ml) 0.002% (v/v),
Regulation pH value 7.0；Ventilating ratio is 1:0.5vvm, tank pressure 0.04Mpa, cultivates 4 days for 25～27 DEG C, fermentation
To fungus ball change is broken, filtrate is muddy；Reducing sugar is down to minimum 0.2% and slightly fluctuates；Microscopy mycelia is in small, broken bits, nothing
Living contaminants, had both obtained Marasmius androsaceus (L.ex Fr.) Fr. fermentation liquid 17700Kg.
2, the separation of Marasmius androsaceus (L.ex Fr.) Fr. fermentation liquid: by above-mentioned Marasmius androsaceus (L.ex Fr.) Fr. fermentation liquid 17700Kg by sleeping spiral shell
Centrifuge separates, and obtains the wet mycelium (water content of Marasmius androsaceus (L.ex Fr.) Fr. filtrate 15540Kg and 1800Kg
It is 75%), wet mycelium directly cold drying can pulverize prepared fermentation Marasmius androsaceus (L.ex Fr.) Fr. mycopowder.Concrete technology bar
Part is: rotating speed controls at 3200r/min, and disengaging time is at 4.5h.
4, cryoconcentration: the Marasmius androsaceus (L.ex Fr.) Fr. filtrate 15540Kg vacuum of step 2 gained is transferred to 3t economic benefits and social benefits
Vaporizer, is 0.09MPa in vacuum, and thickening temperature is concentrated into proportion under the conditions of being 63 DEG C be 1.18 (50
DEG C heat is surveyed) concentrated solution, then concentrated solution vacuum is transferred to 1.5t single-action ball-type concentrator, in vacuum is
0.09MPa, thickening temperature is concentrated into the concentrated pulp that proportion is 1.35 (50 DEG C of heat are surveyed) under the conditions of being 65 DEG C
570kg。
5, precipitate with ethanol, drying: add 95% wine of weight 2300kg in the Alcohol-settling tank filling 570kg concentrated pulp
Essence, with alcohol meter detection mixed liquor alcoholic strength 71% (v/v), stands 10h, separates bottom sediment with true
Empty drying baker is dried, temperature 65 DEG C, vacuum 0.09MPa；Dry 18h and obtain born of the same parents outer sugar 260kg.Inspection
Surveying moisture is 4.5%, and polyoses content is 32%.
Embodiment 3
CNNS is produced in industrialization for the compound prescription of fugitive glucose and long-acting starch in the more preferably checking present invention
The superiority of Marasmius polysaccharide, has done again several contrast test.Test only have adjusted in liquid fermentation medium
Carbon source glucose or (with) consumption of starch, comparative example 1 does not contains starch, concentration of glucose is 1.5%；
Not containing glucose in comparative example 2, starch concentration is 2.5%, and in comparative example 3, glucose content is 2.5%,
Content of starch is 0.5%；Remaining condition is all with embodiment 1, and result of the test is shown in Table 1:
Table 1
Claims (4)
1. the method preparing Marasmius androsaceus (L.ex Fr.) Fr. fermentation liquid, it is characterized in that the fermentation medium after Marasmius androsaceus (L.ex Fr.) Fr. seed culture fluid is accessed sterilizing, inoculum concentration is 10 ~ 20%, ventilating ratio is 1:0.5-1.5vvm, tank pressure 0.02～0.05Mpa, cultivate 3 ~ 5 days at 22 ~ 30 DEG C of bottom fermentations, obtain Marasmius androsaceus (L.ex Fr.) Fr. fermentation liquid；Wherein said fermentative medium formula is: glucose 1-3%, starch 1-3%, yeast powder 0.2-1%, Semen Maydis pulp 0.5-2%, potassium dihydrogen phosphate 0.1-0.5%, Magnesium sulfate heptahydrate 0.1-0.5%, the Thermostable α-Amylase 0.001-0.007%v/v of defoamer 0.01-0.05%, 20000u/ml, regulates pH value 6.5-7.5；
Wherein, described Marasmius androsaceus (L.ex Fr.) Fr. seed culture fluid is prepared by the following method and obtains:
1) first order seed is cultivated: activated shaking flask strain accesses the liquid seed culture medium after sterilizing, inoculum concentration is 0.1 ~ 2%, and ventilating ratio is 1:0.5-1.5vvm, tank pressure 0.02～0.05Mpa, cultivate 5 ~ 6 days at 22 ~ 30 DEG C, obtain Marasmius androsaceus (L.ex Fr.) Fr. first order seed culture fluid；
2) secondary seed is cultivated: the first order seed culture fluid of step 1) gained accesses the liquid seed culture medium after sterilizing, inoculum concentration is 10 ~ 20%, and ventilating ratio is 1:0.5-1.5vvm, tank pressure 0.02～0.05Mpa, cultivate 3 ~ 4 days at 22 ~ 30 DEG C, obtain described Marasmius androsaceus (L.ex Fr.) Fr. seed culture fluid；
Described liquid seed culture medium formula is: glucose 2-4%, dried silkworm chrysalis meal 0.1-0.5%, soybean cake powder 1-3%, potassium dihydrogen phosphate 0.1-0.5%, Magnesium sulfate heptahydrate 0.1-0.5%, defoamer 0.01-0.05%, regulates pH value 6.5-7.5.
2. the extracting method of a Marasmius androsaceus (L.ex Fr.) Fr. extracellular polysaccharide, it is characterised in that the method comprises the steps:
1) Marasmius androsaceus (L.ex Fr.) Fr. fermentation liquid is prepared: prepare Marasmius androsaceus (L.ex Fr.) Fr. fermentation liquid in accordance with the method for claim 1；
2) separate: by separated for the Marasmius androsaceus (L.ex Fr.) Fr. fermentation culture of gained, obtain Marasmius androsaceus (L.ex Fr.) Fr. supernatant；
3) concentrating: by step 2) filtrate of gained carries out using two grade low-temps to concentrate, obtains concentrating the concentrated pulp that proportion is 1.25～1.40；
4) precipitate with ethanol, drying: add 95% ethanol of concentrated pulp weight 3-5 times amount, is 65-80% with alcohol meter detection mixed liquor alcoholic strength, stands 8-16h, precipitate carries out vacuum drying, temperature 50-75 DEG C, vacuum is not less than 0.085MPa；Drying to dried object moisture is 3～8%, and wherein moisture unit is g/100g, obtains the outer sugar of born of the same parents.
The extracting method of Marasmius androsaceus (L.ex Fr.) Fr. extracellular polysaccharide the most according to claim 2, it is characterised in that described step 2) described in separation use horizontal screw centrifuge carry out, rotating speed controls at 2500-3500r/min.
The extracting method of Marasmius androsaceus (L.ex Fr.) Fr. extracellular polysaccharide the most according to claim 2, it is characterized in that the secondary concentration employed in described step 3) is: one-level uses double effect evaporator to concentrate, it is 1.15-1.20 that fermentation liquid is concentrated to proportion, two grades use haplo-effect concentrators to concentrate, and will proportion be that the concentrated solution of 1.15-1.20 continues to be concentrated to the concentrated pulp that proportion is 1.25～1.40；Concentration process is required to control temperature and is not higher than 75 DEG C, and vacuum is not less than 0.085MPa.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410335964.1A CN104073531B (en) | 2014-07-15 | 2014-07-15 | A kind of deep layer liquid fermentation produces the method for Marasmius androsaceus (L.ex Fr.) Fr. extracellular polysaccharide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410335964.1A CN104073531B (en) | 2014-07-15 | 2014-07-15 | A kind of deep layer liquid fermentation produces the method for Marasmius androsaceus (L.ex Fr.) Fr. extracellular polysaccharide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104073531A CN104073531A (en) | 2014-10-01 |
| CN104073531B true CN104073531B (en) | 2016-08-24 |
Family
ID=51595134
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410335964.1A Active CN104073531B (en) | 2014-07-15 | 2014-07-15 | A kind of deep layer liquid fermentation produces the method for Marasmius androsaceus (L.ex Fr.) Fr. extracellular polysaccharide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104073531B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111944860A (en) * | 2019-05-17 | 2020-11-17 | 天津科技大学 | A kind of exopolysaccharide of Anluo fimbriae and its application |
| CN111187723B (en) * | 2019-09-30 | 2022-05-31 | 贵州科学院 | Method for producing mycelium and extracellular polysaccharide by liquid culture of pileus giganteus |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101879189A (en) * | 2009-05-04 | 2010-11-10 | 漯河市方汇药业有限公司 | Method for producing marasmius androsaceus fermented powder by adopting optimized solid culture medium |
| CN102579509B (en) * | 2012-01-18 | 2014-05-14 | 山西康欣药业有限公司 | Selenium-enriched marasmius androsaceus toadstool extract preparation and preparation method thereof |
-
2014
- 2014-07-15 CN CN201410335964.1A patent/CN104073531B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN104073531A (en) | 2014-10-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103478413B (en) | Method for producing protein feed by mixed-strain solid-state fermentation of ginkgo leaf residues | |
| CN104099385B (en) | A kind of deep layer liquid state fermentation produces the method for Inonotus obliquus exocellular polysaccharide | |
| CN104087631B (en) | Method for producing Antrodia camphorata extracellular polysaccharides by deep liquid fermentation | |
| CN103305434A (en) | Microecological preparation with dual functions of probiotics and organic selenium and preparation method of microecological preparation | |
| CN102796675B (en) | Rhodotorula glutinis oil genetic engineering strain and construction method and application thereof | |
| CN104130952A (en) | Rhodotorula mucilaginosa and application in fermentation production of carotenoid and oils | |
| CN103483040B (en) | Chinese caterpillar fungus pilot scale liquid submerged fermentation substratum and fermentation method for producing thereof | |
| CN106387398A (en) | Yeast additive for growth and development promoting and body immunity enhancing feed of piglets and preparation method thereof | |
| CN105193905A (en) | Traditional Chinese medicine processing technology of ganoderma-astragalus membranaceus bidirectional fermentation | |
| CN106754411A (en) | One plant height produces the Aspergillus niger strain and its liquid state fermentation enzyme producing method of β D fructofuranosidases | |
| CN106578802A (en) | Method for preparing fermented beverage by utilizing vinasse | |
| CN102703339A (en) | High-yield arginine deiminase bacterial strain and method for producing L-citrulline by same | |
| CN104846018A (en) | Cordyceps enzyme deep layer liquid fermentation and expanding culture method | |
| CN105754925B (en) | A method of improving Pichia kudriavezii thermo-tolerance | |
| CN102342218A (en) | Method for producing cordyceps sinensis hyphae by virtue of solid-state fermentation | |
| CN101933439A (en) | A kind of method of using vegetable oil to increase the amount of mycelium of Phellinus liquid culture | |
| CN104087632B (en) | A kind of deep layer liquid fermentation produces the method for Phellinus igniarius (L. ex Fr.) Quel. extracellular polysaccharide | |
| CN102948325A (en) | Cordyceps militaris efficient quick cultivation technology | |
| CN109429911A (en) | A kind of fermentation cordyceps sinensis culture solution and preparation method thereof prepared with jerusalem artichoke | |
| CN105725195A (en) | Composite cordyceps militaris probiotic enzyme and preparation method thereof | |
| CN104073531B (en) | A kind of deep layer liquid fermentation produces the method for Marasmius androsaceus (L.ex Fr.) Fr. extracellular polysaccharide | |
| CN103045487B (en) | Bacterial strain for producing citric acid and method for fermenting and producing critic acid through fermentation of bacterial strain | |
| CN103299823A (en) | Method for improving yield and trace element enrichment of liquid fermentation mycelium of lucid ganoderma | |
| CN109729912A (en) | A kind of rejuvenation method of the wild selenium-rich mushroom strain of poplar sawdust cultivating in bag | |
| CN101735329B (en) | Pholiota adiosapose polysaccharide and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |