CN1398978A - Fermentation process of Agaricus Blazei murrill and production process of its polyglycopeptide - Google Patents
Fermentation process of Agaricus Blazei murrill and production process of its polyglycopeptide Download PDFInfo
- Publication number
- CN1398978A CN1398978A CN 02136516 CN02136516A CN1398978A CN 1398978 A CN1398978 A CN 1398978A CN 02136516 CN02136516 CN 02136516 CN 02136516 A CN02136516 A CN 02136516A CN 1398978 A CN1398978 A CN 1398978A
- Authority
- CN
- China
- Prior art keywords
- agaricus blazei
- zymotechnique
- blazei murrill
- polysaccharide peptide
- production method
- 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.)
- Pending
Links
- 241001327634 Agaricus blazei Species 0.000 title claims abstract description 57
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 20
- 238000000855 fermentation Methods 0.000 title claims abstract description 19
- 230000004151 fermentation Effects 0.000 title claims abstract description 19
- 239000007788 liquid Substances 0.000 claims abstract description 20
- 239000000843 powder Substances 0.000 claims abstract description 10
- 229920002472 Starch Polymers 0.000 claims abstract description 6
- 239000008107 starch Substances 0.000 claims abstract description 6
- 235000019698 starch Nutrition 0.000 claims abstract description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 4
- 239000002028 Biomass Substances 0.000 claims abstract description 3
- 239000001963 growth medium Substances 0.000 claims abstract description 3
- 240000008042 Zea mays Species 0.000 claims abstract 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims abstract 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims abstract 2
- 235000005822 corn Nutrition 0.000 claims abstract 2
- 229920001231 Polysaccharide peptide Polymers 0.000 claims description 43
- 108010022457 polysaccharide peptide Proteins 0.000 claims description 43
- 238000000034 method Methods 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 239000002609 medium Substances 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 13
- 238000000605 extraction Methods 0.000 claims description 9
- 239000007787 solid Substances 0.000 claims description 9
- 235000015099 wheat brans Nutrition 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 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 8
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 8
- 229930006000 Sucrose Natural products 0.000 claims description 8
- 230000001580 bacterial effect Effects 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 238000011534 incubation Methods 0.000 claims description 8
- 239000005720 sucrose Substances 0.000 claims description 8
- 241000233866 Fungi Species 0.000 claims description 6
- 238000010899 nucleation Methods 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 6
- 229910019142 PO4 Inorganic materials 0.000 claims description 5
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 5
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 5
- 239000002054 inoculum Substances 0.000 claims description 5
- 231100000350 mutagenesis Toxicity 0.000 claims description 5
- 238000002703 mutagenesis Methods 0.000 claims description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 5
- 239000010452 phosphate Substances 0.000 claims description 5
- 229910052700 potassium Inorganic materials 0.000 claims description 5
- 239000011591 potassium Substances 0.000 claims description 5
- 238000010992 reflux Methods 0.000 claims description 5
- 239000002023 wood Substances 0.000 claims description 5
- 235000001674 Agaricus brunnescens Nutrition 0.000 claims description 4
- 229920000742 Cotton Polymers 0.000 claims description 4
- 240000007594 Oryza sativa Species 0.000 claims description 4
- 235000007164 Oryza sativa Nutrition 0.000 claims description 4
- 239000010440 gypsum Substances 0.000 claims description 4
- 229910052602 gypsum Inorganic materials 0.000 claims description 4
- 235000012204 lemonade/lime carbonate Nutrition 0.000 claims description 4
- 231100000219 mutagenic Toxicity 0.000 claims description 4
- 230000003505 mutagenic effect Effects 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 235000009566 rice Nutrition 0.000 claims description 4
- 239000010902 straw Substances 0.000 claims description 4
- 238000002955 isolation Methods 0.000 claims description 3
- 238000011068 loading method Methods 0.000 claims description 3
- 238000011218 seed culture Methods 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 241000283986 Lepus Species 0.000 claims description 2
- 244000061458 Solanum melongena Species 0.000 claims description 2
- 235000002597 Solanum melongena Nutrition 0.000 claims description 2
- 238000010564 aerobic fermentation Methods 0.000 claims description 2
- 238000009395 breeding Methods 0.000 claims description 2
- 230000001488 breeding effect Effects 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 239000012141 concentrate Substances 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 239000000706 filtrate Substances 0.000 claims description 2
- 238000005286 illumination Methods 0.000 claims description 2
- 230000001678 irradiating effect Effects 0.000 claims description 2
- 230000035800 maturation Effects 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 210000000582 semen Anatomy 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 claims description 2
- 238000004659 sterilization and disinfection Methods 0.000 claims description 2
- 238000000967 suction filtration Methods 0.000 claims description 2
- 238000009423 ventilation Methods 0.000 claims description 2
- 239000002244 precipitate Substances 0.000 claims 2
- 244000068988 Glycine max Species 0.000 claims 1
- 235000010469 Glycine max Nutrition 0.000 claims 1
- 238000013467 fragmentation Methods 0.000 claims 1
- 238000006062 fragmentation reaction Methods 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 2
- 241000222518 Agaricus Species 0.000 abstract 1
- -1 bran Substances 0.000 abstract 1
- 150000004676 glycans Chemical class 0.000 description 8
- 239000005017 polysaccharide Substances 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- 229920001282 polysaccharide Polymers 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 239000001965 potato dextrose agar Substances 0.000 description 6
- 239000000047 product Substances 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 4
- 244000061456 Solanum tuberosum Species 0.000 description 4
- 235000002595 Solanum tuberosum Nutrition 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 230000036528 appetite Effects 0.000 description 3
- 235000019789 appetite Nutrition 0.000 description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 206010047700 Vomiting Diseases 0.000 description 2
- 102000011759 adducin Human genes 0.000 description 2
- 108010076723 adducin Proteins 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000005138 cryopreservation Methods 0.000 description 2
- 230000036737 immune function Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 231100000956 nontoxicity Toxicity 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 0.000 description 1
- 229920002498 Beta-glucan Polymers 0.000 description 1
- 208000000419 Chronic Hepatitis B Diseases 0.000 description 1
- 235000014493 Crataegus Nutrition 0.000 description 1
- 241001092040 Crataegus Species 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 108010006464 Hemolysin Proteins Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 231100000215 acute (single dose) toxicity testing Toxicity 0.000 description 1
- 238000011047 acute toxicity test Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 231100000132 chronic toxicity testing Toxicity 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000000306 component Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000003228 hemolysin Substances 0.000 description 1
- 208000014617 hemorrhoid Diseases 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 230000002584 immunomodulator Effects 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 230000000527 lymphocytic effect Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000010907 mechanical stirring Methods 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 208000004296 neuralgia Diseases 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- ZRSNZINYAWTAHE-UHFFFAOYSA-N p-methoxybenzaldehyde Chemical compound COC1=CC=C(C=O)C=C1 ZRSNZINYAWTAHE-UHFFFAOYSA-N 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 238000011127 radiochemotherapy Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention belongs to the field of bioengineering technology, especially to a fermentation process of Agaricus Blazi murrill and a production process of polyglycopeptide. After its tissue separated, Agaricus Blazei murrill AB strain is purified, and then the strain is mutagenized physical and screened to obtain high-quality strain AB103-51 with great amount of liquid fermented mycelium biomass and high polyglycopeptide yield. The strain is made to pass through the fermentation process flow-path to obtain slant strain, shaking bottle seed, first-order seed, second-order seed and fermented Agaricus Blazei murrill liquid containing great amount of mycelium and polyglycopeptide successively by using corn powder, bran, starch, yeast powder, etc. as culture medium.
Description
Technical field
The invention belongs to technical field of bioengineering.Be specifically related to a kind of production method of zymotechnique and polysaccharide peptide thereof of Agaricus blazei Murrill.
Background technology
Agaricus blazei Murrill claims Brazilian mushroom, Xiao Song mushroom again, is the rare edible fungus of a kind of precious cutting edge of a knife or a sword.Its sporophore contains the multiple amino acids of various active compositions such as polysaccharide and sterols and various trace element, needed by human, to tumour, hemorrhoid, neurodynia tool curative effect, the effect that also has reducing blood-fat and decreasing cholesterol is a kind of medicinal fungi that the exploitation future is arranged very much.Declared patent in the U.S., Japanese multinomial achievement in research.
The main active ingredient of Agaricus blazei Murrill is an Agaricus Blazei Murrill polysaccharide, based on glucose, is primary structure with β-(1 → 3) dextran with β-(1 → 6) side chain in the polysaccharide component.Result of study shows, Agaricus Blazei Murrill polysaccharide is mainly by activating body immune system, strengthen the phagolysis of scavenger cell, stimulate the formation of Interferon, rabbit, activate natural killer cell in the body, promote the generation of immunoglobulin (Ig), interleukin-, improve lymphocytic transformation efficiency etc. and bring into play its antitumor action, stronger anti-chemomorphosis effect is arranged on gene level, can be used for behind chronic hepatitis B and the concurrent chemoradiotherapy of malignant tumor weak, oligoleukocythemia, the complex therapy that immunologic function reduces.
Summary of the invention
The objective of the invention is to improve production technique, improve the output of mycelial yield and polysaccharide peptide.
The invention provides a kind of production method of zymotechnique and polysaccharide peptide thereof of Agaricus blazei Murrill.This method comprises the following steps: the breeding and the feature of (1) Agaricus blazei Murrill
Obtain the pure strains A B103 of Agaricus blazei Murrill with tissue isolation, it is seeded on PDA or the wort constant inclined surface substratum, after 7～10 days, place the refrigerator about 4 ℃ to preserve standby in 25～28 ℃ of constant temperature culture.Adopt the ultraviolet mutagenesis method that hyphal cell is carried out mutagenesis, the uv irradiating distance is 20～30cm, and irradiation time is 5～15 seconds.The mycelial growth ability of primary dcreening operation mutagenic fungi on solid medium sieved mycelial growth amount and polysaccharide peptide output in the mutagenic fungi liquid medium within again, obtains quality strains AB103-51.This strain classification called after Brazilian mushroom (Agaricus blazei Murrill) Agaricus blazei, this bacterial strain have been stored in Chinese microbial preservation management committee common micro-organisms center on July 16th, 2002, the preservation center be numbered CGMCCNo.07722001325B.And detect the result on July 16th, 2002 and be survival.
Solid culture is with 40～50% straw (or wood chip, cotton seed hull), 1% sucrose or glucose, 10～20 wheat bran or rice bran, 1% gypsum or lime carbonate, poach, and water content 60%, the PH nature, bottled, at 1.5kg/m
2Down sterilization 1 hour of pressure, after the cooling, shake-flask seed is inoculated in the solid medium under aseptic technique, 22～26 ℃ of culture temperature, mycelium was covered with full bottle in 20～30 days.After original hase is sprouted, reduce by 22～25 ℃ of cultivation temperature, keep suitable humidity, illumination and ventilation, after 30～40 days, the sporophore maturation; (2) the zymotechnique flow process of bacterial strain
The present invention adopts the aerobic fermentation method to produce the Agaricus blazei filament, and fermention medium is with low cost, and fermentation period is short.The zymotechnique flow process is as follows:
Slant strains or eggplant bottle bacterial classification → shake-flask seed → 50-100L
3Seeding tank (one-level) → 500-1000L
3Seeding tank (secondary) → 5000-10 ton fermentor tank.
Loading amount 75～the 150ml/250ml of shake-flask seed or 150～150ml/250ml substratum, reciprocating type shaking table hunting speed 80～150 times/minute, or 100～200 rev/mins of rotary shaking table hunting speeds, 24～28 ℃ of culture temperature.Incubation time 2～3 days.Seeding tank and fermentor tank charging capacity 50～70%, inoculum size 8～10%, stirring velocity 100～200rpm, air flow 1: 0.5～1: 1v/vmin, fermented incubation time 4～7 days.Along with fermented incubation time prolongs, substratum is become clearly gradually by muddiness.Reducing sugar, ammonia-state nitrogen, PH reduce gradually in the fermented liquid, and mycelial biomass constantly increases, and polysaccharide peptide output reaches and stops fermentation when the highest during to the 5th～6 day.(3) extraction of Agaricus Blazei Murrill polysaccharide peptide
The polysaccharide peptide extraction process of Agaricus blazei Murrill AB103-51 of the present invention is: Agaricus blazei Murrill fermented liquid sintered filter funnel suction filtration, and separate fermentation liquid and mycelium, factory can use board and frame machine separate fermentation liquid and mycelium.Wet mycelium is added 10～20 times of water loggings bubble spend the night,, filter, add 0～4 ℃ of precipitation of 95% ethanol 8～10 hours of 2～5% times after filtrate is concentrated, the dry Agaricus Blazei Murrill polysaccharide peptide that gets with 90～100 ℃ of water-bath refluxing extraction 2～5 hours.
The AB103 bacterial classification that obtains through tissue isolation of the present invention, the sporophore meat, most single, grow thickly individually.The cap semisphere is extremely open and flat, diameter 3～10cm, and light brown to brown, there is fibrous scale on the cap surface, and there is the velum fragment at the edge.The nearly cylindrical cap central authorities of being born in of stem, the initial stage is solid, the middle and later periods is loose to hollow.Collarium easily comes off.The sporidium light red is to brown, and is smooth, short oval.The mycelia branch has tabula, no clamp connexion.The strains A B103-51 of physical mutagenesis gained cultivates on solid wood chip bran mass, and its sporophore shape is identical with natural entity.
The cultivation proterties of Agaricus blazei Murrill bacterial strain AB103-51 of the present invention is: 1. is seeded on the potato dextrose agar, and 24～28 ℃ of temperature, it is vigorous to grow, and mycelium pure white, velvet-like is inoculated ten days colony diameters and is reached 15-20mm.2. be seeded on the sawdust medium, its cultural characteristic as mentioned above.3. when shaking bottle shaking culture, use reciprocating type shaking table, hunting speed 80～150 times/minute, or rotary shaking table, 100～200 rev/mins of hunting speeds form bacterial strain, and when bacterium formed in a large number, fermented liquid became clear by muddiness, and viscosity increases gradually, distributes strong fruital flavor.4. in fermention medium, when cultivating 5～6 days, mycelial growth and polysaccharide peptide output reach the highest, can stop fermentation.
The feature of Agaricus blazei mycelium polysaccharide peptide is: 1. polysaccharide peptide, and polysaccharide content is 20～40%, protein content is 15～30%.2. grey is to the beige powder.3. major ingredient all is a beta-glucan.4. infrared extinction spectrum peak position is 883～892cm
-1
Medium component and the equal nontoxicity of all kinds of SOLVENTS that above-mentioned technological process is used, the Agaricus blazei filament of fermentative production and Agaricus Blazei Murrill polysaccharide peptide prove the equal nontoxicity of product in the product of the present invention through the acute toxicity test and the chronic toxicity test of national regulation.This product of life-time service not only can not produce any toxic side effect, can increase body's immunological function on the contrary.
The Agaricus blazei filament and the polysaccharide Toplink that are obtained from the foregoing invention method obviously promote phagocytosis of macrophages, can resist the white cell that is caused by endoxan and descend and appetite stimulator; Can improve the content of immunoglobulin (Ig), complement and the serum hemolysin of tumor mice; Can suppress the growth of tumour cell.Can be used for clinical antitumorly, the caused side effect of ameliorate tumor chemotherapy of patients and radiotherapy as symptoms such as nausea and vomiting, poor appetite, leucocytes reduction, improves life quality.And can be used as the healthcare products of subhealth state personage enhance immunity function.
Present method fermentation period is short, and production cost is low, product mycelium and polysaccharide peptide safety non-toxic.The polysaccharide peptide that extracts from the Agaricus blazei filament has the adjusting body's immunity, suppress growth of tumour cell, to symptom effects of having clear improvement such as the nausea and vomiting of the tumour patient of chemotherapy and radiation, poor appetite, leucocytes reduction, be a kind of novel biological immunomodulator.
Embodiment
Embodiment 1
The Agaricus blazei Murrill sporophore is through separate tissue, obtains purebredly, carries out the inclined-plane cryopreservation or liquid nitrogen is preserved with purebred, is seeded on the PDA slant medium, cultivates 7～10 days for 24～26 ℃.
The PDA prescription is as follows:
Potato 200g
Glucose 20g
Agar 20g
Water 1000ml
The peeling of bell potato, stripping and slicing added water boil after 20～30 minutes, and 4 layers of filtered through gauze are got juice.
Slant strains is seeded in the 500ml triangular flask that the 100ml liquid nutrient medium is housed shaking culture.Adopt reciprocating type shaking table (hunting speed 80～150 times/minute) or rotary shaking table (100～200 rev/mins of hunting speeds), 24～28 ℃ of shaking culture degree.Cultivate and little bacterium ball occurred in second day, the microscopy mycelial growth is in great numbers, multi-branched, tool tabula, the five colors, transparent, tool clamp connexion, and fermentation is to the 5th～7 day fermented liquid thickness, this moment mycelial growth amount maximum, polysaccharide peptide output is the highest, can stop fermentation.
Fermentative medium formula is as follows:
Starch 3%
Wheat bran 0.3%
Sucrose 1%
Yeast powder 0.2%
Sal epsom 0.05%
Potassium primary phosphate 0.1%
Fermented liquid after centrifugal, the Agaricus blazei filament, add 10 times in water after, smash cell with the cell stamp mill, add 90 ℃ of hot water refluxing extraction 3～5 hours then, extracting solution is concentrated through film under vacuum again, get 1 part and concentrate and to add 3 part of 95% ethanol, alcohol was analysed 8 hours under 0～4 ℃ of condition.After vacuum-drying or lyophilize, can obtain the Agaricus Blazei Murrill polysaccharide peptide of brown powder shape.After getting polysaccharide peptide and adding water (1: 100 weight ratio) dissolving, (Sdphadex-G75) carries out chromatographic separation with glucose gel, with the polysaccharide and the polypeptide of ultraviolet absorption method and phenol sulfuric acid mensuration chromatographic solution, sample polysaccharide and polypeptide occur in collecting test tube, prove the Agaricus Blazei Murrill polysaccharide peptide.
Embodiment 2
The Agaricus blazei Murrill sporophore is through separate tissue, obtains purebredly, carries out the inclined-plane cryopreservation or liquid nitrogen is preserved with purebred, is seeded on the comprehensive PDA slant medium, cultivates 7～10 days for 24～26 ℃.
The comprehensive PDA prescription is as follows:
Potato 200g
Sucrose 20g
Wheat bran 100g
KH
2PO
4 3g
MgSO
47H
2O 1.5g
Agar 20g
Water 1000ml
Peeling potatoes, stripping and slicing added water boil after 20～30 minutes, and 4 layers of filtered through gauze are got juice.Wheat bran added water boil after 20～30 minutes, and 4 layers of filtered through gauze are got juice.
Slant strains is seeded in the 500ml triangular flask that the 100ml seed culture medium is housed shaking culture.Adopt compound shaking table (hunting speed 80～150 times/minute) or rotary shaking table (100～200 rev/mins of hunting speeds), 24～28 ℃ of shaking culture temperature.Go in 50～100L seeding tank with the pressure differential method culture transferring after two days, canned amount 70% (v/v), inoculum size 10% (v/v), aerated culture moves into 500～1000L fermentor tank after 2 days.Fermentor tank loading amount 70% (v/v), inoculum size 10% (v/v), 24～30 ℃ of jar temperature, 1: 1 (v/v) mechanical stirring of air flow speed, 100～200rpm.5～7 days fermented liquid thickness of fermentation to the, this moment mycelial growth amount maximum, polysaccharide peptide output is the highest, can stop fermentation.
The seed culture based formulas is as follows:
Semen Maydis powder 3%
Starch 2%
Potassium primary phosphate 0.1%
Sal epsom 0.05%
Fermentative medium formula is as follows:
Starch 3%
Wheat bran 0.3%
Sucrose 1%
Yeast powder 0.2%
Sal epsom 0.05%
Potassium primary phosphate 0.1%
Fermented liquid behind filter press, the Agaricus blazei filament, add 10 times in water after, smash cell with the cell stamp mill, add 95 ℃ of hot water refluxing extraction 4 hours then, the extracting solution film under vacuum is concentrated, add 95% ethanol of 3 times of volumes of water, low temperature alcohol is analysed.Alcohol is analysed thing and is washed respectively 3 times with anhydrous propanone and anhydrous diethyl ether, in 50 ℃ of oven dry down, obtains the Agaricus Blazei Murrill polysaccharide peptide of brown powder shape.After getting polysaccharide peptide and adding water (1: 100 weight ratio) dissolving, carry out the TLC chromatography with thin the analysing of two blocks of silica gel, with triketohydrindene hydrate and aubepine colour developing, the Rf value unanimity that two boards shows proves that the sample that is extracted is the Agaricus Blazei Murrill polysaccharide peptide respectively.
Claims (13)
1, the production method of a kind of zymotechnique of Agaricus blazei Murrill and polysaccharide peptide thereof is characterized in that this method comprises the following steps: the breeding and the feature of (1) Agaricus blazei Murrill
Obtain the pure strains A B103 of Agaricus blazei Murrill with tissue isolation, it is seeded on PDA or the wort constant inclined surface substratum, in 25～28 ℃ of constant temperature culture after 7～10 days, place the refrigerator about 4 ℃ to preserve standby, adopt the ultraviolet mutagenesis method that hyphal cell is carried out mutagenesis, the uv irradiating distance is 20～30cm, irradiation time is 5～15 seconds, the mycelial growth ability of primary dcreening operation mutagenic fungi on solid medium, sieve mycelial growth amount and polysaccharide peptide output in the mutagenic fungi liquid medium within again, obtain quality strains AB103-51;
Solid culture is with 40～50% straw or wood chip, cotton seed hull, 1% sucrose or glucose, and 10～20% wheat brans or rice bran, 1% gypsum or lime carbonate, poach, water content 60%, the PH nature, bottled, at 1.5kg/m
2Pressure down sterilization 1 hour with, after the cooling, shake-flask seed is inoculated in the solid medium under aseptic technique, 22～26 ℃ of culture temperature, mycelium was covered with full bottle in 20～30 days, after original hase is sprouted, reduce by 22～25 ℃ of cultivation temperature, keep proper temperature, illumination and ventilation, after 30～40 days, the sporophore maturation; (2) the zymotechnique flow process of bacterial strain
The present invention adopts the aerobic fermentation method to produce the Agaricus blazei filament, and fermention medium is with low cost, and fermentation period is short, and the zymotechnique flow process is as follows:
Slant strains or eggplant bottle bacterial classification → shake-flask seed → 50-100L
3Seeding tank, one-level → 500-1000L
3Seeding tank, secondary → 5000-10 ton fermentor tank,
Loading amount 75～the 150ml/250ml of shake-flask seed or 150～150ml/250ml substratum, reciprocating type shaking table hunting speed 80～150 times/minute, or 100～200 rev/mins of rotary shaking table hunting speeds, culture temperature 100～200rpm, air flow 1: 0.5～1: 1v/v min, fermented incubation time 4～7 days, along with fermented incubation time prolongs, substratum is become clearly gradually by muddiness, reducing sugar, ammonia-state nitrogen, PH reduce gradually in the fermented liquid, mycelial biomass constantly increases, polysaccharide peptide output during to the 5th～6 day; (3) extraction of Agaricus Blazei Murrill polysaccharide peptide
The polysaccharide peptide extraction process of Agaricus blazei Murrill AB103-51 of the present invention is: Agaricus blazei Murrill fermented liquid sintered filter funnel suction filtration, separate fermentation liquid and mycelium, factory can use board and frame machine separate fermentation liquid and mycelium, wet mycelium is added 10～20 times of water logging bubbles to spend the night, with 90～100 ℃ of water-bath refluxing extraction 2～5 hours, filter, 95% ethanol that adds 2～5% times after filtrate is concentrated precipitates 8～10 hours for 0～4 ℃, the dry Agaricus Blazei Murrill polysaccharide peptide that gets.
2, the production method of the zymotechnique of a kind of Agaricus blazei Murrill according to claim 1 and polysaccharide peptide thereof, it is characterized in that the classification called after Brazilian mushroom Agaricus blazei of wherein said quality strains AB 103-51, deposit number is CGMCC No.07722001325B.
3, the production method of the zymotechnique of a kind of Agaricus blazei Murrill according to claim 1 and polysaccharide peptide thereof, the zymotechnique that it is characterized in that wherein said quality strains AB103-51, the feature of its seed culture medium are substratum Semen Maydis powder 1～3g, starch 1～2g, sal epsom 0.05～0.1g, the potassium primary phosphate 0.1～0.2g of 100ml.
4, the production method of the zymotechnique of a kind of Agaricus blazei Murrill according to claim 1 and polysaccharide peptide thereof, the zymotechnique that it is characterized in that wherein said quality strains AB103-51, the feature of its fermention medium are that the proportioning of the substratum of 100ml is starch 1～3g, wheat bran 0.1～0.6g, glucose or sucrose 0.5～1g, yeast powder 0.1～0.5g, sal epsom 0.05～0.2g, potassium primary phosphate 0.1～0.3g.
5, the production method of the zymotechnique of a kind of Agaricus blazei Murrill according to claim 1 and polysaccharide peptide thereof, the feature that it is characterized in that wherein said zymotechnique be fermentor cultivation can add bubble enemy or soya-bean oil an amount of.
6, the production method of the zymotechnique of a kind of Agaricus blazei Murrill according to claim 1 and polysaccharide peptide thereof, the feature that it is characterized in that wherein said zymotechnique are can add corn steep liquor in the substratum, and addition is 0.1～1%.
7, the production method of the zymotechnique of a kind of Agaricus blazei Murrill according to claim 1 and polysaccharide peptide thereof, the feature that it is characterized in that wherein said zymotechnique is that the fermentation condition 250ml that shakes the bottle kind shakes bottled amount 75～150ml substratum, 500ml shakes bottled amount 150～250ml substratum, adopt reciprocating type shaking table, hunting speed 80～150 times/minute, or rotary shaking table, 100～200 rev/mins of hunting speed degree, 24～28 ℃ of culture temperature, incubation time 2～3 days.
8, the production method of the zymotechnique of a kind of Agaricus blazei Murrill according to claim 1 and polysaccharide peptide thereof, the feature that it is characterized in that wherein said zymotechnique is a fermentation condition: inoculum size 8～10%, stirring velocity 100～200rpm, air flow 1: 0.5～1: 1v/v min, leavening temperature is 24～28 ℃, tank pressure 0.3～0.5kg/cm
2, fermented incubation time 2～3 days.
9, the production method of the zymotechnique of a kind of Agaricus blazei Murrill according to claim 1 and polysaccharide peptide thereof, the feature that it is characterized in that wherein said zymotechnique is a fermentation condition: inoculum size 8～10%, stirring velocity 100～200rpm, air flow 1: 0.5～1: 1v/v min, leavening temperature is 24～28 ℃, tank pressure 0.3～0.5kg/cm
2, fermented incubation time 4～7 days.
10, the production method of the zymotechnique of a kind of Agaricus blazei Murrill according to claim 1 and polysaccharide peptide thereof, the solid culture that it is characterized in that wherein a kind of Agaricus blazei Murrill G strains A B103-51 is with straw or wood chip, cotton seed hull, sucrose or glucose, the substratum that wheat bran or rice bran, gypsum or lime carbonate are formed.
11, the production method of the zymotechnique of a kind of Agaricus blazei Murrill according to claim 1 and polysaccharide peptide thereof, the feature that it is characterized in that wherein said culture process is that the percentage composition of substratum is straw or wood chip, cotton seed hull 40～50%, sucrose or glucose 1%, wheat bran or rice bran 10～20%, gypsum or lime carbonate 1%.
12, the production method of the zymotechnique of a kind of Agaricus blazei Murrill according to claim 1 and polysaccharide peptide thereof, it is characterized in that wherein said Agaricus blazei Murrill culture filter the mycelium of AB103-51, after the hyphal cell fragmentation, the temperature mycelium adds 10-20 times of water, refluxing extraction is 2～5 hours in 90～100 ℃ of hot water, gets the mycelium extracting solution.
13, the production method of the zymotechnique of a kind of Agaricus blazei Murrill according to claim 1 and polysaccharide peptide thereof, it is characterized in that adding 2～5 times 95% ethanol after wherein said mycelium extracting solution concentrates, precipitate 8～10 hours at 0～4 ℃, the chromatography thing is dry must dun Agaricus Blazei Murrill polysaccharide peptide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02136516 CN1398978A (en) | 2002-08-16 | 2002-08-16 | Fermentation process of Agaricus Blazei murrill and production process of its polyglycopeptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02136516 CN1398978A (en) | 2002-08-16 | 2002-08-16 | Fermentation process of Agaricus Blazei murrill and production process of its polyglycopeptide |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1398978A true CN1398978A (en) | 2003-02-26 |
Family
ID=4748678
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 02136516 Pending CN1398978A (en) | 2002-08-16 | 2002-08-16 | Fermentation process of Agaricus Blazei murrill and production process of its polyglycopeptide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1398978A (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1320102C (en) * | 2005-03-31 | 2007-06-06 | 章克昌 | Method for culturing edible fungus jisong rong zinc enriched liquid |
| CN101015379B (en) * | 2006-12-28 | 2010-05-12 | 沈阳化工学院 | A kind of preparation method of Agaricus blazei health care liquid |
| CN102191297A (en) * | 2011-04-06 | 2011-09-21 | 黑龙江省轻工科学研究院 | Method for making tricholoma matsutake polysaccharides |
| CN102771764A (en) * | 2012-07-20 | 2012-11-14 | 黄晓青 | Pine mushroom health product and preparation method thereof |
| TWI400334B (en) * | 2010-11-29 | 2013-07-01 | Univ Dayeh | Methods of enhancing extracellular polysaccharopeptides and intracellular polysaccharopeptides from trametes versicolor lh1 with mycelium immobilization |
| CN104543666A (en) * | 2014-12-04 | 2015-04-29 | 徐立华 | Composite polysaccharide heath food with anti-radiation function and preparation process of composite polysaccharide heath food |
| CN104312920B (en) * | 2014-10-18 | 2017-07-07 | 福建农林大学 | A kind of Agaricus blazei liquid nitrogen preservation method |
| CN114667890A (en) * | 2021-12-20 | 2022-06-28 | 延边圣泽方圆生物科技有限公司 | A method for separating and cultivating strains and mycelium of pine mushroom fruit bodies, and preparation method of pine mushroom mycelium powder and concentrate |
-
2002
- 2002-08-16 CN CN 02136516 patent/CN1398978A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1320102C (en) * | 2005-03-31 | 2007-06-06 | 章克昌 | Method for culturing edible fungus jisong rong zinc enriched liquid |
| CN101015379B (en) * | 2006-12-28 | 2010-05-12 | 沈阳化工学院 | A kind of preparation method of Agaricus blazei health care liquid |
| TWI400334B (en) * | 2010-11-29 | 2013-07-01 | Univ Dayeh | Methods of enhancing extracellular polysaccharopeptides and intracellular polysaccharopeptides from trametes versicolor lh1 with mycelium immobilization |
| CN102191297A (en) * | 2011-04-06 | 2011-09-21 | 黑龙江省轻工科学研究院 | Method for making tricholoma matsutake polysaccharides |
| CN102771764A (en) * | 2012-07-20 | 2012-11-14 | 黄晓青 | Pine mushroom health product and preparation method thereof |
| CN102771764B (en) * | 2012-07-20 | 2015-09-02 | 北京厚文知识产权顾问有限公司 | A kind of pine mushroom health product and preparation method thereof |
| CN104312920B (en) * | 2014-10-18 | 2017-07-07 | 福建农林大学 | A kind of Agaricus blazei liquid nitrogen preservation method |
| CN104543666A (en) * | 2014-12-04 | 2015-04-29 | 徐立华 | Composite polysaccharide heath food with anti-radiation function and preparation process of composite polysaccharide heath food |
| CN114667890A (en) * | 2021-12-20 | 2022-06-28 | 延边圣泽方圆生物科技有限公司 | A method for separating and cultivating strains and mycelium of pine mushroom fruit bodies, and preparation method of pine mushroom mycelium powder and concentrate |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1273004C (en) | Method for producing hedgehog fungus and special culture medium thereof | |
| CN103330258A (en) | Cordyceps militaris health-care beverage prepared by liquid submerged fermentation and preparation method thereof | |
| EP1280543A2 (en) | Process for producing, methods and compositions of glucuronoxylomannan as nutriceutical agent from higher basidiomycetes mushroom | |
| CN1252245C (en) | Method of artificial planting northerly Chinese caterpillar fungus for increasing content of physiologically active substance | |
| CN1398979A (en) | Fermentation process of ash tree flower and production process of its polyglycopeptide | |
| CN1796539A (en) | Ferment for producing aweto in large scale and technique for processing power of fungus | |
| CN104285667A (en) | Cultivation method for improving polysaccharide content of grifola frondosa | |
| Dulay et al. | Optimization of submerged culture conditions for mycelial biomass production of fourteen Lentinus isolates from Luzon Island, Philippines | |
| CN110129216A (en) | A kind of Bacillus subtilis mutagenesis strain suitable for solid fermentation to produce γ-polyglutamic acid and its cultivation method | |
| CN106978353A (en) | Te Shi Xylaria sp. fungus and its cultural method | |
| CN1398978A (en) | Fermentation process of Agaricus Blazei murrill and production process of its polyglycopeptide | |
| CN110923281B (en) | Edible fungus polysaccharide extraction method, edible fungus polysaccharide and edible fungus beverage | |
| CN1478886A (en) | Chinese beimao spore liquid culture fermentationi technology | |
| CN104429621B (en) | A kind of production method of the hericium mycelium for extracting Hericium Erinaceus Polysaccharide | |
| CN113875496B (en) | Rejuvenation method of cordyceps militaris strains | |
| CN101836596A (en) | New strain of high-cellulose Pleurotus djamor | |
| CN108770592A (en) | A kind of cultural method of lemon squama umbrella cultivation culture medium and lemon squama umbrella | |
| CN1271737A (en) | Process for preparing amylovorin of sea bacteria and its application | |
| CN1279159C (en) | CZ-81 strain of 'Baibachi' bacterium and artificial culture method | |
| CN1109508C (en) | Hericium erinaceus noodles and industrialized liquid fermentation producing technology for Hericium erinaceus baterial strain | |
| CN1303905C (en) | Microbial technology deep process method for tuber preliminary working leftover | |
| CN101270337A (en) | A method for increasing the yield of diosgenin in cultured cells by utilizing endophytic Fusarium oligosaccharides from Dioscorea scutellaria | |
| CN115039633B (en) | A method for artificially cultivating the fruiting body of Corymborhinus japonicus | |
| CN1109507C (en) | Edible mushroom Huishuhua noodles and industrialized liquid fermentation producing technology for bacterial strain | |
| CN1944626A (en) | Microbial bacteria for preparing ginkalide by microbial fermenting and method for preparing ginkalide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |