CN116254306A - Method for fermenting high-yield tremella polysaccharide - Google Patents
Method for fermenting high-yield tremella polysaccharide Download PDFInfo
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- 241001506047 Tremella Species 0.000 title claims abstract description 73
- 150000004676 glycans Chemical class 0.000 title claims abstract description 58
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 58
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 58
- 238000000034 method Methods 0.000 title claims description 28
- 238000000855 fermentation Methods 0.000 claims abstract description 54
- 230000004151 fermentation Effects 0.000 claims abstract description 54
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 11
- 238000001694 spray drying Methods 0.000 claims abstract description 10
- 238000004519 manufacturing process Methods 0.000 claims abstract description 9
- 239000012535 impurity Substances 0.000 claims abstract description 8
- 239000012528 membrane Substances 0.000 claims abstract description 8
- 239000000049 pigment Substances 0.000 claims abstract description 7
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 7
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 7
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 30
- 239000007788 liquid Substances 0.000 claims description 25
- 239000001888 Peptone Substances 0.000 claims description 15
- 108010080698 Peptones Proteins 0.000 claims description 15
- 239000001963 growth medium Substances 0.000 claims description 15
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 15
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 15
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 15
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 15
- 235000019319 peptone Nutrition 0.000 claims description 15
- JZRWCGZRTZMZEH-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 12
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 12
- 229930006000 Sucrose Natural products 0.000 claims description 9
- 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 9
- 239000005720 sucrose Substances 0.000 claims description 9
- 239000000047 product Substances 0.000 claims description 7
- 229920001817 Agar Polymers 0.000 claims description 6
- 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 6
- 239000008272 agar Substances 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 238000011081 inoculation Methods 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- 238000009630 liquid culture Methods 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 238000009423 ventilation Methods 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 241000908178 Tremella fuciformis Species 0.000 claims 2
- 238000005119 centrifugation Methods 0.000 claims 2
- 230000001954 sterilising effect Effects 0.000 claims 1
- 238000000605 extraction Methods 0.000 abstract description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 4
- 238000002360 preparation method Methods 0.000 abstract description 4
- 230000007613 environmental effect Effects 0.000 abstract description 2
- 238000005189 flocculation Methods 0.000 abstract description 2
- 230000016615 flocculation Effects 0.000 abstract description 2
- 238000009395 breeding Methods 0.000 abstract 1
- 230000001488 breeding effect Effects 0.000 abstract 1
- 238000004321 preservation Methods 0.000 description 13
- 238000012216 screening Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 4
- 238000007865 diluting Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 239000003471 mutagenic agent Substances 0.000 description 2
- 231100000707 mutagenic chemical Toxicity 0.000 description 2
- 230000003505 mutagenic effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 1
- 239000004909 Moisturizer Substances 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000003471 anti-radiation Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- DENRZWYUOJLTMF-UHFFFAOYSA-N diethyl sulfate Chemical compound CCOS(=O)(=O)OCC DENRZWYUOJLTMF-UHFFFAOYSA-N 0.000 description 1
- 229940008406 diethyl sulfate Drugs 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
- 230000000694 effects Effects 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 229940097043 glucuronic acid Drugs 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000001333 moisturizer Effects 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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Abstract
The invention relates to a preparation method of tremella polysaccharide with high yield by fermentation. The tremella polysaccharide high-yield strain is obtained by breeding tremella fruiting bodies, and the tremella polysaccharide yield is up to 40-50g/L by fermenting the strain; concentrating with ultrafiltration membrane, removing impurities such as protein and pigment, and spray drying to replace conventional multiple alcohol flocculation product extraction process to obtain white tremella polysaccharide with total sugar content up to 99.5% or more, protein content below 0.3% and molecular weight of 100-3000 kDa; the technology has the advantages of high polysaccharide yield, high total sugar content of the product, low production cost, simple and safe operation, environmental protection and the like, and has great industrialized significance.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a preparation method of tremella polysaccharide with high fermentation yield.
Background
Tremella polysaccharides are important active substances present in the fruiting bodies, mycelia and fermentation broth of silver ear. The tremella polysaccharide is formed by connecting xylose, mannose and glucuronic acid through alpha-1, 3-glycosidic bond, and the side chain is composed of galactose, arabinose and a small amount of fucose. During the last decades, many studies have shown that tremella polysaccharides have moisturizing, gelling, anti-inflammatory, wound healing promoting, antioxidant, anti-aging and anti-radiation effects. Tremella polysaccharide is used as a safe and nontoxic natural active product and has been clinically used as an anti-tumor drug; in the cosmetic industry, tremella polysaccharides can be used as moisturizers.
The most of the methods for obtaining tremella polysaccharide in the market are fruiting body extraction methods, and the extraction methods have obvious defects and mainly comprise the following steps: the raw materials are limited, the extraction steps are complicated, the energy consumption is high, the yield is low, and the cost is high; compared with the extraction method, the tremella spore fermentation method has the advantages of short production period, high polysaccharide purity, easy industrialization and the like. However, the existing fermentation method for producing tremella polysaccharide still has the problems of low yield, complex production procedures, high cost and the like, and can not meet the increasing demands of tremella polysaccharide in the market.
Disclosure of Invention
The invention aims to solve the problems of low yield, complicated production procedures and high cost of the existing tremella polysaccharide, and provides a preparation method of the tremella polysaccharide with high yield, simple process steps and low cost.
The invention has the following overall technical concept:
the tremella polysaccharide is produced by a fermentation method, and the method comprises the following process steps:
(1) Tremella is preparedTremella fuciformis) Strain (strain number used: CGMCC No.23287, strain preservation address: beijing city, chaoyang district, north Chen Xili No. 1,3, date of preservation: 2021, 10 and 12. ) Slant culture medium inoculated into test tube to prepare slant bacteriaSeed;
(2) Performing enlarged culture on the inclined plane strain to obtain seed liquid;
(3) Inoculating the seed liquid into a fermentation tank for fermentation to produce tremella polysaccharide;
(4) Separating and purifying tremella polysaccharide.
The specific process steps and process conditions of the invention are as follows:
the slant culture medium in the step (1) consists of the following components in parts by weight:
10-20 parts of glucose, 1-5 parts of peptone, 1-2 parts of magnesium sulfate, 1-5 parts of monopotassium phosphate and vitamin B 1 0.01-0.03 part, 15 parts of agar, and adding water to 1000 parts;
the process conditions for preparing the inclined plane strain are as follows: the culture temperature is 20-30deg.C, and the culture time is 12-48h.
The seed liquid in the step (2) and the fermentation liquid in the step (3) consist of the following components in parts by weight:
20-80 parts of sucrose, 1-5 parts of peptone, 1-2 parts of magnesium sulfate, 1-5 parts of monopotassium phosphate and vitamin B 1 0.01-0.03 part and 1000 parts of water;
the seed liquid culture conditions are as follows: the culture temperature is 20-30 ℃, the rotation speed of the shaking table is 100-200r/min, and the culture time is 12-48h; then the mixture is connected into a fermentation tank, and the fermentation process conditions of the fermentation tank are as follows: the inoculation amount is 10%, the fermentation temperature is 20-30 ℃, the ventilation amount is 0.5-2vvm, the stirring speed is 100-900r/min, the culture time is 48-96h, and the tremella polysaccharide yield is 40-50g/L.
The separation and purification of the tremella polysaccharide product in the step (4) comprises the following three steps:
a. centrifuging the fermented liquid after fermentation for 20min at 8000r/min to remove thalli;
b. concentrating the supernatant obtained in step a by adopting an ultrafiltration membrane with the molecular weight cut-off of 60-80kDa, and removing impurities such as protein, pigment and the like;
c. drying the concentrated solution obtained in the step b in a spray dryer with the temperature of a feed inlet of 140-160 ℃; the total sugar of the dried tremella polysaccharide reaches more than 99.5%, the protein content is less than 0.3%, and the molecular weight is 100-3000kDa.
Tremella used in the inventionTremella fuciformis) The CGMCC No.23287 strain is preserved in China general microbiological culture Collection center (CGMCC No. 23287) and the preservation date is 2021, 10 and 12.
Screening of tremella CGMCC No.23287 strain (the used strain number is CGMCC No.23287, the strain preservation address is 1 rd hospital No. 3 of North western road in the Korean area of Beijing, the preservation date is 2021, 10 months and 12 days), which comprises the following process steps:
(1) Collecting mature fresh wild tremella fruiting body with large flower, white color, thick flesh, no impurity bacteria and infection of diseases and insects, inoculating slant to obtain tremella slant strain;
(2) Selecting tremella inclined plane strains to perform plate coating to obtain tremella plate colonies;
(3) Inoculating tremella plate colony to shake flask for fermentation culture, and performing primary screening and secondary screening;
(4) And (3) carrying out composite mutagenesis on the tremella strains after re-screening, and screening high-yield tremella strains.
The slant culture medium in the step (1) and the plate culture medium in the step (2) consist of the following components in parts by weight:
glucose 20 parts, peptone 2 parts, magnesium sulfate 1.5 parts, potassium dihydrogen phosphate 3 parts, and vitamin B 1 0.02 part of agar 15 parts, and adding water to 1000 parts;
the process conditions for preparing the inclined plane strain are as follows: the culture temperature is 25 ℃ and the culture time is 24 hours.
The fermentation liquor in the step (3) consists of the following components in parts by weight:
60 parts of sucrose, 3 parts of peptone, 1.5 parts of magnesium sulfate, 3 parts of monopotassium phosphate and vitamin B 1 0.02 parts and 1000 parts of water.
The fermentation process conditions are as follows: the inoculation amount of the fermentation broth is 10%, the culture temperature is 25 ℃, the rotation speed of a shaking table is 150r/min, and the fermentation time is 72h.
The mutagenesis in the step (4) sequentially comprises the preparation of seed bacterial suspension, the addition amount of a mutagen and the ultraviolet irradiation time
Inoculating the inclined plane strain into a test tube containing 5mL of physiological saline, adding diethyl sulfate mutagen with the volume percentage of 0.5% -2.0% into the test tube, oscillating for 1-2min on a vortex oscillator, taking 0.5mL of the strain suspension to coat a flat plate, placing the flat plate in an ultraviolet lamp for irradiation for 20-60s, and then culturing the flat plate in a dark state. The culture conditions are as follows: the culture temperature is 25 ℃ and the culture time is 24 hours.
Finally, obtaining tremellaTremella fuciformis) Strain (strain number used: CGMCC No.23287, strain preservation address: beijing city, chaoyang district, north Chen Xili No. 1,3, date of preservation: 2021, 10, 12).
The technical progress of the invention is that:
(1) The yield is high: by utilizing the technology, each liter of fermentation liquor contains 40-50g of tremella polysaccharide;
(2) The purity is high: the project adopts ultrafiltration membrane to remove impurities such as protein, pigment and the like, and the total sugar content of the obtained tremella polysaccharide is up to more than 99.5%, so that a foundation is laid for the application of tremella polysaccharide in the fields of medicine and daily chemicals;
(3) The operation is safe: compared with the traditional ethanol multiple flocculation purification process, the spray drying technology adopted in the project has the advantages of simple process, safe operation, low cost and the like.
In conclusion, the technology has the advantages of high yield, high polysaccharide content in the product, low production cost, simple and safe operation, environmental friendliness and the like.
Examples: the invention is further described below with reference to examples.
Example 1
(1) Slant culture of strain
Inoculating tremella CGMCC No.23287 strain (the strain number is CGMCC No.23287, the strain preservation address is 1 # 3 of North western road of the Korean area of Beijing, and the preservation date is 2021, 10 months and 12 days) to a slant culture medium in a test tube to prepare slant strain;
wherein the strain inclined plane culture medium consists of the following components in parts by weight:
10 parts of glucose, 5 parts of peptone, 1 part of magnesium sulfate, 5 parts of potassium dihydrogen phosphate and vitamin B 1 0.03 part of agar 15 parts, and adding water to 1000 parts;
the process conditions for preparing the inclined plane strain are as follows: the culture temperature is 25 ℃ and the culture time is 12 hours.
(2) Seed liquid fermentation
Performing enlarged culture on the inclined plane strain to obtain seed liquid;
the seed liquid culture medium consists of the following components in parts by weight:
20 parts of sucrose, 5 parts of peptone, 1 part of magnesium sulfate, 5 parts of monopotassium phosphate and vitamin B 1 0.03 parts and 1000 parts of water.
The culture condition of the prepared seed liquid is that the culture temperature is 25 ℃, the rotation speed of a shaking table is 150r/min, and the culture time is 12h.
(3) Fermentation production of tremella polysaccharide
Inoculating the seed liquid into a fermentation tank for fermentation to produce tremella polysaccharide;
wherein the fermentation liquor consists of the following components in parts by weight:
80 parts of sucrose, 3 parts of peptone, 1.5 parts of magnesium sulfate, 3 parts of monopotassium phosphate and vitamin B 1 0.02 parts and 1000 parts of water.
The fermentation process conditions are as follows: the inoculation amount is 10%, the fermentation temperature is 25 ℃, the ventilation amount is 2vvm, the stirring speed is 100-900r/min, the fermentation time is 72h, and the tremella polysaccharide yield is 48g/L.
(4) The separation and purification of the tremella polysaccharide product comprises the following three steps:
a. diluting the fermented liquid after fermentation for 2 times, and centrifuging for 20min at 8000 r/min;
b. carrying out ultrafiltration concentration on the supernatant fluid obtained in the step a by adopting an ultrafiltration membrane with the molecular weight cut-off of 60-80kDa, and simultaneously removing impurities such as protein, pigment and the like;
c. and c, spray-drying the concentrated solution obtained in the step b to obtain white tremella polysaccharide powder with the total sugar content of more than 99.5 percent, the protein content of less than 0.3 percent and the molecular weight of 100-3000kDa, wherein the air inlet temperature of spray drying is 150 ℃.
Example 2
(1) Slant culture of strain
Inoculating tremella strain (CGMCC No.23287, strain preservation address: north Xili No. 1, no. 3, preservation date: 2021, 10 months and 12 days) into slant culture medium in test tube to obtain slant strain;
wherein the strain inclined plane culture medium consists of the following components in parts by weight:
15 parts of glucose, 3 parts of peptone, 2 parts of magnesium sulfate, 3 parts of monopotassium phosphate and vitamin B 1 0.01 part of agar 15 parts, and water is added to 1000 parts.
The process conditions for preparing the inclined plane strain are as follows: the culture temperature is 20 ℃ and the culture time is 48 hours.
(2) Seed liquid fermentation
Performing enlarged culture on the inclined plane strain to obtain seed liquid;
the seed liquid culture medium consists of the following components in parts by weight:
25 parts of sucrose, 3 parts of peptone, 2 parts of magnesium sulfate, 3 parts of monopotassium phosphate and vitamin B 1 0.01 part and 1000 parts of water.
The culture conditions of the prepared seed liquid are that the culture temperature is 20 ℃, the rotation speed of a shaking table is 100r/min, and the culture time is 48 hours.
(3) Fermenting to produce tremella polysaccharide;
inoculating the seed liquid into a fermentation tank for fermentation to produce tremella polysaccharide;
wherein the fermentation liquor consists of the following components in parts by weight:
70 parts of sucrose, 1 part of peptone, 2 parts of magnesium sulfate, 5 parts of monopotassium phosphate and vitamin B 1 0.03 parts and 1000 parts of water.
The fermentation process conditions are as follows: the inoculation amount is 10%, the fermentation temperature is 30 ℃, the ventilation amount is 0.5vvm, the stirring speed is 100-900r/min, the fermentation time is 96h, and the tremella polysaccharide yield is 45g/L.
(4) The separation and purification of the tremella polysaccharide product comprises the following three steps:
a. diluting the fermented liquid after fermentation for 2 times, and centrifuging for 30min under 10000 r/min;
b. carrying out ultrafiltration concentration on the supernatant fluid obtained in the step a by adopting an ultrafiltration membrane with the molecular weight cut-off of 60-80kDa, and simultaneously removing impurities such as protein, pigment and the like;
c. and c, spray-drying the concentrated solution obtained in the step b to obtain white tremella polysaccharide powder with the total sugar content of more than 99.5 percent, the protein content of less than 0.3 percent and the molecular weight of 100-3000kDa, wherein the air inlet temperature of spray drying is 160 ℃.
Example 3
(1) Slant culture of strain
Inoculating tremella strain (CGMCC No.23287, strain preservation address: north Xili No. 1, no. 3, preservation date: 2021, 10 months and 12 days) into slant culture medium in test tube to obtain slant strain;
wherein the strain inclined plane culture medium consists of the following components in parts by weight:
glucose 20 parts, peptone 1 part, magnesium sulfate 1.5 parts, potassium dihydrogen phosphate 1 part, and vitamin B 1 0.02 part of agar 15 parts, and water is added to 1000 parts.
The process conditions for preparing the inclined plane strain are as follows: the culture temperature is 25 ℃ and the culture time is 24 hours.
(2) Seed liquid fermentation
Performing enlarged culture on the inclined plane strain to obtain seed liquid;
the seed liquid culture medium consists of the following components in parts by weight:
20 parts of sucrose, 1 part of peptone, 1 part of magnesium sulfate, 5 parts of monopotassium phosphate and vitamin B 1 0.03 parts and 1000 parts of water.
The culture condition of the prepared seed liquid is that the culture temperature is 25 ℃, the rotation speed of a shaking table is 150r/min, and the culture time is 24 hours.
(3) Fermenting to produce tremella polysaccharide;
inoculating the seed liquid into a fermentation tank for fermentation to produce tremella polysaccharide;
wherein the fermentation liquor consists of the following components in parts by weight:
60 parts of sucrose, 3 parts of peptone, 1 part of magnesium sulfate, 1 part of monopotassium phosphate and vitamin B 1 0.01 part and 1000 parts of water.
The fermentation process conditions are as follows: the inoculation amount is 10%, the fermentation temperature is 20 ℃, the ventilation amount is 1vvm, the stirring speed is 100-900r/min, the fermentation time is 48h, and the tremella polysaccharide yield is 40g/L.
(4) The separation and purification of the tremella polysaccharide product comprises the following three steps:
a. diluting the fermented liquid after fermentation for 2 times, and centrifuging for 20min at 8000 r/min;
b. carrying out ultrafiltration concentration on the supernatant fluid obtained in the step a by adopting an ultrafiltration membrane with the molecular weight cut-off of 60-80kDa, and simultaneously removing impurities such as protein, pigment and the like;
c. and c, spray-drying the concentrated solution obtained in the step b to obtain white tremella polysaccharide powder with the total sugar content of more than 99.5 percent, the protein content of less than 0.3 percent and the molecular weight of 100-3000kDa, wherein the air inlet temperature of spray drying is 140 ℃.
Claims (9)
1. A method for producing tremella polysaccharide by fermenting high yield is characterized in that tremella polysaccharide is produced by fermenting tremella bacteria, and the production process comprises the following process steps:
(1) Inoculating tremella (Tremella fuciformis) CGMCC No.23287 strain to a slant culture medium in a test tube to prepare slant strain;
(2) Performing enlarged culture on the inclined plane strain to obtain seed liquid;
(3) Inoculating the seed liquid into a fermentation tank for fermentation to produce tremella polysaccharide;
(4) Separating and purifying tremella polysaccharide.
2. The method for fermenting high-yield tremella polysaccharide according to claim 1, which is characterized in that: the slant culture medium in the step (1) comprises the following components in parts by weight: 10-20 parts of glucose, 1-5 parts of peptone, 1-2 parts of magnesium sulfate, 1-5 parts of monopotassium phosphate and vitamin B 1 0.01-0.03 part, 15 parts of agar and water to 1000 parts.
3. A method for fermenting high-yield tremella polysaccharide according to claim 1 or 2, characterized in that: the process conditions for preparing the inclined plane strain are as follows: the culture temperature is 20-30deg.C, and the culture time is 12-48h.
4. The method for fermenting high-yield tremella polysaccharide according to claim 1, which is characterized in that: the seed liquid in the step (2) and the hair in the step (3)The fermentation liquid consists of the following components in parts by weight: 20-80 parts of sucrose, 1-5 parts of peptone, 1-2 parts of magnesium sulfate, 1-5 parts of monopotassium phosphate and vitamin B 1 0.01-0.03 parts and 1000 parts of water.
5. The method for fermenting high-yield tremella polysaccharide according to claim 1, which is characterized in that: the seed liquid culture conditions of the step (2) are as follows: the culture temperature is 20-30 ℃, the rotation speed of the shaking table is 100-200r/min, and the culture time is 12-48h.
6. A method for fermenting high-yield tremella polysaccharide according to claim 1 or 4, characterized in that: the fermentation process conditions of the fermentation tank are as follows: the inoculation amount is 10%, the fermentation temperature is 20-30 ℃, the ventilation amount is 0.5-2vvm, the stirring speed is 100-900r/min, the fermentation time is 48-96h, and the tremella polysaccharide yield is 40-50g/L.
7. The method for fermenting high-yield tremella polysaccharide according to claim 1, which is characterized in that: the separation and purification of the tremella polysaccharide product in the step (4) comprises the following three steps:
a. centrifugally sterilizing the fermented liquid after fermentation;
b. concentrating the supernatant obtained in the step a by using an ultrafiltration membrane to remove impurities such as protein, pigment and the like;
c. and c, spray drying the concentrated solution obtained in the step b.
8. The method for fermenting high-yield tremella polysaccharide according to claim 7, which is characterized in that: the centrifugation condition in the step a is 8000r/min, and the centrifugation time is 20min; step b, the molecular weight cut-off of the ultrafiltration membrane is 60-80kDa; the spray drying condition in the step c is that the temperature of a feed inlet is 140-160 ℃; the white tremella polysaccharide with the total sugar content of more than 99.5 percent, the protein content of less than 0.3 percent and the molecular weight of 100-3000kDa can be obtained after drying.
9. Tremella (Tremella fuciformis) CGMCC No.23287.
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