CN116376705A - Eurotium cristatum strain for producing fungal polysaccharide and application thereof - Google Patents
Eurotium cristatum strain for producing fungal polysaccharide and application thereof Download PDFInfo
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- CN116376705A CN116376705A CN202111587984.4A CN202111587984A CN116376705A CN 116376705 A CN116376705 A CN 116376705A CN 202111587984 A CN202111587984 A CN 202111587984A CN 116376705 A CN116376705 A CN 116376705A
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- fermentation
- eurotium cristatum
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- polysaccharide
- fungal
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 86
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 86
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 86
- 241001205401 Aspergillus cristatus Species 0.000 title claims abstract description 74
- 230000002538 fungal effect Effects 0.000 title claims abstract description 48
- 238000000855 fermentation Methods 0.000 claims abstract description 78
- 230000004151 fermentation Effects 0.000 claims abstract description 78
- 238000004519 manufacturing process Methods 0.000 claims abstract description 9
- 238000004321 preservation Methods 0.000 claims abstract description 7
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- 238000000034 method Methods 0.000 claims description 26
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 5
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 5
- 235000013312 flour Nutrition 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
- 235000013361 beverage Nutrition 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- 230000036541 health Effects 0.000 claims description 4
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 4
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- 229940041514 candida albicans extract Drugs 0.000 claims description 3
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- 235000013305 food Nutrition 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 2
- 239000001888 Peptone Substances 0.000 claims description 2
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- 244000046052 Phaseolus vulgaris Species 0.000 claims description 2
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 2
- 229920002472 Starch Polymers 0.000 claims description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 2
- 230000001133 acceleration Effects 0.000 claims description 2
- 238000005273 aeration Methods 0.000 claims description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 2
- 239000003524 antilipemic agent Substances 0.000 claims description 2
- 239000002246 antineoplastic agent Substances 0.000 claims description 2
- 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 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 2
- 239000004202 carbamide Substances 0.000 claims description 2
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- 239000002054 inoculum Substances 0.000 claims description 2
- 150000002632 lipids Chemical class 0.000 claims description 2
- 235000013379 molasses Nutrition 0.000 claims description 2
- 230000010355 oscillation Effects 0.000 claims description 2
- 235000019319 peptone Nutrition 0.000 claims description 2
- 229910052698 phosphorus Inorganic materials 0.000 claims description 2
- 239000011574 phosphorus Substances 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 claims description 2
- 239000004317 sodium nitrate Substances 0.000 claims description 2
- 235000010344 sodium nitrate Nutrition 0.000 claims description 2
- 239000008107 starch Substances 0.000 claims description 2
- 235000019698 starch Nutrition 0.000 claims description 2
- 238000012869 ethanol precipitation Methods 0.000 claims 1
- 238000012262 fermentative production Methods 0.000 claims 1
- 230000000055 hyoplipidemic effect Effects 0.000 claims 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims 1
- 241000233866 Fungi Species 0.000 abstract description 18
- 239000000243 solution Substances 0.000 description 16
- 239000000047 product Substances 0.000 description 14
- 239000000284 extract Substances 0.000 description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- 241000699670 Mus sp. Species 0.000 description 10
- 206010028980 Neoplasm Diseases 0.000 description 10
- 239000000725 suspension Substances 0.000 description 10
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- 239000007864 aqueous solution Substances 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 7
- 239000012086 standard solution Substances 0.000 description 7
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 6
- 244000269722 Thea sinensis Species 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
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- 239000008223 sterile water Substances 0.000 description 5
- 235000013616 tea Nutrition 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000007790 scraping Methods 0.000 description 4
- 239000013049 sediment Substances 0.000 description 4
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 description 3
- 206010003445 Ascites Diseases 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
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- 150000001875 compounds Chemical class 0.000 description 3
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- 230000005764 inhibitory process Effects 0.000 description 3
- 201000007270 liver cancer Diseases 0.000 description 3
- 208000014018 liver neoplasm Diseases 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical compound O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 description 3
- 235000020195 rice milk Nutrition 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 238000012449 Kunming mouse Methods 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
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- HYBBIBNJHNGZAN-UHFFFAOYSA-N furfural Chemical class O=CC1=CC=CO1 HYBBIBNJHNGZAN-UHFFFAOYSA-N 0.000 description 2
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- 150000002772 monosaccharides Chemical class 0.000 description 2
- 229940054441 o-phthalaldehyde Drugs 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
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- 108090000695 Cytokines Proteins 0.000 description 1
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- 241000238631 Hexapoda Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 description 1
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 description 1
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Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/062—Ascomycota
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Chemical & Material Sciences (AREA)
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- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Mycology (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Nutrition Science (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Obesity (AREA)
- Botany (AREA)
- Natural Medicines & Medicinal Plants (AREA)
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Abstract
The invention relates to a Eurotium cristatum strain for producing fungus polysaccharide and application thereof. The invention provides a Eurotium cristatum BC-43 strain (Eurotium cristatum BC-43), which has a preservation number of CCTCC NO: M20211417. The invention also relates to application of the Eurotium cristatum BC-43 strain in fermentation production of fungal polysaccharide and a fermentation product rich in fungal polysaccharide, which is produced by fermentation of the Eurotium cristatum BC-43 strain. The Eurotium cristatum BC-43 strain (Eurotium cristatum BC-43) provided by the invention has higher content of fungal polysaccharide.
Description
Technical Field
The invention relates to the technical field of microorganism application, in particular to a Eurotium cristatum strain capable of producing fungal polysaccharide by fermentation and application thereof.
Background
Polysaccharides are one of four basic substances constituting living bodies, are formed by condensing a plurality of monosaccharide molecules and losing water, and are sugar substances with complex and huge molecular structures. The active polysaccharide has obvious biological effect on normal organism, can produce effective therapeutic effect on diseased organism, and has safe administration and almost no toxicity.
Polysaccharides are widely found in animals, plants, large fungi and microorganisms, and have various biological activities and functions. The animal and plant polysaccharide and the large fungus polysaccharide have longer production period, more limiting factors, low yield, large consumption of resources and higher large-scale industrial production cost, and have different degrees of influence on the application and development of the animal and plant polysaccharide and the large fungus polysaccharide.
The microbial polysaccharide is a polysaccharide produced by microorganisms such as bacteria and fungi (without large fungi). Microbial polysaccharides have advantages over other polysaccharides. The method has the advantages of short production period, no limitation of seasons, regions and insect pest conditions, abundant and cheap raw material sources, relatively low cost, capability of carrying out large-scale industrial production under the manual control condition, high yield, simple purification and no pollution to the environment, thereby having stronger market competitiveness and wide development prospect.
The Eurotium cristatum (Eurotium cristatum) is a kind of natural probiotics produced in the process of Fuzhuan tea flowering, and is commonly called as "Jinhua bacteria" because it produces golden yellow closed capsule shell in the process of growth and propagation. Toxicology studies carried out in various animals show that the golden flower fungus belongs to non-toxic microorganisms, and fermentation products of the golden flower fungus have good activities of resisting oxidation, inhibiting bacteria, regulating balance of intestinal flora, improving immunity, resisting aging, resisting tumors and the like, which are related to various compounds with health care effects produced by fermentation metabolism of the eurotium cristatum, wherein fungus polysaccharide is one of the most important metabolites, researchers have found that various extracellular polysaccharides can be extracted from tea-derived eurotium cristatum fermentation liquid, and the fungus polysaccharide can activate immune cells and regulate secretion of cytokines through various ways, so that the cellular immunity and humoral immunity functions of an organism are improved.
CN103999946a discloses a health care beverage of eurotium cristatum fermented rice milk and a preparation method thereof, which comprises the steps of using eurotium cristatum for the production of fermented rice milk beverage and preparing novel rice milk beverage with unique flavor and rich health care factors such as fungus polysaccharide, wherein the content of the fungus polysaccharide reaches 210-260 mug/ml.
Disclosure of Invention
Aiming at the problem of low content of fungal polysaccharide produced by Eurotium cristatum in the prior art, the invention provides a novel Eurotium cristatum.
Specifically, the invention provides the following technical scheme.
In a first aspect, the invention provides a Eurotium cristatum BC-43 strain (Eurotium cristatum BC-43), which is characterized in that the Eurotium cristatum BC-43 strain (Eurotium cristatum BC-43) is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M20211417.
Preferably, the ITS gene sequence of the Eurotium cristatum BC-43 strain (Eurotium cristatum BC-43) is shown in SEQ ID NO. 1.
In a second aspect, the invention provides an application of the Eurotium cristatum BC-43 strain in the fermentation production of fungal polysaccharide.
In a third aspect, the present invention provides a fungal polysaccharide enriched fermentation broth produced by fermentation of said strain Eurotium cristatum BC-43.
Preferably, the rate of cholesterol removal by the fermentate is 59% -65%.
In a fourth aspect, the present invention provides a method for preparing the fungal polysaccharide-enriched fermentation broth, comprising culturing the Eurotium cristatum BC-43 strain.
Preferably, the method comprises the steps of:
(1) Seed culture is carried out on the Eurotium cristatum BC-43 strain to obtain a Eurotium cristatum BC-43 strain seed culture solution;
(2) Adding the seed culture solution of the Eurotium cristatum BC-43 strain into a fermentation culture medium, fermenting for 3-6 days, and extracting to obtain the fermentation product rich in fungal polysaccharide.
Preferably, the temperature of the seed culture in step (1) is 28-30 ℃;
preferably, the seed culture is shaking culture,
preferably, the oscillation frequency is 100-250rpm;
preferably, the seed culture time is 20-70h.
Preferably, the fermentation in step (2) is a solid fermentation or a liquid fermentation;
preferably, the fermentation temperature in the step (2) is 28-30 ℃.
Preferably, the liquid fermentation speed is 150-200rpm;
preferably, the liquid fermentation aeration is 600L/h to 3000L/h.
Preferably, the culture medium for liquid fermentation contains 15-60 parts of carbon source, 1.5-5 parts of nitrogen source, 0.1-0.5 part of phosphorus source and ZnSO 4 0.03-1 part of FeSO 4 .7H 2 O0.03-1 part, KCl 0-10 parts and MgSO 4 .7H 2 O1-10 parts;
preferably, the carbon source is selected from one or more than two of glucose, sucrose, maltose, molasses and starch;
preferably, the nitrogen source comprises one or more than two of yeast extract powder, peptone, corn steep liquor, bean cake powder, ammonium sulfate, ammonia water, sodium nitrate and urea.
Preferably, the liquid fermentation comprises a feed fermentation process;
preferably, the feed fermentation medium comprises 15-30wt% sucrose, preferably, the feed fermentation medium comprises 20wt% sucrose;
preferably, the feed fermentation medium flow acceleration is 50-200ml/h.
Preferably, the culture medium for solid fermentation comprises, by mass, 45% -55% of water, 0.5% -4% of glucose, 2% -8% of ammonium sulfate, 0.5% -1.5% of monopotassium phosphate, 0% -0.5% of magnesium sulfate and the balance of bran and corn flour, wherein the mass ratio of the bran to the corn flour is 2-5:1.
Preferably, the inoculation amount in the step (2) is 1-10% by weight.
Preferably, in the step (2), the fungal polysaccharide is precipitated with ethanol for extraction.
In a fifth aspect, the present invention provides a fungal polysaccharide produced by fermentation of said strain of Eurotium cristatum BC-43.
In a sixth aspect, the invention provides the use of the fermentation product rich in the fungus polysaccharide or the fungus polysaccharide in preparing hypolipidemic drugs and antitumor drugs, and foods, drinks, health-care products and medicines for reducing blood lipid and losing weight.
The Eurotium cristatum strain belongs to aspergillus of traditional fermented tea, and does not have the risk of food safety. The Eurotium cristatum strain can be fermented to produce a large amount of fungus polysaccharide by using a common culture medium, and the fermentation yield is high. The Eurotium cristatum strain has high growth speed, is easy to culture, and can shorten the production time of fungus polysaccharide and improve the production efficiency. The crude polysaccharide extract obtained by fermenting the Eurotium cristatum strain has the effects of reducing blood fat and resisting tumors, and has extremely high economic value and use value.
Strain preservation information
The Eurotium cristatum BC-43 strain (Eurotium cristatum BC-43) is preserved in China Center for Type Culture Collection (CCTCC) in the year 2021 and 11 and 15, and the preservation number is CCTCC NO: m20211417, deposit address: chinese, wuhan, university of Wuhan, postal code: 430072; telephone: (027) -68754052.
Detailed Description
As described above, the present invention provides a Eurotium cristatum BC-43 strain (Eurotium cristatum BC-43) capable of producing fungal polysaccharide by fermentation, which is preserved in China Center for Type Culture Collection (CCTCC) of university of Wuhan in China, with a preservation number of CCTCC NO: m20211417.
In the embodiment of the invention, the content of the fungus polysaccharide is measured by using a phenol-sulfuric acid method
The measurement principle is as follows: the polysaccharide is hydrolyzed into monosaccharide under the action of concentrated sulfuric acid, and is rapidly dehydrated to generate furfural derivatives, and then the furfural derivatives are combined with phenol to generate colored compounds, and then the absorbance of the colored compounds is measured by a spectrophotometer, and the polysaccharide content in a sample is quantitatively measured by a standard curve.
(1) Drawing a standard curve:
phenol (pure phenol is needle-shaped colorless crystals) is dissolved after being heated in a water bath at 100 ℃, poured into a distillation flask, heated and distilled to a boiling point of 182 ℃ on an electric furnace for 2-3 minutes, and a fraction at 182 ℃ is collected. 80g of the collected phenol fraction was dissolved in 20g of water to prepare an 80% phenol aqueous solution, which was stored in a refrigerator protected from light for a long period of time. Before use, the 80% phenol aqueous solution is diluted into 5% phenol aqueous solution.
And (3) drying a proper amount of glucose (analytically pure) in an oven (105 ℃) to constant weight, accurately weighing 20.0g, adding distilled water for dissolution, and fixing the volume to 100mL to obtain a glucose standard solution with the concentration of 0.2 g/mL. Glucose standard solutions of 40mg/L, 80mg/L, 120mg/L, 160mg/L and 200mg/L were prepared from the glucose standard solutions. Then, 1mL of glucose standard solution with different concentrations is taken, 1mL of freshly prepared 5% phenol aqueous solution is added respectively, and 5mL of 98% concentrated sulfuric acid is added and mixed uniformly. After 10min, the resulting mixture was placed in a 25℃water bath for 20min. Then, distilled water is used as a blank control group, the absorbance value of the blank control group and the absorbance value of the treated glucose standard solution at 490nm are measured, a standard curve is drawn by taking the absorbance value A as an ordinate and the glucose concentration as an abscissa, and regression treatment is carried out.
(2) 1mL of the solution to be measured was taken, 1mL of 5% phenol aqueous solution was added, and 5mL of 98% concentrated sulfuric acid was added and mixed well. After 10min, the resulting mixture was placed in a 25℃water bath for 20min. And then, taking distilled water as a blank control group, measuring the absorbance values of the blank control group and the treated solution to be measured at 490nm, and calculating the polysaccharide content in the solution to be measured by using a standard curve.
In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.
The following description of the materials and equipment manufacturers used in the examples, as well as the equipment and analytical methods used in the analysis of the products, are provided by those of ordinary skill in the art, as are reagents, instruments or procedures not described herein.
TABLE 1 information on raw materials and instruments used in the present invention
EXAMPLE 1 obtaining of the Eurotium cristatum BC-43 Strain
Xiang Yifu brick tea was purchased from Tianmao APP Xiang Yi tea flagship (https:// xingyicy. Tmall. Com/.
(1) Weighing 25g of Fuzhuan tea sample, soaking in 225ml of 0.85% sterile physiological saline, adding a proper amount of sterilized glass beads, and placing on a constant temperature oscillator for oscillating for 20min at a speed of 120 r/min.
(2) Diluting with multiple ratio dilution method to obtain diluted solution 10 and diluted solution 10 respectively 2 、10 3 、10 4 Multiple times.
(3) 1mL of 10 was pipetted 4 The diluted liquid is coated on the PDA flat plate and placedCulturing for 3d at 28 ℃ in a constant temperature incubator, and growing a colony, wherein the diameter of the colony is 11-16mm, the colony is round and compact in structure, the newly grown mycelium at the edge of the colony grows vigorously, the colony is milky white to pale yellow, the center is golden yellow to dark brown, a small amount of brown exudates exist, and the back of the colony is dark brown. Meets the colony characteristics of the Eurotium cristatum.
(4) And (3) in a sterile state, scraping golden yellow colonies with dominant quantity and better growth vigor by an inoculating needle, inoculating on a PDA culture medium plate, culturing for 3d at 28 ℃, and repeating the transferring for 3 times, wherein the purified Eurotium cristatum is obtained.
(5) Inoculating the obtained Eurotium cristatum into PDB for culturing at 28 ℃ and 150rpm for 2d to obtain mycelium pellets;
(6) Extracting total DNA of the strain by adopting a liquid nitrogen grinding method, carrying out PCR amplification on the conserved sequence by using an ITS universal primer (ITS 1: TCCGTAGGTGAACCTGCGG, ITS4: TCCTCCGCTTATTGATATGC), and sequencing the obtained product, wherein a sequencing company is October Dingsheng biotechnology (Wuhan) Co. The sequence of the ITS zone is shown as SEQ ID NO. 1.
SEQ ID NO.1 sequence is as follows:
the sequencing results were aligned in NCBI to determine their species. Finally, the strain is confirmed to be the Eurotium cristatum BC-43 strain. The Eurotium cristatum BC-43 strain (Eurotium cristatum BC-43) is preserved in China Center for Type Culture Collection (CCTCC) at the year 2021 and the 11 and 15, and the preservation number is CCTCC NO: M20211417.
Example 2 preparation of a fermented extract enriched in fungal polysaccharides by shake flask fermentation
(1) Activating strains: the frozen sporangium coronarium BC-43 strain spore liquid is scratched on PDA inclined plane and cultured for 3 days at 28 ℃.
(2) Spore suspension preparation: adding sterile water into the activated eurotium cristatum BC-43 strain inclined plane, scraping spores by using a scraper, and preparing spore suspension.
(3) Seed culture: the spore suspension was inoculated into a 250ml Erlenmeyer flask containing 100ml PDA liquid medium at 5% inoculum. Then, the inoculated liquid culture medium is shake-cultured for 5 days at 28 ℃ at 150rpm to obtain fermentation liquor containing fungal polysaccharide.
(4) Determination of fungal polysaccharide content in fermentation broth: the content of fungal polysaccharide in the fermentation broth was measured by the phenol-sulfuric acid method described above to be 10g/L.
(5) Separating the fermentation product enriched in fungal polysaccharide: evaporating and concentrating the fermentation liquor to 1/4 of the original volume, adding 95% ethanol with the volume being 4 times that of the fermentation liquor, standing in a refrigerator at the temperature of 4 ℃ for 10 hours to precipitate polysaccharide, centrifuging at 8000r/min, and obtaining precipitate which is the fermentation product rich in fungal polysaccharide.
EXAMPLE 3 liquid submerged fermentation to prepare a fermented extract enriched in fungal polysaccharide
(1) Activating strains: the frozen sporangium coronarium BC-43 strain spore liquid is scratched on PDA inclined plane and cultured for 3 days at 28 ℃.
(2) Spore suspension preparation: adding sterile water into the activated eurotium cristatum BC-43 strain inclined plane, scraping spores by using a scraper, and preparing spore suspension.
(3) Seed culture: the spore suspension was inoculated at 2% into sterilized 1L PDB liquid medium, followed by shaking culture at 30℃for 5 days at 120rpm to obtain a seed culture solution.
(4) Inoculating and fermenting: 2L of seed culture solution is introduced into a 50L fermentation tank, wherein the fermentation tank is filled with a fermentation base material comprising the following components: 18L water, 400g yeast extract powder, 30g monoammonium phosphate, znSO 4 5g，FeSO 4 ·7H 2 O 3g，KCl 50g，MgSO 4 ·7H 2 O80 g, followed by fermentation at 30℃for 6 days, to obtain a fermentation broth containing fungal polysaccharide. During fermentation 10L of 20wt% sucrose in water was fed at a rate of 100 ml/h.
(5) Determination of fungal polysaccharide content in fermentation broth: the content of the fungal polysaccharide in the fermentation broth was determined to be 18g/L by the phenol-sulfuric acid method described above.
(6) Separating the fermentation product enriched in fungal polysaccharide: evaporating and concentrating the fermentation liquor to 1/4 of the original volume, adding 95% ethanol with the volume being 4 times that of the fermentation liquor, standing in a refrigerator at the temperature of 4 ℃ for 10 hours to precipitate polysaccharide, centrifuging at 8000r/min, and obtaining precipitate which is the fermentation product rich in fungal polysaccharide.
EXAMPLE 4 solid State fermentation production of crude polysaccharide from Eurotium cristatum
(1) Activating strains: the sporangium coronarium cryopreserved spore liquid is scratched on PDA inclined plane and cultured for 3 days at 28 ℃.
(2) Spore suspension preparation: adding sterile water into the activated eurotium cristatum inclined plane, scraping spores by using a scraper, and preparing spore suspension.
(3) Seed culture: the spore suspension was inoculated into 0.5L of the treated seed culture solution and shake-cultured at 28℃and 160rpm for 3d.
(4) Fermentation: the solid culture medium comprises bran: 700g of corn flour mixture with the mass ratio of 5:1 is added with 700g of water, 47g of glucose, 78g of ammonium sulfate, 24g of monopotassium phosphate and 8g of magnesium sulfate; fermenting at 30-35 deg.c for 5d.
(5) Preparing sporoderm-broken Eurotium cristatum sporocyst: after 125g of solid fermentation material was dried, spores were separated from the substrate by gentle rubbing, and sieved through a 200 mesh sieve to obtain 25g of Eurotium cristatum spores. Repeating the freeze thawing grinding method for breaking the walls of spores, weighing 25g of Eurotium cristatum spores, putting the spores into 5 50mL centrifuge tubes, adding 20mL of 70% ethanol, shaking and uniformly mixing, putting the centrifuge tubes into liquid nitrogen for cooling for 5min, taking out the centrifuge tubes, putting the centrifuge tubes into a water bath kettle for water bath at 95 ℃ for 5min, repeating the steps for 3 times, transferring spore suspension into a mortar for grinding for 10min, and drying under reduced pressure to obtain 20g of wall-broken Eurotium cristatum spores.
(6) Separating the fermentation product enriched in fungal polysaccharide: taking 20g of wall-broken spores, adding 300mL of distilled water, carrying out reflux extraction at 55 ℃ for 4 hours, filtering to remove sediment, adding 60mL of Sevage reagent (chloroform: n-butanol=5:1 (V/V)) into the filtrate, mixing and shaking for 30 minutes, centrifuging, taking supernatant, concentrating to 100mL, adding 400mL of 95% ethanol, standing in a refrigerator at 4 ℃ for 24 hours to sediment polysaccharide, centrifuging at 8000r/min, and collecting sediment, wherein the obtained sediment 4g is a fermentation product rich in fungal polysaccharide.
(7) Determination of polysaccharide content in primary fermentation product: dissolving 1g of the precipitate obtained in the step (6) in 100ml of water, measuring the polysaccharide content by a phenol-sulfuric acid method, and measuring 781g/kg of the polysaccharide content in the precipitate, wherein the polysaccharide content in the wall-broken spores is converted into 156.2g/kg of the fungal polysaccharide content in the Eurotium cristatum spores, and the polysaccharide content in the Eurotium cristatum spores is converted into 125g/kg, and the polysaccharide content in the solid fermentation material is converted into 25g/kg.
Example 5 determination of the clearance of cholesterol from fermented extracts enriched in fungal polysaccharides
1g of the fungal polysaccharide-rich fermentate prepared in examples 2, 3 and 4 was accurately weighed and placed in a 50mL centrifuge tube, and 10mL of cholesterol standard solution (6.0 mmol/L) was added. And (3) oscillating and adsorbing for 16h at the constant temperature of 37 ℃, centrifuging at 10000rpm for 6 minutes, and obtaining supernatant as the solution to be detected. A blank group was prepared from 10mL of a cholesterol standard solution without the addition of the fermentation extract enriched in fungal polysaccharide. Cholesterol content in the supernatant was determined by the o-phthalaldehyde method as described below, and the clearance of cholesterol from the fermented extract enriched in fungal polysaccharide was calculated using the following formula.
The calculation formula is as follows:
cholesterol clearance (%) =100% (cholesterol content of the blank group-cholesterol content of the sample group)/cholesterol content of the blank group
The cholesterol content determination method comprises the following steps: o-phthalaldehyde process
(1) Drawing a standard curve:
samples 1-7 were prepared as shown in Table 2 below, where concentrated sulfuric acid should be added slowly and thoroughly mixed. After standing at room temperature for 10min, the OD of the obtained samples 1-7 was measured by a spectrophotometer 550 Values.
TABLE 2
(2) Cholesterol content determination: after diluting the prepared solution to be tested and the blank control group by 5 times, 400 mu L of the solution is sucked into a 15mL centrifuge tube, and 3 parts of the solution are measured in parallel. 2mL of the phthalaldehyde reagent and 1mL of 95% sulfuric acid solution were added respectivelyAnd (3) liquid and uniformly mixing. After standing at room temperature for 10min, the OD of the treated 3 parts of the solution to be tested and the blank control group are respectively measured by a spectrophotometer 550 Values. The cholesterol content of the test solution was calculated from the standard curve, and the cholesterol removal rates of the fungal polysaccharide enriched ferments prepared in examples 2, 3 and 4 were calculated to be 59%, 65% and 63% in this order, according to the formulas described above.
Example 6 determination of the tumor inhibiting Effect of fermented extracts enriched in fungal polysaccharides
(1) Preparing an aqueous solution of a fermentation extract enriched in fungal polysaccharides: the fermented products rich in fungal polysaccharide of examples 2, 3 and 4 were dissolved in distilled water, the crude polysaccharide concentration was 0.5g/ml, and the aqueous solution of the obtained fermented extract rich in fungal polysaccharide was packaged in 10ml in 18mm 180mm sterilized test tubes and stored in a refrigerator at 4℃for further use.
(2) Experimental animals: 43 Kunming mice, male and female halves, had a body weight of 18-22g and were 5-6 weeks old, and were offered by the university of three gorges medical college.
(3) Experimental cells: hepatoma cell line HepG2 was purchased from southern medical university and stored in liquid nitrogen.
(4) Culturing and inoculating a liver cancer cell strain HepG 2: randomly taking 3 mice, injecting liver cancer cell strain HepG2 into the right armpit of the mice, extracting ascites of the mice inoculated for 7 days, taking out the ascites, centrifuging and washing with sterile water for 3 times, and adjusting the cell concentration to 1X 10 7 cfu/g, the resulting cell fluid was inoculated into the next-generation mice, passaged 2 times in this way, and then the ascites of the mice inoculated for 7 days were extracted, milky white, and washed 3 times with sterile water by centrifugation. The resulting cell fluid was inoculated subcutaneously into the right axilla of the remaining 40 Kunming mice, 0.2ml per mouse. And (5) strictly performing aseptic operation in the whole process.
(5) Experimental grouping: 40 mice were randomly divided into 4 groups and housed in cages for free feeding and drinking. Group 1 is control group, and physiological saline is administered; the other 3 groups are experimental groups to which aqueous solutions of the fungal polysaccharide enriched fermented extracts of examples 2, 3 and 4 prepared in step (1) were administered, respectively. The 4 groups of mice were orally administered by gastric lavage, 2 times daily for three consecutive weeks, and the external characteristics and death state of the mice were observed. After the end of the administration, the mice were sacrificed the next day, the tumor tissues were completely exfoliated, the tumor weights were weighed, and the tumor inhibition rate was calculated according to the following formula:
tumor inhibition (%) = 100%. Times.1-average tumor weight in experimental group/average tumor weight in control group
(6) Test results: the tumor inhibition rates of the aqueous solutions of the fungal polysaccharide-enriched fermented extracts of examples 2, 3 and 4 on the liver cancer of mice were 37.85%, 39.62% and 38.03% in this order.
The above description is not intended to limit the invention in any way, but is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Sequence listing
<110> Hubei Angel biological group Co., ltd
<120> a fungus polysaccharide-producing Eurotium cristatum strain and use thereof
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<170> SIPOSequenceListing 1.0
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<211> 526
<212> DNA
<213> Eurotium cristatum (Eurotium cristatum)
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gctacgagtg cgggtctctg ggtcaacctc ccatccgtgt ctatctgtac cctgttgctt 60
cggcgtggcc acggcccgcc ggagactaac atttgaacgc tgtctgaagt ttgcagtctg 120
agtttttagt taaacaatcg ttaaaacttt caacaacgga tctcttggtt ccggcatcga 180
tgaagaacgc agcgaaatgc gataattaat gtgaattgca gaattcagtg aatcatcgag 240
tctttgaacg cacattgcgc cccctggtat tccggggggc atgcctgtcc gagcgtcatt 300
gctgccctca agcacggctt gtgtgttggg cttccgtccc tggcaacggg gacgggccca 360
aaaggcagtg gcggcaccat gtctggtcct cgagcgtatg gggctttgtc acccgctccc 420
gtaggtccag ctggcagcta gcctcgcaac caatcttttt aaccaggttg acctcggatc 480
aggtagggat acccgctgaa cttaagcata tcaaaggcgg gaggaa 526
Claims (17)
1. The Eurotium cristatum BC-43 strain (Eurotium cristatum BC-43) is characterized in that the Eurotium cristatum BC-43 strain (Eurotium cristatum BC-43) is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M20211417.
2. The eurotium cristatum BC-43 strain (Eurotium cristatum BC-43) according to claim 1, wherein ITS gene sequence of said eurotium cristatum BC-43 strain (Eurotium cristatum BC-43) is shown in SEQ ID No. 1.
3. Use of a strain of eurotium cristatum BC-43 according to claim 1 or 2, for the fermentative production of fungal polysaccharides.
4. A fungal polysaccharide-rich ferment produced by fermentation of the eurotium cristatum BC-43 strain of claim 1 or 2.
5. The ferment of claim 4, wherein the ferment has a cholesterol clearance of 59% to 65%.
6. The method for producing a fungal polysaccharide enriched fermentation according to claim 4 or 5, wherein the method comprises culturing the eurotium cristatum BC-43 strain according to claim 1 or 2.
7. The method according to claim 6, comprising the steps of:
(1) Seed culture is carried out on the Eurotium cristatum BC-43 strain according to claim 1 or 2, so as to obtain a Eurotium cristatum BC-43 strain seed culture solution;
(2) Adding the seed culture solution of the Eurotium cristatum BC-43 strain into a fermentation culture medium, fermenting for 3-6 days, and extracting to obtain the fermentation product rich in fungal polysaccharide.
8. The method of claim 7, wherein the temperature of the seed culture in step (1) is 28-30 ℃;
preferably, the seed culture is shaking culture;
preferably, the oscillation frequency is 100-250rpm;
preferably, the seed culture time is 20-70h.
9. The method according to claim 7 or 8, wherein the fermentation in step (2) is a solid fermentation or a liquid fermentation;
preferably, the fermentation temperature in the step (2) is 28-30 ℃.
10. The method according to claim 9, wherein the liquid fermentation speed is 150-200rpm;
preferably, the liquid fermentation aeration is 600L/h to 3000L/h.
11. The method according to claim 9 or 10, wherein the medium for liquid fermentation contains 15-60 parts of carbon source, 1.5-5 parts of nitrogen source, 0.1-0.5 part of phosphorus source, znSO 4 0.03-1 part of FeSO 4 .7H 2 O0.03-1 part, KCl 0-10 parts and MgSO 4 .7H 2 O1-10 parts;
preferably, the carbon source is selected from one or more than two of glucose, sucrose, maltose, molasses and starch;
preferably, the nitrogen source comprises one or more than two of yeast extract powder, peptone, corn steep liquor, bean cake powder, ammonium sulfate, ammonia water, sodium nitrate and urea.
12. The method according to any one of claims 9-11, wherein the liquid fermentation comprises a fed-batch fermentation process;
preferably, the feed fermentation medium comprises 15-30wt% sucrose, preferably, the feed fermentation medium comprises 20wt% sucrose;
preferably, the feed fermentation medium flow acceleration is 50-200ml/h.
13. The method according to claim 9, wherein the solid fermentation medium comprises, by mass, 45% -55% of water, 0.5% -4% of glucose, 2% -8% of ammonium sulfate, 0.5% -1.5% of potassium dihydrogen phosphate and 0% -0.5% of magnesium sulfate, and the balance of bran and corn flour, wherein the mass ratio of bran to corn flour is 2-5:1.
14. The method according to any one of claims 7 to 13, wherein the inoculum size in step (2) is 1 to 10% by weight.
15. The method according to any one of claims 7 to 14, wherein in step (2), the extraction is performed by ethanol precipitation of the fungal polysaccharide.
16. A fungal polysaccharide, characterized in that it is produced by fermentation of the strain BC-43 of eurotium cristatum according to claim 1 or 2.
17. Use of the fungal polysaccharide-enriched fermentation product of claim 4 or 5 or the fungal polysaccharide of claim 16 in the preparation of hypolipidemic and antitumor agents, and foods, beverages, health products and medicines for reducing blood lipid and weight.
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