CN110541016A - Extraction and purification method of lentinan - Google Patents
Extraction and purification method of lentinan Download PDFInfo
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- CN110541016A CN110541016A CN201910853650.3A CN201910853650A CN110541016A CN 110541016 A CN110541016 A CN 110541016A CN 201910853650 A CN201910853650 A CN 201910853650A CN 110541016 A CN110541016 A CN 110541016A
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- 229920001491 Lentinan Polymers 0.000 title claims abstract description 89
- 229940115286 lentinan Drugs 0.000 title claims abstract description 89
- 238000000034 method Methods 0.000 title claims abstract description 53
- 238000000605 extraction Methods 0.000 title claims abstract description 49
- 238000000746 purification Methods 0.000 title claims abstract description 15
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 53
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 42
- 239000004365 Protease Substances 0.000 claims description 33
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 32
- 239000007788 liquid Substances 0.000 claims description 32
- 238000000855 fermentation Methods 0.000 claims description 31
- 230000004151 fermentation Effects 0.000 claims description 31
- 239000001963 growth medium Substances 0.000 claims description 27
- 235000015099 wheat brans Nutrition 0.000 claims description 27
- 108091005804 Peptidases Proteins 0.000 claims description 24
- 239000001888 Peptone Substances 0.000 claims description 24
- 108010080698 Peptones Proteins 0.000 claims description 24
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 24
- 239000000706 filtrate Substances 0.000 claims description 24
- 235000019319 peptone Nutrition 0.000 claims description 24
- 235000019419 proteases Nutrition 0.000 claims description 24
- 239000003513 alkali Substances 0.000 claims description 23
- 238000009630 liquid culture Methods 0.000 claims description 22
- 238000001914 filtration Methods 0.000 claims description 21
- 240000000249 Morus alba Species 0.000 claims description 20
- 235000008708 Morus alba Nutrition 0.000 claims description 20
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 18
- 238000002360 preparation method Methods 0.000 claims description 17
- 238000003756 stirring Methods 0.000 claims description 17
- 238000009210 therapy by ultrasound Methods 0.000 claims description 16
- 238000004108 freeze drying Methods 0.000 claims description 15
- 239000004094 surface-active agent Substances 0.000 claims description 15
- 238000000108 ultra-filtration Methods 0.000 claims description 15
- 238000002835 absorbance Methods 0.000 claims description 14
- 102000004190 Enzymes Human genes 0.000 claims description 10
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- 239000000126 substance Substances 0.000 claims description 8
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- 238000002137 ultrasound extraction Methods 0.000 claims description 3
- 235000001674 Agaricus brunnescens Nutrition 0.000 claims description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 claims 2
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- 229910052799 carbon Inorganic materials 0.000 description 20
- 150000004676 glycans Chemical class 0.000 description 14
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- 230000000259 anti-tumor effect Effects 0.000 description 13
- 240000000599 Lentinula edodes Species 0.000 description 9
- 230000000694 effects Effects 0.000 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 description 7
- 239000008103 glucose Substances 0.000 description 7
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- 239000012137 tryptone Substances 0.000 description 4
- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 208000005718 Stomach Neoplasms Diseases 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 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 description 3
- 238000002474 experimental method Methods 0.000 description 3
- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 description 3
- 229940114124 ferulic acid Drugs 0.000 description 3
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 description 3
- 235000001785 ferulic acid Nutrition 0.000 description 3
- 206010017758 gastric cancer Diseases 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
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- 238000001179 sorption measurement Methods 0.000 description 3
- 201000011549 stomach cancer Diseases 0.000 description 3
- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 description 3
- 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 2
- 229920002498 Beta-glucan Polymers 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 235000001715 Lentinula edodes Nutrition 0.000 description 2
- 239000012670 alkaline solution Substances 0.000 description 2
- 230000003544 deproteinization Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000000874 microwave-assisted extraction Methods 0.000 description 2
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- 101710165028 Alpha-amylase 4 Proteins 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 229920001214 Polysorbate 60 Polymers 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 238000001237 Raman spectrum Methods 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 101710097834 Thiol protease Proteins 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- LWZFANDGMFTDAV-BURFUSLBSA-N [(2r)-2-[(2r,3r,4s)-3,4-dihydroxyoxolan-2-yl]-2-hydroxyethyl] dodecanoate Chemical group CCCCCCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O LWZFANDGMFTDAV-BURFUSLBSA-N 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 108010047754 beta-Glucosidase Proteins 0.000 description 1
- 102000006995 beta-Glucosidase Human genes 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000005422 blasting Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
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- 238000012937 correction Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000035614 depigmentation Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
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- 230000002401 inhibitory effect Effects 0.000 description 1
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- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 230000000144 pharmacologic effect 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
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 238000000790 scattering method Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 235000011067 sorbitan monolaureate Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 125000000185 sucrose group Chemical group 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
-
- 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
- C12P19/08—Dextran
Abstract
the invention discloses a method for extracting and purifying lentinan, which comprises the following steps: the method simplifies the processing process of extraction and purification, effectively improves the extraction efficiency of the lentinan and improves the quality of the lentinan.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine extraction, and particularly relates to a method for extracting and purifying lentinan.
background
lentinan is a polysaccharide extracted from fruiting body of Lentinus edodes (Berk) Sing, and has antitumor and antiviral activities. The pharmacodynamic activity of lentinan is related to the molecular structure and molecular weight. Research shows that in the polysaccharide extracted from shiitake mushroom, the main chain has anti-tumor activity only when the main chain is beta- (1,3) glycosidic bond, and the beta- (1,6) glycosidic bond of the side chain can enhance the pharmacological activity of the beta- (1,3) glycosidic bond main chain; in addition, researchers have found that lentinan has a large relative molecular weight of 40 to 80 × 104 as measured by a gel filtration method (HPLC) and a raman spectrum scattering method, and has a β -triple helical three-strand structure, and when treated with urea or guanidine, the three-dimensional structure of lentinan is destroyed, and the anti-tumor activity is lost. Therefore, researchers should protect the structure of lentinan with good drug effect as much as possible when preparing lentinan.
The prior extraction methods of lentinan are various, such as reflux extraction, ultrasonic extraction, microwave extraction, complex enzyme extraction and the like, but the problems of too small molecular weight, damaged three-dimensional space structure or low content of active ingredients of lentinan and poor anti-tumor effect due to the existence of beta- (1,4) glycosidic bonds of the extracted lentinan because of the problems and the defects of the technical means existing in the extraction process; in addition, the currently used purification means such as alcohol precipitation purification, complex enzyme deproteinization, gel column depigmentation and the like have limited selectivity, so that the purification effect is not ideal, and the obtained lentinan has the problems of low effective polysaccharide content, more impurities and complex operation.
In order to solve these problems, patent No. cn201811368332.x provides a method for extracting lentinan under reduced pressure and elevated temperature, then removing impurities by protease and cellulase treatment, and finally obtaining lentinan by alcohol precipitation. The polysaccharide obtained by the method has high extraction rate, but under the operation of reducing pressure and raising temperature, lentinan is in a high-temperature environment, and the essence is that the lentinan is extracted by high-temperature heating, so that the spatial structure of the lentinan is damaged, the molecular weight is reduced, and the activity of the lentinan is influenced.
The extraction method of lentinan provided by patent CN201710445531.5 focuses on the purification of lentinan solution, and lentinan is obtained by multiple alcohol precipitation, and the lentinan obtained by the method has good anti-tumor activity because the spatial structure is not damaged by high heat treatment, but the extraction rate of the method is low, and multiple alcohol precipitation treatment not only has high requirement on reagent consumables, but also increases the difficulty of post-treatment, and is not suitable for mass production.
Patent CN201810438472.3 proposes that the mushroom is firstly crushed and then blasted by a steam blasting device, and a method for extracting lentinan by an auxiliary microwave extraction method improves the extraction efficiency of the lentinan, but the method has higher requirements on equipment in the extraction process, and the treatment of enzymolysis, inactivation, centrifugation, decoloration and deproteinization in the purification process is more complicated, so that certain difficulty exists in industrial production.
in summary, although the prior art improves the problems in lentinan extraction, the prior art still has respective disadvantages, and with the wider application of lentinan in the medical field, the methods cannot meet the market requirements, so the application intends to provide an extraction and purification method to obtain lentinan with good antitumor activity, less impurities and high purity.
Disclosure of Invention
The invention provides a method for extracting lentinan to solve the technical problems. The method is realized by the following technical scheme:
a method for extracting lentinan comprises the following steps:
(1) Liquid fermentation: firstly, taking 2 parts of shiitake mushroom blocks of 1cm multiplied by 1cm in 200ml of liquid culture medium, and fermenting for 4-5 days in a shaking table at the temperature of 26-30 ℃; secondly, mixing the fermentation liquor obtained in the step I and a liquid culture medium according to the volume ratio of 1: fermenting for 10-12 days at the same condition in a ratio of 10-15, wherein the rotating speed of the shaking table is 120-150 r/min;
(2) ultrasonic treatment: placing the fermentation product obtained in the last step in an ultrasonic instrument, performing ultrasonic treatment for 4-6 hours at the ultrasonic temperature of 45-50 ℃, performing ultrasonic crushing treatment at the ultrasonic frequency of 240-300W, and filtering to obtain a subsequent filtrate;
(3) and (3) decoloring: adding special activated carbon with the mass concentration of 0.5-2% into the subsequent filtrate, adding a surfactant with the mass concentration of 0.3-0.5%, decoloring at 35-40 ℃ for 25-35 min, and collecting a decolored solution;
(4) performing enzyme-alkali extraction, namely adding protease with the mass concentration of 1.5-2.5% into a decolorized solution, stirring for 5-6 h at 45-55 ℃, adding a NaOH solution to be alkaline, filtering, and washing a precipitate to be neutral;
(5) and (3) freeze drying: concentrating the extracting solution in the step (4) until the concentration is 1.05-1.15 mol/L, placing the extracting solution in a novel freeze dryer, wherein the liquid level height is not more than 3cm, the temperature is-20 to-30 ℃, and the freeze-drying time is 7-8 hours;
The liquid culture medium comprises the following substances in percentage by mass: 0.5-1% of mulberry leaf juice, 4-6% of wheat bran leachate, 1-2% of sugar and 1-2% of peptone;
the sugar is one or more of glucose, sucrose and brown sugar;
The peptone is one or more of hydrolyzed peptone, tryptone and soybean peptone;
the preparation method of the mulberry leaf juice comprises the following steps: the feed liquid mass ratio is 1: 10-20, the pH value of the water solution is 7-9, reflux extraction is carried out for 2-3 h at 80-90 ℃, 2-5% of alpha-amylase by mass fraction is added into filtered liquid, stirring is carried out for 1-2 h at 35-45 ℃, then the temperature is raised to 95-100 ℃ and kept for half an hour, then centrifugation is carried out, ultrafiltration treatment is carried out, the cutoff molecular weight of an ultrafiltration membrane is 2500kDa, and the absorbance A of the ultrafiltrate is concentrated to 334nm and is between 0.134-0.173;
The preparation method of the wheat bran leachate comprises the following steps: the wheat bran and the ethanol with the volume fraction of 40-60% are mixed according to the mass ratio of 1: performing ultrasonic extraction at the power of 120-200W for 50-60 min in a proportion of 20-30, filtering, collecting filtrate, volatilizing ethanol, adding protease with the mass fraction of 2-5%, stirring at the temperature of 45-55 ℃ for 2-3 h, and performing high-temperature enzyme killing treatment to obtain the finished product;
The special activated carbon is modified activated carbon, and the modification method is to soak the special activated carbon in an ammonia water solution with the mass concentration of 15-25% for 4-8 hours;
The HLB value of the surfactant is 8-16;
the protease extracted by the enzyme-alkali method is thiol protease such as papain, bromelain and the like;
NaOH is added into fermentation liquor in the enzyme-alkali extraction until the pH value of the solution is 9-10, and if the alkalinity is too strong, the three-dimensional space spiral structure of lentinan can be damaged.
has the advantages that:
The invention provides an extraction and purification method of lentinan, which has the following beneficial effects compared with the prior art:
the wheat bran leachate and the mulberry leaf juice are added into the liquid fermentation culture medium adopted by the invention, because experiments show that the polysaccharide production capacity of thalli in the wheat bran culture medium is obvious in several substances (starch, cane sugar and wheat bran) for supplementing carbon sources, and further researches show that the promotion effect has positive significance on the production of the polysaccharide and ferulic acid in wheat bran, so that the document also provides an extraction method of ferulic acid in wheat bran, the method provides the carbon source and ferulic acid for the liquid culture medium, eliminates protein and lightens the subsequent purification burden; the research result shows that the mulberry leaf DNJ has biological activity of inhibiting beta-glucosidase, and the addition of the mulberry leaf DNJ in a culture medium is helpful for protecting beta- (1,3) glucoside of lentinan in a lentinus edodes fermentation liquor from being degraded and broken, so that the document also provides a method for extracting and purifying the mulberry leaf juice rich in DNJ, wherein a DNJ-containing solution is extracted from an alkaline solution, the pH is controlled to ensure that the extracted DNJ has good activity, and the DNJ is purified by enzymolysis and ultrafiltration. Therefore, the preparation method of the mulberry leaf juice and the wheat bran leachate provided by the document is beneficial to preparing the culture medium which has positive significance on the yield of the lentinan.
The ultrasonic treatment process is beneficial to transferring the lentinan inside the lentinan outside the cell, and the yield of the lentinan is improved.
The invention provides an enzyme-alkali extraction method for extracting lentinan, which realizes the synchronous extraction and purification in the process: most of protein is removed by enzyme treatment, and lentinan is precipitated in alkaline liquor. The extraction process is free from adding of organic reagents, so that the problems of subsequent removal treatment of organic reagents and consequent reduction of lentinan activity caused by destruction of a three-dimensional space structure of lentinan and beta-glucan molecular chain fracture are solved, and an enzyme-alkali extraction method can provide a relatively complete beta-glucan molecular structure for lentinan, so that the extracted lentinan has a larger molecular weight, the pharmaceutical activity of the lentinan is ensured, the organic reagent treatment is not required, and the method is economic and environment-friendly; in addition, because the three-dimensional space helical structure of lentinan is denatured in strong alkaline solution, but lentinan is required to be precipitated in alkaline environment, the pH of the extraction method is required to be controlled in order to protect the molecular structure from being damaged during lentinan extraction.
In the decoloring treatment, the activated carbon decoloring method which is simple and easy to operate is selected, but the activated carbon has an excessively large specific surface area and cannot be decomposed into an aqueous solution as soon as possible, and the activated carbon stays on the surface of the liquid for a long time and cannot be sufficiently contacted with the liquid, so that the activated carbon is difficult to remove pigments in the aqueous solution by adsorption, and the decoloring capacity is reduced. In order to solve the problem, the document provides two solutions, namely, firstly, a compatilizer is added, so that the activated carbon and an aqueous solution are easy to be compatible and soaked, the function is realized, and the adsorption efficiency of the activated carbon is improved; ② modified active carbon is added, and the adsorption capacity of the modified active carbon is greatly improved. In the drying process, the document aims to reduce the loss of lentinan as much as possible, takes the retention rate as an index and screens the most suitable vacuum freeze drying process.
In general, the method provided by the application simplifies the process of extracting and purifying the lentinan, protects the anti-tumor activity of the lentinan, effectively improves the extraction efficiency of the lentinan, has high product purity and few impurities, and improves the quality of the lentinan.
Detailed Description
The following is a detailed description of the embodiments of the present invention, but the present invention is not limited to these embodiments, and any modifications or substitutions in the basic spirit of the embodiments are included in the scope of the present invention as claimed in the claims.
example 1
(1) liquid fermentation: firstly, taking 2 parts of shiitake mushroom blocks of 1cm multiplied by 1cm in 200ml of liquid culture medium, and fermenting for 5 days in a shaking table at the temperature of 26 ℃; secondly, mixing the fermentation liquor obtained in the step I and a liquid culture medium according to the volume ratio of 1:10 for 12 days under the same conditions, and the rotating speed of the shaking table is 120 r/min;
The liquid culture medium comprises the following substances in percentage by mass: 0.5% of mulberry leaf juice, 4% of wheat bran extract, 1% of sugar and 1% of peptone;
the sugar is a mixture of glucose, sucrose and brown sugar in a mass ratio of 1:1: 2;
The peptone is hydrolyzed peptone;
The preparation method of the mulberry leaf juice comprises the following steps: the feed liquid mass ratio is 1:10, the pH value of the water solution is 7, reflux extraction is carried out for 3h at the temperature of 80 ℃, 2% of alpha-amylase by mass is added into the filtered filtrate, the mixture is stirred for 2h at the temperature of 35 ℃, the temperature is raised to 95 ℃ and kept for half an hour, then centrifugation is carried out, ultrafiltration treatment is carried out, the cutoff molecular weight of an ultrafiltration membrane is 2500kDa, and the ultrafiltrate is concentrated to the absorbance A at 334nm of 0.134;
the preparation method of the wheat bran leachate comprises the following steps: ultrasonically extracting wheat bran and 40% ethanol by mass ratio of 1:20 at 200W for 50min, filtering, collecting filtrate, volatilizing ethanol, adding 2% protease, stirring at 45 deg.C for 3 hr, and deactivating enzyme at high temperature;
(2) Ultrasonic treatment: placing the fermentation product obtained in the last step in an ultrasonic instrument, performing ultrasonic treatment for 6h at the ultrasonic temperature of 45 ℃, performing ultrasonic crushing treatment at the ultrasonic frequency of 240W, and filtering to obtain a subsequent filtrate;
(3) and (3) decoloring: adding special active carbon with mass concentration of 0.5% and surfactant with mass concentration of 0.3% into the subsequent filtrate, decolorizing at 35 deg.C for 35min, and collecting decolorized solution;
The special active carbon is modified active carbon, and the modification method is to soak the special active carbon in ammonia water solution with the mass concentration of 15% for 8 hours.
the surfactant is Span 20;
(4) enzyme-alkali extraction, namely adding protease with the mass concentration of 1.5% into a decolorized solution, stirring for 6 hours at 45 ℃, adding NaOH solution to be alkaline, filtering, and washing a precipitate to be neutral;
the protease extracted by the enzyme-alkali method is papain;
NaOH is added into the fermentation liquor in the enzyme-alkali extraction until the pH value of the solution is 9, and if the alkalinity is too strong, the three-dimensional space spiral structure of lentinan can be damaged.
(5) And (3) freeze drying: concentrating the extracting solution in the step (4) to the concentration of 1.05mol/L, placing the extracting solution in a novel freeze dryer, wherein the liquid level height is not more than 3cm, the temperature is-20 ℃, and the freeze-drying time is 8 hours;
Example 2
(1) liquid fermentation: firstly, taking 2 parts of shiitake mushroom blocks of 1cm multiplied by 1cm in 200ml of liquid culture medium, and fermenting for 4 days in a shaking table at the temperature of 30 ℃; secondly, mixing the fermentation liquor obtained in the step I and a liquid culture medium according to the volume ratio of 1:15 for 10 days under the same conditions, and the rotating speed of the shaking table is 150 r/min;
The liquid culture medium comprises the following substances in percentage by mass: 1% of mulberry leaf juice, 6% of wheat bran leachate, 2% of sugar and 2% of peptone;
the sugar is glucose;
The peptone is hydrolyzed peptone, tryptone and soybean peptone, and the mass ratio of the peptone to the peptone is 1: composition is carried out;
The preparation method of the mulberry leaf juice comprises the following steps: the feed liquid mass ratio is 1:20, the pH value of the water solution is 9, reflux extraction is carried out for 3h at 90 ℃, 5% of alpha-amylase by mass is added into the filtered filtrate, the mixture is stirred for 1h at 45 ℃, the temperature is raised to 100 ℃ and kept for half an hour, then centrifugation is carried out, ultrafiltration treatment is carried out, the cutoff molecular weight of an ultrafiltration membrane is 2500kDa, and the ultrafiltrate is concentrated until the absorbance A at 334nm is between 0.173;
The preparation method of the wheat bran leachate comprises the following steps: ultrasonically extracting wheat bran and 60% ethanol by mass ratio of 1:30 at power of 120W for 60min, filtering, collecting filtrate, volatilizing ethanol, adding protease with mass fraction of 5%, stirring at 55 deg.C for 2 hr, and deactivating enzyme at high temperature;
(2) ultrasonic treatment: placing the fermentation product obtained in the last step in an ultrasonic instrument, performing ultrasonic treatment for 4h at the ultrasonic temperature of 50 ℃, performing ultrasonic crushing treatment at the ultrasonic frequency of 300W, and filtering to obtain a subsequent filtrate;
(3) And (3) decoloring: adding special activated carbon with the mass concentration of 2% into the subsequent filtrate, adding surfactant with the mass concentration of 0.5%, decolorizing at 40 deg.C for 35min, and collecting decolorized solution;
The special active carbon is modified active carbon, and the modification method is to soak the special active carbon in an ammonia water solution with the mass concentration of 25% for 4 hours.
the surfactant is Tween 80;
(4) enzyme-alkali extraction, namely adding protease with the mass concentration of 2.5% into a decolorized solution, stirring for 5 hours at 55 ℃, adding NaOH solution to be alkaline, filtering, and washing a precipitate to be neutral;
the protease extracted by the enzyme-alkali method is bromelain;
NaOH is added into the fermentation liquor in the enzyme-alkali extraction until the pH value of the solution is 10, and if the alkalinity is too strong, the three-dimensional space spiral structure of lentinan can be damaged.
(5) And (3) freeze drying: concentrating the extracting solution in the step (4) to the concentration of 1.15mol/L, placing the extracting solution in a novel freeze dryer, wherein the liquid level height is not more than 3cm, the temperature is-30 ℃, and the freeze-drying time is 7 hours;
Example 3
(1) Liquid fermentation: firstly, 2 parts of shiitake mushroom blocks of 1cm multiplied by 1cm are taken to be fermented in 200ml of liquid culture medium for 4 days at the temperature of 28 ℃ in a shaking table; secondly, mixing the fermentation liquor obtained in the step I and a liquid culture medium according to the volume ratio of 1: 12 for 11 days under the same conditions, and the rotating speed of the shaking table is 130 r/min;
the liquid culture medium comprises the following substances in percentage by mass: 0.8% of mulberry leaf juice, 5% of wheat bran extract, 1.5% of sugar and 1.5% of peptone;
The sugar is composed of glucose, sucrose and brown sugar according to a mass ratio of 1:2: 2;
The peptone is soybean peptone;
The preparation method of the mulberry leaf juice comprises the following steps: the feed liquid mass ratio is 1:15, the pH value of the water solution is 7, reflux extraction is carried out at 85 ℃ for 2.5h, 3% of alpha-amylase 4 in mass fraction is added into the filtered liquid, stirring is carried out for 1.5h, then the temperature is raised to 97 ℃ and kept for half an hour, then centrifugation is carried out, ultrafiltration treatment is carried out, the cut-off molecular weight of an ultrafiltration membrane is 2500kDa, and the ultrafiltrate is concentrated to the absorbance A at 334nm of 0.154;
the preparation method of the wheat bran leachate comprises the following steps: ultrasonically extracting wheat bran and 50% ethanol by mass ratio of 1:25 at power of 150W for 55min, filtering, collecting filtrate, volatilizing ethanol, adding protease with mass fraction of 3%, stirring at 50 deg.C for 2.5 hr, and deactivating enzyme at high temperature;
(2) ultrasonic treatment: placing the fermentation product obtained in the last step in an ultrasonic instrument, performing ultrasonic treatment for 5h at the ultrasonic temperature of 48 ℃, performing ultrasonic crushing treatment at the ultrasonic frequency of 270W, and filtering to obtain a subsequent filtrate;
(3) and (3) decoloring: adding special activated carbon with the mass concentration of 1% into the subsequent filtrate, adding surfactant with the mass concentration of 0.4%, decolorizing at 38 deg.C for 30min, and collecting decolorized solution;
the special active carbon is modified active carbon, and the modification method is to soak the special active carbon in an ammonia water solution with the mass concentration of 20% for 6 hours.
the surfactant is Tween 60;
(4) enzyme-alkali extraction, namely adding protease with the mass concentration of 2% into a decolorized solution, stirring at 50 ℃ for 5.5h, adding NaOH solution to be alkaline, filtering, and washing a precipitate to be neutral;
The protease extracted by the enzyme-alkali method is papain;
NaOH is added into the fermentation liquor in the enzyme-alkali extraction until the pH value of the solution is 9, and if the alkalinity is too strong, the three-dimensional space spiral structure of lentinan can be damaged.
(5) And (3) freeze drying: concentrating the extracting solution in the step (4) to the concentration of 1.10mol/L, placing the extracting solution in a novel freeze dryer, wherein the liquid level height is not more than 3cm, the temperature is-25 ℃, and the freeze-drying time is 7.5 hours;
example 4
(1) Liquid fermentation: firstly, taking 2 parts of shiitake mushroom blocks of 1cm multiplied by 1cm in 200ml of liquid culture medium, and fermenting for 5 days in a shaking table at the temperature of 29 ℃; secondly, mixing the fermentation liquor obtained in the step I and a liquid culture medium according to the volume ratio of 1: fermenting for 12 days at the ratio of 10-15 under the same condition, wherein the rotating speed of the shaking table is 140 r/min;
the liquid culture medium comprises the following substances in percentage by mass: 0.7% of mulberry leaf juice, 5% of wheat bran extract, 1.5% of sugar and 1.5% of peptone;
the sugar is brown sugar
the peptone is one or more of tryptone;
The preparation method of the mulberry leaf juice comprises the following steps: the feed liquid mass ratio is 1:18, the pH value of the water solution is 9, reflux extraction is carried out for 2.5h at 90 ℃, 4% of alpha-amylase by mass fraction is added into the filtered liquid, the mixture is stirred for 1.5h at 45 ℃, then the temperature is raised to 95 ℃ and kept for half an hour, then centrifugation is carried out, ultrafiltration treatment is carried out, the molecular weight cutoff of an ultrafiltration membrane is 2500kDa, and the absorbance A of the ultrafiltrate is concentrated to 334nm and is 0.167;
the preparation method of the bran extract comprises the following steps: ultrasonically extracting wheat bran and 55% ethanol by mass ratio of 1:28 at power of 180W for 60min, filtering, collecting filtrate, volatilizing ethanol, adding protease with mass fraction of 3%, stirring at 55 deg.C for 2.5 hr, and deactivating enzyme at high temperature;
(2) ultrasonic treatment: placing the fermented product obtained in the previous step in an ultrasonic instrument, performing ultrasonic treatment for 4.5h at an ultrasonic temperature of 50 ℃, performing ultrasonic crushing treatment at an ultrasonic frequency of 290W, and filtering to obtain a subsequent filtrate;
(3) and (3) decoloring: adding special activated carbon with the mass concentration of 1.8% into the subsequent filtrate, adding surfactant with the mass concentration of 0.3%, decolorizing at 40 deg.C for 25min, and collecting decolorized solution;
the special active carbon is modified active carbon, and the modification method is to soak the special active carbon in an ammonia water solution with the mass concentration of 20% for 8 hours.
the surfactant is Tween 65;
(4) enzyme-alkali extraction, adding protease with mass concentration of 2.5% into decolorized solution, stirring at 52 deg.C for 6 hr, adding NaOH solution to alkaline, filtering, and washing precipitate to neutral;
the protease extracted by the enzyme-alkali method is papain;
NaOH is added into the fermentation liquor in the enzyme-alkali extraction until the pH value of the solution is 9, and if the alkalinity is too strong, the three-dimensional space spiral structure of lentinan can be damaged.
(5) And (3) freeze drying: concentrating the extract obtained in the step (4) to a concentration of 1.12mol/L, placing the concentrate in a novel freeze dryer, wherein the liquid level height is not more than 3cm, the temperature is-20 ℃, and the freeze-drying time is 8 hours;
example 5
(1) liquid fermentation: firstly, taking 2 parts of shiitake mushroom blocks of 1cm multiplied by 1cm in 200ml of liquid culture medium, and fermenting for 4 days in a shaking table at the temperature of 30 ℃; secondly, mixing the fermentation liquor obtained in the step I and a liquid culture medium according to the volume ratio of 1: fermenting for 11 days at the ratio of 10-15 under the same condition, wherein the rotating speed of the shaking table is 140 r/min;
The liquid culture medium comprises the following substances in percentage by mass: 0.8% of mulberry leaf juice, 5% of wheat bran extract, 1% of sugar and 2% of peptone;
the sugar is sucrose;
The peptone is tryptone and soybean peptone, and the mass ratio of the peptone to the soybean peptone is 1: 2;
the preparation method of the mulberry leaf juice comprises the following steps: the feed liquid mass ratio is 1:25, the pH value of the water solution is 7, reflux extraction is carried out for 3h at 85 ℃, 4% of alpha-amylase by mass is added into the filtered filtrate, the mixture is stirred for 1h at 45 ℃, the temperature is raised to 98 ℃ and kept for half an hour, then centrifugation is carried out, ultrafiltration treatment is carried out, the cutoff molecular weight of an ultrafiltration membrane is 2500kDa, and the absorbance A of the ultrafiltrate is concentrated to 334nm and is between 0.148;
The preparation method of the wheat bran leachate comprises the following steps: ultrasonically extracting wheat bran and 55% ethanol by mass ratio of 1:20 at power of 150W for 50min, filtering, collecting filtrate, volatilizing ethanol, adding protease with mass fraction of 3%, stirring at 55 deg.C for 2 hr, and deactivating enzyme at high temperature;
(2) ultrasonic treatment: placing the fermentation product obtained in the last step in an ultrasonic instrument, performing ultrasonic treatment for 4h at the ultrasonic temperature of 50 ℃, performing ultrasonic crushing treatment at the ultrasonic frequency of 280W, and filtering to obtain a subsequent filtrate;
(3) and (3) decoloring: adding special activated carbon with the mass concentration of 2% into the subsequent filtrate, adding surfactant with the mass concentration of 0.5%, decolorizing at 40 deg.C for 35min, and collecting decolorized solution;
the special active carbon is modified active carbon, and the modification method is to soak the special active carbon in an ammonia water solution with the mass concentration of 20% for 7 hours.
the surfactant is Tween 21;
(4) Enzyme-alkali extraction, namely adding protease with the mass concentration of 2% into a decolorized solution, stirring for 5.5 hours at 45 ℃, adding a NaOH solution to be alkaline, filtering, and washing a precipitate to be neutral;
The protease extracted by the enzyme-alkali method is bromelain;
NaOH is added into the fermentation liquor in the enzyme-alkali extraction until the pH value of the solution is 10, and if the alkalinity is too strong, the three-dimensional space spiral structure of lentinan can be damaged.
(5) and (3) freeze drying: concentrating the extracting solution in the step (4) to the concentration of 1.09mol/L, placing the extracting solution in a novel freeze dryer, wherein the liquid level height is not more than 3cm, the temperature is-22 ℃, and the freeze-drying time is 7.5 hours;
Comparative example 1
the procedure of example 1 was repeated, except that no surfactant was added in the decoloring step and that no modified activated carbon was added.
Comparative example 2
the same procedure as in example 1 was repeated except that the liquid medium was not supplemented with the wheat bran extract and the mulberry leaf juice.
Comparing the decolorization rate, the protein removal rate, the polysaccharide content and the polysaccharide purity of the lentinan obtained in the examples 1-5 with those obtained in the comparative examples 1 and 2, wherein the decolorization rate, the protein removal rate, the polysaccharide content and the polysaccharide purity are calculated according to the following formula:
the total sugar content is measured by a phenol-sulfuric acid method; protein content was determined by the Bradford method.
pigment removal rate (%) - (A0-A1)/A0X 100.0% (1)
note: a0 and A1 in formula (1) represent the absorbance at 450nm of the solution before and after decolorization, respectively.
decolorization ratio (%) - (C0-C1)/C0X 100.0% (2)
Note: c0 and C1 in formula (2) represent the absorbance at 450nm of the solution before and after decolorization, respectively.
the polysaccharide purity calculation method comprises the following steps: weighing 10mg of glucose, adding distilled water, dissolving in a beaker, diluting to constant volume in a 250mL volumetric flask, respectively taking 0.4mL, 0.6mL, 0.8mL, 1.0mL, 1.2mL, 1.4mL, 1.6mL and 1.8mL, adding water to 2mL, adding 1mL of 6% phenol, mixing uniformly, slowly adding 5mL of concentrated sulfuric acid, mixing uniformly, standing for 20min, measuring absorbance A at a wavelength of 490nm by using a spectrophotometer, and drawing a standard curve according to the concentration and the absorbance of the glucose. And calculating the purity of the crude polysaccharide according to the absorbance of the lentinan and a glucose standard curve.
The settlement results are shown in table 1.
TABLE 1 decolorization rate, protein removal rate, polysaccharide content, and polysaccharide purity of lentinan prepared in examples and comparative examples
the anti-tumor effect of lentinan prepared by the present application is further illustrated below:
the first group is prepared according to the method provided by the application, the second group is prepared according to the method disclosed by patent document CN201811368332.X, the anti-tumor activity of lentinan is detected according to the following experimental scheme, the anti-tumor activity of the two lentinan is compared, and the result is shown in table 2.
The experimental scheme is as follows: the human gastric cancer cell strain SGC-7901 is cultured by RPMI1640 containing 10% FBS, cells in a logarithmic growth phase are used for experiments, the cells are inoculated in a 96-well plate, lentinan with different concentrations is added after the cells adhere to the wall, 3 multiple wells are arranged for each concentration, and a solvent control with corresponding concentration and a cell-free zeroing well are arranged. And (3) incubating the drug and the cells for 48h, adding MTT, placing the cell and the MTT in an incubator for reaction for 4h, removing the culture medium and the MTT in the hole, adding 100 mu l of DMSO, shaking to dissolve the generated purple crystals, immediately measuring the absorbance (OD value) by using an enzyme-linked immunosorbent assay (ELISA) instrument, wherein the measuring wavelength is 570nm, the correction wavelength is 630nm, and subtracting the blank absorbance from the obtained value. The tumor growth inhibition rate was calculated according to the formula (1-OD-treated group/OD-control group). times.100%.
TABLE 2 in vitro gastric cancer cell inhibition rates of two lentinan
therefore, experimental demonstration proves that the lentinan prepared by the method for extracting and purifying the lentinan has high purity and good anti-tumor activity, and in an in vitro tumor inhibition experiment, the lentinan prepared in the document has 85% inhibition effect on gastric cancer cells only at 50 mu g/ml, and is superior to the prior art. Since the anti-tumor activity of lentinan is related to the molecular structure, it is known from literature reports that when the main chain is a beta- (1,3) glycosidic bond, the molecular weight is large, and the molecular conformation is a three-dimensional helical space structure, the activity of lentinan is better, so that lentinan obtained by the preparation method provided by the application may be rich in such molecular structure and characteristics, but the conclusion needs to be further verified by a spectrogram.
according to the method for extracting and purifying the lentinan, the lentinan with high purity and less impurities is obtained by increasing the source of the lentinan, increasing the extraction rate and improving the purification efficiency, and a measure for protecting the molecular structure of the lentinan is added in the technical scheme, so that the lentinan obtained by preparation has good anti-tumor activity.
Claims (10)
1. a method for extracting and purifying lentinan is characterized by comprising the steps of liquid fermentation, ultrasonic treatment, decoloration, enzyme-alkali extraction and freeze drying; the liquid fermentation comprises the following steps:
Firstly, putting mushroom blocks into a liquid culture medium, and fermenting for 4-5 days by using a shaking table;
secondly, mixing the fermentation liquor obtained in the first step with a liquid culture medium, and fermenting for 10-12 days under the same conditions;
the liquid culture medium consists of the following substances: mulberry leaf juice, wheat bran extract, sugar and peptone.
2. the extraction and purification method of lentinan as claimed in claim 1, wherein the enzyme-alkali extraction is: adding protease with the mass concentration of 1.5-2.5% into a decolorized solution, stirring for 5-6 h at 45-55 ℃, adding a NaOH solution to be alkaline, filtering, and washing a precipitate to be neutral.
3. the extraction and purification method of lentinan as claimed in claim 1 or 2, comprising the steps of:
(1) liquid fermentation;
(2) ultrasonic treatment: placing the fermentation product obtained in the last step in an ultrasonic instrument, performing ultrasonic treatment for 4-6 hours at the ultrasonic temperature of 45-50 ℃, performing ultrasonic crushing treatment at the ultrasonic power of 240-300W, and filtering to obtain a subsequent filtrate;
(3) And (3) decoloring: adding special activated carbon with the mass concentration of 0.5-2% into the subsequent filtrate, adding a surfactant with the mass concentration of 0.3-0.5%, decoloring at 35-40 ℃ for 25-35 min, and collecting a decolored solution;
(4) extracting by an enzyme-alkali method;
(5) and (3) freeze drying: and (4) concentrating the extracting solution in the step (4) until the concentration is 1.05-1.15 mol/L, placing the extracting solution in a novel freeze dryer, wherein the liquid level height is not more than 3cm, the temperature is-20 to-30 ℃, and the freeze-drying time is 7-8 hours.
4. The method for extracting and purifying lentinan according to claim 1 or 3, wherein the preparation method of the mulberry leaf juice comprises the following steps: the feed liquid mass ratio is 1: 10-20, the pH value of the water solution is 7-9, reflux extraction is carried out for 2-3 h at 80-90 ℃, 2-5% of alpha-amylase by mass fraction is added into filtered liquid, stirring is carried out for 1-2 h at 35-45 ℃, then the temperature is raised to 95-100 ℃ for half an hour, centrifugation is carried out, ultrafiltration treatment is carried out, the cutoff molecular weight of an ultrafiltration membrane is 2500kDa, and the absorbance A of the ultrafiltrate is concentrated to 334nm and is between 0.134-0.173.
5. the method for extracting and purifying lentinan according to claim 1 or 3, wherein the preparation method of the wheat bran leachate comprises the following steps: wheat bran and 40-60% ethanol are mixed by a mixing ratio of 1: performing ultrasonic extraction at the power of 120-200W for 50-60 min in a proportion of 20-30, filtering, collecting filtrate, volatilizing ethanol, adding protease with the mass fraction of 2-5%, stirring at the temperature of 45-55 ℃ for 2-3 h, and performing high-temperature enzyme killing treatment to obtain the finished product.
6. the method for extracting and purifying lentinan as claimed in claim 3, wherein the special activated carbon is modified activated carbon, and the modification method comprises soaking in 15-25% ammonia water solution for 4-8 h.
7. the method for extracting and purifying lentinan as claimed in claim 3, wherein the surfactant has an HLB value of 8 to 16.
8. The method for extracting and purifying lentinan according to claim 2 or 3, wherein the protease is thiol-containing protease.
9. the method for extracting and purifying lentinan according to claim 2 or 3, wherein the thiol-group-containing protease is one or a combination of papain and bromelain.
10. The method for extracting and purifying lentinan as claimed in claim 2 or 3, wherein NaOH is added to the fermentation liquid until the pH of the solution is 9-10.
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| CN111744231A (en) * | 2020-06-29 | 2020-10-09 | 漯河医学高等专科学校 | Method for enriching active components for inhibiting growth of candida albicans in mushroom fermentation liquor |
| CN111744231B (en) * | 2020-06-29 | 2021-11-19 | 漯河医学高等专科学校 | Method for enriching active components for inhibiting growth of candida albicans in mushroom fermentation liquor |
| CN112094878A (en) * | 2020-08-18 | 2020-12-18 | 周银根 | Method for extracting lentinan by two-step enzyme, lentinan extract and application thereof |
| CN113564212A (en) * | 2021-07-26 | 2021-10-29 | 浙江树人学院（浙江树人大学） | Method for extracting eucommia ulmoides leaf polysaccharide by using microbial fermentation method |
| CN113564212B (en) * | 2021-07-26 | 2024-01-16 | 浙江树人学院（浙江树人大学） | Method for extracting eucommia ulmoides leaf polysaccharide by utilizing microbial fermentation method |
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Application publication date: 20191206 |