WO2022141957A1 - 一种香菇寡肽的制备方法及用途 - Google Patents

一种香菇寡肽的制备方法及用途 Download PDF

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WO2022141957A1
WO2022141957A1 PCT/CN2021/087726 CN2021087726W WO2022141957A1 WO 2022141957 A1 WO2022141957 A1 WO 2022141957A1 CN 2021087726 W CN2021087726 W CN 2021087726W WO 2022141957 A1 WO2022141957 A1 WO 2022141957A1
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preparation
oligopeptide
extract
filtrate
enzymatic hydrolysis
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伍曾利
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海南云皓生物科技有限公司
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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
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis

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  • the invention relates to the technical field of biomedicine, in particular to a preparation method and application of a mushroom oligopeptide.
  • Mushrooms belong to Basidiomycetes, Agaricales, mushrooms, Lentinus, scientific name Lentinus edodes, originated in my country, is the second largest mushroom in the world, and is also a precious edible fungus with a long-standing reputation in my country.
  • the earliest cultivation of shiitake mushrooms in my country has a history of more than 800 years.
  • Shiitake mushroom is also a famous medicinal fungus in my country.
  • the medicinal properties and functions of shiitake mushrooms have been written by medical scientists of all dynasties. "Daily Use Materia Medica" was used for medicinal purposes. Sweet and flat.
  • the technical problem to be solved by the present invention is to provide a preparation method and application of Lentinus edodes oligopeptide with anti-tumor physiological activity.
  • the preparation method of the mushroom oligopeptide provided by the invention comprises:
  • the shiitake mushroom is extracted with water to obtain an extract
  • the extract is concentrated to a solid content of 30% to 50%, treated with CTAB, and centrifuged to obtain the supernatant;
  • the supernatant is subjected to alkaline protease enzymolysis, flavor protease enzymolysis, and activated carbon decolorization in sequence, and the fraction with a molecular weight of 300 to 1000 Da is taken to prepare Lentinus edodes oligopeptide.
  • oligopeptide preparation method of the present invention dried shiitake mushrooms are subjected to extraction and polysaccharide removal processes to preliminarily increase the protein content of the extract;
  • the Lentinus edodes protein is hydrolyzed into oligopeptides and then purified by nanofiltration to control the molecular weight of the Lentinus edodes oligopeptide between 300-1000 Daltons.
  • the shiitake mushroom is the stipe of dried shiitake mushroom.
  • the preparation of the extracting solution includes: pulverizing the dried shiitake stalk, adding 5-15 times the weight of water, grinding to 200-300 mesh, extracting at 90°C-100°C for 2 hours, and filtering to obtain a filtrate1 and filter residue; add 5 to 12 times the weight of water to the filter residue, extract at 90°C to 100°C for 1.5 hours, and filter to obtain filtrate 2; combine filtrate 1 and filtrate 2, centrifuge at 6000 to 20000 rpm for 5 to 30 min, and get The supernatant is the extract.
  • the preparation of the extracting solution includes: pulverizing the dried mushroom stems, adding 5 times the weight of water, grinding to 200 mesh, extracting at 90° C. for 2 hours, and filtering to obtain a filtrate 1 and a filter residue; the filter residue Add 5 times the weight of water, extract at 90°C for 1.5 hours, filter to obtain filtrate 2; combine filtrate 1 and filtrate 2, centrifuge at 6000 rpm for 5 min, and take the supernatant as the extract.
  • the preparation of the extracting solution includes: pulverizing the dried shiitake stalks, adding 10 times the weight of water, grinding to 250 mesh, extracting at 95°C for 2 hours, and filtering to obtain a filtrate 1 and a filter residue; the filter residue Add 10 times the weight of water, extract at 95°C for 1.5 hours, and filter to obtain filtrate 2; combine filtrate 1 and filtrate 2, centrifuge at 13,000 rpm for 10 min, and take the supernatant as the extract.
  • the preparation of the extracting solution includes: pulverizing the dried shiitake stalks, adding 15 times the weight of water, grinding to 300 mesh, extracting at 100° C. for 2 hours, and filtering to obtain a filtrate 1 and a filter residue; the filter residue Add 15 times the weight of water, extract at 100°C for 1.5 hours, and filter to obtain filtrate 2; combine filtrate 1 and filtrate 2, centrifuge at 20,000 rpm for 15 min, and take the supernatant as the extract.
  • the CTAB processing includes:
  • CTAB was added while stirring until its mass fraction was 0.5% to 1.0%, and after stirring was continued for 5 minutes, it was allowed to stand for 30 to 180 minutes.
  • CTAB hexadecyltrimethylammonium bromide
  • an aqueous solution of CTAB wherein the mass fraction of CTAB is 5% to 10%. That is, the CTAB aqueous solution with a mass fraction of 5% to 10% is added to the extract, until the mass fraction of CTAB in the extract is 0.5% to 1.0%.
  • the CTAB treatment includes: at room temperature, adding CTAB to a mass fraction of 0.5% while stirring, continuing to stir for 5 minutes, and then standing for 30 minutes.
  • the CTAB treatment includes: at room temperature, adding CTAB to a mass fraction of 0.75% while stirring, continuing to stir for 5 minutes, and then standing for 30 minutes.
  • the CTAB treatment includes: at room temperature, adding CTAB to a mass fraction of 1.0% while stirring, continuing to stir for 5 minutes, and then standing for 30 minutes.
  • the centrifugation conditions are 6,000-20,000 rpm for 5-30 minutes. In some embodiments, the conditions of centrifugation are centrifugation at 6000/min for 5 min, or centrifugation at 20000/min for 15 min.
  • the conditions for enzymatic hydrolysis by alkaline protease include: adding alkaline protease with a specific activity of 5,000 to 40,000 international units per kg of the supernatant; ⁇ 120min. After the alkaline protease is enzymatically hydrolyzed, the step of inactivating the enzyme by alkaline protease is also included. The conditions for the inactivation of the enzyme are 90 to 100° C. for 5 to 15 minutes.
  • the conditions for the enzymatic hydrolysis by alkaline protease include: adding 5000 IU of alkaline protease with specific activity per kg of the supernatant; pH value of 8.5, enzymatic hydrolysis at 45°C for 20 minutes, and then inactivation at 90°C 5min.
  • the conditions for the enzymatic hydrolysis by alkaline protease include: adding alkaline protease with a specific activity of 21,500 IU per kg of the supernatant; pH value of 9.0, enzymatic hydrolysis at 50°C for 70 minutes, and then inactivation at 95°C 10min.
  • the conditions of the alkaline protease enzymatic hydrolysis include: adding 40,000 IU of alkaline protease with specific activity per kg of the supernatant; pH value of 9.5, enzymatic hydrolysis at 55°C for 120 minutes, and then inactivation at 100°C 15min.
  • the conditions for the enzymatic hydrolysis of the flavor protease include: adding 5,000-40,000 IU of flavor protease with specific activity per kg of the supernatant; the pH value is 6.5-7.5, and the enzymatic hydrolysis is performed at 45-55°C for 20-120 minutes. After the flavor protease is enzymatically hydrolyzed, the step of inactivating the flavor by the flavor protease is also included. The conditions for the inactivation of the enzyme are 90 to 100° C. for 5 to 15 minutes.
  • the conditions for the enzymatic hydrolysis of the flavor protease include: adding 5000 IU of flavor protease with specific activity per kg of the supernatant; pH value of 6.5, enzymatic hydrolysis at 45°C for 20 minutes, and then inactivation at 90°C for 5 minutes.
  • the conditions for the enzymatic hydrolysis of the flavor protease include: adding 21500 IU of flavor protease with specific activity per kg of the supernatant; pH value of 7.0, enzymatic hydrolysis at 50°C for 70 minutes, and then inactivation at 95°C for 10 minutes.
  • the conditions for the enzymatic hydrolysis of the flavor protease include: adding 40,000 international units of flavor protease with specific activity per kg of the supernatant; pH value of 7.5, enzymatic hydrolysis at 55°C for 20 minutes, and then inactivation at 90°C for 5 minutes.
  • the conditions for the activated carbon decolorization include: adding activated carbon to the enzymatic hydrolysis solution after enzymatic hydrolysis until its mass fraction is 0.1%-1%, stirring for 10-60min at 70°C. After the decolorization, a step of filtering the filtrate is also included.
  • the conditions for decolorization of the activated carbon include: adding activated carbon to the enzymatic hydrolysis solution after enzymatic hydrolysis until its mass fraction is 0.1%, at 70° C., and stirring for decolorization for 10 minutes.
  • the activated carbon decolorization conditions include: adding activated carbon to the enzymatic hydrolysis solution after enzymatic hydrolysis until its mass fraction is 0.55%, at 70° C., and stirring for decolorization for 35 minutes.
  • the activated carbon decolorization conditions include: adding activated carbon to the enzymatic hydrolysis solution after enzymatic hydrolysis until its mass fraction is 1%, 70° C., and stirring for decolorization for 60 minutes.
  • the part with a molecular weight of 300 to 1000 Da includes:
  • the decolorized liquid was passed through a nanofiltration membrane with a molecular weight of 300 Daltons to obtain a retentate;
  • the retentate was passed through a nanofiltration membrane with a molecular weight of 1000 Daltons, and the filtrate was collected.
  • the present invention after taking the part with a molecular weight of 300-1000 Da, it also includes the steps of concentrating to a solid content of 40%-55%, then vacuum-0.08--0.1MPa, and vacuum drying at 40-80°C.
  • the part with a molecular weight of 300-1000 Da after taking the part with a molecular weight of 300-1000 Da, it also includes the steps of concentrating to a solid content of 40%, and then vacuum drying at -0.08MPa and 40°C.
  • the part with a molecular weight of 300-1000 Da after taking the part with a molecular weight of 300-1000 Da, it further includes the steps of concentrating to a solid content of 50%, and then vacuum drying at -0.09MPa and 60°C.
  • the part with a molecular weight of 300-1000 Da after taking the part with a molecular weight of 300-1000 Da, it also includes the steps of concentrating to a solid content of 55%, and then vacuum drying at -0.1 MPa and 80°C.
  • the step of sieving is also included, and the sieving mesh is 20-120 mesh, specifically 20 mesh, 80 mesh or 120 mesh.
  • the mushroom oligopeptide prepared by the preparation method of the present invention.
  • mushroom oligopeptide prepared by the preparation method of the present invention can significantly inhibit the growth of mouse tumors and prolong the lifespan of tumor-bearing mice.
  • the present invention also provides a medicament for treating tumors, which comprises the Lentinus edodes oligopeptide prepared by the preparation method of the present invention.
  • the present invention also provides a method for treating tumors, which comprises administering the Lentinus edodes oligopeptide prepared by the preparation method of the present invention.
  • the method of administration is oral or injection.
  • the tumor is a malignant tumor.
  • the tumor is selected from the group consisting of sarcoma, bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, central nervous system cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastrointestinal cancer tract cancer, external genital cancer, genitourinary tract cancer, head cancer, kidney cancer, throat cancer, liver cancer, lung cancer, muscle tissue cancer, neck cancer, oral or nasal mucosa cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, Cancer of the spleen, small intestine, large intestine, stomach, testicular and/or thyroid.
  • the sarcoma is leiomyosarcoma, liposarcoma, fibrosarcoma, or malignant mesothelioma.
  • S 180 A tumor cells were used as experimental objects to construct NIH 615 pure line mice bearing tumor S 180 A.
  • the water extract of Lentinus edodes is treated with CTAB, followed by alkaline protease enzymolysis, flavor protease enzymolysis, and activated carbon decolorization, and the fraction with a molecular weight of 300-1000 Da is taken to prepare Lentinus edodes oligopeptide.
  • the oligopeptide extracted by the method provided by the present invention has good yield and purity. What's more, compared with the Lentinus edodes obtained by other extraction methods, the Lentinoides oligopeptide obtained by the method of the present invention has significant antitumor activity, and its effect is comparable to that of the positive drug (cyclophosphamide).
  • the invention provides a preparation method and application of a mushroom oligopeptide. , those skilled in the art can learn from the content of this paper and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention.
  • the method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications herein without departing from the content, spirit and scope of the present invention, so as to realize and apply the present invention. Invention technology.
  • the preparation method of the mushroom oligopeptide provided by the invention comprises the following steps:
  • the clarified liquid is passed through a nanofiltration membrane with a molecular weight of 300 Daltons, and the intercepted liquid is taken, and the intercepted liquid is passed through a nanofiltration membrane of 1000 Daltons, and the filtrate is taken for subsequent use;
  • the test materials used in the present invention are all common commercial products and can be purchased in the market.
  • the flavor protease model is Flavourzyme 500MG, from Novozymes (China) Investment Co., Ltd.
  • the alkaline protease model is Alcalase2.4L, from Novozymes (China) Investment Co., Ltd.
  • the dried shiitake mushrooms in the present invention are products obtained by drying fresh shiitake mushrooms, and the drying process includes air-drying, drying, drying or roasting.
  • the dried shiitake mushroom refers to the stipe of the dried shiitake mushroom, that is, the stalk of the fruiting body in the center under the cap of the shiitake mushroom.
  • the room temperature in the present invention refers to indoor temperature, preferably 18 to 30°C.
  • Adjust the pH of the clear liquid to 8.5 with NaOH solution add alkaline protease with a specific activity of 5000 international units per kg, and enzymatically hydrolyze at 45 °C for 20 minutes; after the enzymatic hydrolysis, carry out enzyme inactivation.
  • the inactivation conditions are 90 °C, 5 minutes, and cooling. to 20°C.
  • the pH value of the enzymatic hydrolysis solution was adjusted to 6.5 with NaOH solution, 5000 IU of flavor protease with specific activity per kg of liquid was added, and the enzyme was hydrolyzed at 45 °C for 20 min. After the enzymatic hydrolysis, the enzyme was inactivated.
  • the live time was 5 min, and the temperature was lowered to 20 °C. 0.1% activated carbon was added to the enzymatic hydrolysis solution, heated to 70° C., stirred for decolorization for 10 min, filtered, and 5.6 kg of clear liquid was taken.
  • the clarified liquid was passed through a nanofiltration membrane with a molecular weight of 300 Daltons to obtain 2.2 kg of intercepted liquid, and the intercepted liquid was passed through a nanofiltration membrane of 1000 Daltons to obtain 1.6 kg of filtrate.
  • the filtrate was concentrated to a solid content of 40%,
  • the vacuum degree is -0.08MPa, and the temperature is 40° C. for vacuum drying, and the dried material is pulverized and passed through a 20-mesh sieve to obtain 416 g of Lentinus edodes oligopeptide powder.
  • Oligopeptide purity 96.28% (reference standard: GB18186-2000);
  • Adjust the pH of the clear liquid to 9.0 with NaOH solution add alkaline protease with a specific activity of 21,500 IU per kg, and enzymatically hydrolyze at 50 °C for 70 minutes; after the enzymatic hydrolysis, carry out enzymatic inactivation.
  • the inactivation conditions are 95 °C, 10 minutes, and cooling to 30°C.
  • the pH value of the enzymatic hydrolysis solution was adjusted to 7.0 with NaOH solution. Flavor protease with a specific activity of 21,500 IU was added per kg of liquid, and the enzyme was hydrolyzed at 50 °C for 70 min. After the enzymatic hydrolysis, the enzyme was inactivated.
  • the live time was 10 min, and the temperature was lowered to 30 °C.
  • 0.55% activated carbon was added to the enzymatic hydrolysis solution, heated to 70° C., stirred and decolorized for 35 min, filtered, and 5.8 kg of clear liquid was taken.
  • the clarified liquid is passed through a nanofiltration membrane with a molecular weight of 300 Daltons to obtain 2.4 kg of intercepted liquid, and the intercepted liquid is passed through a nanofiltration membrane of 1000 Daltons to obtain 1.7 kg of filtrate, and the filtrate is concentrated to a solid content of 50%,
  • the vacuum degree is -0.09MPa, the temperature is 60°C, and vacuum drying is performed, and the dried material is pulverized and passed through an 80-mesh sieve to obtain 462 g of Lentinus edodes oligopeptide powder.
  • Oligopeptide purity 98.02% (reference standard: GB18186-2000);
  • the pH value of the clear liquid was adjusted to 9.5 with NaOH solution, alkaline protease with a specific activity of 40,000 IU was added per kg, and enzymatic hydrolysis was carried out at 55 °C for 120 min; after the enzymatic hydrolysis, the enzyme was inactivated.
  • the inactivation conditions were 100 °C, 15min, and cooling to 40°C.
  • the pH value of the enzymatic hydrolysis solution was adjusted to 7.5 with NaOH solution, 40,000 IU of flavor protease with specific activity per kg of liquid was added, and the enzyme was hydrolyzed at 55 °C for 120 min. After the enzymatic hydrolysis, the enzyme was inactivated.
  • the live time was 15 min, and the temperature was lowered to 40 °C. 1% activated carbon was added to the enzymatic hydrolysis solution, heated to 70° C., stirred for decolorization for 60 min, filtered, and 6.4 kg of clear liquid was taken.
  • the clarified liquid was passed through a nanofiltration membrane with a molecular weight of 300 Daltons to obtain 2.5kg of intercepted liquid, and the intercepted solution was passed through a nanofiltration membrane of 1000 Daltons to obtain 1.8 kg of filtrate, and the filtrate was concentrated to a solid content of 55%,
  • the vacuum degree is -0.1MPa, and the temperature is 80° C. to carry out vacuum drying, and the dried material is pulverized and passed through a 120-mesh sieve to obtain 505 g of Lentinus edodes oligopeptide powder.
  • Oligopeptide purity 95.19% (reference standard: GB18186-2000);
  • the mushroom polypeptide was prepared with reference to the method of patent ZL201110155206.8.
  • ammonium bromide solution was brought to a final concentration of 0.8%, stirring was continued for 16 min, and the solution was allowed to stand at room temperature for 20 min. Centrifuge at 5000 rpm for 32 min to remove the precipitate to obtain 9.4 kg of clear liquid.
  • the pH value of the clear liquid was adjusted to 8.0 with NaOH solution, alkaline protease with a specific activity of 4000 IU was added per kg, and enzymatic hydrolysis was carried out at 40 °C for 130 min; after the enzymatic hydrolysis, the enzyme was inactivated, and the inactivation conditions were 85 °C, 20 min, and cooling. to 40°C.
  • the pH value of the enzymatic hydrolysis solution was adjusted to 6.0 with NaOH solution, 4000 IU of flavor protease with specific activity per kg of liquid was added, and the enzymatic hydrolysis was carried out at 40 °C for 130 min. After the enzymatic hydrolysis, the enzyme was inactivated.
  • the live time was 20 min, and the temperature was lowered to 40 °C. 1.5% activated carbon was added to the enzymatic hydrolysis solution, heated to 75° C., stirred for decolorization for 65 min, filtered, and 6.4 kg of clear liquid was taken.
  • the clarified liquid was passed through a nanofiltration membrane with a molecular weight of 300 Daltons to obtain 2.3 kg of intercepted liquid, and the intercepted liquid was passed through a nanofiltration membrane of 1000 Daltons to obtain 1.6 kg of filtrate.
  • the filtrate was concentrated to a solid content of 56%,
  • the vacuum degree is -0.1 MPa, and the temperature is 100° C. for vacuum drying, and the dried material is pulverized and passed through a 120-mesh sieve to obtain 441 g of Lentinus edodes oligopeptide powder.
  • Comparative example 1 (patent ZL201110155206.8) and comparative example 2 samples
  • Control substance cyclophosphamide.
  • Tumor strains SI 180 A, EAC and L 615 mice were provided by the Institute of Hematology, Chinese Academy of Medical Sciences.
  • mice Take NIH pure-line mice, weighing 20 ⁇ 2g , both male and female, and inoculate according to the method of "National Antitumor Drug Screening Regulations in vivo". Sterilized physiological saline was diluted at a ratio of 1:4 to prepare a tumor cell suspension (5 ⁇ 10 6 cells/ml). 0.2ml/ R were inoculated into the right armpit of mice subcutaneously or intraperitoneally.
  • mice in each group were administered intraperitoneally or subcutaneously once a day.
  • the dose of Lentinus edodes oligopeptide was 60 mg/kg, and the dose of cyclophosphamide was 60 mg/kg.
  • the control group was given the same volume of normal saline for continuous After 10 days of administration, the subcutaneously inoculated mice were sacrificed the next day after drug withdrawal, and the tumor mass was taken out and weighed to calculate the tumor inhibition rate. The life span of mice inoculated by intraperitoneal cavity was observed after drug withdrawal, and the life extension rate was calculated. The results are shown in the table below.

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Abstract

一种香菇寡肽的制备方法及用途。香菇水提取物进行CTAB处理后，依次经过碱性蛋白酶酶解、风味蛋白酶酶解、活性炭脱色后，取分子量为300～1000Da的部分，制得香菇寡肽。该寡肽进一步制备成治疗肿瘤的药物。

Description

一种香菇寡肽的制备方法及用途
本申请要求于2020年12月29日提交中国专利局、申请号为202011588758.3、发明名称为“一种香菇寡肤的制备方法及用途”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物医药技术领域，尤其涉及一种香菇寡肽的制备方法及用途。
背景技术
香菇属担子菌纲、伞菌目(Agaricales)、口蘑、香菇属(Lentinus)，学名Lentinus edodes，起源于我国，是世界第二大菇，也是我国久负盛名的珍贵食用菌。我国最早栽培香菇，至今已有800多年历史。香菇也是我国著名的药用菌。历代医药学家对香菇的药性及功用均有著述。《日用本草》始入药用，云：蕈生桐、柳、枳椇木上，紫色者，名香蕈。性味甘，平。具有扶正补虚，健脾开胃，祛风透疹，化痰理气，解毒，抗癌之功效。用于正气衰弱，神倦乏力，纳呆，消化不良，贫血，佝偻病，高血压，高脂血症，慢性肝炎，盗汗，小便不禁，水肿，麻疹透发不畅，荨麻疹，毒菇中毒，肿瘤。
近年来，研究表明，香菇干品的蛋白含量为19.3％,香菇蛋白质中氨基酸种类达16种之多，必需氨基酸为7种。目前对于香菇活性物质的研究主要集中在香菇多糖的研究上。大量的研究表明，香菇多糖具有抗肿瘤的作用。事实上，除多糖外，香菇中还存在着大量的肽类物质，而这些物质也通常具有生理活性，但对于香菇肽的生理活性目前研究尚欠缺。
发明内容
有鉴于此，本发明要解决的技术问题在于提供具有抗肿瘤生理活性的香菇寡肽的制备方法及用途。
本发明提供的香菇寡肽的制备方法包括：
香菇经水提取获得提取液；
所述提取液经浓缩至固形物含量为30％～50％，以CTAB处理后，离心取上清液；
所述上清液依次经碱性蛋白酶酶解、风味蛋白酶酶解、活性炭脱色后，取分子量为300～1000Da的部分，制得香菇寡肽。
本发明所述的寡肽制备方法中，干香菇经提取、除多糖工艺，初步提高提取液的蛋白质含量；提取液经双酶酶解、两步纳滤等工艺提取香菇寡肽，最大限度地将香菇蛋白质水解成寡肽，再经纳滤提纯等工艺，将香菇寡肽的分子量控制在300-1000道尔顿之间。
在本发明中，所述所述香菇为干香菇的菌柄。
在本发明中，所述提取液的制备包括：将干香菇柄粉碎，加5～15倍重量的水，研磨至200目～300目，90℃～100℃提取2小时，经过滤获得滤液①和滤渣；所述滤渣加5～12倍重量的水，90℃～100℃提取1.5小时，经过滤，获得滤液②；合并滤液①和滤液②，6000～20000转/min离心5～30min，取上清为提取液。
一些实施例中，所述所述提取液的制备包括：将干香菇柄粉碎，加5倍重量的水，研磨至200目，90℃提取2小时，经过滤获得滤液①和滤渣；所述滤渣加5倍重量的水，90℃提取1.5小时，经过滤，获得滤液②；合并滤液①和滤液②，6000转/min离心5min，取上清为提取液。
一些实施例中，所述所述提取液的制备包括：将干香菇柄粉碎，加10倍重量的水，研磨至250目，95℃提取2小时，经过滤获得滤液①和滤渣；所述滤渣加10倍重量的水，95℃提取1.5小时，经过滤，获得滤液②；合并滤液①和滤液②，13000转/min离心10min，取上清为提取液。
一些实施例中，所述所述提取液的制备包括：将干香菇柄粉碎，加15倍重量的水，研磨至300目，100℃提取2小时，经过滤获得滤液①和滤渣；所述滤渣加15倍重量的水，100℃提取1.5小时，经过滤，获得滤液②；合并滤液①和滤液②，20000转/min离心15min，取上清为提取液。
本发明中，所述CTAB处理包括：
室温下，边搅拌边加入CTAB至其质量分数为0.5％～1.0％，继续搅拌5min后，静置30～180min。
在本发明实施例中，加入CTAB(十六烷基三甲基溴化铵)时，采用CTAB的水溶液，其中CTAB的质量分数为5％～10％。即向提取液中加入质量分数为5％～10％的CTAB水溶液，至提取液中CTAB的质量分数为0.5％～1.0％。
一些实施例中，所述CTAB处理包括：室温下，边搅拌边加入CTAB至其质量分数为0.5％，继续搅拌5min后，静置30min。
一些实施例中，所述CTAB处理包括：室温下，边搅拌边加入CTAB至其质量分数为0.75％，继续搅拌5min后，静置30min。
一些实施例中，所述CTAB处理包括：室温下，边搅拌边加入CTAB至其质量分数为1.0％，继续搅拌5min后，静置30min。
所述CTAB处理后离心取上清液的步骤中，离心的条件为6000～20000转离心5～30min。一些实施例中，离心的条件为6000/min离心5min，或20000/min离心15min。
在本发明中，所述碱性蛋白酶酶解的条件包括：每kg所述上清液加入5000～40000国际单位比活力的碱性蛋白酶；pH值为8.5～9.0，45～55℃酶解20～120min。所述碱性蛋白酶酶解后，还包括碱性蛋白酶灭酶的步骤。所述灭酶的条件为90～100℃灭活5～15min。
一些实施例中，所述碱性蛋白酶酶解的条件包括：每kg所述上清液加入5000国际单位比活力的碱性蛋白酶；pH值为8.5，45℃酶解20min，然后90℃灭活5min。
一些实施例中，所述碱性蛋白酶酶解的条件包括：每kg所述上清液加入21500国际单位比活力的碱性蛋白酶；pH值为9.0，50℃酶解70min，然后95℃灭活10min。
一些实施例中，所述碱性蛋白酶酶解的条件包括：每kg所述上清液加入40000国际单位比活力的碱性蛋白酶；pH值为9.5，55℃酶解120min，然后100℃灭活15min。
在风味蛋白酶酶解前，将碱性蛋白酶酶解并灭酶后的溶液冷却至 20℃～40℃。
本发明中，所述风味蛋白酶酶解的条件包括：每kg所述上清液加入5000～40000国际单位比活力的风味蛋白酶；pH值为6.5～7.5，45～55℃酶解20～120min。所述风味蛋白酶酶解后，还包括风味蛋白酶灭酶的步骤。所述灭酶的条件为90～100℃灭活5～15min。
一些实施例中，所述风味蛋白酶酶解的条件包括：每kg所述上清液加入5000国际单位比活力的风味蛋白酶；pH值为6.5，45℃酶解20min，然后90℃灭活5min。
一些实施例中，所述风味蛋白酶酶解的条件包括：每kg所述上清液加入21500国际单位比活力的风味蛋白酶；pH值为7.0，50℃酶解70min，然后95℃灭活10min。
一些实施例中，所述风味蛋白酶酶解的条件包括：每kg所述上清液加入40000国际单位比活力的风味蛋白酶；pH值为7.5，55℃酶解20min，然后90℃灭活5min。
在本发明中，所述活性炭脱色的条件包括：经过酶解的酶解液中，加入活性炭至其质量分数为0.1％～1％，70℃，搅拌脱色10～60min。脱色之后还包括过滤取滤液的步骤。
一些实施例中，所述活性炭脱色的条件包括：经过酶解的酶解液中，加入活性炭至其质量分数为0.1％，70℃，搅拌脱色10min。
一些实施例中，所述活性炭脱色的条件包括：经过酶解的酶解液中，加入活性炭至其质量分数为0.55％，70℃，搅拌脱色35min。
一些实施例中，所述活性炭脱色的条件包括：经过酶解的酶解液中，加入活性炭至其质量分数为1％，70℃，搅拌脱色60min。
在本发明中，所述取分子量为300～1000Da的部分包括：
所述脱色后的液体过300道尔顿分子量的纳滤膜，取截留液；
所述截留液过1000道尔顿分子量的纳滤膜，取滤过液。
在本发明中，所述取分子量为300～1000Da的部分之后，还包括浓缩至固形物含量为40％～55％，然后真空度-0.08～-0.1MPa，40～80℃真空干燥的步骤。
一些实施例中，所述取分子量为300～1000Da的部分之后，还包括浓缩至固形物含量为40％，然后真空度-0.08MPa，40℃真空干燥的步骤。
一些实施例中，所述取分子量为300～1000Da的部分之后，还包括浓缩至固形物含量为50％，然后真空度-0.09MPa，60℃真空干燥的步骤。
一些实施例中，所述取分子量为300～1000Da的部分之后，还包括浓缩至固形物含量为55％，然后真空度-0.1MPa，80℃真空干燥的步骤。
所述真空干燥后，还包括过筛的步骤，所述过筛的筛网为20～120目，具体为20目、80目或120目。
本发明所述制备方法制得的香菇寡肽。
经试验验证，本发明制备方法制得的香菇寡肽能够显著抑制小鼠肿瘤的生长，延长荷瘤小鼠的寿命。
本发明所述制备方法制得的香菇寡肽在制备治疗肿瘤的药物中的应用。
本发明还提供了一种治疗肿瘤的药物，其包括本发明所述制备方法制得的香菇寡肽。
本发明还提供了一种治疗肿瘤的方法，其包括给予本发明所述制备方法制得的香菇寡肽。所述给予的方法为口服或注射。
本发明中，所述肿瘤为恶性肿瘤。一些实施例中，所述肿瘤选自肉瘤、膀胱癌、血癌、骨癌、脑癌、乳腺癌、中枢神经系统癌症、宫颈癌、结肠癌、子宫内膜癌、食管癌、胆囊癌、胃肠道癌、外生殖器癌、泌尿生殖道癌、头癌、肾癌、喉癌、肝癌、肺癌、肌肉组织癌症、颈癌、口腔或鼻黏膜癌、卵巢癌、胰腺癌、前列腺癌、皮肤癌、脾癌、小肠癌、大肠癌、胃癌、睾丸癌和/或甲状腺癌。一些实施例中，所述肉瘤为平滑肌肉瘤、脂肪肉瘤、纤维组织肉瘤或恶性间皮瘤。本发明实施例中，以S 180A肿瘤细胞为实验对象，构建荷瘤S 180A的NIH 615纯系小鼠。
本发明提供的方法对香菇水提取物进行CTAB的处理后，依次经过碱性蛋白酶酶解、风味蛋白酶酶解、活性炭脱色后，取分子量为300～1000Da的部分，制得香菇寡肽。研究表明，采用本发明提供方法提取获得的寡肽，具有良好的收率和纯度。更重要的是，相对于其他提取方 法获得的香菇肽，本发明所述方法制得的香菇寡肽具有显著的抗肿瘤活性，其效果与阳性药物(环磷酰胺)相当。
具体实施方式
本发明提供了一种香菇寡肽的制备方法及用途。，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明提供的香菇寡肽的制备方法包括以下步骤：
(1)取干香菇柄，粉碎，加5～15倍质量水，用研磨机研磨至200目～300目，加热至90℃～100℃，提取2小时，过滤，得滤液①，备用；
(2)滤渣加5～12倍质量水，加热至90℃～100℃，提取1.5小时，过滤，得滤液②，备用；
(3)收集滤液①和滤液②，6000～20000转离心5～30min，取澄清液体；
(4)浓缩至固形物至30％～50％，在搅拌情况下加入5％～10％的十六烷基三甲基溴化铵溶液至终浓度为0.5％～1.0％，继续搅拌5～15min，室温静置30min-180min；
(5)6000～20000转离心5～30min，除去沉淀，取澄清液体；
(6)澄清液体用NaOH溶液调节pH值至8.5～9.0，按每kg加入5000～40000国际单位比活力的碱性蛋白酶，45～55℃酶解20～120min；酶解结束后进行酶灭活，灭活条件为90～100℃，5～15min，降温至20～40℃，备用；
(7)酶解液用NaOH溶液调节pH值至6.5～7.5，按每kg液体加入5000～40000国际单位比活力的风味蛋白酶，45～55℃酶解20～120min，酶解结束后进行酶灭活，酶灭活温度为90-100℃，灭活时间为5～15min，降温至20～40℃，备用；
(8)酶解液加入0.1％～1％的活性炭，加热至70℃，搅拌脱色10-60min，过滤，取澄清液体，备用；
(9)澄清液体过300道尔顿分子量的纳滤膜，取截留液体，截留液再过1000道尔顿的纳滤膜，取滤过液，备用；
(10)滤过液浓缩至固形物为40％～55％，真空度为-0.8至-0.1MPa，温度为40℃～80℃进行真空干燥，干燥物进行粉碎，过20～120目筛网，得香菇寡肽。
本发明采用的试材皆为普通市售品，皆可于市场购得。其中风味蛋白酶型号为Flavourzyme 500MG，来自诺维信(中国)投资有限公司。所述碱性蛋白酶型号为Alcalase2.4L，来自诺维信(中国)投资有限公司。本发明中所述干香菇为鲜香菇经干燥制得的产品，干燥工艺包括风干、烘干、晾晒或烤制等。本发明实施例中所述干香菇是指干香菇的菌柄，即为香菇菌盖下面中央的子实体的柄。本发明中所述室温是指室内温度，优选为18～30℃。
下面结合实施例，进一步阐述本发明：
实施例1：
取干香菇10kg，粉碎，加5倍水，用研磨机研磨至200目，加热至90℃，提取2小时，过滤，得滤液30.5kg。滤渣加5倍水，加热至90℃，提取1.5小时，过滤，得滤液35.1kg。收集两次滤液，6000转/min离心5min，得澄清液体55.2kg，澄清液浓缩至固形物至30％，得浓缩液7.4kg，浓缩液在搅拌情况下加入5％的十六烷基三甲基溴化铵溶液至终浓度为0.5％，继续搅拌5min，室温静置30min。6000转/min离心5min，除去沉淀，得澄清液体6.5kg。
澄清液体用NaOH溶液调节pH值至8.5，按每kg加入5000国际单位比活力的碱性蛋白酶，45℃酶解20min；酶解结束后进行酶灭活，灭活条件为90℃，5min，降温至20℃。酶解液用NaOH溶液调节pH值至6.5，按每kg液体加入5000国际单位比活力的风味蛋白酶，45℃酶解20min，酶解结束后进行酶灭活，酶灭活温度为90℃，灭活时间为5min，降温至20℃。酶解液加入0.1％的活性炭，加热至70℃，搅拌脱色10min，过滤， 取澄清液体5.6kg。
澄清液体过300道尔顿分子量的纳滤膜，得截留液体2.2kg，截留液再过1000道尔顿的纳滤膜，得滤过液1.6kg，滤过液浓缩至固形物为40％，真空度为-0.08MPa，温度为40℃进行真空干燥，干燥物进行粉碎，过20目筛网，得香菇寡肽粉416g。
检验结果：
寡肽提取得率：4.16％(寡肽粉重量/干香菇粉重量×100)
寡肽纯度：96.28％(参照标准：GB18186-2000)；
分子量范围：300-1000道尔顿(参照标准：JY/T024-1996)
实施例2：
取干香菇10kg，粉碎，加10倍水，用研磨机研磨至250目，加热至95℃，提取2小时，过滤，得滤液80.2kg。滤渣加10倍水，加热至95℃，提取1.5小时，过滤，得滤液85.5kg。收集两次滤液，13000转/min离心10min，得澄清液体132.8kg，澄清液浓缩至固形物至35％，得浓缩液8.0kg，浓缩液在搅拌情况下加入5％的十六烷基三甲基溴化铵溶液至终浓度为0.75％，继续搅拌5min，室温静置30min。6000转/min离心5min，除去沉淀，得澄清液体7.8kg。
澄清液体用NaOH溶液调节pH值至9.0，按每kg加入21500国际单位比活力的碱性蛋白酶，50℃酶解70min；酶解结束后进行酶灭活，灭活条件为95℃，10min，降温至30℃。酶解液用NaOH溶液调节pH值至7.0，按每kg液体加入21500国际单位比活力的风味蛋白酶，50℃酶解70min，酶解结束后进行酶灭活，酶灭活温度为95℃，灭活时间为10min，降温至30℃。酶解液加入0.55％的活性炭，加热至70℃，搅拌脱色35min，过滤，取澄清液体5.8kg。
澄清液体过300道尔顿分子量的纳滤膜，得截留液体2.4kg，截留液再过1000道尔顿的纳滤膜，得滤过液1.7kg，滤过液浓缩至固形物为50％，真空度为-0.09MPa，温度为60℃进行真空干燥，干燥物进行粉碎，过80目筛网，得香菇寡肽粉462g。
检验结果：
寡肽提取得率：4.62％(寡肽粉重量/干香菇粉重量×100)
寡肽纯度：98.02％(参照标准：GB18186-2000)；
分子量范围：300-1000道尔顿(参照标准：JY/T024-1996)
实施例3：
取干香菇10kg，粉碎，加15倍水，用研磨机研磨至300目，加热至100℃，提取2小时，过滤，得滤液112kg。滤渣加15倍水，加热至100℃，提取1.5小时，过滤，得滤液116kg。收集两次滤液，20000转/min离心15min，得澄清液体193kg，澄清液浓缩至固形物至50％，得浓缩液8.6kg，浓缩液在搅拌情况下加入5％的十六烷基三甲基溴化铵溶液至终浓度为1.0％，继续搅拌5min，室温静置30min。20000转/min离心15min，除去沉淀，得澄清液体8.3kg。
澄清液体用NaOH溶液调节pH值至9.5，按每kg加入40000国际单位比活力的碱性蛋白酶，55℃酶解120min；酶解结束后进行酶灭活，灭活条件为100℃，15min，降温至40℃。酶解液用NaOH溶液调节pH值至7.5，按每kg液体加入40000国际单位比活力的风味蛋白酶，55℃酶解120min，酶解结束后进行酶灭活，酶灭活温度为100℃，灭活时间为15min，降温至40℃。酶解液加入1％的活性炭，加热至70℃，搅拌脱色60min，过滤，取澄清液体6.4kg。
澄清液体过300道尔顿分子量的纳滤膜，得截留液体2.5kg，截留液再过1000道尔顿的纳滤膜，得滤过液1.8kg，滤过液浓缩至固形物为55％，真空度为-0.1MPa，温度为80℃进行真空干燥，干燥物进行粉碎，过120目筛网，得香菇寡肽粉505g。
检验结果：
寡肽提取得率：5.05％(寡肽粉重量/干香菇粉重量×100)
寡肽纯度：95.19％(参照标准：GB18186-2000)；
分子量范围：300-1000道尔顿(参照标准：JY/T024-1996)
对比例1：
参照专利ZL201110155206.8的方法制备香菇多肽。
对比例2：
取干香菇10kg，粉碎，加20倍水，用研磨机研磨至120目，加热至80℃，提取1小时，过滤，得滤液168kg。滤渣加18倍水，加热至80℃，提取1小时，过滤，得滤液145kg。收集两次滤液，5000转/min离心31min，得澄清液体298kg，澄清液浓缩至固形物至52％，得浓缩液10.5kg，浓缩液在搅拌情况下加入6％的十六烷基三甲基溴化铵溶液至终浓度为0.8％，继续搅拌16min，室温静置20min。5000转/min离心32min，除去沉淀，得澄清液体9.4kg。
澄清液体用NaOH溶液调节pH值至8.0，按每kg加入4000国际单位比活力的碱性蛋白酶，40℃酶解130min；酶解结束后进行酶灭活，灭活条件为85℃，20min，降温至40℃。酶解液用NaOH溶液调节pH值至6.0，按每kg液体加入4000国际单位比活力的风味蛋白酶，40℃酶解130min，酶解结束后进行酶灭活，酶灭活温度为85℃，灭活时间为20min，降温至40℃。酶解液加入1.5％的活性炭，加热至75℃，搅拌脱色65min，过滤，取澄清液体6.4kg。
澄清液体过300道尔顿分子量的纳滤膜，得截留液体2.3kg，截留液再过1000道尔顿的纳滤膜，得滤过液1.6kg，滤过液浓缩至固形物为56％，真空度为-0.1MPa，温度为100℃进行真空干燥，干燥物进行粉碎，过120目筛网，得香菇寡肽粉441g。
检验结果：
寡肽提取得率：4.41％(寡肽粉重量/干香菇粉重量×100)
寡肽纯度：65.19％(参照标准：GB18186-2000)；
分子量范围：300-1000道尔顿(参照标准：JY/T024-1996)
试验实施例：香菇寡肽的抗肿瘤功能
1.试验药品：
样品：本发明实施例1、2、3制备的香菇寡肽
对比例1(专利ZL201110155206.8)及对比例2样品
分子量大于1000道尔顿的香菇肽
分子量小于300道尔顿的香菇氨基酸
对照品：环磷酰胺。
2.动物：NIH 615纯系小鼠。
3.瘤株：SI 180A、EAC和L 615小鼠由中国医学科学院血液研究所提供。
4.实验方法与结果
(1)香菇寡肽对小鼠S 180的影响
取NIH纯系小鼠，体重20±2g，雌雄兼用，按《全国抗肿瘤药物体内筛选规程》的方法接种，瘤源为接种6天-9天肿瘤生长良好的S 180A，取出腹水，用灭菌的生理盐水按l:4比例稀释制成肿瘤细胞混悬液(5×10 6细胞/ml)。分别以0.2ml/ R接种于小鼠右侧腋窝皮下或者接种于小鼠腹腔内。
24h后随机分组，每组10只，分别每天腹腔或者皮下注射给药一次，香菇寡肽的剂量为60mg/kg，环磷酰胺的剂量为60mg/kg，对照组给同体积的生理盐水，连续给药10d，停药后次日将接种皮下的小鼠处死，取出瘤块称重，计算抑瘤率。接种腹腔的小鼠停药后观察寿命天数，计算生命延长率。结果见下表。
表1：香菇寡肽对小鼠S 180的影响
Figure PCTCN2021087726-appb-000001
注：与空白对照组比较， *P<0.01；与对比例1、对比例2、分子量大于1000道尔顿的香菇肽样品及分子量小于300道尔顿的香菇氨基酸样品比较， #P<0.01。
表2：香菇寡肽对小鼠S180A的影响
Figure PCTCN2021087726-appb-000002
注：与空白对照组比较， *P<0.01；与对比例1、对比例2、分子量大于1000道尔顿的香菇肽、分子量小于300道尔顿的香菇氨基酸比较， #P<0.01。
从试验结果可以看出，香菇寡肽具有显著的对抗小鼠S 180的作用，无论是对实体瘤S 180还是对腹水型S 180(S 180A)都具有强大的对抗作用，效果比对比例1(专利ZL201110155206.8)及对比例2制备的香菇肽、分子量大于1000道尔顿的香菇肽、分子量小于300道尔顿的香菇氨基酸均好。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种香菇寡肽的制备方法，其特征在于，包括：
    香菇经水提取获得提取液；
    所述提取液经浓缩至固形物含量为30％～50％，以CTAB处理后，离心取上清液；
    所述上清液依次经碱性蛋白酶酶解、风味蛋白酶酶解、活性炭脱色后，取分子量为300～1000Da的部分，制得香菇寡肽。
  2. 根据权利要求1所述的制备方法，其特征在于，所述香菇为干香菇的菌柄。
  3. 根据权利要求1或2所述的制备方法，其特征在于，所述提取液的制备包括：
    将干香菇柄粉碎，加5～15倍重量的水，研磨至200目～300目，90℃～100℃提取2小时，经过滤获得滤液①和滤渣；
    所述滤渣加5～12倍重量的水，90℃～100℃提取1.5小时，经过滤，获得滤液②；
    合并滤液①和滤液②，6000～20000转/min离心5～30min，取上清为提取液。
  4. 根据权利要求1所述的制备方法，其特征在于，所述CTAB处理包括：
    室温下，边搅拌边加入CTAB至其质量分数为0.5％～1.0％，继续搅拌5min后，静置30～180min。
  5. 根据权利要求1所述的制备方法，其特征在于，所述碱性蛋白酶酶解的条件包括：每kg所述上清液加入5000～40000国际单位比活力的碱性蛋白酶；pH值为8.5～9.0，45～55℃酶解20～120min。
  6. 根据权利要求1所述的制备方法，其特征在于，所述风味蛋白酶酶解的条件包括：每kg所述上清液加入5000～40000国际单位比活力的风味蛋白酶；pH值为6.5～7.5，45～55℃酶解20～120min。
  7. 根据权利要求1所述的制备方法，其特征在于，所述活性炭脱色 的条件包括：经过酶解的酶解液中，加入活性炭至其质量分数为0.1％～1％，70℃，搅拌脱色10～60min。
  8. 根据权利要求1所述的制备方法，其特征在于，所述取分子量为300～1000Da的部分之后，还包括浓缩至固形物含量为40％～55％，然后真空度-0.08～-0.1MPa，40～80℃真空干燥的步骤。
  9. 权利要求1～8任一项所述制备方法制得的香菇寡肽。
  10. 权利要求1～8任一项所述制备方法制得的香菇寡肽在制备治疗肿瘤的药物中的应用。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101709320A (zh) * 2009-11-27 2010-05-19 广州市味研生物工程科技有限公司 一种香菇多肽粉的制备方法
CN102224879A (zh) * 2011-06-09 2011-10-26 申健 香菇多肽的制备方法和用途
CN102919823A (zh) * 2011-08-11 2013-02-13 上海冠生园天厨调味品有限公司 一种多肽菌菇精的制备方法
CN106866787A (zh) * 2016-10-27 2017-06-20 北京林业大学 一种香菇醒酒肽及其制备方法与应用
CN110922498A (zh) * 2019-12-09 2020-03-27 河南自然之道生物工程有限公司 一种以残次香菇和香菇柄制备香菇多糖及香菇多肽的方法

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0894126B1 (en) * 1996-03-27 2006-02-01 Novozymes A/S Alkaline protease deficient filamentous fungi
CN102190739A (zh) * 2010-03-12 2011-09-21 南京易亨制药有限公司 一种提取香菇多糖的工艺及香菇多糖分子量分布测定方法
CN102286460B (zh) * 2011-06-21 2012-12-05 湖南省宇秀生物科技有限公司 一种改进ctab法提取杏鲍菇中的dna
CN104004813B (zh) * 2014-06-12 2017-12-29 北京林业大学 一种香菇生物活性肽的制备
CN104207150A (zh) * 2014-08-27 2014-12-17 河南省南街村（集团）有限公司 大豆肽真菌多糖口服液及其制备方法
CN105169371A (zh) * 2015-08-14 2015-12-23 伍曾利 组合物及其用途、保湿制剂
CN106174386B (zh) * 2016-07-11 2018-02-02 北京工商大学 一种天然调味剂及其制备的调味膏和调味料
CN107119096B (zh) * 2017-05-10 2022-11-18 天津市农业科学院 一种滑菇活性肽的制备方法及其应用
CN109953316A (zh) * 2017-12-22 2019-07-02 天津市雅敏科技有限公司 一种富硒香菇多肽的制备方法
CN108191991B (zh) * 2018-01-10 2020-06-30 福建省农业科学院农业工程技术研究所 一种杏鲍菇多糖的提纯方法
CN113106138A (zh) * 2021-03-16 2021-07-13 中国农业科学院农产品加工研究所 一种从香菇中提取分离抗肿瘤活性蛋白的制备方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101709320A (zh) * 2009-11-27 2010-05-19 广州市味研生物工程科技有限公司 一种香菇多肽粉的制备方法
CN102224879A (zh) * 2011-06-09 2011-10-26 申健 香菇多肽的制备方法和用途
CN102919823A (zh) * 2011-08-11 2013-02-13 上海冠生园天厨调味品有限公司 一种多肽菌菇精的制备方法
CN106866787A (zh) * 2016-10-27 2017-06-20 北京林业大学 一种香菇醒酒肽及其制备方法与应用
CN110922498A (zh) * 2019-12-09 2020-03-27 河南自然之道生物工程有限公司 一种以残次香菇和香菇柄制备香菇多糖及香菇多肽的方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LEI QIAN, ZHANG YE-NI; ZHANG ZHI-JUN; LIU JIN-FU: "Preparation of Antioxidant Peptides from Pholiota nameko Protein by Ultrasonic-pretreated Double Enzymatic Fractional Hydrolysis", THE FOOD INDUSTRY - SHIPIN GONGYE (SHANGHAI), SHANGHAI SHI SHIPIN GONGYE YANJIUSUO, JP, vol. 39, no. 2, 20 February 2018 (2018-02-20), JP , pages 98 - 102, XP055948268, ISSN: 1004-471X *
ZHANG LU, CAO HUI; GONG ZHI-QING; ZHANG TING; YU MAN-MAN; GE LIN-LI; WANG WEN-LIANG: "Study on the preparation of antioxidant peptides by enzymatic hydrolysis of letinous edodes stalk protein", SCIENCE AND TECHNOLOGY OF FOOD INDUSTRY, GAI KAN BIANJIBU , BEIJING, CN, vol. 37, no. 23, 20 September 2016 (2016-09-20), CN , pages 233 - 238, XP055948265, ISSN: 1002-0306, DOI: 10.13386/j.issn1002-0306.2016.23.035 *

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