WO2017124921A1 - 一种核桃低聚肽粉及其制备方法和用途 - Google Patents

一种核桃低聚肽粉及其制备方法和用途 Download PDF

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WO2017124921A1
WO2017124921A1 PCT/CN2017/000121 CN2017000121W WO2017124921A1 WO 2017124921 A1 WO2017124921 A1 WO 2017124921A1 CN 2017000121 W CN2017000121 W CN 2017000121W WO 2017124921 A1 WO2017124921 A1 WO 2017124921A1
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walnut
preparation
food
oligopeptide powder
molecular weight
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PCT/CN2017/000121
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English (en)
French (fr)
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WO2017124921A9 (zh
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王昭日
刘明川
杨胜杰
洪达
杨进平
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杏辉天力（杭州）药业有限公司
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Priority to EP17740952.1A priority Critical patent/EP3406625B1/en
Priority to AU2017209066A priority patent/AU2017209066C1/en
Priority to MYPI2018702546A priority patent/MY193780A/en
Priority to CN201780007467.0A priority patent/CN109071599A/zh
Priority to KR1020187023722A priority patent/KR102320465B1/ko
Priority to CA3012063A priority patent/CA3012063C/en
Priority to JP2018557173A priority patent/JP6828054B2/ja
Priority to US16/071,583 priority patent/US11072637B2/en
Publication of WO2017124921A1 publication Critical patent/WO2017124921A1/zh
Publication of WO2017124921A9 publication Critical patent/WO2017124921A9/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2/00—Peptides of undefined number of amino acids; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/14—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds
    • A23J1/148—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds by treatment involving enzymes or microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • A23J3/346—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins
    • A—HUMAN NECESSITIES
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    • 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
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • 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
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/185—Vegetable proteins
    • 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
    • A61K38/011—Hydrolysed proteins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/645—Proteins of vegetable origin; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/145—Ultrafiltration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/147—Microfiltration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/149—Multistep processes comprising different kinds of membrane processes selected from ultrafiltration or microfiltration
    • 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
    • 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
    • C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
    • 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
    • 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
    • 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
    • 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
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/322—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the nervous system or on mental function
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to a high purity, low molecular weight walnut oligopeptide product having a polypeptide content of more than 80% by weight and a molecular weight of less than 1500 Dalton of more than 95%.
  • the invention also relates to a method for preparing a walnut protein and catalyzing enzymatic hydrolysis to produce an oligopeptide powder, which can be used as a medicine, a food, a health care product or a cosmetic.
  • the present invention claims priority to Chinese Patent Application No. CN201610043952.0, the entire disclosure of which is incorporated herein by reference.
  • Juglans regia L also known as walnut, is one of the four nuts and has a high nutritional and medicinal value.
  • Li Shizhen's “Compendium of Materia Medica” describes "invigorating Qi and nourishing blood, moistening and dissipating phlegm, benefiting life door, Li Sanjiao, Wenfei Runchang.
  • Treatment of lungs and intestines. Treatment of cold and asthma Waist and waist pain, heart and abdomen pain, bloody intestinal wind, scattered swollen poison, Song Liu Han “Kai Bao Materia Medica” contained “walnut (ie walnut) sweet, flat, non-toxic.
  • Walnut is rich in protein, fat and other nutrients, and the content is relatively balanced. It is an ideal high-protein, high-fat food. It is reported that walnut kernel contains up to 52%-70% of oil, most of which are unsaturated. Fatty acids additionally contain about 24% protein, 12%-16% carbohydrate, 1.5%-2% cellulose, and 1.7%-2% minerals. Walnut is rich in essential amino acids, and the proportion of amino acids is reasonable. Among them, the content of glutamic acid, aspartic acid and arginine which have important functions on human physiological functions are high, and glutamic acid still affects the human body, especially An important functional substance in the development of intelligence and memory in adolescents.
  • Patent CN 101228918 A pulverizes walnut stalk, ultrasonically extracts walnut protein, vacuums walnut protein, and then uses protease to carry out enzymatic hydrolysis, centrifugation, dialysis of supernatant with dialysis bag, concentration of dialysate and vacuum drying to obtain content 60% to 80% of peptide powder.
  • the method is complicated in operation, the peptide content is low, and the molecular weight distribution of the peptide is not clear.
  • the protein is extracted by ultrasonication, and the dialysis bag is refined, and large-scale preparation cannot be performed.
  • Patent CN 102406050 A extracts protein by alkali extraction and acid precipitation method, and freezes and obtains walnut protein powder. After protein hydrolysis, the protein powder is subjected to high pressure treatment at 300 MPa for 10 min, and freeze-dried to obtain walnut peptide powder.
  • the preparation method requires ultra-high pressure equipment and a freeze dryer, and has high cost and is not suitable for large-scale production; although the invention patent provides the molecular weight distribution of the walnut peptide powder prepared by the method, the peptide content in the product is not clear.
  • Patent CN 103103244 B extracts protein by alkali extraction and acid precipitation, and combines with microwave to ultrasonic treatment to obtain polypeptide powder.
  • Patent CN 104293870 A firstly uses the CO 2 supercritical extractor to remove the walnut pupa obtained after the oil is removed. After the alkali is acid-salted, the walnut protein powder is sprayed, and then the protein powder is made into a suspension and boiled to destroy the structure of the protein. Protease, papain, neutral protease and bromelain were subdivided into four parts of walnut protein solution, refined by 5000Dalton or 8000Dalton ultrafiltration membrane, desalted by ion exchange resin, and finally spray dried to obtain walnut peptide powder.
  • This patent relates to the use of supercritical extractor when removing grease, which increases the cost and is not easy for large production; involves four kinds of enzymes, four steps of enzymatic hydrolysis, the steps are cumbersome and costly; after the enzymatic hydrolysis is completed, the microfiltration is not carried out.
  • Membrane removal direct ultrafiltration membrane, resulting in ultrafiltration membrane clogging, prolonged ultrafiltration time, reducing peptide yield, while shortening the ultrafiltration membrane life; this patent does not cover the percentage of peptides and peptides in peptide products Molecular weight distribution.
  • Another object of the present invention is to provide a process for preparing a high purity, low molecular weight walnut oligopeptide powder.
  • Another object of the present invention is to provide a medicament for the preparation of a medicament, food, health supplement or cosmetic for treating or preventing symptoms caused by excessive free radicals of the walnut oligopeptide powder of the present invention.
  • Another object of the present invention is to provide the use of the walnut oligopeptide powder of the present invention for the preparation of a medicament, food, nutraceutical or cosmetic for improving or treating memory loss.
  • Another object of the present invention is to provide a medicament for the preparation of a medicament, a food, a health supplement or a cosmetic for treating or preventing Parkinson's disease and Alzheimer's disease, relieving brain or sports fatigue, of the walnut oligopeptide powder of the present invention.
  • Another object of the present invention is to provide an application of the walnut oligopeptide powder of the present invention for the preparation of a drug, food, health care product or cosmetic which enhances immunity.
  • Another object of the present invention is to provide a pharmaceutical, food, nutraceutical or cosmetic composition comprising the walnut oligopeptide powder of the present invention.
  • a walnut oligopeptide powder adopts the detection method of Appendix A and Appendix B of GB/T 22492-2008, and the peptide content is above 80 wt%, wherein the molecular weight of more than 95% of the walnut peptide is less than 1500 Dalton, and the molecular weight distribution is as follows:
  • Number average molecular weight range 170 ⁇ 3000
  • the peptide content is 81% by weight or more, and the molecular weight of less than 1500 Dalton accounts for 97% or more; more preferably, the peptide content is 81.3 wt% or more, and the molecular weight of less than 1500 Dalton accounts for 96% or more; more preferably, the peptide content is 85 wt% or more, and the molecular weight is less than 1500 Dalton. More than 97%; most preferably, the peptide content is 85 wt% or more, and the molecular weight of less than 1500 Dalton accounts for 96% or more.
  • the walnut oligopeptide powder is prepared by the following method:
  • Pretreatment of walnut meal The walnut is shelled and cold pressed to deoil the oil to obtain defatted walnut meal.
  • the second extraction is continued; after the second extraction is completed, the filter residue is discarded, and the filtrate is poured into an equal amount of walnut meal (recorded as C), and the pH is adjusted to 9 to 11 at room temperature for 1 to 2 hours; After the second extraction, the filter residue is discarded, and the filtrate is poured into C to complete the first extraction of the filter residue for 1 to 2 hours, and the first extraction of the filtrate is ready for use; after the second extraction of the sample C, the filter residue is discarded.
  • C walnut meal
  • the filtrate is ready for use; finally, the above filtrate is mixed, the pH is adjusted to 3 to 5, the reaction is allowed to stand for 0.5 to 2 hours, the supernatant is discarded, and finally the water is added in a volume ratio of 1:10 to 1:20 to the precipitate, and stirred. Evenly.
  • the protease solution is performed using a microfiltration membrane having a pore diameter of 0.1 to 0.5 ⁇ m. After filtration, the permeate is further treated with 2000 to 20000 Dalton ultrafiltration membrane, and the retentate is concentrated at 50-80 ° C until the solid content is 3 to 5 wt%, and spray-dried, the inlet temperature is 140-160 ° C, and the outlet temperature is 55-65 ° C, high purity, low molecular weight light yellow walnut peptide powder, the yield can reach 20 ⁇ 30wt%.
  • the biological enzyme is selected from a food-grade neutral protease (enzyme activity ⁇ 300,000 u/g), papain (enzyme activity ⁇ 400,000 u/g), bromelain (enzyme activity ⁇ 300,000 u/g), alkali One of protease protease (enzyme activity ⁇ 200,000 u/g), pepsin (enzyme activity ⁇ 500,000 u/g), trypsin (enzyme activity ⁇ 3000 u/g) or a mixture thereof, preferably neutral A protease or a complex enzyme, wherein the mass ratio of the neutral protease to the papain of the complex enzyme is 1:1, the activity of the neutral protease is 300,000 u/g, and the activity of papain is 500,000 u/g.
  • a composition comprising the walnut oligopeptide powder of the present invention and a pharmaceutical, food, nutraceutical or cosmetically acceptable adjuvant.
  • the composition can be prepared into any of the dosage forms, such as a plain tablet, a film coated tablet, a sugar coated tablet, an enteric coated tablet, a dispersible tablet, a capsule, a granule, an oral solution or an oral mixture.
  • Suspensions as well as liquid, lotion, cream, powder, block and other cosmetic dosage forms.
  • the walnut oligopeptide powder of the invention can be used for preparing medicines, foods, health products or cosmetics for treating or preventing symptoms caused by excessive free radicals; for preparing medicines, foods, health products or cosmetics for improving or treating memory loss It is used for the preparation of medicines, foods, health products or cosmetics for treating or preventing Parkinson's disease and Alzheimer's disease, relieving brain or sports fatigue; for preparing medicines, foods, health products or cosmetics for enhancing immunity.
  • the present invention has the following advantages:
  • walnuts are cold-squeezed and degreased to prepare walnut mash.
  • the inventors have conducted related experiments. Compared with the hot-pressing method, the protein extraction rate after cold pressing is about 5 wt%, and the content is about 20 wt%.
  • the present invention uses a high-efficiency countercurrent method to extract walnut protein, and the extraction rate of protein is increased by more than 10% compared with the conventional alkali extraction and acid precipitation method, and the water consumption is reduced, and the production cost is saved.
  • the present invention uses only a protein complex solution for enzymatic hydrolysis, and does not dry the protein, which not only reduces the loss of protein drying, but also simplifies the preparation process.
  • the biological enzyme used in the invention can be ensured in content, molecular weight, yield and activity, and the enzymatic hydrolysis process is stable. Both enzymes are edible enzymes, and the source is wide and the cost is low. When enzymatically, the amount of addition is only The quality of walnut meal is 0.5 to 2%.
  • the present invention uses a microfiltration membrane for filtration to initially remove insoluble matter in the enzymatic hydrolysate, and then Large molecular weight proteins were removed using 2000 to 20000 Dalton ultrafiltration membranes.
  • the present invention does not use a freeze-drying or vacuum drying method to prepare a product, and the spray drying method not only saves drying time, but also has a uniform plasmid.
  • the molecular weight distribution of the polypeptide is determined according to Appendix A of GB/T 22492-2008, and the content of the acid-soluble protein and the free amino acid is first determined according to Appendix B, and the final difference is the peptide content.
  • This method of determining the molecular weight and peptide content is highly accepted.
  • Number average molecular weight range 170 ⁇ 3000
  • Weight average molecular weight range 180 ⁇ 4000
  • the peptide content of the present invention is more than 80% by weight, wherein more than 95% of the walnut peptide has a molecular weight of less than 1500 Dalton, and is a high-purity, low-molecular-weight oligopeptide.
  • Figure 3 Effect of walnut oligopeptide on the inhibition of zebrafish macrophage
  • Figure 4 Promoting effect of walnut oligopeptide on phagocytosis of zebrafish macrophages
  • Figure 5 Typical diagram of the effect of walnut oligopeptides on the central nervous system of zebrafish
  • Figure 6 Promoting effect of walnut oligopeptide on neurite outgrowth of zebrafish embryos
  • FIG. 7 Walnut oligopeptide reduces the toxic effect of human wild-type tau protein on zebrafish embryonic neurons.
  • the filtrate extracted by C for the first time is ready for use; after the second extraction of sample C, the filter residue is discarded, and the filtrate is ready for use; finally, the above filtrate to be used is combined, the pH is adjusted to 5, the reaction is allowed to stand for 6 hours, and the supernatant is discarded. Finally, water having a volume ratio of 1:10 was added to the precipitate and stirred well.
  • the above walnut protein solution was heated to 45 ° C, the pH was adjusted to neutral, 1 kg of neutral protease (enzyme activity was 300,000 u / g) was added, and after enzymatic hydrolysis for 6 h, it was boiled and inactivated for 30 min, centrifuged, and the supernatant was centrifuged.
  • the protease solution was filtered using a microfiltration membrane having a pore size of 0.1 ⁇ m, and the permeate was treated with a 5000 Dalton ultrafiltration membrane, and then concentrated at 80 ° C until the solid content was 3.4%, and spray-dried at an inlet temperature. At 140 ° C, the outlet temperature was 55-65 ° C, and a high-purity, low-molecular-weight pale yellow walnut peptide powder was obtained in a yield of 21% by weight.
  • the peptide content was 81% by weight, and the molecular weight of less than 1500 Dalton was 97%.
  • the molecular weight distribution was as described above.
  • the peptide content results are as follows:
  • the filtrate extracted by C for the first time is ready for use; after the second extraction of sample C, the filter residue is discarded, and the filtrate is ready for use; finally, the above filtrate to be used is combined, the pH is adjusted to 5, the reaction is allowed to stand for 6 hours, and the supernatant is discarded. Finally, water having a volume ratio of 1:10 was added to the precipitate and stirred well.
  • the walnut protein solution was heated to 45 ° C, the pH was adjusted to neutral, and 1 kg of neutral papaya complex protease was added to the weight of the walnut meal (the mass ratio of the two proteases was 1:1, and the activity of the neutral protease was 300,000 u).
  • the activity of papain is 500,000 u/g
  • it is boiled and inactivated for 30 minutes, and centrifuged, and the supernatant is a protease hydrolysate.
  • the protease solution was filtered using a microfiltration membrane having a pore size of 0.1 ⁇ m, and the permeate was further treated with a 5000 Dalton ultrafiltration membrane, and then concentrated at 80 ° C until the solid content was 4.1%, and spray-dried at an inlet temperature. At 140 ° C, the outlet temperature was 55-65 ° C, and a high-purity, low-molecular-weight pale yellow walnut peptide powder was obtained in a yield of 21% by weight.
  • the peptide content was determined to be 81.3 wt%, and the molecular weight of less than 1500 Dalton was 96%, and the molecular weight distribution was as described above.
  • the peptide content of the neutral papain complex enzyme after enzymatic hydrolysis is as follows:
  • auxiliaries including but not limited to any adjuvants, carriers, excipients, glidants, sweeteners, dilutions that have been approved by the U.S. Food and Drug Administration for use in humans or animals.
  • Agents, preservatives, dyes/colorants, flavor enhancers, surfactants, wetting agents, dispersing agents, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers, etc. have no side effects on the composition of the pharmaceutical composition.
  • Various forms of carrier include but not limited to any adjuvants, carriers, excipients, glidants, sweeteners, dilutions that have been approved by the U.S. Food and Drug Administration for use in humans or animals.
  • 1.1DPPH ethanol solution preparation accurately weigh DPPH 4mg, placed in a 100mL brown volumetric flask, add 50mL of ethanol, sonicate for 30s, dilute to volume with ethanol, shake well, set aside. This product must be used now.
  • test solution accurately weigh 10mg of walnut oligopeptide powder, put it in a 50mL brown volumetric flask, add 30mL of ethanol, sonicate for 5min, dilute to the mark with ethanol, shake well, that is.
  • Ai represents the absorbance of the solution after mixing the solution to be tested and DPPH
  • Aj represents the absorbance of the solution after mixing the solution to be tested and the solvent
  • A0 represents the absorbance of the solution after the DPPH and the solvent are mixed.
  • PBS buffer Preparation of PBS buffer: Weigh 8g of sodium chloride, 0.2g of potassium chloride, 0.24g of potassium dihydrogen phosphate, 3.62g of disodium hydrogen phosphate dodecahydrate, put it in a 1000mL beaker, add 800mL of distilled water, stir it Dissolve, adjust the pH to 7.4 with hydrochloric acid or sodium hydroxide, transfer to a 1000 mL volumetric flask, dilute to the mark with distilled water, shake well, and set aside.
  • 2.2ABTS + storage solution preparation accurately weigh ABTS+78mg, placed in a 20mL brown volumetric flask, add 15mL distilled water, sonicate for 5min, dilute to the mark with distilled water, shake. Weigh accurately about 76 mg of potassium persulfate, place it in a 2 mL brown volumetric flask, add 1 mL of distilled water, dissolve it by sonication, dilute to volume with distilled water, and shake well. Accurately pipet 352 ⁇ L of potassium persulfate solution into the ABTS solution, shake well, and let stand overnight.
  • 2.3ABTS + working solution preparation Accurately absorb 1mL of storage solution, add about 65mL of PBS buffer, shake.
  • test solution accurately weigh 20mg of walnut oligopeptide powder, placed in a 20mL brown volumetric flask, add 15mL PBS buffer, sonicate for 5min, dilute to the mark with PBS buffer, shake well, that is.
  • Ai represents the absorbance of the solution after the test solution and ABTS are mixed
  • Aj represents the absorbance of the solution after mixing the solution to be tested and the solvent
  • A0 represents the absorbance of the solution after mixing the ABTS and the solvent.
  • test solution Prepare 1mg/mL walnut oligopeptide powder for testing.
  • Ai represents the absorbance of the solution after mixing the solution to be tested and SRSA
  • Aj represents the absorbance of the solution after mixing the solution to be tested and the solvent
  • A0 represents the absorbance of the solution after the mixture of SRSA and solvent.
  • the concentration of the neutral proteolytic product in Preparation Example 1 was 100 ⁇ g/mL, and the vitamin C was used as a positive control (concentration was 100 ⁇ g/mL).
  • concentration was 100 ⁇ g/mL.
  • the walnut oligopeptide powder prepared by the method of the invention has strong scavenging activity on DPPH and ABTS free radicals, moderate scavenging activity on superoxide anion, and further exhibits better antioxidant activity.
  • PC12 neuroprotective model It is a good model for studying the physiology, pathology and pharmacology of nerve cells. It is the most commonly used in vitro drug screening model for studying Parkinson's disease and nerve fatigue.
  • PCl2 cells were cultured in high glucose DMEM medium containing 10% fetal bovine serum. The cells were digested with 0.25% trypsin for about 50 s. The cells were digested with DMEM medium containing 10% serum, and the cells were blown with fresh medium. Evenly. Passage at a cell density of 10 5 /mL. Add 4 mL of cell-containing medium to each vial of cells. Incubate at 37 ° C, 5% CO 2 .
  • PCl2 cells were grown to a confluent state in a culture flask, digested with 0.25% trypsin solution, and repeatedly pipetted to a cell suspension, and diluted to 1.0 ⁇ 10 5 cells/mL in a high glucose DMEM medium containing 10% FBS. 100 ⁇ L was inoculated into 10 replicate wells of each group in a 96-well culture plate, and cultured at 37 ° C, 5% CO 2 for 24 hours to form a fusion state.
  • the 96-well plates were given 100 ⁇ L of walnut oligopeptide in a certain concentration gradient in each well. After 24 hours of culture, the cell viability was detected by MTT assay. 50 mg of MTT was dissolved in 10 mL of PBS and filtered through a 0.22 ⁇ m micropore filter. Dilute to 0.5 mg/mL before use. The cells in each group were discarded, washed twice with PBS, and added with 0.5 mg/mL MTT, incubated at 37 ° C, 5% CO 2 for 3 h, and the MTT working solution was removed. 150 ⁇ L of DMSO was added to each well to dissolve the crystals, and shaken for 10 min.
  • the OD value of each well was measured (measurement wavelength 570 nm, reference wavelength 650 nm).
  • the cell viability of the model group and the drug-administered group was calculated by taking the average value of the OD value of the control group as 100% cell viability. The measurement results are shown in Table 1.
  • a blank group (1% serum DMEM);
  • B model group (1% serum DMEM cultured for 6 h, then added H 2 O 2 to a final concentration of 100 ⁇ M, stimulated for 12 h);
  • C-positive drug (NAC) group firstly add 1% serum DMEM containing a certain concentration of positive drug NAC for 6h, and then add 100 ⁇ M H 2 O 2 for 12h.
  • D administration group 1% serum of walnut oligopeptides of each concentration gradient was added to DMEM for 6 h, and then stimulated with 100 ⁇ M H 2 O 2 for 12 h.
  • the walnut oligopeptide powder prepared by the present invention did not reduce the proliferation activity of PC12 cells, and the number of PCl2 cells increased significantly with the increase of the concentration. It can be seen from Table 3 that the cell viability was 57.2% after stimulation with H 2 O 2 in the model group; when the 80 ⁇ g/mL positive control drug NAC was added, the cell viability was increased to 88.0%, indicating obvious protective effect; After adding walnut oligopeptide culture, the cell viability increased with the increase of peptide powder concentration, showing concentration-dependent and protective effect. At a concentration of 500 ⁇ g/mL, the cell viability reached 72.0%, compared with the model group, cell viability Increased by 30%.
  • the peptide powder has a strong protective effect on neuronal cells, and thus can be used for preventing or treating diseases related to Parkinson's disease, Alzheimer's disease, and the like, and can also be used as a medicine or health food for relieving brain fatigue.
  • a zebrafish macrophage inhibition model was established by intravenous injection of vinorelbine on zebrafish 2 days after fertilization.
  • the walnut oligopeptide powder and the positive control drug berbamine were respectively dissolved in fish water, the concentration of oligopeptide powder was 500 ⁇ g/mL, and the concentration of berbamine was 1.9 ⁇ g/mL.
  • the model control group and the normal control group were set. (Without any treatment), each experimental group was 30 zebrafish and cultured in a 28 ° C incubator. After treatment to 3dpf, the zebrafish were stained with neutral red in each experimental group.
  • each experimental group randomly selected 10 zebrafish to observe, photograph and save the pictures under the microscope; image analysis software was used for image analysis.
  • Zebrafish giant The number of phagocytes was used to quantitatively evaluate the improvement of oligopeptide powder on the inhibition of zebrafish macrophages.
  • the average number of macrophages in the zebrafish in the normal control group was 27, which was compared with the model control group (15), indicating that the zebrafish macrophage suppression model was successfully established.
  • the concentration of the positive drug berbamine was 1.9 ⁇ g/mL
  • the average number of macrophages was 20.
  • the improvement of zebrafish macrophage inhibition was 41.67%, indicating that Xiaoyan
  • the zebrafish macrophage inhibition was significantly improved.
  • the concentration of walnut oligopeptide powder was 500 ⁇ g/mL
  • the average number of macrophages was 25.
  • the improvement effect on zebrafish macrophage inhibition was 83.33%.
  • the walnut oligopeptide powder prepared by the invention has a significant improvement effect on zebrafish macrophage inhibition.
  • a zebrafish macrophage promoting model was established by intravenous injection of ink on zebrafish 2 days after fertilization.
  • the fish oligopeptide and the positive drug Pidotimod were respectively dissolved in fish water, the concentration of oligopeptide was 2000 ⁇ g/mL, and the concentration of Pidotimod was 200 ⁇ g/mL.
  • the model control group was set, and each experimental group was 30.
  • the tail zebrafish was cultured in a 28 ° C incubator. After treatment to 3dpf, the zebrafish were stained with neutral red in each experimental group.
  • the zebrafish of wild-type AB strains were randomly selected from the 16-day (1dpf) 16-day fertilization in a six-well plate. Each well (experimental group) treated 30 zebrafish and induced zebrafish central nervous system injury with mycophenolate mofetil. When the concentration of oligopeptide powder was 222 and 667 ⁇ g/mL, the positive control drug glutathione (GSH) was 154 ⁇ g/mL, and the normal control group (water treatment zebrafish for fish farming) and model control group were set. The volume of the well (experimental group) was 3 mL. The oligopeptides were treated with mycophenolate mofetil for 24h, then stained with acridine orange.
  • zebrafish were randomly selected from each experimental group to take photos under fluorescence microscope and collect data to analyze the zebrafish central nervous system.
  • the fluorescence intensity of apoptotic cells was evaluated according to the fluorescence intensity of the protective effect of walnut oligopeptide powder on the central nervous system of zebrafish.
  • the fluorescence intensity of apoptotic cells in zebrafish telomeres at 222 and 667 ⁇ g/mL were 395,025 and At 451,259 pixels, the central nervous system protection was 42% and 28%, respectively.
  • the walnut oligopeptide had obvious protective effect on the central nervous system of zebrafish.
  • 4dpf wild-type AB strain zebrafish were randomly selected from six-well plates, 30 wells per well (ie, each test group), respectively, and water-soluble walnut oligopeptide was given, and the positive control drug Zhonghua beat pills was 1.0 mg/mL.
  • a normal control group and a model control group were set, and the volume per well was 3 mL.
  • the other experimental groups were simultaneously hydrolyzed with sodium sulfite to induce the zebrafish fatigue model.
  • 10 zebrafish were randomly selected from each experimental group. The behavioral analysis was used to determine the total distance of the zebrafish (S), and the fatigue zebra induced by sodium sulfite was quantitatively evaluated. The movement of fish improves.
  • 4dpf wild-type AB strain zebrafish were randomly selected from six-well plates, 30 wells per well (ie, each test group), respectively, and water-soluble walnut oligopeptide was given, and the positive control drug Zhonghua beat pills was 1.0 mg/mL.
  • the normal control group and the model control group were set, and the volume per well was 3 mL; three parallel groups were set for each experimental group. After pretreatment for a certain period of time, except for the normal control group, the other experimental groups were simultaneously hydrolyzed with sodium sulfite to induce the zebrafish fatigue model.
  • each experimental group collected the zebrafish in three parallel experimental groups (90 total), and indirectly measured the lactic acid content in the zebrafish using the NanoDrop2000 ultra-micro spectrophotometer.
  • the effects of walnut oligopeptides on the lactic acid content of sulphite-induced fatigue zebrafish were evaluated quantitatively at 2000 ⁇ g/mL.
  • the walnut oligopeptide powder prepared by the invention can significantly improve the zebrafish exercise ability and increase the lactic acid metabolism in the body. It can be seen that the walnut oligomeric powder has obvious anti-fatigue effect and can be used for preventing or relieving fatigue food, health care products or medicines.
  • the A ⁇ 1-42 amyloid protein was formulated in DMSO at 2.5 mg/mL, and the walnut oligopeptide powder was diluted to an appropriate concentration. Then, A ⁇ 1-42 amyloid protein solution (1 ⁇ L) was mixed with walnut oligopeptide solution (9 ⁇ L) to make the final concentration of A ⁇ 1-42 amyloid protein 0.25 mg/mL, and the final concentration of walnut oligopeptide was 10 and 100 ⁇ g/mL.
  • walnuts oligopeptide at a concentration of 10 ⁇ g / mL, anti-aggregation of A ⁇ 1-42 amyloid obvious, but at a concentration of 200 ⁇ g / mL, aggregation of A ⁇ 1-42 amyloid It has a certain inhibitory effect, and it can be speculated that the walnut oligopeptide has a protective effect on the cranial nerve and has the potential to improve the memory.
  • anti-CD3 10 ⁇ g/mL anti-CD3 was coated in a 6-well plate (200 ⁇ L/well) and allowed to stand at 4 ° C for 18 to 24 hours. Activate HPBMC, dilute to 5 ⁇ 10 5 cells/mL with cell suspension, and make a final volume of 36 mL containing anti-CD28 (2 ⁇ g/mL), rhIL-2 (10 ng/mL), rhIL-4 (50 ng/ mL). The 6-well plate that had been coated with anti-CD3 was rinsed with the medium, and the HPBMC dilution was transferred to the 6-well plate and cultured in a carbon dioxide incubator.
  • the 6-well plate of HPBMC dilution was collected and centrifuged, the supernatant was removed, and the medium containing rhIL-2 (10 ng/mL) and rhIL-4 (50 ng/mL) was mixed and mixed to make the cells.
  • the medium concentration was 5 ⁇ 10 5 cells/mL, and then transferred to a cell culture flask, and then cultured in a carbon dioxide incubator.
  • HPBMC was collected, centrifuged to remove the supernatant, washed with a medium, and centrifuged again to remove the supernatant.
  • the medium containing 5 ng/mL PMA was added and dispersed to make a cell concentration of 5 ⁇ 10 5 cells/mL, a final volume of 36 mL, and placed in a carbon dioxide incubator. After 4 hours, the supernatant was centrifuged for CBA analysis. The method of operation was carried out in accordance with the instructions of the BD CBA Human Th1/Th2/Th17Cytokine Kit Instruction Manual, and the changes of IL-10 and IL-17A were analyzed.
  • the walnut oligopeptide can obviously down-regulate the content of IL-10 and increase the content of IL-17A. It can be seen that walnut oligopeptides can regulate inflammatory factors and improve immunity.
  • a green fluorescent specific fusion protein was used as a detection signal using a neuron-specific HuC promoter.
  • the green fluorescent protein of plasmid pHuC-GFP was first injected into cells of the zebrafish embryo that developed into a 1-cell stage.
  • the walnut oligopeptide powder (Preparation Example 1) was dissolved in DMSO and diluted with water to a certain concentration. After 8 hours, it and DMSO (control negative) were injected into zebrafish embryo cells. After 40 hours, based on the number of zebrafish neurite outgrowth, it was judged whether the sample was beneficial to the growth of neurites in the zebrafish embryo.
  • the experimental results showed that the promotion rate of zebrafish embryo neurite outgrowth was only 23% in the control negative DMSO group, and the growth rate of neurite outgrowth was 51% after injection of 1 mg/mL walnut oligopeptide. It can be seen that walnut oligopeptides can significantly promote the growth of zebrafish embryonic neurites.
  • the neural tissue-specific Huc promoter was used to detect the apoptosis of zebrafish embryos induced by wild-type human tau protein using hTau green fluorescent fusion protein (GFP).
  • the expression construct is injected into the cells of the zebrafish embryo that develop into a 1-cell stage.
  • the walnut oligopeptide powder (Preparation Example 1) was dissolved in DMSO and diluted with water to a certain concentration. After 8 hours, it and DMSO (control negative) were injected into zebrafish embryo cells. The 24 and 48 hpf GFP-labeled cells were observed using a fluorescence microscope.
  • walnut oligopeptides have the effect of promoting or improving memory.
  • test animals were clean grade ICR mice weighing 18-22 g, 300 in three batches, provided by Nantong University, experimental animal production license: SCXK (Su) 2014-0001. Feed the regular rat feed and drink freely. Pre-test in a quiet environment for 1 week, free access to drinking water, to maintain room temperature (22 ⁇ 1 ° C), natural day and night rhythm lighting.
  • mice were randomly divided into low, medium and high dose groups (30, 100, 300 mg/kg) of walnut oligopeptide (Preparation Example 1), and equal volume distilled water was given to the blank group and the model group, respectively.
  • the positive control group was given Nemo.
  • the ground level was 30 mg/kg, and each group was intragastrically administered once a day for 7 days.
  • mice were administered per batch, run in parallel, and the second batch of mice was injected 10 minutes later, and so on.
  • each batch of 5 mice was placed in the 5 grids of the platform, first adapted to the environment for 3 minutes, and then energized. After the mice were shocked, most jumped onto the platform to escape the electric shock. When jumping, the mice were exposed to the copper grid at the same time as the electric shock. It was regarded as an error reaction. It was trained for 5 minutes and retested after 24 hours.
  • the mouse was placed on the platform and the timing was started.
  • the first jump time of the mouse was recorded. This is the electric shock latency (that is, the error latency), and the number of jumps within 5 minutes (that is, the number of errors) is recorded. As an indicator of observation.
  • the methods of grouping, administration, and training were the same as in Experiment 2.1. After the end of the training, the blank control group was injected subcutaneously with the same amount of normal saline, and the other groups were immediately subcutaneously injected with sodium nitrite 90 mg/kg, and the test was performed 24 hours later. The test method is also the same as Experiment 2.1.
  • the methods of grouping, administration, and training were the same as in Experiment 2.1. Thirty minutes before the test, the model group and the administration group were intragastrically administered with 40% ethanol 10 mL/kg, and the blank control group was given equal volume of distilled water. The test method is also the same as Experiment 2.1.
  • Example 1 (30, 100, 300 mg/kg) was continuously administered for 7 days, and mice with memory impairment in scopolamine-induced memory were significantly prolonged in the preparation of Example 1 in the high-dose group. Latency, reducing the number of errors. The results are shown in Table 11.
  • Preparation Example 1 (30, 100, 300 mg/kg) was continuously administered for 7 days.
  • the preparation of each of the dose groups of Example 1 can significantly prolong the incubation period. , reduce the number of errors. The results are shown in Table 12.
  • Preparation Example 1 (30, 100, 300 mg/kg) was continuously administered for 7 days.
  • the dosage groups of the preparation example 1 could significantly prolong the latency and reduce the latency. The number of errors. The results are shown in Table 13.
  • Learning and memory functions include spatial learning and memory functions and non-spatial learning and memory functions.
  • the memory impairment model is an effective means to evaluate the effects of drugs on the memory process, and it is also a model used to explore the role of drugs in the treatment of senile dementia and its mechanism of action.
  • Scopolamine is an M receptor blocker that blocks the agonistic effects of acetylcholine on M receptors and mimics learning and memory dysfunction caused by acetylcholine deficiency. Sodium nitrite denatures hemoglobin, causing ischemia and hypoxia in brain tissue, impairing learning and memory processes.
  • Ethanol inhibits the neurological activity of the cerebral cortex, inhibits the conditioned reflex process in animals, blocks the synthesis of proteins and RNA in the brain, changes the system of cholinergic and dopamine, and destroys the learning and memory function, which causes learning and memory reproducibility.
  • the medium and high oligopeptides of walnut can improve the latency of mice with learning and memory impairment caused by scopolamine in different degrees, and the high dose group can reduce the number of missed platform errors.
  • the model of memory consolidation disorder caused by sodium nitrate, walnut oligopeptides in each dose group can extend the incubation period and reduce the number of errors.
  • the walnut oligopeptides in each dose group can also significantly reduce the number of errors and prolong the incubation period. It can be seen that the middle and high dose groups of walnut oligopeptides have less learning and memory barriers caused by scopolamine. The learning and memory ability of rats has obvious improvement effect.
  • the low, medium and high dose groups have obvious learning and memory ability in mice with learning and memory reproduction disorder induced by 40% ethanol and mice with learning and memory consolidation disorder induced by sodium nitrite. Improve the role.

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Abstract

本发明提供了一种核桃低聚肽粉、其制备方法和用途。所述低聚肽粉中肽的含量在80wt％以上，其中95％以上的肽分子量小于1500Da。所述方法包括使用高效逆流法提取核桃蛋白，经过滤、酶解、微滤膜和超滤膜纯化、浓缩、喷雾干燥，得低聚肽粉。所述低聚肽粉具有抗氧化活性，对神经元细胞有保护作用，可改善或治疗记忆力衰退，缓解疲劳。

Description

一种核桃低聚肽粉及其制备方法和用途 技术领域
本发明涉及一种高纯度、低分子量的核桃低聚肽产品，其多肽含量在80wt％以上，分子量小于1500Dalton在95％以上。本发明还涉及到核桃蛋白的制备并催化酶解，生成低聚肽粉的制备方法，该肽粉可用作药物、食品、保健品或化妆品。本发明要求中国专利申请CN201610043952.0的优先权，其内容在此全部引入并作参考。
背景技术
核桃(Juglans regia L)又名胡桃，属于四大坚果之一，具有很高的营养药用价值。在我国古医药书籍中，有明确记载，李时珍《本草纲目》记述“补气养血、润燥化痰、益命门、利三焦，温肺润肠。治肺润肠。治虚寒喘咳，腰脚重疼，心腹疝痛，血痢肠风，散肿毒，宋刘翰《开宝本草》载“胡桃(即核桃)味甘、平、无毒。食之令人肥健，润肌黑发，取瓤烧令黑，未断烟，和松脂，研傅瘰疬疮”。唐化孟诜《食疗本草》中说，核桃仁能“通经脉、黑须发，常服骨肉细腻光润”。崔禹锡的《食经》说它“多食利小便，去五痔”。《医林纂要》一书的评价是，可以“补肾，润命门，固精，润大肠，通热秘，止寒泻虚泻。”等等。
核桃中含有丰富的蛋白质、脂肪等营养成分，含量比较均衡，属于比较理想的高蛋白、高脂肪的食物，据报道，核桃仁中含有高达52％-70％的油脂，其中大部分为不饱和脂肪酸，另外还含有约24％的蛋白质，12％-16％的碳水化合物，1.5％-2％的纤维素，以及1.7％-2％的矿物质。核桃中富含人体必需的氨基酸，且氨基酸比例比较合理，其中对人体生理作用有着重要功能的谷氨酸、天冬氨酸、精氨酸含量均较高，其中谷氨酸还是影响人体特别是青少年智力及记忆发育的重要功能物质。
为了获得比核桃原蛋白在营养、功能性及生物活性上更加优良的低聚肽粉，已尝试了多种制备方法。专利CN 101228918 A将核桃粕粉碎，超声波法提取核桃蛋白，将核桃蛋白真空干燥后，再使用蛋白酶进行酶解，离心，使用透析袋对上清液进行透析，经浓缩透析液并真空干燥得到含量在60％～80％的肽粉。该方法操作复杂，肽含量较低，肽分子量的分布也不明确，同时使用超声提取蛋白，透析袋进行精致，无法进行大规模制备。专利CN 102406050 A使用碱提酸沉方法提取蛋白质，经 冷冻干燥得到核桃蛋白粉，蛋白粉经酶解后，用300Mpa高压处理10min，冷冻干燥得核桃肽粉。此制备方法需超高压设备和冷冻干燥机，成本高，不适宜大规模生产；该发明专利虽然提供了该法制备的核桃肽粉的分子量分布，但产品中肽含量不明确。专利CN 103103244 B使用碱提酸沉的方法提取蛋白质，并用微波结合超声波处理后，酶解得到多肽粉。该法仍不适用大规模生产，并且没有对酶解产品中肽含量进行测定。专利CN 104293870 A先使用CO2超临界萃取仪去除油脂后得到的核桃粕，碱提酸沉后喷雾得到核桃蛋白粉，再将蛋白粉制成悬浊液并煮沸破坏蛋白质的结构，使用碱性蛋白酶、木瓜蛋白酶、中性蛋白酶和菠萝蛋白酶分段四步酶解核桃蛋白液，经5000Dalton或者8000Dalton超滤膜精制后使用离子交换树脂脱盐，最后喷雾干燥得到核桃肽粉。此专利涉及到去除油脂时使用超临界萃取仪，增加成本，不易于大生产；涉及到4种酶分段四步酶解，步骤繁琐、成本高；涉及到酶解完成后，未经过微滤膜除杂，直接进行超滤膜，造成超滤膜易阻塞，超滤时间延长，降低多肽收率，同时缩短超滤膜使用寿命；此专利未涉及到多肽产品中肽的百分含量和肽的分子量分布。
可见，以上核桃多肽的制备条件苛刻，工艺步骤繁琐，很难实现大规模生产，因此寻找一套工艺简单、低成本、高含量、较高活性的核桃多肽的制备方法已经成为寻找大规模生产的研究热点之一。
发明内容
本发明的目的是提供了一种高纯度、低分子量的核桃低聚肽粉。
本发明的另一目的是提供了一种高纯度、低分子量的核桃低聚肽粉的制备方法。
本发明的另一目的是提供了本发明核桃低聚肽粉用于制备治疗或预防自由基过多引起的症状的药物、食品、保健品或化妆品的应用。
本发明的另一目的是提供了本发明核桃低聚肽粉用于制备改善或治疗记忆力衰退的药物、食品、保健品或化妆品的应用。
本发明的另一目的是提供了本发明核桃低聚肽粉用于制备治疗或预防帕金森症和阿尔茨海默症、缓解大脑或者运动疲劳的药物、食品、保健品或化妆品的应用。
本发明的另一目的是提供了本发明核桃低聚肽粉用于制备增强免疫力的药物、食品、保健品或化妆品的应用。
本发明的另一目的是提供了含有本发明核桃低聚肽粉的药物、食品、保健品或化妆品组合物。
本发明的目的是通过下述技术方案实现的：
一种核桃低聚肽粉，采用GB/T 22492-2008附录A与附录B的检测方法，测得肽含量在80wt％以上，其中95％以上核桃肽的分子量小于1500Dalton，其分子量分布如下：
分子量Dalton分布
Figure PCTCN2017000121-appb-000001
数均分子量范围：170～3000
重均分子量范围：180～4000，
优选地，肽含量为81wt％以上，小于1500Dalton分子量占97％以上；更优选，肽含量为81.3wt％以上，小于1500Dalton分子量占96％以上；再优选，肽含量为85wt％以上，小于1500Dalton分子量占97％以上；最优选，肽含量为85wt％以上，小于1500Dalton分子量占96％以上。
所述核桃低聚肽粉是通过下述方法制备得到的：
(1)核桃粕的预处理：将核桃去壳、冷榨脱油，得到脱脂核桃粕。
(2)逆流提取法提取蛋白质：将一定量脱脂后的核桃粕(记录为A)，与水按重量比为1∶5～1∶15混合，调节pH至9～11于室温提取1～2h；提取完成后，过滤，滤渣进行二次提取，滤液倒入等量的核桃粕(记录为B)，调节pH至9～11于室温提取1～2h；B完成第一次提取后，滤液待用，滤渣继续进行第二次提取；A完成二次提取后，滤渣弃去，滤液倒入等量的核桃粕(记录为C)，调节pH至9～11于室温提取1～2h；B完成二次提取后，滤渣弃去，滤液倒入C完成第一次提取的滤渣中提取1～2h，而C第一次提取的滤液待用；样品C完成第二次提取后，滤渣弃去，滤液待用；最后合并以上待用滤液，调节pH为3～5，静置0.5～2h，弃去上清液，最后往沉淀物中加入体积比为1∶10～1∶20的水，搅拌均匀。
(3)蛋白酶解：将上述核桃蛋白液加热至40～55℃，将pH值调节至中性，加入核桃粕重量的0.5～2％的生物酶，搅拌酶解3～6h后，煮沸灭活30min，离心，上清液即为蛋白酶解液。
(4)分离纯化：将蛋白酶解液使用孔径为0.1～0.5μm的微滤膜进行 过滤，透过液再经2000～20000Dalton超滤膜处理后，截留液在50～80℃下浓缩至固含为3～5wt％时，进行喷雾干燥，进口温度为140～160℃，出口温度为55～65℃，得到高纯度、低分子量的淡黄色核桃肽粉，产率可达20～30wt％。
(5)肽含量与分子量分布测定：采用GB/T 22492-2008附录A与附录B的检测方法，测得肽含量在80wt％以上，其中95％以上的核桃肽的分子量小于1500Dalton。
所述生物酶选自食品级的中性蛋白酶(酶活力≥30万u/g)、木瓜蛋白酶(酶活力≥40万u/g)、菠萝蛋白酶(酶活力≥30万u/g)、碱性蛋白酶(酶活力≥20万u/g)、胃蛋白酶(酶活力≥50万u/g)、胰酶(酶活力≥3000u/g)中的一种或者它们的混合物，优选地使用中性蛋白酶或复合酶，所述复合酶的中性蛋白酶与木瓜蛋白酶的质量比为1∶1，中性蛋白酶的活力为30万u/g，木瓜蛋白酶的活力为50万u/g。
一种组合物，其含有本发明所述的核桃低聚肽粉和药物、食品、保健品或化妆品上可接受的助剂。
根据现有技术，本发明可将所述的组合物制备成任意所述剂型，如，素片、薄膜包衣片、糖衣片、肠衣片、分散片、胶囊、颗粒剂、口服溶液或口服混悬液，以及液体、乳液、膏霜、粉、块状等化妆品剂型。
本发明所述的核桃低聚肽粉可用于制备治疗或预防自由基过多引起的症状的药物、食品、保健品或化妆品；用于制备改善或治疗记忆力衰退的药物、食品、保健品或化妆品；用于制备治疗或预防帕金森症和阿尔茨海默症、缓解大脑或者运动疲劳的药物、食品、保健品或化妆品；用于制备增强免疫力的药物、食品、保健品或化妆品。
与现有技术相比，本发明具有如下优点：
(1)本发明将核桃冷榨脱脂制备核桃粕，发明人做了相关实验，与热榨方法相比，冷榨后蛋白质提取率高出5wt％左右，同时含量高出20wt％左右。
(2)本发明使用高效逆流法提取核桃蛋白，相比普通的碱提酸沉法，蛋白质的提取率提高了10％以上，同时减少了用水量，节约生产成本。
(3)本发明仅使用蛋白复溶液进行酶解，没有将蛋白进行干燥，这样不仅减少了蛋白质干燥时的损失，也简化了制备工艺。
(4)本发明使用的生物酶能在含量、分子量、产率、活性上得到保证，酶解工艺稳定，两种酶均为食用酶，来源广泛、成本低，酶解时，添加量仅为核桃粕质量的0.5～2％。
(5)本发明使用微滤膜进行过滤，初步去除酶解液中的不溶物，再 使用2000～20000Dalton超滤膜去除大分子量蛋白。
(6)本发明没有使用冷冻干燥或者真空干燥法制备产品，使用喷雾干燥法不仅节约了干燥时间，而且产品质粒均匀。
(7)本发明根据GB/T 22492-2008附录A测定多肽的分子量分布，根据附录B先测定酸溶性蛋白和游离氨基酸的含量，最后差值即为肽含量。此分子量和肽含量的测定方法认可度高。
分子量Dalton分布
Figure PCTCN2017000121-appb-000002
数均分子量范围：170～3000
重均分子量范围：180～4000
(8)本发明测得肽含量在80wt％以上，其中95％以上的核桃肽的分子量小于1500Dalton，是高纯度、低分子量的低聚肽。
附图说明
图1：制备实施例1核桃低聚肽液相色谱图；
图2：制备实施例2核桃低聚肽液相色谱图；
图3：核桃低聚肽对斑马鱼巨噬细胞抑制的改善作用图；
图4：核桃低聚肽对斑马鱼巨噬细胞吞噬功能的促进作用图；
图5：核桃低聚肽对斑马鱼中枢神经影响的典型图；
图6：核桃低聚肽对斑马鱼胚胎神经突起生长的促进作用图；
图7：核桃低聚肽降低人野生型tau蛋白对斑马鱼胚胎内神经细胞的毒性作用图。
实施例
下面通过实施例对本发明作进一步说明。应该理解的是，本发明实施例所述方法仅仅是用于说明本发明，而不是对本发明的限制，在本发明的构思前提下对本发明制备方法的简单改进都属于本发明要求保护的范围。实施例中用到的所有原料和溶剂如无特别说明，均为商购产品。
制备实施例1：
将100kg冷榨脱脂后的核桃粕(记录为A)，与水按重量比为1∶10混合，调节pH至10于室温提取2h；提取完成后，过滤，滤渣进行二次提取，滤液倒入等量的核桃粕(记录为B)，调节pH至10于室温提取2h；B完成第一次提取后，滤液待用，滤渣继续进行第二次提取；A完成二次提取后，滤渣弃去，滤液倒入等量的核桃粕(记录为C)，调节pH至10于室温提取2h；B完成二次提取后，滤渣弃去，滤液倒入C完成第一次提取的滤渣中提取2h，而C第一次提取的滤液待用；样品C完成第二次提取后，滤渣弃去，滤液待用；最后合并以上待用滤液，调节pH为5，静置6h，弃去上清液，最后往沉淀物中加入体积比为1∶10的水，搅拌均匀。将上述核桃蛋白液加热至45℃，将pH值调节至中性，加入1kg中性蛋白酶(酶活力为30万u/g)，搅拌酶解6h后，煮沸灭活30min，离心，上清液即为蛋白酶解液。将蛋白酶解液使用孔径为0.1μm的微滤膜进行过滤，透过液再经5000Dalton超滤膜处理后，透过液在80℃下浓缩至固含为3.4％时，进行喷雾干燥，进口温度为140℃，出口温度为55～65℃，得到高纯度、低分子量的淡黄色核桃肽粉，产率为21wt％。采用GB/T 22492-2008附录A与附录B的检测方法，测得肽含量为81wt％，小于1500Dalton分子量占97％，分子量分布如前所述。肽含量结果如下所示：
测试结果：
Figure PCTCN2017000121-appb-000003
制备实施例2：
将100kg冷榨脱脂后的核桃粕(记录为A)，与水按重量比为1∶10混合，调节pH至10于室温提取2h；提取完成后，过滤，滤渣进行二次提取，滤液倒入等量的核桃粕(记录为B)，调节pH至10于室温提取2h；B完成第一次提取后，滤液待用，滤渣继续进行第二次提取；A完成二次提取后，滤渣弃去，滤液倒入等量的核桃粕(记录为C)，调节pH至10于室温提取2h；B完成二次提取后，滤渣弃去，滤液倒入C完成第一次提取的滤渣中提取2h，而C第一次提取的滤液待用；样品C完成第二次提取后，滤渣弃去，滤液待用；最后合并以上待用滤液，调节pH为5，静置6h，弃去上清液，最后往沉淀物中加入体积比为1∶10的水，搅拌均匀。将上述核桃蛋白液加热至45℃，将pH值调节至中性，加入核桃粕重量的1kg中性木瓜复合蛋白酶(两种蛋白酶的质量比为1∶1，中性蛋白酶的活力为30万u/g，木瓜蛋白酶的活力为50万u/g)，搅拌酶解6h后，煮沸灭活30min，离心，上清液即为蛋白酶解液。将蛋白酶解液使用孔径为0.1μm的微滤膜进行过滤，透过液再经5000Dalton超滤膜处理后，透过液在80℃下浓缩至固含为4.1％时，进行喷雾干燥，进口温度为140℃，出口温度为55～65℃，得到高纯度、低分子量的淡黄色核桃肽粉，产率为21wt％。采用GB/T 22492-2008附录A与附录B的检测方法，测得肽含量为81.3wt％，小于1500Dalton分子量占96％，分子量分布如前所述。中性木瓜蛋白复合酶酶解后肽含量结果如下所示：
测试结果：
Figure PCTCN2017000121-appb-000004
制备实施例3：组合物的制备
药物或食品上可接受的助剂，包括但不限于已经被美国食品与药品管理局认可的而可用于人类或动物的任何佐剂、载体、赋形剂、助流剂、甜味剂、稀释剂、防腐剂、染料/着色剂、香味增强剂、表面活性剂、润湿剂、分散剂、助悬剂、稳定剂、等渗压剂、溶剂或乳化剂等对组成药物组合物无副作用的各种形式的载体。
本领域技术人员可根据现有技术中任何已知的方法将本发明的核桃低聚肽粉与上述助剂相混合，所制备的任何剂型也是现有技术中已知的。
生物活性实施例1：
1.DPPH自由基清除实验：
1.1DPPH乙醇溶液的配制：精密称取DPPH 4mg，置于100mL棕色容量瓶中，加入50mL乙醇，超声30s，用乙醇定容至刻度，摇匀，待用。本品须现配现用。
1.2供试品溶液的配制：精密称取核桃低聚肽粉10mg，置于50mL棕色容量瓶中，加入30mL乙醇，超声5min，用乙醇定容至刻度，摇匀，即得。
1.3操作步骤：准确吸取2mL供试品溶液和2mL DPPH溶液混合均匀；准确吸取2mL供试品溶液和2mL乙醇混合均匀；准确吸取2mL DPPH溶液和2mL乙醇混合均匀，室温放置30min，在515nm波长处测定吸光度，并根据以下计算公式计算自由基清除率：
IR％＝[1-(Ai-Aj)/A0]*100％；
其中，Ai表示待测溶液和DPPH混合后溶液的吸光度；
Aj表示待测溶液和溶剂混合后溶液的吸光度；
A0表示DPPH和溶剂混合后溶液的吸光度。
2.ABTS+自由基清除实验：
2.1PBS缓冲液的配制：称取氯化钠8g，氯化钾0.2g，磷酸二氢钾0.24g，十二水合磷酸氢二钠3.62g，置于1000mL烧杯中，加入800mL蒸馏水，搅怑使其溶解，用盐酸或氢氧化钠调节pH至7.4，转移至1000mL容量瓶中，加蒸馏水稀释至刻度，摇匀，待用。
2.2ABTS+贮存溶液的配制：精密称取ABTS+78mg左右，置于20mL棕色容量瓶中，加入15mL蒸馏水，超声5min，用蒸馏水定容至刻度，摇匀。精密称取过硫酸钾76mg左右，置于2mL棕色容量瓶中， 加入1mL蒸馏水，超声使其溶解，用蒸馏水定容至刻度，摇匀。精确吸取352μL过硫酸钾溶液加入至ABTS溶液中，摇匀，静置过夜。
2.3ABTS+工作溶液的配制：精确吸取贮存溶液1mL，加入65mL左右PBS缓冲液，摇匀。
2.4供试品溶液的配制：精密称取核桃低聚肽粉20mg，置于20mL棕色容量瓶中，加入15mL PBS缓冲液，超声5min，用PBS缓冲液定容至刻度，摇匀，即得。
2.5操作步骤：准确吸取0.5mL供试品溶液和5mL ABTS工作溶液混合均匀；准确吸取0.5mL供试品溶液和5mL PBS缓冲液混合均匀；准确吸取5mL ABTS工作溶液和0.5mL PBS缓冲液混合均匀，立即在734nm处测定吸光度，并根据以下公式计算自由基清除率：
IR％＝[1-(Ai-Aj)/A0]*100％；
其中，Ai表示待测溶液和ABTS混合后溶液的吸光度；
Aj表示待测溶液和溶剂混合后溶液的吸光度；
A0表示ABTS和溶剂混合后溶液的吸光度。
3.SRSA超氧阴离子自由基清除实验：
3.10.1moL/L PBS缓冲液(pH7.4)的配制：称取氯化钠80g，氯化钾2g，磷酸二氢钾2.4g，三水合磷酸氢二钾23.1g，置于1000mL烧杯中，加入600mL蒸馏水，搅怑使其溶解，用盐酸或氢氧化钠调节pH至7.2，转移至1000mL容量瓶中，加蒸馏水稀释至刻度，摇匀，待用。
3.2 150μmoL/L NBT溶液的配制：准确称取NBT 12.5mg置于100mL棕色容量瓶中，加入蒸馏水，超声使其溶解，并用蒸馏水定容至刻度，摇匀，即得。
3.3 60μmoL/L PMS溶液的配制：准确称取PMS 18.8mg，置于1000mL的容量瓶中，加入蒸馏水，超声使其溶解，并用蒸馏水定容至刻度，摇匀，即得。
3.4 468μmoL/L NADH溶液的配制：准确称取NADH 33.9mg，置于100mL的容量瓶中，加入蒸馏水，超声使其溶解，并用蒸馏水定容至刻度，摇匀，即得。
3.5供试品溶液的配制：配制1mg/mL的核桃低聚肽粉，待测。
3.6工作液的配制：取1mL 0.1moL/L PBS缓冲液(pH7.4)于容量瓶中，加入1mL 150μmoL/L NBT溶液，加入2mL 468μmoL/L NADH溶液，加入1mL 60μmoL/L PMS溶液，搅拌均匀，25℃下反应5min，与560nm波长处测定其吸光度值。
3.7操作步骤：准确吸取0.5mL供试品溶液和5mL上述工作溶液 混合均匀；准确吸取0.5mL供试品溶液和5mL蒸馏水混合均匀；准确吸取5mL上述工作溶液和0.5mL蒸馏水混合均匀，立即在560nm处测定吸光度，并根据以下公式计算自由基清除率：
IR％＝[1-(Ai-Aj)/A0]*100％；
其中，Ai表示待测溶液和SRSA混合后溶液的吸光度；
Aj表示待测溶液和溶剂混合后溶液的吸光度；
A0表示SRSA和溶剂混合后溶液的吸光度。
分别配制制备实施例1中的中性蛋白酶解产品浓度为100μg/mL，以维生素C为阳性对照(浓度为100μg/mL)，测试结果如表1所示：
表1 核桃低聚肽粉的抗氧化活性
Figure PCTCN2017000121-appb-000005
由表1可知，本发明方法所制备的核桃低聚肽粉对DPPH和ABTS自由基具有较强的清除活性，对超氧阴离子具有中等强度的清除活性，进而体现出较好的抗氧化活性。
生物活性实施例2：
PC12神经细胞保护模型：它是一种很好的研究神经细胞生理、病理及药理的模型，是研究帕金森症和神经疲劳的最常用的体外药物筛选模型。
1 PC12细胞的培养
使用含10％胎牛血清的高糖DMEM培养基培养PCl2细胞，细胞传代时用0.25％的胰酶消化约50s，用含10％血清的DMEM培养基终止消化，加入新鲜的培养基将细胞吹打均匀。以105个/mL的细胞密度传代。每瓶细胞加入4mL含细胞的培养液。在37℃，5％CO2条件下培养。
2 细胞接种
PCl2细胞在培养瓶中生长至融合状态，采用0.25％胰蛋白酶溶液消化，并反复吹打至细胞悬液，以含10％FBS的高糖DMEM培养基稀释成1.0×105个/mL，每孔100μL接种于96孔培养板中每组10个复孔，在37℃，5％CO2条件下培养24h，成融合状态。
3 药物对正常的PC12细胞生长活力的影响
96孔板于各孔中以一定浓度梯度分别给予核桃低聚肽100μL，培养24h后，MTT法检测细胞活力。50mg MTT溶解于10mL PBS，0.22μm微孔滤膜过滤。临用前稀释到0.5mg/mL。各组细胞弃去培养基，PBS洗两次，每加入0.5mg/mL MTT，37℃，5％CO2条件下孵育3h，除去MTT工作液，每孔加入150μL DMSO溶解结晶，振摇10min，测定每孔的OD值(测定波长570nm，参比波长650nm)。以对照组OD值平均值为100％细胞活力，计算模型组和给药组的细胞活力。测定结果如表1所示。
表2 核桃低聚肽粉对正常PC12细胞活力的影响
Figure PCTCN2017000121-appb-000006
4 药物对过氧化氢损伤的PC12细胞保护作用考察：(检测药物是否可以清除细胞中的自由基以促进细胞的生长)
A空白组(1％血清的DMEM)；
B模型组(1％血清DMEM培养6h，再加入H2O2使其终浓度为100μM，刺激12h)；
C阳性药(NAC)组：先加入含一定浓度阳性药NAC的1％血清DMEM培养6h，再加100μM H2O2刺激12h。
D给药组：先加入各浓度梯度的核桃低聚肽的1％血清的DMEM培养6h，再加100μM H2O2刺激12h。
上述各组于相同条件下培养，随后进行后续实验。于96孔板中MTT法检测细胞活力。测定结果如表3-5所示。
表3 核桃低聚肽粉对H2O2诱导的PC12细胞氧化损伤的影响
Figure PCTCN2017000121-appb-000007
由表2可知，本发明所制备的核桃低聚肽粉没有降低PCl2细胞的增殖活力，并且随着浓度的提高，PCl2细胞数量显著增加。由表3可以看出，模型组经H2O2刺激后，细胞活力为57.2％；当加入80μg/mL阳性对照药NAC培养后，细胞活力提高到88.0％，显示有明显的保护作用；而加入核桃低聚肽培养后，细胞活力随着肽粉浓度的增加而提高，呈现浓度依赖性，保护作用明显；在浓度为500μg/mL，细胞活力达到72.0％，与模型组相比，细胞活力提高了30％。由此可见此肽粉对神经元细胞具有较强的保护作用，因而可以用于预防或者治疗帕金森症、阿尔茨海默症等相关疾病，也可用作缓解大脑疲劳的药品或者保健食品。
生物活性实施例3：
核桃低聚肽粉对斑马鱼巨噬细胞抑制的改善作用
对受精后2天(2dpf)斑马鱼静脉注射长春瑞滨建立斑马鱼巨噬细胞抑制模型。分别溶于鱼水给予核桃低聚肽粉和阳性对照药小檗胺，低聚肽粉的浓度均为500μg/mL，小檗胺的浓度为1.9μg/mL；同时设置模型对照组以及正常对照组(不做任何处理)，每个实验组为30尾斑马鱼，置于28℃培养箱中培养。处理至3dpf时，对各个实验组斑马鱼进行中性红染色，染色4h后，每个实验组随机选择10尾斑马鱼在显微镜下进行观察、拍照并保存图片；利用图像处理软件进行图像分析，斑马鱼巨 噬细胞数量，分别定量评价核桃低聚肽粉对斑马鱼巨噬细胞抑制的改善作用。
表4.核桃低聚肽粉对马鱼头部巨噬细胞的改善作用(n＝10)
Figure PCTCN2017000121-appb-000008
由表4可知，正常对照组斑马鱼平均的巨噬细胞数量为27个，与模型对照组(15个)比较，说明斑马鱼巨噬细胞抑制模型建立成功。阳性药物小檗胺浓度为1.9μg/mL时，平均的巨噬细胞数量为20个，与模型对照组(15个)比较，对斑马鱼巨噬细胞抑制的改善作用为41.67％，说明小檗胺浓度为1.9μg/mL时，对斑马鱼巨噬细胞抑制有显著的改善作用。而核桃低聚肽粉在浓度为500μg/mL时，平均的巨噬细胞数量为25个，与模型对照组(15个)比较，对斑马鱼巨噬细胞抑制的改善作用为83.33％，说明本发明制备的核桃低聚肽粉对斑马鱼巨噬细胞抑制有显著的改善作用。
2核桃低聚肽粉对斑马鱼巨噬细胞吞噬功能的促进作用
对受精后2天(2dpf)斑马鱼静脉注射墨汁建立斑马鱼巨噬细胞促进模型。分别溶于鱼水给予核桃低聚肽和阳性药匹多莫德，低聚肽的浓度为2000μg/mL，匹多莫德的浓度为200μg/mL；同时设置模型对照组，每个实验组为30尾斑马鱼，置于28℃培养箱中培养。处理至3dpf时，对各个实验组斑马鱼进行中性红染色，染色4h后，每个实验组随机选择10尾斑马鱼在显微镜下进行观察、拍照并保存图片；利用图像处理软件进行图像分析斑马鱼巨噬细胞中的墨汁信号(N)，分别定量评价4种供试品对斑马鱼巨噬细胞吞噬功能的促进作用。
表5 核桃低聚肽粉对斑马鱼头部吞噬墨汁的巨噬细胞的促进作用(n＝10)
Figure PCTCN2017000121-appb-000009
由表5可知，阳性药物匹多莫德浓度为200μg/mL时，平均的已吞噬墨汁的巨噬细胞数量为3.5个，与模型对照组(1.5个)比较，对斑马鱼巨噬细胞吞噬的促进作用为2.3倍，说明匹多莫德浓度为200μg/mL时对斑马鱼巨噬细胞的吞噬功能有显著的促进作用。核桃低聚肽浓度为2000μg/mL时，平均的已吞噬墨汁的巨噬细胞数量为5.1个，与模型对照组(1.5个)比较，对斑马鱼巨噬细胞吞噬的促进作用为3.4倍，说明核桃低聚肽粉对斑马鱼巨噬细胞的吞噬功能有显著的促进作用。
生物活性实施例4：
核桃低聚肽粉对斑马鱼中枢神经保护作用
随机选取180尾受精后16天(1dpf)野生型AB品系斑马鱼于六孔板中，每孔(实验组)均处理30尾斑马鱼，用吗替麦考酚酯诱发斑马鱼中枢神经损伤。分别水溶给予低聚肽粉浓度为222和667μg/mL时，阳性对照药谷胱甘肽(GSH)154μg/mL浓度，同时设置正常对照组(养鱼用水处理斑马鱼)和模型对照组，每孔(实验组)容量为3mL。低聚肽分别与吗替麦考酚酯共同处理24h后，用吖啶橙进行染色，染色后每个实验组随机选取10尾斑马鱼在荧光显微镜下拍照并采集数据，分析统计斑马鱼中枢神经(脑和脊髓)凋亡细胞荧光强度，根据荧光强度评价核桃低聚肽粉对斑马鱼中枢神经的保护作用。
表6 核桃低聚肽粉对斑马鱼中枢神经保护作用(n＝10)
Figure PCTCN2017000121-appb-000010
由表6可知，模型对照组斑马鱼中枢神经凋亡细胞荧光强度(565783像素)与正常对照组(161976像素)比较，表明模型建立成功；阳性对照药GSH 154μg/mL浓度组斑马鱼中枢神经凋亡细胞荧光强度为193900像素，与模型对照组比较，其对斑马鱼中枢神经保护作用为92％，说明GSH对斑马鱼中枢神经有明显的保护作用。低聚肽222和667μg/mL浓度组斑马鱼中枢神经凋亡细胞荧光强度分别为395025和 451259像素，中枢神经保护作用分别为42％和28％，与模型对照组比较，表明核桃低聚肽对斑马鱼中枢神经有明显的保护作用。
生物活性实施例5：
1核桃低聚肽对斑马鱼运动能力的改善作用
随机选取4dpf野生型AB品系斑马鱼于六孔板中，每孔(即每个供试品组)30尾，分别水溶给予核桃低聚肽，阳性对照药中华跌打丸1.0mg/mL浓度，同时设置正常对照组和模型对照组，每孔容量为3mL。供试品预处理一段时间后，除正常对照组外，其余实验组均同时水溶给予亚硫酸钠以诱发斑马鱼疲劳模型。供试品和亚硫酸钠共同处理斑马鱼一段时间后，每个实验组随机选取10尾斑马鱼，利用行为分析，测定斑马鱼的运动总距离(S)，定量评价供试品对亚硫酸钠诱发的疲劳斑马鱼的运动改善作用。
2核桃低聚肽对斑马鱼体内乳酸代谢的影响
随机选取4dpf野生型AB品系斑马鱼于六孔板中，每孔(即每个供试品组)30尾，分别水溶给予核桃低聚肽，阳性对照药中华跌打丸1.0mg/mL浓度，同时设置正常对照组和模型对照组，每孔容量为3mL；每个实验组设置3个平行。供试品预处理一段时间后，除正常对照组外，其余实验组均同时水溶给予亚硫酸钠以诱发斑马鱼疲劳模型。供试品和亚硫酸钠共同处理斑马鱼一段时间后，每个实验组将3个平行实验组中的斑马鱼汇集在一起(共90尾)，利用NanoDrop2000超微量分光光度计间接测定斑马鱼体内乳酸含量，分别定量评价核桃低聚肽在2000μg/mL浓度下对亚硫酸钠诱发的疲劳斑马鱼体内的乳酸含量的影响。
表7 核桃多肽对斑马鱼运动能力的改善作用
Figure PCTCN2017000121-appb-000011
表8 核桃多肽对斑马鱼体内乳酸含量的影响
Figure PCTCN2017000121-appb-000012
Figure PCTCN2017000121-appb-000013
由表7和表8可知，本发明制备的核桃低聚肽粉可明显改善斑马鱼运动能力，提高体内乳酸代谢。由此可见核桃低聚粉具有明显的抗疲劳功效，可以用于预防或缓解疲劳的食品，保健品或药品。
生物活性实施例6：
核桃低聚肽对Aβ1-42淀粉样蛋白聚集的抑制作用
将Aβ1-42淀粉样蛋白以DMSO配置为2.5mg/mL，并将核桃低聚肽粉稀释至适当的浓度。再将Aβ1-42淀粉样蛋白液(1μL)与核桃低聚肽溶液(9μL)混合，使Aβ1-42淀粉样蛋白终浓度为0.25mg/mL，核桃低聚肽终浓度分别为10和100μg/mL。混合液置于37℃反应30分钟后，加入200μL Thioflavin T工作液充分混合，移取200μL置于透明底黑色的96孔板并从盘子底部测量ThT荧光强度(Ex440/Em482)以判定Aβ1-42淀粉样蛋白的聚合程度，未添加任何药物的为阴性对照组。
表9 核桃低聚肽对Aβ1-42淀粉样蛋白聚集的抑制作用
Figure PCTCN2017000121-appb-000014
由表9可知，核桃低聚肽在浓度为10μg/mL时，抗Aβ1-42淀粉样蛋白的聚集作用不明显，而在浓度为200μg/mL时，对Aβ1-42淀粉样蛋白的聚集具有一定的抑制作用，可推测核桃低聚肽对脑神经有保护作用，有潜在的改善记忆力的功效。
生物活性实施例7：
核桃低聚肽对细胞内炎症免疫因子的影响
将10μg/mL anti-CD3包被于6孔板(200μL/孔)，4℃静置18～24小时。活化HPBMC，使用细胞悬浮液稀释至5×105cells/mL，并使终体积为36mL，其中含anti-CD28(2μg/mL)，rhIL-2(10ng/mL)，rhIL-4(50ng/mL)。使用培养基冲洗已经包被anti-CD3的6孔板，并将HPBMC稀释液移至此6孔板，于二氧化碳培养箱内培养。2天后，收集此6孔板的HPBMC稀释液并离心，移除上清液，再加入含有rhIL-2(10ng/mL)和rhIL-4(50ng/mL)培养基打散混匀，使细胞中浓度为5×105cells/mL，再转移至细胞培养瓶，再于二氧化碳培养箱内进行培养。2天后，收集HPBMC，离心去上清液，使用培养基清洗后，再次离心去除上清液。 加入含5ng/mL PMA的培养基打散混匀，使细胞浓度为5×105cells/mL，终体积为36mL，并置于二氧化碳培养箱。4小时后，离心，上清液进行CBA分析。操作方法按照BD CBA Human Th1/Th2/Th17Cytokine Kit Instruction Manual说明书进行，分析IL-10和IL-17A的变化。
表10 核桃低聚肽对细胞内IL-10和IL-17A因子的影响(n＝3)
Figure PCTCN2017000121-appb-000015
由表10可知，核桃低聚肽明显可以下调IL-10的含量，提高IL-17A的含量。可见核桃低聚肽可以调节炎症因子，提高机体免疫力。
生物活性实施例8：
1核桃低聚肽对斑马鱼胚胎神经突起生长的促进作用
利用神经元特异性HuC启动子，使用绿色萤光融合蛋白(GFP)为检测信号。质粒pHuC-GFP的绿色荧光蛋白首先被注射到斑马鱼胚胎的发育为1-细胞期的细胞内。将核桃低聚肽粉(制备实施例1)用DMSO溶解，并用水稀释至一定浓度。8小时后，将其以及DMSO(对照阴性)注射至斑马鱼胚胎细胞内。40小时后，根据斑马鱼神经突起生长的数量，来判断样品是否在斑马鱼胚胎中有利于神经突起的生长。
实验结果表明：对照阴性DMSO组，斑马鱼胚胎神经突起生长的促进率仅为23％，而注射浓度为1mg/mL核桃低聚肽后，神经突起生长的促进率可达51％。可见核桃低聚肽能明显促进斑马鱼胚胎神经突起的生长。
2核桃低聚肽降低人野生型tau蛋白对斑马鱼胚胎内神经细胞的毒性作用
利用神经组织特异性Huc启动子，使用hTau绿色荧光融合蛋白(GFP)检测野生型人tau蛋白诱导的斑马鱼胚胎内神经细胞凋亡情况。表达构建体被注射到斑马鱼胚胎的发育为1-细胞期的细胞内。将核桃低聚肽粉(制备实施例1)用DMSO溶解，并用水稀释至一定浓度。8小时后，将其以及DMSO(对照阴性)注射至斑马鱼胚胎细胞内。使用萤光显微镜观察24和48hpf GFP标记的细胞的情况。在斑马鱼24hpf的神经元细胞中，部分GFP信号可以观察到，而在斑马鱼48hpf的神经元细胞中 发现，部分已经裂解，甚至消失。计算斑马鱼48hpf3至5个GFP神经元细胞的百分量。
实验结果表明：对照阴性DMSO组，仅有17.2％的GFP神经元细胞，而核桃低聚肽以浓度为1mg/mL注射后有高达37.6％的GFP神经元细胞。可见核桃低聚肽明显降低由人野生型tau蛋白对斑马鱼胚胎内神经细胞的毒性。
总之，由以上两种生物活性模型可知，核桃低聚肽具有促进或者改善记忆力的功效。
生物活性实施例9：
核桃低聚肽对化学药物致小鼠学习记忆障碍的作用
1供试动物
供试动物清洁级ICR小鼠，体重18-22g，三批共300只，由南通大学提供，实验动物生产许可证：SCXK(苏)2014-0001。常规鼠饲料喂养，自由饮水。实验前于安静环境中适应性饲养1周，自由摄食饮水，保持室温(22±1℃)，自然昼夜节律光照。
2实验方法
2.1对东莨菪碱所致小鼠记忆获得障碍的影响
小鼠随机分组，设核桃低聚肽(制备实施例1)低、中、高剂量组(30、100、300mg/kg)，空白组与模型组分别给予等容积蒸馏水，阳性对照组给予尼莫地平30mg/kg，各组每天分别灌胃1次，连续给药7d。
跳台训练。将XT-911型小鼠跳台条件反射测试仪电压控制在36V，于末次给药1小时后进行小鼠学习记忆功能的测试。训练前10分钟模型组与给药组分别腹腔注射氢溴酸东莨菪碱3mg/kg，空白对照组腹腔注射等容量生理盐水。每批分别有5只小鼠给药，平行操作，10分钟后再给第2批小鼠注射，以此类推。训练时将每批5只小鼠分别放入跳台仪的5个格子中，先适应环境3分钟，然后通电，小鼠受电击后，多数跳上跳台，逃避电击。跳下时以小鼠双足同时接触铜栅为触电，视为错误反应，训练5分钟，24小时后重新测试。
测试时先将小鼠放在跳台上，同时开始计时，记录小鼠第一次跳下时间，此为触电潜伏期(即为错误潜伏期)，并记录5分钟内跳下次数(即为错误次数)，作为观察指标。
2.2对亚硝酸钠所致小鼠记忆巩固障碍的影响
分组、给药和训练方法同实验2.1。训练结束后除空白对照组皮下注射等量生理盐水，其余各组立即皮下注射亚硝酸钠90mg/kg，24小时后进行测试。测试方法亦同实验2.1。
2.3对40％乙醇所致小鼠记忆再现障碍影响
分组、给药和训练方法同实验2.1。测试前30分钟，模型组与给药组分别灌胃40％乙醇10mL/kg，空白对照组给予等容积蒸馏水。测试方法亦同实验2.1。
3实验结果
3.1核桃低聚肽对东莨菪碱所致小鼠记忆获得障碍的影响
连续给予7d制备实施例1(30、100、300mg/kg)，在东莨菪碱所致小鼠记忆获得障碍小鼠，在小鼠跳台记忆成绩中，制备实施例1中、高剂量组均能明显延长潜伏期，减少错误次数。结果见表11。
表11 核桃低聚肽对东莨菪碱所致小鼠记忆获得障碍的影响(n＝10)
Figure PCTCN2017000121-appb-000016
注：#P＜0.05，##P＜0.01，与空白组比较；*P＜0.05，**P＜0.01，与模型组比较
3.2核桃低聚肽对亚硝酸钠所致小鼠记忆巩固障碍的影响
连续给予7d制备实施例1(30、100、300mg/kg)，在亚硝酸钠所致小鼠记忆巩固障碍中，在小鼠跳台记忆成绩中，制备实施例1各剂量组均能明显延长潜伏期，减少错误次数。结果见表12。
表12 核桃低聚肽对亚硝酸钠所致小鼠记忆巩固障碍的影响(n＝10)
Figure PCTCN2017000121-appb-000017
Figure PCTCN2017000121-appb-000018
注：##P＜0.01，与空白组比较；**P＜0.01，与模型组比较
3.3核桃低聚肽对40％乙醇所致小鼠记忆再现障碍的影响
连续给予7d制备实施例1(30、100、300mg/kg)，在乙醇所致小鼠记忆再现障碍中，在小鼠跳台记忆成绩中，制备实施例1各剂量组均能明显延长潜伏期，减少错误次数。结果见表13。
表13 核桃低聚肽对40％乙醇所致小鼠记忆再现障碍的影响(n＝8)
Figure PCTCN2017000121-appb-000019
注：##P＜0.01，与空白组比较；*P＜0.05，**P＜0.01，与模型组比较
学习、记忆功能包括空间学习记忆功能和非空间学习记忆功能。记忆障碍模型是评价药物对记忆过程影响的有效手段，也是探讨药物治疗老年痴呆作用及作用机制常采用的模型。东莨菪碱为M受体阻断剂，能阻断乙酰胆碱对M受体的激动作用，可模拟乙酰胆碱不足而导致的学习记忆功能障碍。亚硝酸钠可使血红蛋白变性，使脑组织缺血缺氧，损害学习和记忆过程。乙醇抑制大脑皮质的神经功能活动，抑制动物的条件反射过程，使脑内蛋白质和RNA合成受阻，胆碱能和多巴胺等系统发生改变，破坏学习记忆功能造成学习记忆再现障碍。在本实验所选模型及剂量范围内，核桃低聚肽中、高组均能不同程度改善东莨菪碱所致学习记忆获得障碍模型小鼠的潜伏期，高剂量组可减少跳下平台错误次数；对亚硝酸钠所致记忆巩固障碍模型，核桃低聚肽各剂量组均可延长潜伏期，减少错误次数。对40％乙醇所致小鼠记忆获得障碍小鼠跳台实验中，核桃低聚肽各剂量组也能明显减少错误次数，延长潜伏期。由此可知，核桃低聚肽中、高剂量组对东莨菪碱导致的学习记忆获得障碍小 鼠学习记忆能力具有明显改善作用，其低、中、高剂量组对40％乙醇所致小鼠学习记忆再现障碍模型和亚硝酸钠所致小鼠学习记忆巩固障碍模型小鼠学习记忆能力具有明显改善作用。

Claims (8)

  1. 一种核桃低聚肽粉，其特征在于：采用GB/T22492-2008附录A与附录B的检测方法，测得肽含量在80wt％以上，其中95％以上核桃肽的分子量小于1500Dalton，其分子量分布如下：
    分子量Dalton分布
    Figure PCTCN2017000121-appb-100001
    优选地，肽含量为81wt％以上，小于1500Dalton分子量占97％以上；更优选，肽含量为81.3wt％以上，小于1500Dalton分子量占96％以上；再优选，肽含量为85wt％以上，小于1500Dalton分子量占97％以上；最优选，肽含量为85wt％以上，小于1500Dalton分子量占96％以上。
  2. 权利要求1所述核桃低聚肽粉的制备方法，其特征包括下述步骤：将冷榨脱脂后的核桃粕，使用高效逆流提取法提取蛋白质，过滤、酶解，再将蛋白酶解液依次经过微滤膜与超滤膜高效分离纯化，最后浓缩喷雾，得到核桃低聚肽粉。
  3. 根据权利要求2所述的制备方法，其特征在于包括下述步骤：
    核桃粕的预处理：将核桃去壳、冷榨脱油，得到脱脂核桃粕；
    高效逆流提取法提取蛋白质：将一定量脱脂后的核桃粕，记录为A，与水按重量比为1∶5～1∶15混合，调节pH至9～11于室温提取1～2h；提 取完成后，过滤，滤渣进行二次提取，滤液倒入等量的核桃粕，记录为B，调节pH至9～11于室温提取1～2h；B完成第一次提取后，滤液待用，滤渣继续进行第二次提取；A完成二次提取后，滤渣弃去，滤液倒入等量的核桃粕，记录为C，调节pH至9～11于室温提取1～2h；B完成二次提取后，滤渣弃去，滤液倒入C完成第一次提取的滤渣中提取1～2h，而C第一次提取的滤液待用；样品C完成第二次提取后，滤渣弃去，滤液待用；最后合并以上待用滤液，调节pH为3～5，静置0.5～2h，弃去上清液，最后往沉淀物中加入体积比为1∶10～1∶20的水，搅拌均匀，得到核桃蛋白液；
    蛋白酶解：将上述核桃蛋白液加热至40～55℃，将pH值调节至中性，加入核桃粕重量0.5～2％的生物酶，搅拌酶解3～6h后，煮沸灭活30min，离心，上清液即为蛋白酶解液。
    分离纯化：将蛋白酶解液使用孔径为0.1～0.5μm的微滤膜进行过滤，透过液再经2000～20000Dalton超滤膜处理后，截留液在50～80℃下浓缩至固含为3～5wt％时，进行喷雾干燥，进口温度为140～160℃，出口温度为55～65℃，得到核桃低聚肽粉，产率为20～30wt％。
  4. 根据权利要求3所述的制备方法，其特征在于：所述生物酶选自食品级的中性蛋白酶(酶活力≥30万u/g)、木瓜蛋白酶(酶活力≥40万u/g)、菠萝蛋白酶(酶活力≥30万u/g)、碱性蛋白酶(酶活力≥20万u/g)、胃蛋白酶(酶活力≥50万u/g)、胰酶(酶活力≥3000u/g)中的一种或者它们的混合物，优选地使用中性蛋白酶或复合酶，所述复合酶的中性蛋白酶与木瓜蛋白酶的质量比为1∶1，中性蛋白酶的活力为30万u/g，木瓜蛋白酶的活力为50万u/g。
  5. 一种组合物，其特征在于：含有权利要求1所述的核桃低聚肽粉和药物、食品、保健品或化妆品上可接受的助剂。
  6. 根据权利要求5所述的组合物，其特征在于：其剂型选自素片、 薄膜包衣片、糖衣片、肠衣片、分散片、胶囊、颗粒剂、口服溶液或口服混悬液，以及液体、乳液、膏霜、粉、块状等化妆品剂型。
  7. 权利要求1所述的核桃低聚肽粉用于制备治疗或预防自由基过多引起的症状的药物、食品、保健品或化妆品的应用；用于制备改善或治疗记忆力衰退的药物、食品、保健品或化妆品的应用；用于制备治疗或预防帕金森症和阿尔茨海默症、缓解大脑或者运动疲劳的药物、食品、保健品或化妆品的应用；用于制备增强免疫力的药物、食品、保健品或化妆品的应用；优选用于制备改善或治疗记忆力衰退的药物、食品、保健品或化妆品的应用。
  8. 权利要求1所述的核桃低聚肽粉用于制备对中枢神经保护的药物、食品或保健品的应用；用于制备对运动能力的改善作用的药物、食品、保健品或化妆品的应用；用于制备对体内乳酸代谢的药物、食品、保健品或化妆品的应用；用于制备对Aβ1-42淀粉样蛋白聚集的药物、食品、保健品或化妆品的应用；用于制备调节细胞内炎症免疫因子的药物、食品、保健品或化妆品的应用；用于制备胚胎神经突起生长的药物、食品、保健品或化妆品的应用；用于制备降低人野生型tau蛋白对胚胎内神经细胞的毒性的药物、食品、保健品或化妆品的应用。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109589400A (zh) * 2018-12-30 2019-04-09 杏辉天力（杭州）药业有限公司 一种具有神经保护功效的组合物
CN110915926A (zh) * 2019-11-22 2020-03-27 河北绿蕾农林科技有限公司 一种复合核桃蛋白粉及其制备方法
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CN114044811B (zh) * 2022-01-13 2022-04-12 中食都庆（山东）生物技术有限公司 一种核桃肽及其制备方法和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101228918A (zh) * 2007-01-24 2008-07-30 西北农林科技大学 一种核桃多肽粉的制作方法
CN104293870A (zh) * 2014-09-25 2015-01-21 汾州裕源土特产品有限公司 一种抗氧化核桃多肽保健品的制备方法
CN105567774A (zh) * 2016-01-22 2016-05-11 杏辉天力（杭州）药业有限公司 一种核桃低聚肽粉及其制备方法和用途

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1329411C (zh) 2002-08-05 2007-08-01 不二制油株式会社 大豆蛋白的制造方法
US7354616B2 (en) 2003-11-25 2008-04-08 Solae, Llc Modified oilseed material with a high gel strength
JPWO2005120244A1 (ja) 2004-06-07 2008-04-03 不二製油株式会社 大豆蛋白加水分解物の製造法
CN101280327B (zh) * 2008-05-26 2012-03-21 昆明理工大学 核桃油和核桃蛋白肽的同步水酶超声提取法
CN102406050B (zh) * 2011-11-17 2013-08-07 中国农业大学 一种核桃小分子多肽及其制备方法
CN103103244B (zh) * 2013-02-22 2014-12-03 北京市农林科学院 核桃降血压活性肽及其制备方法与应用
CN103109971B (zh) * 2013-03-04 2014-09-10 四川省均易润泽食品有限公司 制备高精氨酸核桃肽的方法
CN103125735A (zh) * 2013-03-11 2013-06-05 武汉工业学院 一种核桃多肽的制备方法
CN103497985B (zh) 2013-09-06 2015-10-28 华南理工大学 核桃粕抗氧化肽及其制备方法
CN103484518B (zh) * 2013-09-11 2016-03-30 邹远东 一种核桃蛋白肽及其制备方法和其应用
CN104031968B (zh) 2014-06-27 2016-10-19 广西壮族自治区中国科学院广西植物研究所 核桃多肽的应用
CN105002247A (zh) * 2015-07-30 2015-10-28 北京天肽生物科技有限公司 一种小分子核桃肽及其制备方法
CN105936927B (zh) * 2016-05-19 2020-02-14 杏辉天力（杭州）药业有限公司 一种核桃低聚肽及其制备工艺和用途
CN106520872A (zh) * 2016-10-13 2017-03-22 杏辉天力（杭州）药业有限公司 一种具有抗疲劳功效的核桃低聚多肽

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101228918A (zh) * 2007-01-24 2008-07-30 西北农林科技大学 一种核桃多肽粉的制作方法
CN104293870A (zh) * 2014-09-25 2015-01-21 汾州裕源土特产品有限公司 一种抗氧化核桃多肽保健品的制备方法
CN105567774A (zh) * 2016-01-22 2016-05-11 杏辉天力（杭州）药业有限公司 一种核桃低聚肽粉及其制备方法和用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GAO, JIANPING ET AL.: "Extraction of Soybean Protein Isolate Using Multi-stage Countercurrent Solid-Liquid Extraction Method", THE CHINESE JOURNAL OF PROCESS ENGINEERING, vol. 11, no. 2, 15 April 2011 (2011-04-15), pages 311 - 317, XP009512091, ISSN: 1009-606X *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109589400A (zh) * 2018-12-30 2019-04-09 杏辉天力（杭州）药业有限公司 一种具有神经保护功效的组合物
CN110915926A (zh) * 2019-11-22 2020-03-27 河北绿蕾农林科技有限公司 一种复合核桃蛋白粉及其制备方法
CN114751957A (zh) * 2022-03-15 2022-07-15 吉林农业大学 一种高抗氧化可穿透血脑屏障的改善记忆力衍生肽及其应用
CN114751957B (zh) * 2022-03-15 2024-02-02 吉林农业大学 一种高抗氧化可穿透血脑屏障的改善记忆力衍生肽及其应用

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