CN105063152A - Polypeptide raw material prepared by enzymolysis of walnuts and application thereof - Google Patents
Polypeptide raw material prepared by enzymolysis of walnuts and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biological medicines and particularly relates to a polypeptide raw material prepared by enzymolysis of walnuts and an application thereof. The polypeptide raw material is characterized in that the combination of an alkaline-dissolving and acid-precipitating technology and a biological enzymolysis technology is adopted for extracting protein in the walnuts with high efficiency and degrading polysaccharide in alkaline-dissolving matters; multienzyme synergetic hydrolysis is adopted, the content of target amino acid with the function of improving the memory, such as serine and lysine and the like, in the polypeptide raw material is enriched. The polypeptide raw material and the application have the advantages that two-stage ultrafiltration membranes are adopted for separating walnut protein hydrolysates, on one hand, the polypeptide can be separated with high efficiency, the polypeptide with the molecular weight being between 1000-10000Da is enriched, simultaneously the purpose of concentrating the target polypeptide is also achieved, and the flow of the production process is simplified; the process operation is simple, the production cost is low, no any pollution is caused, the protein recovery rate is high, and the prepared polypeptide raw material is strong in activity, has the function of improving the memory action and can be widely applied in the field of foods.
Description
Technical field
The present invention relates to biomedicine technical field, particularly a kind of walnut is through the polypeptide raw material of enzyme-squash techniqued and application thereof.
Background technology
The reason of memory decay is caused to have three kinds: the aging memory decay caused, the amnesia that pressure etc. cause, the memory impairment that congenital or physical abuse causes.Nowadays, social pressures are increasing, and amnesia day of people is aobvious outstanding, make more researcher turn one's attention to improvement aspect to memory.
In present stage research, Improving memory is placed on extraction and the research of active substance in herbal medicine by most scholar, and this method relates to the residual of complex process and organic reagent, easily introduces safety problem.Another study hotspot is exactly Yelkin TTS and polyunsaturated fatty acid fish oil etc., and these raw material of substance source difficulty, cost intensive, limits its suitability for industrialized production and popularization.In recent years, the demand of people to heath food is more urgent, researcher is made to turn one's attention to food endogenous binding protein peptide in having physiological function, pharmaceutical factory as large in Austrian Yi Biwei with pig brain for raw material, combined acid hydrolysis and enzymic hydrolysis prepare mixture---the cerebrolysin of 85% amino acid and 15% small peptide, use it in clinical treatment senile dementia, there is obvious result for the treatment of.But cerebrolysin is a kind of injectable drug, expensive and use inconvenient, make it not be suitable for the demand of general population to memory improvement.Therefore, urgently to be resolved hurrily is seek one at present, safety, generally, is efficiently easy to the Improving memory peptide prepared.
Biologically active peptides be in protein 20 kinds of natural amino acids with difference composition and arrangement mode formed linearly or the general name of cyclic peptide class, be the functional compound coming from protein.Cytodifferentiation in vital movement, the adjustment of neurohormone mediator, immunomodulatory etc. all have certain dependency with biologically active peptides.Nowadays, aspect widely, such as anti-oxidation peptide, antibacterial peptide, immune peptide, blood pressure lowering peptide etc. relate to the research of biologically active peptides.Preparation method's mainly proteolysis method of current biologically active peptides, be divided into chemical hydrolysis and biological enzyme hydrolysis, biologic enzymolysis method is because having working condition gentleness, hydrolytic process is controlled, the advantage that peptide yield is high and safe, instead of traditional chemical method gradually, become the common method preparing biologically active peptides.
Walnut (JuglansregiaL) has another name called English walnut, Qiang peach, and belonging to Juglandaceae walnut, is the precious fruit tree that a kind of nutritive value and economic worth are all very high.As a kind of economic tree, walnut is long at the cultivation history of China, and distribution is relatively extensive, especially in Yunnan, Sichuan, the southwest such as Xinjiang and the Northwest's plantation more.Walnut is also a kind of high energy food, and every 100 grams of walnuts can provide 631kcal energy, and its protein and lipid account for whole walnut kernel weight more than 84%, and wherein lipid accounts for about 60%.Walnut protein is made up of four kinds of protein, is respectively white protein, sphaeroprotein, and prolamine and gluten account for 6.81%, 17.57%, 5.33% and 70.11% of walnut protein total amount respectively.Research shows, vegetable-protein is aboundresources not only, cheap, and compared with animal proteinum, amino acid composition balances more, and essential amino acids content is higher, and not containing cholesterol.Containing 18 seed amino acids in walnut, wherein have 8 kinds of indispensable amino acids, arginine and content of glutamic acid are all quite high, are a kind of protein resources of high-quality.In addition, in all benefit brain food, walnut is exactly the best tonic of " brain tonic and intelligence development " since ancient times, but what people utilized is walnut oil always, does that walnut protein also have the effect of intelligence development? there are some researches show that walnut protein is also a kind of quality protein raw material preparing Improving memory, can strengthen brain activity, memory, control that is overstrain one's nerves to teenager, person in middle and old age's amnesia has good effect.But owing to containing a large amount of Mierocrystalline celluloses in the dregs of rice, and in the process extracting grease, cause certain protein denaturation, greatly add the technology difficulty utilizing walnut protein to prepare Improving memory peptide.
Therefore, take walnut dregs as raw material, the protein in high efficiency extraction walnut, then extraction and the preparation of carrying out Improving memory peptide, have larger economy and social effect.
Summary of the invention
In view of this, the invention provides a kind of walnut through the polypeptide raw material of enzyme-squash techniqued and application thereof.The walnut protein peptide of Improving memory effect that what the present invention obtained have may be used for preparing Improving memory healthcare products and medicine.Can be used alone or with other combined Chinese herbs with Improving memory effect conveniently preparation process make protective foods.
In order to realize foregoing invention object, the invention provides following technical scheme:
The invention provides a peptide species, it is prepared by following steps:
Step 1: get walnut mix with water after grind, through the first acid-base modifier adjusted to ph to 8.0 ~ 9.0, stirring, centrifugal, collecting by filtration filtrate; Get described filtrate mix with lytic enzyme after enzymolysis, through the second acid-base modifier adjusted to ph to 4.0 ~ 4.2, stir, centrifugal, collecting by filtration filter residue, through alcohol precipitation, washing, obtain walnut protein;
Step 2: get the described walnut protein that step 1 prepares and mix with water, through Alcalase2.4L proteolytic enzyme and compound protease, under the condition of 50-55 DEG C, enzymolysis is to degree of hydrolysis 8-12%, and go out enzyme, centrifugal, collecting by filtration filtrate, obtains walnut protein enzymolysis solution;
Step 3: get the described walnut protein enzymolysis solution that step 2 prepares and sieve, collect permeate after the ultrafiltration membrance filter of 10000Da; By described permeate again through the ultrafiltration membrance filter of 1000Da, collect trapped fluid, obtain the walnut peptide of molecular weight at 1000-10000Da.
In specific embodiments more of the present invention, described walnut can be the walnut dregs that can obtain for walnut fruit or walnut fruit.
In specific embodiments more of the present invention, in preparation method's step 1 of polypeptide, lytic enzyme comprises α-amylase and/or cellulase.
In specific embodiments more of the present invention, the quality of the described walnut protein prepared with step 1 is for Calculation Basis, and the add-on of Alcalase2.4L proteolytic enzyme is 2000-3000U/g albumen, and the add-on of compound protease is 1200-2500U/g albumen.
In specific embodiments more of the present invention, the addition of α-amylase described in preparation method's step 1 of polypeptide is 1% ~ 2% (w/w) of walnut protein quality, and the addition of described cellulase is 0.5% ~ 1.0% (w/w) of walnut quality.
In specific embodiments more of the present invention, described in preparation method's step 1 of polypeptide, the condition of enzymolysis for stir 120-180min at 50-55 DEG C.
In specific embodiments more of the present invention, the aqueous ethanolic solution of the alcohol that alcohol precipitation described in preparation method's step 1 of polypeptide adopts to be volumetric concentration be 45-60%.
In specific embodiments more of the present invention, described stirring after adding the first acid-base modifier in preparation method's step 1 of polypeptide, centrifugal being specially stir 60-90min at 40-50 DEG C, then centrifugal 10min under 3500r/min.
In specific embodiments more of the present invention, in preparation method's step 1 of polypeptide, add the and described stirring after acid-base modifier, centrifugal being specially stir 30-60min, then centrifugal 10min under 3500r/min.
In specific embodiments more of the present invention, described in preparation method's step 1 of polypeptide, the first acid-base modifier is NaOH solution, and described second acid-base modifier is HCl solution.
Preferably, the first acid-base modifier is the NaOH solution of 1mol/L.
Preferably, the second acid-base modifier is the HCl solution of 1mol/L.
In specific embodiments more of the present invention, the enzyme that goes out described in step 2 is specially and goes out enzyme at 100 DEG C of heating 15min.
In specific embodiments more of the present invention, in preparation method's step 1 of polypeptide, the add-on of water is 10 ~ 15 times of walnut quality; In step 2, the add-on of water is 7 ~ 10 times of walnut protein quality; Vacuum concentration is also comprised to solid substance 30-40%, spray-dired step after collecting trapped fluid described in step 3.
Present invention also offers described polypeptide and prepare the application in medicine, food and/or the healthcare products preventing and/or treating memory decay.
In specific embodiments more of the present invention, the formulation of medicine, food and/or healthcare products described in the application of polypeptide is oral liquid, capsule, tablet, pulvis or granule.
The present invention has following advantage and effect relative to prior art:
(1) the present invention adopts alkali extraction and acid precipitation combine with technique biological enzymolysis technology, the polysaccharide in degraded alkali-soluble substance, the protein of release polysaccharide combination on the one hand, and the viscosity on the other hand in reduction system improves the extraction yield of the heavy albumen of acid; Utilize ethanolic soln to clean the heavy albumen of acid, the effect except oil decolorization can be reached, and appropriate sex change can be carried out to protein, be conducive to the carrying out of later stage enzymolysis.
(2) the present invention adopts multienzyme synergism to be hydrolyzed, enrichment is improved the Serine of effect to memory, the amino acid such as Methionin, improves the desired amino acid content in finished product polypeptide raw material, in addition walnut protein Glutamic Acid content is higher, can play the effect of content of glutamic acid in stable brain.
(3) the present invention adopts two-stage Ultra filtration membrane walnut protein enzymolysis product, on the one hand can high efficiency separation peptide, and the peptide of enrichment molecular weight between 1000-10000Da, also reaches the object of concentrated target peptide simultaneously, simplify the flow process of production technique.
(4) present invention process is simple to operate, production cost is low, without any pollution, protein recovery is high, and the memory improvement of gained Improving memory peptide is active strong, can be widely used in field of food.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below.
The peptide molecular weight distribution color atlas of Fig. 1 walnut protein peptide;
Fig. 2 shows Morris water maze schematic diagram.
Embodiment
The invention discloses a peptide species and application thereof, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
In the embodiment of the present invention, the measuring method of protein recovery and peptide content is as follows:
Protein content adopts Kjeldahl nitrogen determination/GB5009.5-85, protein recovery calculation formula:
The mensuration of peptide content adopts the method in soy peptide powder/GB/T22492-2008, nitrogen content calculation formula:
In the present invention, the measuring method of the peptide molecular weight distribution of walnut protein peptide is as follows:
Adopt gel chromatography.Chromatographic condition is as follows: Amersham protein analysis purification system, Superdex_peptide_10/300_GL glass column (Vt=24.0mL, V0=8.0mL), elutriant is the phosphate buffered saline buffer (pH7.2) of 0.25MNaCl, sampling volume is 40 μ L, flow velocity 0.5mL/min, monitoring wavelength is 214nm.Then according to the molecular weight distribution situation going out cutting edge of a knife or a sword Time Calculation protein peptide sample of standard substance.
Walnut protein peptide of the present invention and PC12 cell H2O2 injury protection experimental technique as follows:
PC12 cell is that PC12 cells strain is bought by Shanghai cell bank.Adopt Dulbecco ' sModifiedEagleMedium culture medium culturing, wherein add 10% foetal calf serum (FBS), the penicillin of 1%.Culture environment is 5%CO237 DEG C.Go down to posterity after three times, start to carry out Hydroperoxide injury test.Adopt 96 orifice plate culturing cells, cell concn is in 1.5 × 105 cells/well.First cell carries out co-cultivation after 12 hours with the peptide sample of different concns, exchanges fresh substratum for.And then adopt phase microscope observation of cell form after adopting Hydroperoxide injury 2h, and mtt assay is adopted to detect cell survival rate.
Mtt assay, also known as MTT colorimetry, is a kind of method detecting cell survival and growth.Its Cleaning Principle is that the succinodehydrogenase in viable cell plastosome can make exogenous 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) is reduced to water-insoluble bluish voilet crystallization first a ceremonial jade-ladle, used in libation (Formazan) and is deposited in cell, and dead cell is without this function.First a ceremonial jade-ladle, used in libation in dimethyl sulfoxide (DMSO) (DMSO) energy dissolved cell, measures its absorbance value with enzyme-linked immunosorbent assay instrument at 490nm wavelength place, can indirectly reflect viable cell quantity.Within the scope of certain cell count, the amount that MTT crystallization is formed is directly proportional to cell count.
Take the MTT of 250 μ L (final concentration is at 0.5mg/mL) to join in 96 orifice plates, with testing sample lucifuge Dual culture 4h, afterwards, by substratum sucking-off old in 96 orifice plates, add 250 μ LDMSO and dissolve first a ceremonial jade-ladle, used in libation.Adopt microplate reader 570nm to detect first a ceremonial jade-ladle, used in libation concentration, calculate cell survival rate.
Wherein:
A experiment is for having added the cell of sample and hydrogen peroxide;
A contrast is the PC12 cell only having added same volume PBS.
The efficacy validation test method that walnut protein peptide of the present invention improves mouse memory is as follows:
(1) study subject
SPF level Kunming mouse, male and female half and half, 20-22g, purchased from Traditional Chinese Medicine University Of Guangzhou's Experimental Animal Center, conformity certification number: SCXK (Guangdong) 2008-0020
(2) positive drug
Piracetam: GuangDong HuaNan Pharmacy Group Co., Ltd, lot number 121004;
(3) test reagent and instrument
Scopolamine hydrobromide injection: Guangzhou Baiyunshan Mingxing Pharmaceutical Co., Ltd., lot number 130401;
Physiological saline agent: Jiangxi Cologne Medicine Co., Ltd, lot number D121018E;
Dehydrated alcohol: Tianjin Fu Yu Fine Chemical Co., Ltd (analytical pure), lot number GB/T678-2002
Acetylcholine ester enzyme reagent kit: biotinylated biomolecule Graduate School of Engineering is built up in Nanjing, lot number 20130703
L-glutamic acid test kit: biotinylated biomolecule Graduate School of Engineering is built up in Nanjing, lot number 20130705
Electronics refiner: IKA company, model: T10BS25
Electronic analytical balance: plum Teller-Tuo benefit Instrument Ltd., model EL204
XZC-5A type mouse diving tower instrument: Shandong Academy of Medical Sciences's equipment station, numbering 0012;
Morris water maze video analytic system: Shanghai shift Science and Technology Ltd., numbering 0017625.
(4) experimental technique:
The screening of grouping advance action thing, shaves except not being able to swim or swimming abnormal and insensitive to electric shock, latent period is greater than 60s animal.Get the KM mouse that screening is qualified, male and female half and half, are divided into Normal group, Scopolamine model group, positive controls and 4 sample sets (embodiment 1-3 and comparative example 1) at random, often organize 15 animals.Gastric infusion effective dose is 1.0g/kg/d, continuous 21d.Self administration of medication starts 14d, carries out the directed swimming test of Morris water maze.Model control group and by reagent group respectively abdominal injection scopolamine hydrobromide 3.0mg/kg, Normal group abdominal injection respective volume physiological saline, enters water training after 20min.During training, pond be divided into 4 quadrants (the Ith, II, III, IV quadrant), platform position is the IIth quadrant (as figure) 1cm place, underwater.During experiment according to the Ith, III, the order of IV quadrant such as figure marked position by mouse towards and put into water near pool wall, computer software record mouse is from entering water to the swimming time of climbing up platform and stay more than 3s, for escape latency (escapelatency, EL).Find the mouse less than platform more than 120s, count 120s latent period and guided to platform stop 10s.Whole mouse carries out the test of lower 1 quadrant again after completing 1 quadrant test, until complete the test of 3 quadrants, and continuous 5d.6d, removes platform, measures in 120s: (1) escape latency: animal enters the time that water arrives platform position for the first time; (2) platform traversing times; (3) mouse is swum distance and time in central zone and platform peripheral region, count ratio.
In polypeptide provided by the invention and application thereof, raw materials used and reagent all can be buied by market.
Below in conjunction with embodiment, set forth the present invention further:
Embodiment 1 utilizes walnut to prepare Improving memory peptide
(1) in 1 part of walnut, add the water of 10 parts by weight, cross colloidal mill and obtain walnut slurries, add 1mol/LNaOH solution adjustment walnut slurries pH to 8.0, at 50 DEG C, stir 60min, then centrifugal 10min under 3500r/min, filter and obtain supernatant liquor; In supernatant liquor, add the α-amylase of walnut protein quality 1.0% (w/w) and the cellulase of walnut quality 1.0% (w/w), at 55 DEG C, stir 120min; Add 1mol/LHCl solution afterwards and pH is adjusted to 4.2, stir 30min, under 3500r/min, centrifugal 10min, is precipitated thing; Then adopt 45% concentration ethanol to stir, and clean with water, spraying dry, obtain Walnut protein powder.
(2) water adding powdery walnut protein 10 times stirs, add the Alcalase2.4L of Novozymes Company by 2000U/g albumen and add the compound protease of Novozymes Company by 2500U/g albumen, degree of hydrolysis 12% is hydrolyzed at 55 DEG C, 100 DEG C of heating 15min go out enzyme, centrifugation, obtaining supernatant liquor, is walnut protein enzymolysis product.
(3) walnut protein enzymolysis product is crossed 100 order filter clothes, then by the ultra-filtration membrane of membrane flux 10000Da, collect permeate, again by the ultra-filtration membrane of membrane flux 1000Da, collect trapped fluid, obtain walnut peptide solution, vacuum concentration is to solid substance 30%, carry out spraying dry, obtain Improving memory peptide 1.
The enzymolysis product protein recovery of Improving memory peptide 1 and peptide content are in table 1.
The H of Improving memory peptide 1
2o
2damage PC12 cell survival rate result is as table 2.
The animal efficacy validation test-results of Improving memory peptide 1 is as table 3 and table 4.
Embodiment 2 utilizes walnut to prepare Improving memory peptide
(1) in 1 part of walnut, add the water of 15 parts by weight, cross colloidal mill and obtain walnut slurries, add 1mol/LNaOH solution adjustment walnut slurries pH to 9.0, at 40 DEG C, stir 90min, then centrifugal 10min under 3500r/min, filter and obtain supernatant liquor; In supernatant liquor, add the α-amylase of walnut protein quality 2.0% (w/w) and the cellulase of walnut quality 0.5% (w/w), at 50 DEG C, stir 180min; Add 1mol/LHCl solution afterwards and pH is adjusted to 4.2, stir 60min, under 3500r/min, centrifugal 10min, is precipitated thing; Then adopt 60% concentration ethanol to stir, and clean with water, spraying dry, obtain Walnut protein powder.
(2) water adding powdery walnut protein 7 times stirs, add the Alcalase2.4L of Novozymes Company by 3000U/g albumen and add the compound protease of Novozymes Company by 1200U/g albumen, degree of hydrolysis 8% is hydrolyzed at 50 DEG C, 100 DEG C of heating 15min go out enzyme, centrifugation, obtaining supernatant liquor, is walnut protein enzymolysis product.
(3) walnut protein enzymolysis product is crossed 100 order filter clothes, then by the ultra-filtration membrane of membrane flux 10000Da, collect permeate, again by the ultra-filtration membrane of membrane flux 1000Da, collect trapped fluid, obtain walnut peptide solution, vacuum concentration is to solid substance 40%, carry out spraying dry, obtain Improving memory peptide 2.
The enzymolysis product protein recovery of Improving memory peptide 2 and peptide content are in table 1.
The H of Improving memory peptide 2
2o
2damage PC12 cell survival rate result is as table 2.
The animal efficacy validation test-results of Improving memory peptide 2 is as table 3 and table 4.
Embodiment 3 utilizes walnut to prepare Improving memory peptide
(1) in 1 part of walnut, add the water of 12 parts by weight, cross colloidal mill and obtain walnut slurries, add 1mol/LNaOH solution adjustment walnut slurries pH to 8.5, at 45 DEG C, stir 80min, then centrifugal 10min under 3500r/min, filter and obtain supernatant liquor; In supernatant liquor, add the α-amylase of walnut protein quality 1.5% (w/w) and the cellulase of walnut quality 0.8% (w/w), at 55 DEG C, stir 150min; Add 1mol/LHCl solution afterwards and pH is adjusted to 4.2, stir 45min, under 3500r/min, centrifugal 10min, is precipitated thing; Then adopt 50% concentration ethanol to stir, and clean with water, spraying dry, obtain Walnut protein powder.
(2) water adding powdery walnut protein 8 times stirs, add the Alcalase2.4L of Novozymes Company by 2500U/g albumen and add the compound protease of Novozymes Company by 1800U/g albumen, degree of hydrolysis 10% is hydrolyzed at 55 DEG C, 100 DEG C of heating 15min go out enzyme, centrifugation, obtaining supernatant liquor, is walnut protein enzymolysis product.
(3) walnut protein enzymolysis product is crossed 100 order filter clothes, then by the ultra-filtration membrane of membrane flux 10000Da, collect permeate, again by the ultra-filtration membrane of membrane flux 1000Da, collect trapped fluid, obtain walnut peptide solution, vacuum concentration is to solid substance 35%, carry out spraying dry, obtain Improving memory peptide 3.
The enzymolysis product protein recovery of Improving memory peptide 3 and peptide content are in table 1.
The H of Improving memory peptide 3
2o
2damage PC12 cell survival rate result is as table 2.
The animal efficacy validation test-results of Improving memory peptide 3 is as table 3 and table 4.
Embodiment 4 utilizes walnut dregs to prepare Improving memory peptide
(1) in 1 part of walnut dregs, add the water of 10 parts by weight, cross colloidal mill and obtain walnut dregs slurries, add 1mol/LNaOH solution adjustment walnut dregs slurries pH to 8.0, at 50 DEG C, stir 60min, then centrifugal 10min under 3500r/min, filters and obtains supernatant liquor; In supernatant liquor, add the α-amylase of walnut dregs protein mass 1.0% (w/w) and the cellulase of walnut dregs quality 1.0% (w/w), at 55 DEG C, stir 120min; Add 1mol/LHCl solution afterwards and pH is adjusted to 4.2, stir 30min, under 3500r/min, centrifugal 10min, is precipitated thing; Then adopt 45% concentration ethanol to stir, and clean with water, spraying dry, obtain Walnut protein powder.
(2) water adding powdery walnut protein 10 times stirs, add the Alcalase2.4L of Novozymes Company by 2000U/g albumen and add the compound protease of Novozymes Company by 2500U/g albumen, degree of hydrolysis 12% is hydrolyzed at 55 DEG C, 100 DEG C of heating 15min go out enzyme, centrifugation, obtaining supernatant liquor, is walnut protein enzymolysis product.
(3) walnut protein enzymolysis product is crossed 100 order filter clothes, then by the ultra-filtration membrane of membrane flux 10000Da, collect permeate, again by the ultra-filtration membrane of membrane flux 1000Da, collect trapped fluid, obtain walnut peptide solution, vacuum concentration is to solid substance 30%, carry out spraying dry, obtain Improving memory peptide 1.
The enzymolysis product protein recovery of Improving memory peptide 1 and peptide content are in table 1.
The H of Improving memory peptide 1
2o
2damage PC12 cell survival rate result is as table 2.
The animal efficacy validation test-results of Improving memory peptide 1 is as table 3 and table 4.
Embodiment 5 utilizes walnut dregs to prepare Improving memory peptide
(1) in 1 part of walnut dregs, add the water of 15 parts by weight, cross colloidal mill and obtain walnut dregs slurries, add 1mol/LNaOH solution adjustment walnut dregs slurries pH to 9.0, at 40 DEG C, stir 90min, then centrifugal 10min under 3500r/min, filters and obtains supernatant liquor; In supernatant liquor, add the α-amylase of walnut dregs protein mass 2.0% (w/w) and the cellulase of walnut dregs quality 0.5% (w/w), at 50 DEG C, stir 180min; Add 1mol/LHCl solution afterwards and pH is adjusted to 4.2, stir 60min, under 3500r/min, centrifugal 10min, is precipitated thing; Then adopt 60% concentration ethanol to stir, and clean with water, spraying dry, obtain Walnut protein powder.
(2) water adding powdery walnut protein 7 times stirs, add the Alcalase2.4L of Novozymes Company by 3000U/g albumen and add the compound protease of Novozymes Company by 1200U/g albumen, degree of hydrolysis 8% is hydrolyzed at 50 DEG C, 100 DEG C of heating 15min go out enzyme, centrifugation, obtaining supernatant liquor, is walnut protein enzymolysis product.
(3) walnut protein enzymolysis product is crossed 100 order filter clothes, then by the ultra-filtration membrane of membrane flux 10000Da, collect permeate, again by the ultra-filtration membrane of membrane flux 1000Da, collect trapped fluid, obtain walnut peptide solution, vacuum concentration is to solid substance 40%, carry out spraying dry, obtain Improving memory peptide 2.
The enzymolysis product protein recovery of Improving memory peptide 2 and peptide content are in table 1.
The H of Improving memory peptide 2
2o
2damage PC12 cell survival rate result is as table 2.
The animal efficacy validation test-results of Improving memory peptide 2 is as table 3 and table 4.
Embodiment 6 utilizes walnut dregs to prepare Improving memory peptide
(1) in 1 part of walnut dregs, add the water of 12 parts by weight, cross colloidal mill and obtain walnut dregs slurries, add 1mol/LNaOH solution adjustment walnut dregs slurries pH to 8.5, at 45 DEG C, stir 80min, then centrifugal 10min under 3500r/min, filters and obtains supernatant liquor; In supernatant liquor, add the α-amylase of walnut dregs protein mass 1.5% (w/w) and the cellulase of walnut dregs quality 0.8% (w/w), at 55 DEG C, stir 150min; Add 1mol/LHCl solution afterwards and pH is adjusted to 4.2, stir 45min, under 3500r/min, centrifugal 10min, is precipitated thing; Then adopt 50% concentration ethanol to stir, and clean with water, spraying dry, obtain Walnut protein powder.
(2) water adding powdery walnut protein 8 times stirs, add the Alcalase2.4L of Novozymes Company by 2500U/g albumen and add the compound protease of Novozymes Company by 1800U/g albumen, degree of hydrolysis 10% is hydrolyzed at 55 DEG C, 100 DEG C of heating 15min go out enzyme, centrifugation, obtaining supernatant liquor, is walnut protein enzymolysis product.
(3) walnut protein enzymolysis product is crossed 100 order filter clothes, then by the ultra-filtration membrane of membrane flux 10000Da, collect permeate, again by the ultra-filtration membrane of membrane flux 1000Da, collect trapped fluid, obtain walnut peptide solution, vacuum concentration is to solid substance 35%, carry out spraying dry, obtain Improving memory peptide 3.
The enzymolysis product protein recovery of Improving memory peptide 3 and peptide content are in table 1.
The H of Improving memory peptide 3
2o
2damage PC12 cell survival rate result is as table 2.
The animal efficacy validation test-results of Improving memory peptide 3 is as table 3 and table 4.
Comparative example 1
Adopt ordinary method (non-invention method) to be hydrolyzed walnut protein and prepare walnut peptide:
(1) in 1 part of walnut dregs, add the water of 12 parts by weight, cross colloidal mill and obtain walnut dregs slurries.
(2) water adding walnut dregs 10 times stirs, by 4000U/g albumen add Novozymes Company Alcalase2.4L and by 3000U/g albumen add Novozymes Company compound protease, degree of hydrolysis 10% is hydrolyzed at 55 DEG C, 100 DEG C of heating 15min go out enzyme, centrifugation, obtaining supernatant liquor, is walnut protein enzymolysis product.
(3) walnut protein enzymolysis product is crossed 100 order filter clothes, then by the ultra-filtration membrane of membrane flux 10000Da, collect permeate, again by the ultra-filtration membrane of membrane flux 1000Da, collect trapped fluid, obtain walnut peptide solution, vacuum concentration is to solid substance 35%, carry out spraying dry, obtain contrast walnut peptide.
The enzymolysis product protein recovery of contrast walnut peptide and peptide content are in table 1.
The H of contrast walnut peptide
2o
2damage PC12 cell survival rate result is as table 2.
The animal efficacy validation test-results of contrast walnut peptide is as table 3 and table 4.
The protein recovery of table 1 walnut protein peptide and peptide content
Note: mark different footmark person in same column and there is significant difference (P<0.05)
Get walnut protein peptide prepared by walnut protein peptide and comparative example prepared by the embodiment of the present invention 1 to 6 and carry out molecular weight detection, the measuring method that the peptide molecular weight of walnut protein peptide distributes is as follows:
Adopt gel chromatography.Chromatographic condition is as follows: Amersham protein analysis purification system, Superdex_peptide_10/300_GL glass column (Vt=24.0mL, V0=8.0mL), elutriant is the phosphate buffered saline buffer (pH7.2) of 0.25MNaCl, sampling volume is 40 μ L, flow velocity 0.5mL/min, monitoring wavelength is 214nm.Then according to the molecular weight distribution situation going out cutting edge of a knife or a sword Time Calculation protein peptide sample of standard substance.
The results are shown in Figure 1.The peptide molecular weight of the walnut protein peptide (embodiment 1 to 6) adopting the present invention to prepare as seen from Figure 1 is mainly distributed between 1000Da-10000Da, and walnut protein peptide prepared by comparative example has moieties amount at more than 10000Da.And the main technique difference of embodiment 1 to 6 and comparative example is that first embodiment adopts enzyme process to assist alkali extraction and acid precipitation technology to extract walnut protein, and utilize ethanolic soln to clean the heavy albumen of acid, then carry out enzymolysis, illustrate and adopt patented technology of the present invention can obtain the walnut protein peptide product of the higher molecular weight of purity at 1000-10000Da.
Table 2 walnut protein peptide and PC12 cell H
2o
2injury protection experiment is compared
First patent of the present invention extracts protein from walnut or walnut dregs, and then carry out biological enzymolysis and prepare walnut protein peptide, the essence of the method take walnut protein as hydrolysis material, as shown in Table 1, the walnut protein peptide protein recovery adopting patent of the present invention (embodiment 1-6) to prepare is higher (all more than 66%), peptide content reaches more than 67%, is significantly higher than protein recovery and the too content of comparative example respectively; And the enzyme solution of routine is that raw material carries out enzymolysis with walnut dregs, owing to containing a large amount of polysaccharide in walnut dregs, polysaccharide combines a large amount of protein, limits disengaging of protein, causes the protein recovery of comparative example to only have about 45%, peptide content 50.11%.
As seen from Table 2, the cell survival rate of the Improving memory peptide adopting the inventive method (embodiment 1-6) to prepare improves more than the cell survival rate adopting ordinary method (comparative example 1) to be hydrolyzed the walnut protein peptide obtained.Mainly because extract walnut protein by alkali extraction and acid precipitation to carry out multienzyme synergism hydrolysis, make to remember related amino acid containing more hydrophobic amino acid and Serine, Methionin etc. in enzymolysis solution, the provide protection of PC12 cell is promoted obviously.And by two-stage ultrafiltration membrance filter, being enriched the peptide section of 1000-10000Da, the anti-oxidant value of peptide section between this molecular weight is comparatively strong to have a large amount of documents to show at present, effectively can improve the damaging action of oxidative stress to PC12 cell., finding by detecting meanwhile, adopting the inventive method resulting improvement memory polypeptide cell survival rate to have the cell survival rate of the gsh of strong antioxidant action higher than bibliographical information.
Table 3 sample is removed platform experiment escape latency to Scopolamine model mice Morris water maze and is worn the impact of platform number of times
Note:
##represent that model group compares P<0.01 with normal group; * represents that administration group compares P<0.01 with model control group, and * represents that administration group compares P<0.05 with model control group.
Table 4 sample removes the impact in platform experimental activity region to Scopolamine model mice Morris water maze
Note:
##represent that model group compares P<0.01 with normal group,
#represent that model group compares P<0.05 with normal group; * represents that administration group compares P<0.01 with model control group, and * represents that administration group compares P<0.05 with model control group.
The mouse memory damage model that the present invention adopts Scopolamine to induce, its principle is that m receptor blocker Scopolamine can destroy cholinergic function, and before training, administration can make animal produce acquired dysmnesia.This test evaluates walnut protein peptide sample to the improvement result of Scopolamine model mice learning and memory function with the classical behavioral indexes of Morris water maze.Table 3 and the display of table 4 result, embodiment 1-6 all can significantly can improve Scopolamine model mice spatial memory capacity, comprise shortening escape latency, increase and wear platform number of times, improve its distance in platform periphery activity and time ratios (P<0.05-0.01), compared with model group, the walnut protein peptide (comparative example 1) adopting conventional enzyme solution to prepare also tool has some improvement Scopolamine model mice spatial memory capacity, but improvement result is strong not as the effect of embodiment.
Comprehensive above-mentioned experimental result, the Improving memory peptide adopting the inventive method to prepare has good application prospect.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. walnut is through a polypeptide for enzyme-squash techniqued, it is characterized in that, it is prepared by following steps:
Step 1: get walnut mix with water after grind, through the first acid-base modifier adjusted to ph to 8.0 ~ 9.0, stirring, centrifugal, collecting by filtration filtrate; Get described filtrate mix with lytic enzyme after enzymolysis, through the second acid-base modifier adjusted to ph to 4.0 ~ 4.2, stir, centrifugal, collecting by filtration filter residue, through alcohol precipitation, washing, obtain walnut protein;
Step 2: get the described walnut protein that step 1 prepares and mix with water, through Alcalase2.4L proteolytic enzyme and compound protease, under the condition of 50 ~ 55 DEG C, enzymolysis is to degree of hydrolysis 8 ~ 12%, and go out enzyme, centrifugal, collecting by filtration filtrate, obtains walnut protein enzymolysis solution;
Step 3: get the described walnut protein enzymolysis solution that step 2 prepares and sieve, collect permeate after the ultrafiltration membrance filter of 10000Da; By described permeate again through the ultrafiltration membrance filter of 1000Da, collect trapped fluid, obtain the walnut peptide of molecular weight at 1000-10000Da.
2. polypeptide according to claim 1, it is characterized in that, the addition of α-amylase described in step 1 is 1% ~ 2% (w/w) of walnut protein quality, and the addition of described cellulase is 0.5% ~ 1.0% (w/w) of walnut quality.
3. polypeptide according to claim 1 and 2, it is characterized in that, the quality of the described walnut protein prepared with step 1 is for Calculation Basis, and the add-on of Alcalase2.4L proteolytic enzyme is 2000 ~ 3000U/g albumen, and the add-on of compound protease is 1200 ~ 2500U/g albumen.
4. the polypeptide according to any one of claims 1 to 3, is characterized in that, the condition of enzymolysis described in step 1 for stir 120-180min at 50-55 DEG C.
5. the polypeptide according to any one of Claims 1-4, is characterized in that, the aqueous ethanolic solution of the alcohol that alcohol precipitation described in step 1 adopts to be volumetric concentration be 45-60%.
6. the polypeptide according to any one of claim 1 to 5, is characterized in that, described stirring after adding the first acid-base modifier in step 1, centrifugal being specially stir 60-90min at 40-50 DEG C, then centrifugal 10min under 3500r/min;
30-60min, then centrifugal 10min under 3500r/min are stirred in described stirring after adding the second acid-base modifier in step 1, centrifugal being specially.
7. the polypeptide according to any one of claim 1 to 6, is characterized in that, described in step 1, the first acid-base modifier is NaOH solution, and described second acid-base modifier is HCl solution.
8. the polypeptide according to any one of claim 1 to 7, is characterized in that, in step 1, the add-on of water is 10 ~ 15 times of walnut quality; In step 2, the add-on of water is 7 ~ 10 times of walnut protein quality; Vacuum concentration is also comprised to solid substance 30-40%, spray-dired step after collecting trapped fluid described in step 3.
9. the polypeptide according to any one of claim 1 to 8 prevents and/or treats the application in the medicine of memory decay, food and/or healthcare products in preparation.
10. application according to claim 9, is characterized in that, the formulation of described medicine, food and/or healthcare products is oral liquid, capsule, tablet, pulvis or granule.
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| CN108201140A (en) * | 2018-01-31 | 2018-06-26 | 无限极（中国）有限公司 | A kind of walnut peptide colon-specific pellets and preparation method thereof |
| CN109576330A (en) * | 2018-11-23 | 2019-04-05 | 南京中医药大学 | Brush-cherry seed polypeptide and its preparation method and application |
| CN110710592A (en) * | 2019-11-16 | 2020-01-21 | 四川农业大学 | Method for improving antioxidant activity of walnut cake protein |
| CN111011567A (en) * | 2019-11-28 | 2020-04-17 | 程红 | Tabletting candy with memory improving function and preparation method thereof |
| CN111838666A (en) * | 2020-08-10 | 2020-10-30 | 桑迪(武汉)生物科技有限公司 | Composition with auxiliary memory improving function and application thereof |
| CN113774101A (en) * | 2021-07-30 | 2021-12-10 | 海南大学 | Animal and plant mixed protein peptide with memory improving function and preparation method and application thereof |
| CN113774101B (en) * | 2021-07-30 | 2022-11-08 | 海南大学 | Animal and plant mixed protein peptide with memory improving function and preparation method and application thereof |
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