CN109306367A - The preparation method and pilose antler phosphoeptide and application of a kind of pilose antler phosphoeptide - Google Patents
The preparation method and pilose antler phosphoeptide and application of a kind of pilose antler phosphoeptide Download PDFInfo
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- 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
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Abstract
The present invention relates to a kind of preparation method and applications of pilose antler phosphoeptide.The present invention establishes the preparation method of pilose antler phosphoeptide, and prepared pilose antler phosphoeptide, which has, promotes calcium uptake, can be applied to the fields such as food, health care product, drug, has broad application prospects.
Description
Technical field
The present invention relates to pilose antler phosphoeptide, specifically a kind of preparation method and application of pilose antler phosphoeptide.
Background technique
Metal ion plays an important role in human physiological activity, can lead when the metallic element deficiency in food
Various diseases are caused, need to additionally supplement and meet Human Physiology demand.The absorption of metallic element is a complicated process in human body, no
It is only dependent upon the physiology, biology and hormone situation of host, is also influenced by diet and metallic element absorbing state.Most gold
Belong to ion to be absorbed in human small intestine, pH drops sharply to 5.5 from 7 when reaching small intestine from digestive system, last when reaching ileum
PH is gradually increased to 7.5 or so again when end, therefore solubility of the metal ion at different pH directly determines it in the suction of human body
Receive situation.
Pilose antler is the children of the unossified dense villus of stag of the animal sika deer or red deer of Chordata Mammalia Cervidae
Angle.Pilose antler is used as medicine existing more than 2000 years history, Compendium of Material Medica record pilose antler " production of sperm mends marrow, blood-nourishing Yiyang, strengthening the muscles and bones,
Control all are deficient, deaf, mesh is dark, the empty dysentery of dizziness ".Recent study discovery, pilose antler have immunological regulation, anti-inflammatory, anti-oxidant, anti-
The multiple biological functions such as aging.Pilose antler contains protein, glycosaminoglycan, phosphatide, fatty acid, hormonelike substance, nucleic acid, polyamines
And various inorganic substances, wherein pilose antler albumen is most important chemical component, accounts for the 53.9%~76.4% of dry weight.On protein
The amino acid such as serine form phosphoeptide in combination with phosphate radical, and the above calcium can be improved after phosphoeptide and calcium, zinc, iron plasma chelating
Ion human body alimentary canal solubility, thus promote calcium, zinc, iron plasma human body absorption.The present invention is enriched with preparation deer
Phosphoeptide in young pilose antler, prepared pilose antler phosphoeptide can be used for human calcium's zinc, iron plasma replenishers.
Summary of the invention
The object of the present invention is to provide a kind of preparation method and applications of pilose antler phosphoeptide, and the present invention is by digesting deer bone
Middle protein degradation, wherein phosphoeptide, prepared pilose antler phosphoeptide can be used as human body calcium complement agent to concentration and separation.
To achieve the above object, The technical solution adopted by the invention is as follows:
By pilose antler enzymatic hydrolysis, the enrichment of pilose antler phosphoeptide, purifying, it is prepared into pilose antler phosphoeptide.
Above-described pilose antler phosphoeptide preparation method will crush after the freeze-drying of fresh pilose antler, pilose antler quality 10 be added
PH is adjusted to 1.0~5.0 with the HCl of 1~6M by~50 times of deionized water, be stirring evenly and then adding into pilose antler quality 0.1~
5.0% protease A digests 0.5~24 hour at a temperature of 20~80 DEG C；With the NaOH of 1~6M by pH tune after enzymatic hydrolysis
Section adds the Cathepsin B of pilose antler quality 0.1~5.0%, it is small that 0.5~24 is digested at a temperature of 20~80 DEG C to 4.0~8.0
When, temperature is risen to 80~100 DEG C after reaction and keeps the temperature 10~30 minutes.
Enzymolysis liquid is cooled to room temperature, adjusts pH with the HCl of 1~6M and is adjusted to 3.0~6.0, after mixing evenly with 10000~
The speed of 30000 × g is centrifuged 10~20 minutes, collects supernatant.
The CaCl that concentration is 10~30% is added in supernatant2Solution, until CaCl in solution2Final mass concentration be
0.5~5%, it stirs evenly.
Isometric ice ethyl alcohol is added in above-mentioned solution, is placed in and is kept for 3~30 minutes on ice, then with 10000~30000
The speed of × g is centrifuged 10~20 minutes in -10~10 DEG C, collects precipitating, and freeze-drying is pilose antler phosphoeptide.
The proteolytic enzyme A be pepsin, trypsase, neutral proteinase, flavor protease, papain,
The combination of one or more of protease such as bromelain；Proteolytic enzyme B is trypsase, chymotrypsin, carboxylic peptide
The combination of one or more of enzyme, papain, bromelain, flavor protease, neutral proteinase.Proteolytic enzyme A
It cannot be identical with Cathepsin B.
The present invention establishes the preparation method of pilose antler phosphoeptide, and prepared pilose antler phosphoeptide can be with calcium, zinc, iron plasma knot
Conjunction, which is prepared into, is used as the replenishers such as human calcium, zinc, iron, can be applied to the fields such as food, health care product, drug, has wide
Application prospect.
The present invention has the advantage that
1. pilose antler phosphoeptide prepared by all has good dissolubility in different pH, is remarkably improved calcium in human body
In bioavailability.
2. reaction condition is mild, method is simple and feasible.The present invention prepares deer bone peptide by enzymatic isolation method, prepared by ethanol precipitation
Pilose antler phosphoeptide, reaction condition is mild, reaction method simple possible, can be mass-produced.
3. having a good application prospect.The effect of present invention is according to traditional nutrient health deer bone, prepared pilose antler
Phosphoeptide can be prepared into conjunction with calcium, zinc, iron plasma is used as the replenishers such as human calcium, zinc, iron, can increase these ions and exist
The intracorporal solubility of people to be easier to be absorbed by the body, is edible more convenient, therefore has as drug, functional food, food
Broad application prospect.
Specific embodiment
Embodiment 1
Using fresh sika deer velvet antler as raw material, prepared according to following technique:
It will be freeze-dried 1 kilogram of pilose antler, crushed as 100 mesh powders, the deionized water of 10 liters of addition, with the HCl general of 6M
PH is adjusted to 2.0, is stirring evenly and then adding into 1 gram of pepsin, digests 2 hours in 37 DEG C；PH is adjusted to 7.0 after enzymatic hydrolysis,
1 gram of trypsase is added, is reacted at 37 DEG C 5 hours, temperature is risen to 100 DEG C after reaction and keeps the temperature 10 minutes.
Enzymolysis liquid is cooled to room temperature, adjusts pH with the HCl of 6M and is adjusted to 3.0, after mixing evenly with the speed of 10000 × g from
The heart 10 minutes, collect supernatant.
The CaCl that mass concentration is 10% is added in supernatant2Solution, until CaCl in solution2Final mass concentration be
5%, it stirs evenly.
Isometric ice ethyl alcohol is added in above-mentioned solution, is placed in and is kept for 30 minutes on ice, then with the speed of 10000 × g
It is centrifuged 10 minutes in 4 DEG C, collects precipitating, freeze-drying is pilose antler phosphoeptide.
Prepared pilose antler phosphoeptide measures peptide content, phosphorus content and the sequence for identifying phosphoeptide in accordance with the following methods.
One, experimental method
(1) peptide content:
1. instrument and reagent
Reagent: bicinchoninic acid, crystal carbonate, two hydration sodium tartrates, sodium hydroxide, sodium bicarbonate, five hydrations
Copper sulphate etc. is that analysis is pure；Bovine serum albumin(BSA)；Distilled water.
Instrument: ultraviolet-visible spectrophotometer.
2. solution is prepared
Reagent A (1L): weighing 10g bicinchoninic acid (BCA), 20g crystal carbonate respectively, and 1.6g bis- is hydrated tartaric acid
Sodium, 4g sodium hydroxide, 9.5g sodium bicarbonate add distilled water to 1L, adjust pH to 11.25 with sodium hydroxide.
Reagent B (50mL): 2g Salzburg vitriol is taken, adds distilled water to 50mL.
BCA reagent: before use with reagent A: the ratio of reagent B=50:1 (v/v) is uniformly mixed.
Standard protein solution: weighing the bovine serum albumin(BSA) of 50mg, is dissolved in distilled water and is settled to 100mL, obtains 500 μ g/
The solution of mL.
Sample solution: with distilled water by sample preparation at the solution of about 50 μ g/mL of protein concentration.
3. experimental procedure
(1) standard curve is drawn
96 clean orifice plates are taken, according to the form below sequentially adds reagent.
It after mentioned reagent adds, mixes, 37 DEG C of heat preservation 30min, after being cooled to room temperature, with No. 1 pipe for blank determination 562nm
Under absorbance, with bovine serum albumin content (μ g) be abscissa, using absorbance be ordinate draw standard curve its return
Equation is y=0.0621x+0.0964 (R2=0.997).
(2) sample measures
250 μ L sample solution are accurately pipetted, BCA reagent 5mL is added, shakes up, in 37 DEG C of heat preservation 30min, is cooled to room temperature
Afterwards, with the absorbance that standard curve 1 pipe is under blank determination 562nm, and pass through regression equation calculation sample peptide content.
(2) method for measuring phosphor content
1. instrument and reagent
Instrument: microwave dissolver；Ultraviolet-visible spectrophotometer
Reagent: ammonium molybdate, hydroquinone, sulfuric acid, potassium dihydrogen phosphate, sodium sulfite, sodium hydroxide, nitric acid, hydrogen peroxide are equal
It is pure to analyze.Experimental water is ultrapure water.
2. the preparation of reagent and sample solution
(1) solution is prepared
100.00 μ g/mL phosphorus titers: 0.4394g potassium dihydrogen phosphate adds water to be settled to 1000mL.
15% sulfuric acid solution: taking 15mL sulfuric acid to be added in 80mL water slowly and mix, and after cooling plus water is settled to 100mL.
Ammonium molybdate solution: 0.50g ammonium molybdate is weighed before use with 15% (v/v) sulfuric acid and is settled to 100mL.
Quinol solution: weighing 0.50g hydroquinone before use and be dissolved in 100mL water, and a drop sulfuric acid is added, and protects
It is stored in dark place.
Sodium sulfite solution: 20g sodium sulfite is weighed before use and is dissolved in 100mL water.
(2) sample treatment
Treatments of the sample: precision weighs 0.2g sample, adds 4mL nitric acid, 2mL hydrogen peroxide.With power 600W, rate of pressure rise
0.3bar/s heating 15min, maintains 30min when rising to 190 DEG C of temperature, pressure 40bar.Gained digestive juice is transferred to tool scale
Test tube in, heating makes sour evolution, and distilled water is added constantly to ensure pH value of solution > 1 after acid evolution in midway.It is molten that blank is done with method
Liquid.
3. measuring method
Will 100.00 μ g/mL phosphorus titers dilute 10 times after measure 0,50.0,100.0,200.0,300.0,400.0,
500.0 μ L, mix after being separately added into 0.2mL ammonium molybdate solution, then are successively separately added into 0.2mL 20% (w/v) sodium sulfite water
Solution, 0.1mL 0.5% (w/v) hydroquinone aqueous solution stand 30min, measure 660nm absorbance.With the absorbance measured
Standard curve is drawn to phosphorus content (μ g), regression equation is y=0.2373x+0.0311 (R2=0.999).
It is accurate to draw test solution or 500.0 μ L of blank solution, it is measured in the same method, calculates phosphorus content according to standard curve.
(3) Sequence Identification of Phosphorylated Peptide
1. instrument and reagent
Instrument: RPLC-MS/MS system by Finnigan Surveyor liquid chromatography pump, linear ion hydrazine (LTQ,
Linear trap quadrupole mass spectrometer, Thermo) mass spectrum composition.
Reagent: formic acid, acetonitrile, ultrapure water.
2. experimental method
0.1% aqueous formic acid of sample is redissolved into the solution at 4mg/mL, using freezing after waters HLB column desalination
It is dry, then the solution of 0.1% aqueous formic acid weight melt into, 0.4 μ g/ μ L is used, progress LC-MS/MS analysis.
One end of capillary is pulled into the tip that internal diameter is about 5 μm, C18AQ filler is pressed into column by air pressure, filling
Column length about 15cm.The tip of capillary is connected with mass spectrum.Mobile phase A used is the aqueous solution of 0.1%FA, Mobile phase B
For the acetonitrile solution of 0.1%FA, gradient elution process are as follows: 0%B (0min), 2%B (2min), 25%B (87min), 35%B
(97min), 90%B (99min), 90%B (109min), 2%B (110min), 2%B (120min).60 μ L/min of flow velocity.
It is arranged 200 DEG C of temperature of ion transfer capillary, electron spray voltage 1.8KV, normalizes collision energy 35.0%.?
Map acquisition is carried out to MS and MS/MS using data dependence mode (data-dependent mode).The setting of scanning of the mass spectrum condition
Are as follows: select 6 highest abundance quasi-molecular ions to carry out MS/MS scanning from the full scan of each m/z=400~2000, wherein dynamic
Exclude (dynamic exclusion) setting are as follows: number of repetition (repeat count) is 2, repeats patient time (repeat
Duration) 30s, dynamic exclude time (exclusion duration) 90s.Utilize Xcalibur software (Version
2.2) (Thermo) carries out system control and data collection.
By the * .raw file data of the acquisition of sample Thermo Proteome Discoverer Daemon (v1.4)
It is converted into * .mgf format, then with Mascot (version 2.3.0, Matrix Science, London, UK) in Bovidae data
Library (bovine, albumen number 17890, under be downloaded from http://www.uniprot.org/) retrieved, retrieval parameter is as follows:
It is not provided with restriction enzyme site, several and fixed modification is cut in maximum leakage；Methionine residue, proline residue add turning revisionism for 15.9949Da
Decorations, serine residue, threonine residues add the variable modification of 97.9769Da；Quality tolerance (the peptide of parent ion
It tolerance) is 20ppm, the quality tolerance (fragment ions tolerance) of fragment ion is 0.8Da.Export peptide
Score > 20 are set when section result, and adjustment significant difference P makes peptide fragment false positive rate (FDR, false discovery rate)
Control is within 1%.
Two, experimental result
After measured, peptide content is 0.201g/g, phosphorus content 0.0848g/g in prepared pilose antler phosphoeptide.By matter
Spectrum is identified, following phosphoeptide is contained in pilose antler phosphoeptide:
KDS#DGGCDS#PAGPPELRLD
RDICSHASTSPS#S#T#LS#S#VS#PPT#
AVPGPPPLPGLPS#A
LLGT#GTLGPSP
AQRRGLPT#G
LLGTGT#LGPSP
FIS#SVIK
S#S#SS#S#T#T#S#
INTPIAATLPITT#
KMDIHKKVT#DPSVA
FISS#VIK
PQGAT#GPLGPK
LLATLSPRGVPTT#
RELEELNVPGEIVES#LS#S#S#EESITRINK
INTPIAATLPIT#T
RELEELNVPGEIVESLSSS#EESITRIN
Wherein S#、T#It respectively indicates with phosphorylation serine, phosphorylation threonine.
The above result shows that containing phosphoeptide in the deer antler extract prepared using this method.
Embodiment 2
Using fresh cervus elaphus linnaeus as raw material, prepared according to following technique:
It will be freeze-dried 1 kilogram of pilose antler, crushed as 100 mesh powders, the deionized water of 10 liters of addition, with the HCl general of 1M
PH is adjusted to 3.0, is stirring evenly and then adding into 10 grams of pepsins, digests 2 hours in 37 DEG C；PH is adjusted to 5.0 after enzymatic hydrolysis,
10 grams of bromelains react 5 hours at 50 DEG C, temperature are risen to 80 DEG C after reaction and keeps the temperature 20 minutes.
Enzymolysis liquid is cooled to room temperature, adjusts pH with the HCl of 1M and is adjusted to 4.0, after mixing evenly with the speed of 20000 × g from
The heart 10 minutes, collect supernatant.
The CaCl that mass concentration is 20% is added in supernatant2Solution, until CaCl in solution2Ultimate density quality be
5%, it stirs evenly.
Isometric ice ethyl alcohol is added in above-mentioned solution, is placed in and is kept for 30 minutes on ice, then with the speed of 20000 × g
It is centrifuged 10 minutes in 4 DEG C, collects precipitating, freeze-drying is pilose antler phosphoeptide.
Prepared pilose antler phosphoeptide is identified using the method for embodiment 1, the results showed that peptide in pilose antler phosphoeptide
Content is 0.325g/g, phosphorus content 0.0985g/g.Mass spectral results show the phosphoeptide containing embodiment 1.
Embodiment 3
Using fresh sika deer velvet antler as raw material, prepared according to following technique:
It will be freeze-dried 1 kilogram of pilose antler, crushed as 100 mesh powders, the deionized water of 50 liters of addition, with the HCl general of 6M
PH is adjusted to 5.0, is stirring evenly and then adding into 50 grams of pepsins, digests 4 hours in 40 DEG C；PH is adjusted to 5.0 after enzymatic hydrolysis,
50 grams of papains react 10 hours at 50 DEG C, temperature are risen to 100 DEG C after reaction and keeps the temperature 30 minutes.
Enzymolysis liquid is cooled to room temperature, adjusts pH with the HCl of 2M and is adjusted to 5.0, after mixing evenly with the speed of 30000 × g from
The heart 10 minutes, collect supernatant.
The CaCl that mass concentration is 30% is added in supernatant2Solution, until CaCl in solution2Final mass concentration be
5%, it stirs evenly.
Isometric ice ethyl alcohol is added in above-mentioned solution, is placed in and is kept for 30 minutes on ice, then with the speed of 20000 × g
It is centrifuged 10 minutes in 4 DEG C, collects precipitating, freeze-drying is pilose antler phosphoeptide.
Prepared pilose antler phosphoeptide is identified using the method for embodiment 1, the results showed that peptide in pilose antler phosphoeptide
Content is 0.186g/g, phosphorus content 0.109g/g.Mass spectral results show the phosphoeptide containing embodiment 1.
Embodiment 4
Using fresh sika deer velvet antler as raw material, prepared according to following technique:
It will be freeze-dried 1 kilogram of pilose antler, crushed as 100 mesh powders, the deionized water of 30 liters of addition, with the HCl general of 6M
PH is adjusted to 3.0, is stirring evenly and then adding into 1 gram of pepsin, digests 8 hours in 35 DEG C；PH is adjusted to 7.0 after enzymatic hydrolysis,
1 gram of neutral proteinase is added, is reacted at 50 DEG C 5 hours, temperature is risen to 90 DEG C after reaction and keeps the temperature 30 minutes.
Enzymolysis liquid is cooled to room temperature, adjusts pH with the HCl of 3M and is adjusted to 4.0, after mixing evenly with the speed of 30000 × g from
The heart 10 minutes, collect supernatant.
The CaCl that mass concentration is 30% is added in supernatant2Solution, until CaCl in solution2Final mass concentration be
5%, it stirs evenly.
Isometric ice ethyl alcohol is added in above-mentioned solution, is placed in and is kept for 30 minutes on ice, then with the speed of 20000 × g
It is centrifuged 10 minutes in 4 DEG C, collects precipitating, freeze-drying is pilose antler phosphoeptide.
Prepared pilose antler phosphoeptide is identified using the method for embodiment 1, the results showed that peptide in pilose antler phosphoeptide
Content is 0.560g/g, phosphorus content 0.0735g/g.Mass spectral results show the phosphoeptide containing embodiment 1.
The present invention is established using deer bone as the preparation method of the deer bone peptide chelated metal ions of raw material, is prepared using the present invention
The metal ion-chelants such as deer bone chelating peptide and iron, copper, zinc, calcium are prepared into replenishers, promote metal ion in the intracorporal suction of people
It receives, can be applied to the fields such as food, health care product, drug, have broad application prospects.
Claims (4)
1. a kind of preparation method of pilose antler phosphoeptide, it is characterised in that: by pilose antler enzymatic hydrolysis, the enrichment of pilose antler phosphoeptide, purifying, preparation
At pilose antler phosphoeptide；
Detailed process is as follows:
(1) it will be crushed after the freeze-drying of fresh pilose antler, going for 10~50 times of pilose antler quality (optimized scope being 10~20 times) be added
PH is adjusted to 1.0~5.0 (optimized scope is 1.5~4.0) with the HCl of 1~6M, is stirring evenly and then adding into pilose antler matter by ionized water
The protease A for measuring 0.1~5% (W/W) (optimized scope is 0.5~4.0%), it is small to digest 0.5~24 at a temperature of 20~80 DEG C
When；PH is adjusted to 4.0~8.0 with the NaOH of 1~6M after enzymatic hydrolysis, adds the protease of pilose antler quality 0.1~5.0%
B digests 0.5~24 hour (optimized scope is 2~10 hours) at a temperature of 20~80 DEG C, temperature is risen to 80 after reaction
~100 DEG C and heat preservation 10~30 minutes；
(2) enzymolysis liquid is cooled to room temperature, adjusts pH with the HCl of 1~6M and is adjusted to 3.0~6.0 (optimized scope is 3.0~5.0), stirs
It is centrifuged 10~20 minutes after mixing uniformly with the speed of 10000~30000 × g, collects supernatant；
(3) CaCl that concentration is 10~30% (W/W) (optimized scope is 15~20%) is added in supernatant2Solution, until solution
Middle CaCl2Final mass concentration be 0.5~5%, stir evenly；
(4) isometric ice ethyl alcohol is added in above-mentioned solution, is placed in and is kept for 3~30 minutes on ice, then with 10000~30000
The speed of × g is centrifuged 10~20 minutes in -10~10 DEG C, collects precipitating, and freeze-drying is pilose antler phosphoeptide.
2. preparation method described in accordance with the claim 1, it is characterised in that: the proteolytic enzyme A is pepsin, tryptose
One of protease such as enzyme, neutral proteinase, flavor protease, papain, bromelain or two kinds of combination of the above；
Proteolytic enzyme B be trypsase, chymotrypsin, carboxypeptidase, papain, bromelain, flavor protease, in
One of property protease or two kinds of combination of the above.Proteolytic enzyme A and Cathepsin B cannot be identical, i.e. proteolytic enzyme A and egg
Protease in the difference of protease used by white enzyme B or used combined protein enzyme is different.
3. a kind of pilose antler phosphoeptide that preparation method as claimed in claim 1 or 2 prepares.
4. the application of pilose antler phosphoeptide described in a kind of claim 3, it is characterised in that: the pilose antler phosphoeptide can be with calcium, zinc, iron
Deng one of or two kinds or more ions bindings be prepared into and be used as one of human calcium, zinc, iron etc. or two kinds or more and supplement
Agent.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111407779A (en) * | 2020-04-16 | 2020-07-14 | 山东省现代中药研究院有限公司 | Pilose antler beverage and preparation method thereof |
| CN111549084A (en) * | 2020-05-12 | 2020-08-18 | 营家健康科技(广东)有限公司 | Method for preparing protein small molecule peptide by simulating human body digestive tract enzymolysis |
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