CN114214385A - Preparation method of cornu cervi pantotrichum root ossified tissue source peptide chelated calcium - Google Patents
Preparation method of cornu cervi pantotrichum root ossified tissue source peptide chelated calcium Download PDFInfo
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- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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Abstract
The invention is suitable for the technical field of biology, and provides a preparation method of cornu cervi pantotrichum root ossified tissue source peptide chelated calcium, which comprises the following steps of 1) carrying out enzymolysis reaction on cornu cervi pantotrichum root ossified tissue through flavourzyme (derived from aspergillus oryzae), and carrying out freeze drying treatment on an obtained enzymolysis product; 2) separating and purifying the enzymolysis product in the step 1) to obtain a peptide with higher calcium binding capacity; 3) carrying out chelation reaction on the peptide and calcium ions to obtain a peptide chelated calcium product; the peptide obtained by the invention has higher calcium binding capacity, and the peptide chelated calcium product prepared after the peptide is bound with calcium ions has a crystal structure.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method of cornu cervi pantotrichum root ossified tissue source peptide chelated calcium.
Background
The peptide chelated calcium is a nutrition enhancer which is obtained by taking small molecular peptides obtained by hydrolyzing proteins as raw materials and combining the small molecular peptides with calcium ions. The small molecular peptide can be directly used as a carrier for calcium ion transmembrane transport, and has the advantages of low energy consumption, high transport speed, low carrier saturation tendency and the like.
Currently, the existing calcium peptide chelate is mostly derived from food-derived protein hydrolysates, such as casein phosphopeptide, whey protein peptide, soy protein peptide, wheat germ protein peptide, pig bone protein peptide and the like. The peptides from different sources can provide essential amino acids for human bodies, so that the amino acid balance in the human bodies is better kept, and negative effects on the human bodies are avoided.
The invention aims to provide a preparation method of deer antler root ossified tissue source peptide chelated calcium by taking deer antler root ossified tissue enzymolysis polypeptide as a raw material, so as to obtain three peptides with high calcium binding capacity, and prove that the peptides can perform chelation reaction with calcium ions to form a peptide chelated calcium product.
Disclosure of Invention
The invention aims to provide a preparation method of cornu cervi pantotrichum root ossified tissue source peptide chelated calcium, and aims to provide a preparation method of the cornu cervi pantotrichum root ossified tissue source peptide chelated calcium.
For raw materials, Os Cervi, Carnis Cervi, deer tray and deer placenta containing bioactive components can also be selected; the invention adopts cartialgenous root ossified tissue as a raw material, peptides are obtained by separation and purification through enzymolysis, hydroxyapatite chromatography, gel filtration chromatography and reversed-phase high performance liquid chromatography, and the sequences are respectively TKLGTQLQL (TL9), LETVILGLLKT (LT11) and KMVFLMDLLK (KK10) through LC-MS/MS identification, the three peptides have good calcium binding activity, and the formed peptide chelated calcium product has a certain crystal structure.
The invention is realized in such a way that the preparation method of the antler root ossified tissue source peptide chelated calcium comprises the following steps:
1) performing enzymolysis reaction on ossified tissues of the root of the pilose antler through flavourzyme (derived from aspergillus oryzae), and performing freeze drying treatment on obtained enzymolysis products;
2) separating and purifying the enzymolysis product in the step 1) to obtain a peptide with higher calcium binding capacity;
3) and (3) carrying out chelation reaction on the peptide and calcium ions to obtain a peptide chelated calcium product.
In a further technical scheme, in the enzymolysis reaction in the step 1): the enzymolysis time is 4-6 h; the enzymolysis temperature is 40-60 ℃; the pH value is 7.5-9.5; the concentration of the substrate is 2-10%; the amount of the flavourzyme (from aspergillus oryzae) is 6000-; the obtained enzymolysis product is pre-frozen at-20 deg.C for 10-12h, then pre-frozen at-80 deg.C for 10-12h, and finally freeze-dried under 1-10Pa to completely dry state and stored.
In the further technical scheme, in the step 2), the enzymolysis product is purified by hydroxyapatite chromatography, and in the process: controlling the flow rate to be 1.8-2.0ml/min, selecting 200-400mM phosphate buffer solution as eluent concentration, setting the time interval of a collector to be 2min, sequentially collecting peak components, detecting the wavelength at 215nm, and collecting the component with the highest calcium binding activity for next purification.
In the step 2), the components of the enzymolysis product separated by the hydroxyapatite chromatography are further separated by using gel filtration chromatography, and in the process: controlling the flow rate at 0.3-0.5ml/min, eluting with deionized water, setting the time interval of the collector at 4min, collecting the peak components, detecting the wavelength at 215nm, and collecting the component with the highest calcium binding activity for further purification.
In the step 2), the components after the gel filtration chromatography are further separated by using a reversed-phase high performance liquid chromatography, wherein the process comprises the following steps: controlling the flow rate to be 0.8-1.0ml/min, wherein the mobile phase is as follows: the solution A is deionized water (containing 0.05 percent of trifluoroacetic acid), and the solution B is acetonitrile (containing 0.05 percent of trifluoroacetic acid); eluting with gradient of 0-25% B for 20min, collecting peak components, detecting wavelength at 215nm, and collecting the component with the highest calcium binding activity for peptide sequence identification.
In the step 3), the calcium ions are derived from anhydrous calcium chloride; in the chelation reaction, the mass ratio of the peptide to the anhydrous calcium chloride is 4:1-6: 1; the temperature of the chelation reaction is 45-55 ℃; the pH value of the chelation reaction is 7.0-8.0; the chelating reaction time is 50-70 min.
The further technical scheme is that the peptides TL9, LT11 and KK10 separated by the reversed-phase high performance liquid chromatography are identified by LC-MS/MS, and the sequences are TKLGTQLQL, LETVILGLLKT and KMVFLMDLLK respectively; through database comparison, the genes are respectively derived from toll-like receptor 3 short type, annexin A2 and DNA helicase ERCC 6L; the calcium binding capacity is 87.68 +/-2.86%, 80.72 +/-0.93% and 67.97 +/-0.98%, respectively.
The further technical scheme is that the product after the chelation reaction is added into absolute ethyl alcohol with a certain volume, then the mixture is refrigerated at low temperature for a period of time, after peptide and calcium ions form flocculent precipitate, the flocculent precipitate is centrifugally separated under the conditions of 6200rpm and 4 ℃, and the supernatant is discarded to obtain precipitate, namely the peptide chelated calcium product.
Compared with the prior art, the invention has the following beneficial effects:
(1) the raw materials adopted by the method are taken from the ossified part of the root of the pilose antler, the ossification of the part is serious, the part belongs to the part with poor quality in the pilose antler, but the growth speed of the part can reach 1.7cm/day, the part is the only mammal organ which can be regenerated, and the parts determine that the peptide or the protein chelated with the calcium is rich, and the part is the source of the high-quality calcium chelated peptide.
(2) The invention provides a preparation method of cornu cervi pantotrichum root ossified tissue source peptide chelated calcium, which is characterized in that peptides TL9, LT11 and KK10 are obtained, the sequences of the peptides are identified as TKLGTQLQL, LETVILGLLKT and KMVFLMDLLK, and the calcium binding capacities of the peptides are 87.68 +/-2.86%, 80.72 +/-0.93% and 67.97 +/-0.98% respectively; wherein the calcium binding capacity of TL9 and LT11 is higher than that of the porcine ossein peptide obtained by the prior separation technology.
(3) Through sequence identification, the peptide obtained by the invention is found for the first time and has higher calcium binding capacity; the peptide obtained by the invention can be used as a carrier of calcium ions, and can be subjected to chelation reaction with the calcium ions to prepare the peptide chelated calcium product.
Drawings
Fig. 1 and 2 are graphs showing different components collected after separation by hydroxyapatite chromatography and binding energy thereof to calcium in the present invention.
FIGS. 3 and 4 are graphs showing different fractions collected after separation by gel filtration chromatography and binding energy thereof to calcium in the present invention.
FIGS. 5 and 6 are graphs showing different fractions collected after separation by reversed-phase high performance liquid chromatography and binding energy thereof to calcium in the present invention.
FIGS. 7, 8, 9 and 10 show three peptide sequences identified by LC-MS/MS and their binding ability to calcium in the present invention.
FIG. 11 is an infrared spectrum of the peptide TL9 of the present invention before and after binding to calcium ion.
FIG. 12 is a graph showing infrared spectra of a peptide LT11 of the present invention before and after binding to calcium ion.
FIG. 13 is a graph of the UV-Vis spectra of the peptide TL9 of the present invention before and after binding to calcium ion.
FIG. 14 is a graph showing the ultraviolet-visible spectrum of the peptide LT11 of the present invention before and after binding to calcium ion.
FIG. 15 is an X-ray diffraction pattern of the peptide TL9 of the present invention before and after binding to calcium ions.
FIG. 16 is an X-ray diffraction pattern of the peptide LT11 of the present invention before and after binding to calcium ion.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Specific implementations of the present invention are described in detail below with reference to specific embodiments.
The invention provides a preparation method of cornu cervi pantotrichum root ossified tissue source peptide chelated calcium, which comprises the following steps:
step 1): preparing an enzymolysis product of ossified tissues of the root of the pilose antler:
taking fresh cornu Cervi Pantotrichum root ossified tissue slices, washing with deionized water to neutrality, drying at room temperature, pulverizing, and sieving with 100 mesh sieve; the influence of enzyme type, pH value, time, temperature, enzyme dosage and substrate concentration on the hydrolysis degree and calcium binding capacity of the enzymolysis product is examined. The method comprises the following steps:
taking 2g of cornu Cervi Pantotrichum root ossified tissue powder, adding 20ml of deionized water, preparing into solution with substrate concentration of 10%, enzyme dosage of 7000u/g, enzymolysis time of 5h, examining influence of neutral protease (50 deg.C, pH 7.5), alkaline protease (45 deg.C, pH 9.5), papain (55 deg.C, pH 6.0), trypsin (50 deg.C, pH 8.0), and flavourzyme (derived from Aspergillus oryzae) (50 deg.C, pH 7.5) on hydrolysis degree and calcium binding ability of enzymolysis product, and setting 3 parallel samples in each group.
Taking 2g of cornu Cervi Pantotrichum root ossified tissue powder, adding 20ml of deionized water, preparing into solution with substrate concentration of 10%, enzyme dosage of 7000u/g, enzymolysis time of 5h, temperature of 50 deg.C, adjusting pH value to 7.5, 8.5 and 9.5 with 0.5mol/l NaOH solution, and setting 3 parallel samples in each group; the degree of hydrolysis and calcium binding capacity of the enzymatic hydrolysate were determined.
Taking 2g of cornu Cervi Pantotrichum root ossified tissue powder, adding 20ml of deionized water, preparing into solution with substrate concentration of 10%, enzyme dosage of 7000u/g, adjusting pH to 8.5, setting temperature to 50 deg.C, setting enzymolysis time to 4, 5 and 6h, setting 3 parallel samples in each group, and determining hydrolysis degree and calcium binding capacity of enzymolysis product.
Taking 2g of cornu Cervi Pantotrichum root ossified tissue powder, adding 20ml of deionized water, preparing into solution with substrate concentration of 10%, enzyme dosage of 7000u/g, enzymolysis time of 5h, adjusting pH to 8.5, setting temperature to 40, 50 and 60 deg.C respectively, setting 3 parallel samples in each group, and determining hydrolysis degree and calcium binding capacity of enzymolysis product.
Taking 2g of cornu Cervi Pantotrichum root ossified tissue powder, adding 100 ml, 33 ml and 20ml of deionized water respectively, preparing into solution with substrate concentration of 2%, 6% and 10%, enzyme dosage of 7000u/g, enzymolysis time of 5h, adjusting pH value to 8.5, temperature to 50 deg.C, setting 3 parallel samples in each group, and determining hydrolysis degree and calcium binding capacity of enzymolysis product.
Taking 2g of cornu Cervi Pantotrichum root ossified tissue powder, adding 20ml of deionized water, preparing into solution with substrate concentration of 10%, adjusting pH to 8.5, setting temperature to 50 deg.C, setting enzymolysis time to 5h, adjusting enzyme dosage to 6000, 7000 and 8000u/g, setting 3 parallel samples in each group, and determining hydrolysis degree and calcium binding capacity of enzymolysis product.
Specifically, the calculation formula of the degree of hydrolysis is as follows:
(DH is hydrolysis degree determined by the percentage of alpha-amino nitrogen to total protein nitrogen; TPN is total protein nitrogen, total protein nitrogen is detected by Kjeldahl method, protein is total nitrogen amount multiplied by 6.25; AN is alpha amino nitrogen; V is total volume of enzymolysis liquid.)
Determination of AN by a neutral formaldehyde method: titrating the enzymolysis product (5ml) with 0.05mol/l NaOH titration solution to pH value of 8.2; adding 10ml of neutral formaldehyde, and titrating with 0.05mol/l NaOH titration solution to a pH value of 9.2; the consumption of 0.05mol/l NaOH in the blank (the sample was replaced by deionized water) was recorded as V0. The volume of 0.05mol/l NaOH consumed after the addition of formaldehyde was recorded as V1;
wherein, the determination method of the calcium binding capacity comprises the steps of equivalently mixing 5mg/ml peptide and 1mg/ml calcium ions, shaking and shaking uniformly for 60min at the temperature of 50-55 ℃, keeping the pH value at 7.0-7.5, detecting the total calcium content in the solution by a flame atomic absorption spectrophotometer, then adding 9 times of absolute ethyl alcohol by volume, centrifuging for 10min at the temperature of 4 ℃ and 6200rpm, and then taking the supernatant to determine the calcium content in the supernatant.
Formula for calculation of calcium binding capacity:
pre-freezing the obtained enzymolysis product under the optimal enzymolysis condition at-20 deg.C for 12h and-80 deg.C for 12h, freeze-drying under 1-10Pa for 24-48h to completely dry state, and storing.
Step 2): hydroxyapatite chromatography separation and purification peptide
Separating the enzymolysis product obtained in the step 1) by using a ceramic hydroxyapatite chromatography chromatographic column with the pore diameter of 80 mu m, wherein the separation process comprises the following steps:
column assembling: vertically fixing a chromatographic column, taking hydroxyapatite filler powder with certain mass, soaking and swelling the hydroxyapatite filler powder with deionized water, stirring the column material in a J shape until the hydroxyapatite filler powder is uniformly dispersed, draining the mixture by using a glass rod, pouring the mixture into the chromatographic column, screwing a valve at the upper end of the column when the column material is uniformly precipitated to the bottom, and finishing column packing;
balancing: setting the flow rate of a peristaltic pump to be 1.8-2.0ml/min, balancing the chromatographic column by using a balance liquid (5mM phosphate) with the volume of 5-10 times of the column volume until the light absorption value measured under 215nm is equal to the balance liquid, and finishing the balancing;
loading and eluting: dissolving 200-500mg of enzymolysis product freeze-dried powder in 1ml of deionized water, filtering the solution through a 0.22 mu m filter membrane for later use, controlling the flow rate to be 1.8-2.0ml/min, and selecting 200mM-400mM phosphate buffer solution for elution, wherein the sample loading amount is 18-20 ml;
collecting and measuring: collecting one tube every 2min by a collector, measuring light absorption value at 215nm, drawing an elution curve, sequentially collecting peak components H1, H2 and H3, and detecting the calcium binding capacity of the peak components, wherein the results are shown in figure 1 and figure 2; freeze drying the component with highest calcium binding capacity, and storing for use.
Step 3): separation and purification of peptide by gel filtration chromatography
Separating the H3 fraction obtained in step 2) by using a Sephadex G-25(Bio-rad) gel filtration chromatography chromatographic column; the separation process comprises the following steps:
pretreatment of the gel: taking 300g of gel, adding 1l of deionized water, boiling for 2h, stirring intermittently to fully swell the gel, avoiding excessive stirring to avoid particle crushing, standing for settling, pouring out deionized water and unsettled particles, repeating the steps for several times until no suspended matters exist, and filling the gel into a column;
column assembling: the chromatographic column is fixed vertically, the valve at the bottom is screwed, and deionized water with the volume equivalent to 1/3 of the height of the column is injected to prevent bubbles from being generated in the process of gel sedimentation. Draining the gel with a glass rod, slowly pouring into the chromatographic column along the wall at a constant speed, sucking part of supernatant when the colloid in the column is settled to a certain height, stirring the interface, continuously filling the rest of the colloid, repeatedly circulating until the distance between the colloid surface and the uppermost port of the chromatographic column is about 5cm, screwing a valve at the upper end of the column, and finishing column filling;
balancing: setting the flow rate of the peristaltic pump to be 0.3-0.5ml/min, balancing the chromatographic column by using deionized water with 2-3 times of column volume until the measured light absorption value under 215nm is equal to that of the deionized water, and finishing the balancing.
Loading and eluting: 800-1000mg of the component H3 was weighed out and dissolved in 1ml of deionized water, the amount of the sample was 1ml, and the solution was filtered through a 0.22 μm filter for further use. Closing the peristaltic pump, opening a valve at the upper end of the chromatographic column, sucking off deionized water on the upper layer of the gel surface until the deionized water is tangent to the surface of the gel, carefully and slowly adding the treated sample into the chromatographic column along the wall, opening the peristaltic pump to allow the sample solution to completely enter the gel surface, washing residual sample on the wall of the tube with a small amount of deionized water, filling the deionized water when the liquid level is tangent to the gel surface again, covering the valve to ensure screwing, and eluting with deionized water at the flow rate of 0.3-0.5 ml/min;
collecting and measuring: setting a collector to collect one tube every 2min, using deionized water to zero at 215nm, measuring the light absorption value, drawing an elution curve, and sequentially collecting different sample components S1, S2 and S3, wherein the results are shown in FIG. 3 and FIG. 4; collecting the fraction with highest calcium binding ability, freeze drying, and storing.
Step 4): separating and purifying peptide by reversed phase high performance liquid chromatography
Separating the S3 fraction obtained in step 3) by C-18 semi-preparative reverse phase high performance liquid chromatography; the separation process comprises the following steps:
the experiment adopts a C18 semi-preparative RP-HPLC chromatographic column (Zorbax 9.4X 250mm), and the sample is processed by dissolving the S3 component in deionized water, wherein the sample concentration is 600mg/ml, the sample injection amount is 200 mu l, the sample is reserved after passing through a 0.22 mu m filter membrane, and the flow rate is 1 ml/min. Mobile phase: the solution A is deionized water (containing 0.05% of trifluoroacetic acid), and the solution B is acetonitrile (containing 0.05% of trifluoroacetic acid). Eluting with gradient of 0-25% B for 20min at detection wavelength of 215nm, sequentially collecting peak components F1 and F2, and detecting calcium binding capacity, with the results shown in FIG. 5 and FIG. 6; collecting the fraction with highest calcium binding ability, freeze drying, and storing.
Step 5): identification of peptide sequences by LC-MS/MS
Using the F1 fraction obtained in step 4) for identifying peptide sequences, this example provides a method for LC-MS/MS identification of peptide sequences comprising the steps of: the peptide fragment was dissolved in 20. mu.l of a dissolving solution (0.1% formic acid, 5% acetonitrile), vortexed sufficiently with shaking, centrifuged at 6200rpm at 4 ℃ for 20min, and the supernatant was transferred to a sample tube and 8. mu.l of the supernatant was aspirated for mass spectrometric identification.
Setting mass spectrum parameters:
database retrieval was performed using PEAKS software.
As shown in FIGS. 7-9, the present invention identifies three peptides with high calcium binding ability derived from the ossified tissue of the root of velvet antler, whose sequences are TKLGTQLQL, LETVILGLLKT and KMVFLMDLLK, and the three peptides are novel peptides with high calcium binding ability according to database search. As shown in FIG. 10, the calcium binding capacity of the three peptides was 87.68. + -. 2.86%, 80.72. + -. 0.93% and 67.97. + -. 0.98%, respectively.
Step 6): preparation of the peptide chelated calcium product and structural characterization
The method for preparing the peptide chelated calcium comprises the following steps: the peptide and the anhydrous calcium chloride are shaken and shaken for 50-70min at the temperature of 45-55 ℃ according to the mass ratio of 4:1-6:1, the pH value is kept at 7.0-8.0, and the chelating time is 50-70 min; adding 9 times volume of anhydrous ethanol to precipitate chelate, centrifuging at 4 deg.C and 6200rpm for 10min, removing supernatant to obtain flocculent precipitate, pre-freezing at-20 deg.C for 12 hr and-80 deg.C for 12 hr, and freeze-drying in a freeze-drying machine under 1-10 Pa;
fourier transform infrared spectrum analysis is carried out on the prepared peptide chelated calcium: peptide or peptide chelated calcium 1mg was mixed homogeneously with 100mg of KBr and then loaded on FTIR instrument. The absorption spectrum was recorded at 400-4000cm-1And (4) a region. The peak signals in the spectra were analyzed by the oncom software, and the results are shown in fig. 11 and 12. In FIG. 11, 3291.41cm of the spectrum of TL9-1The absorption peak is caused by N-H stretching vibration, and the absorption peak in the map of the peptide chelated calcium is red-shifted to 3426.28cm-1Description of Ca2+and-NH2Binding occurs; at 1664.75cm-1The characteristic absorption peak is mainly due to the stretching vibration of amido bond (C ═ O), and the original 1664.7cm after chelation-1Blue shifted to 1644.50cm-1The change in position indicates that the carboxylic acid group is covalently bound to the calcium ion; 1539.40cm after TL9 is combined with calcium ions-1The absorption peak at (A) is shifted to 1552.42cm-1This suggests that C-N may be involved in the chelation of calcium ions; in the fingerprint area, 1139.14cm-1The absorption peak at (A) is shifted to 1150.33cm-1Such shift of absorption peak may be caused by formation of-COOCa through chelation reaction of carbonyl oxygen and calcium ion. In FIG. 12, LT11 is in contact with Ca2+After combination, originally 3281.77cm-1The absorption peak at (A) is shifted to 3425.44cm-1This is due to stretching of the N-H bond; LT11 at 1629.55cm-1And 1535.54cm-1The absorption peaks observed were amide-I vibration caused by C ═ O bond stretching vibration and amide-II vibration caused by C-N bond, respectively, and LT11-Ca moved to 1637.75cm at amide-I-1The absorption peak at amide-II was shifted to 1550.49cm-1This indicates that C ═ O and C-NPossibly participating in the binding of calcium ions. 1000-500cm-1The change of multiple absorption peaks can be caused by C-H stretching and N-H bending coupling vibration. FIGS. 11 and 12 illustrate that the peptide undergoes a chelation reaction with calcium ions, and that the chelation reaction may be with respect to the carboxyl oxygen atom and the amino nitrogen atom;
performing ultraviolet and visible light spectrum analysis on the prepared peptide chelated calcium: the peptide and the peptide chelated calcium were dissolved in deionized water, respectively, at a concentration of 1 mg/ml. And recording the spectrum of the sample by using an ultraviolet-visible spectrophotometer, wherein the wavelength range is 200-400 nm. Before measurement, the ultraviolet-visible spectrophotometer was blank-corrected with deionized water, and the results are shown in fig. 13 and 14. In fig. 13, TL9 shows a high-intensity absorption peak near 200nm, and shows a red shift phenomenon when chelated with calcium ions, which indicates a characteristic peak resulting from n → pi transition of the amide bond C ═ O in the peptide chain. In FIG. 14, LT11 produced a strong absorption peak near 220nm, a possible explanation being that the peptide's chromosphere and co-pigment undergo chiral changes upon addition of calcium. The peptide bound to calcium results in a change in the electron cloud of the amide bond and the light absorption properties of the ligand;
the prepared peptide chelated calcium is subjected to X-ray diffraction pattern analysis: 2mg of peptide and peptide chelated calcium were ground uniformly, and then the sample was loaded into a sample plate and placed in a holder, and the diffraction data was collected by a diffractometer. The scanning angle was 10-80 deg., and the scanning speed was 5 deg.C/min, the results are shown in FIGS. 15 and 16. As shown in fig. 15, the strongest peak in TL9 occurs between 17 ° and 21 ° in 2 θ, being a widely dispersed diffraction peak, indicating that the peptide does not form an ordered arrangement in the structure, being an amorphous structure. TL9-Ca appears with a plurality of sharp diffraction peaks at a plurality of positions, which shows that TL9 and Ca2+The chelation reaction is carried out, the self structure is greatly changed, and a new crystal structure is formed. As shown in fig. 16, before and after the LT11 is chelated with calcium ions, the X-ray diffraction pattern is significantly changed, LT11 as an organic matter has a broad peak at 20 °, and after the LT11 is combined with calcium ions, the X-ray diffraction pattern of peptide chelated calcium has a plurality of weak diffuse type diffraction peaks, which indicates that after the peptide is combined with calcium ions, the structure of the peptide is greatly changed, and a stable structure similar to crystals is formed; FIGS. 15 and 16 illustrateThe peptide forms a stable crystal structure upon binding to calcium ions.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (7)
1. A preparation method of cornu cervi pantotrichum root ossified tissue source peptide chelated calcium is characterized by comprising the following steps:
1) performing enzymolysis on ossified tissue of cornu Cervi Pantotrichum root with flavourzyme (from Aspergillus oryzae), and freeze drying the obtained enzymolysis product;
2) separating and purifying the enzymolysis product in the step 1) to obtain a peptide with higher calcium binding capacity;
3) and (3) carrying out chelation reaction on the peptide and calcium ions to obtain a peptide chelated calcium product.
2. The method for preparing the cornu cervi pantotrichum root ossified tissue source peptide chelated calcium according to claim 1, wherein in the enzymolysis reaction in the step 1): the enzymolysis time is 4-6 h; the enzymolysis temperature is 40-60 ℃; the pH value is 7.5-9.5; the concentration of the substrate is 2-10%; the amount of the flavourzyme (from aspergillus oryzae) is 6000-; the obtained enzymolysis product is pre-frozen at-20 deg.C for 12h, then pre-frozen at-80 deg.C for 12h, and finally freeze-dried under 1-10Pa to completely dry state and stored.
3. The method for preparing the cornu cervi pantotrichum root ossified tissue-derived peptide chelated calcium according to claim 1, wherein in step 2), the enzymolysis product is purified by hydroxyapatite chromatography, and in the process: controlling the flow rate to be 1.8-2.0ml/min, selecting 200-400mM phosphate buffer solution as eluent concentration, setting the time interval of a collector to be 2min, collecting each peak component, detecting the wavelength at 215nm, and collecting the component with the highest calcium binding activity for next purification.
4. The method for preparing the cornu cervi pantotrichum root ossified tissue-derived peptide chelated calcium according to claim 3, wherein in step 2), the components of the enzymolysis product separated by hydroxyapatite chromatography are further separated by gel filtration chromatography, in the process: controlling the flow rate at 0.3-0.5ml/min, eluting with deionized water, setting the time interval of the collector at 4min, collecting the peak components, detecting the wavelength at 215nm, and collecting the component with the highest calcium binding activity for further purification.
5. The method for preparing cornu Cervi Pantotrichum root ossified tissue source peptide chelated calcium as claimed in claim 4, wherein in step 2), the components after gel filtration chromatography are further separated by reversed phase high performance liquid chromatography, during which: controlling the flow rate to be 0.8-1.0ml/min, wherein the mobile phase is as follows: the solution A is deionized water (containing 0.05 percent of trifluoroacetic acid), and the solution B is acetonitrile (containing 0.05 percent of trifluoroacetic acid); eluting with gradient of 0-25% B for 20min, collecting peak components, detecting wavelength at 215nm, collecting components with highest calcium binding activity, and identifying peptide sequences of TKLGTQLQL, LETVILGLLKT and KMVFLMDLLK.
6. The method for preparing the cornu cervi pantotrichum root ossified tissue-derived peptide chelated calcium according to claim 1, wherein in step 3), the calcium ion is derived from anhydrous calcium chloride; in the chelation reaction, the mass ratio of the peptide to the anhydrous calcium chloride is 4:1-6: 1; the temperature of the chelation reaction is 45-55 ℃; the pH value of the chelation reaction is 7.0-8.0; the chelating reaction time is 50-70 min.
7. The method for preparing the peptide-chelated calcium from the ossified tissue of pilose antler root as claimed in claim 6, wherein the product of the chelation reaction is added into a certain volume of absolute ethanol, then refrigerated at a low temperature for a period of time, after the peptide and calcium ions form flocculent precipitate, the peptide-chelated calcium product is obtained by centrifugal separation at 6200rpm and 4 ℃, and the supernatant is discarded.
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