CN112812175A - Application of high-glutamic acid donkey collagen active peptide - Google Patents
Application of high-glutamic acid donkey collagen active peptide Download PDFInfo
- Publication number
- CN112812175A CN112812175A CN202110143314.7A CN202110143314A CN112812175A CN 112812175 A CN112812175 A CN 112812175A CN 202110143314 A CN202110143314 A CN 202110143314A CN 112812175 A CN112812175 A CN 112812175A
- Authority
- CN
- China
- Prior art keywords
- donkey
- collagen peptide
- peptide
- glutamic acid
- donkey collagen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Links
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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Abstract
The invention discloses application of a high-glutamic acid donkey collagen active peptide, and belongs to the technical field of functional product development. The invention adopts a method of composite enzymolysis and affinity column chromatography to separate and obtain the high-glutamic acid donkey collagen active peptide from donkey collagen, the proportion of glutamic acid is 50-75% relative to glycine, the donkey collagen active peptide has strong metal chelating capacity, the chelating activity to ferrous ions is 4.7-32.8 mug/mg, the chelating activity to calcium ions is 0.9-24.6 mug/mg, and the chelating activity to zinc ions is 1.2-23.2 mug/mg. The donkey collagen peptide iron chelate is obtained by chelating donkey collagen active peptide with metal ions, has obvious improvement effect on iron deficiency anemia, and can enhance the immunity level of an organism. The high-glutamic acid donkey collagen active peptide prepared by the invention can be used for producing health care products, foods and medicines, has high nutritive value, has various effects of enriching blood and benefiting qi, nourishing yin and moistening dryness, resisting aging, enhancing immunity and the like, and opens up a new way for promoting the diversification and quality improvement of the traditional donkey collagen product.
Description
Technical Field
The invention relates to application of a high-glutamic acid donkey collagen active peptide, and belongs to the technical field of functional product development.
Background
The growth and metabolism of human body are closely related to the existence of various essential elements, and the deficiency, excess or imbalance of the element intake can cause the abnormality of the physiological function of the body or the occurrence of diseases to different degrees. In recent years, inorganic metal salts, including ferrous sulfate, calcium carbonate, and zinc chloride, have become common element supplements. However, these inorganic metal supplements tend to form insoluble materials in the alkaline intestinal environment, which severely affects the absorption and utilization efficiency of the human body. Researchers have been searching for calcium, iron, zinc and other nutrition enhancers with low side effects and high bioavailability. A third generation amino acid metal element supplement, such as ferrous glycinate, appears after a second generation organometallic iron supplement, exemplified by ferrous lactate, zinc gluconate. They are stable, increase the solubility of minerals in the intestine, and are believed to potentially promote the absorption of metal ions and increase their bioavailability. However, these amino acid chelates are costly and cause fat oxidation with side effects, and are not effective enough. Compared with amino acids, the biological value and the nutritional value of the peptide are higher than those of single amino acid. The peptide can be transported to intestinal epithelial cells through a cell bypass channel, does not need to consume energy, and is easier to be absorbed by the body. The peptide can be used as a carrier of metal ions, forms a chelate with the metal ions, plays a role in protecting the metal ions and simultaneously promotes the absorption of the metal ions. The metal peptide chelate can avoid the damage of directly absorbing inorganic ions to the gastrointestinal tract, simultaneously keep the physiological activity of metal ions and enhance the bioavailability of calcium, iron and zinc ions.
The metal binding activity of a peptide can be influenced by the type of protease, the molecular weight of the peptide, the amino acid composition, and the specific amino acid content. Glutamic acid is used as dicarboxylic acid amino acid, is rich in chelating group, has strong metal chelating activity, can protect liver and improve intelligence development, and the reaction of the glutamic acid and the metal ion is more complex than the chelating reaction of glycine and the metal ion. The contents of 17 amino acids in 18 donkey-hide gelatins of enterprises are respectively measured by a full-automatic amino acid analyzer, and the results show that the contents of various amino acids in the donkey-hide gelatins of different enterprises have certain differences, but the contents of the amino acids are respectively the highest glycine content and the next proline content; wherein, the content of glutamic acid is 37-42 percent relative to glycine (Geshenyu, Poncirus, Prunus serrulata, Zhengjie, Youlong, Yanhuan, Shenyuping, 18 family donkey-hide gelatin content analysis and comparative study [ J ] Chinese pharmacy 2017,28(01): 122-.
In the prior art in the field, different peptide fragments are separated from donkey collagen peptide only by enzymolysis and ultrafiltration interception according to molecular weight, namely collagen is hydrolyzed into small molecular peptide to be beneficial to digestion and absorption, but the content of the obtained peptide is low and the functionality is not obviously improved. Patent CN 107904274A provides a preparation method of colla Corii Asini active peptide, and 1000-2000 Da peptide is considered to play a main active role. However, the peptide alone has no blood-replenishing effect, and from the viewpoint of the traditional Chinese medicine coordination theory, the traditional donkey-hide gelatin is ignored to be taken in the form of mixed meal (such as donkey-hide gelatin cake), and the synergistic effect of the peptide and metal ions in the digestion process is ignored.
Disclosure of Invention
Aiming at the problems existing at present, the high-glutamic acid donkey collagen active peptide is obtained by separation by adopting a composite enzymolysis and column chromatography method, in the amino acid composition, the content of glutamic acid can reach 50-75% and the peptide content reaches 95-99% relative to glycine, so that the high-glutamic acid donkey collagen active peptide has strong divalent metal chelating capacity, and has excellent effects on the aspects of improving anemia and calcium deficiency, supplementing trace elements, improving body immunity and the like.
The invention aims to provide a high-glutamic acid donkey collagen active peptide, a preparation method of the high-glutamic acid donkey collagen active peptide, a donkey collagen polypeptide metal chelate prepared by the preparation method, and application of the donkey collagen polypeptide metal chelate in the aspects of improving anemia, supplementing elements and improving body immunity. Provides theoretical support for improving the economic value, the product nutrient utilization rate and the reasonable resource utilization of the donkey industry and developing a novel dietary supplement.
The invention provides donkey collagen peptide, wherein the content of glutamic acid in the donkey collagen peptide is 50-75%, the content of peptide with the relative molecular mass of 180-2000 Da in the donkey collagen peptide is more than 70% of the total donkey collagen peptide, and the content of the peptide is 95-99%.
The present invention provides a method for preparing the donkey collagen peptide according to claim 1, comprising the steps of:
(1) enzymolysis and digestion: selecting donkey-derived collagen, carrying out in-vitro digestion by adopting a composite enzymolysis technology, inactivating enzymes, centrifuging, and taking supernatant fluid, namely collagen polypeptide enzymolysis liquid;
(2) separation preparation: and (3) performing column chromatography and elution by using eluent on the collagen polypeptide hydrolysate, separating components with different adsorption strengths, collecting peptide components with strong adsorption force, performing nanofiltration, concentration, desalination and freeze drying to obtain the high-glutamic acid donkey collagen peptide.
In one embodiment, the donkey-derived collagen of step (1) is one or more of donkey-hide gelatin, donkey hide, donkey bone, and donkey hoof.
In one embodiment, the compound enzymolysis technology in step (1) is enzymolysis using two or more of pepsin, trypsin, alkaline protease, papain, and neutral protease.
In one embodiment, when the enzymes are papain and alkaline protease, stepwise enzymatic hydrolysis is used: the concentration of an enzymolysis substrate is 5-10%, papain with the final concentration of 1000-5000U/mL is added into an enzymolysis system, the pH is kept at 7.0-9.0, the temperature is kept at 40-60 ℃, enzymolysis is carried out for 2-4 hours, alkaline protease with the final concentration of 500-3000U/mL is added into the enzymolysis system, the pH is kept at 9.0-12.0, the temperature is kept at 40-60 ℃, and enzymolysis is carried out for 2-4 hours.
In one embodiment, when the enzymes are pepsin and trypsin, stepwise enzymatic hydrolysis is used: the concentration of an enzymolysis substrate is 5-10%, pepsin with the final concentration of 1000-3000U/mL is added into an enzymolysis system, the pH is kept at 2.5-3.5, the temperature is kept at 30-40 ℃, enzymolysis is carried out for 2-4 hours, trypsin with the final concentration of 300-600U/mL is added into the enzymolysis system, the pH is kept at 6.0-8.0, the temperature is kept at 30-40 ℃, and enzymolysis is carried out for 2-4 hours.
In one embodiment, when the enzymes are neutral and alkaline proteases, stepwise enzymatic hydrolysis is used: the concentration of an enzymolysis substrate is 5-10%, neutral protease with the final concentration of 1000-3000U/mL is added into an enzymolysis system, the pH is kept at 6.0-8.0, the temperature is kept at 40-50 ℃, enzymolysis is carried out for 2-4 hours, alkaline protease with the final concentration of 2000-4000U/mL is added into the enzymolysis system, the pH is kept at 8.0-10.0, the temperature is kept at 40-50 ℃, and enzymolysis is carried out for 2-4 hours.
In one embodiment, the enzyme deactivation temperature in the step (1) is 90-100 ℃, and the enzyme deactivation time is 3-15 min; the centrifugal rotating speed is 6000-10000 r/min, and the centrifugal time is 1-30 min.
In one embodiment, the column chromatography of step (2) utilizes immobilized metal affinity chromatography, and the medium base comprises agarose, chitosan, or dextran.
In one embodiment, 40-80% of dimethyl sulfoxide is used as a detergent, epichlorohydrin is used as an activator, the activation temperature is 30-60 ℃, and the activation time is 2-4 h; iminodiacetic acid is used as a grafting ligand, the grafting temperature is 50-70 ℃, and the grafting time is 8-16 h.
In one embodiment, the step (2) of separating the components with different adsorption strengths and collecting the peptide components with strong adsorption strength comprises: eluting with buffer solution A until the baseline is stable, and eluting with buffer solution B (0.02mol/L disodium hydrogen phosphate, 0.05mol/L NaCl) to obtain peptide component with strong adsorption.
In one embodiment, the buffer A contains 0.05 to 0.1mol/L sodium acetate, 0.05 to 0.1mol/L NaCl.
In one embodiment, buffer B comprises 0.02 to 0.1mol/L disodium hydrogen phosphate, 0.05 to 0.1mol/L NaCl.
The invention provides a donkey collagen peptide metal chelate prepared by using the donkey collagen peptide, and the donkey collagen peptide is chelated with metal ions to obtain the donkey collagen peptide metal chelate.
The invention provides a method for preparing donkey collagen peptide metal chelate, which comprises the steps of adjusting the pH of collagen peptide liquid to be 5.0-7.0, adjusting the mass ratio of collagen peptide to metal ions to be 1: 1-4: 1, carrying out chelation reaction at the temperature of 30-60 ℃ for 30-90 min, filtering, taking supernatant, and carrying out freeze drying to obtain the donkey collagen peptide metal chelate.
In one embodiment, the metal ion is one or more of iron, calcium, zinc.
The invention provides application of the donkey collagen peptide, or the donkey collagen peptide metal chelate compound of claim 5, or a product containing the donkey collagen peptide metal chelate compound in improving body immunity and iron deficiency anemia.
In one embodiment, the product may be used to include a medicament, a pharmaceutical composition, or a pharmaceutical excipient.
In one embodiment, the medicament or the pharmaceutical composition further comprises microcapsules, microspheres, nanoparticles and liposomes, and the pharmaceutic adjuvant comprises an excipient and an additive.
In one embodiment, the product includes, but is not limited to, a food product, a nutraceutical beverage, an enteral nutritional formulation, a dietary supplement, a veterinary drug, or a feed additive.
The invention has the beneficial effects that: according to the high-glutamic acid donkey collagen active peptide provided by the invention, through amino acid composition analysis, the content of glutamic acid is 45% -75% relative to glycine, the molecular mass is intensively distributed in 180-2000 Da, the high-glutamic acid donkey collagen active peptide has strong divalent metal ion chelating capacity, and a donkey collagen peptide metal chelate prepared by chelating with metal ions can effectively improve the symptoms of iron-deficiency anemia, obviously improve the immune function of an organism, can be used for developing a novel dietary supplement, provides theoretical support for improving the economic value of donkey industry, improving the product nutrient utilization rate, reasonably utilizing resources and developing novel dietary supplements, and can be applied to the aspects of medicine, food and health care product development.
Drawings
FIG. 1 is a diagram showing the molecular weight distribution of a peptide of donkey-hide gelatin with homoglutamic acid;
FIG. 2 is an infrared spectrum of a homoglutamic acid donkey-hide gelatin peptide and a peptide iron chelate;
FIG. 3 is a fluorescence spectrum of different concentrations of ferrous ion and high glutamic acid colla Corii Asini peptide after action.
Detailed Description
Buffer a used at elution: 0.05mol/L sodium acetate and 0.05mol/L NaCl;
and (3) buffer solution B: 0.02mol/L disodium hydrogen phosphate and 0.05mol/L NaCl.
Example 1
1. Preparation and application of high-glutamic acid donkey collagen peptide I
(1) Uniformly mixing colla Corii Asini powder and ultrapure water until the substrate concentration is 5%, digesting the solution at 37 deg.C for 2min, adjusting pH to 3.0 with hydrochloric acid, adding pepsin solution (enzyme addition amount is 2000U/mL), oscillating at 37 deg.C and 200rpm in a desktop constant temperature oscillator for 2h, detecting pH, and maintaining pH at 3.0. The pH was adjusted to 7.0 with NaOH, and trypsin solution (enzyme addition amount: 500U/mL) was added and the mixture was shaken at 37 ℃ and 200rpm for 2 hours in a tabletop constant temperature shaker. Inactivating enzyme at 95 deg.C for 15min, cooling to room temperature, centrifuging at 6000r/min for 30min, and collecting supernatant to obtain colla Corii Asini polypeptide enzymatic hydrolysate.
(2) Washing the agarose microspheres with ultrapure water for multiple times, sequentially adding 20%, 40% and 60% of dimethyl sulfoxide to clean the agarose microspheres, transferring the cleaned medium to a conical flask, adding 60% of dimethyl sulfoxide solution and 30% of epoxy chloropropane as a reaction environment system, and adding NaOH to enable the concentration of the agarose microspheres to be 0.8 mol/L. The conical flask is sealed and then placed on a constant temperature air bath shaker at 40 ℃ and 160rpm for activation for 3.5 h. After activation, the agarose microspheres and iminodiacetic acid were stirred at a mass ratio of 1:0.6 at 60 ℃ for 16h at 150rpm for coupling. After the coupling is finished, filling materials into a column, washing 4 column volumes by using ultrapure water, and then using 0.1mol/L FeSO4Washing 6 column volumes with solution flow to make the filler fully adsorb Fe2+Washing with ultrapure water until the effluent liquid is free of Fe2+Detection, using buffer A (0.05mol/L sodium acetate, 0.05mol/L NaCl) balance, control the flow rate of 1 mL/min. The colla Corii Asini polypeptide enzymolysis solution (10mg/ml) is applied to the balanced affinity chromatography column, and the absorbance at 220nm is monitored. After loading, elution was carried out with buffer A until the baseline was stable, and then the fraction with strong affinity was eluted with buffer B (0.02mol/L disodium hydrogenphosphate, 0.05mol/L NaCl) to give A, B two peptide fractions. The trichloroacetic acid is adopted to precipitate the protein, the peptide content of the component B (namely the high glutamic acid donkey collagen peptide I) is determined to be 97.6 percent by an absorption difference method, and the glutamic acid content is 52.71 percent relative to the glycine by detecting the amino acid composition analysis after acid hydrolysis by a high performance liquid chromatograph.
(3) Adjusting the pH of A, B component polypeptide liquid to 5.5, and mixing with Fe2+、Ca2+、Zn2+Solution reaction of peptide with Fe2+、Ca2 +、Zn2+The mass ratio of the components is 4:1, the components are subjected to ultrasonic treatment for 15min, the components are subjected to constant-temperature oscillation chelation for 30min at 37 ℃, then the supernatant is obtained by filtration, and the iron chelation activity of the component A is respectively measured by adopting ICP-MS to be 6.3ug/mg, the calcium chelation activity is 2.1ug/mg, and the zinc chelation activity is 3.4 ug/mg; the iron chelating activity of the component B (i.e. the homoglutamic acid donkey collagen peptide I) is 23.6 mu g/mg, the calcium chelating activity is 12.7ug/mg, and the zinc chelating activity is 14.5ug/mg。
2. High glutamic acid donkey collagen peptide I molecular weight distribution
The molecular weight distribution was measured by high performance liquid chromatography. The results are shown in FIG. 1 and Table 1.
TABLE 1 relative molecular weight distribution of homoglutamic acid donkey collagen peptide I
As can be seen from Table 1, the relative molecular weight distribution of homoglutamic acid donkey collagen peptide I is mainly concentrated in 180-2000 Da. After enrichment by an affinity chromatographic column, more peptides with low molecular weight are reserved, and the small molecular peptides can generate strong chelation with metal ions, have biological nutritive value and promote the absorption of the metal ions.
3. Preliminary identification of high-glutamic acid donkey-hide gelatin peptide and high-glutamic acid donkey-hide gelatin peptide iron chelate
(1) Fourier transform infrared spectroscopy
Respectively weighing high-glutamic acid donkey-hide gelatin peptide and donkey-hide gelatin peptide iron chelate samples and dried KBr powder at a ratio of 1:100 in an agate mortar, grinding, uniformly mixing, tabletting, and carrying out Fourier infrared spectrum scanning within a scanning range of 4000cm-1～400cm-1。
The results are shown in FIG. 2, where the high glutamic acid donkey-hide gelatin peptide is 3428.81cm-1The high-frequency absorption is the expansion vibration peak of the amide N-H; 1654cm-1A stretching vibration peak at which C ═ O is located, and C ═ O shifts to a low wave number due to the influence of an amino group; 1390cm-1The absorption peak of stretching vibration at carboxyl C-O is 1249cm-1And 1085cm-1The weak absorption peaks of (a) are C-N stretching vibration and C-H bending vibration. After the donkey-hide gelatin peptide chelated iron is generated by reaction with iron, some main absorption peaks are obviously shifted, and the relative intensity is also changed. 3428cm-1The absorption peak is shifted to 3409cm-1And the amido participates in the chelation reaction after the iron is added, so that the steric hindrance is increased, the original conjugated system is damaged, and the absorption peak moves to a short wavelength. Stretching vibration of 1654cm-1Is moved to 1639cm-1At lower wavenumbers, it is indicated that the carboxyl oxygen participates in the chelation reaction; 1249cm-1And 1085cm-1The absorption peak at (C) disappeared, and 1124cm-1And a new larger absorption peak appears, which shows that the C-N stretching vibration and the N-H bending vibration absorption are changed after the iron ions are acted. The infrared absorption result shows that iron ions mainly have chelation reaction with carboxyl oxygen and amino nitrogen atoms of the donkey-hide gelatin peptide to generate a new substance.
(2) Fluorescence spectroscopy
Setting different concentration gradients of Fe2+The solution was added to the homo-glutamic acid donkey-hide gelatin peptide (EP) solution and the two were mixed well to a final concentration of 0.1mg/mL of peptide. The excitation wavelength of a fluorescence spectrophotometer is set to be 335nm, and the emission wavelength is set to be 365-500 nm.
As a result, Fe was added to the peptide as shown in FIG. 32+After the solution, the fluorescence intensity at 335nm excitation is significantly reduced, with Fe2+The fluorescence intensity is continuously reduced when the concentration is increased, and the emission wavelength under the maximum fluorescence intensity has a red shift trend, which shows that the interaction between the chromophore and the iron ions in the donkey-hide gelatin peptide causes the energy change of the excited state and the change of the fluorescence intensity. Fe2+The chelation reaction with the peptide results in quenching of the chelate fluorescence, and the reduction in fluorescence intensity is a typical indication of the occurrence of a change in the structure of the polypeptide.
Example 2
1. Preparation and application of high-glutamic acid donkey collagen peptide II
(1) Mixing donkey bone powder with ultrapure water until the substrate concentration is 6%, adjusting pH to 7.0, adding neutral protease (with enzyme amount of 3000U/mL) in a table type constant temperature oscillator at 40 deg.C, oscillating at 200rpm for 2 hr, detecting pH, and maintaining pH at 7.0. The pH was adjusted to 9.0 with NaOH, alkaline protease (2000U/mL) was added, and the mixture was shaken at 45 ℃ and 200rpm for 2 hours in a tabletop shaker. Inactivating enzyme at 90 deg.C for 10min, cooling to room temperature at 8000r/min, centrifuging for 20min, and collecting supernatant to obtain donkey bone collagen peptide enzymatic hydrolysate.
(2) Washing agarose microspheres with ultrapure water for multiple times, sequentially adding 40% and 60% dimethyl sulfoxide, and washingThe cleaned medium is transferred to a conical flask, 60% of dimethyl sulfoxide solution and 30% of epoxy chloropropane are used as a reaction environment system, and NaOH is added to ensure that the concentration of the reaction environment system is 0.8 mol/L. The conical flask is sealed and then placed on a constant temperature air bath shaker at 50 ℃ and 160rpm for activation for 3 h. After activation, the agarose microspheres and iminodiacetic acid were stirred at a mass ratio of 1:0.6 at 50 ℃ for 12h at 150rpm for coupling. After the coupling is finished, filling materials into a column, washing 4 column volumes by using ultrapure water, and then using 0.1mol/L FeSO4Washing 6 column volumes with solution flow to make the filler fully adsorb Fe2+Washing with ultrapure water until the effluent liquid is free of Fe2+Detection, using buffer A (0.05mol/L sodium acetate, 0.05mol/L NaCl) balance, flow rate of 1 mL/min. The donkey collagen peptide enzymatic hydrolysate (10mg/ml) was applied to an equilibrated affinity chromatography column and the absorbance at 220nm was monitored. After loading, elution was carried out with buffer A until the baseline was stable, and then the fraction with strong affinity was eluted with buffer B (0.02mol/L disodium hydrogenphosphate, 0.05mol/L NaCl) to give A, B two peptide fractions. The trichloroacetic acid is adopted to precipitate the protein, the peptide content of the component B (namely the high glutamic acid donkey collagen peptide II) is determined to be 98.1 percent by an absorption difference method, and the glutamic acid content is 62.38 percent relative to the glycine by detecting the amino acid composition analysis after acid hydrolysis by a high performance liquid chromatograph.
(3) Adjusting the pH of A, B component polypeptide liquid to 6.0, and mixing with Fe2+、Ca2+、Zn2+Solution reaction of peptide with Fe2+、Ca2 +、Zn2+The mass ratio of the components is 3:1, the components are subjected to ultrasonic treatment for 15min, the components are subjected to constant-temperature oscillation chelation for 60min at the temperature of 30 ℃, then supernatant liquid is obtained through filtration, and the iron chelation activity, the calcium chelation activity and the zinc chelation activity of the component A are respectively measured by ICP-MS to be 5.5ug/mg, 1.9ug/mg and 1.2 ug/mg; the iron chelating activity of the component B (i.e. the homoglutamic acid donkey collagen peptide II) is 27.4 mu g/mg, the calcium chelating activity is 18.9ug/mg, and the zinc chelating activity is 17.5 ug/mg.
2. High glutamic acid donkey collagen peptide II molecular weight distribution
The molecular weight distribution was measured by high performance liquid chromatography. The results are shown in Table 2.
TABLE 2 relative molecular weight distribution of homoglutamic acid donkey collagen peptide II
As can be seen from Table 2, the relative molecular weight distribution of homoglutamic acid donkey collagen peptide II is mainly concentrated in 180-2000 Da. After enrichment by an affinity chromatographic column, more peptides with low molecular weight are reserved, and the small molecular peptides can generate strong chelation with metal ions, have biological nutritive value and promote the absorption of the metal ions.
Example 3
1. Preparation and application of high-glutamic acid donkey collagen peptide III
(1) Mixing dried donkey hide gelatin raw powder with ultrapure water until the substrate concentration is 8%, melting at 95 deg.C for 30min, cooling to 45 deg.C, adjusting pH to 7.5, adding papain solution (enzyme addition amount 4000U/mL), shaking at 45 deg.C and 200rpm in a table type constant temperature oscillator for 3h, detecting pH, and keeping pH at 7.5. Adjusting pH to 9.0 with NaOH, adding alkaline protease solution (enzyme amount of 2000U/mL), and shaking at 50 deg.C and 200rpm in a table type constant temperature shaker for 3 hr. Inactivating enzyme at 100 deg.C for 5min, cooling to room temperature at rotation speed of 10000r/min, centrifuging for 15min, and collecting supernatant to obtain donkey skin polypeptide enzymolysis solution.
(2) Washing the agarose microspheres with ultrapure water for multiple times, adding 60% dimethyl sulfoxide to clean the agarose microspheres for multiple times, transferring the cleaned medium to a conical flask, taking 60% dimethyl sulfoxide solution and 30% epoxy chloropropane as a reaction environment system, and adding NaOH to enable the concentration of the agarose microspheres to be 0.8 mol/L. The conical flask is sealed and then placed on a constant temperature air bath shaker at 55 ℃ and 160rpm for activation for 2.5 h. After activation, the agarose microspheres and iminodiacetic acid were stirred at a mass ratio of 1:0.6 at 65 ℃ for 8h at 150rpm for coupling. After the coupling is finished, filling materials into a column, washing 4 column volumes by using ultrapure water, and then using 0.1mol/L FeSO4Washing 6 column volumes with solution flow to make the filler fully adsorb Fe2+Washing with ultrapure water until the effluent liquid is free of Fe2+Detection, using buffer A (0.05mol/L sodium acetate, 0.05mol/L NaCl) balance, control the flow rate of 1 mL/min. Adding donkey hide collagen peptide enzymolysis solution (10mg/ml) to the balanced affinity chromatography column, and monitoring absorbance at 220nm. After loading, elution was carried out with buffer A until the baseline was stable, and then the fraction with strong affinity was eluted with buffer B (0.02mol/L disodium hydrogenphosphate, 0.05mol/L NaCl) to give A, B two peptide fractions. The trichloroacetic acid is adopted to precipitate the protein, the peptide content of the component B (namely the high glutamic acid donkey collagen peptide II) is determined to be 98.7 percent by an absorption difference method, and the glutamic acid content is 74.15 percent relative to the glycine by detecting the amino acid composition analysis after acid hydrolysis by a high performance liquid chromatograph.
(3) Adjusting the pH of A, B component polypeptide liquid to 6.5, and mixing with Fe2+、Ca2+、Zn2+Solution reaction of peptide with Fe2+、Ca2 +、Zn2+The mass ratio of the components is 2:1, the components are subjected to ultrasonic treatment for 15min, the components are subjected to constant-temperature oscillation chelation for 60min at 40 ℃, then the supernatant is obtained through filtration, and the iron chelation activity of the component A is respectively measured to be 4.7ug/mg, the calcium chelation activity is 0.9ug/mg, and the zinc chelation activity is 2.1ug/mg by adopting ICP-MS; the iron chelating activity of the component B (homoglutamic acid donkey collagen peptide III) is 32.8 mu g/mg, the calcium chelating activity is 24.6ug/mg, and the zinc chelating activity is 23.2 ug/mg.
2. High glutamic acid donkey collagen peptide III molecular weight distribution
The molecular weight distribution was measured by high performance liquid chromatography. The results are shown in Table 3.
TABLE 3 relative molecular weight distribution of homoglutamic acid donkey collagen peptide III
As can be seen from Table 2, the relative molecular weight distribution of homoglutamic acid donkey collagen peptide II is mainly concentrated in 180-2000 Da. After enrichment by an affinity chromatographic column, more peptides with low molecular weight are reserved, and the small molecular peptides can generate strong chelation with metal ions, have biological nutritive value and promote the absorption of the metal ions.
3. Analysis and comparison of relative amino acid content of high-glutamic acid donkey collagen peptide
TABLE 4 analysis of the relative amino acid content of homoglutamic acid donkey collagen peptides I, II, III
Amino acid composition has an important influence on the function of the peptide. The donkey collagen contains amino acid such as glycine (Gly), proline (Pro), alanine (Ala) and glutamic acid (Glu) in sequence, and after enzymolysis and separation preparation, the content of the glutamic acid (Glu) is obviously improved, and the content of the proline (Pro) and the alanine (Ala) is obviously reduced. Glutamic acid is used as dicarboxylic acid amino acid, is rich in chelating group, has strong metal chelating activity, can enrich and expose more glutamic acid through enzymolysis digestion and affinity column chromatography, simultaneously enhances the metal chelating capacity of peptide, and improves the functional activity of polypeptide.
The first experimental example: the high-glutamic acid donkey collagen peptide iron chelate provided by the invention can improve the symptoms of iron-deficiency anemia of mice
The high-glutamic acid donkey collagen peptides prepared in the embodiments 1, 2 and 3 are respectively chelated with ferrous ions to obtain iron chelates I, II and III of the high-glutamic acid donkey collagen peptides, and compared with the traditional iron supplement agent ferrous sulfate, the test method and the result are as follows:
the experimental method comprises the following steps: after the primary weaning mice are fed and stayed, the mice are randomly divided into 1 group of normal control groups and 6 groups of anemia building groups according to the body weight, the normal groups are fed with normal feed (the iron content is 45mg/kg), the building groups are fed with low-iron feed (the iron content is 10mg/kg), after 4 weeks of model building, the hemoglobin HB content is measured, and HB <100g/L is taken as the success of model building. The IDA model mice successfully molded are randomly divided into 6 groups according to the hemoglobin value, each group comprises 10 mice, namely a normal group, a model group, an iron sulfate group, a donkey-hide gelatin group and a homoglutamic acid donkey collagen peptide iron chelate (I group, II group and III group), wherein the iron adding amount of each experimental group is consistent. On the 2 nd day after the molding is successful, the components are respectively and simultaneously subjected to intragastric administration for 1 time every day, the intragastric volume is 0.2mL/40g.bw, and the mice in a control group are intragastric with normal saline with the same volume. After 4 weeks of continuous administration, after determining the rise of the hemogram index, fasting for 12h, collecting blood and analyzing the hemogram value and biochemical index.
The experimental results are as follows: the influence of the high-glutamic acid donkey collagen peptide iron chelate compound on the blood picture value of mice with iron deficiency anemia is shown in a table 5.
TABLE 5 Effect of high glutamic acid donkey collagen peptide iron chelate on iron deficiency anemia mouse hemogram index
As can be seen from table 5, the iron deficiency anemia model group has significant differences in the red blood cell content, hemoglobin content, hematocrit, and mean red blood cell volume (p <0.05) compared to the normal group of mice. After different forms of iron supplement agents are continuously administrated by stomach irrigation for 4 weeks, compared with an anemia model group, the high-glutamic acid donkey collagen peptide iron chelate I, II and III can obviously improve the red blood cell content, the hemoglobin content, the hematocrit and the average red blood cell volume in the blood of an anemia mouse. Compared with the ferrous sulfate group, the administration can obviously improve the red blood cell content and the hemoglobin value in the blood of the mice, and the II group and the III group have no difference compared with the normal group. This indicates that the high glutamic acid donkey collagen peptide iron chelate has an improvement effect on iron deficiency anemia.
Experiment example two: the influence of the high-glutamic acid donkey collagen peptide iron chelate on the body immunity of anemic mice
In order to explore the influence of the high-glutamic acid donkey collagen peptide iron chelate prepared by the invention on the body immunity of anemic mice, the high-glutamic acid donkey collagen peptide iron chelate is compared with ferrous sulfate to respectively detect the expression level of T lymphocytes of each group of mice.
The experimental results are as follows: the influence of the high glutamic acid donkey collagen peptide iron chelate compound on the body immunity of anemic mice is shown in table 6.
TABLE 6 Effect of homoglutamic acid donkey collagen peptide iron chelate on T lymphocyte levels in anemic mice
T lymphocytes are an important part of the immune system, CD4 +T cells are induced cells, CD8 +T cells are cytotoxic cells. As shown in the Table, administrationCD of each group administered after different iron supplementation4 +Increased proportion of T cells, CD8 +The proportion of T cells is reduced, under the same dosage level, the high glutamic acid donkey collagen peptide iron chelate group and the ferrous sulfate group have significant difference, and the proportion of the group III T cells is close to that of the normal group. Shows that the administration of the high-glutamic acid donkey collagen peptide iron chelate is beneficial to the recovery of the immunologic function of the donkey collagen peptide iron chelate so as to improve the anemia symptom, and the supplement of iron can increase CD (compact disc) by supplementing iron4 +Proportion of T cells, and reduced CD8 +T cell ratio, and enhancing immunity.
The influence of the high-glutamic acid donkey collagen peptide iron chelate on the improvement of the hematological state and the body immunity of an anemic mouse is researched through an animal experiment, and the high-glutamic acid donkey collagen peptide iron chelate prepared from the high-glutamic acid donkey collagen active peptide can restore the hematological index of the anemic mouse to a normal level, and has a better effect than that of the ferrous sulfate of the traditional iron supplement; the anemia-improving health-care food has the beneficial effects of improving the anemia state, simultaneously recovering the collective immune function, positively influencing the immunity of the organism, improving the immunity of the organism and being applied to the production of foods, medicines and health-care products.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. The donkey collagen peptide is characterized in that the content of glutamic acid in the donkey collagen peptide is 50-75% relative to glycine, the content of peptides with the relative molecular mass of 180-2000 Da accounts for more than 70% of the total donkey collagen peptide, and the content of peptides is 95-99%.
2. A method for preparing the donkey collagen peptide according to claim 1, characterized by comprising the following steps:
(1) enzymolysis and digestion: selecting donkey-derived collagen, carrying out in-vitro digestion by adopting a composite enzymolysis technology, inactivating enzymes, centrifuging, and taking supernatant fluid, namely collagen polypeptide enzymolysis liquid;
(2) separation preparation: and (3) performing column chromatography and elution by using eluent on the collagen polypeptide hydrolysate, separating components with different adsorption strengths, collecting peptide components with strong adsorption force, performing nanofiltration, concentration, desalination and freeze drying to obtain the high-glutamic acid donkey collagen peptide.
3. The method according to claim 2, wherein the donkey-derived collagen of step (1) is one or more of donkey-hide gelatin, donkey skin, donkey bone, donkey teeth and donkey hoof.
4. Donkey collagen peptide metal chelate prepared by using donkey collagen peptide according to claim 1, characterized in that the donkey collagen peptide is chelated with metal ions to obtain donkey collagen peptide metal chelate.
5. The method for preparing the donkey collagen peptide metal chelate complex as claimed in claim 4, wherein the pH of a collagen peptide solution is adjusted to 5.0-7.0, the mass ratio of collagen peptide to metal ions is 1: 1-4: 1, the temperature is 30-60 ℃, the chelating reaction is carried out for 30-90 min, and the donkey collagen peptide metal chelate complex is obtained by filtering, taking supernatant and freeze-drying.
6. The method of claim 5, wherein the metal ions comprise iron, calcium, and/or zinc.
7. Use of the donkey collagen peptide according to claim 1, or the donkey collagen peptide metal chelate according to claim 4, or a product containing the donkey collagen peptide metal chelate for improving the body immunity and iron deficiency anemia.
8. Use according to claim 7, wherein the product is intended to comprise a medicament, a pharmaceutical composition or a pharmaceutical excipient.
9. The use of claim 8, wherein the medicament or pharmaceutical composition further comprises microcapsules, microspheres, nanoparticles, and liposomes, and the pharmaceutical excipient comprises an excipient and an additive.
10. Use according to claim 9, wherein the product comprises, but is not limited to, a food product, a nutraceutical beverage, an enteral nutritional preparation, a dietary supplement, a veterinary drug or a feed additive.
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