CN113817019B - Prolyl endopeptidase natural inhibitor, and preparation method and application thereof - Google Patents
Prolyl endopeptidase natural inhibitor, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a prolyl endopeptidase natural inhibitor, a preparation method and application thereof, wherein the preparation method comprises the following steps: coarse extraction and heat treatment: heating the crude extract, cooling to room temperature, and centrifuging to obtain supernatant, namely crude protease inhibitor solution; separating by ultrafiltration membrane: separating the crude protease inhibitor solution by adopting an ultrafiltration membrane with the molecular weight cut-off of 14-20kDa, and collecting permeate; separating the permeate by using an ultrafiltration membrane with the molecular weight cutoff of 5-10kDa, and collecting concentrated solution which does not permeate the ultrafiltration membrane to obtain concentrated solution of the protease inhibitor with the molecular weight of 11-13 kDa; drying and pulverizing. The natural prolyl endopeptidase inhibitor contains a prolyl endopeptidase inhibiting component having a molecular weight of 11-13 kDa; the natural prolyl endopeptidase inhibitor has the activity inhibition rate of more than or equal to 80% on prolyl endopeptidase under the environment with the temperature of 40-90 ℃ and the pH value of 5-12, has the effect of inhibiting abalone autolysis, and has excellent market application prospect in abalone preservation.
Description
Technical Field
The invention relates to the field of protease inhibitors, in particular to a prolyl endopeptidase natural inhibitor, a preparation method and application thereof.
Background
Prolyl endopeptidase (Prolyl endopeptidase, PEP) is a special protease in the serine protease family that only degrades peptide chains containing proline residues and less than 33 amino acid residues. This property is clearly distinguished from the serine protease family, indicating that PEP protein conformations are different from other enzymes of the serine protease family, belonging to the novel serine proteases.
At present, PEPs have been isolated from a variety of different sources such as mammals, aquatic animals, plants, fungi, and the like, and PEPs from different biological sources have certain similarities and also have differences. At present, PEP has a common property in that it plays a role in learning and memory, DNA synthesis, cell differentiation and signal transduction by degrading peptide hormones and neuropeptides containing proline residues. PEPs of specific origin also have unique properties, for example, PEPs of microbial origin can be utilized to debitterize milk and whey products and to reduce beer turbidity in the fermentation of beer.
Prolyl endopeptidase inhibitors, abbreviated as PEPI, as the name implies, inhibit prolyl endopeptidase activity. At present, prolyl endopeptidase inhibitors have been commercialized and widely used clinically, for example: can degrade vasopressin, angiotensin, neurotensin, oxytocin, thyrotropin releasing hormone, etc.
Disclosure of Invention
The invention aims to provide a preparation method and application of a prolyl endopeptidase natural inhibitor, wherein the prolyl endopeptidase natural inhibitor is obtained from abalone viscera, and the prolyl endopeptidase natural inhibitor from the source has good heat stability and pH stability and has a strong inhibition effect on PEP. The specific expression is that the inhibitor can keep more than 90 percent of inhibition activity at 40-90 ℃, and still has 80 percent of relative inhibition activity when heated to 90 ℃. Can maintain the inhibition activity of 80% and above in the pH range of 5.0-12.0, and the inhibition activity is still 60% and above under the condition of strong acid of pH 3.0-4.0.
The inventors found that prolyl endopeptidase exists in abalone through research, and thought that the corresponding inhibitor may exist in abalone meat based on the self-balance mechanism of organisms, and finally an abalone-derived prolyl endopeptidase natural inhibitor (PEPI) is extracted from abalone viscera through continuous attempts. SDS-PAGE research shows that the molecular weight of PEPI is about 12kDa, and the PEPI is smaller than serine protease inhibitors from other aquatic animals reported before, and belongs to low molecular weight serine protease inhibitors. The PEPI is determined to be glycoprotein through PAS staining, which shows that the PEPI contains oligosaccharide chains, and the PEPI is combined with colorless fuchsin after being oxidized by iodic acid to form a purplish red dye to be deposited on the protein, so that the protein presents purplish red.
At present, the PEP inhibition drugs chemically synthesized by pracetin, JTP-4819, S-17092, KYP-2047 and the like have good clinical treatment effects in the aspects of neuroprotection and cognitive enhancement. Bioactive peptides of animal and plant origin have also been reported to have PEP inhibiting activity. The present inventors have found that natural inhibitors of prolyl endopeptidase derived from abalone have a specific inhibitory effect only on prolyl endopeptidase of abalone, and are not prolyl endopeptidase inhibitors disclosed in the market and prior art.
Based on accidental factors, the inventor immerses fresh abalone meat in a solution consisting of the natural prolyl endopeptidase inhibitor and the metalloprotease inhibitor (EDTA) of the abalone source extracted by the invention, discovers that the autolysis of abalone is obviously slowed down, and further researches find that the natural prolyl endopeptidase inhibitor and the metalloprotease inhibitor obtained by the preparation method have a brand new effect after being combined, namely, the autolysis of abalone is inhibited.
In the storage process of abalone, collagen in abalone muscle is degraded and autolyzed, which causes abalone deterioration and is a great difficulty in the current abalone preservation. The invention creatively mixes the abalone source prolyl endopeptidase natural inhibitor (PEPI) and the matrix metalloproteinase inhibitor (such as EDTA) for preserving abalone, and inhibits the self degradation of collagen in muscle in the storage process of abalone, thereby preventing the degradation of muscle in the storage process of abalone and finally achieving the preservation effect.
The specific scheme is as follows:
a method of preparing a natural inhibitor of prolyl endopeptidase comprising:
coarse extraction: mashing abalone viscera tissue, adding water, centrifuging, and collecting supernatant to obtain crude extract;
and (3) heat treatment: heating the crude extract, cooling to room temperature, and centrifuging to obtain supernatant, namely crude protease inhibitor solution;
separating by ultrafiltration membrane: separating the crude protease inhibitor solution by adopting an ultrafiltration membrane with the molecular weight cut-off of 14-20kDa, and collecting permeate; separating the permeate by using an ultrafiltration membrane with the molecular weight cutoff of 5-10kDa, and collecting concentrated solution which does not permeate the ultrafiltration membrane to obtain concentrated solution of the protease inhibitor with the molecular weight of 11-13 kDa;
drying and pulverizing: drying and pulverizing the protease inhibitor concentrate to obtain powder, namely the natural prolyl endopeptidase inhibitor.
Further, in the crude extraction, the volume ratio of abalone viscera to water is 1:3-5, the abalone viscera are placed in water for tissue trituration, the gravity acceleration of centrifugal treatment is 8000-12000g, and the time is 15-30min.
Further, the heating temperature of the heat treatment is 80-90 ℃ and the heating time is 10-20min; cooling to room temperature, and centrifuging at a gravitational acceleration of 8000-12000g for 15-30min.
Further, the ultrafiltration membrane separation is that the crude protease inhibitor solution is firstly separated by adopting an ultrafiltration membrane with the molecular weight cut-off of 14-16kDa, and permeate liquid is collected; and separating the permeate by using an ultrafiltration membrane with the molecular weight cutoff of 8-10kDa, and collecting concentrated solution which does not permeate the ultrafiltration membrane to obtain concentrated solution of the protease inhibitor with the molecular weight of 11-13 kDa.
Further, the drying powder preparation is one of freeze drying powder preparation and spray drying powder preparation.
The invention also protects a prolyl endopeptidase natural inhibitor, which is prepared by using the preparation method of the prolyl endopeptidase natural inhibitor, wherein the prolyl endopeptidase natural inhibitor contains a prolyl endopeptidase inhibition component with the molecular weight of 11-13 kDa; the natural prolyl endopeptidase inhibitor has an inhibition rate of 80% or more on the activity of abalone prolyl endopeptidase in an environment with a temperature of 40-90 ℃ and a pH=5-12.
The invention also protects the application of the prolyl endopeptidase natural inhibitor in abalone preservation.
The invention also provides an abalone preservative, which comprises the natural prolyl endopeptidase inhibitor and also comprises a matrix metalloproteinase inhibitor.
Further, the matrix metalloproteinase inhibitor is EDTA, and the mass ratio of the prolyl endopeptidase natural inhibitor to the matrix metalloproteinase inhibitor in the abalone preservative is 1-2:1-2.
The invention also provides a method for preserving abalone, which comprises the steps of adopting the abalone preservative, and dissolving the abalone preservative in water to obtain preservative solution with the mass concentration of 0.1-10%; then the abalone meat to be preserved is put into the preservative solution for soaking at the temperature of 0-8 ℃ for 20-60min; finally, the abalone meat is fished out, drained and stored in the environment of 0-4 ℃.
The beneficial effects are that:
(a) The prolyl endopeptidase natural inhibitor (PEPI) prepared from abalone viscera has good thermal stability and pH stability, and has a strong inhibition effect on abalone PEP.
(b) The invention mixes the prolyl endopeptidase natural inhibitor (PEPI) and the matrix metalloproteinase inhibitor (such as EDTA) for preserving abalone, solves the problems of preservation, storage, transportation and processing caused by autolysis of abalone due to collagen degradation, improves the quality of abalone products, prolongs the shelf life and has high popularization and application value.
(c) The invention uses abalone viscera as raw materials to develop the prolyl endopeptidase natural inhibitor, has low cost, and is beneficial to the efficient utilization of abalone processing byproducts.
Drawings
In order to more clearly illustrate the technical solutions of the present invention, the following brief description will be made on the accompanying drawings, which are given by way of illustration only and not limitation of the present invention.
FIG. 1 is a SDS-PAGE diagram of purified endogenous inhibitors of prolyl endopeptidase provided by one embodiment of the invention;
FIG. 2 is a graph of the effect of temperature on PEPI activity provided by one embodiment of the present invention;
FIG. 3 is a graph showing the effect of pH on PEPI activity provided by one embodiment of the present invention;
FIG. 4 is a graph showing the inhibition activity of an endogenous inhibitor of Prolyl Endopeptidase (PEPI) against PEP according to one embodiment of the present invention.
Detailed Description
Definitions of some of the terms used in the present invention are given below, and other unrecited terms have definitions and meanings well known in the art:
in the preparation method of the prolyl endopeptidase natural inhibitor, the crude extraction is used for extracting the prolyl endopeptidase natural inhibitor from abalone viscera, the heat treatment is used for removing foreign proteins through thermal denaturation, and the ultrafiltration membrane separation is used for purifying and enriching to obtain the prolyl endopeptidase natural inhibitor (PEPI) with the molecular weight of 11-13 kDa. The drying and pulverizing function is to dry the protease inhibitor concentrate and make into powder.
Specifically, the heat treatment is preferably carried out at 80-90 ℃ for 10-20min, and at the temperature, the protein which cannot bear the heating temperature is denatured, and the target object with good heat stability can be screened out, so that the separation of PEPI and heat-labile protease is realized.
In the ultrafiltration membrane separation process, it is critical to determine the molecular weight of the target object to be screened, and thereby select an appropriate membrane. Indeed, the inventors were not initially aware of the molecular weight range of natural inhibitors of prolyl endopeptidase. The inventor finally confirms that the crude protease inhibitor solution is separated by adopting an ultrafiltration membrane with the molecular weight cutoff of 14-20kDa through repeated experiments, and permeate liquid is collected; and separating the permeate by using an ultrafiltration membrane with the molecular weight cutoff of 5-10kDa, and collecting concentrated solution which does not permeate the ultrafiltration membrane to obtain concentrated solution of the protease inhibitor with the molecular weight of 11-13 kDa. Subsequent experiments prove that the product with the molecular weight range has excellent prolyl endopeptidase inhibition effect.
Preferred embodiments of the present invention will be described in more detail below. While the preferred embodiments of the present invention are described below, it should be understood that the present invention may be embodied in various forms and should not be limited to the embodiments set forth herein. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. In the examples below, "%" refers to weight percent, unless explicitly stated otherwise.
Example 1: preparation of natural inhibitors of prolyl endopeptidase
First, crude extraction: mashing abalone viscera in distilled water with volume of 3 times, centrifuging with gravity acceleration of 12000g for 15min to obtain supernatant, which is crude extract;
second, heat treatment: heating the crude extract at 80deg.C for 10min, immediately cooling to room temperature, centrifuging with gravity acceleration of 9500g, centrifuging for 25min to obtain supernatant, and collecting the supernatant as crude protease inhibitor solution;
third, ultrafiltration membrane separation: separating the crude protease inhibitor solution by adopting an ultrafiltration membrane with a molecular weight cutoff of 15kDa, collecting a permeate, separating and concentrating the permeate by using an ultrafiltration membrane with a molecular weight cutoff of 10kDa, and collecting an un-permeate to obtain a protease inhibitor concentrated solution with a molecular weight of 11-13 kDa;
and fourthly, drying to prepare powder, namely freeze-drying the concentrated solution of the protease inhibitor with the molecular weight of 11-13kDa to prepare powder of the natural inhibitor of the prolyl endopeptidase.
The method for testing the performance of the natural prolyl endopeptidase inhibitor (PEPI) comprises the steps of carrying out ammonium sulfate fractional precipitation on the crude protease inhibitor solution obtained in the second step in the example 1, removing a large amount of impurity proteins through Q-Sepharose strong anion column chromatography, eluting the target proteins by using a buffer solution (25 mmol/L Tris-HCl, pH8.0 and containing 4mmol/L beta-mercaptoethanol), collecting components with inhibition activity, carrying out ultrafiltration membrane separation in the third step, and loading the obtained protease inhibitor concentrate onto Superdex-200 to remove impurity proteins with larger molecular weight and collect inhibition activity peaks and parts with fewer impurity proteins. After SP-Sepharose cation column chromatography, a large amount of unadsorbed protein is removed by washing, thereby obtaining PEPI with higher purity.
1. Molecular weight
Molecular weight identification the molecular weight of PEPI was determined by SDS-PAGE electrophoresis analysis, in combination with the molecular weight of the standard protein.
The purified PEPI was analyzed by SDS-PAGE and its molecular weight was about 12kDa, as shown in FIG. 1.
2. Inhibition activity assay
PEPI inhibitory activity is indicated by inhibition of PEP activity. For the measurement, 50. Mu.L of PEP (commercially available product) and inhibitor sample (purified PEPI) were each thoroughly mixed in 850. Mu.L of 50mmol/L phosphate buffer (pH 6.5). After incubation at 4℃for 30min, 50. Mu.L of 10. Mu. Mol/L of Suc-Gly-Pro-MCA fluorogenic substrate was added, and after thorough mixing, the reaction was stopped by adding 1.5mL of methanol-isopropanol-distilled water (35:30:35, V/V) solution for 30min in a 37℃water bath. The fluorescence intensity of 7-amino-4-methylcoumarin (AMC) released from the fluorogenic substrate was measured at an excitation wavelength of 380nm and an emission wavelength of 450 nm. PEP activity without PEPI was used as a control group under the same conditions. The inhibition activity units of PEPI are defined as the amount of inhibition required to reduce the PEP activity units by 1. PEP activity units are defined as the amount of enzyme required to release 1nmoL AMC per minute.
2-1, thermal stability analysis
The method comprises the following steps: after incubation of the purified PEPI in 0.1mol/L phosphate buffer (pH 6.5) at different temperatures (40-90 ℃) for 30min, it was immediately cooled to room temperature. The inhibition activity of PEPI after treatment at different temperatures was determined as described in "inhibition Activity assay". The relative inhibition activity of PEPI is plotted on the abscissa, with the relative inhibition activity being 100% of that of the untreated sample, with the incubation temperature on the abscissa.
2-2, pH stability analysis
After incubation of the purified PEPI in 0.1mol/L buffer with different pH values (2.0-12.0) for 60min at 4 ℃. The PEP inhibitor inhibition activity after treatment at different pH was determined as described in "inhibition activity assay". The relative inhibition activity of PEP inhibitors is plotted on the abscissa, with the inhibition activity of untreated samples being 100% on the ordinate.
The buffer solutions are glycine-hydrochloric acid buffer solutions (pH 2.0-3.0) respectively; acetic acid-sodium acetate buffer (pH 4.0-5.0); disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH 6.0-7.0); tris-HCl buffer (pH 8.0-9.0); sodium carbonate-sodium hydroxide buffer (pH 10.0-11.0); potassium chloride-sodium hydroxide buffer (pH 12.0).
The test results show that the natural inhibitor prepared in example 1 has good thermal stability and pH stability after purification, and can maintain 80% inhibition activity in the range of pH 5-12 at 40-90deg.C, as shown in figures 2 and 3.
The effect of PEPI on PEP activity was measured at various concentrations, and the results are shown in FIG. 4, which shows that PEP activity gradually decreases as PEPI concentration increases. When the activity of PEP is reduced to 50%, the concentration of PEPI is 5.76 mu mol/L, namely the IC50 is 5.76 mu mol/L, which indicates that the PEPI of the viscera of the abalone has strong inhibition effect on the PEP.
In the above-mentioned inhibitory activity assay, the purification operation was combined with the preparation process of example 1 for the convenience of the test, and the properties of PEPI in the natural inhibitor powder of prolyl endopeptidase were not changed, so that it was found that the natural inhibitor powder of prolyl endopeptidase prepared in example 1 had equivalent heat stability and pH stability by the heat stability and pH stability of PEPI after purification.
Therefore, based on the inventive research, the invention creatively extracts the prolyl endopeptidase natural inhibitor with higher purity from abalone viscera through crude extraction, heat treatment and ultrafiltration membrane separation, and prepares the prolyl endopeptidase natural inhibitor into powder through drying, thereby being convenient for storage and transportation.
Example 2: preparation of natural inhibitors of prolyl endopeptidase
First, crude extraction: mashing abalone viscera in distilled water with volume of 5 times, centrifuging with gravity acceleration of 8000g, centrifuging for 30min to obtain supernatant, which is crude extract;
second, heat treatment: heating the crude extract at 80deg.C for 20min, immediately cooling to room temperature, centrifuging at a gravitational acceleration of 8000g for 30min to obtain supernatant which is crude protease inhibitor solution;
third, ultrafiltration membrane separation: separating the crude protease inhibitor solution by adopting an ultrafiltration membrane with a cut-off molecular weight of 16kDa, collecting a permeate, separating and concentrating the permeate by using an ultrafiltration membrane with a cut-off molecular weight of 10kDa, and collecting an un-permeate to obtain a protease inhibitor concentrated solution with a molecular weight of 11-13 kDa;
and fourthly, drying to prepare powder, namely freeze-drying the concentrated solution of the protease inhibitor with the molecular weight of 11-13kDa to prepare powder of the natural inhibitor of the prolyl endopeptidase.
Example 3: preparation of natural inhibitors of prolyl endopeptidase
First, crude extraction: mashing abalone viscera in 4 times volume of distilled water, centrifuging with gravity acceleration of 10000g, centrifuging for 20min to obtain supernatant, which is crude extract;
second, heat treatment: heating the crude extract at 85deg.C for 15min, immediately cooling to room temperature, centrifuging at gravity acceleration of 9000g for 20min to obtain supernatant, which is crude protease inhibitor solution;
third, ultrafiltration membrane separation: separating the crude protease inhibitor solution by adopting an ultrafiltration membrane with a cut-off molecular weight of 14kDa, collecting a permeate, separating and concentrating the permeate by using an ultrafiltration membrane with a cut-off molecular weight of 10kDa, and collecting an un-permeate to obtain a protease inhibitor concentrated solution with a molecular weight of 11-13 kDa;
and fourthly, drying to prepare powder, namely spray-drying the concentrated solution of the protease inhibitor with the molecular weight of 11-13kDa to prepare powder of the natural inhibitor of the prolyl endopeptidase.
Example 4: abalone preservative and application method thereof
Removing viscera and shells of fresh abalone, and cleaning to obtain abalone meat;
uniformly mixing the natural prolyl endopeptidase inhibitor prepared in the embodiment 1 with the EDTA (ethylene diamine tetraacetic acid) serving as a matrix metalloproteinase inhibitor according to a mixing ratio of 1:1, and dissolving the abalone preservative in water to prepare a preservative solution with the concentration of 0.1 wt%;
soaking abalone meat in preservative solution at a temperature below 8deg.C for 20min;
fishing out the abalone meat, and placing the abalone meat on a gauze for draining;
placing the drained abalone meat in a low-temperature environment of 0-4 ℃ for storage.
According to the evaluation index of the shelf life of the volatile basic nitrogen content in the fresh and frozen animal aquatic products (GB 2733-2015) of the national food safety standard (the volatile basic nitrogen content of shellfish is less than or equal to 15mg/100 g), the shelf life of the refrigerated abalone meat which is not treated by the preservative (abalone meat is directly refrigerated) is 8 days, and the shelf life of the refrigerated abalone meat which is subjected to the preservative treatment is prolonged to 15 days.
Example 5: abalone preservative and application method thereof
Removing viscera and shells of fresh abalone, and cleaning to obtain abalone meat;
uniformly mixing the natural prolyl endopeptidase inhibitor prepared in the embodiment 2 with the EDTA (ethylene diamine tetraacetic acid) serving as a matrix metalloproteinase inhibitor according to a mixing ratio of 1:2, and dissolving the abalone preservative in water to prepare a preservative solution with the concentration of 0.3 wt%;
soaking abalone meat in preservative solution at a temperature below 8deg.C for 40min;
fishing out the abalone meat, and placing the abalone meat on a gauze for draining;
the drained abalone meat is stored in a low-temperature environment of 0-4 ℃ for fresh selling and processing.
According to the evaluation index of the shelf life of the volatile basic nitrogen content in the fresh and frozen animal aquatic products (GB 2733-2015) of the national food safety standard (the volatile basic nitrogen content of shellfish is less than or equal to 15mg/100 g), the shelf life of the refrigerated abalone meat which is not treated by the preservative (abalone meat is directly refrigerated) is 8 days, and the shelf life of the refrigerated abalone meat which is subjected to the preservative treatment is prolonged to 18 days.
Example 6: abalone preservative and application method thereof
Removing viscera and shells of fresh abalone, and cleaning to obtain abalone meat;
the natural prolyl endopeptidase inhibitor prepared in the embodiment 3 and the EDTA matrix metalloproteinase inhibitor are uniformly mixed according to the mixing ratio of 2:1, and the abalone preservative is dissolved in water to prepare preservative solution with the concentration of 0.5 wt%;
soaking abalone meat in preservative solution at a temperature below 8deg.C for 60min;
fishing out the abalone meat, and placing the abalone meat on a gauze for draining;
the drained abalone meat is stored in a low-temperature environment of 0-4 ℃ for fresh selling and processing.
According to the evaluation index of the shelf life of the volatile basic nitrogen content in the fresh and frozen animal aquatic products (GB 2733-2015) of the national food safety standard (the volatile basic nitrogen content of shellfish is less than or equal to 15mg/100 g), the shelf life of the refrigerated abalone meat which is not treated by the preservative (abalone meat is directly refrigerated) is 8 days, and the shelf life of the refrigerated abalone meat which is subjected to the preservative treatment is prolonged to 20 days.
Comparative example
Referring to example 1, the difference is that in the third step, an ultrafiltration membrane with a molecular weight cut-off of 21kDa is used for separation, and permeate is collected; and separating the permeate by using an ultrafiltration membrane with the molecular weight cut-off of 4kDa, and collecting concentrated solution which does not permeate the ultrafiltration membrane to obtain concentrated solution of the protease inhibitor. And drying and pulverizing the protease inhibitor concentrate to obtain comparative powder.
Referring to example 4, the comparative powder and the matrix metalloproteinase inhibitor EDTA were prepared into abalone preservative in the same manner as in example 4, and the same preservation test was performed, and it was found that the shelf life of the treated chilled abalone meat was prolonged to 10 days.
Comparative example 2
Referring to example 4, the prolyl endopeptidase natural inhibitor prepared in example 1 was dissolved alone in water to prepare a preservative solution at a concentration of 0.1wt%, and the same preservation test was performed to find that the shelf life of the treated chilled abalone meat was 8 days.
Comparative example 3
Referring to example 4, the matrix metalloproteinase inhibitor EDTA alone was dissolved in water to prepare a preservative solution at a concentration of 0.1wt% and the same preservation test was performed, and it was found that the shelf life of the treated chilled abalone meat was prolonged to 10 days.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner without contradiction. The various possible combinations of the invention are not described in detail in order to avoid unnecessary repetition.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.
Claims (6)
1. A method for preparing a natural inhibitor of prolyl endopeptidase, which is characterized by comprising the following steps: comprising the following steps:
coarse extraction: mashing abalone viscera tissue, adding water, centrifuging, and collecting supernatant to obtain crude extract; in the crude extraction, the volume ratio of abalone viscera to water is 1:3-5, the abalone viscera are placed in water for tissue trituration, the gravity acceleration of centrifugal treatment is 8000-12000g, and the time is 15-30 min;
and (3) heat treatment: heating the crude extract, cooling to room temperature, and centrifuging to obtain supernatant, namely crude protease inhibitor solution; the heating temperature of the heat treatment is 80-90 ℃ and the heating time is 10-20min; cooling to room temperature, and centrifuging at a gravitational acceleration of 8000-12000-g for 15-30 min;
separating by ultrafiltration membrane: separating the crude protease inhibitor solution by adopting an ultrafiltration membrane with the molecular weight cut-off of 14-16kDa, and collecting permeate; separating the permeate by using an ultrafiltration membrane with the molecular weight cutoff of 8-10kDa, and collecting concentrated solution which does not permeate the ultrafiltration membrane to obtain concentrated solution of the protease inhibitor with the molecular weight of 11-13 kDa;
drying and pulverizing: drying and pulverizing the protease inhibitor concentrate to obtain powder, namely the natural prolyl endopeptidase inhibitor.
2. The method for preparing the natural inhibitor of prolyl endopeptidase according to claim 1, wherein: the drying powder preparation is one of freeze drying powder preparation and spray drying powder preparation.
3. The use of the prolyl endopeptidase natural inhibitor prepared by the preparation method of the prolyl endopeptidase natural inhibitor according to claim 1 or 2 in abalone preservation.
4. An abalone preservative comprising the prolyl endopeptidase natural inhibitor prepared by the preparation method of the prolyl endopeptidase natural inhibitor according to claim 1 or 2, which is characterized in that: matrix metalloproteinase inhibitors are also included.
5. The abalone preservative according to claim 4, characterized in that: the matrix metalloproteinase inhibitor is EDTA, and the mass ratio of the prolyl endopeptidase natural inhibitor to the matrix metalloproteinase inhibitor in the abalone preservative is 1-2:1-2.
6. The abalone fresh-keeping method is characterized by comprising the following steps of: adopting the abalone preservative according to claim 4 or 5, dissolving the abalone preservative in water to obtain preservative solution with the mass concentration of 0.1-10%; then the abalone meat to be preserved is put into the preservative solution for soaking at the temperature of 0-8 ℃ for 20-60min; finally, the abalone meat is fished out, drained and stored in the environment of 0-4 ℃.
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| CN103255186A (en) * | 2013-04-23 | 2013-08-21 | 集美大学 | Combined production method for abalone polysaccharide, lipid and protein peptide |
| CN105272887A (en) * | 2015-10-20 | 2016-01-27 | 集美大学 | Method for extracting taurine and polysaccharides from abalone's viscera simultaneously |
| KR20160058538A (en) * | 2014-11-17 | 2016-05-25 | 전라남도 | The manufacturing method of collagen peptide using abalone |
| CN109303789A (en) * | 2017-07-28 | 2019-02-05 | 中国科学院大连化学物理研究所 | A kind of pilose antler active component and preparation and application with prolyl inscribe enzyme inhibition activity |
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| CN103255186A (en) * | 2013-04-23 | 2013-08-21 | 集美大学 | Combined production method for abalone polysaccharide, lipid and protein peptide |
| KR20160058538A (en) * | 2014-11-17 | 2016-05-25 | 전라남도 | The manufacturing method of collagen peptide using abalone |
| CN105272887A (en) * | 2015-10-20 | 2016-01-27 | 集美大学 | Method for extracting taurine and polysaccharides from abalone's viscera simultaneously |
| CN109303789A (en) * | 2017-07-28 | 2019-02-05 | 中国科学院大连化学物理研究所 | A kind of pilose antler active component and preparation and application with prolyl inscribe enzyme inhibition activity |
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| Characterization and crystal structure of prolyl endopeptidase from abalone (Haliotis discus hannai);Wan-Yu Li 等;《Food Chem .》;第1-2页 * |
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