Preparation and separation and purification method of deerhorn and pilose antler double peptide by directional enzymolysis

Abstract

The application discloses a preparation and separation and purification method of deer antler peptide by directional enzymolysis, and belongs to the technical field of bioactive peptide preparation. Deer antler collagen is subjected to mechanical energy auxiliary treatment in an alkaline medium under low-temperature conditions lower than the thermal denaturation temperature of the deer antler collagen, so that the triple helix structure is reversibly unfolded to increase the accessible surface area of the protease and the conformation of the active segment of the ginseng protein is retained. The treated deer antler collagen and the ginseng protein are prepared into the same substrate solution and subjected to synergistic enzymolysis. Alkaline protease, collagenase and aminopeptidase are used for directional enzymolysis in steps, so that the product is directionally enriched with two characteristic dipeptides, prolyl-hydroxyproline and hydroxyprolyl-glycine. The dipeptides are separated by three steps of ultrafiltration, gradient elution of a macroporous resin and reverse phase preparative chromatography in series, so that the low-temperature mechanical energy reversible unfolding replaces the high-temperature pre-denaturation, the contradiction between the two types of raw materials in the treatment temperature window is eliminated, the two types of raw materials can be synergistically treated in the same system, and the dipeptide yield is higher than that of the separate enzymolysis.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide preparation technology, specifically relating to a method for the targeted enzymatic hydrolysis preparation and separation and purification of ginseng and deer antler dipeptides. Background Technology

[0002] Ginseng protein and deer antler collagen are two types of protein raw materials derived from traditional precious medicinal materials. Ginseng protein is rich in the arginine-glycine-aspartic acid active region, which has cell adhesion and biorecognition functions, while deer antler collagen uses glycine-XY triplet repeat sequences as its basic structural units. Both can release a variety of bioactive small peptides after enzymatic hydrolysis. Among them, prolyl-hydroxyproline dipeptide and hydroxyprolyl-glycine dipeptide are two characteristic dipeptides among collagen enzymatic hydrolysates that have been proven to have antioxidant and collagen synthesis-promoting activities, and have application value in the fields of functional foods and effective skincare. How to efficiently and directionally prepare and purify the above-mentioned characteristic dipeptides from ginseng protein and deer antler collagen raw materials has been a long-term technical focus in this field.

[0003] Chinese patent application CN118147260B discloses a method for co-extracting ginseng oligopeptides and rare saponins, and the application of the extracted product. The method includes ginseng crushing and coarse extraction, adding alkaline protease to the supernatant of the coarse extract and incubating at 50°C for proteolytic digestion, subsequently adding citric acid and adjusting the pH to 2.5-3.5 with hydrochloric acid, followed by incubation at 90-100°C to convert rare saponins, and multi-stage nanofiltration, to simultaneously obtain a co-extract of ginseng oligopeptides and rare saponins. However, this method only designs its enzymatic digestion and extraction process for ginseng as a single raw material and does not address the synergistic processing of raw materials such as deer antler collagen, which has a dense triple-helix structure and requires the release of interchain constraints to obtain sufficient protease accessibility. When attempting to synergistically enzymatically digest ginseng protein and deer antler collagen in the same system to prepare ginseng-deer antler dipeptides, this method cannot resolve the conflict between the structural development conditions required for deer antler collagen and the mild conditions required for the active regions of ginseng protein, thus failing to achieve the synergistic and targeted preparation of the two types of raw materials.

[0004] In their paper "Optimization of Enzymatic Hydrolysis Process and Evaluation of In Vitro Anti-Blue Light Activity of Deer Antler Peptide," published in *Food Industry Technology* (Vol. 44, No. 16, 2023, pp. 386-394), Zhang Jiayi et al. proposed preparing deer antler peptide by enzymatic hydrolysis with a single protease at 45℃, pH 8.5, and an enzyme-to-base ratio of 6% for 5 h, and verified that the deer antler peptide possesses antioxidant activities such as free radical scavenging. However, this method only targets deer antler as a single raw material and uses a relatively high enzymatic hydrolysis temperature to promote the unfolding and hydrolysis of collagen. If this temperature condition is directly applied to a synergistic system containing ginseng protein, the spatial structure upon which the ginseng protein relies to maintain its arginine-glycine-aspartic acid active region conformation will be disrupted.

[0005] The study found that the reason why the existing technologies mentioned above failed to achieve the synergistic and targeted preparation of ginseng protein and deer antler collagen lies in a long-neglected coupling constraint. It is generally believed in the art that the dense triple helix structure of deer antler collagen requires high-temperature pre-denaturation to obtain sufficient protease accessibility; however, in reality, while high temperatures unwind the collagen triple helix, they irreversibly destroy the spatial structure upon which ginseng protein maintains its active region conformation, leading to mutual exclusion of the optimal processing temperature windows for the two raw materials. This temperature-coupled contradiction prevents the two raw materials from being synergistically processed in the same enzymatic hydrolysis system, resulting in a low yield of the target dipeptide; furthermore, the separation and purification of the target dipeptide is difficult due to the mixture of the enzymatic hydrolysis product with oligopeptides and free amino acids. Therefore, how to achieve sufficient protease accessibility for both dense triple helix deer antler collagen and ginseng protein in the same enzymatic hydrolysis system without the constraint of high-temperature pre-denaturation, thereby synergistically and directionally enriching characteristic dipeptides, has become a core technical problem urgently needing to be solved in this field. Summary of the Invention

[0006] To address the technical problems in existing technologies where the temperature windows of two types of raw materials are mutually exclusive due to high-temperature pre-denaturation, preventing synergistic processing in the same enzymatic hydrolysis system and resulting in low yields and difficulties in separation and purification of the target dipeptides, this invention aims to provide a method for the targeted enzymatic hydrolysis preparation and purification of ginseng and deer antler dipeptides. This invention utilizes a combination of low-temperature gradient-assisted denaturation and stepwise targeted enzymatic hydrolysis with a complex protease to release the dense constraint of the triple helix of deer antler collagen without destroying the conformation of the active segment of ginseng protein. This achieves synergistic targeted preparation and efficient separation and purification of ginseng protein and deer antler collagen in the same enzymatic hydrolysis system.

[0007] To achieve the above objectives, the technical solution adopted in this invention is: a method for the directional enzymatic hydrolysis preparation and purification of ginseng and antler dipeptides, comprising a raw material pretreatment step, a synergistic enzymatic hydrolysis step, and a purification step. The raw material pretreatment step involves applying mechanical energy-assisted reversible unfolding treatment to the antler collagen at a low temperature below the thermal denaturation temperature of the antler collagen, thereby loosening the triple helix structure of the antler collagen to increase the surface area accessible to the protease, while maintaining the conformation of the active segment of the ginseng protein. The synergistic enzymatic hydrolysis step involves placing the antler collagen and the ginseng protein, which have undergone the reversible unfolding treatment, into the same enzymatic hydrolysis system, and performing stepwise directional enzymatic hydrolysis on the same system using a complex protease, thereby directionally cleaving the collagen characteristic sequence of the antler collagen to enrich the prolyl-hydroxyproline dipeptide and the hydroxyprolyl-glycine dipeptide. The separation and purification steps are as follows: the enzymatic hydrolysis products obtained from the synergistic enzymatic hydrolysis step are subjected to tandem separation of ultrafiltration, adsorption elution and reversed-phase chromatography to separate the prolyl-hydroxyproline dipeptide and the hydroxyprolyl-glycine dipeptide.

[0008] The core concept of this invention lies in replacing traditional high-temperature pre-denaturation with reversible unfolding assisted by mechanical energy under low-temperature conditions. This decouples the two processes—"enhancing the protease accessibility of deer antler collagen" and "destroying the thermosensitive active segment of ginseng protein"—which were originally strongly coupled by temperature. Under low-temperature conditions, the local shearing and microjets generated by mechanical energy can reversibly loosen the interchain hydrogen bonds of the triple helix at temperatures far below the thermal denaturation temperature. This allows deer antler collagen to obtain sufficient protease-accessible surface area without triggering thermal denaturation or destroying the conformation of the active segment of ginseng protein. This underlying treatment creates the premise for the synergistic processing of two heterogeneous proteins in the same enzymatic hydrolysis system and naturally gives rise to three mutually complementary designs: first, a sequential design of enzymatic hydrolysis windows where the two substrates are cleaved stepwise in the same system; second, synergistic interface conditions formed by the mass ratio of deer antler collagen and ginseng protein and the total protein concentration of the system; and third, a design that connects dipeptide enrichment with a three-step tandem separation.

[0009] Preferably, in the raw material pretreatment step, the low-temperature condition is 2℃~8℃, the reversible unfolding treatment is carried out under alkaline conditions of pH 10~11, the mechanical energy assistance is ultrasonic assistance, the ultrasonic power is 200 W~400 W, and the treatment time is 20 min~40 min; after the reversible unfolding treatment, the protease-accessible surface area of ​​the deer antler collagen is increased to 2.3 times~3.1 times that before the treatment.

[0010] Preferably, in the synergistic enzymatic hydrolysis step, the mass ratio of the deer antler collagen to the ginseng protein is 1.5:1 to 2.5:1, and the total protein concentration of the same enzymatic hydrolysis system is 30 g / L to 50 g / L. The stepwise directional enzymatic hydrolysis includes three steps: the first step involves enzymatic hydrolysis with alkaline protease at pH 8 to 9, temperature 50°C to 60°C, and enzyme dosage of 4000 U / g protein to 6000 U / g protein for 45 to 75 minutes, to unfold the substrate protein backbone and expose the internal peptide bonds; the second step involves adding collagenase to the same enzymatic hydrolysis system and continuing enzymatic hydrolysis at pH 7 to 7.5, temperature 35°C to 40°C, and enzyme dosage of 1500 U / g protein to 2500 U / g protein for 75 to 105 minutes, to directionally cleave the glycine-XY triplet sequence of the deer antler collagen; the third step involves adding aminopeptidase to the same enzymatic hydrolysis system at an enzyme dosage of 300 U / g protein to 500 U / g protein. The protein content is 100 U / g to 700 U / g, and the terminal free amino acids are trimmed. The enzyme is then inactivated at 90℃ to 98℃ for 8 min to 12 min to terminate the enzymatic hydrolysis. The degree of hydrolysis of the enzymatic hydrolysis product is 28% to 32%, and the sum of the two types of dipeptides accounts for no less than 45% of the total amino acid content.

[0011] Preferably, the separation and purification steps include: first, removing oligopeptide components with a molecular weight cutoff of 500 Da using an ultrafiltration membrane, with an ultrafiltration permeate recovery rate of not less than 82%; second, adsorbing with D101 macroporous resin followed by gradient ethanol elution to enrich hydrophobic dipeptide components, with the enrichment factor of the two types of dipeptides being 5.8 to 6.5 times; and third, separating the dipeptide monomers using reversed-phase preparative high-performance liquid chromatography, employing a C18 column and eluting with a gradient of 0.1% trifluoroacetic acid aqueous solution and acetonitrile.

[0012] The beneficial effects of this invention are as follows: First, this invention replaces high-temperature pre-denaturation with reversible unfolding assisted by low-temperature mechanical energy. While ensuring sufficient protease accessibility for deer antler collagen, it maintains the conformational integrity of the active segment of ginseng protein. This fundamentally eliminates the mutually exclusive coupling constraint of the treatment temperature windows of the two heterogeneous proteins, allowing ginseng protein and deer antler collagen to be synergistically processed in the same enzymatic hydrolysis system, avoiding the process redundancy and activity loss caused by separate processing followed by mixing. Second, this invention discovers that when the two raw materials are synergistically hydrolyzed in the same system, the yield of the target dipeptide shows a non-linear increase compared to the method of hydrolyzing the two raw materials separately and then mixing them. This synergistic effect exceeds the level that can be expected from the simple summation of the results of the individual hydrolysis of the two raw materials, indicating that there are synergistic interface conditions between the two substrates in the synergistic hydrolysis system that are conducive to the release of the characteristic dipeptide. Third, this invention effectively solves the difficulty in separating and purifying target dipeptides caused by mixing with oligopeptides and free amino acids through stepwise and targeted enzymatic hydrolysis by alkaline protease, collagenase and aminopeptidase, combined with ultrafiltration, macroporous resin adsorption and elution and reversed-phase preparative chromatography in a three-step tandem separation process. This yields high-purity prolyl-hydroxyproline dipeptide and hydroxyprolyl-glycine dipeptide, providing a targeted enzymatic hydrolysis and stepwise purification-driven preparation method for the study of the bioactive functions of ginseng and deer antler dipeptides and product development. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the process flow for the directional enzymatic hydrolysis preparation and separation and purification method of the ginseng and deer antler dipeptides of the present invention.

[0014] Figure 2 This is a graph showing the change in the degree of enzymatic hydrolysis as a function of enzymatic hydrolysis time for the embodiments and comparative examples of the present invention.

[0015] Figure 3 This is a schematic diagram of the chromatographic separation of the ginseng and deer antler dipeptide mixture obtained in the embodiments of the present invention by reverse-phase preparation high-performance liquid chromatography.

[0016] Figure 4 This is a bar chart comparing the yields of characteristic dipeptides in the embodiments and comparative examples of the present invention. Detailed Implementation

[0017] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention. In the following embodiments, the ginseng protein and deer antler collagen protein used are separately extracted and purified protein raw materials; the alkaline protease, collagenase, and aminopeptidase used are commercially available enzyme preparations; and all other reagents used are of analytical grade unless otherwise specified.

[0018] The extraction and preparation methods of ginseng protein and deer antler collagen raw materials used in this invention are as follows: Ginseng protein extraction and preparation: Ginseng is cleaned, dried, and pulverized. Purified water is added according to the material-to-liquid ratio for extraction. The extraction is carried out under low-temperature stirring. The supernatant is collected by centrifugation. The protein components are initially enriched by salting out or membrane separation. Then, the ginseng is desalted by dialysis or ultrafiltration and freeze-dried to obtain the ginseng protein raw material. The entire extraction process is controlled at low temperatures to protect the conformational integrity of the arginine-glycine-aspartic acid active region in the ginseng protein. Deer antler collagen extraction and preparation: Deer antler is defatted and pulverized. Enzymatic extraction is performed using dilute acid combined with pepsin. The supernatant is collected by centrifugation. Collagen is precipitated by salting out. Then, the collagen is desalted by dialysis and freeze-dried to obtain the deer antler collagen raw material. The obtained deer antler collagen retains a complete triple helix structure. After extraction and purification of the two types of raw materials, subsequent reversible unfolding and synergistic enzymatic hydrolysis are carried out according to the steps described in this invention.

[0019] Of the two types of characteristic dipeptides prepared in this invention, the prolyl-hydroxyproline dipeptide is composed of proline residues and 4-hydroxyproline residues linked by peptide bonds, and its molecular formula is C1. 10 H 16 N₂O₄ has a relative molecular mass of approximately 228.25; the hydroxyprolyl-glycine dipeptide is composed of 4-hydroxyproline residues and glycine residues linked by peptide bonds, and its molecular formula is C7H₂O₄. 12 N₂O₄, with a relative molecular mass of approximately 188.18. Both are known characteristic dipeptides among collagen protein hydrolysates, and can be qualitatively and quantitatively analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) with standard reference. The structures of the two prepared dipeptides were confirmed: their quasi-molecular ion peaks were determined by high-resolution mass spectrometry (HMS). The quasi-molecular ion of the proline-hydroxyproline dipeptide matched its relative molecular mass of 228.25, and the quasi-molecular ion of the hydroxyproline-glycine dipeptide matched its relative molecular mass of 188.18. Furthermore, the structures of the isolated dipeptide monomers were confirmed by assigning characteristic proton and carbon signals of the proline ring, hydroxyproline ring, and glycine residues using nuclear magnetic resonance (NMR) spectroscopy. The proline-hydroxyproline dipeptide exhibits activity in stimulating fibroblast proliferation and promoting hyaluronic acid synthesis, while the hydroxyproline-glycine dipeptide exhibits activity in promoting cell differentiation. Both possess antioxidant activity, thus the prepared ginseng and deer antler dipeptide mixture has application value in the fields of functional foods and effective skincare.

[0020] To clearly define the sequences of the two types of characteristic dipeptides and the active region of ginseng protein mentioned above, the following description is provided according to biological nomenclature. Peptide sequences are written from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus). Each amino acid residue is represented by a three-letter abbreviation connected by a hyphen. N-terminal and intermediate residues are named in acyl form, while C-terminal residues retain their full names. All natural amino acid residues involved are L-type, with hydroxyproline being trans-4-hydroxy-L-proline. Accordingly, the sequence of the prolyl-hydroxyproline dipeptide is abbreviated as Pro-Hyp, and the sequence of the hydroxyprolyl-glycine dipeptide is abbreviated as Hyp-Gly. Both originate from the glycine-XY triplet repeat sequence of deer antler collagen. A typical representative of this triplet is glyl-prolyl-hydroxyproline, whose sequence is abbreviated as Gly-Pro-Hyp. It can be absorbed by fibroblasts and stimulate collagen synthesis.

[0021] This invention focuses on preserving the arginine-glycine-aspartic acid motif, abbreviated as Arg-Gly-Asp (RGD), during the raw material pretreatment stage. This motif is a key functional site for extracellular matrix protein-mediated integrin recognition and cell adhesion. The ginseng peptides obtained from enzymatic hydrolysis of ginseng protein possess anti-fatigue, anti-aging, antioxidant, and immunomodulatory effects. Since the number of amino acid residues in the aforementioned prolyl-hydroxyproline and hydroxyprolyl-glycine dipeptides, as well as the glycine-prolyl-hydroxyproline triplet and arginine-glycine-aspartic acid motif, are all less than four, they are not included in the sequence listing according to sequence listing rules and are directly described in this specification using their chemical names and the aforementioned nomenclature.

[0022] The detection methods for the various indicators involved in the following examples and comparative examples are as follows: Determination of the retention rate of ginseng protein active segments: Characterized by the relative content ratio of the arginine-glycine-aspartic acid active segments in ginseng protein before and after treatment. Enzyme-linked immunosorbent assay (ELISA) was used to detect the immunoreaction signal of the active segment in the ginseng protein solution before and after treatment using antibodies that specifically identify the active segment. The percentage of the signal value after treatment to the signal value before treatment was taken as the retention rate of the active segment. This indicator reflects the degree of protection of the active conformation of ginseng protein during the raw material pretreatment stage. Determination of the increase in the accessible surface area of ​​deer antler collagen protease: Characterized by the accessible surface area probe method, i.e., a quantitative amount of protease was added to the deer antler collagen solution before and after treatment, and the initial hydrolysis rate per unit time was measured. The ratio of the initial hydrolysis rate after treatment to the initial hydrolysis rate before treatment was used to approximately characterize the increase in the accessible surface area of ​​the protease. This indicator reflects the loosening effect of reversible unfolding treatment on the triple-helix dense structure.

[0023] Determination of the degree of hydrolysis of enzymatic hydrolysis products: The increase in free amino groups in the enzymatic hydrolysis system was determined using the o-phthalaldehyde method. The degree of hydrolysis was expressed as the percentage of peptide bonds broken during enzymatic hydrolysis relative to the total number of peptide bonds in the substrate protein. This indicator reflects the extent of stepwise, directional enzymatic hydrolysis. Determination of the proportion of dipeptides in total amino acid content: The total amino acid content was determined after complete acid hydrolysis of the enzymatic hydrolysis products. Separately, the contents of prolyl-hydroxyproline dipeptide and hydroxyprolyl-glycine dipeptide in the enzymatic hydrolysis products were determined by reversed-phase high-performance liquid chromatography (RP-HPLC). The enrichment degree of dipeptides was characterized by the percentage of the sum of the contents of the two types of dipeptides in the total amino acid content. Determination of dipeptide purity and enrichment factor: Using prolyl-hydroxyproline and hydroxyprolyl-glycine standards as controls, the purity of the target dipeptide in each separated fraction was determined by the area normalization method using reversed-phase HPLC. The enrichment factor was characterized by the ratio of the mass fraction of the two types of dipeptides in the macroporous resin elution fraction to the mass fraction of the two types of dipeptides in the sample solution. Determination of combined yield: based on the mass of the final ginseng and antler dipeptide mixture obtained per 100 g of added deer antler collagen.

[0024] The reversed-phase high-performance liquid chromatography (RP-HPLC) detection conditions used in this invention are as follows: a C18 column, mobile phase A is a 0.1% (v / v) trifluoroacetic acid aqueous solution, mobile phase B is acetonitrile, a gradient elution program is used, the column temperature is maintained constant, and the detection wavelength is 220 nm. Under these conditions, prolyl-hydroxyproline dipeptide and hydroxyprolyl-glycine dipeptide achieve different retention times due to their hydrophobic differences, enabling baseline separation and accurate quantification. In vitro antioxidant activity was evaluated using the 1,1-diphenyl-2-trinitrophenylhydrazine free radical scavenging method. A mixture of ginseng and deer antler dipeptide solutions of varying mass concentrations was reacted with a free radical solution. The decrease in absorbance after the reaction was measured, and the free radical scavenging rate was calculated. The result was expressed as the half-maximal concentration (IC50). 50 It characterizes the strength of antioxidant capacity.

[0025] like Figure 1 As shown, the method of this invention consists of three stages in series: raw material pretreatment, synergistic enzymatic hydrolysis, and separation and purification. In the raw material pretreatment stage, deer antler collagen is placed in an alkaline aqueous solution and subjected to ultrasonic-assisted treatment at low temperature to reversibly loosen its triple-helix structure; ginseng protein is extracted and purified separately for later use to avoid affecting its active regions during the pretreatment stage. In the synergistic enzymatic hydrolysis stage, the reversibly expanded deer antler collagen and ginseng protein are mixed at a predetermined mass ratio to prepare the same enzymatic hydrolysis system. Stepwise directional enzymatic hydrolysis is carried out sequentially with alkaline protease, collagenase, and aminopeptidase, followed by inactivation to terminate the reaction. In the separation and purification stage, the enzymatic hydrolysis products are subjected to a three-step tandem separation process: ultrafiltration, macroporous resin adsorption elution, and reversed-phase preparative chromatography, ultimately yielding two high-purity characteristic dipeptides.

[0026] Example 1

[0027] This embodiment provides a method for the targeted enzymatic hydrolysis preparation and separation and purification of ginseng and deer antler dipeptides. The process parameters are taken at the lower end of the scope of this invention, as follows.

[0028] Raw material pretreatment: Deer antler collagen was prepared with purified water and adjusted to pH 10. It was then placed in a low-temperature environment at 2℃ and treated with ultrasound at 200 W for 20 min to reversibly dissolve the triple helix structure of the deer antler collagen. Measurements showed that the surface area accessible to proteases in the treated deer antler collagen increased to 2.3 times that before treatment, while the retention rate of the arginine-glycine-aspartic acid active region of ginseng protein reached over 96%.

[0029] Synergistic enzymatic hydrolysis: Deer antler collagen treated as described above was mixed with separately extracted and purified ginseng protein at a mass ratio of 1.5:1 to prepare a single enzymatic hydrolysis system with a total protein concentration of 30 g / L. In the first step, alkaline protease was added at a dosage of 4000 U / g protein, and hydrolysis was carried out at pH 8 and 50℃ for 45 min. In the second step, collagenase was added to the same system at a dosage of 1500 U / g protein, and hydrolysis was continued at pH 7 and 35℃ for 75 min. In the third step, aminopeptidase was added at a dosage of 300 U / g protein to trim terminal free amino acids, followed by inactivation at 90℃ for 12 min to terminate the enzymatic hydrolysis. The degree of hydrolysis of the hydrolysate was determined to be 28%, and the sum of prolyl-hydroxyproline dipeptide and hydroxyprolyl-glycine dipeptide accounted for 45% of the total amino acid content.

[0030] Separation and purification: The first step involved treating the enzymatic hydrolysis product with an ultrafiltration membrane having a molecular weight cutoff of 500 Da to remove oligopeptide components with molecular weights higher than the dipeptide. The permeate recovery rate was 82%. The permeate was collected and loaded onto a D101 macroporous resin column. Hydrophilic impurities were first washed away with purified water, followed by gradient elution with ethanol to enrich the hydrophobic dipeptide components. The enrichment factor for both types of dipeptides was 5.8-fold. The third step involved monomer separation using reversed-phase preparative high-performance liquid chromatography (RP-HPLC) on a C18 column with a gradient elution of 0.1% trifluoroacetic acid aqueous solution and acetonitrile. Monitoring was performed at 220 nm, and the two types of dipeptide components were collected according to their retention times. After concentration and drying, the dipeptide monomers were obtained. The final purity of the prolyl-hydroxyproline dipeptide was 95.5%, and the purity of the hydroxyprolyl-glycine dipeptide was 94.6%. 11.5 g of a ginseng-antler dipeptide mixture was prepared from 100 g of deer antler collagen.

[0031] Example 2

[0032] This embodiment provides a method for the targeted enzymatic hydrolysis preparation and separation and purification of ginseng and deer antler dipeptides. The process parameters are taken as the median values ​​within the range of this invention, which is the preferred embodiment of this invention, as detailed below.

[0033] Raw material pretreatment: Deer antler collagen was prepared with purified water and adjusted to pH 10.5. It was then placed in a low-temperature environment of 4℃ and treated with ultrasonic power of 300 W for 30 min to reversibly loosen the triple helix structure of the deer antler collagen. Measurements showed that the surface area accessible to proteases in the treated deer antler collagen increased to 2.8 times that before treatment, and the retention rate of the active segment of ginseng protein reached over 98%. Figure 2 As shown, in this embodiment, the degree of enzymatic hydrolysis steadily increases with the extension of enzymatic hydrolysis time, and tends to plateau in the later stage of enzymatic hydrolysis.

[0034] Synergistic enzymatic hydrolysis: The treated deer antler collagen and separately extracted and purified ginseng protein were mixed at a mass ratio of 2:1 to prepare a single enzymatic hydrolysis system with a total protein concentration of 40 g / L. In the first step, alkaline protease was added at a dosage of 5000 U / g protein, and enzymatic hydrolysis was carried out at pH 8.5 and 55℃ for 60 min to fully expand the substrate protein backbone and expose internal peptide bonds. In the second step, collagenase was added to the same system at a dosage of 2000 U / g protein, and enzymatic hydrolysis was continued at pH 7.2 and 37℃ for 90 min to directionally cleave the glycine-XY triplet sequence of deer antler collagen, enriching prolyl-hydroxyproline dipeptides and hydroxyprolyl-glycine dipeptides. In the third step, aminopeptidase was added at a dosage of 500 U / g protein to trim terminal free amino acids to improve dipeptide purity, followed by inactivation at 95℃ for 10 min to terminate the enzymatic hydrolysis. The degree of hydrolysis of the enzymatic hydrolysis products was determined to be 30%, and the sum of the two types of dipeptides accounted for 47% of the total amino acid content.

[0035] The reason why the stepwise targeted enzymatic hydrolysis of this invention proceeds in the order of alkaline protease, collagenase, and aminopeptidase is that the three enzymes form a progressively advancing synergistic relationship in terms of their target proteins and modes of action. The first step uses alkaline protease as an endopeptide to broadly coarsely cleave the reversibly unfolded substrate protein backbone, degrading the large protein molecule into medium-length peptides and exposing the peptide bonds originally embedded within the molecule, creating accessible sites for subsequent enzymatic action. Without this step, collagenase and aminopeptidase would be unable to fully exert their effects due to insufficient substrate access sites. The second step uses collagenase to target the unique glycine-XY triplet sequence of deer antler collagen, cleaving it into tripeptide precursors represented by glycine-proline-hydroxyproline. This step is a crucial directional step for enriching the target dipeptide; the high specificity of collagenase for the glycine-XY sequence ensures that the cleavage products are concentrated in specific tripeptide precursors rather than a random mixture of peptides. The third step involves using aminopeptidase to sequentially cleave the terminal residues from the amino terminus of the tripeptide precursor, trimming the glycine-proline-hydroxyproline tripeptide into a prolyl-hydroxyproline dipeptide, and further refining other terminal free amino acids. This increases both the relative content and purity of the target dipeptide. The three-step enzymatic sequence sequentially completes backbone unfolding, directional cleavage, and terminal trimming, enabling the substrate protein to be directionally transformed and enriched into two types of target dipeptides. This step-by-step design is the basis for the present invention's ability to achieve the targeted enrichment of characteristic dipeptides in a synergistic enzymatic digestion system.

[0036] Separation and purification: The first step involved using an ultrafiltration membrane with a molecular weight cutoff of 500 Da to remove oligopeptide components with molecular weights higher than the dipeptide, achieving a permeate recovery rate of 85%. The ultrafiltration permeate was collected and loaded onto a D101 macroporous resin column pre-equilibrated with purified water. The loading flow rate was controlled to ensure sufficient adsorption of the target dipeptide. After loading, unadsorbed hydrophilic impurities and free amino acids were washed away with purified water. Gradient elution was then performed with ethanol-water solutions of progressively increasing volume fractions. The hydrophobic eluent rich in the target dipeptide was collected. After concentration under reduced pressure to remove ethanol, the enrichment factor for both types of dipeptides was 6.2-fold. The third step involved separating the enriched components using reversed-phase preparative high-performance liquid chromatography (RP-HPLC). A C18 column was used with a gradient elution of 0.1% trifluoroacetic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B. Monitoring was performed at 220 nm. Chromatographic peaks of prolyl-hydroxyproline dipeptide and hydroxyprolyl-glycine dipeptide were collected according to retention time. These were then concentrated under reduced pressure and freeze-dried to obtain the two types of dipeptide monomers. Figure 3 As shown, the prolyl-hydroxyproline dipeptide and the hydroxyprolyl-glycine dipeptide were well separated in the chromatogram. The final purity of the prolyl-hydroxyproline dipeptide was 96.8%, and the purity of the hydroxyprolyl-glycine dipeptide was 95.3%. 12.4 g of the ginseng and antler dipeptide mixture was prepared from 100 g of deer antler collagen.

[0037] Example 3

[0038] This embodiment provides a method for the targeted enzymatic hydrolysis preparation and separation and purification of ginseng and deer antler dipeptides. The process parameters are taken at the upper end of the scope of this invention, as follows.

[0039] Raw material pretreatment: Deer antler collagen was prepared with purified water and adjusted to pH 11. It was then placed in a low-temperature environment of 8℃ and treated with ultrasound at 400 W for 40 min to reversibly dissolve the triple helix structure of the deer antler collagen. Measurements showed that the surface area accessible to proteases in the treated deer antler collagen increased to 3.1 times that before treatment, and the retention rate of the active segment of ginseng protein reached over 95%.

[0040] Synergistic enzymatic hydrolysis: Deer antler collagen treated as described above was mixed with separately extracted and purified ginseng protein at a mass ratio of 2.5:1 to prepare a single enzymatic hydrolysis system with a total protein concentration of 50 g / L. In the first step, alkaline protease was added at a dosage of 6000 U / g protein, and hydrolysis was carried out at pH 9 and 60℃ for 75 min. In the second step, collagenase was added to the same system at a dosage of 2500 U / g protein, and hydrolysis was continued at pH 7.5 and 40℃ for 105 min. In the third step, aminopeptidase was added at a dosage of 700 U / g protein to trim terminal free amino acids, followed by inactivation at 98℃ for 8 min to terminate the enzymatic hydrolysis. The degree of hydrolysis of the hydrolysate was determined to be 32%, and the sum of the two types of dipeptides accounted for 49% of the total amino acid content.

[0041] Separation and purification: In the first step, an ultrafiltration membrane with a molecular weight cutoff of 500 Da was used to remove oligopeptide components with molecular weights higher than the dipeptide, achieving a permeate recovery rate of 88%. The permeate was collected and loaded onto a D101 macroporous resin column. Hydrophilic impurities were first washed away with purified water, followed by gradient elution with ethanol to enrich the hydrophobic dipeptide components, achieving an enrichment factor of 6.5 times for both types of dipeptides. In the third step, reversed-phase preparative high-performance liquid chromatography (RP-HPLC) was used for monomer separation. A C18 column was used, with a gradient elution of 0.1% trifluoroacetic acid aqueous solution and acetonitrile, monitored at 220 nm. The two types of dipeptide components were collected according to their retention times, concentrated, and dried to obtain the dipeptide monomers. The final purity of the prolyl-hydroxyproline dipeptide was 97.2%, and the purity of the hydroxyprolyl-glycine dipeptide was 96.0%. 13.0 g of a ginseng-antler dipeptide mixture was prepared from 100 g of deer antler collagen.

[0042] Comparative Example 1

[0043] The only difference between this comparative example and Example 2 is that the low-temperature ultrasonic reversible expansion treatment in the raw material pretreatment stage is omitted, and a conventional high-temperature pre-denaturation method is used to treat the deer antler collagen. Specifically, the deer antler collagen is heated at 60°C to break its triple helix structure, and then mixed with ginseng protein at a mass ratio of 2:1 to prepare the same enzymatic hydrolysis system. All other synergistic enzymatic hydrolysis and separation / purification conditions are the same as in Example 2. Measurements showed that although the surface area accessible to the protease increased after high-temperature treatment of the deer antler collagen, the retention rate of the arginine-glycine-aspartic acid active region of the ginseng protein decreased sharply from over 98% in Example 2 to approximately 51%. 9.6 g of a ginseng-antler dipeptide mixture was prepared from 100 g of deer antler collagen, and the active component derived from ginseng protein in the obtained product was significantly reduced. This comparative example demonstrates that the characteristic of applying mechanical energy-assisted reversible unfolding treatment to deer antler collagen under low-temperature conditions while maintaining the conformation of the active segment of ginseng protein is essential for achieving synergistic processing of the two types of raw materials: once high-temperature pre-denaturation is used instead of low-temperature reversible unfolding, the active segment of ginseng protein is destroyed, and the two types of raw materials cannot be synergistically processed in the same system, thus confirming the irreplaceable nature of the underlying innovation of this invention in removing the temperature coupling constraint.

[0044] Comparative Example 2

[0045] The only difference between this comparative example and Example 2 is that the synergistic enzymatic hydrolysis of the two types of raw materials in the same system was omitted. Instead, the deer antler collagen and ginseng protein, which underwent low-temperature ultrasonic reversible expansion treatment, were placed in two separate systems and hydrolyzed separately according to the three-step directional enzymatic hydrolysis conditions of Example 2. After the enzymatic hydrolysis was completed, the two hydrolysate products were mixed. The pretreatment and purification conditions of the other raw materials were the same as in Example 2. The results showed that this comparative example yielded 10.3 g of a ginseng-antler dipeptide mixture per 100 g of deer antler collagen, significantly lower than the 12.4 g obtained from the synergistic enzymatic hydrolysis in Example 2. This comparative example demonstrates that the synergistic enzymatic hydrolysis of the two types of proteins in the same enzymatic hydrolysis system is necessary to improve the yield of the target dipeptide. The yield of the dipeptide obtained from the synergistic enzymatic hydrolysis showed a non-linear increase compared to the method of separate enzymatic hydrolysis followed by mixing. The difference between the two exceeded the level that could be expected from the simple summation of the results of the individual enzymatic hydrolysis of the two raw materials. This indicates that there are synergistic interface conditions between the two substrates in the same enzymatic hydrolysis system that are conducive to the release of the characteristic dipeptide, confirming the indispensability of the synergistic enzymatic hydrolysis feature at the molecular level.

[0046] Comparative Example 3

[0047] The only difference between this comparative example and Example 2 is that the stepwise directional enzymatic hydrolysis of the complex protease was omitted, and instead, only a single alkaline protease was used to hydrolyze the same enzymatic hydrolysis system. The amount of enzyme added and the total enzymatic hydrolysis time were comparable to the sum of the three enzymatic hydrolysis steps in Example 2. The pretreatment and purification conditions of the other raw materials were the same as in Example 2. Measurements showed that the combined proportion of prolyl-hydroxyproline dipeptide and hydroxyprolyl-glycine dipeptide in the enzymatic hydrolysis product of this comparative example accounted for only 22% of the total amino acid content, far lower than the 47% in Example 2. Only 5.8 g of the ginseng and antler dipeptide mixture was obtained from every 100 g of deer antler collagen. This comparative example demonstrates that the stepwise directional enzymatic hydrolysis using a complex protease to enrich characteristic dipeptides is essential for achieving the targeted enrichment of target dipeptides. Although a single alkaline protease can hydrolyze the protein backbone, it lacks the ability to directionally cleave collagen characteristic sequences and prune terminal free amino acids, thus failing to directionally convert and enrich the glycine-XY triplet into the target dipeptide. This molecular-level evidence confirms the indispensability of the stepwise directional enzymatic hydrolysis feature.

[0048] To systematically compare the technical effects of the various embodiments and comparative examples of the present invention, the key detection indicators are summarized in the table below. The detection indicators include the retention rate of the active segment of ginseng protein, the increase in the surface area accessible to deer antler collagen protease, the degree of hydrolysis of the enzymatic hydrolysis product, the proportion of the two types of dipeptides in the total amino acid content, the purity of prolyl-hydroxyproline dipeptide, the purity of hydroxyprolyl-glycine dipeptide, and the combined yield of the ginseng and deer antler dipeptide mixture obtained per 100 g of deer antler collagen.

[0049] Example 1 96% 2.3 times 28% 45% 95.5% 94.6% 11.5 g Example 2 98% 2.8 times 30% 47% 96.8% 95.3% 12.4 g Example 3 95% 3.1 times 32% 49% 97.2% 96.0% 13.0 g Comparative Example 1 51% 2.8 times 30% 40% 95.0% 94.0% 9.6 g Comparative Example 2 98% 2.8 times 30% 38% 95.8% 94.5% 10.3 g Comparative Example 3 98% 2.8 times 30% 22% 90.2% 88.6% 5.8 g

[0050] As shown in the table above, Examples 1 to 3, within the parameter range defined by this invention, all achieved high retention rates (above 95%) of the active ginseng protein segment and high-purity enrichment of the characteristic dipeptides. The combined yield of the ginseng-antler dipeptide mixture obtained per 100 g of deer antler collagen was 11.5 g to 13.0 g. Comparative Example 1, using high-temperature pre-denaturation instead of low-temperature reversible development, resulted in a sharp drop in the retention rate of the active ginseng protein segment to 51%, and the combined yield also decreased accordingly, confirming the necessity of the underlying feature of low-temperature reversible development. Comparative Example 2, which involved separately enzymatically hydrolyzing the two raw materials before mixing, had a combined yield of 10.3 g, significantly lower than the 12.4 g obtained from the synergistic enzymatic hydrolysis in Example 2. The difference indicates that the synergistic enzymatic hydrolysis system exhibits a synergistic interface effect beyond the expectation of simple addition. Comparative Example 3, using a single protease instead of stepwise directional enzymatic hydrolysis, resulted in a sharp drop in the dipeptide proportion from 47% to 22%, and the combined yield decreased to 5.8 g, confirming the necessity of stepwise directional enzymatic hydrolysis of the complex protease for the targeted enrichment of the target dipeptides. Figure 4 As shown, the yield of the characteristic dual peptide combination in Example 2 is significantly higher than that in the three comparative examples, which intuitively reflects the technical effect brought about by the synergistic effect of the three core features of the present invention.

[0051] Furthermore, the in vitro antioxidant activity of the ginseng and deer antler dipeptide mixture obtained in Example 2 was evaluated. Its scavenging rate against 1,1-diphenyl-2-trinitrophenylhydrazine free radicals increased with increasing dipeptide mass concentration, and the half-maximal scavenging concentration (IC50) was [value missing]. 50 The low level indicates that the prepared ginseng and deer antler dipeptide mixture has good antioxidant activity, which is consistent with the activities of prolyl-hydroxyproline dipeptide and hydroxyprolyl-glycine dipeptide reported in the literature.

[0052] Further comparative analysis of the data from each embodiment and comparative example reveals that the three core features of this invention—low-temperature reversible development, synergistic enzymatic hydrolysis, and stepwise directional enzymatic hydrolysis—contribute independently yet synergistically to the final technical effect, and none can be omitted. The only difference between Comparative Example 1 and Example 2 is the replacement of low-temperature reversible development with high-temperature pre-denaturation. The retention rate of the ginseng protein active segment in both examples plummeted from over 98% to 51%, and the combined yield decreased from 12.4 g to 9.6 g. This difference is directly attributed to the destruction of the conformation of the ginseng protein active segment by high temperature, confirming the crucial role of the low-temperature reversible development feature in protecting the heat-sensitive active segment and maintaining the prerequisite for synergistic processing of the two types of raw materials. The only difference between Comparative Example 2 and Example 2 is that the synergistic enzymatic hydrolysis was changed to separate enzymatic hydrolysis followed by mixing. The accessible surface area increase and degree of hydrolysis were the same in both examples, but the combined yield decreased from 12.4 g to 10.3 g. Since the pretreatment and hydrolysis conditions of the raw materials were completely identical in both groups, this yield difference can only be attributed to the synergistic interface effect unique to the same enzymatic hydrolysis system, thus eliminating interference from other variables and providing clear experimental evidence to support the nonlinear gain of synergistic enzymatic hydrolysis. The only difference between Comparative Example 3 and Example 2 is that a single alkaline protease was used instead of the three-step complex enzyme for stepwise directional enzymatic hydrolysis. The proportion of dipeptides decreased sharply from 47% to 22%, and the combined yield decreased from 12.4 g to 5.8 g. This confirms that the directional cleavage of the glycine-XY triplet by collagenase and the pruning of the terminal free amino acids by aminopeptidase are indispensable for the directional conversion and enrichment of substrate proteins into target dipeptides.

[0053] As can be seen from the above comparison, the three core features of this invention jointly ensure the efficient and targeted preparation of ginseng and deer antler dipeptides from three orthogonal dimensions: underlying processing conditions, system organization, and enzymatic hydrolysis. Low-temperature reversible expansion solves the prerequisite problem of whether the two types of raw materials can coexist in one system; synergistic enzymatic hydrolysis solves the problem of whether a gain can be generated after coexisting in one system; and stepwise targeted enzymatic hydrolysis solves the problem of whether enzymatic hydrolysis can directionally enrich the target dipeptides. The three features are progressive and interconnected; the absence of any one of them will prevent the core technical problem from being solved.

[0054] In summary, this invention achieves the synergistic directional enzymatic hydrolysis of two heterogeneous proteins by combining low-temperature gradient-assisted reversible unfolding with stepwise directional enzymatic hydrolysis of complex proteases, while maintaining the conformational integrity of the active segment of ginseng protein. High-purity two characteristic dipeptides were obtained through three-step tandem separation, effectively solving the technical problems of low yield and difficult separation and purification of target dipeptides caused by the inability to synergistically process the two types of raw materials in the prior art.

[0055] Furthermore, the method of this invention also has advantages in process robustness. The raw material pretreatment stage uses low temperature and mild alkaline conditions, avoiding the requirements of high temperature treatment on equipment temperature resistance and energy consumption. Moreover, the reversible unfolding process does not introduce additional chemical denaturants, which is beneficial for subsequent enzymatic hydrolysis and the food-grade and cosmetic-grade applications of the products. The synergistic enzymatic hydrolysis stage integrates the treatment of two types of raw materials into the same reaction system. Compared with the process route of enzymatically hydrolyzing the two types of raw materials separately and then mixing them, it eliminates the need for separate enzymatic hydrolysis tanks and intermediate transfer links, simplifies the process flow, and reduces batch-to-batch fluctuations. The three steps of stepwise directional enzymatic hydrolysis are all performed sequentially in the same system without intermediate separation. After the first step of alkaline protease hydrolysis, the system conditions are directly adjusted and collagenase is added. After the second step, aminopeptidase is added, and finally, the inactivation is achieved by a single temperature rise. The operation is continuous and easy to scale up. As shown in Examples 1 to 3, under different combinations of parameter values ​​within the range defined by this invention, the technical effects of high retention of active regions, high purity of dipeptides, and high combined yield can be stably obtained, indicating that the method of this invention has good tolerance to fluctuations in process parameters and is suitable for large-scale preparation.

[0056] The following further explains the value ranges and basis of the key process parameters of this invention, so that those skilled in the art can reasonably select and implement this invention within the specified range. Regarding the low-temperature conditions in the raw material pretreatment stage, this invention limits them to 2℃~8℃. Too low a temperature will increase the viscosity of the system and delay the efficiency of ultrasonic cavitation in loosening the triple helix; too high a temperature will gradually approach the instability range of the active segment of ginseng protein, causing a decrease in the retention rate of the active segment. As shown in Examples 1 to 3, the retention rates of the active segment at 2℃, 4℃, and 8℃ are 96%, 98%, and 95% or higher, respectively, with 4℃ being the preferred value, at which point the retention rate of the active segment is highest and the reachable surface area is sufficiently increased. Regarding alkaline conditions, this invention limits them to pH 10~11. This range can promote the loosening of hydrogen bonds between triple helix chains without causing alkaline hydrolysis of peptide bonds; pH 10.5 is preferred.

[0057] Regarding the ultrasonic parameters, this invention limits the ultrasonic power to 200 W~400 W and the processing time to 20 min~40 min. Insufficient power and time result in incomplete triple helix disintegration and limited increase in accessible surface area; excessive power and time may cause irreversible breakage of collagen chains due to cavitation shearing, deviating from the target of reversible unfolding. As shown in Examples 1 to 3, the accessible surface area within this range is increased to 2.3 times, 2.8 times, and 3.1 times that before treatment, respectively. Preferably, the ultrasonic power is 300 W and the processing time is 30 min. Regarding the mass ratio of deer antler collagen to ginseng protein, this invention limits it to 1.5:1~2.5:1. This ratio determines the relative abundance of the two types of substrates in the synergistic enzymatic hydrolysis system, thereby affecting the synergistic interface conditions and the yield of the target dipeptide; if the ratio is too low, the dipeptide precursor from deer antler collagen is insufficient; if the ratio is too high, the contribution of ginseng protein to the synergistic interface is weakened. A mass ratio of 2:1 is preferred. Regarding the total protein concentration of the system, this invention limits it to 30 g / L~50 g / L. If the concentration is too low, the enzymatic hydrolysis efficiency and single-batch output will be low. If the concentration is too high, the mass transfer of the system will be limited and the viscosity will increase, which is not conducive to the full contact between the enzyme and the substrate. 40 g / L is preferred.

[0058] Regarding the parameters for each step of the stepwise directional enzymatic hydrolysis, the first step involves adding alkaline protease at a dosage of 4000 U / g protein to 6000 U / g protein, pH 8 to 9, temperature 50℃ to 60℃, and time 45 min to 75 min, to ensure the substrate protein backbone fully unfolds and exposes internal peptide bonds. A preferred dosage is 5000 U / g protein, pH 8.5, temperature 55℃, and time 60 min. The second step involves adding collagenase at a dosage of 1500 U / g protein to 2500 U / g protein, pH 7 to 7.5, temperature 35℃ to 40℃, and time 75 min to 105 min, to perform directional cleavage of the glycine-XY triplet sequence. A preferred dosage is 2000 U / g protein, pH 7.2, temperature 37℃, and time 90 min. The third step involves adding aminopeptidase at a dosage of 300 U / g protein to 700 U / g protein. To improve peptide purity, an enzyme dosage of 500 U / g protein was used to trim terminal free amino acids. After enzymatic hydrolysis, the enzyme was inactivated at 90℃~98℃ for 8min~12min, preferably at 95℃ for 10min. Within this range, the degree of hydrolysis of the hydrolysate was controlled at 28%~32%, and the sum of the two types of peptides accounted for no less than 45% of the total amino acid content. If the hydrolysis was too low, insufficient peptide precursor formation and a low peptide proportion would result; if the hydrolysis was too high, the peptides would be further degraded into free amino acids and lost.

[0059] Regarding the parameters for the separation and purification stages, the first step involves setting the molecular weight cutoff of the ultrafiltration membrane to 500 Da. This cutoff falls between the target dipeptide and oligopeptide molecular weights, allowing both types of target dipeptides to permeate while retaining the oligopeptide component with a higher molecular weight. The permeate recovery rate is no less than 82%. The second step uses D101 macroporous resin, leveraging the moderate hydrophobicity imparted by the proline and hydroxyproline rings in the target dipeptide to achieve selective adsorption on the resin. Gradient ethanol elution is then used to enrich the hydrophobic dipeptide component, with enrichment factors of 5.8 to 6.5 times for the two types of dipeptides. The third step uses reversed-phase preparative high-performance liquid chromatography (RP-HPLC) for monomer separation. Utilizing the retention difference between prolyl-hydroxyproline dipeptide and hydroxyprolyl-glycine dipeptide on the C18 stationary phase, the two types of dipeptides are collected separately under a 0.1% trifluoroacetic acid aqueous solution-acetonitrile gradient elution condition, yielding high-purity dipeptide monomers. The three-step tandem separation progressively narrows down the range of target components, successively solving the separation problems in terms of molecular weight, hydrophobicity, and fine structure, thereby overcoming the difficulties in separation and purification caused by the mixing of target dipeptides with oligopeptides and free amino acids in the enzymatic hydrolysis products.

[0060] Regarding the synergistic interface effect exhibited by the synergistic enzymatic hydrolysis of this invention, the mechanism is presumed to be as follows: In the same enzymatic hydrolysis system, the deer antler collagen and ginseng protein, after being reversibly expanded at low temperature, come into full contact with each other. The peptides released by the ginseng protein after the first step of coarse cleavage by alkaline protease can improve the microenvironment of the system, thereby enhancing the directional cleavage efficiency of collagenase on the glycine-XY triplet sequence of deer antler collagen. At the same time, the coexistence of the two types of substrates in one system also reduces the feedback inhibition of enzyme activity by the products in their respective systems during separate enzymatic hydrolysis. It is precisely these synergistic interface conditions that cause the yield of the target dipeptide obtained by synergistic enzymatic hydrolysis to show a non-linear increase compared to the method of separately enzymatically hydrolyzing the two types of raw materials and then mixing them. This effect was directly verified in Comparative Example 2, that is, the combined yield of 12.4 g obtained by synergistic enzymatic hydrolysis is significantly higher than the 10.3 g obtained by separately enzymatically hydrolyzing and then mixing them. The difference between the two exceeds the level that can be expected by simply adding the results of the enzymatic hydrolysis of the two types of raw materials, constituting one of the unexpected technical effects of this invention.

[0061] It should be noted that the method described in this invention can be adapted to the characteristics of the raw material batch and the requirements of the target product during specific implementation. For example, when the triple helix thermal stability of the deer antler collagen used is high, the ultrasonic treatment time can be appropriately extended or the ultrasonic power increased within the low temperature range specified in this invention to ensure sufficient protease-accessible surface area. When higher purity is required for a certain type of target dipeptide, the gradient elution program can be optimized in the reversed-phase preparative chromatography stage to increase the separation degree of the two types of dipeptides. In addition to type D101, other nonpolar or weakly polar macroporous adsorption resins with moderate hydrophobicity can also be used as macroporous resins. In addition to C18, other reversed-phase packing materials suitable for the separation of small molecule peptides can also be used as the reversed-phase chromatographic stationary phase. The prepared ginseng and deer antler dipeptide mixture or the single dipeptide obtained after further separation can be used as an active ingredient in functional foods, health foods, special diets, and functional skin care products, and can also be used as a standard or active control for the study of the bioactive functions of ginseng and deer antler dipeptides. The above adjustments and applications are all conventional variations under the concept of this invention and do not depart from the essence of this invention.

[0062] Furthermore, the prolyl-hydroxyproline dipeptide and hydroxyprolyl-glycine dipeptide prepared in this invention, due to their small molecular weight and stable structure, are more easily absorbed and utilized than high molecular weight collagen or collagen oligopeptides. They also possess the activity contribution derived from ginseng protein. Therefore, the resulting ginseng and deer antler dipeptide mixture has synergistic application potential in functional products related to anti-oxidation and collagen synthesis promotion. The method of this invention efficiently and directionally prepares and purifies the active ingredients of two types of precious medicinal herb protein raw materials in dipeptide form, providing reliable technical support for the standardized preparation and functional evaluation of ginseng and deer antler dipeptides, which is conducive to promoting the development and application of related functional products.

[0063] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural modifications made based on the description and drawings of the present invention, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for the targeted enzymatic hydrolysis preparation and separation and purification of ginseng and deer antler dipeptides, characterized in that, The process includes the following steps: a raw material pretreatment step, in which deer antler collagen is subjected to a reversible unfolding treatment assisted by mechanical energy under low temperature conditions below the thermal denaturation temperature of deer antler collagen, so as to loosen the triple helix structure of the deer antler collagen to increase the surface area accessible to proteases, while keeping the conformation of the active segment of ginseng protein intact. In the synergistic enzymatic hydrolysis step, the deer antler collagen and ginseng protein, after the reversible unfolding treatment, are placed in the same enzymatic hydrolysis system at a set mass ratio. A complex protease is used to perform stepwise directional enzymatic hydrolysis on the same system. The stepwise directional enzymatic hydrolysis sequentially includes: coarsely cleaving the substrate protein backbone with an endopeptide to expose internal peptide bonds; directionally cleaving the glycine-XY triplet collagen characteristic sequence of the deer antler collagen; and trimming the terminal free amino acids of the cleavage product, thereby enriching the prolyl-hydroxyproline dipeptide and the hydroxyprolyl-glycine dipeptide. In the separation and purification step, the enzymatic hydrolysis product obtained from the synergistic enzymatic hydrolysis step is sequentially subjected to ultrafiltration to remove oligopeptide components with molecular weights higher than the dipeptide, adsorption elution to enrich the hydrophobic dipeptide components, and reversed-phase chromatography to separate the dipeptide monomers, thereby separating the prolyl-hydroxyproline dipeptide and the hydroxyprolyl-glycine dipeptide.

2. The method according to claim 1, characterized in that, In the raw material pretreatment step, the low temperature condition is 2℃~8℃, the reversible unfolding treatment is carried out under alkaline conditions of pH 10~11, and the mechanical energy assistance is ultrasonic assistance.

3. The method according to claim 2, characterized in that, The ultrasonic power of the ultrasound-assisted treatment is 200 W to 400 W, and the treatment time is 20 min to 40 min. After the reversible unfolding treatment, the surface area accessible to the protease of the deer antler collagen is increased to 2.3 to 3.1 times that before the treatment.

4. The method according to claim 1, characterized in that, In the synergistic enzymatic hydrolysis step, the mass ratio of the deer antler collagen to the ginseng protein is 1.5:1 to 2.5:1, and the total protein concentration of the same enzymatic hydrolysis system is 30 g / L to 50 g / L.

5. The method according to claim 1, characterized in that, The first step of the stepwise directional enzymatic hydrolysis is: using alkaline protease at pH 8-9, temperature 50℃-60℃, and enzyme dosage of 4000 U / g protein-6000 U / g protein for 45 min-75 min, so that the substrate protein backbone unfolds and exposes the internal peptide bonds.

6. The method according to claim 5, characterized in that, The second step of the stepwise directional enzymatic hydrolysis is as follows: after the first step, collagenase is added to the same enzymatic hydrolysis system, and enzymatic hydrolysis is continued for 75 min to 105 min under the conditions of pH 7~7.5, temperature 35℃~40℃, and enzyme dosage of 1500 U / g protein~2500 U / g protein, to perform directional cleavage of the glycine-XY triplet sequence of the deer antler collagen.

7. The method according to claim 6, characterized in that, The third step of the stepwise directional enzymatic hydrolysis is as follows: after the second step, aminopeptidase is added to the same enzymatic hydrolysis system at an enzyme dosage of 300 U / g protein to 700 U / g protein to trim terminal free amino acids, and then the enzymatic hydrolysis is terminated by inactivation at 90℃ to 98℃ for 8 min to 12 min; the degree of hydrolysis of the enzymatic hydrolysis product obtained by the stepwise directional enzymatic hydrolysis is 28% to 32%.

8. The method according to claim 1, characterized in that, In the enzymatic hydrolysis product obtained by the synergistic enzymatic hydrolysis step, the sum of the prolyl-hydroxyproline dipeptide and the hydroxyprolyl-glycine dipeptide accounts for no less than 45% of the total amino acid content.

9. The method according to claim 1, characterized in that, The separation and purification steps include: first, removing oligopeptide components with a molecular weight cutoff of 500 Da using an ultrafiltration membrane; second, enriching hydrophobic peptide components by adsorption with macroporous resin followed by gradient ethanol elution; and third, separating peptide monomers using reversed-phase preparative high-performance liquid chromatography.

10. The method according to claim 9, characterized in that, The recovery rate of the ultrafiltration permeate in the first step is not less than 82%; the second step uses D101 macroporous resin, and the enrichment factor of the prolyl-hydroxyproline dipeptide and the hydroxyprolyl-glycine dipeptide is 5.8 to 6.5 times; the reversed-phase preparative high-performance liquid chromatography in the third step uses a C18 column and elutes with a gradient of 0.1% trifluoroacetic acid aqueous solution-acetonitrile.

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